UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Regulation of primary, immortalized, and metatstatic human pancreatic ductal cells by insulin Chan, Michelle Tsz Ting 2012

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
24-ubc_2013_spring_chan_michelletszting.pdf [ 2.3MB ]
Metadata
JSON: 24-1.0073475.json
JSON-LD: 24-1.0073475-ld.json
RDF/XML (Pretty): 24-1.0073475-rdf.xml
RDF/JSON: 24-1.0073475-rdf.json
Turtle: 24-1.0073475-turtle.txt
N-Triples: 24-1.0073475-rdf-ntriples.txt
Original Record: 24-1.0073475-source.json
Full Text
24-1.0073475-fulltext.txt
Citation
24-1.0073475.ris

Full Text

REGULATION OF PRIMARY, IMMORTALIZED, AND METASTATIC HUMAN PANCREATIC DUCTAL CELLS BY INSULIN  by Michelle Tsz Ting Chan B. Sc. (H.), QUEEN’S UNIVERSITY, 2010  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Cell and Developmental Biology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) December 2012  © Michelle Tsz Ting Chan, 2012  Abstract Epidemiological studies have reported positive correlation between type 2 diabetes and risk of pancreatic cancer. Hyperinsulinemia occurring during early diabetes has been postulated to be a possible pathophysiological mechanism in promoting pancreatic cancer, but the mechanisms by which elevated insulin levels contribute to pancreatic carcinogenesis are still unknown. Here, the effects of insulin and its associated mechanism on cellular viability were examined in three cell models that are meant to represent three stages in pancreatic cancer progression. Furthermore, using small molecule inhibitors, the role of RAF/ERK pathway and PI3K/AKT pathway on cellular viability were also compared across three cell models. Different stages of pancreatic cancer progression were modeled in vitro with primary human pancreatic ductal cells, an immortalized pre-malignant human pancreatic ductal cell line (HPDE), and an advanced metastatic human pancreatic adenocarcinoma cell line (PANC1). All cell types were serum starved and treated with insulin, IGF1, or small molecule inhibitors targeting RAF1, MEK, AKT, or PI3K, then subjected to cell viability assays, cell death assays, and Western blot analysis for ERK and AKT activation. We observed that cell viability was promoted by exogenous insulin in PANC1 cells, and not in primary cells. In PANC1 cells, the insulin-mediated enhancement of cell viability was associated with sustained AKT activation. Furthermore, insulin-mediated reduction in cell death was not observed. When comparing the roles of RAF/ERK and PI3K/AKT pathways on cell viability, we observed an increase in cell death in primary cells when AKT was inhibited. In contrast, cell death was induced in HPDE and PANC1 cells when the RAF1 or MEK were inhibited. If extrapolated, the data suggest that hyperinsulinemia may not play a role in initiating pancreatic cancer, but high levels of insulin may accelerate the cancer progression. ii  Table of Contents Abstract .................................................................................................................................... ii Table of Contents ................................................................................................................... iii List of Tables .......................................................................................................................... vi List of Figures ........................................................................................................................ vii List of Abbreviations ........................................................................................................... viii Acknowledgements ................................................................................................................ xi Dedication .............................................................................................................................. xii Chapter 1: Introduction ........................................................................................................ 1 1.1  Pathogenesis of diabetes mellitus ............................................................................. 1  1.2  Insulin and IGF1 signalling ...................................................................................... 2  1.3  Cancer risks in individuals with type 2 diabetes ....................................................... 5  1.4  Pathogenesis of pancreatic cancer ............................................................................ 8  1.5  Hyperinsulinemia, insulin/IGF1 signalling and pancreatic cancer ......................... 11  1.6  Thesis investigation ................................................................................................ 13  Chapter 2: Material and methods ...................................................................................... 22 2.1  Reagents .................................................................................................................. 22  2.2  Cell models of this study......................................................................................... 23  2.2.1  Human mixed pancreatic exocrine and ductal cell culture ................................. 23  2.2.1.1  Culturing procedures for primary human non-islet tissue .......................... 24  2.2.1.2  Cell dissociation of human unsorted pancreatic exocrine cells .................. 24  2.2.1.3  Magnetic activated cell sorting (MACS) .................................................... 25  2.2.1.4  Primary cell treatment procedures .............................................................. 26  iii  2.2.2  HPDE cell culture ............................................................................................... 26  2.2.3  PANC1 cell culture ............................................................................................. 26  2.2.4  HPDE and PANC1 cells treatment procedures ................................................... 27  2.3  Cell count and cell death assays.............................................................................. 27  2.4  Immunoblotting and densitometry analysis ............................................................ 28  2.5  Statistical Analysis .................................................................................................. 29  Chapter 3: Characterization of insulin signalling in human pancreatic ductal cells .... 31 3.1  Insulin signalling of primary human ductal cells .................................................... 31  3.1.1  Inhibition of AKT and RAF1 in human primary pancreatic sorted cells ........... 31  3.1.2  Actions of insulin in primary pancreatic unsorted and sorted cells .................... 32  3.1.3  Conclusion regarding primary human pancreatic ductal cells ............................ 32  3.2  Insulin signalling in HPDE cells ............................................................................. 33  3.2.1  Inhibition of PI3K/AKT or RAF/ERK pathway in HPDE cells ......................... 33  3.2.2  Effects of exogenous insulin on AKT and ERK activation in HPDE cells ........ 33  3.2.3  Conclusion regarding HPDE cells ...................................................................... 34  3.3  Insulin signalling in PANC1 cells........................................................................... 34  3.3.1  Effects of AKT and ERK inhibition on cellular viability in PANC1 cells ......... 34  3.3.2  Effects of exogenous insulin in PANC1 cells ..................................................... 36  3.3.3  Conclusion regarding insulin and IGF1 signalling on PANC1 cells .................. 37  Chapter 4: Discussion and conclusion................................................................................ 52 4.1  Insulin signalling in primary human pancreatic ductal cells .................................. 52  4.2  Insulin signalling in HPDE cells ............................................................................. 55  4.3  Insulin signalling in PANC1 cells........................................................................... 57  iv  4.4  Conclusion .............................................................................................................. 63  References .............................................................................................................................. 65  v  List of Tables  Table 1-1. Relative cancer risks of individuals with type 2 diabetes...................................... 20 Table 2-1. List of antibodies used in this study ...................................................................... 30 Table 2-2. Information of the donor primary tissues. ............................................................. 30 Table 3-1. Concentration of insulin across the culture media used in this study.................... 43  vi  List of Figures  Figure 1-1 Working model of type 2 diabetes pathogenesis................................................... 16 Figure 1-2. Transmembrane receptors involved in insulin and IGF1 signalling .................... 17 Figure 1-3. Schematic of insulin and IGF1 signalling. ........................................................... 18 Figure 1-4. Multi-step progression to pancreatic ductal adenocarcinoma. ............................. 19 Figure 1-5. In vitro model of pancreatic cancer progression. ................................................. 21 Figure 3-1. AKT and RAF-1 inhibition in primary human pancreatic sorted cells. ............... 39 Figure 3-2. The effects of insulin in primary human pancreatic unsorted cells. .................... 40 Figure 3-3. Effects of insulin on cell viability of primary human pancreatic sorted cells...... 41 Figure 3-4. The effects of small molecule inhibitors on cellular viability of HPDE cells. .... 42 Figure 3-5. Effects of insulin and IGF1 on AKT and ERK phosphorylation in HPDE cells. 44 Figure 3-6. Inhibition of the AKT and ERK pathway in PANC1 cells. ................................. 45 Figure 3-7. Effects of inhibitors on ERK and AKT levels in PANC1 cells. .......................... 47 Figure 3-8. Effects of insulin and IGF1 cellular viability in PANC1 cells............................. 48 Figure 3-9. Effects of insulin or IGF1 on cell death in PANC1 cells. .................................... 50 Figure 3-10. Effects of insulin on AKT and ERK phosphorylation in PANC1 cells. ............ 51  vii  List of Abbreviations  Adenosine triphosphate  ATP  Analysis of variance  ANOVA  B-cell lymphoma 2 protein  BCL2  Bcl-2-associated death promoter  BAD  Breast cancer type 2 susceptibility protein  BRCA2  Bromodeoxyuridine  BrdU  Cytokeratin 19  CK19  Deoxyribonucleic acid  DNA  Dimethyl sulfoxide  DMSO  Dulbecco’s modified eagle medium  DMEM  E twenty-six-like transcription factor  ELK  Ethylenediaminetetracetic acid  EDTA  Extracellular signal-regulated kinases  ERK  Fetal bovine serum  FBS  Forkhead Box subfamily O protein  FOXO  Glucose transporter type 4  GLUT4  Glycogen synthase kinase 3  GSK3  Growth-factor-receptor-bound-protein 2  GRB2  Guanosine triphosphate  GTP  Hank’s balanced salt solution  HBSS  Human epidermal growth factor receptor 2  HER2  viii  Human pancreatic ductal epithelial  HPDE  Hydroxyethyl piperazineethanesulfonic acid  HEPES  Insulin receptor  IR  Insulin-like-growth-factor-1  IGF1  Insulin-receptor-substrate  IRS  Keratinocyte serum free medium  KSF  Magnetic activated cell sorting  MACS  Mammalian target of rapamycin  mTOR  Mitogen-activated protein kinase kinase  MEK  Packed cell volume  PCV  Pancreatic and duodenal homeobox 1  PDX1  Pancreatic ductal adenocarcinoma  PDAC  Pancreatic intraepithelial neoplasia  PanIN  Phosphatase and tensin homolog  PTEN  Phosphate buffered saline  PBS  Phosphatidylinositol 3,4,5-triphosphate  PIP3  Phosphatidylinositol 4,5-bisphosphate  PIP2  Phosphoinositide 3-kinase  PI3K  Phosphoinositide-dependent kinase-1  PDK1  Polyacrylamide gel electrophoresis  PAGE  Polyoma virus middle T antigen  PyVmT  Propidium iodide  PI  Roswell Park Memorial Institute medium  RPMI  ix  Serum free  SF  Severe combined immunodeficiency  SCID  Sodium dodecyl sulfate  SDS  Son-of-sevenless  SOS  Src-homology-2-domain-containing-transformating proteins  SHC  x  Acknowledgements  I would like to express my gratitude to my supervisor Dr. James Johnson for his patience, understanding, and mentorship. Throughout this difficult time, the completion of this degree would not be possible without my supporting supervisor and my committee members, Dr. Sandra Dunn, Dr. Calvin Roskelley, and Dr. Garth Warnock.  I like to extend this gratitude to the present and previous members of the Johnson laboratory, for their technical assistance, expertise and support. I owe particular thanks to Dr. Kwan-Yi (Connie) Chu, Dr. Gareth Lim, and Carol Yang for providing me with critical feedback on my research, for their tremendous support, and broadening my vision of science and research.  I thank the University of British Columbia for financially supporting my study.  Special thanks to my parents for morally, financially, and emotionally supporting me through many years of post-secondary education.  I owe a special thanks to Vladimir Crown for supporting me throughout all these years of education. I appreciate his patience in tolerating my ups and down during my hardships.  xi  Dedication I would like to dedicate this body of work to my father who is currently battling colon cancer, and to those who have been affected by this terrible disease.  xii  Chapter 1: Introduction Cancer and diabetes mellitus are the leading causes of death in North America1,2. In 2008, the World Health Organization estimated that cancer and diabetes have globally contributed to 7.6- and 1.3-million deaths, respectively. By 2030, a doubling of cancer and diabetes incidences are projected to occur as the growing population ages around the world3. Alarmingly, epidemiological studies have reported increased cancer risks in individuals affected by type 2 diabetes. With the rising incidence of type 2 diabetes, it is important to decipher the mechanisms behind this correlation to prevent further increase in cancer incidences and morbidity.  1.1  Pathogenesis of diabetes mellitus The glucose homeostasis of healthy individuals is mainly regulated by glucagon and  insulin, and these hormones are secreted by α-cells and β-cells of the islets of Langerhans, respectively. In the postprandial state of a healthy individual, elevated glucose levels stimulate insulin secretion and production from the β-cells. The secreted circulating insulin then promotes lipogenesis, glyconeogenesis, glucose uptake, and decrease lipolysis in insulin-sensitive-peripheral tissues. Examples of the main insulin-sensitive peripheral tissues include; liver, adipose tissue, and the skeletal muscle. Conversely, during starvation, blood glucose levels are maintained via glucagon-stimulated gluconeogenesis and fatty acid oxidation in the liver and adipose tissues4,5. In individuals with diabetes, glucose homeostasis is found to be disrupted due to dysregulated insulin production, secretion, and sensitivity4-6. Type 1 diabetes and type 2 diabetes are two of the multiple forms of diabetes mellitus. Type 1 diabetes is a disease in which the body is unable to produce insulin due to the loss of pancreatic β-cells4-6. Type 2  1  diabetes is a chronic, multi-factorial and progressive disease that is characterized by the inability of β-cells to produce sufficient insulin, as well as the inability of several tissues to respond efficiently to circulating insulin6,7. Both of these physiological conditions will lead to elevated blood glucose level or hyperglycemia. The current working model for the aetiology of type 2 diabetes have been proposed (Figure 1-1). Environmental factors, such as obesity, chronic stress, and sedentary lifestyles, have been identified to increase the risk of type 2 diabetes by predisposing individuals to insulin resistance. Once the individual with insulin resistance displays dysregulated glucose homeostasis, the individual may experience brief episodes of hyperglycemia after a meal. To counteract hyperglycemia, β-cells hypersecrete insulin to lower blood glucose levels, this is also known as compensatory hyperinsulinemia6,7. As the individual progressively becomes more insulin resistant, the body will continue to strive for glucose homeostasis by compensatory hyperinsulinemia. Over time, the enhancement of insulin production from β-cells imposes stress on the cell. This stress can greatly reduce β-cell mass by triggering cell death, and irreversibly affect β-cell function. Without sufficient β-cells, glucose homeostasis can no longer be maintained, thus leading to sustained hyperglycemia, also classified as clinical type 2 diabetes4-7. If chronic hyperglycemia is left untreated, it can lead to several complications such as stroke, heart diseases, and microvascular illnesses (peripheral neuropathy, blindness, and renal failure).  