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Crosstalk and modulation of signaling between somatostatin and growth factor receptors in human breast… Zhou, Jiemin 2017

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CROSSTALK AND MODULATION OF SIGNALING BETWEEN SOMATOSTATIN AND GROWTH FACTOR RECEPTORS IN HUMAN BREAST CANCER CELL LINES  by Jiemin Zhou  MD., Nanjing Medical University, China, 2011  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in  The Faculty of Graduate and Postdoctoral Studies  (Pharmaceutical Sciences)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  August 2017  ©Jiemin Zhou, 2017 ii  Abstract The molecular mechanisms of breast cancer are poorly understood, which present serious therapeutic problems and complicates drug design. Cell surface receptors belonging to G-protein-coupled receptor (GPCR) and receptor tyrosine kinase (RTK) families are potential drug targets relevant to pathological conditions, and have attracted great interest from pharmaceutical industry.  Recent studies have suggested that somatostatin (SST) receptors (SSTR1-5) belonging to GPCR family may interact with human epidermal growth factor (EGF) receptors (ErbB 1-4) from RTK family in pathophysiological conditions, exerting antiproliferative effects that may be useful in the treatment of breast cancers. An understanding of molecular mechanisms responsible for these effects reveal new approaches to the design of efficient breast cancer therapies that would significantly improve the lives of patients.  The work presented in this thesis was conducted to investigate crosstalk between SSTR and ErbB proteins in BT-474 and SK-BR-3 breast cancer cell lines upon SST and/or EGF treatment, to clarify the underlying molecular mechanisms, and to explore their implications for cancer therapy. Several pairs of SSTR and ErbB proteins exhibited strong membrane coexpression and crosstalk in the presence of the tested ligands, including SSTR2/ErbB1, SSTR3/ErbB2, and SSTR5/ErbB3 in BT-474 cells, and SSTR5/ErbB1 in SK-BR-3 cells. New crosstalk processes between SSTR and ErbB subtypes were observed in both cell lines. In BT-474 cells, there were substantial reductions in the membrane expression of ErbB1 (degradation and termination of signaling) and ErbB2, as well as moderately reduced expression of ErbB3 and greatly enhanced activation of SSTR1 and SSTR4. Similarly, SK-BR-3 cells exhibited strong reductions in the expression of ErbB1 (degradation), ErbB3, and ErbB2 expression (partial degradation), while enhanced activation of SSTR1 and SSTR2 expression at the cell surface. The activated SSTRs were shown to antagonize ErbB-mediated MAPK signaling and tumor-promoting signaling pathways, resulting in pronounced antiproliferative effects. In BT-474 cells, they inhibited ERK1/2, p38 and PI3K, and enhanced PTEN pathways, while in SK-BR-3 cells they promoted ERK1/2 and p38, inhibited PI3K and maintained PTEN pathways. These results show that the activated SSTRs exert antiproliferative effects in breast cancer cells via mechanisms that resemble those determined for drugs modulating cancer-related signaling pathways.  iii  Lay Summary Breast cancer has become the most common cancer among women, with a high mortality rate worldwide. The lack of clearly defined molecular mechanisms for pathological conditions makes it difficult to identify appropriate treatments. This thesis presents research on the crosstalk and modulation of signaling between SSTR and ErbB subtypes in human breast cancer cell lines that sheds new light on the molecular mechanisms underpinning the antiproliferative activity of SSTRs and the proliferative activity of ErbBs.  Overall, SSTR subtypes engaged in functional crosstalk and modulated multiple signaling pathways affecting apoptosis and proliferation, suppressing or eliminating the deleterious effects of signaling by ErbB subtypes. Our investigations on the crosstalks between SSTR and ErbB receptors in the BT-474 and SK-BR-3 breast cancer cells upon SST and/or EGF treatments are of great significance for clarifying the underlying molecular mechanisms, and exploring their implications for cancer therapy.  iv  Preface I hereby declare that the degree thesis submitted is my research work and achievements under the guidance of my supervisor. My supervisor proposed the title.  I did the study design for investigating the crosstalk between SSTR1-5 and ErbB1-4 in BT-474 and SK-BR-3 cells, and its effects on intracellular signaling under the guidance of my supervisor. I did and finished all experimental researches, analysis of the research data, writing this the degree thesis submitted. My supervisor revised this thesis.  All experiments, data and related materials are true in this thesis submitted. In this thesis, except for references and acknowledgements, there are no studies published or written by others or other organizations. The contributions of others to the research have been made in the thesis and expressed thanks.  An ethics certificate was not required for the research conducted. v  Table of Contents Abstract ........................................................................................................................................................ ii Lay Summary ............................................................................................................................................. iii Preface ......................................................................................................................................................... iv Table of Contents ........................................................................................................................................ v List of Figures ........................................................................................................................................... viii List of Abbreviations .................................................................................................................................. x Acknowledgements ................................................................................................................................... xii Dedication ................................................................................................................................................. xiii 1 Introduction .............................................................................................................................................. 1 1.1 Morbidity and mortality of breast cancer ............................................................................................ 1 1.1.1 Epidemiology, morbidity and mortality of breast cancer ............................................................. 1 1.1.2 Breast cancer subtypes, risk of death, and prognosis ................................................................... 2 1.1.3 A brief introduction to GPCRs ..................................................................................................... 4 1.2 SST and SSTRs ................................................................................................................................... 6 1.2.1 Definition, distribution, and physiological function of SST ........................................................ 6 1.2.2 Structure, molecular signaling and function of SSTRs ................................................................ 7 1.2.2.1 Definition, distribution and physiological functions of SSTRs ............................................ 7 1.2.2.2 Molecular signaling of SSTRs .............................................................................................. 8 1.2.2.3 SSTRs trafficking ................................................................................................................ 11 1.2.2.4 SSTR dimerization .............................................................................................................. 12 1.2.2.5 Heterodimerization of SSTR subtypes with other GPCRs – a brief introduction ............... 16 1.2.3 SSTRs and breast cancer ............................................................................................................ 17 1.2.3.1 Expression and biological functions of SSTRs in breast cancer ......................................... 17 1.2.3.2 Studying the functional crosstalk between SSTRs and other GPCRs in breast cancer by coexpression .................................................................................................................................... 19 1.3 EGF and ErbBs ................................................................................................................................. 20 vi  1.3.1 Definition and classification of EGF.......................................................................................... 20 1.3.2 Structure and function of ErbBs ................................................................................................. 21 1.3.3 Molecular signaling of ErbBs .................................................................................................... 23 1.3.4 ErbBs and breast cancer ............................................................................................................. 26 1.4 Possible crosstalk between SSTRs and ErbBs in breast cancer ........................................................ 29 1.4.1 The coexpression and heterodimerization of SSTRs and ErbBs in breast cancer cells ............. 29 1.4.2 The role of activated SSTRs in breast cancer ............................................................................ 31 1.4.2.1 Activated SSTRs antagonize ErbB-mediated MAPK signaling pathways ......................... 31 1.4.2.2 Activated SSTRs antagonize ErbB-mediated tumor-promoting signaling pathways ......... 34 1.5 BT-474 and SK-BR-3 cell line models ............................................................................................. 35 1.6 Study rationale and hypotheses ......................................................................................................... 36 2 Materials and Methods .......................................................................................................................... 40 2.1 Cell lines and culture ........................................................................................................................ 40 2.2 Immunocytochemistry ...................................................................................................................... 40 2.3 Western blot analysis ........................................................................................................................ 41 2.4 MTT cell proliferation Assay ............................................................................................................ 42 2.5 Statistical Analysis ............................................................................................................................ 43 3 Experimental Results ............................................................................................................................. 43 3.1 Coexpression and colocalization of SSTR1-5 and ErbB1-4 in BT-474 and SK-BR-3 cells ............ 43 3.1.1 Patterns of SSTR 1-5 and ErbB1-4 expression in BT-474 and SK-BR-3 cells ......................... 43 3.1.1.1 Patterns of SSTR 1-5 expression in BT-474 and SK-BR-3 cells ........................................ 44 3.1.1.2 Patterns of ErbB1-4 expression in BT-474 and SK-BR-3 cells .......................................... 45 3.1.2. Time-dependent changes in SSTR 1-5 and ErbB1-4 expression in BT-474 and SK-BR-3 cells upon SST and/or EGF treatment ......................................................................................................... 49 3.1.2.1 Time-dependent changes in SSTR 1-5 expression in BT-474 and SK-BR-3 cells upon SST and/or EGF treatment ...................................................................................................................... 49 3.1.2.2 Time-dependent changes in ErbB 1-4 expression in BT-474 and SK-BR-3 cells after treatment with SST and/or EGF ...................................................................................................... 61 vii  3.2 Time-dependent changes in ERK1/2 and p38 phosphorylation in BT-474 and SK-BR-3 cells upon SST and/or EGF treatment ...................................................................................................................... 75 3.2.1 Time-dependent changes in ERK1/2 phosphorylation after treatment with SST and/or EGF in BT-474 and SK-BR-3 cells ................................................................................................................. 75 3.2.2 Time-dependent changes in p38 phosphorylation in BT-474 and SK-BR-3 cells upon SST and/or EGF treatment .......................................................................................................................... 76 3.3 Time-dependent changes in PI3K and PTEN phosphorylation in BT-474 and SK-BR-3 cells upon SST and/or EGF treatment. ..................................................................................................................... 79 3.3.1 Time-dependent changes in PI3K phosphorylation in BT-474 and SK-BR-3 cells upon SST and/or EGF treatment .......................................................................................................................... 80 3.3.2 Time-dependent changes in PTEN phosphorylation in BT-474 and SK-BR-3 cells upon SST and/or EGF treatment .......................................................................................................................... 81 3.4 Time-dependent changes in cell proliferation in BT-474 and SK-BR-3 cells upon SST and/or EGF treatment ................................................................................................................................................. 85 3.4.1 Time-dependent changes in proliferation of BT-474 cells upon SST and/or EGF treatment .... 86 3.4.2 Time-dependent changes in proliferation of SK-BR-3 cells upon SST and/or EGF treatment . 86 4 Discussion................................................................................................................................................ 90 5 Conclusion and Future Directions ...................................................................................................... 102 References ................................................................................................................................................ 108    viii  List of Figures Figure 1.1 The study design for investigating the crosstalk between SSTR and ErbB proteins in BT-474 and SK-BR-3 breast cancer cells, and its effects on intracellular signaling. ............................................... 39  Figure 3.1 Representative photomicrographs illustrating the immunofluorescence localization of SSTR1-5 in BT-474 (A) and SK-BR-3 (B) breast cancer cells. .............................................................................. 46 Figure 3.2 Semi-quantitative analyses of SSTR1-5 protein expression in BT-474 (A) and SK-BR-3 (B) breast cancer cells and their patterns (C). ................................................................................................... 47 Figure 3.3 Semi-quantitative analyses of ErbB1-4 protein expression in BT-474 (A) and SK-BR-3 (B) breast cancer cells and their patterns (C).. .................................................................................................. 48 Figure 3.4 Time-dependent changes in SSTR1 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF. ............ 56 Figure 3.5 Time-dependent changes in SSTR2 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF ............. 57 Figure 3.6 Time-dependent changes in SSTR3 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF. ............ 58 Figure 3.7 Time-dependent changes in SSTR4 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF. ............ 59 Figure 3.8 Time-dependent changes in SSTR5 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF ............. 60 Figure 3.9 Time-dependent changes in ErbB1 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF, and possible crosstalk between ErbB1 with SSTRs in BT-474 (E, Upper panel) and SK-BR-3 (E, Lower panel) cells... .......................................................................................................................................................... 69 Figure 3.10 Time-dependent changes in ErbB2 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF, and possible crosstalk between  ErbB2 with SSTRs in BT-474 (E) cells.......................................................... 71 Figure 3.11 Time-dependent changes in ErbB3 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF. ............ 73 Figure 3.12 Time-dependent changes in ErbB4 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF. ............ 74 ix  Figure 3.13 Time-dependent changes in the phosphorylation of ERK1/2 in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF .. 78 Figure 3.14 Time-dependent changes in the phosphorylation of p38 in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF........ 79 Figure 3.15 Time-dependent changes in the phosphorylation of PI3K in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF........ 83 Figure 3.16 Time-dependent changes in the phosphorylation of PTEN in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF........ 84 Figure 3.17 Time-dependent changes in cell proliferation induced by SSTR and ErbB expression in BT-474 breast cancer cells following 12, 24, 36, 48 and 72 hours treatments with 1 µM SST and/or 10 nM EGF. ............................................................................................................................................................ 88 Figure 3.18 Time-dependent changes in cell proliferation induced by SSTR and ErbB expression in SK-BR-3 breast cancer cells following 12, 24, 36, 48 and 72 hours treatments with 1 µM SST and/or 10 nM EGF ............................................................................................................................................................. 89 Figure 5.1 SSTR subtypes via functional crosstalk of SSTR/ErbB subtypes and modulation of signaling resulting in antagonizing EGF-mediated effects on cell survival pathways and subsequently diverting the deleterious effects of ErbB subtypes in BT-474 breast cancer cells. ........................................................ 106 Figure 5.2 SSTR subtypes via functional crosstalk of SSTR/ErbB subtypes and modulation of signaling resulting in antagonizing EGF-mediated effects on cell survival pathways and subsequently diverting the deleterious effects of ErbB subtypes in SK-BR-3 breast cancer cells. ..................................................... 107            x  List of Abbreviations  AC         Adenylyl cyclase  AR          Adrenergic receptors β2AR      β2-adrenergic receptor cAMP     Adenosine 3, 5,-monophosphate Ca2+            Calcium ion CDK2     Cyclin-dependent kinase 2 CHO-1    Chinese hamster ovary-1  cGMP     Cyclic guanosine monophosphate c-tail       Cytoplasmic tail DEP        Density-enhanced phosphatase DFS        Disease-free survival D2R        Dopamine receptor 2 DR          Dopamine receptors  EGF        Epidermal growth factor ER           Estrogen receptor  ErbB        Epidermal growth factor receptor ERK1/2   Extracellular signal-regulated kinase 1 and 2 GDP         Guanosine diphosphate GPCR      G-protein-coupled receptor  GTP         Guanosine triphosphate HEK-293 Human embryonic kidney 293 HER2      Human epidermal growth factor 2  JNK         Janus kinase K+            Potassium ion MAPK     Mitogen-activated protein kinase MOR1     μ-opioid receptor NHEs      Sodion ion/ hydrogen ion ( Na+/H+) exchangers OS          Overall survival OR          Opioid receptors xi  PI3K       Phosphatidylinositol 3-hydroxy kinase  PKA        Protein kinase  PKC        Protein kinase C PLCγ       Phospholipase C gamma PR           Progesterone receptor  PTEN      Phosphatase and tensin homolog PTP         Phosphotyrosine phosphatase PTX         Pertussis toxin Raf           Rapidly accelerated fibrosarcoma Ras           Rat sarcoma RTK         Receptor tyrosine kinase RT-PCR   Real-time polymerase chain reaction SST          Somatostatin SSTR       Somatostatin receptor STAT       Signal transducers and activators SHP-1      Src-homology phosphatase types 1  SHP1       Src-homology phosphatase 1 TM          Transmembrane WHO       World Health Organisation      xii  Acknowledgements • I owe a debt of gratitude to my supervisor, Dr. Ujendra Kumar for welcoming me into his lab and training me in the theory and practice of molecular biology and oncology research. I owe particular thanks to Dr.  David Dayong Chen, whose penetrating questions taught me to question more deeply. I offer my sincere gratitude to all the members of my Advisory Committee, they have inspired me to continue my work in this field. I thank Dr Weihong Song, for extending my vision of science and providing thoughtful answers to my endless questions. • I thank the financial support from Mitacs-Accelerate Graduate Research Internship Program. • I would also like to thank all the members of our lab and the faculty, staff and my fellow students at the UBC for welcoming and helping me and the good times we enjoyed together over the past three years.  • Special thanks are owed to my great grandfather (Dr. Zhou, Chuandian) and my parents, who have supported me throughout my years of education, both morally and financially. xiii  Dedication I dedicate this work to my great grandfather, Dr. Zhou, Chuandian, who is a kind old man of one hundred and three years old. When my dear old great granddad was one hundred years old, I came to UBC from China and began the task of preparing this thesis. He encouraged me to meet the challenges of the next few years, and wished me to be a productive and effective researcher. I particularly want to thank him for his love and encouragement over the course of my graduate studies. 1  1 Introduction 1.1 Morbidity and mortality of breast cancer  1.1.1 Epidemiology, morbidity and mortality of breast cancer Cancer is a devastating and complex pathology that presents a major challenge to modern health care systems and societies. Worldwide, it is expected to cause the deaths of 13-17 million people in 2030 [1, 2]. Breast cancer has become the second most common cancer among women, and has a high mortality rate [3]. The World Health Organisation (WHO) has ranked breast cancer as the most common cause of cancer death among women: in 2012, it accounted for 25% of all new cancer cases in women (1.63 million incidences) and 6.4% of all cancer deaths (0.522 million deaths worldwide) [4, 5]. Approximately 60% of all breast cancer mortality occurs in less developed countries, and incidences of breast cancer are expected to increase at a rate of 3-5% annually [4-6]. It has been predicted that one in nine Canadian women will ultimately suffer from breast cancer, and one in thirty will die of the condition [7]. The American Cancer Society estimates that there will be around 0.25 million new cases of breast cancer among American women in 2017, and around 0.04 million deaths from breast cancer in the United States (corresponding to a mortality rate of around 16%) [8]. The 5-year mortality rate for breast cancer in American women is about 15% [8, 9]. Breast cancer mortality is even higher in developing countries [10, 11]: the five-year mortality rates for women with breast cancer are around 50% in India, and 25-30% in China and Costa Rica [9].   2  1.1.2 Breast cancer subtypes, risk of death, and prognosis Rapid cell division is associated with fast-growing and aggressive cancers. The Ki-67 test is used to estimate the rate of cancer cell division; a higher Ki-67 index corresponds to faster division [12]. Clinical experience has shown breast cancer to be a very heterogeneous condition with a variety of histological subtypes including the luminal A and B subtypes, human epidermal growth factor 2 (HER2)-driven, and basal-like tumors [13-15]. Information on a breast cancer patient’s histological subtype has been shown to significantly help clinicians when making therapeutic decisions [16-20]. Several breast cancer subtypes are characterized by mutations affecting the activity and/or expression of hormone receptors such as the progesterone receptor (PR), estrogen receptor (ER), or HER2. Several hormone signalling pathways that are essential for normal development become accelerants of malignant neoplasia if their activity becomes misregulated [21]. The luminal A breast cancer subtype is characterized as being ER- and/or PR-positive, HER2-negative, and having a Ki-67 index of <10%. The human breast cancer cell lines MCF-7 and TD47 represent this subtype [22-25]. The prognosis for luminal A breast cancers are poor, especially among younger patients: the 5-year overall survival (OS)  and 5-year disease-free survival (DFS) rates for patients aged 40 or less are around 87% and 72%, respectively [8, 26-29]. The luminal B subtype is characterized as being ER- and/or PR-positive (or negative), HER2-positive (or negative), and having a Ki-67 index of >10%. Breast cancer cell lines representing this subtype include BT-474 and ZR-75 [22-25, 30]. The Luminal B subtype of breast cancer is similar to Luminal A subtype but is more frequently ER-positive/PR-negative, and has a worse prognosis: the risks of death and relapse among patients with luminal B breast cancer are 2.5 and 3.6 times higher, respectively, than those for patients with the luminal A 3  subtype [8, 26-28, 31]. The HER2-driven subtype is characterized as being ER- and/or PR- negative with HER2 overexpression and strong proliferation; it is represented by human breast cancer cell lines such as SK-BR-3 and MDA-MB-453 [24, 25]. HER2-driven breast cancers are very aggressive, have high histological grades, and pose higher risks to young patients (those aged 39 or less) than the luminal B subtype [8, 27, 32]. Basal-like tumors (or the basal-like subtype) are characterized as being ER- and/or PR- negative, and HER2-negative; they are represented by the human breast cancer cell lines MDA-MB-231 and BT549 [25, 28, 29, 33]. Basal-like subtype cancers are aggressive with high histological grades, high mitotic rates, and pose high risks was aggression, high grade histology, high mitotic rate and risk at young age (<40 year) [8, 27]. ER-positive tumors, which include all those belonging to the luminal A and B subtypes, account for around 68% of all breast cancer cases, whereas ER-negative tumors (which include those belonging to the HER2-driven and basal-like subtypes) account for around 32% [8, 13, 25, 27]. HER2 is overexpressed in approximately 20% of all patients with ER-negative breast cancer [34].  This diversity necessitates the use of a wide range of treatment modalities, including endocrinotherapy, treatment with neoadjuvant chemotherapeutics prior to surgical resectioning, and radiotherapy [22, 23, 30, 35]. Several instances of breast cancers with resistance to chemotherapeutic drugs have been reported (especially among hormone-independent cancers), leading to a strong increase in patient mortality [21]. In addition, the molecular mechanisms associated with tumor initiation, progression and failure of treatment are currently poorly understood [36, 37]. The lack of clearly defined molecular mechanisms for pathological conditions makes it difficult to identify appropriate treatments; this difficulty is exacerbated by 4  the heterogeneous nature of breast cancer and the involvement of several tightly integrated signaling pathways in cancer progression and therapeutic failure. 1.1.3 A brief introduction to GPCRs  G-protein-coupled receptors (GPCRs) are important membrane proteins that play critical roles in several pathological conditions and modulate many signaling pathways associated with disease progression [38]. Although about 50% of all currently available drugs target GPCRs, many GPCRs remain unexplored as potential therapeutic targets. Consequently, there are probably still many undiscovered roles of GPCRs in pathophysiological conditions [39-44]. Studies on GPCR signaling and the effects of GPCR agonists and antagonists have shown that heterotrimeric guanine nucleotide—binding proteins (i.e. G proteins) are signal transducers, relaying signals delivered via various hormones, neurotransmitters, chemokines, and autocrine/paracrine factors [40, 45, 46]. The first four G proteins to be discovered (Gs, Gt, Gi, and Go) were identified by biochemical purification; following their discovery, many more G proteins and their subunits were identified by cDNA cloning [45, 47]. These discoveries provided a sound structural basis for clarifying the mechanisms by which GPCRs modulate disease-related signaling pathways.  Experiments with agonists and antagonists have yielded important insights into the workings of GPCR signaling pathways in recent years [39, 44, 46, 48, 49]. Agonist binding induces activating conformational changes in GPCRs, causing them to bind to heterotrimeric G proteins, which are denoted Gαβγ because they consist of tightly bound α, β and γ subunits. The resulting complex converts guanosine diphosphate (GDP) into guanosine triphosphate (GTP), which is accompanied by the dissociation of the Gαβγ unit into Gα and Gβγ subunits that are not bound to the GPCR. Because the subunits are not bound to the GPCR, they can diffuse within 5  the membrane, activating downstream signaling systems [39, 46, 48]. The Gα proteins are divided into Gαs, Gαi, Gαq and Gα12 subclasses based on sequence similarity, and are powerful regulators of downstream GPCR signaling. For example, GPCR coupling with Gαs activates adenylyl cyclase (AC) isoforms 1–9, enhancing the production of the second messenger adenosine 3, 5,-monophosphate (cAMP), whereas GPCR coupling with Gαi inhibits AC isoforms 5-6 [39, 45, 46]. Furthermore, recent studies have shown that a single GPCR can associate with multiple Gα proteins (including Gαi/s, Gαq/s, Gαi/q, Gαq/i/s) and Gβγ proteins involved in downstream signaling, triggering the activation, production, or liberation of effector molecules including AC, cAMP,  protein kinase A (PKA), phosphotyrosine phosphatases (PTPs), mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-hydroxy kinase (PI3K), and ion channels such as calcium  (Ca2+) or potassium (K+) channels, among others [39, 50, 51]. Investigators initially believed that cellular responses were regulated by monomeric GPCRs, but advances made over the last decade have prompted revisions of this belief [52], showing that homo- and/or hetero-dimerization of GPCRs plays a central role in mediating cellular responses and modulating receptors’ functions and responses to pharmacological interventions [21, 39]. Interestingly, some GPCRs were shown to form heterodimers not just with other GPCRs but also with receptors from other protein families. Most importantly, GPCRs have been shown to play important roles in many diseases at different stages [39]. However, it is not clear how GPCRs and their downstream cellular signaling pathways affect the development and survival of cancers (tumors). A notable group of GPCRs that undergo homo/heterodimerization are somatostatin receptors (SSTRs), particularly SSTR 1-5, which play critical roles in controlling the proliferative properties of both normal and cancerous (tumor) cells [53, 54]. Among the proteins 6  with which they form heterodimers are homoplastic receptor tyrosine kinases (RTKs) - another class of cell surface proteins that have been linked to several human diseases and are activated by growth factor ligands [52, 55]. Several members of the RTK family are known to form homo- and heterodimers, including the EGF receptors ErbB1-4, which play major roles in regulating cell survival, proliferation, migration and differentiation in normal and cancer cells [52, 56, 57]. It has been suggested that crosstalk and modulation of signaling between SSTR/ErbB subtypes in cancer cells has important clinical implications [21, 52, 58]. The aim of this thesis is to clarify the molecular mechanisms involved in the crosstalk between SSTRs and ErbBs, to explain how this crosstalk modulates the corresponding signaling pathways, and to thereby help explain their involvement in pathological conditions in general and breast cancer specifically.  1.2 SST and SSTRs  1.2.1 Definition, distribution, and physiological function of SST SST is a tetradecapeptide that inhibits growth hormone release [59, 60]. It exists in two naturally occurring bioactive forms: SST-14 and SST-28 [61]. SST-14 was originally identified by studying ovine hypothalamus extracts; a congener extended at the N-terminus was later discovered and designated SST-28 [62]. SST is widely distributed throughout the central nervous system and in peripheral organs including the pituitary, stomach, pancreas, kidneys, gut, thyroid, adrenals, submandibular glands, prostateand placenta [63]. SST can be easily found in endocrine, inflammatory, neuropeptides, and immune cells in response to ions, etc. [51, 61]. SST is now known to be a multi-functional regulatory peptide [54, 64]. Today, SST is best known as a major biological system, and acts to regulate a variety of physiological functions both locally as a neuromodulator, paracrine/autocrine regulator, neurotransmitter, and systemically as a true 7  inhibition of hormonal secretion, cell proliferation,  smooth muscle contractility, etc.[49, 65]. Studies conducted over the last four decades have provided important insights into the dynamic effects of SST on physiological functions and the pathophysiology of diseases such as Alzheimer’s disease, acquired immune deficiency syndrome, Huntington’s disease, epilepsy, diabetes mellitus, inflammation, and neoplasia [52, 59, 66-72]. The actions of SST-14 and SST-28 are mediated by a family of seven transmembrane (TM) domain proteins known as of SSTR1-5, which are encoded by separate genes that are segregated on different chromosomes [61]. Aside from SSTR5 (which has a greater affinity for SST-28 than SST-14), the SSTRs bind their natural agonistic ligands SST-14 and SST-28 with similar high affinities [73]. The development of ligand selective for individual SSTR subtypes has provided important insights into their effects and physiological functions [49]. Several SST analogs (manely SSAs) with varying selectivities for SSTR1-5 have been prepared [44, 74]. Because all SSTRs bind their natural ligands, SST-14 and SST-28, with very similar low nanomolar affinities, they were classified into subfamilies on the basis of their coupling to different effector pathways and their affinity for selected SST analogs (e.g. short synthetic octapeptide and hexapeptide analogs). Subfamily 1 includes SSTR2 (A and B), SSTR3, and SSTR5, which all display nanomolar affinity for the tested SST analogs, whereas the members of subfamily 2 (SSTR1 and SSTR4) bound with micromolar affinities [58, 61]. 1.2.2 Structure, molecular signaling and function of SSTRs 1.2.2.1 Definition, distribution and physiological functions of SSTRs  As noted above, SSTR1-5 are heptahelical transmembrane GPCRs that present high affinity SST-binding domains at the cell surface [65]. Five membrane-bound SSTR subtypes have been 8  shown to be widely expressed in normal and abnormal human tissues; SSTR1, SSTR2, and SSTR3 are the dominant subtypes found in endocrine organs including the pancreas, pituitary, thyroid, and parathyroid [49, 61]. SSTR1 is overwhelmingly expressed in the brain, pituitary, islets, and adrenals. SSTR2 is also predominantly expressed in the brain, as well as in the pituitary, islets and adrenals, and in diseased organs. SSTR3 is mainly expressed in the cerebellum, but expressed at moderate levels in other parts of the brain and is also found in the spleen, kidneys and liver. SSTR4 is poorly expressed in the brain but is expressed strongly in the heart and moderately in the lungs and islets. Finally, SSTR5 is sparsely expressed in the brain but strongly expressed in the pituitary, islets, and intestines [51, 53, 75-78]. 1.2.2.2 Molecular signaling of SSTRs Upon binding to SST or an SST analog, SSTRs activate various intracellular signaling pathways. This process is predominantly initiated by the activation of specific Gi proteins (although occasionally Go or Gq proteins are involved) in Gαβγ heterotrimers, which function as signal transducers and modulate the activity of various key enzymes including AC, PTPs, and MAPKs via G-protein-dependent mechanisms [45, 50, 51, 79, 80]. The Gβγ subunits of Gαβγ heterotrimers are major regulators of voltage-dependent ion channels, and play a critical role in coupling SSTRs to diverse effectors. It has been established that Gβγ subunit directly activate these effectors in vivo via protein–protein interactions, but it is not wholly clear how the activation of these effectors is spatially and temporally coordinated [45, 50]. The situation is made more complex by the fact that mammalian cells express two major forms of the β subunit (Gβ36 and β35) and at least four forms of γ subunit (Gγ1, Gγ2, Gγ3 and Gγ4), each combination of which coordinates the activation of many different effectors [81]. 9  It has been shown that the SSTR 1-5 subtypes bind to pertussis toxin (PTX)-sensitive G-proteins (i.e. Gαβγ heterotrimers) that incorporate the subunits Giα (e.g.  Gαi1, Gαi3), Go (e.g. Gαo2), and Gq (e.g. Gα14), as well as Gβ (e.g. Gβ36) and Gγ (e.g. Gγ3). Upon binding in this way, they downregulate AC and thus suppress cAMP formation, which in turn downregulates the PKA pathway (which has been shown to be important in human pituitary adenomas, among other things) in a receptor-specific manner [39, 65, 80-82]. After binding to agonists (i.e. SST and SST analogs), the SSTR1-5 can couple with G-proteins featuring three different Gαi subunits (Gαi1, Gαi2, and Gαi3), activating cell signaling pathways and downstream biological functions in a receptor-specific fashion. Among other things, this causes potent AC inhibition and reductions in cAMP synthesis [39, 45, 46]. Specially, SSTR1 is coupled to AC via Gαi3; SSTR2 can combine with Gαi1, Gαi2, Gαi3, Gαo2, and Gα14; and SSTR3 interacts with Gαi1, Gαi2, Gα14, and Gα16 [45, 60, 80, 83]. SSTRs induced by SST have been found to inhibit Ca2+ currents by activating cyclic guanosine monophosphate (cGMP)-dependent  protein kinases, which alter the phosphorylation of Ca2+ channels [84]. However, SSTR subtypes activated by agonists such as SST-14, octreotide- OCT, pasireotide or lanreotide can also activate K+ channels (SSTR1, SSTR2, SSTR5) and inhibit voltage-gated Ca2+ channels (SSTR1-5) via efficient coupling to the PTX-sensitive subunits Gαi/o and Gβ/γ, leading to the inhibition of hormone secretion [45, 83, 85]. In addition to regulating ion channels, SSTRs are also coupled to sodion ion/ hydrogen ion ( Na+/H+) exchangers (NHEs) [53]. The inhibition of NHE activity was mediated by SSTRl, SSTR3, and SSTR4 in a PTX-independent fashion, highlighting the pharmacological and functional differences between these three SSTR proteins and SSTR2 (or SSTR5) [81, 86, 87]. The human SSTR1-5 also stimulates PTPs via a PTX -sensitive pathway involving Gαi2. However, this behavior varies between species: the SSTR5 protein from rates does not affect 10  PTP activity [60, 88]. There are three known enzymes in the PTP family: Src-homology phosphatase types 1 (SHP-1) and 2 (SHP-2), and a protein known as density-enhanced phosphatase (DEP-1) in humans or PTPη in the rat thyroid [83]. Each SSTR subtype activates different PTPs upon agonist binding, leading to changes in the levels and activities of various downstream signalling proteins including MAPKs such as extracellular signal-regulated kinase (ERK). Notably, ERK1 and ERK2 were shown to induce the activation of cyclin-dependent kinase inhibitors such as p27Kip1 and p21Cip1/Waf1 in response to SSTR signaling [49, 53]. In addition to PTPs, PI3K recruitment appears to play a fundamental role in MAPK-mediated signaling induced by activated SSTRs such as SSTR2 and SSTR4 [53, 89-91]. There is also evidence that some MAPK signalling cascades are inhibited in the presence of activated SSTRs, particularly SSTR2, SSTR3, and SSTR5 [60]. Overall, the available evidence indicates that the SSTR subtypes bind to SST with nanomolar affinities, and upon binding, interact with Gα/β/γ heterotrimers to mediate receptor-specific biological functions that involve suppressing cAMP production, modulating the activity of several key enzymes (including AC, PTPs and MAPKs), changing the intracellular levels of Ca2+and K+ ions by manipulating Ca2+ and K+ channel activation, regulating NHE antiporter activity, and ultimately inhibiting cell proliferation and hormone secretion [49, 53]. SSTR1 primarily couples to Gαi3-containing heterotrimers, and activates MAPK and PTP enzymes. The remaining SSTRs either activate or inhibit MAPK enzymes and activate PTPs (specifically, p27Kip1 and p21Cip1/Waf1 in the case of SSTR2). SSTR2 primarily couples with Gαo2, Gαi1, Gαi2, Gαi3 and Gα14; SSTR3 primarily couples with Gαi1, Gαi2, Gα14, and Gα16; SSTR4 primarily couples with Gαi1, Gαi2 and Gαi3; and SSTR5 primarily couples with Gαi1, Gαi2 and Gαi3 [45, 52, 60, 80]. 11  The downstream cellular effects induced by agonist-activated SSTR subtypes (the agonists in question being SST and receptor-specific ligands including SST analogs) have been summarized by Kumar from our lab and previous researchers; they include (1)  inhibition of Ca2+ influx and hormonal secretions; (2) inhibition of cAMP production, leading to inhibition of cell proliferation; (3) receptor-specific modulation of MAPK (ERK1/2 and p38) and PI3K/AKT signalling pathways via the coupling of activated SSTRs with Gi/o/q, leading to inhibition of cell proliferation, survival and migration [49, 52, 83]. 1.2.2.3 SSTRs trafficking In addition to its activating effects, the binding of SST to all SSTRs other than SSTR1 eventually causes their uncoupling from Gα/β/γ heterotrimers and their internalization into the cell [79]. Internalization removes the SSTR subtype from the cell surface, which is necessary to terminate its signaling activity or to desensitize the receptor to sustained agonist signaling [53]. Van Koppen et al. developed a model of this process based on their studies on β-arrestin-regulated internalization [92]. The key steps in this model are: (1) the SSTR subtype binds to an agonist and induces signaling via Gα/β/γ-proteins; (2) prolonged stimulation induces phosphorylation of the SSTR via Gα/βγ (i.e. Gαq and Gβγ) protein-coupled receptor kinases as well as kinases activated by second messengers such as cAMP; (3) the phosphorylated SSTR recruits cytosolic -arrestins; and  (4) interactions between the cytosolic β-arrestin and the phosphorylated SSTR uncouple the SSTR from the Gα/β/γ proteins, causing desensitization. In addition, β-arrestin interacts with clathrin and the clathrin adaptor complex, causing immobilization of the receptor in a clathrin-coated pit (i.e. internalization). Depending on its affinity of binding, the β-arrestin may dissociate from the SSTR either before the clathrin-coated vesicle pinches off from the plasma membrane or at a later stage during intracellular trafficking; 12  Importantly, β-arrestin bound to the receptor in the clathrin-coated vesicle may also act as an adaptor/scaffold for SSTR-mediated activation of ERK 1/2, Janus kinases (JNK3) and p38 in the cytoplasm; Finally, after the β-arrestin is released, intracellular phosphatases dephosphorylate the SSTR (inducing resensitization), causing it be immediately recycled back to the cell surface for another round of stimulation [60, 92, 93]. The agonist-regulated internalization of SSTRs thus exhibits characteristics common to most GPCRs, many of which undergo similar cyclical processes of signaling, desensitization, internalization, resensitization, and recycling to the plasma membrane [60]  Generally speaking, subfamily 1 receptors (SSTR2, SSTR3 and SSTR5) are readily internalized in response to agonist stimulation, whereas subfamily 2 receptors (SSTR1, SSTR4)  are quite resistant to agonist-induced internalization, and especially to agonist-induced upregulation expression of SSTR1 at the cell surface [53]. After endocytosis, SSTR2 and SSTR5 are primarily recycled to the plasma membrane whereas SSTR3 is predominantly downregulated at the plasma membrane and targeted for degradation [79, 93, 94]. Unlike SSTR1, SSTR4 also showed moderate or low levels of internalization in Chinese hamster ovary (CHO-K1), human embryonic kidney 293 (HEK-293), and rat insulinoma cells, indicating that neither of these proteins depend on the internalizing interaction with β-arrestin [53, 83, 94]. The varied internalization and trafficking behaviors of SSTR1-5 reflect the diversity and dynamic nature of protein-protein interactions at cell surfaces [53]. 1.2.2.4 SSTR dimerization Homo-and heterodimerization are common consequences of protein-protein interactions at cell surfaces, and have been shown to modulate the ligand binding, signaling, and trafficking properties of SSTRs [95]. The coexpression of SSTRs in cell membranes may favor their 13  interaction, leading to the formation of homodimers and heterodimers consisting of either two different SSTR subtypes or an SSTR and some other GPCR, producing new receptor units with distinct pharmacological and functional profiles [60]. Using a combination of pharmacological, biochemical, and biophysical techniques (including transfected mutant and wild type receptors, as well as endogenous receptors and fluorescence resonance energy transfer analysis), Rocheville et al. showed that ligand activation induces the formation of SSTR5 homodimers and SSTR5/SSTR1 heterodimers in CHO-K1 cells [96, 97]. Moreover, the homo- and heterodimerization of SSTR subtypes has been shown to modify the receptors’ functional properties by enhancing their ligand binding affinity and altering their agonist-induced trafficking and expression at the cell surface [96, 97]. Importantly, agonist-induced dimerization of SSTR1/SSTR5 in CHO-K1 cells expressing both proteins caused the dimer to be internalized and colocalized in cytoplasmic vesicles [96]. The observed dimerization of SSTR subtypes with other members of possibly related receptor families revealed a new level of molecular crosstalk between receptors [61]. Since 2000, many researchers (including some from our lab) have shown that SSTR subtypes can form homo- and/or heterodimers with a variety of closely and more distantly related receptors [39, 58, 65, 75, 77, 98-112]. The formation and dissociation of SSTR homodimers has been observed for all SSTR subtypes other than SSTR1 in various cell types, including CHO-K1 cells, HEK-293 cells, liver cells, and human pancreatic islet cells [96, 100, 113, 114]. SSTR1 reportedly remains monomeric independently of agonist binding, although agonist exposure did increase its abundance at the cell surface and provoke inhibition of cAMP synthesis and cell proliferation [75, 96, 100, 101, 105, 115]. SSTR2 exists as a constitutive dimer (SSTR2/SSTR2) that dissociates into a pair of monomers upon agonist binding [116, 117]. The formation and dissociation of SSTR2 14  homodimers has been observed in cells from multiple species (humans, rats, and pigs), and is important in agonist-induced receptor trafficking because SSTR2 homodimer dissociation is a prerequisite for receptor internalization in all species studied to date [116-118]. SSTR3 exists as a preformed dimer (SSTR3/SSTR3) that partially dissociates into separate monomers upon agonist treatment, and whose intracellular signaling functions (which include apoptosis induction) are mediated by its cytoplasmic terminus, or C-tail [108, 118]. SSTR4 exists as a homodimer (SSTR4/SSTR4) that is stabilized by agonist binding [105]. Finally, SSTR5 exists as monomer in the absence of agonist treatment, but forms an SSTR5/SSTR5 homodimer upon agonist binding [39, 75, 77, 79, 96, 105]. In general, the C-tails of the different SSTR subtypes play central and receptor-specific roles in controlling their dimerization [75, 105]. The formation of heterodimers involving SSTRs is typically controlled by the C-tail and can exhibit either positive or negative cooperativity in response to ligand binding. This can enhance agonist-dependent regulatory responses and increase signaling efficiency, particularly in terms of the modulation of downstream signaling pathways. In addition, it can change patterns of receptor trafficking, desensitization and internalization behaviors [83, 96, 98, 105, 118]. Interestingly, replacing the C-tail of SSTR1 with that of SSTR5 was found to yield an SSTR1 derivative that formed a heterodimer with SSTR5, resulting in the formation of SSTR1/SSTR5 dimers on the plasma membrane upon treatment with agonists such as SST-14 [75, 96, 100]. Grant et al. found that the activation of SSTR5 but not SSTR1 is necessary for the heterodimeric formation of SSTR1/SSTR5, indicating that the heterodimerization of SSTR1/SSTR5 is a subtype-selective process that depends on ligand-induced conformational changes in SSTR5 [75]. In fact, SSTR1 was only internalized in the form of the SSTR1/SSTR5 heterodimer, indicating that heterodimerization is an important component of its agonist-dependent regulatory responses [96, 15  97]. This result demonstrated that agonist-mediated heterodimerization can occur as the result of a ligand binding to just one receptor subtype but may nevertheless enhance the signaling efficiency of both components of the resulting heterodimer (in the specific case of SSTR1 and SSTR5, this resulted in stronger inhibition of  cell proliferation via the suppression of forskolin-induced cAMP production) or change their patterns of receptor trafficking [75, 119]. Earlier studies also showed that the SSTR2-selective agonist L-779,976 (but not SST-14) induced the formation of SSTR2/SSTR5 heterodimers even when SSTR5 was not activated by SST-14 [77, 106]. The activation of SSTR2 results in the recruitment and stable association of β-arrestin, followed by receptor internalization and intracellular receptor pooling; the heterodimerization of SSTR2 with SSTR5 increases the recycling rate of the internalized SSTR2 by destabilizing its interaction with β-arrestin [77]. Heterodimerization with SSTR5 also greatly increases the efficiency of SSTR2’s G-protein coupling and the activation of MAPK (ERK1/2)  signaling, resulting in stronger inhibition of cell proliferation together with increased expression of p27Kip1 and reduced cAMP synthesis [77, 79].  