1.2  Insulin and insulin-growth-factor-1 signalling Insulin and insulin-like-growth-factor-1 (IGF1) are circulating hormones with  metabolic and mitogenic actions. Insulin is produced mainly by the pancreatic β-cells6,7, while IGF1 is mainly produced by the liver8-11. Insulin predominately regulates carbohydrate metabolism, whereas IGF1 predominately regulates growth and differentiation8-11. Although 2  both hormones elicit distinct physiological effects, insulin and IGF1 share many components of their signalling pathways. The specificity of insulin and IGF1 actions is mediated by tissue-specific and differential signalling capabilities of the receptors of insulin and IGF18-13. There are four receptor proteins in the signalling pathway, insulin receptors (IR), IRrelated receptors (IRR), type 1 and 2 IGF receptors (IGF1R, IGF2R) (Figure 1-2)8-11. IRs are tyrosine kinase receptors that are alternatively-spliced into two structurally and functionally different isoforms, IR-A and IR-B12. Activation of IR-A favors proliferative effects and IR-B favors metabolic effects14. IRs are expressed ubiquitously, higher expression of IR-A is found during fetal development and cancer15, whereas IR-B expression is found in muscle, liver and adipose tissue of healthy adults. IRRs are orphan tyrosine kinase receptors that are involved in regulation of sex differentiation13. IGF1Rs are also tyrosine kinase receptors that are expressed almost ubiquitously, except for the liver and adipose tissue8-11. IGF2Rs or mannose-6-phosphate are receptors lacking tyrosine kinase domain that are predominately expressed during development10,11,16. IGF2R regulate circulating IGF2 levels by targeting IGF2 for lysosomal degradation. Activation of the insulin/IGF1 signalling pathway involves ligand-mediated autophosphorylation and dimerization of two receptor proteins. Insulin has the highest and equal affinity for both IR-A and IR-B, and IGF1 has the highest affinity for IGF1R8-14,16. IGF2 can bind to both IGF2R and IR-A. To add another layer of complexity, IR isoforms can also form hybrid receptors with IGF1R. From studies that performed radioligand labeling of receptor complexes, hybrid receptors were found to have a lower affinity for insulin and higher affinity for IGF112,14 (Figure 1-2). The physiological role for hybrid receptors is currently unclear10,16.  3  Proliferative and metabolic effects of insulin/IGF1 signalling pathway are transduced through the canonical RAS-ERK and phosphatidylinositol-3-kinase (PI3K)-AKT pathway (Figure 1-3)8-13. Binding of insulin or IGF1 to the α-subunit of their respective receptors will elicit a conformational change and subsequent auto-phosphorylation of the tyrosine kinase receptor. The phosphorylated tyrosine domain then serves as a docking site for adaptor proteins such as insulin receptor substrates (IRS1-6)17 and src-homology-2-domaincontaining-transformating proteins (SHC)18. In the RAS/ERK signalling arm (Figure 1-3), docking of IRS or SHC will recruit proteins containing the Src-homology-2 (SH2) domain8-14,16, such as SHP2 or growth-factorreceptor-bound-protein (GRB2)19. GRB2 is constitutively bound to a guanine exchange factor named son-of-sevenless (SOS)20. Thus the docking of GRB2 will bring SOS in closer proximity to RAS (a small GTPase), and induce a nucleotide exchange of GDP-RAS to the activated  GTP-RAS20,21.  Activated  RAS  then  elicits  successive  kinase-mediated  phosphorylation and activation beginning with RAF, then MEK1/2, then ERK1/2 in mammals8-11,20,21. Activation of ERK will enhance the expression of genes involved in mitogenic signals (such as, cyclin D1, ELK), embryogenesis, differentiation, and apoptosis811,16,19-22  . To enhance signal specificity and regulation, each node of the signalling cascade  can form complex interactions with various types of proteins. Specifically, temporal and spatial activation of ERK signalling pathway are highly regulated by several scaffolding proteins such as kinase suppressor of RAS 1 (KSR1)21,23. In the PI3K/AKT signalling arm (Figure 1-3), docking of pIRS1/2 will activate PI3K24. PI3K complex is composed of a regulatory (p85) and catalytic subunit (p110). The phosphorylation of p85 by pIRS1/2 will elicit a conformational change in p85 subunit  4  thereby releasing the p110 catalytic subunit from the p110-p85 complex. Activated PI3K phosphorylates PIP2 to form PIP3 at the plasma membrane. PIP3 then recruits phosphoinositide-dependent kinase-1 (PDK1) and AKT to the plasma membrane, PDK1 will then phosphorylate and activate AKT. Activation of AKT elicits downstream signals to promote cellular metabolism, apoptosis, protein synthesis, and cell division8-11,25-34. Activated AKT mediates insulin-stimulated-glucose uptake via GLUT4 translocation, glycogen synthesis via Glycogen Synthase Kinase 3 (GSK3) inhibition, and cell survival by inhibiting pro-apoptotic actions of the Forkhead Box subfamily O protein (FOXO), and phosphorylation of BAD8-11,26-35. Similar to the RAS/ERK pathway, PI3K/AKT pathway is also highly regulated. PTEN is one of the main phosphatases that negatively regulate the cascade by de-phosphorylating AKT and PDK1. PTEN is a tumor suppressor and it is found to be highly dysregulated in several types of cancer, including pancreatic cancer26.  1.3  Cancer risks in individuals with type 2 diabetes Aside from the numerous complications that individuals affected by type 2 diabetes  have to face, there are growing epidemiological evidences that suggest type 2 diabetes is an independent risk factor for cancer. Cancer of the liver35-39, breast40-45, colorectal46, endometrial47,48, and pancreas49-51 were found to be associated with type 2 diabetes (Table 11). Among these observations, the highest associated risks were found in cancers of the liver, endometrial, and pancreas35-43,46-49,51 (Table 1-1). Aside from increased susceptibility to cancer, association studies have shown that patients with type 2 diabetes have poorer cancer prognosis, worsened response to chemotherapy, and more complications during cancer therapy when compared to people without type 2 diabetes49,50,52,53. The molecular mechanism underlying this phenomenon remains elusive. However, various researchers have suggested 5  compensatory hyperinsulinemia and insulin resistance during pre-diabetes may accelerate or initiate cancer development via the mitogenic effects of insulin5,35,36. During the multi-step progression of cancer, neoplastic cells acquire variety of biological adaptations to achieve carcinogenesis. Exposure of cells to chronic hyperinsulinemia may promote several characteristics of cancer. Some of the biological adaptations that are acquired by cancer cells include: sustained proliferative signalling, evasion of growth suppression, resistance to cell death, and replicative immortality54,55. The production and release of growth factors are carefully orchestrated in tissues to ensure appropriate timing of cell growth, division, and ultimately the maintenance of cellular functions and architecture integrity. When the functionality of normal cells are dysregulated, the cells may become more susceptible to external growth cues leading to chronic proliferation, evasion of growth suppressors, and cell death54,55. It is plausible that chronic hyperinsulinemia may provide additional growth advantages to pre-cancerous cells to accelerate cancer progression8-11. Supporting this hypothesis, a recent meta-analysis summarizing epidemiological studies prior to 2007 has correlated higher levels of circulating insulin and C-peptide in colorectal and pancreatic cancers patients39. C-peptide is a byproduct of insulin production. Furthermore, the association between higher circulating C-peptide and higher mortality rate in non-diabetic breast cancer patient was reported with a multivariable-adjusted hazard ratio of 2.3944. The risk of mortality was significantly enhanced in populations with type 2 diabetes at a hazard ratio of 2.8344. Several studies examining the expression of insulin receptors in cancer cells have also supported the notion that hyperinsulinemia and insulin resistance may initiate or accelerate  6  cancer progression. Independent reports have observed an increased expression of insulin receptors in human samples of breast, lung, and colon cancer15,56,57. IGF1R and IGF2R were also found to be overexpressed in tumors of prostate58, breast59 and pancreas60. Moreover, a study by Law et al. reported a positive correlation between poor survival rates in breast cancer patients with higher protein levels of IR, phosphorylated IR and phosphorylated IGF1R59. Several in vitro IR over-expression studies have demonstrated a ligand-mediated malignant transformation of Kaposi sarcoma, fibroblast, ovary cells, and human mammary epithelial cells61-63. Overexpression of IGF1R in vitro can also lead to ligand-dependent cellular transformation,64,65. Recently, Zhang et al. also observed a suppression of cancer cell proliferation and metastasis in two different breast cancer cell types (LCC6 and T47D) by siRNA knockdown of IR66. These findings were also recapitulated in animal models of hyperinsulinemia and insulin resistance. Streptozotocin or alloxan-induced type 1 diabetes (near complete loss of circulating insulin) in mice and rats with pre-existing mammary carcinoma have resulted in fewer tumors, and the observed anti-tumorigenic effects were reverted by insulin administration8,67. In the MKR mouse model of non-obese type 2 diabetes (a transgenic mouse expressing dominant-negative IGF1Rs in skeletal muscle), the mammary gland development of animals with hyperinsulinemia were found to be accelerated, when compared with the littermate control41. Furthermore, those investigators reported a notable increase in ductal hyperplasia in this mouse model of type 2 diabetes and breast cancer (PyVmT+/MKR+/+ mice). Both of these observations were linked to increased phosphorylation of IR, IGF1R and AKTS473 levels, but changes to phosphorylated ERK levels were not detected. This group further analyzed the effects of type 2 diabetes on later stages of breast cancer  7  progression. They investigated this by inoculating Met-1 and MCNeuA cell-lines into inguinal mammary fat of 8-weeks old hyperinsulinemic and glucose-intolerant MKR mice to generate an orthotopic tumor model. Tumor progressions were accelerated in both orthotopic (Met-1 and MCNeuA) models, and this was linked to increased IR/IGF1R and phosphorylated AKTS473 levels. The insulin-promoted acceleration of tumor growth and tumor weight were reversed with targeted inhibition of IR/IGF1R, PI3K-mTOR, and PI3K. This strongly supports the importance of PI3K/mTOR pathway in murine breast cancer progression that is influenced by hyperinsulinemia41,45,68. Altogether, the current literature suggests that elevated insulin levels and insulin receptor activation may trigger abnormal proliferation, replicative immortality, and resistance to growth suppression and cell death. With the rise of individuals with type 2 diabetes reaching to an estimated 588 million in 2030, a greater understanding of the cancer risk associated with patients affected by type 2 diabetes is much needed.  1.4  Pathogenesis of pancreatic cancer The pancreas is a complex organ that provides exocrine and endocrine  functionality69,70. The endocrine portion contains the islets of Langerhans. Hormones secreted by the islets of Langerhans act together to regulate whole-body glucose homeostasis69,70, these hormones include insulin, glucagon, somatostatin, and pancreatic polypeptide. The exocrine pancreas is composed of clusters of acinar cells that feed their secretions into an intricate branched ductal network. Acinar cells produce and secrete zymogen granules. These zymogen granules contain various proteases, amylolytic enzymes, lipases, nucleases, and pancreatic hydrolases69,70. Pancreatic ductal cells secrete bicarbonate juices into the ductal lumen to neutralize acidic chyme from the stomach. Zymogens secreted 8  by the acinar cells travel through the bicarbonate-enriched pancreatic ducts and then into the duodenum of the gastrointestinal tract. The secretions of pancreatic juices are highly regulated by gastric hormones (secretin and cholecystokinin) in the post-prandial state69,70. Neoplasms (tumors) that arise from the pancreatic epithelium are classified based on the cellular lineage or cell type that the neoplasm recapitulates. Tumors can arise from exocrine, endocrine, or the supportive tissue of the pancreas. 90% of all pancreatic cancers are exocrine tumors and less than 3% of all pancreatic cancer incidences are endocrine tumors69-75. Pancreatic ductal adenocarcinoma (PDAC) is the most common and aggressive type of pancreatic cancer. It accounts for approximately 85% of all pancreatic cancer incidences71. PDAC can arise from pre-invasive lesions that arise from the epithelial cells (pancreatic intraepithelial neoplasia, PanIN) or mass-forming cysts (intraductal papillary mucinous neoplasm, and mucinous cystic neoplasm)71. The most common pre-invasive lesion, PanIN, has been extensively studied, and this pre-cancer lesion model is graded into three different stages based on histological accumulation of dysplastic growth (Figure 14)69,72. Several studies have established the molecular aberrations that occur during each stage of PanIN, as shown in Figure 1-472-75. Similar to other cancers, telomere shortening and KRAS gain-of-function mutations are the earliest detected genetic lesions before PDAC. This is followed by the loss or mutated CDNK2A detected at PanIN1b, then the loss of a tumor suppressor p53 and mutated SMAD4 at PanIN3. The KRAS gain-of-function mutation at codon 12 is found in 90% of PDAC and 30% of early stages pancreatic cancer69-75. KRAS is a weak GTPase that requires the presence of GTP exchange factor such as SOS to propagate RAS/ERK signalling. The KRAS mutant is  9  constitutively active, because G12D point mutation greatly reduces the GTPase activity of the protein, thus leading to an irreversible formation of GTP-KRAS complex76. Constitutively active KRAS may cause sustained activation of the insulin/IGF signalling pathway to stimulate proliferation and pro-survival effects76. As mentioned previously, these are some of the biological adaptations that are required for carcinogenesis. Despite the recent advances in understanding the molecular pathogenesis behind pancreatic cancer, early detection methods and treatment for pancreatic cancer are still very limited. This is reflected in the low survival rate in pancreatic cancer patients, the 5-yearsurvival rate of pancreatic cancer remains at 5%39,49,69,73,77. Even if pancreatic cancer is detected early (10 – 20% of cancer incidences), treatment is still limited to pancreaticoduodenectomy followed by adjuvant chemotherapy78-81. The median survival for those detected at early stage PDAC is ~20.1 – 23.6 months. However, the majority of individuals with pancreatic cancer (80 - 90%) are detected at the advanced unresectable or metastatic stage78-81. Palliative chemotherapy then becomes their only option to minimize tumor progression and pain. The median survival for these patients is only 5 - 9 months78-81. Currently, 5-fluorouracil and/or gemcitabine are the only effective chemotherapy regimen and the gold standard of care against pancreatic cancer78-81. Fluorouracil and gemcitabine both targets rapidly dividing cells and interfere with DNA synthesis and replication to induce cell death82. It is important to note that chemotherapy will only increase the survival of advanced/metastatic patients by one month79. This further emphasizes the importance of investigating independent risk factors of pancreatic cancer to provide any potential preventative options for high-risk individuals.  