Just like the heterodimerization of SSTR2/SSTR5, the SSTR2 is also mainly coexpressed and dimerized with SSTR3 in normal ( or tumor) cells (e.g., HEK-293 cells, rostate and gastric cancer cells), and noteworthy, which only SSTR2/SSTR3 are responsible for stimulating apoptosis in normal and tumor cells through regulation of the two main signaling pathways mentioned above [102, 118, 120]. Pfeiffer et al. reported coimmunoprecipitation experiments using differentially epitope-tagged receptors, which provided direct evidence for the heterodimerization of SSTR2 (A) and SSTR3. An SSTR2/SSTR3 heterodimer formed from rat proteins exhibited a high affinity for SST-14 and the SSTR2-selective ligand L-779,976, but not for the SSTR3-selective ligand L-796,778 [118]. However, while both SSTR2/SSTR2(A) and SSTR3/SSTR3 homodimers underwent agonist-16  induced endocytosis upon SST-14 treatment, the SSTR2/SSTR3 heterodimer was separated at the plasma membrane and only SSTR2 (but not SSTR3) underwent agonist-induced endocytosis in the presence of SST-14 [118]. By investigating the role of the SSTR2/SSTR3 heterodimerization, internalization, signaling, cell proliferation and apoptosis in HEK-293 cells after treatment with SST and specific agonists of SSTR2 and SSTR3, War et al. showed that agonist activation immediately reduced the cell surface concentration of SSTR2/SSTR3, with a parallel increase in their intracellular colocalization. These observations provided the first evidence that the heterodimerization and coexpression of SSTR2/SSTR3 may modulate antiproliferative signaling and apoptosis [108, 120]. Using immunocytochemistry, western blotting, photobleaching-fluorescence resonance energy transfer, and co-immunoprecipitation,  Somvanshi et al. demonstrated that SSTR4/SSTR1 and SSTR4/SSTR5 also form heterodimers that are trafficked and coupled to AC in HEK-293 cells although the dimerization of SSTR4 has been less extensively studied than that of the other SSTRs [49, 105] However, it has been shown that the heterodimerization of SSTR4/SSTR5 (but not SSTR4/SSTR1) induces significant changes in the receptors’ functions, which are similar to those of the SSTR4 homodimer and include signaling and inhibition of cell proliferation. Moreover, heterodimerization is associated with stronger ligand binding, increased expression of the cyclin-dependent-kinase p27kip1, inhibition of cAMP synthesis, and agonist-dependent changes in MAPK (ERK) activity [105]. 1.2.2.5 Heterodimerization of SSTR subtypes with other GPCRs – a brief introduction Many researchers, including workers from our lab, have found that SSTR subtypes form heterodimers with one-another (e.g. SSTR1/SSTR5, SSTR2/SSTR3, SSTR2/SSTR5, SSTR5/SSTR5 and SSTR4/SSTR1), but also with other closely related GPCRs such as dopamine receptors (DR) [97, 104], opioid receptors (OR) [95], and adrenergic receptors (AR) 17  [107, 109]. Interestingly, the heterodimerization of SSTR subtypes with DR (e.g., dopamine receptor 2, D2R), OR (e.g. the μ-opioid receptor, MOR1), or AR (e.g. the β2-adrenergic receptor, β2AR), generates novel receptors (e.g. SSTR2/D2R, SSTR2/ MOR1 and SSTR5/β2AR) with unique properties, resulting in distinct signaling and pharmacological behavior [79, 95, 104, 109]. For instance, the coexpression of SSTR2 and D2R at the cell surface in CHO-K1 and HEK-293 cells can lead to functional interactions between these two proteins in response to agonist treatment, leading to an increased affinity for dopamine and augmented D2R signalling, as well as prolonged  SSTR2 internalization [104]. Conversely, the coexpression of  SSTR2 and MOR1 at the cell surface in the same cells led to SSTR2/MOR1 heterodimerization, which cross-modulated phosphorylation, internalization, and desensitization [95]. 1.2.3 SSTRs and breast cancer 1.2.3.1 Expression and biological functions of SSTRs in breast cancer  Breast cancers express both SST and SSTRs [102, 121], and the expression and distributional patterns of all five SSTRs in primary breast cancers have been characterized [3, 102, 122-124]. Levels of SSTRs in primary breast cancer cells have been determined using classical biochemical cross-linking techniques, in vitro autoradiography, in vivo scintigraphy, in situ hybridization, immunocytochemistry, and RT-PCR [121, 123, 125, 126]. Binding analyses indicate that 15-66 % of primary breast tumors are SSTRs-positive, whereas in vivo receptor imaging using scintigraphy with the indium-labeled OCT analog [111In-DTPA-DPhe1]–OCT suggests that around 75% of all breast cancers are SSTR positive [127-130]. Preclinical data showed that SSTRs are expressed in a large proportion of breast cancers, with the most frequently expressed subtype (at the protein level) being SSTR2, followed by SSTR1 and SSTR5 [102]. Previous investigators have also found that SSTR2 and SSTR5 were the predominant 18  subtypes, and that SSTR2 is the most strongly expressed in breast tumors [95, 103, 121, 131]. The mRNA- and protein-level expression of SSTRs in breast cancers is quite similar [123]. Kumar et al. used peroxidase immunohistochemistry to generate representative photomicrographs showing the variable localizations of SSTRs in sections from 12 ductal NOS breast tumor tissues [121]. Meanwhile, Kumar and colleagues found that all breast tumors displayed expressing more than one SSTR subtype (e.g. SSTR2 and SSTR5), which SSTRs that are differentially expressed within the same tissues were further demonstrated [65, 121]. Some groups have reported correlations between individual SSTRs and various markers in breast tumors [123, 132]. For instance, cellular levels of SSTR1, SSTR 2 and SSTR4 have been correlated with ER levels, SSTR2 expression correlates with PR levels, and SSTR3 expression is positively correlated with tumor grade [121, 133]. Comparisons of SSTR expression in solid tumors and breast cancer cells have also revealed variable distributions of SSTRs in cultured cell lines [58, 111, 121, 134, 135]. Like solid tumors, breast cancer cell lines generally express multiple SSTRs, although the level of differential SSTR expression in cell lines is lower than in solid tumors [121]. Therefore, human breast cancer cell lines have been studied extensively to better understand how the behaviors of SST and SSTRs affect the biology of breast cancer [21, 53]. Watt et al. observed significant expression of multiple SSTRs as both membrane and cytoplasmic proteins in the MCF-7 and MDA-MB-231 breast cancer cell lines [58]. Interestingly, SSTR1 and SSTR4 were expressed more strongly in MCF-7 cells than in MDA-MB-231 cells, while SSTR3 was weakly expressed in both lines [58]. Overexpressing SSTR3 in both lines revealed that SSTR3 promoted apoptosis by activating downstream signaling molecules (e.g. ERK1/2, p38, PI3K and p27Kip1) associated with cytostatic and cytotoxic effects in MCF-7 cells (which represent the luminal A breast cancer subtype) and 19  induced cell cycle arrest in MDA-MB-231 cells (which represent the basal-like breast cancer subtype) [21].  SST analogs including OCT, lanreotide, pasireotid, and SOM230 have been shown to exert antiproliferative effects in breast cancer cells via two mechanisms: interactions with SSTRs at the cell surface directly induced cell cycle arrest and apoptotic pathways, and indirectly inhibited the production of secretory products such as growth hormone and downregulated angiogenic factors [3, 102, 136]. For example, the anti-hormonal agent OCT has been studied extensively for the treatment of different types of tumors, especially breast cancers, because of its ability to modify signaling in cancer cells by interacting with SSTR2 [137-141]. However, the specific functions of SSTRs in breast cancers remain unclear, and there is a need for better diagnostic methods and therapeutic approaches [39, 141]. 1.2.3.2 Studying the functional crosstalk between SSTRs and other GPCRs in breast cancer by coexpression As mentioned above, coexpression of SSTRs with other GPCRs such as DR, OR and AR in cells has been shown to cause heterodimer formation, which alters receptor internalization and downstream signaling. Multiple previous studies have suggested that the heterodimerization of SSTRs and OR may be important in the development of breast cancer [142-144]. Kharmate et al. found that the agonist treatment of MCF-7, T47D and  MDA-MB-231 cells that coexpressed SSTR2 and ORs (μ, δ and κ) led to heterodimerization and the modulation of signaling pathways involved in cancer progression [111]. Furthermore, the activation of SSTR2 and ORs modulated MAPK (ERK1/2 and p38) activity in a cell-dependent manner, activated the tumor suppressor proteins phosphatase and tensin homolog (PTEN) and p53, and antagonized the PI3K/AKT cell survival pathway, ultimately increasing necrosis (as opposed to apoptosis) in MCF-7 and T47D 20  cells relative to ER-negative MDA-MB231 cells [111]. In the 2009 years ago, no significant progress in uncovering role of SSTRs stimulated by SST has been considered in breast cancer [83, 123]. It is now believed that SSTRs participate in functional crosstalk with other GPCRs in breast cancer as a result of their coexpression and heterodimerization, and that these behaviors contribute significantly to the modulation of clinically relevant cancer-related signaling pathways [111]. It has also been suggested that the heterodimerization of SSTR subtypes with more distantly related receptor proteins may have similar effects on various cancer-related signaling pathways [131]. Unfortunately, much remains to be learned about the crosstalk between SSTRs and other GPCRs in cancers, including breast cancer although some important results have been presented [3, 49, 112, 114].  1.3 EGF and ErbBs   1.3.1 Definition and classification of EGF  EGF is a mitogenic polypeptide with 53 amino acid residues and 3 intramolecular disulfide bonds [145]. Unlike SST, EGF and its relatives (i.e. EGF family ligands) bind to proteins known as ErbB1-4 (collectively referred to as ErbBs) that belong to the RTK family. This binding activates a rich network of signalling pathways, culminating in responses including angiogenesis, cell adhesion, cell motility, development, and organogenesis [56, 145-148]. To date, four classes of EGF family ligands have been identified  [147]. The first class includes EGF, epigen (EPG), transforming growth factor-α (TGFα) and amphiregulin (AR), which bind specifically to ErbB1; the second class includes betacellulin (BTC), heparin-binding epidermal growth-factor like growth factor (HB-EGF), and epiregulin (EPR), and bind specifically to ErbB1 and ErbB2; the third class includes neuregulin 1 (Nrg-1) and neuregulin 2 (Nrg-2), and bind specifically to 21  ErbB3 and ErbB4; and the fourth class includes neuregulin 3 (Nrg-3) and neuregulin 4 (Nrg-4), which bind specifically to ErbB4 [56, 148]. 1.3.2 Structure and function of ErbBs ErbBs are typical RTKs and belong to subclass I of the RTK superfamily. ErbB1 was discovered in the early 1980s, and is also known as EGFR or ErbB1/HER [56, 149]. Another three members of the ErbB family have since been discovered: ErbB2 (also known as HER2), ErbB3 (aka HER3), and ErbB4 (aka HER4). All four ErbBs (ErbB1-4) exhibit strong structural homology [149]. Since their discovery, several studies have been conducted to unravel the normal and pathological functions of ErbBs and their ligands in organogenesis and adulthood [56]. The four ErbB proteins all have a common extracellular segment (region) containing two cysteine-rich domains (II, IV) that mediates ligand binding interspersed with two unique domains (I , III), followed by a single membrane-spanning region, and a cytoplasmic region containing multiple phosphorylation sites that respond to ligand binding and activation [52, 149, 150]. Ligand binding mainly promotes conformational change in the extracellular region, opening up the extracellular ErbB domains and removing a hairpin-loop dimerization arm from a sleeve in domain IV, allowing the dimerization arm to enter a different sleeve in domain II of a second ligand-bound ErbB molecule [148]. As a consequence, ErbB domains I and III participate in ligand binding (except in the case of ErbB2), while domain II participates in dimer formation [148, 151]. Interestingly, the extracellular region of ErbB2 lacks domains I and III regions, and the structure of ErbB2 is not compatible with binding to any of the EGF family ligands [145, 148, 151]. In addition, the kinase domain of ErbB3 in the cytoplasmic region is kinase-impaired [148]. 22  It has therefore been concluded that ErbB2/ErbB2 and ErbB3/ErbB3 homodimers have no function in cells [148]. In the absence of agonists, ErbB1 exists in a monomeric state, but ligand activation causes it to form homo- and hetero-dimers [52]. Ligand binding has been reported to induce the formation of an asymmetric ErbB1/ErbB1 homodimer resembling the heterodimer formed between cyclin-dependent kinase 2 (CDK2) and cyclin A, its activating protein [152]. Additionally, the binding affinity of EGF towards ErbB1 is modulated by coexpressing it with either ErbB2 or ErbB3, neither of which can directly bind to EGF [150]. On the other hand, simultaneously coexpressing ErbB1-3 leads to extensive formation of an ErbB3/ErbB2 heterodimer that strongly induced cell growth and various transformations [153]. The heterodimerization of  ErbB1 with ErbB2, ErbB3, strongly enhances the tyrosine phosphorylation of ErbB1-3 [150]. Ligand binding to ErbB1 may induce its rapid internalization and degradation [65, 154, 155], but ErbB2, ErbB3 and ErbB4 do not exhibit this behavior or internalization followed by recycling to the plasma membrane [146, 153]. Although ErbB2's structure is not compatible with binding any of the EGF family ligands, it appears to be primed and ready to interact with other ligand-bound receptors, which may be why it does not require an activating ligand [145, 151, 156]. So ErbB2 is the favored heterodimerization with ErbB1, ErbB3 and ErbB4, respectively [151, 156]. Uniquely among the ErbBs, ErbB3 contains mutations in the cytoplasmic domain that block the activity of its tyrosine kinase domain [52, 56]. Moreover, Shi et al. reported that the kinase domain of ErbB3 is about 1,000-fold less active than ErbB1, but they proposed that in the context of a heterodimer, this may be sufficient for transphosphorylation of, e.g., ErbB2 [145, 157]. ErbB4 is similar to ErbB1 in that it exists as an inactive monomer in the absence of a ligand, but forms active homo- and hetero-dimers upon ligand binding [158]. The ErbB2/ErbB4 heterodimer formed upon ligand 23  binding is essential for heart development [145]. The heterodimers of the ErbB proteins have varied and important responses to specific ligands. Binding to EGF induces the formation of the following dimers: ErbB1/ErbB3, ErbB1/ErbB2, ErbB1/ErbB1, ErbB1/ErbB4, ErbB4/ErbB2, and ErbB3/ErbB2 [56, 150]. Of these, ErbB1/ErbB2, ErbB2/ErbB3, ErbB1/ErbB1, ErbB1/ErbB3, ErbB3/ErbB4 and ErbB1/ErbB4 fulfill important physiological roles and functions in various pathological states in the presence of EGF [145, 148, 150]. 1.3.3 Molecular signaling of ErbBs Many researchers such as Riese et al., Yarden et al., Kumar et al, Roskoski Jr., and Hynes have summarized the signalling networks formed by ErbBs under physiological and pathological conditions [52, 56, 65, 145, 148, 159]. The components of each ErbB subtype’s signalling pathways that are active under physiological conditions appear to have developed rather early in evolutionary history; these pathways look very much like typical simple growth factor signaling pathways [56, 148]. For instance, ligand binding to a monomeric RTK promotes its dimerization and self-phosphorylation at tyrosine, which activates the catalytic function of its cytoplasmic domain; the self-phosphorylated tyrosine residues serve as docking sites for various adaptor proteins or enzymes, allowing it to simultaneously initiate various signalling cascades to generate physiological effects [56, 148]. A model of the ErbBs signalling network featuring input, signal processing, and output layers has been proposed by Yarden et al. [145, 148]. The input layer consists of the ErbBs and their ligands, including all the possible ErbB homo- and heterodimers, which function as signaling complexes [56]. The signal processing layers consist of a vast array of phosphotyrosine-binding (PTB) proteins that interact with the cytoplasmic tails of the ErbB proteins after dimer formation. The main downstream signaling systems included in the model are the PI3K/Akt, Ras/Raf/MEK/ERK1/2, and phospholipase C (PLC) pathways  [56, 24  148]. Crosstalk between the ErbB network and GPCR signalling pathways has been demonstrated by Yarden et al., who showed that GPCRs exert positive effects on ErbB signalling via a range of mechanisms [56]. The outputs of the ErbBs signaling network are effects on cellular processes such as cell division and migration, adhesion, differentiation and apoptosis [56]. The molecular signaling processes of the different ErbB subtypes are clearly intertwined in a range of signaling cascades. Only a few of the pathways and transcription factors involved ErbB-mediated molecular signaling have been characterized in depth, and the associated signaling mechanisms are generally poorly understood [145, 148, 160]. Two researchers from our lab, Kumar and Kharmate, have presented an overview of the ErbB1 signaling pathway by aggregating results from various published works. ErbB1 regulates four major pathways: the JNK/STAT (Janus kinases/signal transducers and activators of transcription), PLCγ/PKC (phospholipase Cγ/protein kinase C), MAPK (ERK/p38), and PI3K/AKT signaling pathways [65, 161]. Specifically, ErbB1 binding to EGF leads to the formation of the asymmetric ErbB1 homodimer and/or the heterodimers discussed above, followed by tyrosine phosphorylation of the ErbB cytoplasmic domain, increased phosphorylation, and the activation of the MAPK and PI3K/AKT cell survival pathways, which in turn induces cell proliferation, invasion, and migration [52, 65, 110]. The generic MAPK signaling pathway is activated via at least four distinct cascades, of which the ERK1/2 cascade is the most the most extensively studied and well characterized pathway that is activated by ErbB1. This pathway regulates various cellular processes, including proliferation, migration and differentiation, invasion, and suppression of apoptosis [65, 161]. Notably, the activation of ERK1/2 by ErbB1 in presence of EGF inhibited p27Kip1 and p21Cip1/Waf1 via a process involving cyclin D1, and promoted cell proliferation [52, 161]. Similarly, the activation of PI3K/AKT via 25  EGF-bound ErbB1 suppresses the expression of PTEN and p53, and promotes cell proliferation [52]. The MAPKs are serine/threonine kinases that control vital cellular functions including cell growth, differentiation and proliferation. Consequently, many researchers have studied the activation of MAPK pathways by ErbB2-4 in response to ligand binding [56, 65, 145]. ErbB subtypes can activate MAPK signaling by directly recruiting the Src homology 2 (SH2) domain linked ErbB-bound protein 2 (Grb2), or by indirectly recruiting the phosphotyrosine-binding domain [145]. Upon being recruited, Grb2 recruits son of sevenless (SOS), a nucleotide exchange factor that activates Ras after exchanging a bound GDP molecule for GTP [65]. The activated Ras subsequently phosphorylates Raf, activating downstream kinases including MAPK kinases (MEK1/2) [65, 161]. MEK1/2 then phosphorylate and activate ERK1/2, which are translocated into the nucleus and initiate the transcription of various genes, ultimately promoting cell growth via gene transcription [65, 145, 161, 162]. Hyperactivation of MAPKs, and particularly the ERK pathway, is a hallmark of cancer. This is especially true for ERK5, which is associated with cell proliferation [65, 145, 161, 163]. Moreover, ligand-induced signaling was less robust and less prolonged in the absence of ErbB2, indicating that the heterodimerization of ErbB2 with ErbB1or ErbB3 enhances this signaling activity [164]. Interestingly, tumor cells overexpressing ErbB2 also exhibited elevated basal expression of ERK5 [145, 163]. It has also been shown that ErbB3 is extremely adept at activating the PI3K pathway because its carboxy-terminal domain has six docking sites for the p85 adaptor subunit of PI3K, a characteristic that has turned out to be very important in tumor biology [145, 165].   26  1.3.4 ErbBs and breast cancer ErbBs are expressed in various epithelial, mesenchymal and neuronal tissues, and 14–65% of all breast cancers are reportedly ErbB1-positive [58, 166]. In ErbB1-positive breast cancers, ErbB1 is often coexpressed with ErbB2 or ErbB3 [148, 167]. The field of ErbB2 biology has exploded in the almost 30 years since the first publications describing ErbB2 overexpression in breast cancer, and around 18–22% of primary invasive breast cancers are reported to be ErbB2-positive [145, 148, 168]. There are some FDA-approved therapeutics targeting ErbB1 or ErbB2, such as neratinib, gefitinib and erlotinib,  trastuzumab, pertuzumab, lapatinib, and trastuzumab–emtansine, an antibody–microtubule polymerization inhibitor conjugate. Unfortunately, despite the availability of these drugs, breast tumors expressing ErbB1and ErbB2 still tend to have poor clinical outcomes [167, 168]. ErbB2 appears to have a greater potential for oncogenic transformation in breast tumors than ErbB1; a range of different tumor types have been shown to result from its overexpression, gene amplification, or mutation [148, 160, 169]. Importantly, the coexpression of ErbB subtypes has been found to enhance the transforming ability of breast cancer cells [65, 148]. The translocation of ErbB1 and ErbB2 from the cell surface to the nucleus has been reported to occur via distinct mechanisms that depend on importin α1/β1 and importin β1, respectively [170, 171]. Additionally, overexpression of ErbB1 and ErbB2 in breast carcinoma cancers is often accompanied by overproduction of their ligands, including EGF and TGFα [172, 173]. Many ErbB1-positive breast cancers exhibit coexpression and dimerization of ErbB1/ErbB2 and/or  ErbB1/ErbB3, and ErbB2-positive breast cancers frequently exhibit coexpression and dimerization of ErbB1/ErbB2 and  ErbB2/ErbB3. By analysing data for 807 breast cancer patients, DiGiovanna et al. found that 87% of ErbB1-positive cancers coexpress ErbB2, and patients whose tumors exhibit ErbB1/ErbB2  coexpression and dimerization had the 27  shortest survival [167]. In keeping with these observations, there is evidence that coexpression of ErbB1/ErbB2 and ErbB1/ErbB3 at low to moderate levels may enable cells to match the responses of high ErbB2 expressers [153]. Interestingly, Holbro et al. clearly showed that the ErbB2/ErbB3 dimer functions as an oncogenic unit that stimulates proliferation, and that ErbB2 requires ErbB3 to drive breast tumor cell proliferation [169]. Previous studies have demonstrated that breast cancers that coexpress ErbB2/ErbB3 or ErbB1/ErbB2/ErbB3 are more aggressive than those that coexpress ErbB3/ErbB4, and have lower changes of patient survival [65, 145, 148, 160, 174]. In addition to the results discussed above, there is also a growing body of evidence indicating that overproduction of ErbB ligands such as EGF and TGFα1or TGFβ1 is often associated with overexpression of ErbBs, especially ErbB1 and ErbB2, and the activation of their downstream signaling cascades [148, 172, 173, 175]. In breast cancer cells, EGF was found to enhance the dimerization of ErbBs (especially e.g., ErbB1/ErbB2, ErbB2/ErbB3, ErbB1/ErbB1, ErbB1/ErbB3) and the phosphorylation and activation of MAPK, leading to sustained activation of MEK and ERK1/2 (including prolonged basal ERK1/2 expression) [176-178]. Circulating tumor cells in breast cancer patients have been found to express the phosphorylated ErbB1/ErbB2 and PI3K/Akt signaling kinases in the early stages of tumor development and in metastatic tumors [179]. Furthermore, high levels of PI3K/AKT phosphorylation were observed in breast cancer cells, and mutations of PI3K and/or AKT are also bound up with losses of PTEN and overexpression of ErbB2 and ErbB1 [56, 174, 180, 181]. Additionally, nuclear translocation of ErbB1, ErbB2, and many other cancer-relevant proteins has been reported in breast tumor cells, resulting in enhanced cell proliferation [182-184]. 28  Over the last three decades, various target-based agents targeting the transcription of genes encoding proteins such as ErbBs, their ligands, and their signal transduction processes have been developed for the treatment of ErbB-positive breast cancer [145, 185]. There are currently two main therapeutic strategies for ErbB-positive breast cancer. The first involves using monoclonal antibodies to block the ligand-binding ErbB subtype domains I and III (which is not an applicable strategy in the case of ErbB2), and/or the dimerization domains IV and II. The second therapeutic strategy involves using small molecule tyrosine kinase inhibitors (TKIs) to inhibit autophosphorylation and modulate downstream intracellular signaling of ErbBs [148, 186]. The FDA-approved antibodies targeting the extracellular domains of ErbB1 and ErbB2 are Trastuzumab/Herceptin® (ErbB1, 1998), Cetuximab/Erbitux® (ErbB2, 2004), Pertuzumab/Omnitarg® (ErbB2, 2012), and Ado-trastuzumab emtansine/Kadcyla® (ErbB2, 2013). Approved small molecule drugs, which mostly target ErbBs kinase domains, include Lapatinib/Tykerb® (ErbB1 and ErbB2, 2007), Afatinib/Gilotrif® (ErbB1, 2013) [145, 148]. For instance, Ado-trastuzumab emtansine/Kadcyla® is an antibody-drug conjugate that delivers a cytotoxic drug to ErbB2-positive metastatic breast cancer cells previously treated with trastuzumab and/or a taxane, whereas Lapatinib/Tykerb® is a monoclonal antibody that targets ErbB1 and ErbB2 and is used in second-line treatments (1) with capecitabine for ErbB2-positive breast cancer patients who have previously received cytotoxic chemotherapy or trastuzumab, and (2) with letrozole in post-menopausal hormone receptor-positive breast cancer patients [148]. Because breast cancers treated with these targeted drugs ultimately become resistant to them, different combinations of  targeted drugs (antibody and small molecule) and targeted drugs with cytotoxic therapies are being explored [145]  29  1.4 Possible crosstalk between SSTRs and ErbBs in breast cancers 1.4.1 The coexpression and heterodimerization of SSTRs and ErbBs in breast cancer cells Since the concept of crosstalk between receptors was first proposed, it has become increasingly clear that specialized crosstalk between different classes of signaling receptors plays significant roles in regulating a variety of cellular functions [21, 83, 96, 97, 107, 112, 187]. As mentioned above, ErbBs have been closely linked to cell proliferation, survival and transformation in breast cancer, and their (over) expression is strongly correlated with tumor grade and malignancies [51, 145, 173]. Conversely, SSTRs have been found to be negative regulators of cell proliferation in breast cancers, and are strongly expressed in lower grade and less aggressive tumors [21, 51, 65, 131]. The activities of SSTR and ErbB subtypes thus appear to be opposed. Remarkably, it has been shown that when coexpressed with other receptors, SSTR proteins form heterodimers and crosstalk with other GPCRs (including DR, OR, and AR) but also with more distantly related receptor proteins such as ErbBs from the RTK family, raising the possibility of direct crosstalk between SSTR and ErbB signaling [58, 65, 110, 123, 131, 188]. The coexpression of SSTR and ErbB subtypes at the cell surface in breast cancer cells has been shown to be receptor-, cell line-, and ER-dependent. Kumar et al. initially proved that all five SSTR and four ErbB subtypes are extensively expressed in breast tumor tissues and cell lines, and that there is extensive coexpression of SSTR and ErbB subtypes in both breast cancer cell lines [103, 121, 189]. For example, by comparing mRNA- and protein-level expression of SSTR1-5 and ErbB1-4 in ER-positive MCF-7 and ERα-negative MDA-MB-231 cells, Watt and 30  Kumar discovered that the coexpression of SSTR and ErbB subtypes in breast cancer cells resulted in the colocalization of the SSTRs and ErbB proteins in a receptor-, cell line- and ER-dependent manner [103]. Moreover, ERα-negative breast cancer cells were shown to express SSTR subtypes more strongly and ErbB subtypes less strongly than ER-positive breast cancer cells [103]. A second line of evidence supporting the potential for crosstalk between SSTRs and ErbB proteins is that they are frequently coexpressed (or colocalized) at cell surfaces and/or intracellularly in breast cancer cells, leading to their functional dimerization. Kumar et al. have reported that functional interactions between ErbB1 and SSTR5 or SSTR1 lead to the agonist-dependent formation of heteromeric SSTR5/ErbB1 complexes in cells [58, 110, 188]. The oncogenic effects of these ErbB subtypes are tightly regulated by their coexpression/colocalization with other ErbB subtypes at the cell surface, the phosphorylation of ErbBs, and the homo and/or heterodimerization between ErbB subtypes [52, 145].The functional coexpression and dimerization of SSTR5/ErbB1 in both breast cancer cells has been shown to block homo- and hetero-dimerization between ErbB subtypes (e.g., ErbB1/ErbB1, ErbB1/ErbB2) in cells [52, 110, 188]. For instance, SSTR5 blocked homo- and hetero-dimerization of ErbB1/ErbB2 in MCF-7 and MDA-MB231 cells [110, 188]. Furthermore, the coexpression/dimerization of ErbB1/ErbB1 was more effective at inducing the  phosphorylation of ErbB1 following activation by EGF than the heterodimerization of ErbB1/ErbB3 following neuregulin treatment [148, 153]. The findings discussed above suggest that the coexpression or dimerization of SSTRs and ErbBs at the cell surface in breast cancer cells could inhibit ErbB1 phosphorylation by blocking the homo-and/or heterodimerization of ErbBs, suppressing the proliferation and differentiation of 31  breast cancer cells [51, 153]. Studies from our lab have provided evidence that SSTR5 induces the dissociation of the ErbB1/ErbB2 heterodimer in HEK-293 cells, and that activated SSTR1 and SSTR1/5 interfere with the formation of ErbB1/ErbB2 heteromers in a receptor-specific manner [110, 131, 188]. These results support the proposed competitive effect of SSTRs on ErbB1 dimerization and phosphorylation, and the hypothesized crosstalk between SSTRs and ErbB1 in breast cancer cells, raising the possibility that the activation of SSTRs could have positive therapeutic effects by suppressing the effects of activated ErbBs [49, 52, 83, 188]. 1.4.2 The role of activated SSTRs in breast cancer 1.4.2.1 Activated SSTRs antagonize ErbB-mediated MAPK signaling pathways A large body of experimental data suggests that when the SSTRs are coexpressed with ligands (SST and SST analogs e.g., OCT,vapreotide, lanreotide and TT-232), they form homodimers and heterodimers with other GPCRs, altering their functional profiles in terms of receptor activation and trafficking, and subsequently activating different signaling pathways [39, 51, 60, 79, 111, 119]. Furthermore, the coexpression and heterodimerization of SSTRs with other GPCRs (as exemplified by the case of SSTR2 and ORs in MCF-7 breast cancer cells) could enhance the regulation of ERK in breast cancer cells because ligand-activated SSTRs regulate ERK activity in a receptor- and cell- dependent manner [83, 91, 111, 119, 190]. Meanwhile, activated SSTRs and/or their heterodimers (e.g. SSTR2 and SSTR2/ORs) can modulate MAPK (e.g. ERK1/2 and p38) signaling pathways, inhibiting cell proliferation and promoting apoptosis in breast cancer cells [60, 111, 119, 191]. Previous studies have suggested that disequilibria in ErbB activating and trafficking have detrimental effects on the activation of signaling pathways by ErbB subtypes [58, 148, 153, 192]. For example, the ErbB1’s cyclical process of trafficking, 32  internalization, degradation, and recycling back to the cell surface is closely related to its mediation of mitogenic signaling in breast cancer cells [145, 153]. Therefore, any factor that interferes with its expression or activity at the cell surface will affect the cellular response [52]. It has been reported that sustained activation of ErbB1 and blockage of its degradation could lead to the activation of ERK in cellular transformations [161, 193]. It is not clear whether interference with the expression of ErbB1 at the cell surface via activating SSTR subtypes will activate or inhibit ERK and thus inhibit cell proliferation in breast cancer cells. Classically, GPCRs have been shown to regulate the phosphorylation of ERK1/2 via directly or indirectly EGF-mediated pathways [58, 91]. Additionally, studies have suggested that ErbB1 plays an important role in regulating GPCR- or SSTR-mediated activation of the MAPK pathway [58, 188, 194]. However, the nature of this role is unclear. For instance, SSTR1- and SSTR4-mediated activating phosphorylation of ERK1/2 inhibited cell growth, whereas cell growth inhibition mediated by SSTR5 appeared to require inhibition of ERK1/2 phosphorylation [195]. Moreover, the SSTR2-mediated inhibition of cell growth seems to involve both inhibiting and promoting the phosphorylation of ERK1/2, depending on the cellular environment [58, 91, 195]. Interestingly, it was suggested that the direct antiproliferative effects of SSTRs activated by SST and its analogs stem from the arrest of cell division induced by blocking EGF-mediated mitogenic signals in tumor cells [49, 60, 102]. As mentioned above, the coexpression and heterodimerization of SSTR5/ErbB1 after ligand stimulation could alter the effective expression of ErbB1 at the cell surface, either activating or inhibiting ERK in breast cancer cells [58, 60]. Kumar et al. found that SST alone or the combination of SST and EGF did not induce any changes in SSTR1 but both treatments upregulated SSTR5 expression and reduced the surface levels of ErbB1 in both breast cancer cell lines and HEK 293 cells [58, 188]. Additionally, SSTR 33  activation that caused losses of ErbB1 at the cell surface did not induce any detectable intracellular accumulation of ErbB1, suggesting that it was degraded in the cell, terminating ErbB1-mediated mitogenic signaling in breast cancer cells [52, 58]. Furthermore, mRNA- and protein-level expression analyses reported by Kumar et al. showed that coexpression of SSTR5/ErbB1 at the cell surface in MCF-7 cells (representing the luminal A breast cancer subtype) and MDA-MB-231 cells (representing the basal-like subtype) induced cell-specific and ligand-selective changes in receptor expression [58]. Importantly, it was found that activated SSTR5 inhibited cell proliferation by inducing ERK1/2 phosphorylation and inhibiting p38 phosphorylation in MCF-7 cells but not in MDA-MB-231 cells [58]. Similarly, activated SSTR5 induced EGF-mediated p38 phosphorylation in breast cancer cells is in a receptor- specific manner, and pronounced inhibition was also observed in the presence of SSTR1 alone [58]. Agonist (or ligand)-dependent association/dissociation of SSTR5/ErbB1 heterodimers has also been reported, and shown to affect the phosphorylation of ERK1/2 and p38 in cells [58, 65]. Taken together, these results strongly suggest that activated SSTRs antagonize ErbB1-mediated MAPK signaling pathways in a cell-specific manner, and that SSTR-mediated inhibition of cell proliferation requires the activation of both ERK1/2 and p38 via the coexpression and heterodimerization of SSTRs/ErbB1 in breast cancer cells, further strengthening the hypothesis that SSTRs and ErbBs interact functionally in cancer cells [52, 58, 65, 110, 119, 188]. The specific functions of activated SSTRs in breast tumors (e.g. suppression of ErbB1-mediated MAPK signaling pathways in different subtypes, especially luminal B and ErbB2-driven subtypes, and antagonism of ErbB2- or ErbB3-mediated MAPK signaling pathways in other tumor subtypes) are still not clear, but could potentially be exploited for diagnostic and therapeutic purposes [13, 51, 79, 145, 148, 160].  34  1.4.2.2 Activated SSTRs antagonize ErbB-mediated tumor-promoting signaling pathways  Aggressive tumor growth and the failure of trastuzumab therapy in breast cancers have frequently been associated with hyperactivation of PI3K/AKT cell survival pathways and the loss or inhibition of PTEN [51, 146, 174, 179]. It has therefore been suggested that inhibitors of PI3K/Akt could be a source of new breast cancers therapies [51, 145, 196, 197]. Biochemical studies have shown that trastuzumab destabilizes ligand-independent constitutive ErbB2/ErbB3 complexes, uncoupling ErbB3 from ErbB2 and blocking downstream PI3K/Akt signaling in breast cancers [174, 196]. Specific antagonistic antibodies or kinase inhibitors of ErbBs can also very strongly inhibit PI3K/AKT [51, 198, 199]. Building on these results, studies conducted in our lab have shown that activated SSTRs can inhibit PI3K signaling in cells [110, 111, 188]. It is not yet clear whether the inhibition of tumor promoting signals (PI3K/AKT, PTEN) by activated SSTRs affects the outcome of trastuzumab therapy in cancers. As the tumor progresses, the gradual loss of SSTR subtypes may lead to trastuzumab treatment failure, which is associated with the loss of PTEN and enhanced PI3K activity in tumor cells [52, 200]. The finding that activated SSTRs can inhibit the phosphorylation of PI3K/AKT and thereby enhance or maintain the phosphorylation of PTEN is the first evidence that it is possible to inhibit tumor-promoting signals using FDA-approved drugs [52, 200]. Importantly, previous studies from our lab have shown that activated SSTRs (SSTR1 or SSTR5) can inhibit the phosphorylation of PI3K/AKT, and that this inhibition is more pronounced in cells expressing the SSTR1/SSTR5 heterodimer, indicating that activated SSTRs may play an important role in the response to trastuzumab therapy in cancers [110, 188]. Studies have also indicated that SSTR inactivation leads to hyperactivation of PI3K/AKT cell survival pathways and the loss or inhibition of PTEN, as well 35  as the loss of trastuzumab responsiveness in tumor cells [51, 111, 145]. In addition, cells that express active SSTR1, SSTR5 and SSTR1/SSTR5 can accelerate the dissociation of ErbB1 from ErbB1/ErbB2 heteromers, leading the inhibition of ErbB1 phosphorylation via the formation of SSTR5/ErbB1 heterodimers and thereby regulating EGF-mediated downstream signaling proteins such as MAPK and PI3K/AKT, as well as in PTP translocation and p27Kip1 expression [110, 111, 188]. While these processes offer potential avenues for developing new therapeutic options for treating cancers (and particularly breast cancer), the development of new therapies is hindered by a lack of information on the underlying molecular mechanisms [13, 51, 145].  Many critical aspects of the pathological biology of SST, SSTRs, EGF, and ErbBs remain unclear, including SSTRs and/or  ErbBs expression and coexpression patterns, the extent and relevance of their heterodimerization, their trafficking dynamics, the functional crosstalk and signaling pathways of different receptor subtypes, and the competing molecular mechanisms of antiproliferative and proliferative effects in breast cancers, among other things [3, 13, 21, 33, 83]. There is thus a clear need for a better understanding of the crosstalk between SST/SSTRs and EGF/ErbBs in breast cancer cells.  1.5 BT-474 and SK-BR-3 cell line models Breast cancer is an extremely complex and heterogeneous pathology that depends strongly on the presence of certain regulatory proteins, hormones, and growth factors, as well as the presence or absence of certain cell surface receptor proteins [201-204]. To help clinicians overcome this complexity, optimal treatment strategies for different breast cancers have been identified by analyzing data from individual institutes and through retrospective analyses of randomized clinical trials of different patient subtypes [204]. Because cell lines generally retain 36  most of the recurrent genomic deregulation of transcription present in primary tumors, breast cancer cell lines are well suited for in vitro evaluation of the functional contributions of genome copy number abnormalities to breast cancer pathophysiologies [205, 206]. Consequently, the four known breast cancer subtypes are each represented by a well-established cell line: MCF-7 for the luminal A subtype, BT-474 for the luminal B subtype, SK-BR-3 for the ErbB2-driven subtype, and MDA-MB-231 for the basal-like subtype [25, 206-208]. The first reports describing crosstalk/interactions between SSTR5 and ErbB1 were based on experiments conducted using the MCF-7 and MDA-MB-231 cell lines, which provided the first evidence that GPCRs (specifically, SSTR5) can influence MAPK signaling mediated by ErbB1 proteins belonging to the RTK family in breast cancer [58]. However, the specific contribution of SSTR-ErbB crosstalk to the antagonism of ErbB-mediated MAPK signaling and tumor promotion by activated SSTRs is unclear; in general, little is known about the crosstalk between SSTRs and ErbBs in human breast cancer cells. The aim of this work was to study the signaling and interactions between SSTRs and ErbBs in two estrogen-dependent human breast cancer cell lines (the ER-positive BT-474 and the ER-negative SK-BR-3) as well as in the lines representing the previously studied luminal B and ErbB2-driven subtypes.  1.6 Study rationale and hypotheses The results discussed in the preceding sections indicate that the binding of appropriate ligands (SST and/or EGF) can induce interactions between SSTRs and ErbBs on the cell surface in a receptor-, cell line- and ER-dependent manner. The coexpression of SSTR and ErbB subtypes at the cell surface may lead to functional (hetero) dimerization of SSTR and ErbB proteins by blocking the homo-and heterodimerization of ErbB subtypes, immediately inhibiting the contribution of ErbB phosphorylation to tumor development and survival. The competition of 37  SSTR binding with ErbB dimerization and phosphorylation suggests that there is crosstalk between SSTRs and ErbBs in cells, raising the possibility that these activated SSTRs could be used to antagonise ErbB-mediated MAPK signaling and ErbB-modulated tumor suppressor protein PTEN and PI3K/AKT cell survival pathways. This could be used to enhance the antiproliferative responses in breast cancer cells or suppress their proliferation.  On the basis of these results, it was proposed that the crosstalk/interactions between SSTRs and ErbBs in breast cancer cells depends on dynamic changes in the  coexpression/heterodimerization of SSTRs and ErbBs at the cell surface after SST and/or EGF stimulation. Moreover, we hypothesized that the specific contribution of crosstalk between SSTRs and ErbBs is that activated SSTRs antagonise ErbB-mediated MAPK activity and tumor promoting signaling pathways. Furthermore, it was suggested that the activated SSTRs antagonize ErbB-mediated downstream MAPK (e.g. ERK1/2 and p38) signaling pathways and modulate the tumor suppressor protein PTEN and PI3K cell survival pathway, resulting in a significant antiproliferative effect in breast cancer cells.  However, these hypotheses were untested and their relevance to breast cancer was unknown. The work presented in this thesis was conducted to address these deficiencies by performing experiments using the BT-474 and SK-BR-3 breast cancer cell lines, which represent the luminal B and ErbB2-driven breast cancer subtypes identified in clinical studies. A key goal of the experiments was to characterize the crosstalk and modulation of signaling between SSTR1-5 and ErbB1-4 in these breast cancer cell lines upon SST and/or EGF stimulation, to shed light on the molecular mechanisms underpinning these behaviors, and to assess their effects in the BT-474 and SK-BR-3 lines. The specific aims are laid out below and in Figure 1.1. 38  First, it was noted that the distributions of SSTRs and ErbBs, and especially those of their active forms, might strongly influence dynamic changes in their coexpression at the cell surface and the crosstalk/interactions between SSTRs and ErbBs. Therefore, the first objective was to characterize the coexpression and colocalization of SSTR1-5 and ErbB1-4 in BT-474 and SK-BR-3 breast cancer cells by determining:  • The patterns of SSTR 1-5 and ErbB1-4 expression and their distribution in BT-474 and SK-BR-3 cells.  • The changes in these patterns over time following treatment with SST and/or EGF. Second, it was suggested that time-dependent changes in the phosphorylation of ERK1/2 and p38 following treatment with SST and/or EGF might have important effects on the ability of activated SSTRs to antagonise ErbB-mediated downstream MAPK (ERK1/2 and p38) signaling pathways. Therefore, the second objective was to characterize time-dependent changes in p38 and ERK1/2 phosphorylation following treatment with SST and/or EGF in BT-474 and SK-BR-3 breast cancer cells. Similarly, time-dependent changes in PI3K and PTEN phosphorylation following treatment with SST and/or EGF could also have important effects on the ability of activated SSTRs to antagonise ErbB-mediated modulation of the tumor suppressor protein PTEN and PI3K/AKT cell survival pathway. Therefore, the third objective was to characterize time-dependent changes in PI3K and PTEN phosphorylation following treatment with SST and/or EGF in BT-474 and SK-BR-3 breast cancer cells. Finally, time-dependent changes in cell proliferation following treatment with SST and/or EGF in BT-474 and SK-BR-3 breast cancer cells could indicate that activated SSTRs antagonise 39  ErbB-mediated MAPK signaling pathways and ErbB-modulated tumor suppressor protein PTEN and PI3K/AKT cell survival pathways. Therefore, the fourth objective was to characterize time-dependent changes in cell proliferation following treatment with SST and/or EGF in BT-474 and SK-BR-3 breast cancer cells.    Figure 1.1  The study design for investigating the crosstalk between SSTR and  ErbB proteins in breast cancer cells, and its effects on intracellular signaling.   40  2 Materials and Methods 2.1 Cell lines and culture Human breast cancer cell lines, BT-474, and SK-BR-3 cells were obtained from Dr. Marcel Bally (BC Cancer Agency). SK-BR-3 cells were maintained in McCoy’s 5A Medium supplemented with 10% (v/v) fetal bovine serum (FBS, Invitrogen, Paisley, UK), 50 U/ml penicillin and 50μg/ml streptomycin (both from TEVA, Debrecen, Hungary) at 37°C, a CO2 free atmosphere [58]. BT-474 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with 10% (v/v) fetal bovine serum (FBS) and 1% antibiotic (penicillin/streptomycin) at 37°C, in an atmosphere with 5% CO2 as previously described [58, 121]. 2.2 Immunocytochemistry Human breast cancer cells were processed for immunocytochemistry to visually monitor the expression patterns of receptors of interest during drug treatment [110]. Cells were seeded on glass coverslips coated with poly-D-lysine and grown until 70% cell confluency. The appropriate concentrations of SST (1uM) and EGF (10nM) were then added to the medium. After culturing for a predefined time period, the cells were washed and fixed with 4% paraformaldehyde, then treated with Triton X-100 for 15 min at room temperature [111]. The treated cells were incubated with polyclonal SSTR1-5 antibodies (1:150-300) overnight at 4 °C and incubated with Cy3-conjugated goat anti-rabbit IgG (1:300-500) for 1 h at room temperature for final color development. They were then observed and photographed using a Leica DMLB microscope attached to a Retiga 2000R camera. The specificity of immunoreactivity was determined in the 41  absence of primary antibodies or in presence of pre-immune serum as previously described by our group [76]. 2.3 Western blot analysis To monitor changes in the expression of all SSTR subtypes (i.e. SSTRs 1-5) upon treatment with SST (1uM), and changes in ErbB (1-4) expression upon treatment with EGF (10nM), BT-474 and SK-BR-3 cells were harvested at different time points (5, 10, 15, 30 min) after the start of incubation with the appropriate ligand. Cell lysates were then prepared in radioimmunoprecipitation assay (RIPA) buffer. The lysates were sonicated using a Misonix Ultrasonic Liquid Processor XL-2000 (Farmingdale, NY) to prepare homogenized cell suspensions. Cytosol samples were then collected to determine the status of downstream signaling molecules, and the remaining material was centrifuged for 1 hr at 4 °C to separate the cell membrane proteins [103]. The resulting samples were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) for 2 hours and transferred to a nitrocellulose HyBond ECL membrane. To determine SSTR expression in the membrane extracts, blots were incubated with primary anti-SSTR antibodies (1:500) overnight, followed by incubation with peroxidase-conjugated goat-anti-rabbit (1:500) secondary antibodies. To determine the status of downstream signaling molecules, blots were incubated with primary antibodies against phosphorylated and total ERK1/2, p38,  PI3K and PTEN (1:1000) [111]. The resulting membranes were then incubated with peroxidase-conjugated goat-anti-rabbit (1:500) secondary antibodies. The bands were characterized by chemiluminescence according to the manufacturer’s instructions. Band images were captured using an Alpha Innotech FluorChem 8800 instrument (San Leandro, CA), and the FluorChem software package was utilized to quantitatively analyze the blots. The β-actin was used as the loading control. 42  Membrane protein samples (25 µg) were fractionated by SDS-PAGE and probed with affinity-purified SSTR antibodies. Major protein bands of 53 (SSTR1), 57 (SSTR2), 60 (SSTR3), 44 (SSTR4) and 58 kDa (SSTR5) were obtained in this way. Cytosolic protein samples (15 µg) were fractionated by SDS-PAGE and probed with affinity-purified SSTR antibodies. Major protein bands of 170 (ErbB1), 185 (ErbB2), 200 (ErbB3) and 175 kDa (ErbB4) were also yielded in this way.  Separate cytosolic protein (15 µg) were fractionated by SDS-PAGE and probed with affinity-purified ErbB antibodies. Major protein bands of 44 and 42 kDa (ERK1/2), 43 kDa (p38), and 42 kDa (β-actin) were yielded. The proteins’ phosphorylation status was determined by densitometric analysis of phosphorylated vs. total ERK1/2, p38, PI3K and PTEN.  2.4 MTT cell proliferation Assay The MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) cell proliferation assay was performed as previously described by our group [108] to characterize the time-dependent antiproliferative functions of SSTRs. Briefly, cells were seeded at a density of 5000 cells/well in 96-well plates to grow for 24 h and then subjected to 24 hours of serum starvation. They were then treated with SST (1µM) and EGF (10nM) [209, 210]. After selected treatment periods (0, 12, 24, 36, 48, 72 hours), the cells were processed and their survival rates were analyzed. The conventional MTT assay protocol was used to determine cell viability. Briefly, 20μl of 5mg/mL MTT solution was added per 1 mL of medium, and the resulting mixture was incubated in a 37 °C humidified incubator. The resulting formazan precipitate was then dissolved in 100 μL of 100% isopropanol, and the solution’s absorbance at 550 nm was determined using a microplate spectrophotometer [108].  43  2.5 Statistical Analysis Changes in SSTR expression were quantified using two-way ANOVA and Dunnett’s or Bonferroni’s post hoc tests [111]. All statistical analyses were performed using Graph Pad Prism 5.0. Differences were considered statistically significant based on a threshold of p < 0.05. Results are presented as means ± S.E based on three independent experiments (n = 3). Differences in the measured levels of the SSTR1-5, ErbB1-4, ERK1/2, p38, PI3K and PTEN proteins were analyzed using two-way ANOVA and Bonferroni's post hoc test to compare all treatment groups against controls (*, p < 0.05). The MTT assay results were analyzed by using two-way ANOVA and Bonferroni's post hoc test to compare all treatment groups to controls (*, #, +, p<0.05). In addition, Bonferroni's test was used to analyze the SST vs. SST+EGF treatment groups where applicable (++, p<0.05).  