10  1.5  Hyperinsulinemia, insulin/IGF1 signalling and pancreatic cancer The insulin/IGF1 signalling pathway has been implicated in cancer progression of  breast, colon and liver8-11,16,35-37,40-43,46. Regardless, the molecular mechanisms of how insulin or IGF1 may accelerate the progression of pancreatic ductal adenocarcinoma are still unclear. Evidences from epidemiological studies describing the correlation between anti-diabetic therapies and cancer risks have provided insights to potential mechanisms. Drugs that enhance circulating insulin were associated with higher mortality rate from pancreatic cancer83-85. Conversely, individuals with type 2 diabetes taking insulin-sensitizing drugs (that reduce circulating insulin) were less likely to develop pancreatic cancer84-89. The use of metformin (an insulin-sensitizing drug) is also currently pursued as an anti-cancer agent in individuals without type 2 diabetes. Metformin was found to improve chemotherapy response, reduce circulating insulin, and decrease Ki-67 positive cells in breast cancer patients without type 2 diabetes90. It was also reported that low doses of metformin can reduce the proliferation and formation of aberrant crypt foci in rectal epithelium of individuals not affected by type 2 diabetes91. Furthermore, clinical association studies have found that IGF1R92, IRS-193 and IRS294 are highly expressed in human pancreatic tumor samples. In addition, IR-A was found to be overexpressed in patients with hyperinsulinemia, insulin resistance, and/or type 2 diabetes9,13,15. It is proposed that overexpression of IR-A may predispose hyperinsulinemic individuals to cancer by sensitizing cancer cells to enhance mitogenic effects 13,15. It was demonstrated that IR-A have increased affinity for the oncogenic growth factor IGF2, thus potentially promoting tumorigenesis9,15. Interestingly, in recent genome wide association studies of pancreatic cancer patients, investigators found that a single nucleotide  11  polymorphism, rs8191754, located in the IGF2R gene may be associated to overall survival of pancreatic cancer95,96. However, this correlation is still highly disputed97. Studies directly investigating the molecular mechanism of hyperinsulinemia on insulin signalling of pancreatic duct cells have not been reported. However, there are other correlative studies that underline the importance of AKT and ERK signalling during pancreatic carcinogenesis. Increased circulating insulin may increase AKT activity, phosphorylation, and expression. Since AKT activation was found to be elevated in breast, and pancreatic cancer27-29, this may be a potential mechanism by which hyperinsulinemia may promote cancer progression. Pancreatic and duodenal homeobox 1 (PDX1) is a downstream effector of AKT25, where it is normally expressed during development and adult endocrine pancreas. Interestingly, elevated PDX1 expression was found in the exocrine pancreas of several pancreatic cancer mouse models30. Moreover, sustained PDX1 expression was reported to initiate acinar-to-ductal metaplasias98, tumor formation in SCID mice99, and increased proliferation and invasion of pancreatic cancer cells MIA PaCa299. As mentioned previously, KRASG12D mutations are found in 95% of pancreatic adenocarcinoma69-75, this mutation can lead to amplified ERK signalling eliciting aberrant growth responses and potentially enhancing cancer cell invasion and metastasis21,23. In fact, the expression of KRASG12D under a pancreas specific promoter in mice was found to cause pancreatic lesions and pancreatic ductal adenocarcinoma100. To further highlight the importance of ERK signalling in pancreatic cancer progression, loss of a metastatic suppressor, RAF1 Kinase Inhibitory Protein (RKIP), expression was reported in 57.1% of the PDAC cases101. The loss of RKIP was also significantly associated poor prognosis, along with greater nodal and distant metastases in pancreatic cancer patients102,103. Recent studies  12  from our lab and others have suggested dual functions of RAF1 in primary pancreatic islet cells104-106. Aside from the mitogenic role of RAF1 through ERK, insulin-stimulated RAF1 can also play an anti-apoptotic role via activating a BCL2 dependent pathway to reduce cell death104,105 Interestingly, constitutive activation of KRASG12D or AKT alone is insufficient to induce tumors or metastasis in mouse models. In glioblastoma and lung carcinoma, the activation of both KRASG12D and AKT is required for tumorigenesis and metastasis107,108. AKT1 overexpression in the mammary gland was only found to be tumorigenic when crossed with a KRAS mouse strain31. The pancreatic ductal specific expression of KRASG12D mouse model does not recapitulate advanced, metastatic pancreatic cancer observed in human100,109. This suggests that multiple players are required to initiate and fully develop pancreatic cancer. Hyperinsulinemia may coordinate synergistic stimulation of both RAF1/ERK and PI3K/AKT pathway in pancreatic ductal cells to enhance proliferative and pro-survival effects to promote pancreatic carcinogenesis. Collectively, the reported observations from epidemiological, clinical, and animal studies have helped emphasize the potential role of elevated levels of insulin in cancer progression.  1.6  Thesis investigation The objective of this thesis was to elucidate the potential cancer promoting effects of  hyperinsulinemia in human pancreatic ductal cells in vitro. Several research groups have characterized  the  role  of  insulin  and  IGF1  signalling  on  pancreatic  ductal  cells26,29,30,32,33,60,92,93,100,109-121. However, these in vitro and in vivo studies only provided information on transformed or immortalized human pancreatic cancer cell lines, or on pancreatic cancer progression in murine models. Furthermore, the supra-physiological 13  concentrations of insulin utilized in these studies may limit the relevance of their results. For future therapeutics and preventative interventions, greater understanding of normal human pancreatic ductal cell physiology is required. Thus, to understand the effects of hyperinsulinemia on pancreatic cancer initiation and progression, it is crucial to also examine the effects of insulin on normal human pancreatic ductal cells in vitro. This research study will highlight the role of insulin/IGF1 pathway on cellular viability and cell death of human pancreatic ductal cells. The objective of this study is to identify and compare the importance of RAF/ERK versus PI3K/AKT pathway by measuring changes in total cell number, viability, propidium iodide incorporation, and cleaved caspase3 protein levels across stages of pancreatic ductal cancer in vitro. The cell types used in this study consist of primary ductal cells, pre-malignant cell line (HPDE), and aggressive malignant cell line (PANC1) as shown in Figure 1-5. Furthermore, the insulin-mediated response on cellular viability and cell death were compared and evaluated across all cell types. The effects elicited by insulin were also evaluated relative to a known tumorigenic growth factor, IGF18-11,16,62,95,96,122, in HPDE and PANC1 cells. HPDE cells were transformed by introduction of the E6E7 retrovirus120,123-125. Although the HPDE cells contain abnormal p53 and RB function, this cell type retains normal cell morphology to that of the human pancreatic ductal cell123-125. Furthermore, these cells are non-tumorigenic and unable to form colonies in a soft-agar assay120. Thus far, effects of insulin or IGF1 on the viability of HPDE cells have not been reported. PANC1 cells are derived from an aggressive human pancreatic adenocarcinoma, and it has been reported to be highly metastatic and tumorigenic126. Fisher et al. reported proliferative effect  14  of exogenous insulin in PANC1 cells110; however, that study did not examine mechanism behind their observations. This is one of the first studies to evaluate the importance of insulin pathway in normal human pancreatic ductal cells in vitro. By using different cell types, this work provides a diverse understanding on how mitogens may differentially affect different stages of pancreatic ductal cancer progression, and thereby provide a greater understanding in how insulin may positively influence human pancreatic ductal cancer cells in vitro.  15  Figure 1-1 Working model of type 2 diabetes pathogenesis. Several environmental factors such as obesity, chronic stress and sedentary lifestyles have been identified as risk factors for type 2 diabetes. These factors may predispose individuals to insulin resistance and hyperinsulinemia, also known as pre-diabetes. During pre-diabetes, the insulin signalling in insulin-responsive tissues (liver, skeletal muscle, and adipose tissue) is impaired, thus leading to reduced glucose disposal. To maintain post-prandial glucose homeostasis, β-cells of the islet of Langerhans hyper-secrete insulin, this physiological condition is known as hyperinsulinemia. Although normal blood glucose levels are maintained, the continual production and secretion of insulin by the β-cells may lead to hypertrophy, irreversible β-cell dysfunction, and eventually β-cell failure and death. Without the β-cells, glucose homeostasis can no longer be maintained by compensatory hyperinsulinemia, leading to sustained hyperglycemia or also known as type 2 diabetes.  16  Figure 1-2. Transmembrane receptors involved in insulin and IGF1 signalling During stimulation of the insulin/IGF1 signalling pathway, insulin receptors (IR), type 1 IGF receptor (IGFIR) and type 2 IGF receptor (IGFIIR) can form combinations of receptor dimers. The yellow circles depict activation and stimulation, whereas the red circle demonstrates inactivation or inhibition.  17  A.  B.  Figure 1-3. Schematic of insulin and IGF1 signalling. The schematic diagram above demonstrates the two major canonical signalling pathways of insulin and IGF1 (A) PI3K/AKT pathway and the (B) RAS/ERK pathway. *IGF1 was not shown in this figure. PM refers to plasma membrane.  18  Figure 1-4. Multi-step progression to pancreatic ductal adenocarcinoma. The figure has been adapted with permission from Macmillan Publishers Ltd: Modern pathology (Maitra et al.72), copyright (2003) to depict the molecular aberrations that are associated with lesions occurring in the pancreatic epithelium prior to pancreatic ductal adenocarcinoma. The horizontal arrows represent the existence of the mutations relative to the PanIN.  19  Table 1-1. Relative cancer risks of individuals with type 2 diabetes.  Cancer Type  Relative Risk  Colon cancer46  1.3  Liver cancer35-37  2.5  Breast cancer40-42,127  1.2  Endometrial cancer47,48  2.1  Pancreatic cancer36,37,40,50-52  1.8  20  4 3  1  2  Figure 1-5. In vitro model of pancreatic cancer progression. The cell models used in this study includes primary human pancreatic sorted (1) and unsorted (2) cell population, HPDE cells (3), and PANC1 cells (4). The cell models will represent normal pancreatic ductal cell type, non-transformed, pre-cancer cell type and pancreatic ductal adenocarcinoma stage shown at the far right. The schematic of PanIN driving to pancreatic adenocarcinoma was adapted with permission from Macmillan Publishers Ltd: Modern pathology (Maitra et al.72), copyright (2003).  21  Chapter 2: Material and methods 2.1  Reagents Human recombinant insulin was purchased from Sigma-Aldrich (St. Louis, Missouri,  USA), human recombinant IGF1 was purchased from R&D Systems (Minneapolis, Minnesota, USA). RAF1 inhibitor (GW5074) was purchased from Life Technologies (Carlsbad, California, USA). MEK inhibitor (U0126) was purchased from Cell Signaling (Danvers, Massachusetts, USA). Akt-inhibitor VIII, isozyme selective inhibitor was purchased from Calbiochem (now known as EMD Biosciences, Darmstadt, Germany) and wortmannin was purchased from EMD Biosciences. GW5074 is an isoform-selective and cell-permeable inhibitor that inhibits the activity of RAF1 by competitively binding to the ATP binding pocket128. The Akt inhibitor VIII (Akti-1/2) is an AKT inhibitor that works by binding to the pleckstrin homology domain of AKT to prevent the release of the kinase domain thus preventing the formation of active AKT conformation129-132. The concentrations of inhibitors used in the current studies were chosen based on the EC50 values listed by the manufacturers and from the literature. U0126 inhibits both MEK1 and MEK2 by inhibiting the catalytic activity of the active enzyme133. Wortmannin is a non-competitive, irreversible inhibitor that specifically targets the catalytic subunit of PI3K complex 134,135. All the small molecule inhibitors were reconstituted in DMSO at concentration of 10 mM, aliquoted and stored at - 20 ˚C. Working solution of 1 X lysis buffer was made from 10 X lysis buffer (Cell Signaling) with 2 X protease inhibitor (Calbiochem) in milliQ-H2O. SDS-PAGE ladders were purchased from Thermo (formerly Fermentas, Waltham, Massachusetts, USA). The concentration and the sources of antibodies used throughout this work are summarized in Table 2-1.  22  2.2 2.2.1  Cell models of this study Human mixed pancreatic exocrine and ductal cell culture Primary pancreatic exocrine cells are by-products of the clinical islet transplantation  program directed by Dr. Garth Warnock at the Ike Barber Human Islet Transplant Laboratory (Vancouver, BC). Information about the origin of the donor tissues is listed in Table 2-1. The isolation of these cells have been modified from the Bonner-Weir et al. (2004) protocol136 and further fine-tuned by previous graduate students, Theresa Liao and Dr. Corinne Hoesli. The conditions for culturing these cells have been previously published137. Upon arrival of these tissues, the cell population consisted of 61% amylase+ cells, 6% CK19+ cells, 12% vimentin+ cells, and approximately 0.01% insulin+ cells137. Amylase, CK19, vimentin, and insulin are markers for acinar cells, ductal cells, fibroblasts and β-cells respectively. Overtime, CK19+ cells and vimentin+ cells begin to dominate the population (On day 2, 25% CK19+ cells and 27% vimentin+ cells; on day 7, 39% CK19+ cells and 33% vimentin+ cells)137. On day 7, both CK19 and vimentin positive population remains BrdU positive, indicating that these cell populations are still proliferative137. Several strategies have been examined to negatively select out the vimentin+ fibroblast-like cells. The use of magnetic cell sorting for CD90- cell population was found to be the most cost-effective and productive method137. CD90 is a cell surface marker for mesenchymal-like cells. Due to the lower-yield from the sorted cell population, both unsorted and sorted cell populations were used in this study. Sorted cell population was used for total cells tracking and cell death assays, whereas the unsorted cell population was used for Western blot analysis.  23  2.2.1.1  Culturing procedures for primary human non-islet tissue Unless otherwise specified, human duct and acinar cells from the human exocrine  pancreas were cultured in CMRL-1066 media (CellGro now known as Corning CellGro, Manassas, VA, USA or GIBCO now Life Technologies, Grand Island, NY, USA) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine and 10% fetal bovine serum (Invitrogen of Life Technologies). Upon arrival (day 0), the supernatant of the homogenized non-islet human pancreatic tissue was pelleted, aspirated, and washed twice with supplemented CMRL-1066 (CMRL) then centrifuged for 5 minutes at 250 g. Packed cell volume (PCV) of the re-suspended pellet was determined by adding 10 μl of cell suspension into PCV tubes (Techno Plastic Product AG, Trasadingen, Switzerland) then centrifuged for 1 minute at 250 g.  2.2.1.2  Cell dissociation of human unsorted pancreatic exocrine cells The following procedure was used to obtain unsorted pancreatic exocrine cells used  for western blot analysis. 20 mL of non-islet human pancreatic homogenate was spun down at 250 g for 5 minutes and further washed twice with 20 ml of dispersion medium (calcium and magnesium free HBSS (Invitrogen), 0.5% bovine serum albumin (Sigma), 10 mM HEPES (Sigma), 1 mM EDTA (Sigma) spun at 250 g for 1 minute. The cells were then resuspended at 0.5 mL PCV in 10 mL of dispersion medium in a T-25 flask. The flask containing the suspension was placed on shaker orbiting at 37˚C at 300 rpm for 6 minutes. Then 5 μg/mL trypsin (Invitrogen) and 4 μg/mL DNase (Qiagen, Venlo, Netherlands) were added, then incubated for an additional 10 minutes on the shaker. The trypsinization was stopped by the addition of 20 mL CMRL. Thereafter, the suspension was filtered through a 40 μm nylon sieve (BD Biosciences, Franklin, New Jersey, USA) into a 50 mL falcon tube 24  containing 10 mL of CMRL media. Cells were stained with trypan blue (GIBCO) and enumerated. To reduce the fibroblast population, the unsorted cells were plated in a T-150 flask at 10 X 106 cells per flask for one hour in the incubator. Then the non-attached cell suspension were then aspirated and re-plated at 6-well plates at cell density of 1.5 X 106 cells per well for further treatment and Western blot analysis.  