3 Experimental Results 3.1 Coexpression and colocalization of SSTR1-5 and ErbB1-4 in BT-474 and SK-BR-3 cells 3.1.1 Patterns of SSTR 1-5 and ErbB1-4 expression in BT-474 and SK-BR-3 cells  To compare patterns of receptor expression and the intensity of receptor expression using immunocytochemistry and Western blot analysis, we determined the distribution and intensity of SSTR1-5 and ErbB1-4 in BT-474 (luminal B subtype: ER-positive and/or PR-positive, ErbB2-positive or negative) and SK-BR-3 (ErbB2-driven subtype: ER-negative and/or PR- negative, 44  ErbB2-overexpression) human breast cancer cells (Figures 3.1-3.3). We observed significant differences between these two cell lines with respect to the expression of SSTR1-5 and ErbB1-4. In the following sections, whenever a protein’s expression or phosphorylation is said to be increased or reduced, or up- or down-regulated, it should be taken to mean that its expression is changed relative to a negative control unless stated otherwise. 3.1.1.1 Patterns of SSTR 1-5 expression in BT-474 and SK-BR-3 cells  Figure 3.1 presents the results of immunofluorescence localization experiments targeting SSTR1-5 in BT-474 (Figure 3.1 A) and SK-BR-3 (Figure 3.1 B) cells. The studied receptors were detected in both the membrane (a-e) and cytoplasm (f-j) samples. The results of semi-quantitative Western blot analyses are shown in Figure 3.2. All five SSTR receptor subtypes were detected in both the membranes (Figure 3.2, Upper panel) and the cytoplasm (Figure 3.2, Lower panel). Major protein bands were identified with masses of 53 (SSTR1), 57 (SSTR2), 60 (SSTR3), 44 (SSTR4) and 58 kDa (SSTR5). As shown in Figure 3.2, SSTR1-5 was clearly expressed at different levels in both BT-474 (Figure 3.2 A) and SK-BR-3 (Figure 3.2 B) cells, respectively. SSTR2 was the most prominent receptor subtype in BT-474 cells, followed by SSTR3, SSTR4, SSTR5 and SSTR1 (Figure 3.2 A, Upper panel).  Conversely, SSTR4 was the most prominent receptor subtype in SK-BR-3 cells, followed by SSTR5, SSTR2, SSTR3 and SSTR1 (Figure 3.2 B, upper panel). Notably, SSTR1 was poorly expressed at the cell surface in both lines (Figure 3.2, upper panel) but was more abundant in the cytosol (Figure 3.2, lower panel), indicating that it had the lowest activity in both lines. These results are consistent with previous studies on SSTR expression in cell lines derived from luminal B and ErbB2-driven breast cancers [33, 51]. The pattern of SSTR1-5 protein expression in BT-474 and SK-BR-3 cells was summarized in Figure 3.2 C. 45  3.1.1.2 Patterns of ErbB1-4 expression in BT-474 and SK-BR-3 cells  The expression/localization of ErbB1-4 in BT-474 and SK-BR-3 human breast cancer cells as well as tumor cells and their surroundings has been reported by multiple groups [207, 208, 211]. The results of semi-quantitative Western blotting experiments revealing the expression and localization of ErbB1-4 in BT-474 and SK-BR-3 cells are shown in Figure 3.3. The major protein bands detected in both cell lines had masses of 170 (ErbB1), 185 (ErbB2), 200 (ErbB3) and 175 kDa (ErbB4). ErbB1 was the most prominent receptor subtype in BT-474 cells, followed by ErbB2, ErbB3 and ErbB4 (Figure 3.3 A, Upper panel), whereas ErbB2 was the most prominent receptor subtype in SK-BR-3 cells, followed by ErbB1, ErbB3 and ErbB4 (Figure 3.3 B, Upper panel). In both cell lines, ErbB1 and ErbB2 were expressed more strongly at the cell surface than ErbB3, and ErbB4 was expressed weakly (Figure 3.3 A, B, Upper panel). Our results are consistent with previous reports about the expression and localization of ErbB1-4 in the luminal B and ErbB2-driven breast cancer subtypes and the corresponding cell lines, i.e. BT-474 and SK-BR-3 cells [208, 211]. The pattern of ErbB1-4 protein expression in BT-474 and SK-BR-3 cells was summarized in Figure 3.3 C.    46   Figure 3.1 Representative photomicrographs illustrating SSTR1-5 expression immunoreactivity in BT-474 (A) and SK-BR-3 (B) breast cancer cells. BT-474 (A) and SK-BR-3 (B) cells were treated as indicated for 15 min at 37 C and processed for immunofluorescence immunocytochemistry. SSTR1-5 expression in BT-474 and SK-BR-3 cells is shown in red. SSTR1-like immunoreactivity was observed in BT-474 and SK-BR-3 cells on cell surface (a) and in cytosol (f), SSTR2-like immunoreactivity was observed in BT-474 and SK-BR-3 cells on cell surface (b) and in cytosol (g), SSTR3-like immunoreactivity was observed in BT-474 and SK-BR-3 cells on cell surface (c) and in cytosol (h), SSTR4-like immunoreactivity was observed in BT-474 and SK-BR-3 cells on cell surface (d) and in cytosol (i), SSTR4-like immunoreactivity was observed in BT-474 and SK-BR-3 cells on cell surface (e) and in cytosol (j). Note strong SSTR2-like immunoreactivity largely confined to the cell surface in BT-474 cells (A).  Conversely, strong SSTR4-like immunoreactivity largely confined to the cell surface in SK-BR-3 cells (B). Results are representative of three independent experiments.  47   Figure 3.2 Western blot analysis illustrating SSTR1-5 expression in BT-474 (A) and SK-BR-3 (B) breast cancer cells and their patterns (C). Membrane protein samples (25 µg) in BT-474 (A) and SK-BR-3 (B) cells were fractionated by SDS-PAGE and probed with affinity-purified SSTR antibodies. Major protein bands of SSTR1 (53 kDa), SSTR2 (57 kDa), SSTR3 (60 kDa), SSTR4 (44 kDa) and SSTR5 (58 kDa) membrane expression in BT-474 cells (A, Upper panel) and SK-BR-3 cells (B, Upper panel) were respectively obtained in this way. Cytosolic protein samples (15 µg) were fractionated by SDS-PAGE and probed with affinity-purified SSTR antibodies, yielding cytosolic fractions of SSTR1-5 in BT-474 cells (A, Lower panel) and SK-BR-3 cells (B, Lower panel). The pattern of SSTR1-5 protein expression in BT-474 cells (C, left) and SK-BR-3 cells (C, right) were also obtained (++++ strong +++ moderate ++ mild + weak). Note strong SSTR2 membrane expression largely confined to the cell surface in BT-474 cells (A).  Conversely, strong SSTR4 membrane expression largely confined to the cell surface in SK-BR-3 cells (B). The status of SSTR1-5 expression was respectively determined by densitometric analysis of SSTR1-5 using β-Actin (42 kDa) as a loading control. Results represent Mean ± S.D. of three independent experiments.    48   Figure 3.3 Western blot analysis illustrating ErbB1-4 expression in BT-474 (A) and SK-BR-3 (B) breast cancer cells and their patterns (C). Membrane protein samples (25 µg) in BT-474 (A) and SK-BR-3 (B) cells were fractionated by SDS-PAGE and probed with affinity-purified ErbB antibodies. Major protein bands of ErbB1 (170 kDa), ErbB2 (185 kDa), ErbB3 (200 kDa) and ErbB4 (175 kDa) membrane expression in BT-474 cells (A, Upper panel) and SK-BR-3 cells (B, Upper panel) were respectively obtained in this way. Cytosolic protein samples (15 µg) were fractionated by SDS-PAGE and probed with affinity-purified ErbB antibodies, yielding cytosolic fractions of ErbB1-4 in BT-474 cells (A, Lower panel) and SK-BR-3 cells (B, Lower panel). The pattern of ErbB1-4 protein expression in BT-474 cells (C, left) and SK-BR-3 cells (C, right) were also obtained (++++ strong +++ moderate ++ mild + weak). Note strong ErbB1 membrane expression largely confined to the cell surface in BT-474 cells (A).  Conversely, strong ErbB2 membrane expression largely confined to the cell surface in SK-BR-3 cells (B). The status of SSTR1-5 expression was respectively determined by densitometric analysis of ErbB1-4 using β-Actin (42 kDa) as a loading control. Results represent Mean ± S.D. of three independent experiments.    49  3.1.2. Time-dependent changes in SSTR 1-5 and ErbB1-4 expression in BT-474 and SK-BR-3 cells upon SST and/or EGF treatment The interactions between membrane proteins at the cell surface in cells are believed to be among the most dynamic of cellular processes [79]. In keeping with previous studies showing SST- and/or EGF-dependent changes in SSTR1-5 and ErbB1-4 expression at the cell surface in breast cancer cells [58, 103, 121], we observed time-dependent changes in the expression and interactions of SSTR 1-5 and ErbB1-4 in BT-474 and SK-BR-3 cells following treatment with SST and EGF. Figures 3.4-2.12 showed the expression and distribution of SSTR 1-5 and ErbB1-4 in the membranes and cytoplasms of these cell lines after treatment with SST and/or EGF, revealing receptor- and cell-specific trends and their changes over time. 3.1.2.1 Time-dependent changes in SSTR 1-5 expression in BT-474 and SK-BR-3 cells upon SST and/or EGF treatment  The time-dependent changes in SSTR1 expression seen in BT-474 (Figure 3.4 A, C) and SK-BR-3 (Figure 3.4 B, D) cells following treatment with SST and/or EGF are presented in Figure 3.4.  In BT-474 cells, treatment with SST and/or EGF for 5, 10, 15, and 30 min significantly increased SSTR1 expression at the membrane (relative to control cells) in a time- and treatment-dependent fashion. The upregulation induced by SST was strongest at 15 minutes (increased by 1294%) after the start of treatment, whereas that induced by EGF was faster, peaking within 5 minutes of treatment (Figure 3.2 A, C, Upper panel) (increased by 1623%). Combined treatment with SST and EGF (SST+EGF) induced a similar pattern of SSTR1 upregulation to treatment with SST alone, producing a peak at 15 min (Figure 3.4 A, C, Upper panel) (powerfully increased by 1038%).  The up- and down-regulation of SSTR1 at the membrane (Figure 3.4 A, C, Upper panel) in cells treated with EGF or SST+EGF for 5, 10, 15, 50  and 30 min produced corresponding changes in the cytoplasm. Notably, the downregulation of SSTR1 expression at the membrane observed after 15 minutes’ treatment with EGF was matched with an increase in the levels of SSTR1 in the cytosolic fraction (Figure 3.4 A, C, Lower panel). Similarly, the upregulation of SSTR1 at the membrane induced by SST+EGF after 15 min (Figure 3.4 A, C, Upper panel) corresponded to a downregulation of SSTR1 in the cytosolic fraction (Figure 3.4 A, C, Lower panel). Together with previous reports indicating that internalized SSTR1 is efficiently recycled to the cell surface in BT-474 breast cancer cells, this suggests that EGF or SST+EGF accelerate the internalization or trafficking of SSTR1 [51, 107]. These findings contradict earlier reports stating that monomeric SSTR1 is resistant to internalization and that agonist treatment increased the expression of SSTR1 at the cell surface, suggesting that EGF or SST+EGF treatment induced SSTR1 dimerization in BT-474 breast cancer cells [52, 79, 96].  In SK-BR-3 cells, as shown in Figure 3.4 B and D, treatment with SST and/or EGF treatment over 30 minutes also produced appreciable time- and treatment-dependent changes in SSTR1 expression at the membrane relative to that seen in control cells. SST treatment maximally upregulated SSTR1 after 10 min (Figure 3.4 B, D, Upper panel) (increased by 90%), whereas the activation of SSTR1 peaked after 15 minutes in BT-474 cells under this treatment (Figure 3.4 A, C, Upper panel). Conversely, the maximal SSTR1 activation in SK-BR-3 cells treated with EGF occurred after 30 minutes (Figure 3.4 B, D, Upper panel) (increased by 77%), compared to 5 minutes in BT-474 cells (Figure 3.4 A, C, Upper panel). The combined SST+EGF treatment induced stronger SSTR1 activation (peaking at 30 min) (strongly increased by 374%) than SST or EGF alone (Figure 3.4 B, D, Upper panel), yielding high levels of membrane expression of SSTR1 at 15-30 min in SK-BR-3 cells; there was such trend in BT-474 cells 51  (powerfully increased by 701%). Additionally, the up- or down-regulation of SSTR1 at the membrane in BT-474 cells treated with SST+EGF (Figure 3.4 A, C, Upper panel) produced corresponding changes in the levels of SSTR1 in the cytosolic fraction (Figure 3.4 A, C, Lower panel). This suggests that the combined SST+EGF strongly activated SSTR1 and promoted its efficient recycling to the cell surface. This result again contradicts earlier reports that monomeric SSTR1 is resistant to internalization and is upregulated at the cell surface upon agonist treatment [52, 96]; Importantly, Figure 3.4 B and D showed that SST+EGF treatment enhanced SSTR1 trafficking in SK-BR-3 cells, reducing levels of this protein in the cytosolic fraction (Figure 3.4 B, D, Lower panel) while increasing those at the membrane (Figure 3.4 B, D, Upper panel). This stands in contrast to earlier reports concerning the effects of SSTR1 agonist treatment in breast cancer cells, and suggests that the SST+EGF treatment induced SSTR1 dimerization in both the studied cell lines [58, 103, 121, 208, 211]. Relative to controls and cells treated with SST or EGF alone, combined treatment with SST+EGF enhanced the coexpression/homodimerization of SSTR1/SSTR1 strongly in BT-474 cells and in SK-BR-3 cells.  Similar time-dependent and treatment-specific changes were observed in the expression of SSTR2 in BT-474 (Figure 3.5 A, C) and SK-BR-3 (Figure 3.5 B, D) cells treated with SST and/or EGF, as shown in Figure 3.5. Treatment with SST, EGF, or SST+EGF maximally upregulated SSTR2 in BT-474 cells by 93% within 30 min, 46% within 10 min, and 47% within 5min (Figure 3.5 A, C, Upper panel), whereas the strongest upregulation observed in SK-BR-3 cells was 166% within 30 min, 246% within 30 min, and 107% within 15min (Figure 3.5 B, D, Upper panel) relative to controls. The combined SST+EGF treatment activated SSTR2 at most slightly more strongly in BT-474 (Figure 3.5 A, C, Upper panel) and SK-BR-3 cells (Figure 3.5 B, D, Upper panel) relative to that seen in control cells. The increases in membrane SSTR2 52  expression in cells treated with SST+EGF were matched by corresponding decreases in cytosolic SSTR2 levels in both cell lines. These results are consistent with previous reports that SSTR2 activation by agonist treatment causes dissociation of SSTR2 homodimers at the cell surface [116, 117]. SSTR2 activation at the surfaces of BT-474 cells treated with SST+EGF apparently triggered the increases in the desensitization and internalization of SSTR2 after 30 min, but the receptor’s cytosolic levels were stable, indicating that the treatment did not induce its degradation. This result is consistent with previous reports showing that the activation of SSTR2 in breast cancer cells coexpressing SSTR2 and OR resulted in increased receptor phosphorylation, desensitization, and endocytosis of both receptors, inhibiting tumor promoting signals [52, 95, 111]. The SSTR2 activation induced by the SST+EGF treatment after 5-10 min, and the inhibition observed after 15-30 min in BT-474 cells (Figure 3.5 A, C, Upper panel) are also consistent with previous reports that SSTR2 exists as preformed dimers that dissociate upon agonist treatment prior to internalization [65]. Interestingly, the changes in the membrane levels of SSTR2 (Figure 3.5, Upper panel) and SSTR1 (Figure 3.4, Upper panel) over time under the SST+EGF treatment are not synchronized, showing that the two proteins are not coexpressed or colocalized at the plasma membrane in BT-474 or SK-BR-3 cells. Figure 3.6 shows time-dependent changes in SSTR3 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) cells upon SST and/or EGF treatment. SSTR3 was expressed much less strongly than either SSTR1 (Figure 3.4, Upper panel) or SSTR2 (Figure 3.5, Upper panel) in both studied cell lines. It was weakly expressed at the membrane in BT-474 (Figure 3.6A, C, Upper panel) and SK-BR-3 (Figure 3.6 B, D, Upper panel) cells under all SST and/or EGF treatments, in keeping with previous reports based on studies using MCF-7 cells and MDA-MB-231 cells [103]. However, stimulation with SST or EGF alone did increase its expression and 53  activation at the cell surface in BT-474 cells by 22% and 44%, respectively, after 30 min (Figure 3.6 A, C, Upper panel). These treatments had stronger effects in SK-BR-3 cells, increasing the expression and activation of SSTR3 at the cell surface by 139% and 124% after 15 min (Figure 3.6 A, C, Upper panel). The combined SST+EGF treatment induced moderate SSTR3 expression at the membrane in SK-BR-3 cells, giving a maximum increase of 41% after five minutes, but reducing its expression by 12% relative to controls after 30 min (Figure 3.6 B, D, Upper panel). In BT-474 cells, the combined treatment mildly downregulated SSTR3, reducing its membrane levels by 25% after 30 min (Figure 3.6 A, C, Upper panel) relative to controls. The results for both cell lines are consistent with previous reports stating that SSTR3 activation leads to agonist-induced receptor trafficking as a result of the dissociation of SSTR3/SSTR3 homodimers, which mediate intracellular signaling and regulate apoptosis via their C-tails [108, 118].  Figure 3.7 shows that agonist treatment induced striking time-dependent and treatment-specific changes in the cell surface expression of SSTR4 in both cell lines. Treatment with SST or EGF alone strongly upregulated SSTR4 in BT-474 cells, increasing its expression by as much as 339% and 159% relative to controls after 15 and 30 minutes, respectively (Figure 3.7 A, C, Upper panel). The increases seen in SK-BR-3 cells were less pronounced but still substantial: 71% after 15 min, and 44% after 15 min (Figure 3.7 B, D, Upper panel) upon SST or EGF treatment, respectively. The SST+EGF treatment induced similar prolonged activation of SSTR4 in BT-474 cells between 5 and 15 minutes, when its upregulation relative to control cells rose from 33% to 159% (Figure 3.7 A, C, Upper panel). The combined treatment also delayed the internalization of SSTR4 at 30 min (Figure 3.7 A, C, Lower panel). These results are consistent with earlier reports stating that SSTR4 dimerization at the cell surface is stabilized by agonist binding [79]. The combined treatment thus powerfully enhanced the 54  expression/homodimerization of SSTR4 in BT-474 cells relative to controls (Figure 3.7 A, C, Upper panel). Conversely, in SK-BR-3 cells, the combined SST+EGF treatment only induced the downregulation of SSTR4 expression at the cell surface (Figure 3.7 B, D, Upper panel), and enhanced its internalization (Figure 3.7 B, D, Lower panel), even though treatment with SST or EGF alone strongly increased its expression. These time-dependent changes in the expression of SSTR4 are completely different to those observed for SSTR1 (Figure 3.4), SSTR2 (Figure 3.5), and SSTR3 (Figure 3.6) in both cell lines, demonstrating that there is no coexpression (or colocalization) of SSTR4 with SSTR1, SSTR2, or SSTR3 at the plasma membrane in BT-474 (Figure 3.7 A, C, Upper panel) and SK-BR-3 (Figure 3.7 B, D, Upper panel) cells after treatment with SST and/or EGF.  Figure 3.8 shows that SSTR5 exhibited interesting and unusual time-dependent and treatment-specific changes in expression in BT-474 (Figure 3.8 A, C) and SK-BR-3 (Figure 3.8 B, D) cells following treatment with SST and/or EGF. SST stimulation activated SSTR5 in BT-474 very rapidly, increasing its abundance at the membrane by 506% after 5 min and 592% after 30 min (Figure 3.8 A, C, Upper panel) while SST stimulated the activation of SSTR5 in SK-BR-3 cells at 10 min, causing the levels of this protein at the cell surface to peak at 66% above the control value after 15 min (Figure 3.8 B, D, Upper panel). Despite this, only a small proportion of the SSTR5 at the cell surface was respectively internalized into BT-474 (Figure 3.8 A, C, Lower panel) or SK-BR-3 (Figure 3.8 B, Lower panel) cells following treatment with SST. Whereas EGF treatment caused a strong increase in the levels of SSTR5 at the cell surface (149%) within 5 minutes (Figure 3.8 A, C, Upper panel) in BT-474 cells, it caused decrease in the abundance of SSTR5 at the cell surface within 5 minutes in SK-BR-3 cells (Figure 3.8 B, D, Upper panel). However, it appreciably increased the abundance of SSTR5 at the surface of SK-  55  BR-3 cells (67%) after 15 minutes (Figure 3.8 B, D, Upper panel). The combined SST+EGF treatment induced similar levels of SSTR5 activation at the cell surface in both the BT-474 (increased by 431%) and SK-BR-3 (increased by 40%) cells, peaking after 5 and 10 minutes, respectively. Interestingly, SSTR5 at the cell surface was gradually internalized into BT-474 (Figure 3.8 A, C, Lower panel) and SK-BR-3 (Figure 3.8 B, D, Lower panel) cells after treatment with SST+EGF, indicating that the combined treatment both activated SSTR5 and induced its trafficking in both tested cell lines. These findings contradict earlier reports indicating that SSTR5 is coexpressed with other receptors (e.g. SSTR1, SSTR2, or SSTR4), and formed heterodimers with these proteins upon binding to SST [58, 96]. But these time-dependent changes in the expression of SSTR5 are difference from that of those observed SSTR1 (Figure 3.4), SSTR2 (Figure 3.5), SSTR3 (Figure 3.6) and SSTR4 (Figure 3.7) in both cell lines, indicating that has no coexpression (or colocalization) of SSTR5/SSTR1, SSTR5/SSTR2, SSTR5/ SSTR3 or SSTR5/SSTR4 at the cell surface in BT-474 (Figure 3.8 A, C, Upper panel) and SK-BR-3 (Figure 3.8 B, D, Upper panel) cells upon SST and/or EGF treatment. These effects have not been reported previously [33, 51, 202-204, 208, 211].    56   Figure 3.4 Time-dependent changes in SSTR1 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF. Membrane protein samples (25 µg) in BT-474 (A) and SK-BR-3 (B) cells were fractionated by SDS-PAGE and probed with affinity-purified SSTR1 antibodies. Major protein bands of SSTR1 (53 kDa) membrane expression in BT-474 cells (A, Upper panel) and SK-BR-3 cells (B, Upper panel) were respectively obtained in this way. Cytosolic protein samples (15 µg) were fractionated by SDS-PAGE and probed with affinity-purified SSTR1 antibodies, yielding cytosolic fractions of SSTR1 in BT-474 cells (A, Lower panel) and SK-BR-3 cells (B, Lower panel). In subfigures C and D, the topmost graphs (blue bars) show SSTR1 expression in the membrane fraction of BT-474 (C,  Upper panel) and SK-BR-3 (D, Upper panel)  cells and the bottom graphs (orange bars) show SSTR1 expression in the cytosolic fraction of BT-474 (C, Lower panel) and SK-BR-3 (D, Lower panel) cells. Note time-dependent increases in SSTR1, expression at the membrane following SST or SST+EGF treatment in both cell lines. The status of SSTR1 expression was respectively determined by densitometric analysis of SSTR1 using β-Actin (42 kDa) as a loading control. Results represent Mean ± S.D. of three independent experiments. Data analysis was performed by using two-way ANOVA and post hoc Bonferroni's test to compare all treatment groups against control (*, p < 0.05). 57   Figure 3.5 Time-dependent changes in SSTR2 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF. Membrane protein samples (25 µg) in BT-474 (A) and SK-BR-3 (B) cells were fractionated by SDS-PAGE and probed with affinity-purified SSTR2 antibodies. Major protein bands of SSTR2 (57 kDa) membrane expression in BT-474 cells (A, Upper panel) and SK-BR-3 cells (B, Upper panel) were respectively obtained in this way. Cytosolic protein samples (15 µg) were fractionated by SDS-PAGE and probed with affinity-purified SSTR2 antibodies, yielding cytosolic fractions of SSTR2 in BT-474 cells (A, Lower panel) and SK-BR-3 cells (B, Lower panel). In subfigures C and D, the topmost graphs (blue bars) show SSTR2 expression in the membrane fraction of BT-474 (C,  Upper panel) and SK-BR-3 (D, Upper panel)  cells and the bottom graphs (orange bars) show SSTR2 expression in the cytosolic fraction of BT-474 (C, Lower panel) and SK-BR-3 (D, Lower panel) cells. Note time-dependent increases in SSTR2 expression at the membrane following SST or SST+EGF treatment in BT-474 (A, C, Upper panel) and SK-BR-3 (B, D, Upper panel) cells. The status of SSTR2 expression was respectively determined by densitometric analysis of SSTR2 using β-Actin (42 kDa) as a loading control. Results represent Mean ± S.D. of three independent experiments. Data analysis was performed by using two-way ANOVA and post hoc Bonferroni's test to compare all treatment groups against control (*, p < 0.05). 58   Figure 3.6 Time-dependent changes in SSTR3 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF. Membrane protein samples (25 µg) in BT-474 (A) and SK-BR-3 (B) cells were fractionated by SDS-PAGE and probed with affinity-purified SSTR3 antibodies. Major protein bands of SSTR3 (60 kDa) membrane expression in BT-474 cells (A, Upper panel) and SK-BR-3 cells (B, Upper panel) were respectively obtained in this way. Cytosolic protein samples (15 µg) were fractionated by SDS-PAGE and probed with affinity-purified SSTR3 antibodies, yielding cytosolic fractions of SSTR3 in BT-474 cells (A, Lower panel) and SK-BR-3 cells (B, Lower panel). In subfigures C and D, the topmost graphs (blue bars) show SSTR3 expression in the membrane fraction of BT-474 (C,  Upper panel) and SK-BR-3 (D, Upper panel)  cells and the bottom graphs (orange bars) show SSTR3 expression in the cytosolic fraction of BT-474 (C, Lower panel) and SK-BR-3 (D, Lower panel) cells. Note time-dependent increases in SSTR3 expression at the membrane following SST or SST+EGF treatment in BT-474 (A, C, Upper panel) and SK-BR-3 (B, D, Upper panel) cells. The status of SSTR3 expression was respectively determined by densitometric analysis of SSTR3 using β-Actin (42 kDa) as a loading control. Results represent Mean ± S.D. of three independent experiments. Data analysis was performed by using two-way ANOVA and post hoc Bonferroni's test to compare all treatment groups against control (*, p < 0.05). 59   Figure 3.7 Time-dependent changes in SSTR4 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF. Membrane protein samples (25 µg) in BT-474 (A) and SK-BR-3 (B) cells were fractionated by SDS-PAGE and probed with affinity-purified SSTR4 antibodies. Major protein bands of SSTR4 (44 kDa) membrane expression in BT-474 cells (A, Upper panel) and SK-BR-3 cells (B, Upper panel) were respectively obtained in this way. Cytosolic protein samples (15 µg) were fractionated by SDS-PAGE and probed with affinity-purified SSTR4 antibodies, yielding cytosolic fractions of SSTR4 in BT-474 cells (A, Lower panel) and SK-BR-3 cells (B, Lower panel). In subfigures C and D, the topmost graphs (blue bars) show SSTR4 expression in the membrane fraction of BT-474 (C,  Upper panel) and SK-BR-3 (D, Upper panel)  cells and the bottom graphs (orange bars) show SSTR4 expression in the cytosolic fraction of BT-474 (C, Lower panel) and SK-BR-3 (D, Lower panel) cells. Note time-dependent increases in SSTR4 expression at the membrane following SST or SST+EGF treatment in BT-474 (A, C, Upper panel) and SK-BR-3 (B, D, Upper panel) cells. The status of SSTR4 expression was respectively determined by densitometric analysis of SSTR4 using β-Actin (42 kDa) as a loading control. Results represent Mean ± S.D. of three independent experiments. Data analysis was performed by using two-way ANOVA and post hoc Bonferroni's test to compare all treatment groups against control (*, p < 0.05). 60   Figure 3.8 Time-dependent changes in SSTR5 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF. Membrane protein samples (25 µg) in BT-474 (A) and SK-BR-3 (B) cells were fractionated by SDS-PAGE and probed with affinity-purified SSTR5 antibodies. Major protein bands of SSTR5 (58 kDa) membrane expression in BT-474 cells (A, Upper panel) and SK-BR-3 cells (B, Upper panel) were respectively obtained in this way. Cytosolic protein samples (15 µg) were fractionated by SDS-PAGE and probed with affinity-purified SSTR5 antibodies, yielding cytosolic fractions of SSTR5 in BT-474 cells (A, Lower panel) and SK-BR-3 cells (B, Lower panel). In subfigures C and D, the topmost graphs (blue bars) show SSTR5 expression in the membrane fraction of BT-474 (C, Upper panel) and SK-BR-3 (D, Upper panel)  cells and the bottom graphs (orange bars) show SSTR5 expression in the cytosolic fraction of BT-474 (C, Lower panel) and SK-BR-3 (D, Lower panel) cells. Note time-dependent increases in SSTR5 expression at the membrane following SST or SST+EGF treatment in BT-474 (A, C, Upper panel) and SK-BR-3 (B, D, Upper panel) cells. The status of SSTR5 expression was respectively determined by densitometric analysis of SSTR5 using β-Actin (42 kDa) as a loading control. Results represent Mean ± S.D. of three independent experiments. Data analysis was performed by using two-way ANOVA and post hoc Bonferroni's test to compare all treatment groups against control (*, p < 0.05).. 61  3.1.2.2 Time-dependent changes in ErbB 1-4 expression in BT-474 and SK-BR-3 cells after treatment with SST and/or EGF Our group has shown that SSTRs and ErbBs can be coexpressed in ER-positive MCF-7 cells and ERa-negative MDA-MB-231 breast cancer cells, and exhibit receptor-, cell line-, and ER-dependent colocalization following treatment with SST and/or EGF [65]. It is not known whether SSTR1-5 and ErbB1-4 are coexpressed in BT-474 and SK-BR-3 cells. Figure 3.9 shows time-dependent changes in ErbB1 protein expression in BT-474 (Figure 3.9 A, C) and SK-BR-3 (Figure 3.9 B, D) breast cancer cells after treatment with SST and/or EGF. Both cell lines exhibited significant time- and treatment-dependent changes in ErbB1 expression (relative to controls) at the membrane under these conditions. The strongest upregulation of ErbB1 expression at the cell surface across all treatments in BT-474 cells was 266% after 30 min, 118% after 10 min, and 128% after 5 min (Figure 3.9 A, C, Upper panel) upon SST, EGF, SST+EGF treatment, respectively. The corresponding values for SK-BR-3 cells were 187% after 15 min, 177% after 15 min, and 89% after 15 min (Figure 3.9 B, D, Upper panel) upon SST, EGF, SST+EGF treatment, respectively. The SST+EGF treatments maximally downregulated ErbB1 expression at the cell surface in BT-474 cells by 78% after 15 min and 92% after 30min, respectively (Figure 3.9 A, C, Upper panel); the corresponding downregulated values for SK-BR-3 cells were 20% after 15 min and  57% after 30 min, respectively (Figure 3.9 B, D, Upper panel). More importantly, the SST+EGF treatment caused ErbB1 at the membrane to be gradually internalized (Figure 3.9 A, C, Lower panel), with around 92% internalization after 30 min in BT-474 cells (Figure 3.9 B, D, Lower panel) while around 57% internalized into SK-BR-3 cells. Interestingly, the time-dependent changes in ErbB1 membrane expression in BT-474 cells treated with SST and/or EGF (Figure 3.9 A, C) closely mirrored those seen for SSTR2 62  expression (Figure 3.5 A, C). For example, both ErbB1 and SSTR2 exhibited maximal upregulation at 30, 10, and 5 minutes in BT-474 cells under the SST, EGF, and SST+EGF treatments, respectively. Moreover, both proteins exhibited similar patterns of downregulation under all treatments (except at 30 minutes under the SST+EGF treatments), suggesting that they participate in the same cyclical processes of sensitization, desensitization, internalization, and recycling to the plasma membrane, and are synchronously coexpressed and colocalized in BT-474 cells under the test conditions. This result is consistent with previous reports showing that activation resulting from ligand binding induces SSTR2 heterodimerization with other receptor proteins [58, 96, 103], suggesting that SSTR2/ErbB1 coexpression and heterodimerization may occur in breast cancer cells upon stimulation by SST and/or EGF. Importantly, ErbB1 (Figure 3.9 A, C, Upper panel) and SSTR2 (Figure 3.5 A, C, Upper panel) simultaneously underwent strong membrane downregulation and internalization after 15 minutes in BT-474 cells under the SST+EGF treatment (Figure 3.9 and 3.5 A, C, Upper panel, Lower panel). The loss of ErbB1 loss at the cell surface (Figure 3.9 A, C, Upper panel) was not accompanied by any corresponding intracellular accumulation (Figure 3.9   A, C, Lower panel), but SSTR2 (Figure 3.5 A, C, Lower panel) did accumulate in the cytosolic fraction, suggesting that SST+EGF induced ErbB1 degradation in BT-474 cells [58, 65]. It is interesting that the ErbB1 in the BT-474 cells was rapidly degraded after 30 minutes under the SST+EGF treatment rather than being recycled back to the cell surface (Figure 3.9 A, C, Lower panel); this suggests that SST+EGF induced ErbB1 degradation and signal termination in BT-474 cells, in keeping with previously reports that degradation of ErbB1 mediated by receptor ubiquitination in the internal compartments is a mechanism of signal termination [58, 153]. The most important finding, however, is that the observed time-dependent changes in SSTR2/ErbB1 expression at the cell 63  surface (Figure 3.5 and 3.9 A, C) in BT-474 cells induced by SST and/or EGF treatment indicate that these two proteins exhibit very significant coexpression and may form heterodimers. In particular, the SST+EGF treatment seemingly induced the coexpression and heterodimerization of SSTR2/ErbB1 within 5-10 min, followed by the dissociation of the SSTR2/ErbB1 heterodimers after 15-30 min. This in turn led to the internalization of SSTR2 and ErbB1, with SSTR2 accumulating in the cytosol and ErbB1 undergoing intracellular degradation (Figure 3.5 and 3.9 A, C, Lower panel). These results clearly suggest the existence of direct interactions and crosstalk between SSTR2 and ErbB1 in BT-474 cells, in keeping with previously reported results [52, 96].  Equally interestingly, the time-dependent changes in ErbB1 expression (Figure 3.9 B, D) observed in SK-BR-3 cells closely mirror those for the expression of SSTR5 in SK-BR-3 cells (Figure 3.8 B, D). In particular, the SST, EGF and SST+EGF induced to maximally upregulate both receptors (SSTR5/ErbB1) at 15, 15, and 10 min, respectively. This is consistent with coexpression and potentially heterodimerization of SSTR5 and ErbB1 in the SK-BR-3 cell line, and with earlier reports from our group showing that these proteins form heterodimers in MDA-MB-231 cells [58]. The results obtained also suggest that SSTR5 and ErbB1 were coexpressed and formed heterodimers at the surface of SK-BR-3 cells within 5-10 minutes of SST+EGF treatment (Figures 3.8-3.9 B, D, Upper panel), and that the heterodimers then dissociated between 15 and 30 minutes, with both proteins being internalized into the cells (Figures 3.7-3.9 B, D, Lower panel). The internalized SSTR5 appears to have been recycled back to the cell surface (Figure 3.9 B, D, Upper panel), whereas the ErbB1 was degraded inside the cell (Figure 3.9 B, D, Lower panel). These results strongly suggest that SST and/or EGF treatment activated SSTR5 and/or the SSTR5/ErbB1 heterodimer, leading to coexpression of SSTR5 and ErbB1 at 64  the surface of SK-BR-3 cells. As such, these observations suggest that there is crosstalk between SSTR5 and ErbB1 in SK-BR-3 cells treated with SST and EGF [58, 110, 188]. The possible crosstalk of ErbB1 between with SSTR in both cell lines was summarized in Figure 3.9 E. EGF treatment has been reported to induce strong coexpression of ErbB1/ErbB2, as well as including  ErbB1/ErbB3 and ErbB2/ErbB3 coexpression, at the surface of BT-474 cells via the formation of ErbB1/ErbB2, ErbB1/ErbB3 and ErbB2/ErbB3 heterodimers [145, 167],. Similarly, in SK-BR-3 cells, EGF treatment was reported to induce strong coexpression of ErbB2/ErbB3, as well as including ErbB1/ErbB2 coexpression, at the cell surface via the formation of ErbB2/ErbB3 and ErbB1/ErbB2 heterodimers [145, 167, 208, 211-214]. To characterize the expression and interactions of ErbB2 in the membrane and cytosolic fractions of BT-474 and SK-BR-3 cells, we performed additional Western blotting experiments; the results obtained are shown in Figure 3.10. EGF upregulated ErbB2 expression at the cell surface by 30% over 30min in BT-474 cells (Figure 3.10 A, C, Upper panel). Similarly, EGF induced ErbB2 membrane expression increased by 76% in SK-BR-3 cells (Figure 3.10 B, D, Upper panel). This behavior was time-dependent and treatment-specific. Surprisingly, the expression of ErbB2 at the cell surface was respectively induced to observably downregulated at cell surface by SST+EGF treatments in BT-474 cells, suggesting that ErbB1/ErbB2 are werk coexpression at the membrane in this line (Figures 3.9-3.10 A, C, Upper panel), and that the coexpression of SSTR2/ErbB1 (Figure 3.5, 3.10, A, C) at the cell surface induced by treatment with SST and/or EGF reduces the heterodimerization of ErbB1 with ErbB2 [145, 208]. More surprisingly, the changes in the expression of ErbB2 in the membranes of BT-474 cells treated with SST and/or EGF (Figure 3.10 A, C, Upper panel) were synchronized with those of SSTR3 (Figure 3.6, A, C, Upper panel). Like SSTR3 (Figure 3.6, A, C, Upper panel), ErbB2 exhibited EGF-induced 65  upregulation after 5 min (by 7% in the case of ErbB2 and 8% in the case of SSTR3), downregulation (by 27% and 17%, respectively) after 10 min, further downregulation (37% and 22%, respectively) after 15 min, and finally upregulation (30% and 44%, respectively) after 30 min relative to controls (Figure 3.6, 3.10, A, C, Upper panel). Similarly, the SST+EGF treatment downregulated the membrane expression of ErbB2 and SSTR3 in BT-474 cells by 22% and 15%, respectively, after 5 min; by31% and 23%, respectively, after 10 min; and by 17% and 5%, respectively, after 15 min (relative to controls). Both proteins were then downregulated (by 22% and 25%) over the final 15 minutes of the experiment (Figure 3.6, 3.10, A, C, Upper panel), respectively.  Possible crosstalk of ErbB2 between with SSTR in this cell line was displayed in Figure 3.10 E. These results show that SST and/or EGF induces the coexpression and possibly the heterodimerization of ErbB2 with SSTR3 at the plasma membrane in BT-474 cells, suggesting the existence of crosstalk between these receptors in this cell line. This conclusion is consistent with previously reported results [52, 96, 108, 118].    Interestingly, the decrease in the expression of ErbB2 at the surfaces in SK-BR-3 cells induced by SST or SST+EGF was stronger than that induced by EGF alone (Figure 3.10 B, D, Upper panel). The changes in the expression of ErbB2 at the membrane in SK-BR-3 cells treated with SST+EGF was very different to those induced by SST or EGF alone, e.g.  EGF treatment induced the upregulation of ErbB2 (increased by 36%) at 5 min, downregulation at 10 min (decreased by 2%with internalization), and then upregulation at 15 and 30 min (increased by 75% and 76%, i.e. sustained membrane expression) in SK-BR-3 cells. Conversely, the SST+EGF treatment caused ErbB2 to be downregulated by 8% at 5 min, downregulated by 53% and 62% at 10 min and 15 min (with very little recycling to the plasma membrane, suggesting extensive cytosolic degradation), and finally upregulated by 12% at 30 min (Figure 3.10 B, D, Lower panel, 66  Upper panel). This indicates that the SST+EGF treatment significantly downregulated ErbB2 at the cell surface in SK-BR-3 cells by enhancing ErbB2 internalization, delaying its recycling to the plasma membrane, and inducing its intracellular partial degradation. These responses were accompanied by reduced heterodimerization of ErbB1 with ErbB2 and coexpression of ErbB1 with SSTR5 at the cell surface, with potential ErbB1/SSTR5 i.e. SSTR5/ErbB1 heterodimer formation and crosstalk as discussed above (Figures 3.8-3.10 B, D, Lower panel, Upper panel). Previous studies have showed that the heterodimerization of ErbB3 with other members of the ErbB family (producing the ErbB3/ ErbB2, ErbB3/ ErbB1, and ErbB3/ ErbB4 heterodimers) is a prerequisite for ErbB3 signaling because ErbB3 is kinase-impaired, in that it has mutations in its cytoplasmic domain that block its tyrosine kinase activity [52, 56, 148]. As shown in Figure 3.11, the EGF treatment downregulated the expression of ErbB3 at the cell surface by 32% in BT-474 (Figure 3.11 A, C, Upper panel) cells over 5 min, and upregulated by 12% in SK-BR-3 cells (Figure 3.11 B, D, Upper panel) over 15 min. Conversely, the SST+EGF treatments strongly downregulated the membrane expression of ErbB3 in both cell lines - by 42% and 44% in BT-474 and SK-BR-3 cells after 30 min, respectively. These results are inconsistent with earlier reports stating that EGF treatment increased the expression of ErbB3 in the membranes of breast carcinoma cells [211]. The SST and/or EGF treatments also shown to reduce  the coexpression of ErbB3/ErbB2, ErbB3/ErbB1 in BT-474 (Figures 3.9-3.11 A, C, Upper panel) and SK-BR-3 (Figure 3.9 -3.11 B, D, Upper panel) cells, suggesting that the heterodimerization of these proteins may be blocked or suppressed in breast cancer cells [167, 211, 212].  More interestingly, the SST+ EGF -induced changes in the membrane expression of ErbB3 (Figure 3.11 A, C, Upper panel) paralleled those seen for SSTR5 (Figure 3.8 A, C, Upper 67  panel), indicating that SST+ EGF also induced coexpression of ErbB3 and SSTR5 at the membrane in BT-474 cells. This result is consistent with previous reports showing approximately 41% colocalization of ErbB3 with SSTR5 in MCF-7 cells [103]. Importantly, the SST+EGF treatment desensitized both of these receptors (ErbB3 and SSTR5), inducing their internalization and downregulation at the membrane after 30 min (Figure 3.8 and 11 A, C, Upper panel). This suggests that membrane coexpression of ErbB3 and SSTR5 leads to partial heterodimerization of SSTR5/ErbB3 in BT-474 cells upon SST+EGF stimulation, which is consistent with previous reports showing that the SST-induced internalization and subsequent downregulation of SSTR2-5 at the membrane may release ErbBs from heterodimers [101, 103, 215, 216]. More notably, the expression of ErbB3 (Figure 3.11, Upper panel), ErbB2 (Figure 3.10, Upper panel) and ErbB1 (Figure 3.9 , Upper panel) at the plasma membrane in BT-474 (A, C) and SK-BR-3 (B, D) cells under the SST+EGF treatment was clearly weaker than in control cells. This finding is consistent with reduced expression of ErbB1/ErbB2, ErbB1/ErbB3, and ErbB2/ErbB3 at the membrane as a consequence of their dissociation and the competing formation of heterodimers with other proteins [55, 148]. These results suggested that the persistence of ErbB3/ErbB2, ErbB3/ErbB1 and ErbB1/ErbB2 heterodimers at the cell surface in the studied cell lines is significantly reduced by the coexpression of and possible crosstalk between SSTR2 and ErbB1, SSTR3 and ErbB2, and SSTR5 and ErbB3 at the cell surface in BT-474 cells, and SSTR5 and ErbB1 in SK-BR-3 cells [58, 110, 188]. In contrast to the results obtained for ErbB1-3 (Figures 3.9-3.11, Upper panel), the membrane expression of ErbB4 in BT-474 and SK-BR-3 cells was not greatly affected by SST, EGF, or SST+EGF treatments (Figure 3.12, Upper panel), although there were modest increases in its expression in both cell lines relative to controls. Furthermore, because the SST+EGF 68  treatment reduced the expression of ErbB1-3 at the cell surface in both cell lines, the coexpression or heterodimerization of ErbB4 with the other ErbB proteins was reduced or eliminated [49, 79, 105]. Taken together, these results indicate that treatment of BT-474 cells with SST and/or EGF induces the coexpression and possible heterodimerization of SSTR2/ErbB1, SSTR3/ErbB2, and SSTR5/ErbB3 at the cell membrane; powerfully enhances the expression of SSTR1 and SSTR4 at the cell surface, and may give rise to crosstalk between SSTR2/ErbB1, SSTR3/ErbB2 and SSTR5/ErbB3, dramatically reducing the membrane expression of ErbB1 (and causing its subsequent intracellular degradation) as well as ErbB2 and ErbB3. In SK-BR-3 cells, treatment with SST and/or EGF induced the membrane coexpression and possible heterodimerization of SSTR5/ErbB1, strongly increased the cell surface expression of SSTR1 and SSTR2, as well as moderately increased that of SSTR3 while potentially inducing crosstalk between SSTR5 and ErbB1, leading to significantly reduced membrane expression of ErbB1 and ErbB2 (both of which underwent intracellular degradation) and also ErbB3.        69    Figure 3.9 Time-dependent changes in ErbB1 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF, and possible crosstalk between ErbB1 with SSTRs in BT-474 (E, Upper panel) and SK-BR-3 (E, Lower panel) cells. Membrane protein samples (25 µg) in BT-474 (A) and SK-BR-3 (B) cells were fractionated by SDS-PAGE and 70  probed with affinity-purified ErbB1 antibodies. Major protein bands of ErbB1 (170 kDa) membrane expression in BT-474 cells (A, Upper panel) and SK-BR-3 cells (B, Upper panel) were respectively obtained in this way. Cytosolic protein samples (15 µg) were fractionated by SDS-PAGE and probed with affinity-purified ErbB1 antibodies, yielding cytosolic fractions of ErbB1 in BT-474 cells (A, Lower panel) and SK-BR-3 cells (B, Lower panel). In subfigures C and D, the topmost graphs (blue bars) show ErbB1 expression in the membrane fraction of BT-474 (C,  Upper panel) and SK-BR-3 (D, Upper panel)  cells and the bottom graphs (orange bars) show ErbB1 expression in the cytosolic fraction of BT-474 (C, Lower panel) and SK-BR-3 (D, Lower panel) cells. Note time-dependent decreases in ErbB1 expression at the membrane following SST or SST+EGF treatment in BT-474 (A, C, Upper panel) and SK-BR-3 (B, D, Upper panel) cells. The blue arrow indicates a rise in value of membrane protein expression whereas the red arrow represents the drop value of membrane protein expression in BT-474 (E, Upper panel) and SK-BR-3 (E, Lower panel) cells upon SST and/or EGF treatment compared with control group.  The status of ErbB1 expression was respectively determined by densitometric analysis of ErbB1 using β-Actin (42 kDa) as a loading control. Results represent Mean ± S.D. of three independent experiments. Data analysis was performed by using two-way ANOVA and post hoc Bonferroni's test to compare all treatment groups against control (*, p < 0.05).    71    Figure 3.10 Time-dependent changes in ErbB2 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF, and possible crosstalk between ErbB2 with SSTRs in BT-474 (E) cells. Membrane protein samples (25 µg) in BT-474 (A) and SK-BR-3 (B) cells were fractionated by SDS-PAGE and probed with affinity-purified ErbB2 antibodies. Major protein bands of ErbB2 (185 kDa) membrane expression in BT-474 cells (A, Upper panel) and SK-BR-3 cells (B, Upper panel) were respectively obtained in this way. Cytosolic protein samples (15 µg) were fractionated by SDS-PAGE and probed with affinity-purified ErbB2 antibodies, yielding cytosolic fractions of ErbB2 in BT-474 cells (A, Lower panel) and SK-BR-3 cells (B, Lower panel). In subfigures C and D, the topmost graphs (blue bars) show 72  ErbB2 expression in the membrane fraction of BT-474 (C,  Upper panel) and SK-BR-3 (D, Upper panel)  cells and the bottom graphs (orange bars) show ErbB2 expression in the cytosolic fraction of BT-474 (C, Lower panel) and SK-BR-3 (D, Lower panel) cells. Note time-dependent decreases in ErbB2 expression at the membrane following SST or SST+EGF treatment in BT-474 (A, C, Upper panel) and SK-BR-3 (B, D, Upper panel) cells. The blue arrow indicates a rise in value of membrane protein expression whereas the red arrow represents the drop value of membrane protein expression in BT-474 (E) cells upon SST and/or EGF treatment compared with control group. The status of ErbB2 expression was respectively determined by densitometric analysis of ErbB2 using β-Actin (42 kDa) as a loading control. Results represent Mean ± S.D. of three independent experiments. Data analysis was performed by using two-way ANOVA and post hoc Bonferroni's test to compare all treatment groups against control (*, p < 0.05).            73   Figure 3.11 Time-dependent changes in ErbB3 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF. Membrane protein samples (25 µg) in BT-474 (A) and SK-BR-3 (B) cells were fractionated by SDS-PAGE and probed with affinity-purified ErbB3 antibodies. Major protein bands of ErbB3 (200 kDa) membrane expression in BT-474 cells (A, Upper panel) and SK-BR-3 cells (B, Upper panel) were respectively obtained in this way. Cytosolic protein samples (15 µg) were fractionated by SDS-PAGE and probed with affinity-purified ErbB3 antibodies, yielding cytosolic fractions of ErbB3 in BT-474 cells (A, Lower panel) and SK-BR-3 cells (B, Lower panel). In subfigures C and D, the topmost graphs (blue bars) show ErbB3 expression in the membrane fraction of BT-474 (C,  Upper panel) and SK-BR-3 (D, Upper panel)  cells and the bottom graphs (orange bars) show ErbB3 expression in the cytosolic fraction of BT-474 (C, Lower panel) and SK-BR-3 (D, Lower panel) cells. Note time-dependent decreases in ErbB3 expression at the membrane following SST or SST+EGF treatment in BT-474 (A, C, Upper panel) and SK-BR-3 (B, D, Upper panel) cells. The status of ErbB3 expression was respectively determined by densitometric analysis of ErbB3 using β-Actin (42 kDa) as a loading control. Results represent Mean ± S.D. of three independent experiments. Data analysis was performed by using two-way ANOVA and post hoc Bonferroni's test to compare all treatment groups against control (*, p < 0.05). 74   Figure 3.12 Time-dependent changes in ErbB4 protein expression in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF. Membrane protein samples (25 µg) in BT-474 (A) and SK-BR-3 (B) cells were fractionated by SDS-PAGE and probed with affinity-purified ErbB4 antibodies. Major protein bands of ErbB4 (175 kDa) membrane expression in BT-474 cells (A, Upper panel) and SK-BR-3 cells (B, Upper panel) were respectively obtained in this way. Cytosolic protein samples (15 µg) were fractionated by SDS-PAGE and probed with affinity-purified ErbB4 antibodies, yielding cytosolic fractions of ErbB4 in BT-474 cells (A, Lower panel) and SK-BR-3 cells (B, Lower panel). In subfigures C and D, the topmost graphs (blue bars) show ErbB4 expression in the membrane fraction of BT-474 (C,  Upper panel) and SK-BR-3 (D, Upper panel)  cells and the bottom graphs (orange bars) show ErbB4 expression in the cytosolic fraction of BT-474 (C, Lower panel) and SK-BR-3 (D, Lower panel) cells. Note time-dependent decreases in ErbB4 expression at the membrane following SST or SST+EGF treatment in BT-474 (A, C, Upper panel) and SK-BR-3 (B, D, Upper panel) cells. The status of ErbB4 expression was respectively determined by densitometric analysis of ErbB4 using β-Actin (42 kDa) as a loading control. Results represent Mean ± S.D. of three independent experiments. Data analysis was performed by using two-way ANOVA and post hoc Bonferroni's test to compare all treatment groups against control (*, p < 0.05). 75  3.2 Time-dependent changes in ERK1/2 and p38 phosphorylation in BT-474 and SK-BR-3 cells upon SST and/or EGF treatment  The results presented in the preceding sections show that SSTRs interfere with ErbB heterodimerization via coexpression with ErbBs at the cell surface in the BT-474 and SK-BR-3 human breast cancer cells upon SST and/or EGF stimulation (Figures 3.5-3.12). Previous studies have shown that ERK1/2 plays a central and cell-specific role in controlling cell proliferation [49, 52, 83]. Similarly, p38 is known to play a key role in tumor development. We therefore studied changes in the phosphorylation of ERK1/2 and p38 after treatment with SST and/or EGF in the BT-474 and SK-BR-3 breast cancer cell lines (see Figures.3.13-3.14).  