2.2.1.3  Magnetic activated cell sorting (MACS) The following procedures were used to obtain human sorted pancreatic ductal cells  for cellular viability and cell death studies. Primary unsorted pancreatic exocrine cells were plated at 1 PCV/cm2 in T-175 flask on day 0. Cells were washed with MACS buffer (1X PBS, 1% FBS, 2 mM glutamine), trypsinized for 3 minutes, then spun down at 250 g for 5 minutes. The pellet was re-suspended in CMRL and the cells were enumerated. Cell suspensions were then labeled and incubated with CD90 antibody (final concentration 1:500) with a cell density of 10 X 106 cells/ml for 25 minutes at 4˚C on a shaker. The cells were then washed twice in MACS buffer and re-suspended at the cell density of 1 X 106 cells/ml. Anti-rat IgG MicroBeads (Miltenyi Biotec, Cologne, Germany) were then added at 20 μl per 10 X 106 cells and incubated for 15 minutes at 4˚C on a shaker. The suspension was then washed with MACS buffer and re-suspended at 4 X 106 cells/ml. This was then loaded onto the MACS machine (Miltenyi Biotec) at Biomedical Research Center, University of British Columbia for sorting using the depletes program on the MACS machine to negatively select for CD90- cells. After sorting, cells were plated at cell density of 0.04 X 106 cells/well in a 96-well plate for viability and cell death assays or 1.5 X 106 cells/well in a 6-well plate for Western blot analysis.  25  2.2.1.4  Primary cell treatment procedures Human unsorted and sorted pancreatic exocrine cells were all serum-starved in the  basal media (0.5 mg/L transferrin, 10 mM nicotinamide, 0.5 μg/L sodium selenium, 0.5% BSA, 2 mM glutamine) for 6 hours and treated with the indicated doses of insulin or small molecule inhibitors in the basal media. Serum-starved sorted primary cells were treated with control (basal media), 5 μM GW5074 (RAF1 inhibitor), or 100 nM Akti-1/2 (AKT inhibitor) for 24 hours.  2.2.2  HPDE cell culture Human pancreatic ductal epithelium cells (HPDE) were generously provided by Dr.  Sylvia Ng from BC Cancer Agency and were used under material transfer agreement from Dr. Ming Tsao (University of Toronto). HPDE cells are human pancreatic ductal cells that were immortalized by transfection of E6E7 protein from human papilloma virus 16123-125. These cells were cultured in keratinocyte-serum-free medium (KSF) supplemented with 50 µg/ml of bovine pituitary extract and 5 ng/ml of epidermal growth factor (Invitrogen).  2.2.3  PANC1 cell culture PANC1 cells were purchased from ATCC (Manassas, Virginia, USA). PANC1 cell  line was derived from an aggressive pancreatic tumor at the head of the pancreas from a 56year old Cauasian male126. This cell line contains HER-oncogene, mutated SMAD4, and abnormal p53 signalling126,138,139. PANC1 cells were cultured in DMEM with 4.5 mg/ml Dglucose (GIBCO/Sigma) supplemented with 10% FBS and 1% penicillin/streptomycin.  26  2.2.4  HPDE and PANC1 cells treatment procedures HPDE and PANC1 cells were washed with 1X PBS and starved in non-supplemented  KSF media for 6 hours or DMEM with 1 mg/ml glucose, and 1 % penicillin/streptomycin (GIBCO/Sigma) for 24 hours, respectively. Starved cells were then treated with the indicated doses of insulin, IGF1, or small molecule inhibitors in the starvation media. The inhibitors used in this experiment include 10 μM GW5074 (RAF1 inhibitor), 10 μM U0126 (U0126), 200 nM Akti-1/2 (AKT inhibitor) or 1 μM wortmannin.  2.3  Cell count and cell death assays Proliferation was indirectly assessed by total cell count and colourimetric formation  of formazan from XTT as an indication of metabolic activity and cellular viability. Total cells were tracked over time with the addition of Hoechst 33342 (Hoechst; Invitrogen) at 0.5 μg/ml, and then imaged by at the DAPI channel (excitation and emission wavelengths are 377 nm and 447, respectively) with ImageXpressMicro (Molecular Devices, Sunnyvale, California, USA) with a 10x Plan Fluor objective using the program MetaXpress 3.1 (Molecular Devices). The Hoechst-positive cell population was analyzed in Acuity Xpress 2.0 (Molecular Devices). The changes in metabolic activity or cellular viability were assessed via the XTT assay (ATCC). XTT reagent was added at indicated time-point according to the manufacturer’s recommendations, and the absorbance was measured at A660nm and A460nm. This assay takes advantage of the cellular redox machinery and an intermediate electron acceptor PMS in the kit. The XTT dye is reduced into a brightly orange formazan product at the cell surface. The degree of colorimetric reduction of XTT is indicative of the number of viable cells and the pyridine nucleotide redox status of cells140.  27  Cell death was evaluated by propidium iodide (PI; Sigma) incorporation and cleaved caspase-3 protein levels. Serum-starved cells were incubated with media containing 0.5 μg/ml Hoechst and 5 μg/ml propidium iodide one hour prior to imaging, cells were then imaged at DAPI and Texas red channel (excitation and emission wavelengths are 562 nm and 624 nm, respectively) at one-hour interval for 24 hours at 37ºC and 5 % CO2 using an ImageXpressMicro high content imaging system. PI incorporation were analyzed by Acuity Xpress 2.0, cells were scored PI positive when PI co-localizes with the nucleus. In most cases, PI incorporation was expressed as the number of PI and Hoechst co-positive cells in a population of Hoechst-positive cells as % PI+ cells. PI incorporation was also expressed as a fold change in % PI+ cells at a given time point relative to % PI+ cells at t = 0 h. The relative protein levels of cleaved caspase-3 were measured by Western blot analysis as described below.  2.4  Immunoblotting and densitometry analysis Treatment media were aspirated, then the monolayer was washed thrice with cold 1 X  PBS (1.058 nM KH2PO4, 1.54 mM NaCl, 5.6 nM Na2HPO4, these were all purchased from Sigma-Aldrich). 1 X lysis buffer (Cell Signaling) diluted in milliQ water, and supplemented with 2 X protease inhibitor (Calbiochem) were added onto the monolayer and incubated on ice for 15 minutes. The cell lysates were then collected by scraping and frozen down at 20˚C. Frozen cell lysates were then thawed, sonicated and centrifuged at 10 000 rpm for 10 minutes at 4ºC. The supernatant was used to determine the protein concentrations by the BCA assay (Thermo-PIERCE). Protein samples were prepared with 1 X loading buffer (Cell Signaling) and separated by SDS electrophoresis in 1 X running buffer (2.5 µM Tris Base, 1.92 mM Glycine, 3.47 nM SDS (all were purchased from Bio-Rad, Berkeley, California, 28  USA)). 12% SDS-gel were then transferred to a 0.22 μm polyvinylidene fluoride membrane (EMD Biosciences) with semi-dry transfer with transfer buffer (48 mM Tris, 39 mM glycine, 1.3 nM SDS, 20% methanol, Fischer) for 70 minutes at 20 V. After observing the transfer quality with Ponceau S (Sigma) staining, membranes were incubated with I-Block (Tropix, Bedford, Massachusetts) for 1 hour at room temperature. Membrane were then transferred into primary antibody solution and incubated overnight at 4˚C. Thereafter, membranes were washed thrice with 1 X PBS with 0.1% v/v Tween (Sigma) and incubated with a horseradish peroxidase-linked secondary antibody (Cell Signaling) for 1 hour at room temperature. The addition of chemiluminescent reagent (Thermo-PIERCE) cleaves the horseradish peroxidase and emits a luminescence signal that is proportional to the amount of protein present. The luminescence signals were detected by film. Each immunoblot was subjected to densitometry analysis using Photoshop (Adobe Systems Inc., San Jose, CA) and expressed as a fold change of mean pixels of treated cells relative to non-treated control ± standard error (serumfree).  2.5  Statistical Analysis The results from densitometry, XTT, and single time point cell death studies were  analyzed using one-way ANOVA, followed by post-hoc tests (Dunnett’s analysis or Bonferrini analysis). Kinetic data on cell death and total cells were analyzed using repeated measure two-way ANOVA, followed by Bonferroni post-hoc test. Results were considered significant if they yielded a p-value of less than 0.05.  29  Table 2-1. List of antibodies used in this study  Antibody  Source  Concentration  Rabbit monoclonal anti-phospho-ERK  Cell Signaling (4370)  1:2000  Rabbit monoclonal anti-ERK  Cell Signaling (4695)  1:2000  Rabbit monoclonal anti-phospho-AKTS473  Cell Signaling (4060)  1:1000  Rabbit monoclonal anti-AKT  Cell Signaling (4691)  1:1000  Mouse polyclonal anti-insulin receptor β  Cell Signaling (3020)  1:1000  Rabbit monoclonal anti-cleaved caspase-3  Cell Signaling (9654)  1:1000  Mouse monoclonal anti-beta-actin  Novus Biologicals (Littleton, Colorado, USA)  1:10 000  Rabbit polyclonal anti-beta-tubulin  Cell Signaling (2146)  1:1000  Mouse monoclonal anti-human CD90 (Thy1)  BD Pharmingen (Franklin, New Jersey, USA)  1:500  Anti-mouse IgG, HRP-linked antibody  Cell Signaling  For Actin (1:15 000 to 1:20 000) Other (1:3000)  Anti-rabbit IgG, HRP-linked antibody  Cell Signaling  1:3000  Table 2-2. Information of the donor primary tissues.  Donor ID: H148 H155 H172  Age 44 30 32  30  Chapter 3: Characterization of insulin signalling in human pancreatic ductal cells Numerous investigators have examined the effects of insulin on metastatic or transformed human pancreatic cancer cell lines66,85,110,114,116,141. However, only a limited number of studies have looked specifically at the effects of insulin on primary human pancreatic ductal cells. The goal of this study is to provide further understanding of the role of AKT versus ERK signalling in the regulation of cellular viability in primary human pancreatic non-islet cells, HPDE cells, and PANC1 cells. In addition, the effects of exogenous insulin on cellular viability and its potential mechanism were explored.  3.1 3.1.1  Insulin signalling of primary human ductal cells Inhibition of AKT and RAF1 in human primary pancreatic sorted cells The requirement of the two main downstream pathways of insulin, ERK and AKT  was investigated to determine their relative roles in cellular viability of sorted primary pancreatic ductal cells. The effects of 24-hour inhibition of RAF1 and AKT on changes in total cell number and propidium iodide (PI) incorporation were measured under serum-free conditions. Inhibition of RAF1 activity by GW5074 did not alter the total cell number or PI incorporation when compared to serum-free control (Figure 3-1), whereas inhibition of AKT activity by Akti-1/2 led to a significant increase in PI-positive cells by 1.5 fold (Figure 3-1C). This suggests that the AKT pathway may be involved in regulating cell death in primary human pancreatic sorted cells.  31  3.1.2  Actions of insulin on cellular viability of primary pancreatic unsorted and  sorted cells The effects of exogenous insulin on AKT and ERK activation were determined by measuring phosphorylated levels of AKT at S473 (pAKTS473) and phosphorylated levels of ERK at T202/Y204 (pERK). The phosphorylation of these residues relative to the total protein levels is an indication of activation in the respective protein. Although the doses of insulin used in this study did not elicit significant changes in pAKTS473 levels, there was a trend towards insulin-mediated increase in pAKTS473 levels after both 5-minute and 24-hour treatments (Figure 3-2A,C). On the other hand, acute 200 nM insulin treatment elicited a significant 1.4 fold increase in the pERK/ERK ratio (Figure 3-2B). Since exogenous insulin elicited acute ERK activation in unsorted primary pancreatic unsorted cells, we next determined whether exogenous insulin affects total cell number and PI incorporation in sorted primary pancreatic cells under serum-free conditions. When compared to non-treated sorted cells, changes in total cell number (Figure 3-3A-B) or PI incorporation (Figure 3-3C-D) in sorted cells treated with 0.2 to 200 nM insulin were not observed.  3.1.3  Conclusion regarding primary human pancreatic ductal cells These experiments suggest that AKT may be involved in regulating cell death in  sorted primary ductal cells under serum-free conditions. Although there was an acute insulinmediated increase in ERK activity in primary unsorted pancreatic cells, the doses of exogenous insulin used in this study did not affect total cell number or PI incorporation in sorted primary pancreatic duct cells under our experimental conditions.  32  3.2 3.2.1  Insulin signalling in HPDE cells Inhibition of PI3K/AKT or RAF/ERK pathway in HPDE cells The importance of AKT and ERK pathway in mediating cellular viability of HPDE  cells was evaluated in this study. This was done by inhibiting RAF1, MEK, PI3K, or AKT inhibition via small molecule inhibitors and evaluating their effects on cellular viability using the XTT assay after 24 hours or 7 days of incubation. The viability of HPDE cells was attenuated by 10 µM GW5074 after 24-hour and 7-day treatment (Figure 3-4A,C). Interestingly, 10 µM U0126 attenuated the viability of HPDE cells only after 7 days of treatment but not after 24 hours of treatment (Figure 3-4A,C). Attenuation of HPDE cellular viability by 10 µM GW5074 after 24 hours was consistent with the GW5074-induced elevation in PI incorporation at 24 hours (Figure 3-4B). Contrary to the observation in primary human sorted cells, inhibition of AKT pathway by 200 nM Akti-1/2 or 1 µM wortmannin did not decrease the viability of HPDE cells after 24 hours or 7 days of treatment (Figure 3-4A,C). This indicates the RAF/ERK pathway may play a role in regulating viability and cell death of HPDE cells under our experimental conditions.  3.2.2  Effects of exogenous insulin on AKT and ERK activation in HPDE cells After establishing the importance of the RAF/ERK signalling arm on HPDE cell  viability, we further investigated whether these cells would respond to insulin. HPDE cells are typically grown in a specific culture media called keratinocyte-serum-free (KSF) media. KSF is a proprietary media, therefore, the ingredients in KSF media were not readily available or immediately obvious. We were suspicious that this media might contain insulin, and may obscure the effects of our added insulin. Therefore, the concentration of insulin across all media used in this study was determined by radioimmunoassay (RIA). Our analysis  33  revealed that very high levels of insulin (779 ± 87 nM) were present in the non-supplemented KSF media (Table 3-1). Unfortunately this was only discovered after the study on the effects of added insulin on HPDE cells was completed. This may explain our observations that in spite of insulin receptor β and IGFIR expression in HPDE cells (Figure 3-5E), acute and chronic treatment with insulin or IGF1 treatments did not alter pAKTS473/AKT and pERK/ERK levels when compared to control (Figure 3-5A-D).  3.2.3  Conclusion regarding HPDE cells In the HPDE cell line, we observed RAF/ERK signalling arm maybe required for  cellular viability and reduction in cell death under serum-free conditions. Due to the presence of insulin in the KSF media, the effects of insulin on AKT and ERK activation could not be deciphered.  3.3 3.3.1  Insulin signalling in PANC1 cells Effects of AKT and ERK inhibition on cellular viability in PANC1 cells Small molecule inhibitors targeting the canonical downstream targets (AKT, PI3K,  RAF1 and MEK) of the insulin signaling pathway were used to evaluate the importance of AKT versus ERK signalling pathway on cell viability in PANC1 cells. Under serum-free conditions, 24 hour-inhibition of RAF1 by 10 μM GW5074 significantly decreased the viability of PANC1 cells by 0.4 fold relative to non-treated control (Figure 3-6A). This is consistent with increased cleaved caspase-3 protein levels after 24 hours of treatment (Figure 3-6B). Whereas, inhibition for 24 hours of another canonical target of ERK pathway, MEK, by 10 μM U0126 did not elicit changes in cellular viability or cleaved caspase-3 protein levels relative to serum free control (Figure 3-6A-B). The cleaved product of caspase-3 is a  34  marker of apoptotic cell death25. Furthermore, 24-hour inhibition of PI3K or AKT by 1 μM wortmannin and 200 nM Akti-1/2, respectively, did not alter PANC1 cellular viability or cleaved caspase-3 protein levels in PANC1 cells. Further analysis on the effects of small molecule inhibitors on PI incorporation of PANC1 cells over 48 hours revealed similar results. Treatment with GW5074 induced a significant fold increase in PI incorporation of PANC1 cells starting at 10 hours relative to DMSO control. Moreover, PI incorporation in PANC1 cells was significantly enhanced by U0126 and Akti-1/2 after 42 hours and 39 hours of incubation, respectively (Figure 3-6C). Area under the curve analysis of PI incorporation over 48 hours suggested that treatment with 10 μM GW5074 elicited the most significant increase in PI incorporation of PANC1 cells under our experimental conditions (Figure 3-6D). Inhibition of PI3K by 1 μM wortmannin did not alter PI incorporation in PANC1 cells (Figure 3-6CD). The significant increase in PI incorporation by GW5074 and U0126 during the kinetic analysis was consistent with the reduced cellular viability of PANC1 cells after 5 days treatment (Figure 3-6E). To verify the actions of the small molecule inhibitors, the phosphorylation levels of downstream effectors were determined by Western blot (Figure 3-8). Inhibitors targeting the ERK pathway (U0126 and GW5074) have both effectively decreased pERK levels after incubation. Furthermore, inhibitors targeting the AKT pathway (wortmannin and Akti-1/2) have both effectively decreased the downstream effector of AKT, pFOXO3a. Altogether, these observations may indicate the role of RAF/ERK pathway in regulating cellular viability of PANC1 cells under our experimental conditions.  35  3.3.2  Effects of exogenous insulin on cellular viability, AKT and ERK activation in  PANC1 cells To address the potential cancer promoting effects of hyperinsulinemia on existing pancreatic adenocarcinoma, we attempted to explore the potential mechanism behind the insulin-mediated effects on cellular viability, total cell number, and cell death relative to a known proliferation promoting growth factor, IGF18-11,16,63,96,97,123,111. Fisher et al. reported insulin receptor expression and proliferative effect of exogenous insulin in PANC1 cells; however, the mechanisms behind these observations were not provided. PANC1 cells contain constitutively active KRASG12D, thus there is a possibility that insulin-mediated actions in the RAS/ERK pathway could be blunted or desensitized. Thus, IGF1 was included in these studies due to its established proliferative role in various cancer cell types8-11,16,63,96,97,123. Serum-starved PANC1 cells were treated with exogenous insulin or IGF1 at concentration between 0.2 nM and 200 nM, and the number of Hoechst-positive cells was assessed over time. Under serum-free condition, treatment of PANC1 cells with 20 nM insulin for 24 hours elicited a small, but significant increase in the number of cells relative to t = 0 h (1.055) when compared to non-treated control (1.004) as shown in Figure 3-8A. This trend was not observed from the area the curve calculation over 48 hours (Figure 3-8B). After 24 hours of treatment, 20 nM and 200 nM of insulin or IGF1 significantly enhanced cellular viability of PANC1 cells (Figure 3-8D). Treatment of 20 and 200 nM of insulin for 5 days significantly enhanced cellular viability of PANC1 cells (Figure 3-8E). Treatment with 2, 20 200 nM IGF1 on PANC1 cells also enhanced cellular viability after 5 days (Figure 3-8E). Interestingly, Bonferroni post-hoc analysis of Figure 3-8E revealed that 200 nM insulin promoted a greater increase in cellular viability than 200 nM IGF1 after 5 days of incubation in PANC1 cells (indicated by #). Altogether the results suggest that 20 nM of exogenous 36  insulin can promote an increase in total cell number over time and cellular viability in PANC1 cells under our experimental setting. To test whether insulin-mediated elevation in total cell number and cellular viability were due to the reduction in cell death under serum-free conditions, the effects of exogenous insulin on PI incorporation and cleaved caspase-3 protein levels were measured. Under our experimental conditions, concentrations of insulin and IGF1 used in this study did not reduce PI incorporation when compared to the non-treated cells (Figure 3-9A-B). In agreement with the kinetic PI incorporation data in treated PANC1 cells, changes in cleaved caspase-3 protein level were not induced after 24 hours of insulin treatment (Figure 3-9C). Collectively, the insulin-stimulated increases in PANC1 cell number and viability were not associated with the reduction in PI incorporation and cleaved caspase-3 protein levels in PANC1 cells. After confirming the positive effects of insulin on total cell number and cellular viability in PANC1 cells under our experimental conditions, we attempted to examine the effects of insulin on AKT and ERK activation. When compared to control (0 nM insulin), acute incubation of exogenous insulin significantly elevated pAKTS473/AKT by 73.5 fold, and pERK/ERK levels by 1.77 fold (Figure 3-10A-B). Relative to non-treated conditions, chronic exposure to 20 nM insulin elevated pAKTS473/AKT level by 6.99 fold in PANC1 cells, but significant changes in pERK/ERK levels were not detected (Figure 3-10C). Instead, lower concentrations of insulin (0.2 and 2 nM) significantly enhanced pERK/ERK levels by 2.83- and 2.72- fold, respectively (Figure 3-10D).  3.3.3  Conclusion regarding insulin and IGF1 signalling on PANC1 cells This study suggests that through our small molecule inhibitor study, RAF/ERK  pathway may play a predominate role in regulating cellular viability and cell death in PANC1 37  cells under our experimental conditions. Furthermore, 20 nM of exogenous insulin increased both total cell number and viability of PANC1 cells without reducing PI incorporation or cleaved caspase-3 protein levels under serum-free conditions. Acute exogenous insulin treatment at higher concentrations induced a significant increase in pAKTS473/AKT and pERK/ERK levels. While chronic incubation of insulin at higher concentrations elicited a significant increase in pAKTS473/AKT, pERK/ERK was only significantly enhanced by chronic insulin treatment at lower concentrations.  38  Figure 3-1. AKT and RAF-1 inhibition in primary human pancreatic sorted cells. Primary sorted cells were serum-starved and treated with GW5074 (RAF1 inhibitor), Akti-1/2 (AKT inhibitor) for 24 hours. (A) Fold change in total (Hoechst-positive) cells at that hour relative to total cells at t = 0 hour. (B) Area under the curve (AUC) of the treatment groups are shown in (A) expressed in arbitrary units (AU). (C) Fold change in PI and Hoechst co-positive cells relative to SF observed at 24th hour after treatment. SF denotes serum-free. The data above are representative of biological replicates from three human donors.  39  Figure 3-2. The effects of insulin in primary human pancreatic unsorted cells. Primary human unsorted cells were serum-starved then treated at the indicated dose of insulin for 5 minutes (A, B) and 24 hour (C, D) as described in Methods and Material (n = 3). The effects of insulin on phosphorylated AKTS473 (A,C) and phosphorylated-ERK (B, D) relative to total AKT and ERK were quantified and expressed as fold change relative to non-treated cells. β-actin was used as the loading control. Data above are representative of biological replicates from three human donors.  40  Figure 3-3. Effects of insulin on cell viability of primary human pancreatic sorted cells. Primary human pancreatic sorted cells were serum-starved and treated as described in Materials and Methods. (A) Effects of indicated doses of insulin on total cell count (Hoechst-stained nuclei) were expressed as fold change in total cell number relative to t = 0 hour over 24 hours. (B) Area under the curve of (A) depicted in arbitrary units (AU). (C) The effects of insulin on propidium iodide (PI) incorporation of primary pancreatic sorted cells were expressed as the percent of PI and Hoechst co-positive cells over total Hoechst positive cells over 24 hours. (D) Area under the curve of (C) is depicted in arbitrary units (AU). The errors bars are the representative of standard error of the mean. The data above were collected from preparation from three human donors.  41  HPDE Cell Survival Inhibitors  HPDE 24h inhibitors for SF  A  B  HPDE 7d inhibitors  C 40  1.5  1.5  1.0  0.5  *  Cell Viability Relative to DMSO (Fold Change)  Percent of PI + Cells  Cell Viability Relative to DMSO (Fold Change)  * 30  20  10  0.5  * * 0.0  0  0.0  1.0  W t.  or  /2  26  i-1  t Ak  74  50 W  1 U0  G  /2  SO DM  t or W  6  i-1  t Ak  4  2  74 50 W  12 U0  G  t.  or  1/  SO DM  W  6  7 50  tiAk  W  SO  12 U0  G  DM  Figure 3-4. The effects of small molecule inhibitors on cellular viability of HPDE cells. HPDE cells were serum-starved, and the effects of 10 µM RAF1 (GW5074), 10 µM MEK (U0126), 200 nM AKT (Akti-1/2), or 1 µM PI3K (wort. = wortmannin) inhibition on cellular viability of HPDE cells were assessed by XTT assay (A,C) and propidium iodide (PI) incorporation (B). After 24 hours (A) or 7 days (C) of inhibitor treatment, the effects on cellular viability were measured and expressed as the fold change in mean absorbance of treated cells relative to DMSO control. (B) The effects of different small molecule inhibitors on PI incorporation of HPDE cells were expressed as the percent of PI and Hoechst co-positive cells over total Hoechst positive cells (Percentage of PI+ cells) after 24 hours of incubation. The data above are representative of three independent biological replicates.  42  Table 3-1. Concentration of insulin across the culture media used in this study Media samples were taken from the product bottles and subjected to an insulin radioimmunoassay to determine the concentration of insulin.  !  = below detection range. The graph below was used as a standard curve to  determine the concentration of insulin of the media samples in Table 3-1. Values were converted from ng/ml, to nanomolar.  Insulin RIA standard curve 1.2  CPM  1 0.8 0.6 0.4  0.2 0 0.01  0.1  1  10  [Log ng/ml]  MEDIA  Insulin (nM)  KSF CMRL - GIBCO  779.1± 87.43 ND!  CMRL - CELLGRO  ND  Low Glucose DMEM  ND  ! !  43  Figure 3-5. Effects of insulin and IGF1 on AKT and ERK phosphorylation in HPDE cells. HPDE cells were serum-starved, and then treated with doses of exogenous insulin or IGF1 in non-supplemented KSF media for 5 minutes (A, B) and 24 hours (C, D). Activation of AKT and ERK is shown by fold change in mean phosphorylated -AKTS473 (A, C) and -ERK (B, D) levels relative to total AKT and ERK, respectively. The data above are representative of three independent biological replicates. (E) HPDE cells were harvested without serum starvation or treatments to measure the protein levels of IGF1R and insulin receptor (INSRβ) present. Each lane represents a biological replicate. (A-E) Beta-actin is the loading control.  44  ed  Figure 3-6. Inhibition of the AKT and ERK pathway in PANC1 cells. PANC1 cells were serum starved and treated with either DMSO, GW5074, U0126, Akti-1/2 and wortmannin (wort.) for 24 hours (A-B), 48 hours (C-D), or 5 days (E). Cell viability of PANC1 cells was expressed as the fold change of the treated relative to control (DMSO) (A, n = 5; E, n = 4). (B) The effect of 24 hours treatment  45  on cleaved capsase-3 protein levels in PANC1 cells. This is a representative immunoblot of three independent biological replicates. (C-D) The effects of different small molecule inhibitors on propidium incorporation (PI) in PANC1 cells were tracked and expressed as the fold change in the percent of PI and Hoechst co-positive cells over total Hoechst positive cells at that hour relative to t = 0 hour. Kinetic data were analyzed relative to serumfree control by two-way ANOVA (n = 3). (D) Area-under-curve (AUC) was also calculated from the kinetic data (C) and analyzed with one way ANOVA relative to SF (n = 3).  46  A  B  Figure 3-7. Effects of inhibitors on ERK and AKT levels in PANC1 cells. PANC1 cells were serum-starved and treated with either DMSO, GW5074, U0126, Akti-1/2 and wortmannin (wort.) for 1 hour (A) or 24 hour (B). The effects of small molecule inhibitors on the downstream effectors of ERK pathway (A) and AKT pathway (B).  47  Figure 3-8. Effects of insulin and IGF1 cellular viability in PANC1 cells. (A-C) PANC1 cells were serum-starved and treated with indicated doses of insulin and IGF1 and tracked over 48 hours. The change in total cell number was expressed as fold change in Hoechst positive cells at that time point relative to t = 0 hour. Kinetic data were analyzed relative to serum-free control by two-way ANOVA (n =  48  3). (C) Area-under-the-curve (AUC) values were calculated from the kinetic data (A), and analyzed with one way ANOVA relative to control “0”. (D-E) PANC1 cellular viability was also assessed by XTT at 24 hours (D) or 5 days of incubation (E), and expressed as fold change in mean absorbance treatment relative to control. (n = 6) “#” indicates a statistical significant when comparing insulin and IGF1 at 200 nM with a one-way ANOVA and post-hoc Bonferroni test p <0.05.  49  Figure 3-9. Effects of insulin or IGF1 on cell death in PANC1 cells. (A-B) Cell death of serum-starved and insulin or IGF1 treated PANC1 cells were tracked by propidium iodide and Hoechst co-positive cells over 48 hours. (n = 5) Kinetic data were analyzed relative to non-treated cells “0” using two-way ANOVA. (B) Area under the curve (AUC) was also calculated from the kinetic data, and analyzed with one way ANOVA relative to 0. (C) Cell lysates of serum-starved PANC1 cells were collected after 24 hours of insulin treatment, protein levels of cleaved caspase-3 was expressed as the fold change of cleaved caspase-3/ β-actin relative to 0 nM insulin. (n = 5)  50  Figure 3-10. Effects of insulin on AKT and ERK phosphorylation in PANC1 cells. PANC1 cells have been serum-starved for 24 hours then incubated with indicated doses insulin for 5 minutes (A-B) or 24 hours (C-D) at 37˚C. Protein lysates were collected and subjected to immunoblotting as described in the Material and Methods for pAKTS473, pERK, AKT, ERK, beta-actin, and beta-tubulin. β-actin, β-tubulin are loading control. Activation of AKT and ERK were shown by fold change in mean phosphorylated -AKTS473 (A, C) and -ERK (B, D) levels relative to total AKT and ERK, respectively. The immunoblots shown above are representative of independent biological replicates. (A-B) n =12, (C) n = 11 (D) n = 7.  51  Chapter 4: Discussion and conclusion Insulin and IGF1 are growth factors with putative roles in regulating proliferation, survival, development, and cancer142. Hyperinsulinemia has been identified as an independent risk factor for pancreatic cancer39,49,87,143. It has been proposed that elevated insulin levels may accelerate pancreatic cancer progression by promoting aberrant proliferation and resisting cell death85,87. To date, most of the reported proliferative effects of insulin have originated from animal studies or human metastatic cell lines66,85,110, thus not much is known about the action of insulin on normal human pancreatic exocrine and ductal cells. Furthermore, direct comparison on the effects of insulin and its associated mechanism across different stages of pancreatic cancer has not been reported. In this study, we attempted to decipher the role of insulin and its associated mechanism across a proposed in vitro human cell model of pancreatic cancer progression. The cell model consists of primary pancreatic ductal cells from healthy individuals, a near-normal HPDE cell line, and a metastatic pancreatic adenocarcinoma, PANC1 cell line (Figure 1-5).  