3.2.1 Time-dependent changes in ERK1/2 phosphorylation after treatment with SST and/or EGF in BT-474 and SK-BR-3 breast cancer cells As shown in Figure 3.13, BT-474 (Figure 3.13 A) and SK-BR-3(Figure 3.13 B) cells exhibited differential phosphorylation of ERK1/2 after SST and/or EGF treatment. The SST, EGF, SST+EGF treatments maximally upregulated the phosphorylation of ERK1/2 by 19%, 88%, and 8%, respectively, in BT-474 cells after 5 min, indicating that the SST and SST+EGF treatments did not delay the activation of ERK1/2 when compared to treatment with EGF alone. Similarly, the SST, EGF, and SST+EGF treatments downregulated the levels of ERK1/2 phosphorylation in BT-474 cells by 93%, 90%, and 82%, respectively, after 30 min (Figure 3.13 A, C), indicating that SST and/or EGF stimulation eventually induces strong downregulation of ERK 1/2 phosphorylation. These findings differ from earlier reports based on experiments with MCF-7 cells, in which there was a time-dependent increase in ERK1/2 phosphorylation upon treatment with SST, EGF, or SST+EGF [58].  76  The SST, EGF, and SST+EGF treatments upregulated the phosphorylation of ERK1/2 by 114%, 194%, and 381%, respectively in SK-BR-3 cells after 5 min (Figure 3.13 B, D), again showing that the ERK1/2 activation induced by SST or SST+EGF treatment was not delayed relative to that seen with EGF alone. The upregulation of ERK1/2 phosphorylation was sustained over the experimental period under all treatments, with upregulation of 381-491% being observed after 30 min. These observations with SK-BR-3 breast cancer cells differ from results previously obtained for MDA-MB-231 cells, which exhibited no time-dependent changes in ERK1/2 phosphorylation under any of the tested treatments [58].  There were clear differences between the BT-474 and SK-BR-3 cell lines in terms of their time-dependent changes in ERK1/2 phosphorylation upon SST, EGF, or SST+EGF treatment: the main downregulation of ERK1/2 phosphorylation were observed in BT-474 cells, whereas only upregulation was observed in SK-BR-3 cells. However, both sets of results demonstrate that SST and/or EGF binding strongly affected ERK1/2 signaling in these cell lines. This conclusion is consistent with previous studies showing that ERK1/2 inhibition or activation has important effects on cell proliferation [21, 58].  3.2.2 Time-dependent changes in p38 phosphorylation in BT-474 and SK-BR-3 cells upon SST and/or EGF treatment As shown in Figure 3.14, BT-474 (Figure 3.14 A, C) and SK-BR-3 (Figure 3.14 B, D) cells also exhibited differential p38 phosphorylation responses after treatment with SST and/or EGF.  Treatment with SST or EGF alone induced inhibited p38 phosphorylation by 18% or 17% within 5 min in BT-474 cells (Figure 3.14 A, C) but increased p38 phosphorylation by 133% and 104%, respectively, in SK-BR-3 cells after 5 min (Figure 3.14 B, D). Interestingly, the SST+EGF treatment caused p38 phosphorylation to be upregulated by 18% after 10 min and then 77  downregulated by 40% after 30 min in BT-474 cells (Figure 3.14 A, C) whereas it upregulated p38 phosphorylation by 154% after 5 min and by 212% after 30 min in SK-BR-3 cells (Figure 3.14 B, D). Overall, then, treatment with SST and/or EGF induced time-dependent reductions in p38 phosphorylation in BT-474 cells (Figure 3.14 A, C) but caused time-dependent increases in p38 phosphorylation in SK-BR-3 cells (Figure 3.14 B, D). This indicates that both ligands have strong effects on p38 signaling and activation in both cell lines [51].   Because treatment with SST and/or EGF strongly reduced the phosphorylation of ERK1/2 and p38 in BT-474 cells, these treatments would have significantly inhibited MAPK (ERK1/2 and p38) signaling. Conversely, these treatments significantly increased the phosphorylation of ERK1/2 and p38 in SK-BR-3 cells, which would have significantly enhanced MAPK (ERK1/2 and p38) signaling [21, 58]. The observed time-dependent changes in the phosphorylation of ERK1/2 and p38 in both cell lines upon SST and/or EGF treatment suggest that the regulation of ERK1/2 and p38 activity is both cell-specific and agonist-dependent [21, 58].       78   Figure 3.13 Time-dependent changes in the phosphorylation of ERK1/2 in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF. Cell lysate was processed for total and phospho-ERK1/2 using Western blot analysis. Note showing that significantly increased or decreased the phosphorylated levels of ERK1/2 in BT-474 (A) and SK-BR-3 (B) cells in comparison to control group. In subfigures C and D, the graphs of blue bars respectively show the phosphorylated ERK1/2 levels in BT-474 (C) and SK-BR-3 (D)  cells. Results represent three experiments performed independently. The phosphorylation status was determined by densitometric analysis of phosphorylated vs. total ERK1/2. Data analysis was done by using two-way ANOVA and post hoc Bonferroni's test to compare all treatment groups against control (*, p < 0.05).  79   Figure 3.14 Time-dependent changes in the phosphorylation of p38 in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF. Cell lysate was processed for total and phospho-p38 using Western blot analysis. Note showing that significantly increased or decreased the phosphorylated levels of p38 in BT-474 (A) and SK-BR-3 (B) cells in comparison to control group. In subfigures C and D, the graphs of blue bars respectively show the phosphorylated p38 levels in BT-474 (C) and SK-BR-3 (D) cells. Results represent three experiments performed independently. The phosphorylation status was determined by densitometric analysis of phosphorylated vs. total p38. Data analysis was done by using two-way ANOVA and post hoc Bonferroni's test to compare all treatment groups against control (*, p < 0.05).  3.3 Time-dependent changes in PI3K and PTEN phosphorylation in BT-474 and SK-BR-3 cells upon SST and/or EGF treatment.  To determine whether the interference with ErbB heterodimerization by activated SSTRs inhibits PI3K signaling and PTEN upregulation, we studied the time-dependent changes in PI3K 80  and PTEN phosphorylation in BT-474 and SK-BR-3 cells after treatment with SST and/or EGF (see Figures 3.15-3.16).  3.3.1 Time-dependent changes in PI3K phosphorylation in BT-474 and SK-BR-3 cells upon SST and/or EGF treatment  As shown in Figure 3.15, treatment of BT-474 (Figure 3.15 A, C) and SK-BR-3(Figure 3.15 B, D) cells with SST and/or EGF induced pronounced changes in PI3K phosphorylation. The SST treatment reduced PI3K phosphorylation by 60% within 15 min in BT-474 cells (Figure 3.15 A, C) and by 47% within 5 min in SK-BR-3 (Figure 3.15 B, D) cells. The SST-induced downregulation of PI3K phosphorylation persisted in BT-474 cells (Figure 3.15 A, C) (54 % downregulation at 10 min and 60% at 15 min but not in SK-BR-3 cells (see Figure 3.15 B, D). Conversely, EGF treatment downregulated PI3K phosphorylation by 21% after 5 min, while upregulated it by 7% after 10 min, and finally downregulated it by 1% after 15 min and 32% after 30 min in BT-474 cells (Figure 3.15 A, C).  In SK-BR-3 cells, EGF treatment downregulated PI3K phosphorylation by 48% after 5 min, 56% after 10 min, and 34% after 15 min, before upregulating it by 35% after 30 min (Figure 3.15 B, D).  Importantly, the SST+EGF treatment strongly reduced PI3K phosphorylation in both cell lines, reducing it by 50% after 5 min, 20% after 10 min, 46% after 15 min, and 53% after 30 min in BT-474 cells (Figure 3.15 A, C). In the SK-BR-3 line, the SST+EGF treatment reduced PI3K phosphorylation by 60% after 5 min, 68% after 10 min, 47% after 15 min, and 42% after 30 min (Figure 3.15 B, D).  These results  indicate that the PI3K cell survival pathway was significantly inhibited by treatment with SST and/or EGF in the BT-474 (Figure 3.15 A, C) and SK-BR-3 (Figure 3.15 B, 81  D) cell lines [51]. This conclusion is consistent with previous studies showing that these ligands inhibited PI3K phosphorylation in ER-positive MCF-7 and T47D cells, and increased necrosis in MCF-7 and T47D cells but not in ER-negative MDA-MB231 cells [111]. It is also consistent with results showing that activated SSTRs modulated the expression of PTP- 1C and the status of ERK1/2, p38 and PI3K phosphorylation in breast cancer cells in a cell-specific manner. For example, activated SSTR3 induced apoptosis in MCF-7 and cell cycle arrest in MDA-MB-231 [21, 51].  3.3.2 Time-dependent changes in PTEN phosphorylation in BT-474 and SK-BR-3 cells upon SST and/or EGF treatment As shown in Figure 3.16, the SST, EGF, and SST+EGF treatments all substantially increased PTEN phosphorylation in BT-474 cells (Figure 3.16 A, C), but reduced slightly it in SK-BR-3 (Figure 3.16 B, D) cells. Across all three treatments, the maximal observed increases in PTEN phosphorylation in BT-474 cells were 213% after 10 min, 187% after 5 min, and 206% after 15 min (Figure 3.16 A, C) upon SST, EGF and SST+EGF treatment, respectively. Interestingly, the increases in PTEN phosphorylation (Figure 3.16 A, C) were more pronounced than those in PI3K phosphorylation (Figure 3.15 A, C) in BT-474 cells treated with SST+EGF for 15 and 30 min.  Conversely, the level of PTEN phosphorylation was relatively steady in SK-BR-3 cells treated with SST and/or EGF (Figure 3.16 B, D), and remained higher than that of PI3K (Figure 3.15 B, D) in this line. The decrease in phosphorylation level of PI3K (Figure 3.15 B, D) was far more than that of PTEN (Figure 3.16 B, D) under the SST and SST+EGF treatments in SK-BR-3 cells. The results observed in both cell lines provide the first clear evidence that SST and/or EGF 82  can inhibit PI3K signaling and upregulate or maintain PTEN via a crosstalk mechanism involving SSTRs/ErbBs in breast cancer cells [52, 65].  Overall, the results presented above show that changes in the expression of and interactions between SSTR1-5 (Figures 3.4-3.8 A, C) and ErbB1-4 (Figures 3.9 -3.12 A, C) at the surfaces of BT-474 and SK-BR-3 breast cancer cells exposed to SST and/or EGF can induce significant time-dependent changes in the phosphorylation of PI3K (Figure 3.15 A, C), PTEN (Figure 3.16 A, C), ERK1/2 (Figure 3.13 A, C), and p38 (Figure 3.14 A, C). For instance, the combined treatment of BT-474 cells with SST and EGF induced the expression of activated SSTR1, SSTR2, SSTR3, SSTR4, and SSTR5 at the cells’ surfaces and dramatically reduced that of ErbB1-3. This reduced the presence of ErbB homodimers and heterodimers consisting of two different ErbB proteins, and induced the formation of SSTR/ErbB heterodimers, blocking ErbB heterodimer-mediated cell survival signaling and suppressing the phosphorylation of PTEN and PI3K [52, 65]. Similarly, in SK-BR-3 cells treated with SST and EGF, the expression of SSTR1, SSTR2, SSTR3 and SSTR5 increased at the cell surface while that of ErbB1-3 decreased. This reduced the abundance of ErbB homodimers and heterodimers comprising two different ErbB proteins at the cell surface, and increased the levels of SSTR/ErbB heterodimers, causing the phosphorylation of PTEN and PI3K to be maintained and reduced, respectively.  Put more simply, these results demonstrate that activated SSTRs can interfere with the heterodimerization of ErbBs and thereby antagonise ErbB-modulated tumor suppressor protein PTEN and PI3K cell survival signaling pathways [52, 65]   83   Figure 3.15 Time-dependent changes in the phosphorylation of PI3K in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF. Cell lysate was processed for total and phospho-PI3K using Western blot analysis. Note showing that significantly decreased the phosphorylated levels of PI3K in BT-474 (A) and SK-BR-3 (B) cells in comparison to control group. In subfigures C and D, the graphs of blue bars respectively show the phosphorylated PI3K levels in BT-474 (C) and SK-BR-3 (D)  cells. Results represent three experiments performed independently. The phosphorylation status was determined by densitometric analysis of phosphorylated vs. total PI3K. Data analysis was done by using two-way ANOVA and post hoc Bonferroni's test to compare all treatment groups against control (*, p < 0.05).   84   Figure 3.16 Time-dependent changes in the phosphorylation of PTEN in BT-474 (A, C) and SK-BR-3 (B, D) breast cancer cells following 5, 10, 15 or 30 min treatments with 1 µM SST and/or 10 nM EGF. Cell lysate was processed for PTEN using Western blot analysis. Note showing that significantly increased or maintained the PTEN levels in BT-474 (A) and SK-BR-3 (B) cells in comparison to control group. In subfigures C and D, the graphs of blue bars respectively show the PTEN levels in BT-474 (C) and SK-BR-3 (D) cells.Results represent three experiments performed independently. The phosphorylation status was determined by densitometric analysis of phosphorylated vs. total PTEN. Data analysis was done by using two-way ANOVA and post hoc Bonferroni's test to compare all treatment groups against control (*, p < 0.05).      85  3.4 Time-dependent changes in cell proliferation in BT-474 and SK-BR-3 cells upon SST and/or EGF treatment The results presented above clearly show that activated SSTR2, SSTR3, SSTR5, SSTR1 and SSTR4 in BT-474, and activated SSTR5, SSTR1, SSTR2 and SSTR3 in SK-BR-3 cells impeded the heterodimerization of the ErbB proteins via coexpression and through the formation of SSTR/ErbB heterodimers in a complex agonist-, time-, and cell-dependent manner (Figures 3.1-3.12). The coexpression of and interactions between SSTR and ErbB subtypes strongly suggest the occurrence of crosstalk and modulation of signaling between SSTR and ErbB subtypes in the studied human breast cancer cell lines, allowing the activated SSTRs to interfere with ErbB-heterodimerization-mediated regulation of ERK1/2, p38, PI3K and PTEN signaling upon stimulation by SST and/or EGF (Figures 3.13-3.16).  However, the physiological significance of this interference had not been determined. The most prominent effect of SST is its capacity to inhibit tumor cell proliferation, whereas EGF promotes tumor cell proliferation. Therefore, ErbBs stimulated by EGF are important cell proliferation regulators that promote tumor growth, whereas SSTRs stimulated by SST inhibit cell proliferation and tumor growth, making them attractive targets for the development of new cancer therapies [188, 217, 218]. To delineate the physiological significance of SSTR interference with ErbB heterodimerization, cell proliferation assays were performed as previously described by our groups, using EGF as a positive inducer of proliferation [21]. The key findings of these experiments are presented in Figures 3.17-3.18.  86  3.4.1 Time-dependent changes in proliferation of BT-474 cells upon SST and/or EGF treatment As shown in Figure 3.17, the activation of SSTR2, SSTR3, SSTR5, SSTR1 and SSTR4 and the resulting blockage of ErbB heterodimerization substantially reduced the proliferation of BT-474 cells. The SST treatment had clear but modest antiproliferative effects, reducing the rate of cell proliferation by around 1%, -2%, 5%  and 5% relative to controls after 12, 24, 36, and 48 hours, respectively. However, the SST+EGF treatment strongly reduced the cells’ proliferative activity, reducing their rates of proliferation by -2%, 18%, 24%  and 35% relative to controls after 12, 24, 36, and 48 hours, respectively. Finally, the rate of proliferation under the SST+EGF treatment after 36 hours treatment strongly reduced the cells’ proliferation: the rate of proliferation at 36 h was around half that seen for cells treated with EGF alone at the same time point. These results indicate that activated SSTR proteins interfere with ErbB heterodimerization in BT-474 cells, causing dramatic inhibition of cell proliferation in breast cancer cells of the luminal B subtype (ER-positive and/or PR-positive, ErbB2-positive or negative). This has not previously been reported [49, 52, 65, 145, 188, 208]. 3.4.2 Time-dependent changes in proliferation of SK-BR-3 cells upon SST and/or EGF treatment Figure 3.18 shows the effects of treatment with SST and/or EGF on SK-BR-3 cell proliferation. Treatment with SST alone had a modest antiproliferative effect; reducing the rate of cell proliferation by around 3%, 9%, 9%, and 10% relative to controls after 12, 24, 36, and 48 h, respectively. Treatment with SST+EGF strongly reduced the cells’ proliferative activity: the rates of cell proliferation at 12, 24, 36 and 48 h were reduced by 5%, 16%, 26% and 38%, respectively, relative to controls. Finally, the rate of cell proliferation in cells treated with 87  SST+EGF after 36 hours was around 56% of that for cells treated with EGF alone at the same time point. These results indicate that activated SSTR1, SSTR2, SSTR3 and SSTR5 interfered with ErbB homo- and heterodimerization in SK-BR-3 cells, dramatically reducing proliferation in this breast cancer line (ErbB2-driven subtype: ER-negative and/or PR-negative, ErbB2-overexpressing). As above, this behavior has never previously been reported [49, 52, 65, 145, 188, 208].               88   Figure 3.17 Time-dependent changes in cell proliferation induced by SSTR and ErbB expression in BT-474 breast cancer cells following 12, 24, 36, 48 and 72 hours treatments with 1 µM SST and/or 10 nM EGF. BT-474 cells were first serum-deprived for 24 h, and then treated as indicated for 24 h before processing for MTT assay. In comparison to control cells, BT-474 cells display dramatic inhibition of cell proliferation in response to SST, SST +EGF.  Data are presented as mean±SD from 3 experiments performed in triplicate. The results were analyzed by using two-way ANOVA and Bonferroni's post hoc test to compare all treatment groups to controls (*, #, +, p<0.05). In addition, Bonferroni's test was used to analyze the SST vs. SST+EGF treatment groups where applicable (++, p<0.05).  89   Figure 3.18 Time-dependent changes in cell proliferation induced by SSTR and ErbB expression in SK-BR-3 breast cancer cells following 12, 24, 36, 48 and 72 hours treatments with 1 µM SST and/or 10 nM EGF.  SK-BR-3 cells were first serum-deprived for 24 h, and then treated as indicated for 24 h before processing for MTT assay. In comparison to control cells, SK-BR-3 cells display dramatic inhibition of cell proliferation in response to SST, SST +EGF.  Data are presented as mean±SD from 3 experiments performed in triplicate. The results were analyzed by using two-way ANOVA and Bonferroni's post hoc test to compare all treatment groups to controls (*, #, +, p<0.05). In addition, Bonferroni's test was used to analyze the SST vs. SST+EGF treatment groups where applicable (++, p<0.05).      90  4 Discussion The molecular mechanisms involved in the onset and progression of breast cancer are poorly understood, which presents significant problems in the treatment of this disease and the design of improved drugs. Cell surface receptors belonging to the GPCR and RTK families are potential targets for drugs designed to treat many different pathological conditions and have attracted great interest from the pharmaceutical industry [52]. A better understanding of the roles of SSTRs from the GPCR family and ErbBs from the RTK family in pathophysiological conditions such as breast cancer could potentially reveal important new ways of improving the lives and prognosis of breast cancer patients. Some malignant breast cancers have been shown to exhibit unrestrained growth and the ability to evade tumor suppression mechanisms by downregulating SSTRs [21, 145]. SSTRs have been shown to inhibit cell proliferation in malignancies and cancers. Conversely, ErbBs promote cell proliferation and differentiation [153], and have been linked to poor prognosis in epithelial cancers [153]. Previous studies have shown that homo- and/or heterodimerization of SSTRs and ErbBs in malignancies play crucial roles in controlling the behavior of these proteins, including their coexpression, trafficking, and signal transduction [52, 55]. The coexpression and homo and/or heterodimerization of ErbB proteins (particularly the formation of ErbB1/ErbB2, ErbB1/ErbB3, ErbB2/ErbB3, and ErbB1/ErbB1 dimers) at the surface of cancer cells have been reported to activate aberrant downstream signaling pathways associated with breast tumor progression [65, 148]. Significantly, recent studies have shown that SSTR subtypes can form dimers with other members of the SSTR family or closely related GPCRs, and also with more distantly related receptor proteins. In particular, it was found that SSTR5 and ErbB1 interacted when coexpressed, leading to the formation of SSTR5/ErbB1 heterodimers; this heterodimerization changed the proteins’ 91  pharmacological and functional profiles in cancer cells, including those derived from breast cancers [51, 60, 85]. There is compelling evidence that SST-mediated interactions and crosstalk between SSTRs and ErbBs play key roles in inhibiting and promoting cell proliferation in breast cancer. However, the molecular mechanisms underlying these effects are not well understood. This thesis presents research on the crosstalk and modulation of signaling between SSTR and ErbB subtypes in human breast cancer cell lines that sheds new light on the molecular mechanisms underpinning the antiproliferative activity of SSTRs and the proliferative activity of ErbBs. Our group showed for the first time that SSTR5 and ErbB1 are coexpressed in MCF-7 cells (representing the luminal A breast cancer subtype) and MDA-MB-231 cells (representing the basal-like subtype) and form SSTR5/ErbB1 heterodimers that modulate downstream MAPK signalling in both cell lines [58]. However, it was not known whether modulation of downstream MAPK signalling caused by SSTR5/ErbB1 coexpression and heterodimerization occurred in other breast cancer cell types such as BT-474 (representing the luminal B subtype) or SK-BR-3 (representing the ErbB2 -driven subtype). In addition, there was an unexplored possibility that other SSTR and ErbB proteins might engage in similar crosstalk and modulation of downstream signaling in breast cancer cells. The results presented in this thesis show that other kinds of crosstalk between different SSTR and ErbB subtypes do indeed occur in BT-474 and SK-BR-3 cells (Figures 3.1-3.12), and that the activated SSTRs antagonize ErbB-mediated MAPK signaling via ERK1/2 and p38 as well as ErbB -modulated tumor suppressor protein PTEN and PI3K cell survival pathways in BT-474 and SK-BR-3 cells (Figures 3.13-3.16). The antiproliferative mechanisms of activated SSTRs via the functional crosstalk between SSTRs/ErbBs in both cell lines resemble those responsible for antitumor activity in various 92  FDA-approved antibodies and small molecule drugs, including SST analogs. To our knowledge, this is the first attempt to investigate all-sided functional crosstalk and modulation of signaling between SSTR1-5 and ErbB1-4 in human breast cancer cells following stimulation with SST and/or EGF, and to characterize the molecular biology of SSTRs/ErbBs that governs their modulation of signaling pathways and the inhibition of proliferation in breast cancer cell lines.  A well-established strategy for investigating the possibility of crosstalk between different receptor families is to examine their membrane expression, coexpression and colocalization, and the changes in their levels in the presence of various agonists and in different cell lines [21, 52, 58]. Therefore, the first step towards characterizing the crosstalk between SSTRs and ErbBs, and the associated modulation of downstream signaling, was to determine the expression of the different SSTR and ErbB subtypes at the surfaces of appropriate cell lines and to assess their coexpression/colocalization. Both BT-474 (Figures 3.1-3.12 A, C) and SK-BR-3 (Figures 3.1-3.12 B, D) cells were found to display various SSTR and ErbB receptors on their surfaces, and to exhibit agonist-, time- and cell-dependent changes in their external and internal distributions of these proteins in response to treatment with SST and/or EGF. Treatment with SST and/or EGF induced time-dependent dynamic changes in SSTR 1-5 and ErbB1-4 expression in BT-474 and SK-BR-3 cells (Figures 3.3-3.12).   