4.1  Insulin signalling in primary human pancreatic ductal cells We observed that under serum-free conditions, propidium iodide (PI) incorporation in  primary human pancreatic sorted cells was increased when AKT was inhibited, but not when RAF1 was inhibited. PI incorporation is a marker of cell death, thus this may suggest that AKT may play a role in regulating primary pancreatic ductal cell survival. These preliminary data are in agreement with the current literature on the pro-survival role of AKT25. Specifically, AKT was found to regulate cell survival by mediating several of anti-apoptotic proteins such as BCL and FOXO in neurons, T-cells, B-cells and murine embryonic fibroblasts25. Furthermore, studies on the regulatory role of AKT on pancreatic ductal cell 52  differentiation have illustrated the importance of AKT in the pancreas. Through lineage tracing studies, Elghazi et al. demonstrated that expression of constitutively-active AKT enhanced the expansion of ductal cell populations from mature β-cells and mature acinar cells115. Moreover, sustained overexpression of AKT induced precancerous lesions and malignant transformation of the pancreas that recapitulates invasive pancreatic ductal adenocarcinoma115. As a follow-up to our findings, future studies using siRNA and other small molecule inhibitors targeting both AKT and RAF1 should be performed to confirm the role of RAF1 or AKT in mediating cell death of sorted primary cells. Collectively, our data hints that AKT may play a role in mediating cell death in primary pancreatic ductal cells. The acute treatment of unsorted cells with 200 nM insulin elicited a significant increase in ERK phosphorylation at T202 and Y204 relative to total ERK levels, which is indicative of ERK activation (Figure 3-2B). Moreover, there was a trend towards an increase in pAKTS473/AKT with acute treatment (Figure 3-2A); however this trend was not significant. Due to the variability in sample preparation and donor-to-donor variability, the current study examining the acute and chronic insulin effects on pAKTS473/AKT was underpowered, thus it is difficult to draw any conclusions on insulin’s effects on AKT activation. These data suggest that the unsorted primary ductal cells are responsive to insulin. Interestingly, although insulin elicited acute response in pERK/ERK levels at 200 nM in unsorted cells, insulin did not affect total cell number or PI incorporation over time in sorted cells under serum-free conditions (Figure 3-3). The change in total cell number over time is an indirect measure of proliferation. This suggests that under serum-free conditions, insulin alone may not be sufficient to promote proliferation or resist basal level cell death. These results are consistent with the data in a pilot study from our laboratory, where seven  53  days of insulin exposure did not increase bromodeoxyuridine (BrdU) incorporation of primary sorted ductal cells. The incorporation of BrdU into the DNA of proliferating cells is also another method to measure cellular proliferation106. Since the insulin-mediated ERK activation was not associated with changes in total cells number or PI incorporation, future studies should examine other possible biological consequences that can be associated with ERK activation after an acute insulin treatment. The discordance between ERK activation and cellular viability in primary cells may be due to use of different cell populations (sorted versus unsorted cells). A large fraction of the unsorted cell population contain cells with fibroblastic characteristics, thus the observed insulin stimulated ERK activation may reflect on the biological effects of insulin on fibroblastic-like cells. Additional studies investigating the effects of insulin on total cell number and cellular viability in the pure fibroblastic-like cell population may help elucidate the discrepancy between insulin-mediated ERK activation and lack of changes in total cell number over time. We did not observe significant changes in Hoechst-positive cells over time in Figure 3-1 and Figure 3-3 when compared with control. However, during the first four hours of the experiments, there was a slight increase in Hoechst-positive cells across all treatments conditions. According to the estimated proliferative rates of primary ductal cells at 0.5% per day144, the increase in Hoechst positive cells may be due to the ongoing process of dye incorporation. The incubation time for Hoechst incorporation were pre-determined in cell lines, this suggests future studies in primary cells may require longer dye incubation period prior to treatment conditions.  54  Our results suggest that insulin does not enhance total cell number over time or reduce PI incorporation in primary human pancreatic sorted cells under our experimental conditions. Insulin is a circulating hormone that is normally secreted in response to the rise of circulating blood glucose4-11,16. Thus, it is highly plausible that insulin alone is unable to promote sufficient cancer promoting effects, such as proliferation and pro-survival cues, to initiate pancreatic cancer. Instead, hyperinsulinemia may increase pancreatic cancer risk by enhancing proliferation and pro-survival cues in cells containing certain driver mutations. This is supported by several epidemiological studies demonstrating that individuals that were newly diagnosed with type 2 diabetes have a higher risk of pancreatic cancer than those with long standing diabetes49. Perhaps, in patients with normal pancreatic ductal cells, insulin would not promote cancer progression. However, in patients with existing lesions in their pancreatic ducts, the presence of elevated insulin may further accelerate their pancreatic cancer progression. The effects of insulin and the role of insulin signalling have not previously been investigated in primary human pancreatic ductal cells to the best of our knowledge. Our in vitro experiments on the effects of insulin in primary cells may argue against a prominent role for hyperinsulinemia during pancreatic cancer initiation and early cancer progression.  4.2  Insulin signalling in HPDE cells In primary human ductal cells, total cell number and PI incorporation were not altered  by insulin, thus we hypothesized that insulin may promote cell survival and/or growth in premalignant cells. Using a near normal human ductal cell line containing loss of RB gene, enhanced expression of EGFR, and loss of functional p53 pathway124,125, the role of  55  RAF/ERK and PI3K/AKT were compared. Moreover, we also attempted to examine the effects of insulin on cellular viability and PI incorporation in HPDE cells. Contrary to the results obtained from the primary sorted cells, the inhibition of AKT did not alter cellular viability or PI incorporation in HPDE cells (Figure 3-4). Instead, inhibition of both RAF1 and MEK activity attenuated the viability of HPDE cells (Figure 34A, C). Moreover, when compared to MEK inhibition, RAF1 inhibition by GW5074 was more potent in enhancing PI incorporation of HPDE cells, which is indicative of cell death (Figure 3-4B). The discrepancy between the regulators of cellular viability in primary cells versus HPDE cells may be due to several factors. First, according to Ouyang et al, they reported a higher success rate when culturing these cells with KSF media rather than RPMI media (non-insulin supplemented media)125. Thus, the basal proliferation of HPDE cells may be mediated by insulin in the KSF media. Secondly, immortalization of non-humanpapilloma-virus host cells by E6E7 oncogene has been shown to reduce growth-factormediated AKT signalling145,146. Moreover, the difference in cell death mediators of primary cells versus HPDE cells may be a consequence of the genetic alteration of the RB and p53 pathway, as well as overexpression of EGFR in HPDE cells124,125. In a recent report of immunoassay-based proteome profiling of 24 pancreatic cancer cell lines, Alhamdani et al. found that the overall protein levels of HPDE cells are more comparable to pancreatic cancer cell line than a normal control cell line, HUVEC, human umbilical vein endothelial cells147. This further emphasized that HPDE cells may deviate from near-normal state of pancreatic ductal cells. Further studies are required to directly compare the changes between primary ductal cells and the near-normal HPDE cells. Altogether, our data suggest the RAF/ERK pathway may play a role in mediating cellular viability in HPDE cells.  56  After establishing the importance of the RAF/ERK cascade in HPDE cells, we attempted to examine the effects of exogenous insulin on HPDE cells relative to IGF1. IGF1 has a well-established autocrine role in promoting tumorigenesis8-11,16,62,95,96,122. In this study, IGF1 was included to examine and compare the relative effects of insulin on cellular viability. Due to the scarcity of primary human cells, IGF1 treatments were not performed in the primary cells. Chronic and acute exposure of HPDE cells to insulin or IGF1 did not elicit any significant changes to pERK/ERK and pAKTS473/AKT levels (Figure 3-5A-D). The presence of ~ 700nM insulin in the media may desensitize the machinery for signal propagation in the insulin signalling pathway148-151. As shown by Fucini et al., insulinstimulated-CHO cells could not elicit a second insulin-mediated ERK activation 120 minutes after an acid wash151. The acid wash procedure used by the authors ensured a near-complete removal of insulin from the insulin receptors. The insulin-mediated ERK activation was only partial restored 240 minutes after the acid wash. Since insulin was unexpectedly included in the media in our experiment, the 6-hour starvation prior to treatment may not be sufficient to reset insulin signalling for further insulin stimulation. Our data suggests that RAF/ERK pathway may be one of the main mediators of cell viability in HPDE cells. Beyond our control, the effects of insulin could not be deciphered due to the presence of insulin in the KSF media. Thus further studies examining the effects of exogenous insulin in the absence of insulin-supplemented-media are required.  4.3  Insulin signalling in PANC1 cells To further address the hypothesis that hyperinsulinemia may accelerate pancreatic  cancer by promoting cellular viability in cancer cells, the role of PI3K/AKT and RAF1/MEK on cellular viability was examined on a metastatic and aggressive pancreatic adenocarcinoma 57  cell line, PANC1. PANC1 cell line is heterogeneous and express constitutively active KRAS oncogene and dysregulated p53 pathway126. Similar to HPDE cells, under serum-free conditions 24 hours inhibition of RAF1 by GW5074 (Figure 3-6A) and 5 days inhibition of RAF1 and MEK activity by GW5074 and U0126 reduced PANC1 cellular viability and enhanced cell death as indicated by PI incorporation (Figure 3-6B-E). Further in-depth characterization of cell death kinetics, measured by PI incorporation over time, revealed that after 10 hours of GW5074 treatment, PI incorporation significantly increased relative to control (Figure 3-6C). Treatment of U0126 elicited significant increase in PI incorporation relative to control only after 40 hours incubation. This suggests that RAF/ERK pathway may be important in mediating cellular viability of PANC1 cells under serum-free conditions. RAF1 is one of the immediate downstream effectors of the constitutively active KRAS in the ERK pathway, thus it was expected that inhibition of RAF1 activity would elicit a faster increase in PI incorporation. The later onset of PI incorporation by MEK inhibition is consistent with the fact that MEK is downstream of KRAS and RAF1. To confirm the role of PI3K/AKT pathway in regulating viability and cell death of PANC1 cells, small molecule inhibitors, wortmannin and Akt-1/2 were used. Inhibition of PI3K by 1 μM wortmannin did not affect cellular viability, PI incorporation, or cleaved caspase-3 levels when compared to DMSO control after 24 hours and 7 days of incubation (Figure 3-6). Our data were not consistent with the study by Yao et al. on the role of PI3K in PANC1 cells121. In that study, they reported an increase in DNA laddering in cells treated with 100 nM wortmannin for 48 hours and cells infected with dominant-negative subunit of PI3K. In this study, as expected wortmannin reduced insulin stimulated pAKT levels. The inconsistent results may reflect on the differences in experimental designs. Yao et al. did not  58  provide a vehicle control for the wortmannin treatment, or a control for the p110overexpression experiment, thus it is difficult to directly compare our results121. Consistent with our results, Ng et al. described an anti-proliferative and anti-survival effects of 50 - 400 nM wortmannin in the presence of 20 μM gemcitabine in pancreatic cancer cell lines, PK1 and PK8, as well as human pancreatic cancer xenografts in immune-deficient mice119,152. However, they also found that wortmannin alone was unable to induce cell death which was assessed by the loss of mitochondrial inner membrane potential and propidium iodide (PI) uptake in PK1 and PK8 cells119. Our findings revealed that PI3K may not mediate cellular viability or survival in PANC1 cells under our experimental settings. Consistent with the effects of wortmannin on PANC1 cells, 24 hours and 5 days AKT inhibition by Akti-1/2 did not affect PANC1 cellular viability as assessed by XTT assay. Whereas a significant but slight increase in PI positive cells was observed after 40 hours of AKT inhibition when compared to DMSO control (Figure 3-6C). Again, our results disagreed with the study performed in PANC1 cells by Yao et al. The overexpression of kinase-dead dominant negative AKT (DN-AKT) in their studies resulted in the attenuation of anchorage-dependent growth after 7 days of incubation, as well as inhibition of anchorage– independent growth after 14 days of incubation when compared to wildtype AKT control121. Moreover, they reported that expression of DN-AKT resulted in less apoptotic cell death when compared to wildtype AKT in PANC1 cells121. The discordance among the observations may be due to the method and site of inhibition. The kinase domain of AKT was specifically targeted by Yao et al., whereas the AKT inhibitor VIII used in this study specifically inhibits AKT by binding to the pleckstrin homology domain of AKT129-131. The binding of AKT inhibitor VIII (Akti-1/2) will prevent the release of the kinase domain to  59  obstruct the formation of the active AKT129-131. Furthermore, the process of overexpressing specific proteins may dysregulate endogenous cellular machinery, thus providing a possible explanation for the discordance among observations. Overexpression studies of wildtype AKT, AKT without the kinase domain, and/or the PH domain in PANC1 cells in the future to address the observed disagreement. In this study, our results suggest that under serum-free conditions, compared to the PI3K/AKT pathway, RAF/ERK pathway may be the prominent pathway mediating cellular viability. After investigating the role of the RAF/ERK cascade in PANC1 cells, we investigated the effects of exogenous insulin on cellular viability and PI incorporation in PANC1 cells relative to IGF1. As mentioned previously, IGF1 has a well-established autocrine role in promoting tumorigenesis8-11,15,55,86-88. Both insulin and IGF1 promoted cellular viability after 24 hours and 5 days of treatment in PANC1 cells. This is consistent with Fisher et al. and Nair et al. demonstrating the proliferative effects of insulin and IGF1 in pancreatic cancer cells110,118. The treatment of 20 nM insulin was the only condition that elicited significant enhancement of viability in both assays of cellular viability after 24 hours of incubation (Figure 3-8A-D). IGF1 was found to enhance cellular viability at both 24-hour and 5-day treatment (Figure 3-8D,E). However, IGF1 did not significantly affect total cell number over 48 hours of incubation. The sample size of the experiment in Figure 3-3A may be underpowered to detect small differences between IGF1-treated and non-treated conditions. Interestingly, Bonferroni post-hoc analysis of Figure 3-8E revealed that 200 nM insulin promoted a greater increase in cellular viability than 200 nM IGF1 after 5 days of incubation in PANC1 cells (indicated by #). The cellular viability assays used in this study may not allow an accurate examination of IGF1 proliferative effects. The use of three-  60  dimensional matrices has been demonstrated to increase the proliferative potency of IGF1 in vitro153. Future studies should address this discrepancy between the mitogenic potency of IGF1 in two-dimensional and three-dimensional cultures. Interestingly, both insulin and IGF1 did not reduce cell death under our serum-free experimental conditions. This is inconsistent with the recently published data112 investigating the importance of IR and IGF1R in mediating survival in both wild type and mutant KRAS murine pancreatic ductal epithelial cells. Under cycloheximide-induced stress, cell survival of pancreatic ductal epithelial cells was attenuated by small chemical inhibitors and shRNA targeting IR and IGF1R. This may be due to the difference in stress inducers where insulin or IGF1 may be more efficient in promoting survival in a greater stress-inducing environment. It is important to note that our data do not suggest that insulin is more potent than IGF1 in accelerating cancer-promoting effects in pancreatic cancer cells. Instead, it illustrates the potential tumor promoting capability of insulin, and it may be as potent as IGF1. To explore the potential mechanism associated with insulin-stimulated increase in total cells number and cellular viability, we examined the acute and chronic effects of insulin on AKT and ERK activation. Incubation of exogenous insulin in PANC1 cells elicited changes in AKT and ERK activation (Figure 3-10). While acute treatment of 200 nM insulin elicited AKT and ERK activation (Figure 3-10A-B), chronic insulin treatment at higher concentration (20 nM) led to AKT activation and not ERK activation (Figure 3-10C). This indicates that chronic exposure insulin at 20 nM can elicit sustained AKT activation in PANC1 cells. Moreover, this finding was also associated with increased total cell number and cellular viability at that concentration, but not with the reduction of PI incorporation in PANC1 cells (Figure 3-8 to 3-8). From this association, it may suggest that insulin may  61  enhance cellular viability via the AKT pathway. Sustained activation of AKT has been shown to be highly involved in carcinogenesis25-34. However, as mentioned previously, activation of AKT during carcinogenesis alone may not be sufficient to promote cancer progression107,108. In majority of KRAS mutated tumors, the dysregulation of PI3K pathway leading to sustained AKT activation are common. For example loss of tumor suppressor PTEN are found in 60% of pancreatic ductal cancers32, AKT1 is overexpressed in 20 - 70% of PDAC, and AKT2 are amplified in 10 - 20% PDAC27,29,32,33. In fact, activation of both KRAS and AKT are required for some cancer progression, such as glioblastoma104 and lung adenocarinomas103. Furthermore, several groups have hypothesized that chronic aberrant AKT activation during pancreatic carcinogenesis is required for the development of advanced metastatic pancreatic cancer26,34. Firstly, the neoplasm observed in tissue-specific transgenic murine model of KRASG12D did not recapitulate advanced pancreatic cancer in humans, suggesting that additional passenger mutations were required100. Secondly, the presence of KRAS alone was shown to elicit a strong irreversible oncogene-induced senescence109,154, and this anti-proliferative defense must be suppressed for the continuation of carcinogenesis. Oncogene-induced senescence is a process by which cells are placed under replicative arrest due to oncogene-mediated stress144. During this period, cells will undergo irreversible growth arrest without undergoing cell death. In the pancreatic cancer field, there is a paradox in which mutations in both KRAS and PI3K pathways have been observed143. Previously, the presence of mutations in both KRAS and AKT pathway may induce unfavourable senescence during cancer progression. Nonetheless, Kennedy et al. has suggested the presence of both oncogenes will help in bypassing of tumor barrier initiated by this oncogene-induced senescence143. This was supported by their recent work in murine  62  models, PTEN inactivation in KRASG12D murine models decreased the episodes of senescence seen in KRASG12D mice26, and enhanced the expression of proliferative markers such as Ki6734. They proposed that the addition of AKT-induced senescence is a requirement to bypass RAS-induced senescence to enable the occurrence of cancer and its progression to invasive adenocarcinomas. Our current in vitro study may suggest that insulin may promote cellular viability of cancer cells by promoting sustained AKT activation. Supplementary studies comparing the effects of insulin on cells not containing KRASG12D and cells containing KRASG12D are required to decipher whether and how insulin may participate in decreasing KRAS-induced senescence during pancreatic cancer.  4.4  Conclusion Evidence derived from epidemiological and clinical studies have suggested a  correlation between hyperinsulinemia and pancreatic cancer. To elucidate the etiology behind this correlation, we employed an in vitro model meant to represent three different stages of pancreatic cancer: primary ductal cells, immortalized HPDE cell line, and metastatic PANC1 cells. Inhibition of AKT enhanced cell death in primary cells, while inhibition of RAF1 and MEK elevated cell death in HPDE and PANC1 cells. Our data suggest that rewiring of cell survival pathways may occur during pancreatic carcinogenesis. As suggested earlier, the rewiring of pro-survival signal may reflect on the impaired RB and p53 pathway of HPDE and PANC1 cells. If the findings of this pilot study can be confirmed, this could be a potential avenue for exploring future therapeutic drug design that selectively targets pancreatic cells containing driver mutations. The role of insulin during cancer progression has been long debated. There is clinical evidence for and against the concept that high levels of insulin may have cancer-promoting 63  properties155-157. The present study has attempted to examine the actions of hyperinsulinemia on cellular viability across different stages of pancreatic cancer in vitro. If the cell models chosen in this study faithfully recapitulate the natural history of the disease, our experimental data may suggest that hyperinsulinemia may not play a role in initiating pancreatic cancer, but high levels of insulin may accelerate the cancer progression. Future studies elucidating how insulin may selectively enhance proliferation of cells containing driver mutations in vivo can provide valuable understanding in growth-factor-mediated cancer biology.  64  References 1. 2. 3. 4. 5. 6.  7. 8. 9.  10. 11. 12.  13. 14.  15.  16. 17.  18. 19.  Canada, S. Leading Causes of Death in Canada. Vol. 84-215-X2011001 (2008). Heron, M. Deaths: Leading Causes for 2006. National vital statistics reports. Hyattsville, MD: National Center for Health Statistics 58, 1-100 (2010). Organization, W.H. Description of the global burden of NCDs, their risk factors and determinants. Global status report on noncommunicable diseases 2010, 176 (2011). Biddinger, S.B. & Kahn, C.R. From mice to men: insights into the insulin resistance syndromes. Annual review of physiology 68, 123-58 (2006). Vollenweider, P. Insulin resistant states and insulin signaling. Clinical chemistry and laboratory medicine : CCLM / FESCC 41, 1107-19 (2003). Muoio, D.M. & Newgard, C.B. Mechanisms of disease: molecular and metabolic mechanisms of insulin resistance and beta-cell failure in type 2 diabetes. Nature reviews. Molecular cell biology 9, 193-205 (2008). Ashcroft, F.M. & Rorsman, P. Diabetes mellitus and the beta cell: the last ten years. Cell 148, 1160-71 (2012). Belfiore, A. & Malaguarnera, R. Insulin receptor and cancer. Endocrine-related cancer 18, R125-47 (2011). Belfiore, A., Frasca, F., Pandini, G., Sciacca, L. & Vigneri, R. Insulin receptor isoforms and insulin receptor/insulin-like growth factor receptor hybrids in physiology and disease. Endocrine reviews 30, 586-623 (2009). Belfiore, A. & Frasca, F. IGF and insulin receptor signaling in breast cancer. Journal of mammary gland biology and neoplasia 13, 381-406 (2008). Belfiore, A. The role of insulin receptor isoforms and hybrid insulin/IGF-I receptors in human cancer. Current pharmaceutical design 13, 671-86 (2007). Bailyes, E.M. et al. Insulin receptor/IGF-I receptor hybrids are widely distributed in mammalian tissues: quantification of individual receptor species by selective immunoprecipitation and immunoblotting. The Biochemical journal 327 ( Pt 1), 20915 (1997). Mosthaf, L. et al. Functionally distinct insulin receptors generated by tissue-specific alternative splicing. The EMBO journal 9, 2409-13 (1990). Blanquart, C., Achi, J. & Issad, T. Characterization of IRA/IRB hybrid insulin receptors using bioluminescence resonance energy transfer. Biochemical pharmacology 76, 873-83 (2008). Frasca, F. et al. Insulin receptor isoform A, a newly recognized, high-affinity insulinlike growth factor II receptor in fetal and cancer cells. Molecular and cellular biology 19, 3278-88 (1999). Frasca, F. et al. The role of insulin receptors and IGF-I receptors in cancer and other diseases. Archives of physiology and biochemistry 114, 23-37 (2008). White, M.F. & Yenush, L. The IRS-signaling system: a network of docking proteins that mediate insulin and cytokine action. Current topics in microbiology and immunology 228, 179-208 (1998). Sasaoka, T. & Kobayashi, M. The functional significance of Shc in insulin signaling as a substrate of the insulin receptor. Endocrine journal 47, 373-81 (2000). Skolnik, E.Y. et al. The function of GRB2 in linking the insulin receptor to Ras signaling pathways. Science 260, 1953-5 (1993).  65  20.  21. 22. 23.  24. 25. 26. 27.  28.  29.  30. 31.  32.  33.  34.  35.  36.  Simon, M.A., Dodson, G.S. & Rubin, G.M. An SH3-SH2-SH3 protein is required for p21Ras1 activation and binds to sevenless and Sos proteins in vitro. Cell 73, 169-77 (1993). Pearson, G. et al. Mitogen-activated protein (MAP) kinase pathways: regulation and physiological functions. Endocrine reviews 22, 153-83 (2001). Bonni, A. et al. Cell survival promoted by the Ras-MAPK signaling pathway by transcription-dependent and -independent mechanisms. Science 286, 1358-62 (1999). Krueger, J.S., Keshamouni, V.G., Atanaskova, N. & Reddy, K.B. Temporal and quantitative regulation of mitogen-activated protein kinase (MAPK) modulates cell motility and invasion. Oncogene 20, 4209-18 (2001). Backer, J.M. et al. Phosphatidylinositol 3'-kinase is activated by association with IRS-1 during insulin stimulation. The EMBO journal 11, 3469-79 (1992). Manning, B.D. & Cantley, L.C. AKT/PKB signaling: navigating downstream. Cell 129, 1261-74 (2007). Hill, R. et al. PTEN loss accelerates KrasG12D-induced pancreatic cancer development. Cancer research 70, 7114-24 (2010). Ruggeri, B.A., Huang, L., Wood, M., Cheng, J.Q. & Testa, J.R. Amplification and overexpression of the AKT2 oncogene in a subset of human pancreatic ductal adenocarcinomas. Molecular carcinogenesis 21, 81-6 (1998). Bellacosa, A. et al. Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas. International journal of cancer. Journal international du cancer 64, 2805 (1995). Cheng, J.Q. et al. Amplification of AKT2 in human pancreatic cells and inhibition of AKT2 expression and tumorigenicity by antisense RNA. Proceedings of the National Academy of Sciences of the United States of America 93, 3636-41 (1996). Stanger, B.Z. et al. Pten constrains centroacinar cell expansion and malignant transformation in the pancreas. Cancer cell 8, 185-95 (2005). Hutchinson, J., Jin, J., Cardiff, R.D., Woodgett, J.R. & Muller, W.J. Activation of Akt (protein kinase B) in mammary epithelium provides a critical cell survival signal required for tumor progression. Molecular and cellular biology 21, 2203-12 (2001). Asano, T. et al. The PI 3-kinase/Akt signaling pathway is activated due to aberrant Pten expression and targets transcription factors NF-kappaB and c-Myc in pancreatic cancer cells. Oncogene 23, 8571-80 (2004). Semba, S., Moriya, T., Kimura, W. & Yamakawa, M. Phosphorylated Akt/PKB controls cell growth and apoptosis in intraductal papillary-mucinous tumor and invasive ductal adenocarcinoma of the pancreas. Pancreas 26, 250-7 (2003). Kennedy, A.L. et al. Activation of the PIK3CA/AKT pathway suppresses senescence induced by an activated RAS oncogene to promote tumorigenesis. Molecular cell 42, 36-49 (2011). El-Serag, H.B., Hampel, H. & Javadi, F. The association between diabetes and hepatocellular carcinoma: a systematic review of epidemiologic evidence. Clinical gastroenterology and hepatology : the official clinical practice journal of the American Gastroenterological Association 4, 369-80 (2006). El-Serag, H.B., Richardson, P.A. & Everhart, J.E. The role of diabetes in hepatocellular carcinoma: a case-control study among United States Veterans. The American journal of gastroenterology 96, 2462-7 (2001). 66  37. 38. 39. 40.  41.  42.  43. 44.  45.  46.  47. 48.  49. 50.  51.  52.  53.  El-Serag, H.B., Tran, T. & Everhart, J.E. Diabetes increases the risk of chronic liver disease and hepatocellular carcinoma. Gastroenterology 126, 460-8 (2004). El-Serag, H.B. & Rudolph, K.L. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 132, 2557-76 (2007). Pisani, P. Hyper-insulinaemia and cancer, meta-analyses of epidemiological studies. Archives of physiology and biochemistry 114, 63-70 (2008). Larsson, S.C., Mantzoros, C.S. & Wolk, A. Diabetes mellitus and risk of breast cancer: a meta-analysis. International journal of cancer. Journal international du cancer 121, 856-62 (2007). Novosyadlyy, R. et al. Insulin-mediated acceleration of breast cancer development and progression in a nonobese model of type 2 diabetes. Cancer research 70, 741-51 (2010). Schairer, C. et al. Serum concentrations of IGF-I, IGFBP-3 and c-peptide and risk of hyperplasia and cancer of the breast in postmenopausal women. International journal of cancer. Journal international du cancer 108, 773-9 (2004). Kaaks, R. Nutrition, hormones, and breast cancer: is insulin the missing link? Cancer causes & control : CCC 7, 605-25 (1996). Irwin, M.L. et al. Fasting C-peptide levels and death resulting from all causes and breast cancer: the health, eating, activity, and lifestyle study. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 29, 47-53 (2011). Fierz, Y., Novosyadlyy, R., Vijayakumar, A., Yakar, S. & LeRoith, D. Insulinsensitizing therapy attenuates type 2 diabetes-mediated mammary tumor progression. Diabetes 59, 686-93 (2010). Elwing, J.E., Gao, F., Davidson, N.O. & Early, D.S. Type 2 diabetes mellitus: the impact on colorectal adenoma risk in women. The American journal of gastroenterology 101, 1866-71 (2006). Friberg, E., Orsini, N., Mantzoros, C.S. & Wolk, A. Diabetes mellitus and risk of endometrial cancer: a meta-analysis. Diabetologia 50, 1365-74 (2007). Nagamani, M. & Stuart, C.A. Specific binding and growth-promoting activity of insulin in endometrial cancer cells in culture. American journal of obstetrics and gynecology 179, 6-12 (1998). Chari, S.T. et al. Probability of pancreatic cancer following diabetes: a populationbased study. Gastroenterology 129, 504-11 (2005). Calle, E.E., Murphy, T.K., Rodriguez, C., Thun, M.J. & Heath, C.W., Jr. Diabetes mellitus and pancreatic cancer mortality in a prospective cohort of United States adults. Cancer causes & control : CCC 9, 403-10 (1998). Huxley, R., Ansary-Moghaddam, A., Berrington de Gonzalez, A., Barzi, F. & Woodward, M. Type-II diabetes and pancreatic cancer: a meta-analysis of 36 studies. British journal of cancer 92, 2076-83 (2005). Saydah, S.H., Loria, C.M., Eberhardt, M.S. & Brancati, F.L. Abnormal glucose tolerance and the risk of cancer death in the United States. American journal of epidemiology 157, 1092-100 (2003). Coughlin, S.S., Calle, E.E., Teras, L.R., Petrelli, J. & Thun, M.J. Diabetes mellitus as a predictor of cancer mortality in a large cohort of US adults. American journal of epidemiology 159, 1160-7 (2004). 67  54. 55. 56. 57.  58.  59.  60.  61.  62.  63.  64.  65.  66. 67.  68.  69.  Hanahan, D. & Weinberg, R.A. The hallmarks of cancer. Cell 100, 57-70 (2000). Hanahan, D. & Weinberg, R.A. Hallmarks of cancer: the next generation. Cell 144, 646-74 (2011). Huang, J. et al. Altered expression of insulin receptor isoforms in breast cancer. PloS one 6, e26177 (2011). Mulligan, A.M., O'Malley, F.P., Ennis, M., Fantus, I.G. & Goodwin, P.J. Insulin receptor is an independent predictor of a favorable outcome in early stage breast cancer. Breast cancer research and treatment 106, 39-47 (2007). Hellawell, G.O. et al. Expression of the type 1 insulin-like growth factor receptor is up-regulated in primary prostate cancer and commonly persists in metastatic disease. Cancer research 62, 2942-50 (2002). Law, J.H. et al. Phosphorylated insulin-like growth factor-i/insulin receptor is present in all breast cancer subtypes and is related to poor survival. Cancer research 68, 10238-46 (2008). Ioannou, N., Seddon, A.M., Dalgleish, A., Mackintosh, D. & Modjtahedi, H. Expression pattern and targeting of HER family members and IGF-IR in pancreatic cancer. Frontiers in bioscience : a journal and virtual library 17, 2698-724 (2012). Frittitta, L., Vigneri, R., Stampfer, M.R. & Goldfine, I.D. Insulin receptor overexpression in 184B5 human mammary epithelial cells induces a liganddependent transformed phenotype. Journal of cellular biochemistry 57, 666-9 (1995). Giorgino, F. et al. Overexpression of insulin receptors in fibroblast and ovary cells induces a ligand-mediated transformed phenotype. Molecular endocrinology 5, 452-9 (1991). Luttichau, H.R., Johnsen, A.H., Jurlander, J., Rosenkilde, M.M. & Schwartz, T.W. Kaposi sarcoma-associated herpes virus targets the lymphotactin receptor with both a broad spectrum antagonist vCCL2 and a highly selective and potent agonist vCCL3. The Journal of biological chemistry 282, 17794-805 (2007). Kaleko, M., Rutter, W.J. & Miller, A.D. Overexpression of the human insulinlike growth factor I receptor promotes ligand-dependent neoplastic transformation. Molecular and cellular biology 10, 464-73 (1990). Kato, H. et al. Paradoxical biological effects of overexpressed insulin-like growth factor-1 receptors in Chinese hamster ovary cells. Journal of cellular physiology 156, 145-52 (1993). Zhang, H. et al. Inhibition of cancer cell proliferation and metastasis by insulin receptor downregulation. Oncogene 29, 2517-27 (2010). Heuson, J.C. & Legros, N. Influence of insulin deprivation on growth of the 7,12dimethylbenz(a)anthracene-induced mammary carcinoma in rats subjected to alloxan diabetes and food restriction. Cancer research 32, 226-32 (1972). Gallagher, E.J. et al. Inhibiting PI3K reduces mammary tumor growth and induces hyperglycemia in a mouse model of insulin resistance and hyperinsulinemia. Oncogene 31, 3213-22 (2012). Hezel, A.F., Kimmelman, A.C., Stanger, B.Z., Bardeesy, N. & Depinho, R.A. Genetics and biology of pancreatic ductal adenocarcinoma. Genes & development 20, 1218-49 (2006).  