We found that SSTR2 (or ErbB1) was the most prominent receptor subtype followed by SSTR3 (or ErbB2) in BT-474 breast cancer cells (Figures 3.1-3.2 A), while SSTR4 (or ErbB2) was the most prominent receptor subtype, followed by SSTR5 (or ErbB1) in SK-BR-3 (Figure 3.1, 3.3 B) breast cancer cells [33, 51, 207, 208, 211]. Treatment with EGF upregulated ErbB1, ErbB3 and ErbB2 expression at the cell surface in BT-474 cells, leading to ErbB1/ErbB2, ErbB1/ErbB3, and ErbB2/ErbB3 coexpression/heterodimerization. This finding is interesting 93  because BT-474 cells and ErbB1-positive breast cancers (luminal B subtype) are known to overproduce ligands including EGF [167, 172, 173]. EGF treatment also increased ErbB2, ErbB1 and ErbB3 expression at the cell surface in SK-BR-3 cells, leading to ErbB2/ErbB3, ErbB1/ErbB2 coexpression/heterodimerization, which is likely to also occur in ErbB2-positive breast cancer (ErbB2-driven subtype) patients whose tumors overproduce ligands such as EGF [167, 172, 173]. There is evidence that prolonged expression of ErbB1 at the cell surface and blockage of ErbB1 degradation in cells can enhance ErbB-mediated signaling by promoting the formation of ErbB1/ErbB2, ErbB1/ErbB3, and ErbB2/ErbB3 heteromers in breast cancers [153, 168, 174, 219]. Prolonged ErbB1 expression at the cell surface leading to enhanced ErbB1 phosphorylation has been identified as an important mechanism of treatment failure in FDA-approved drugs targeting ErbB1 or ErbB2, which significantly reduces the likelihood of survival in luminal B or ErbB2-driven breast cancer patients [167, 168, 174, 219]. Surprisingly, we found that treatment with SST and/or EGF synchronously induced the expression and localization of SSTR2 and ErbB1 at the cell surface in BT-474 cells, causing SSTR2 and ErbB1 to exhibit near-identical time-dependent dynamic changes in expression and cyclical sensitization, desensitization, internalization, intracellular trafficking and recycling to the plasma membrane(Figure 3.5 A, C and Figure 3.9 A, C). The two proteins were coexpressed and colocalized at the surfaces of BT-474 cells and in their interior. This creates the potential for heterodimerization and crosstalk between SSTR2 and ErbB1 in BT-474 cells [58, 111]. The binding of a ligand (SST and/or EGF) induced time-dependent dynamic changes in SSTR2/ErbB1 coexpression at the cell surface, significantly reducing the duration of ErbB1 expression at the cell surface (Figure 3.5, 3.9 A, C) and reducing the heterodimerization of ErbB1/ErbB2, ErbB1/ErbB3 and ErbB2/ErbB3 in BT-474 cells [168, 174, 219]. Very 94  surprisingly, SST+EGF induced SSTR2/ErbB1 coexpression at the cell surface and the trafficking of both proteins into the cells’ interior, where ErbB1 was degraded, terminating its signaling activity (Figure 3.9 A, C). The crosstalk between SSTR2 and ErbB1 (Figure 3.5, 3.9, A, C) thus appears to directly affect the trafficking and recycling of ErbB1 in BT-474 cells, and to terminate ErbB1-mediated mitogenic signaling [58, 168]. In addition, because of the coexpression of ErbB1/ErbB2, ErbB1/ErbB3, and ErbB2/ErbB3 in BT-474 cells[168], the degradation of ErbB1 and the alteration of its trafficking also indirectly impeded the trafficking of ErbB2 and ErbB3 in BT-474 cells (see e.g. the results for the 30 min data point in Figures 3.9-3.11 A, C). Interestingly, the SST and/or EGF treatments also induced time-dependent dynamic changes in SSTR3/ErbB2 (Figure 3.6, 3.10, A, C) coexpression at the cell surface, and that of SSTR5/ErbB3 (Figure 3.7, 3.11, A, C) to a lesser extent, which also impeded the trafficking of ErbB2 and ErbB3 (see e.g. the results for the 30 min data point in Figures 3.10-3.11 A, C) by disfavoring the coexpression of ErbB2/ErbB3 and ErbB3/ErbB1, and their interaction/heterodimerization at the cell surface [56, 220, 221]. The activation of SSTR2, SSTR3 and SSTR5 by SST and/or EGF in BT-474 cells thus reduced ErbB1 (maximumly decreased by 92%), ErbB2 (maximumly decreased by 31%) and ErbB3 (maximumly decreased by 42%) expression at the cell surface (Figures 3.9 -3.11, A, C) via by significantly reducing the coexpression and heterodimerization of ErbB1/ErbB2, ErbB1/ErbB3 and ErbB2/ErbB3, which may suppress or block the tumor-promoting effects of ErbB1-3 in breast cancer cells [65, 148, 168]. The SST and/or EGF treatments also induced the synchronized SSTR5/ErbB1 expression and localization at the cell surface in SK-BR-3 cells, leading to near-identical time-dependent dynamic changes in the expression of SSTR5 and ErbB1 (Figures 3.8-3.9, B, D): the two receptors exhibited identical cyclic patterns of sensitization, desensitization, internalization, 95  intracellular trafficking and recycling to the plasma membrane in SK-BR-3 cells, resulting in weakly expressed of ErbB1 (maximumly decreased by 57%), ErbB2 (maximumly decreased by 62%) and ErbB3 (maximumly decreased by 44%) at the membrane (Figures 3.8-3.11, B, D) in this cell line. It can be suggested that SSTR5/ErbB1 coexpression/colocalization at the cell surface and in the cytosolic fraction promoted heterodimerization and crosstalk between SSTR5 and ErbB1 in SK-BR-3 cells [58, 111, 168, 174, 219]. More interestingly, the potential SSTR5/ErbB1 heterodimerization and crosstalk in SK-BR-3 cells treated with SST and/or EGF is very similar to that seen for SSTR5/ErbB1 in ER-positive MCF-7 or ERα-negative MDA-MB-231 breast cancer cells, which causes ErbB1 degradation and reduces the coexpression and heterodimerization of ErbB1/ErbB2, leading to agonist-dependent modulation of downstream MAPK signalling [58]. The activation of SSTR5 by SST and/or EGF in SK-BR-3 cells immediately reduced the (co)expression of ErbB3, ErbB2, and ErbB1 at the cell surface by reducing the abundance of ErbB2/ErbB3 and ErbB1/ErbB2 heterodimers, which may significantly weaken the tumor-promoting effects of ErbB1-3 in conditions including breast cancers [65, 148, 168]. To summarize, SST and/or EGF induced the coexpression and possible heterodimerization of SSTR2/ErbB1, SSTR3/ErbB2 and SSTR5/ErbB3 in BT-474 cells while reducing the membrane coexpression of ErbB1/ErbB2, ErbB1/ErbB3 and ErbB2/ErbB3, interfering with the trafficking of ErbB1-3, and promoting ErbB1 degradation and the termination of ErbB1-mediated mitogenic signaling. Additionally, in SK-BR-3 cells, these ligands induced the coexpression and possible heterodimerization of SSTR5/ErbB1, reduced the membrane coexpression of ErbB2/ErbB3, ErbB1/ErbB2, and ErbB1/ErbB3, and interfered with the trafficking of ErbB1-3. All of these results support the hypothesis that there is crosstalk between SSTRs and ErbBs in breast cancer cells [52, 58, 65].  96  In addition to the effects described above, the ligand-induced activation of SSTR proteins and their possible crosstalk with ErbB receptors may have blocked the formation of the asymmetric ErbB1/ErbB1 homodimer in both BT-474 (Figure 3.9  A, C) and SK-BR-3 (Figure 3.9 B, D) cells, due to ligand-activated ErbB1 kinase domains in this asymmetric homodimer have been implicated in the development and progression of breast cancers [145, 152]. The reduced membrane expression of ErbB1, ErbB2 and ErbB3 induced by the SST+EGF treatment thus inactivated and blocked the homodimerization of ErbB1/ErbB1 in addition to the heterodimerization of ErbB1/ErbB2, ErbB1/ErbB3 and ErbB2/ErbB3 in BT-474 cells (Figures 3.9-3.11 A, C), and the homodimerization of ErbB1/ErbB1 and the heterodimerization of ErbB2/ErbB3, ErbB1/ErbB2 in SK-BR-3 cells (Figures 3.9 -3.11 B, D), potentially reducing the duration of ErbB1-3 phosphorylation in both cell lines [168, 174, 219], and promoting the degradation of ErbB proteins and the termination of their signaling. Phosphorylated ErbB1-3 proteins have been linked to tumor progression, so antagonizing their phosphorylation may suppress tumor growth and cell proliferation [52, 153, 168].  Both SSTR1 and SSTR4 (Figure 3.4 A, C and Figure 3.7 A, C) were observed to exhibit very powerfully increased expression accompanying the possible crosstalk of SSTR2/ErbB1, SSTR3/ErbB2 and SSTR5/ErbB3 in BT-474 cells treated with SST and/or EGF. Similarly, the strongly increased SSTR1 and SSTR2, and moderately enhanced SSTR3 expression were observed to accompany the possible SSTR5/ErbB1 crosstalk in SK-BR-3 cells under the studied conditions (Figures 3.4-3.6 B, D). Both cell lines also exhibited time-dependent changes in trafficking of SSTR1, SSTR2, SSTR3, SSTR4 and SSTR5 (Figures 3.4-3.8). These changes could enhance the coupling to AC and signaling in the breast cancer cells, which would reinforce the treatments’ effects on cell growth [49, 52, 105, 153, 168]. Taken together, the results 97  discussed above show that the binding of SST to SSTRs and EGF to ErbBs induces coexpression of SSTR/ErbB subtypes that blocks the functional homo-and heterodimerization of ErbB subtypes at the cell surface and changes the trafficking patterns of SSTRs and ErbBs in two different breast cancer cell lines. These behaviors indicate that SSTR activation can reduce or terminate ErbB signaling, and suggests that crosstalk between SST/SSTRs and EGF/ErbBs may be important in breast cancer cells [60, 148]. Reduced membrane expression of ErbBs and enhanced or extended membrane expression of SSTRs leading to the enhancement of SSTRs’ antiproliferative activity has been reported in various kinds of tumors [13, 51, 60, 148, 202, 204].  Previous studies have shown that agonist-dependent activation of ErbBs and their functional dimerization could activate downstream signalling systems such as MAPK (ERK1/2) pathways, leading to increased tumor growth, cell proliferation, and resistance to apoptosis [52, 148]. Conversely, the binding of agonists to SSTRs modulates ERK1/2 phosphorylation in a receptor- and cell-dependent manner [60]. SSTR2- and SSTR5-mediated inhibition of ERK1/2 phosphorylation was shown to inhibit proliferation in certain cells, and SSTR1- and SSTR4-mediated activation of ERK1/2 phosphorylation inhibited cancer cell proliferation [91, 102]. Treating BT-474 or SK-BR-3 breast cancer cells with SST and/or EGF caused significant time-dependent reductions in the phosphorylation of ERK 1/2 - by as much as 60-82%% in BT-474 cells whereas increase in the phosphorylation of ERK 1/2 - by as much as 463-491% in SK-BR-3 cells, respectively (see e.g. the results for the 5-30 min data points in Figure 3.13 treated with SST+EGF). The SST and/or EGF treatments also increased the phosphorylation of p38 in SK-BR-3 cells (e.g. by as much as 212% after 30 min under the SST+EGF treatment (Figure 3.14 B, D), but significantly reduced the phosphorylation of p38 in BT-474 cells (e.g. by as much as 40% after 30 min under the SST+EGF treatment) (Figure 3.14 A, C). These results strongly 98  suggest that SST and EGF have cell-specific effects on the regulation of MAPK signalling, and are consistent with previous reports showing that SSTR-mediated inhibition of cell proliferation required the activation or inhibition of both ERK1/2 and p38 [58]. A mechanism for this effect can be identified by noting that the activation of SSTRs antagonized ErbB-mediated ERK signaling pathways in both BT-474 (Figure 3.13 A, C) and SK-BR-3 (Figure 3.13 B, D) cells. Specifically, SSTR2 activation induced apparent crosstalk between SSTR2 and ErbB1 in BT-474 breast cancer cells, leading to the degradation of ErbB1 and the termination of its signaling. This is significant because ErbB1 regulates the activation of ERK signaling pathways in BT-474 cells [65, 161]. In keeping with these observations, we found that the crosstalk between SSTR2 and ErbB1 resulting from their coexpression and heterodimerization at the cell surface in BT-474 cells led to the inactivation and degradation of ErbB1 (Figure 3.9 A, C), terminating ErbB1-mediated ERK signaling [188, 192]. We also observed evidence for potential SSTR3/ErbB2 and SSTR5/ErbB3 crosstalk resulting from SST-mediated activation of SSTR3 and SSTR5, followed by similar coexpression and heterodimerization processes at the cell surface. These behaviors apparently reduce the activation of ErbB2 and ErbB3 (Figures 3.10-3.11 A, C) in BT-474 cells. The decreased activation of ErbB2 and ErbB3 reduced their phosphorylation and the abundance of ErbB2/ErbB1, ErbB3/ErbB1, and ErbB1/ErbB1 homo- and heterodimers, blocking downstream ErbB-mediated ERK signaling pathways and suppressing the translocation of activated ERK1/2 into the nucleus [65, 145, 161, 162, 188, 192, 198]. This suppresses cell growth.  Similar results were observed in SK-BR-3 cells: treatment with SST and/or EGF activated SSTR5, leading to apparent coexpression of SSTR5 and ErbB1 and crosstalk between these two receptors. This in turn reduced the abundance of ErbB1/ErbB1, ErbB1/ErbB2, ErbB1/ErbB3 and 99  ErbB2/ErbB3 homo- and heterodimers at the cell surface, reducing the activation of ErbB1, ErbB2 and ErbB3 (Figures 3.9-3.11 B, D). The decreased activation of these ErbB proteins increased the phosphorylation of ERK1/2 (Figure 3.13 B, D) and may have blocked ErbB-mediated ERK signaling [65, 145, 161, 162, 188, 192]. We also observed activation of SSTR1, SSTR2 or SSTR3 via greatly enhanced SSTR1 or SSTR2 (including moderately enhanced SSTR3) trafficking in SK-BR-3 cells treated with SST+EGF (Figures 3.4-3.6 B, D). This resulted in SSTR1-, SSTR2- and SSTR3-mediated ERK1/2 activation (Figure 3.13 B), which has been shown to inhibit cell proliferation in some cancer cells [49, 58, 61]. The inhibition of ErbB-mediated p38 signaling was by activated ErbB-antagonizing signaling [49, 58, 61]. The inhibition of ErbB-mediated p38 signaling by activated ErbB-antagonizing SSTRs was also observed in SK-BR-3 cells (Figure 3.14 B, D) in addition to BT-474 cells (Figure 3.14 A, C). Specifically, activated SSTR2, SSTR5, SSTR3, SSTR1 and SSTR4 engaged in crosstalk with ErbB proteins (particularly via the SSTR2/ErbB1, SSTR3/ErbB2 and SSTR5/ErbB3 pairs) and thereby inhibited p38 signaling in BT-474 cells (Figure 3.14 A, C), suggesting that a major consequence of the activation of SSTRs by SST and/or EGF is the antagonism of ErbB-mediated MAPK (ERK 1/2, p38) signaling pathways in BT-474 cells. Conversely, the activation of SSTR5, SSTR1, SSTR2 and SSTR3 in SK-BR-3 cells induced SSTR5/ErbB1 crosstalk that significantly increased p38 phosphorylation (Figure 3.14 B, D), indicating that p38 signaling pathways in breast cancer cells can be activated by SST and/or EGF stimulation. This is important because p38 signaling inhibits cell proliferation and induces apoptosis in breast cancer cells [21, 65].  On the other hand, EGF regulates tumor cell proliferation by controlling the phosphorylation and  dimerization of ErbB proteins, and thus the downstream activation of MAPK signaling and PI3K/AKT cell survival pathways [188, 222]. PTEN dysfunction plays a 100  significant role in the pathogenesis of hereditary and sporadic cancers [147]. As a phospholipid phosphatase, PTEN catalyzes the hydrolysis of the second messenger PIP3 and counteracts the activation of the PI3K/AKT signaling pathway, thereby regulating cellular growth, proliferation, and metabolism [147, 223]. Tumor progression and losses of trastuzumab responsiveness during tumor treatment have been linked to the activation of the PI3K/AKT cell survival pathway and the loss of PTEN [52, 146]. In contrast, SSTR activation by SST has been shown to reduce cell proliferation, making these proteins attractive targets in tumor therapy [188]. Recent studies have shown that SSTRs can also contribute to the inhibition of PI3K signaling in insulinoma, pituitary and pancreatic tumors, and breast cancers, in which PI3K activation was linked to tumor growth and therapeutic failure [221, 224]. Therefore, inhibiting PI3K is a potentially interesting way of treating diverse tumors [21]. A SST analog, OCT, was shown to induce PTP-1C dephosphorylation and inhibit PI3K pathway signaling, reducing cell proliferation [33]. Additionally, some studies have suggested that trastuzumab destabilizes ErbB2/ErbB3 dimers or uncouples ErbB3 from ErbB2, lead to the blockage of PI3K/Akt signaling pathways in breast cancers [174, 196]. Surprisingly, the results presented in this thesis indicate that the activation of SSTR2, SSTR3 and SSTR5 induces SSTR2/ErbB1, SSTR3/ErbB2, and SSTR5/ErbB3 crosstalk, which uncouples dimers of ErbB1 with ErbB1-3, and those of ErbB2 with ErbB3, leading to strong inhibition of PI3K signaling in BT-474 cells treated with SST+EGF (Figure 3.15 A, C). The crosstalk between SSTR2 and ErbB1 also promoted the degradation of ErbB1 in BT-474 cells treated with SST+EGF, terminating or weakening ErbB1-, ErbB2, and ErbB3-mediated tumor-promoting signaling pathways [21]. The activation of SSTR2 and SSTR2/ErbB1, SSTR3 and SSTR3/ErbB2, SSTR5 and SSTR5/ErbB3, SSTR1, and SSTR4 in BT-474 cells also activated PTEN (Figure 3.16 A, C), which antagonized ErbB1-, ErbB2 and ErbB3- mediated 101  promotion of PI3K cell survival pathways (Figure 3.15 A, C). This reinforces the notion that crosstalk between SSTR2 and ErbB1, SSTR3 and ErbB2, or SSTR5 and ErbB3 modulates the activity of breast cancer-related PI3K signaling pathways in a potentially clinically significant manner [145]. In SK-BR-3 cells treated with SST+EGF, activated SSTR5 and SSTR5/ErbB1, SSTR1, SSTR2, and SSTR3 maintained a steady level of PTEN phosphorylation (Figure 3.16 B, C), which similarly antagonized ErbB3-, ErbB1- and ErbB2-mediated promotion of PI3K cell survival pathways (Figure 3.15 B, D). Once again, this suggests that crosstalk between SSTR5 and ErbB1 modulates breast cancer-related PI3K signaling pathways [145]. Furthermore, the enhenced activation of SSTR1, SSTR4, SSTR2 or SSTR3 and the crosstalk between SSTRs and ErbBs in both BT-474 and SK-BR-3 breast cancer cells treated with SST+EGF upregulated or maintained PTEN phosphorylation, and antagonized ErbB3-, ErbB1- and ErbB2- mediated promotion of PI3K cell survival pathways in both cell lines [65, 145]. EGF-activated ErbB proteins are known regulators of cell proliferation that promote tumor growth, whereas SSTRs (activated by SST) are known to inhibit cell proliferation and promote tumor cell apoptosis, making them potential targets in tumor therapy [188, 217, 218]. Our results indicate that ligand binding and crosstalk between these receptor families modulates their activity, causing time-dependent changes in the proliferation of BT-474 and SK-BR-3 breast cancer cells following SST and/or EGF treatment. Crosstalk between SSTR and ErbB subtypes in both BT-474 and SK-BR-3 cells treated with SST+EGF drastically inhibited their proliferation (Figures 3.17-3.18). In BT-474 cells treated with SST+EGF, this occurred as a result of functional crosstalk between SSTR2 and ErbB1, SSTR3 and ErbB2, or SSTR5 and ErbB3 that led to the degradation of ErbB1 and reduced the membrane expression of ErbB2 and ErbB3 by interfering with the trafficking of ErbB1-3 and suppressing the homodimerization of ErbB1 and 102  the membrane coexpression/heterodimerization of ErbB1/ErbB2, ErbB1/ErbB3, and ErbB2/ErbB3. This in turn modified the activity of can     cer-related signaling pathways, increasing the phosphorylation of PTEN and reducing that of PI3K, ERK1/2, and p38, leading to substantial reductions in proliferative activity (e.g. decreased by 64% at 36 h in comparison with EGF treatment in Figure 3.17). These results are consistent with previous reports that reducing the phosphorylation of PI3K, ERK1/2, and p38 reduces proliferative activity [13, 21, 33, 49, 83, 145, 221]. The effects observed in BT-474 cells are similar to those induced by the FDA-approved antibodies Trastuzumab/Herceptin® (which targets the extracellular domain of ErbB1) and Pertuzumab/Omnitarg® (which targets ErbB2 ), as well as the small molecule drugs Lapatinib/Tykerb® (which target ErbB1 and ErbB2) [145, 221]. The antiproliferative effects of treatment with SST+EGF also closely resembled those of treatment with the SST analogs pasireotide and lanreotide, which was recently claimed to improve clinical outcomes in cancer patients [49, 51, 208, 224]. The results obtained in SK-BR-3 breast cancer cells fully supported these conclusions.   5 Conclusion and Future Directions Functional crosstalk between activated SSTR receptors and ErbB proteins has been demonstrated in the membrane expressions of the BT-474 and SK-BR-3 human breast cancer cell lines. This crosstalk affected downstream ErbB-mediated signaling and negated or reduced its proliferation-promoting effects via four different pathways. In BT-474 cells, the interactions between activated antiproliferative SSTRs (SSTR2, SSTR3 SSTR5, SSTR1, and SSTR4) and proliferative ErbBs (ErbB1, ErbB2, ErbB3, and ErbB4), 103  resulted in functional SSTR2/ErbB1, SSTR3/ErbB2, and SSTR5/ErbB3 crosstalk that reduced the signaling activity of ErbB1, ErbB2 and ErbB3. The SSTRs were activated by treatment with SST and/or EGF, which induced strong coexpression of SSTR2/ErbB1 and SSTR5/ErbB3 together with moderate coexpression of SSTR3/ErbB2 at the cell surface. This in turn caused the uncoupling of ErbB1 from ErbB1/ErbB2 heterodimers, ErbB1/ErbB3 heterodimers, and ErbB1/ErbB1 homodimers. In addition, ErbB2 was uncoupled from ErbB2/ErbB3 heterodimers and ErbB3 from ErbB2/ErbB3 or ErbB3/ErbB4 heterodimers. This strongly reduced the coexpression of ErbB1, ErbB2, ErbB3, and ErbB4 at the cell surface and the duration of their phosphorylation, inhibiting their downstream signaling activity. The crosstalk also strongly increased the activation and trafficking of SSTR1 and SSTR4. The combined effect of these responses was to antagonize ErbB-mediated MAPK signaling and significantly inhibit the phosphorylation of ERK1/2, thereby inhibiting cell proliferation, invasion, and migration. This process corresponds to pathway 1 in Figure 5.1. The crosstalk of SSTR2/ErbB1, SSTR3/ErbB2 and SSTR5/ErbB3 also strongly inhibited p38 phosphorylation by suppressing the ErbB1-, ErbB2- and ErbB3-mediated P38 MAPK signaling pathway, leading to the increased induction of apoptosis (pathway 2). The SSTR2/ErbB1, SSTR3/ErbB2 and SSTR5/ErbB3 crosstalk markedly increased the phosphorylation of PTEN, which inhibits cell survival pathways and thus reduces cell proliferation and promotes apoptosis (pathway 3). Finally, the crosstalk reduced the phosphorylation of PI3K and thus suppressed signaling via the PI3K/AKT cell survival pathway, which again disfavored cell proliferation (pathway 4). Furthermore, the increased activation and trafficking of SSTR1 and SSTR4 induced by the SSTR2/ErbB1, SSTR3/ErbB2 and SSTR5/ErbB3 crosstalk directly inhibited cell proliferation, presumably via a mechanism similar to that of antitumor SST analogs. The mechanisms by which the SSTR subtypes and their 104  crosstalk with ErbBs suppress the deleterious effects of ErbB signaling in BT-474 cells are very similar to those responsible for the antitumor effects of some FDA-approved antibodies and small molecule drugs. Figure 5.1 summarizes these mechanisms.  Similar effects were observed in SK-BR-3 cells. Treatment with SST and/or EGF induced apparent functional crosstalk between SSTR5 and ErbB1, causing strong coexpression of these proteins SSTR5/ErbB1 at the membrane together with strongly enhanced expression of SSTR1 and SSTR4, and moderately enhanced expression of SSTR3 at the cell surface. This in turn suppressed the dimerization of ErbB1/ ErbB1, ErbB1/ ErbB2, and ErbB1/ErbB3, and greatly reduced the membrane (co)expression of ErbB1, ErbB2, and ErbB3.  This induced significant inactivation and intracellular degradation of ErbB1 and partial degradation of ErbB2, as well as the uncoupling of ErbB1 from ErbB1/ErbB2 heterodimers, ErbB2 from ErbB2/ErbB3 heterodimers, ErbB1 from ErbB1/ErbB3 heterodimers, ErbB1 from ErbB1/ErbB1 homodimers, and ErbB3 from ErbB3/ErbB4 heterodimers. The overall effect was to greatly reduce the (co)expression of ErbB1, ErbB2 and ErbB3 at the cell surface and to strongly inhibit their signaling activity. Treatment with SST and/or EGF also greatly reduced the levels of phosphorylated ErbB1, ErbB2 and ErbB3 in SK-BR-3 cells, contributing to the dissociation of the ErbB1/ErbB2 and ErbB2/ErbB3 heterodimers observed in SK-BR-3 cells treated with EGF alone. The SSTR5/ErbB1 crosstalk and the strongly enhanced activation of SSTR1 and SSTR2,  moderately enhanced activation of SSTR3 also caused the activated SSTRs to antagonize ErbB-mediated MAPK signaling by significantly increasing the phosphorylation of ERK1/2 (this effect corresponds to pathway 1 in Figure 5.2), which inhibited proliferation, invasion and migration in SK-BR-3 cells. The SSTR5/ErbB1 crosstalk and enhanced activation of SSTR1, SSTR2 and SSTR3 also increased the phosphorylation of p38, promoting the induction of apoptosis 105  (pathway 2). Another effect was that the phosphorylation of PTEN remained steady, inhibiting the PTEN cell survival pathway. This inhibited cell proliferation and induced apoptosis (i.e. pathway 3). The final effect was a strong reduction in PI3K phosphorylation, which inhibited the cell survival pathway of PI3K/AKT and thus inhibited proliferation (pathway 4). These conclusions are summarized in Figure 5.2.  Overall, the induction of apoptosis and inhibition of cell proliferation resulting from the modulation of ErbB1-, ErbB2- and ErbB3-mediated ERK1/2, p38, PTEN and PI3K signaling (pathways 1-4) could be more pronounced in BT-474 cells than in SK-BR-3 cells. However, in both cell lines it was clear that SSTR subtypes engaged in functional crosstalk and modulated multiple signaling pathways affecting apoptosis and proliferation (specifically, MAPK and cell survival pathways), suppressing or eliminating the deleterious effects of signaling by ErbB subtypes. Our investigations on the crosstalks between SSTR and ErbB receptors in the BT-474 and SK-BR-3 breast cancer cell lines upon SST and/or EGF treatments are of great significance for clarifying the underlying molecular mechanisms, and exploring their implications for cancer therapy.     106   Figure 5.1 SSTR subtypes via functional crosstalk of SSTR/ErbB subtypes and modulation of signaling resulting in antagonizing EGF-mediated effects on cell survival pathways and subsequently diverting the deleterious effects of ErbB subtypes in BT-474 breast cancer cells. The dotted line represents the EGF-mediated effects on cell survival pathways (blue dotted line: activation; red dotted line: inhibition). The solid line represents the aforesaid SSTR subtypes antagonizing EGF-mediated effects on cell survival pathways (green solid line: activation; red solid line: inhibition). This illustration of the dotted line is constructed from references [52, 65]. 107   Figure 5.2 SSTR subtypes via functional crosstalk of SSTR/ErbB subtypes and modulation of signaling resulting in antagonizing EGF-mediated effects on cell survival pathways and subsequently diverting the deleterious effects of ErbB subtypes in SK-BR-3 breast cancer cells. The dotted line represents the EGF-mediated effects on cell survival pathways (blue dotted line: activation; red dotted line: inhibition). The solid line represents the aforesaid SSTR subtypes antagonizing EGF-mediated effects on cell survival pathways (green solid line: activation; red solid line: inhibition). This illustration of the dotted line is constructed from references [52, 65].    108  References 1.     Gobbo, O. L., Sjaastad, K., Radomski, M. W., Volkov, Y., Prina-Mello, A., 2015. Magnetic Nanoparticles in Cancer Theranostics. Theranostics 5(11): 1249-63 2.     Orecchioni, M., Cabizza, R., Bianco, A., Delogu, L. G., 2015. 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