68  70.  71.  72.  73.  74. 75. 76. 77.  78.  79. 80. 81. 82. 83.  84. 85. 86.  Rovasio, R.A. Development and Structure of the Pancreas. in Pancreatic Cancer, Vol. 1 (ed. Neoptolemos, J.L., Urrutia, R., Abbruzzese, J.L., Buchler, M.W.) 27-38 (Springer Science + Business Media, 2010). Basturk, O., Coban, I., Adsay, V.A. Pathological Classification and Biological Behavior of Pancreatic Neoplasia. in Pancreatic Cancer, Vol. 1 (ed. Neoptolemos, J.L., Urrutia, R., Abbruzzese, J.L., Buchler, M.W.) 40-64 (Springer Science + Business Media, 2010). Maitra, A. et al. Multicomponent analysis of the pancreatic adenocarcinoma progression model using a pancreatic intraepithelial neoplasia tissue microarray. Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc 16, 902-12 (2003). Mihaljevic, A.L., Michalski, C.W., Friess, H. & Kleeff, J. Molecular mechanism of pancreatic cancer--understanding proliferation, invasion, and metastasis. Langenbeck's archives of surgery / Deutsche Gesellschaft fur Chirurgie 395, 295-308 (2010). Longnecker, D.S. The quest for preneoplastic lesions in the pancreas. Archives of pathology & laboratory medicine 118, 226 (1994). Rozenblum, E. et al. Tumor-suppressive pathways in pancreatic carcinoma. Cancer research 57, 1731-4 (1997). Malumbres, M. & Barbacid, M. RAS oncogenes: the first 30 years. Nature reviews. Cancer 3, 459-65 (2003). Matrisian, L.M., Alzenberg, R., Rosenzwelg, A. . The alarming rise of pancreatic cancer deaths in the United States: Why we need to stem the tide today. in Pancreatic Cancer Action Network 1-12 (Manhattan Beach, CA, 2012). Neoptolemos, J.P. et al. Adjuvant therapy in pancreatic cancer: historical and current perspectives. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 14, 675-92 (2003). Bond-Smith, G., Banga, N., Hammond, T.M. & Imber, C.J. Pancreatic adenocarcinoma. BMJ 344, e2476 (2012). Alexakis, N. et al. Current standards of surgery for pancreatic cancer. The British journal of surgery 91, 1410-27 (2004). Neoptolemos, J.P. Adjuvant treatment of pancreatic cancer. European journal of cancer 47 Suppl 3, S378-80 (2011). Huang, P., Chubb, S., Hertel, L.W., Grindey, G.B. & Plunkett, W. Action of 2',2'difluorodeoxycytidine on DNA synthesis. Cancer research 51, 6110-7 (1991). Bowker, S.L., Majumdar, S.R., Veugelers, P. & Johnson, J.A. Increased cancerrelated mortality for patients with type 2 diabetes who use sulfonylureas or insulin. Diabetes care 29, 254-8 (2006). Currie, C.J., Poole, C.D. & Gale, E.A. The influence of glucose-lowering therapies on cancer risk in type 2 diabetes. Diabetologia 52, 1766-77 (2009). Li, D., Yeung, S.C., Hassan, M.M., Konopleva, M. & Abbruzzese, J.L. Antidiabetic therapies affect risk of pancreatic cancer. Gastroenterology 137, 482-8 (2009). Evans, J.M., Donnelly, L.A., Emslie-Smith, A.M., Alessi, D.R. & Morris, A.D. Metformin and reduced risk of cancer in diabetic patients. BMJ 330, 1304-5 (2005).  69  87.  88.  89. 90.  91. 92.  93.  94. 95. 96.  97.  98. 99. 100. 101. 102.  103.  Bodmer, M., Becker, C., Meier, C., Jick, S.S. & Meier, C.R. Use of antidiabetic agents and the risk of pancreatic cancer: a case-control analysis. The American journal of gastroenterology 107, 620-6 (2012). Sadeghi, N., Abbruzzese, J.L., Yeung, S.C., Hassan, M. & Li, D. Metformin use is associated with better survival of diabetic patients with pancreatic cancer. Clinical cancer research : an official journal of the American Association for Cancer Research 18, 2905-12 (2012). Dowling, R.J., Goodwin, P.J. & Stambolic, V. Understanding the benefit of metformin use in cancer treatment. BMC medicine 9, 33 (2011). Hadad, S. et al. Evidence for biological effects of metformin in operable breast cancer: a pre-operative, window-of-opportunity, randomized trial. Breast cancer research and treatment 128, 783-94 (2011). Hosono, K. et al. Metformin suppresses colorectal aberrant crypt foci in a short-term clinical trial. Cancer prevention research 3, 1077-83 (2010). Bergmann, U., Funatomi, H., Yokoyama, M., Beger, H.G. & Korc, M. Insulin-like growth factor I overexpression in human pancreatic cancer: evidence for autocrine and paracrine roles. Cancer research 55, 2007-11 (1995). Bergmann, U., Funatomi, H., Kornmann, M., Beger, H.G. & Korc, M. Increased expression of insulin receptor substrate-1 in human pancreatic cancer. Biochemical and biophysical research communications 220, 886-90 (1996). Kornmann, M. et al. Enhanced expression of the insulin receptor substrate-2 docking protein in human pancreatic cancer. Cancer research 58, 4250-4 (1998). Dong, X. et al. Insulin-like growth factor axis gene polymorphisms and clinical outcomes in pancreatic cancer. Gastroenterology 139, 464-73, 473 e1-3 (2010). Nakao, M. et al. Interaction between IGF-1 polymorphisms and overweight for the risk of pancreatic cancer in Japanese. International journal of molecular epidemiology and genetics 2, 354-66 (2011). Willis, J.A. et al. A replication study and genome-wide scan of single-nucleotide polymorphisms associated with pancreatic cancer risk and overall survival. Clinical cancer research : an official journal of the American Association for Cancer Research 18, 3942-51 (2012). Miyatsuka, T. et al. Persistent expression of PDX-1 in the pancreas causes acinar-toductal metaplasia through Stat3 activation. Genes & development 20, 1435-40 (2006). Liu, S.H. et al. PDX-1: demonstration of oncogenic properties in pancreatic cancer. Cancer 117, 723-33 (2011). Hingorani, S.R. et al. Preinvasive and invasive ductal pancreatic cancer and its early detection in the mouse. Cancer cell 4, 437-50 (2003). Kim, H.S., Kim, G.Y., Lim, S.J. & Kim, Y.W. Loss of Raf-1 kinase inhibitory protein in pancreatic ductal adenocarcinoma. Pathology 42, 655-60 (2010). Song, S.P. et al. Reduced expression of Raf kinase inhibitor protein correlates with poor prognosis in pancreatic cancer. Clinical & translational oncology : official publication of the Federation of Spanish Oncology Societies and of the National Cancer Institute of Mexico (2012). Kim, H.S., Kim, G.Y., Lim, S.J., Park, Y.K. & Kim, Y.W. Reduced expression of Raf-1 kinase inhibitory protein is a significant prognostic marker in patients with gallbladder carcinoma. Human pathology 41, 1609-16 (2010). 70  104.  105. 106. 107.  108. 109.  110.  111. 112.  113. 114.  115. 116.  117. 118.  119.  Alejandro, E.U. & Johnson, J.D. Inhibition of Raf-1 alters multiple downstream pathways to induce pancreatic beta-cell apoptosis. The Journal of biological chemistry 283, 2407-17 (2008). Alejandro, E.U. et al. Acute insulin signaling in pancreatic beta-cells is mediated by multiple Raf-1 dependent pathways. Endocrinology 151, 502-12 (2010). Beith, J.L., Alejandro, E.U. & Johnson, J.D. Insulin stimulates primary beta-cell proliferation via Raf-1 kinase. Endocrinology 149, 2251-60 (2008). Okudela, K. et al. K-ras gene mutation enhances motility of immortalized airway cells and lung adenocarcinoma cells via Akt activation: possible contribution to noninvasive expansion of lung adenocarcinoma. The American journal of pathology 164, 91-100 (2004). Holland, E.C. et al. Combined activation of Ras and Akt in neural progenitors induces glioblastoma formation in mice. Nature genetics 25, 55-7 (2000). Collins, M.A. et al. Oncogenic Kras is required for both the initiation and maintenance of pancreatic cancer in mice. The Journal of clinical investigation 122, 639-53 (2012). Fisher, W.E., Boros, L.G. & Schirmer, W.J. Insulin promotes pancreatic cancer: evidence for endocrine influence on exocrine pancreatic tumors. The Journal of surgical research 63, 310-3 (1996). Tomizawa, M. et al. Insulin-like growth factor-I receptor in proliferation and motility of pancreatic cancer. World journal of gastroenterology : WJG 16, 1854-8 (2010). Appleman, V.A., Ahronian, L.G., Cai, J., Klimstra, D.S. & Lewis, B.C. KRASG12Dand BRAFV600E-Induced Transformation of Murine Pancreatic Epithelial Cells Requires MEK/ERK-Stimulated IGF1R Signaling. Molecular cancer research : MCR (2012). Bergmann, U. et al. Insulin-like growth factor II activates mitogenic signaling in pancreatic cancer cells via IRS-1. International journal of oncology 9, 487-92 (1996). Dey, P. et al. Overexpression of ecdysoneless in pancreatic cancer and its role in oncogenesis by regulating glycolysis. Clinical cancer research : an official journal of the American Association for Cancer Research 18, 6188-98 (2012). Elghazi, L. et al. Regulation of pancreas plasticity and malignant transformation by Akt signaling. Gastroenterology 136, 1091-103 (2009). Erbel, S. et al. Proliferation of colo-357 pancreatic carcinoma cells and survival of patients with pancreatic carcinoma are not altered by insulin glargine. Diabetes care 31, 1105-11 (2008). Ishiwata, T. et al. Altered expression of insulin-like growth factor II receptor in human pancreatic cancer. Pancreas 15, 367-73 (1997). Nair, P.N., De Armond, D.T., Adamo, M.L., Strodel, W.E. & Freeman, J.W. Aberrant expression and activation of insulin-like growth factor-1 receptor (IGF-1R) are mediated by an induction of IGF-1R promoter activity and stabilization of IGF-1R mRNA and contributes to growth factor independence and increased survival of the pancreatic cancer cell line MIA PaCa-2. Oncogene 20, 8203-14 (2001). Ng, S.S.W., Tsao, M.S., Chow, S. & Hedley, D.W. Inhibition of phosphatidylinositide 3-kinase enhances gemcitabine-induced apoptosis in human pancreatic cancer cells. Cancer research 60, 5451-5 (2000).  71  120.  121. 122.  123.  124.  125.  126.  127.  128. 129.  130. 131. 132.  133. 134. 135.  136.  Qian, J., Niu, J., Li, M., Chiao, P.J. & Tsao, M.S. In vitro modeling of human pancreatic duct epithelial cell transformation defines gene expression changes induced by K-ras oncogenic activation in pancreatic carcinogenesis. Cancer research 65, 5045-53 (2005). Yao, Z. et al. Role of Akt in growth and survival of PANC-1 pancreatic cancer cells. Pancreas 24, 42-6 (2002). Wu, Y. et al. Reduced circulating insulin-like growth factor I levels delay the onset of chemically and genetically induced mammary tumors. Cancer research 63, 4384-8 (2003). Furukawa, T. et al. Long-term culture and immortalization of epithelial cells from normal adult human pancreatic ducts transfected by the E6E7 gene of human papilloma virus 16. The American journal of pathology 148, 1763-70 (1996). Liu, N., Furukawa, T., Kobari, M. & Tsao, M.S. Comparative phenotypic studies of duct epithelial cell lines derived from normal human pancreas and pancreatic carcinoma. The American journal of pathology 153, 263-9 (1998). Ouyang, H. et al. Immortal human pancreatic duct epithelial cell lines with near normal genotype and phenotype. The American journal of pathology 157, 1623-31 (2000). Lieber, M., Mazzetta, J., Nelson-Rees, W., Kaplan, M. & Todaro, G. Establishment of a continuous tumor-cell line (panc-1) from a human carcinoma of the exocrine pancreas. International journal of cancer. Journal international du cancer 15, 741-7 (1975). Kaaks, R. et al. Serum C-peptide, insulin-like growth factor (IGF)-I, IGF-binding proteins, and colorectal cancer risk in women. Journal of the National Cancer Institute 92, 1592-600 (2000). Lackey, K. et al. The discovery of potent cRaf1 kinase inhibitors. Bioorganic & medicinal chemistry letters 10, 223-6 (2000). Calleja, V., Laguerre, M., Parker, P.J. & Larijani, B. Role of a novel PH-kinase domain interface in PKB/Akt regulation: structural mechanism for allosteric inhibition. PLoS biology 7, e17 (2009). Lindsley, C.W. et al. Allosteric Akt (PKB) inhibitors: discovery and SAR of isozyme selective inhibitors. Bioorganic & medicinal chemistry letters 15, 761-4 (2005). Logie, L. et al. Characterization of a protein kinase B inhibitor in vitro and in insulintreated liver cells. Diabetes 56, 2218-27 (2007). Zhao, H. et al. Akt contributes to neuroprotection by hypothermia against cerebral ischemia in rats. The Journal of neuroscience : the official journal of the Society for Neuroscience 25, 9794-806 (2005). Goueli, S.A., Hsiao, K., Lu, T., Simpson, D. U0126: a Novel, Selective and Potent Inhibitor of MAP Kinase Kinase (MEK). Promega Notes 69(1998). Powis, G. et al. Wortmannin, a potent and selective inhibitor of phosphatidylinositol3-kinase. Cancer research 54, 2419-23 (1994). Wymann, M.P. et al. Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction. Molecular and cellular biology 16, 1722-33 (1996). Bonner-Weir, S. et al. The pancreatic ductal epithelium serves as a potential pool of progenitor cells. Pediatric diabetes 5 Suppl 2, 16-22 (2004). 72  137.  138. 139.  140.  141.  142.  143.  144.  145.  146.  147. 148.  149.  150. 151.  152.  Hoesli, C.A., Johnson, J.D. & Piret, J.M. Purified human pancreatic duct cell culture conditions defined by serum-free high-content growth factor screening. PloS one 7, e33999 (2012). Lan, M.S., Hollingsworth, M.A. & Metzgar, R.S. Polypeptide core of a human pancreatic tumor mucin antigen. Cancer research 50, 2997-3001 (1990). Wu, M.C., Arimura, G.K. & Yunis, A.A. Mechanism of sensitivity of cultured pancreatic carcinoma to asparaginase. International journal of cancer. Journal international du cancer 22, 728-33 (1978). Marshall, N.J., Goodwin, C.J. & Holt, S.J. A critical assessment of the use of microculture tetrazolium assays to measure cell growth and function. Growth regulation 5, 69-84 (1995). Li, W., Yuan, Y., Huang, L., Qiao, M. & Zhang, Y. Metformin alters the expression profiles of microRNAs in human pancreatic cancer cells. Diabetes research and clinical practice 96, 187-95 (2012). Novosyadlyy, R. & Leroith, D. Insulin-like growth factors and insulin: at the crossroad between tumor development and longevity. The journals of gerontology. Series A, Biological sciences and medical sciences 67, 640-51 (2012). Osorio-Costa, F., Rocha, G.Z., Dias, M.M. & Carvalheira, J.B. Epidemiological and molecular mechanisms aspects linking obesity and cancer. Arquivos brasileiros de endocrinologia e metabologia 53, 213-26 (2009). Githens, S. The pancreatic duct cell: proliferative capabilities, specific characteristics, metaplasia, isolation, and culture. Journal of pediatric gastroenterology and nutrition 7, 486-506 (1988). Pim, D., Massimi, P., Dilworth, S.M. & Banks, L. Activation of the protein kinase B pathway by the HPV-16 E7 oncoprotein occurs through a mechanism involving interaction with PP2A. Oncogene 24, 7830-8 (2005). Menges, C.W., Baglia, L.A., Lapoint, R. & McCance, D.J. Human papillomavirus type 16 E7 up-regulates AKT activity through the retinoblastoma protein. Cancer research 66, 5555-9 (2006). Alhamdani, M.S. et al. Immunoassay-based proteome profiling of 24 pancreatic cancer cell lines. Journal of proteomics 75, 3747-59 (2012). Cherniack, A.D., Klarlund, J.K., Conway, B.R. & Czech, M.P. Disassembly of Sonof-sevenless proteins from Grb2 during p21ras desensitization by insulin. The Journal of biological chemistry 270, 1485-8 (1995). Klarlund, J.K., Cherniack, A.D. & Czech, M.P. Divergent mechanisms for homologous desensitization of p21ras by insulin and growth factors. The Journal of biological chemistry 270, 23421-8 (1995). Waters, S.B., Yamauchi, K. & Pessin, J.E. Insulin-stimulated disassociation of the SOS-Grb2 complex. Molecular and cellular biology 15, 2791-9 (1995). Fucini, R.V., Okada, S. & Pessin, J.E. Insulin-induced desensitization of extracellular signal-regulated kinase activation results from an inhibition of Raf activity independent of Ras activation and dissociation of the Grb2-SOS complex. The Journal of biological chemistry 274, 18651-8 (1999). Tahir, S.K. et al. A-204197, a new tubulin-binding agent with antimitotic activity in tumor cell lines resistant to known microtubule inhibitors. Cancer research 61, 54805 (2001). 73  153.  154. 155.  156. 157.  Dunn, S.E., Hardman, R.A., Kari, F.W. & Barrett, J.C. Insulin-like growth factor 1 (IGF-1) alters drug sensitivity of HBL100 human breast cancer cells by inhibition of apoptosis induced by diverse anticancer drugs. Cancer research 57, 2687-93 (1997). Collado, M. et al. Tumour biology: senescence in premalignant tumours. Nature 436, 642 (2005). Davidson, J.K. & Eddleman, E.E. Insulin resistance; review of the literature and report of a case associated with carcinoma of the pancreas. A.M.A. archives of internal medicine 86, 727-42 (1950). Renehan, A.G. et al. Diabetes and cancer (2): evaluating the impact of diabetes on mortality in patients with cancer. Diabetologia 55, 1619-32 (2012). Johnson, J.A. et al. Diabetes and cancer (1): evaluating the temporal relationship between type 2 diabetes and cancer incidence. Diabetologia 55, 1607-18 (2012).  74  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.24.1-0073475/manifest

Comment

Related Items