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Role of tumour suppressor ING3 in melanoma pathogenesis Wang, Yemin 2009

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ROLE OF TUMOUR SUPPRESSOR ING3 IN MELANOMA PATHOGENESIS by YEMIN WANG B.Sc., Nanjing University, 2001 M.Sc., Nanjing University, 2003  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY  in  THE FACULTY OF GRADUATE STUDIES (Experimental Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  January 2009 © YEMIN WANG, 2009  ABSTRACT The type II tumour suppressor ING3 has been shown to modulate transcription, cell cycle control, and apoptosis. To investigate the putative role of ING3 in melanoma development, we examined the expression of ING3 in 58 dysplastic nevi, 114 primary melanomas,  and  50  metastatic  melanomas  with  tissue  microarray  and  immunohistochemistry. Overall ING3 was reduced in metastatic melanomas compared with dyslastic nevi and primary melanomas. Reduced nuclear ING3 staining also correlated with melanoma progression, increased cytoplasmic ING3 level, tumour location at sun-exposed sites, and a poorer disease-specific 5-year survival of patients with primary melanoma. Multivariate analysis revealed that nuclear ING3 staining can independently predict patient outcome in primary melanomas. In melanoma cells, ING3 expression was rapidly induced by UV irradiation. Using stable clones of melanoma cells overexpressing ING3, we showed that ING3 significantly promoted UV-induced apoptosis. Unlike its homologues ING1b and ING2, ING3-enhanced apoptosis upon UV irradiation was independent of functional p53. Furthermore, ING3 did not affect the expression of mitochondrial proteins but increased the cleavage of Bid and caspases. Moreover, ING3 upregulated Fas expression and ING3mediated apoptosis was blocked by inhibiting caspase-8 or Fas activation. Knockdown of ING3 expression decreased UV-induced apoptosis remarkably, suggesting that ING3 plays a crucial role in cellular response to UV radiation. To explore how ING3 is deregulated in advanced melanomas, we examined ING3 expression in metastatic melanoma cells and found that ING3 was downregulated due to a rapid protein turnover in these cells. Further studies demonstrated that ING3 undergoes degradation via the ubiquitin-proteasome pathway. We also demonstrate that ING3 interacts with the SCF (Skp1/Cul1/Roc1/Skp2) E3 ligase complex. Knockdown of Cul1 or Skp2 significantly stabilized ING3 in melanoma cells. In addition, lysine residue 96 is essential for ING3 ubiquitination as its mutation to arginine completely abrogated ING3 turnover and enhanced ING3-stimulatd apoptosis upon  ii  UV irradiation. Taken together, ING3 is deregulated in melanomas as a result of both nucleus-to-cytoplasm shift and rapid degradation. The level of ING3 in the nucleus may be an important marker for human melanoma progression and prognosis. Restoration of ING3 expression significantly sensitizes melanoma cells to UV radiation through the activation of Fas/caspase-8 pathway.  iii  TABLE OF CONTENTS  Abstract……………………………………..……………………………………………… ii Table of Contents……………………… …………………………………………………. iv List of Tables……………………………………………………………………………… vii List of Figures…………………………………………………………………………… viii Abbreviations ………………….………………………………………………………...... ix Acknowledgements ……………………………………………………………………….. xi Dedication ………………………………………………………………………………… xii CHAPTER 1 GENERAL INTRODUCTION…………………………………………….1 1.1  1.2  1.3  1.4  1.5  Cutaneous Melanoma ……………………………………………………………. ..1 1.1.1 The Biology of Melanocytes …………..…………………...……………… ..1 1.1.2 Epidemiology of Melanoma……….………………………………………... 3 1.1.3 UV Exposure and Melanoma………………………………………………...4 1.1.4 Staging and Subtypes of Melanoma…..…………………….………………..5 1.1.5 Molecular Progression of Melanoma …………………………………....... ..8 1.1.6 Treatment of Melanoma…………………………………...………………..10 Apoptosis ………….………………………….……………………………………12 1.2.1 Apoptosis versus Necrosis ……………………………………………........ 12 1.2.2 Apoptotic Pathways…………..…………………………………................. 13 1.2.3 UV-induced Apoptosis……………………………………………………...18 Protein Degradation………………………….………………………………… ...19 1.3.1 Pathways of Protein Degradation…………………………………………...19 1.3.2 Ubiquitin-dependent Proteasome Degradation…………………………….. 22 1.3.3 SCFSkp2 E3 Ligase Complex-mediated Protein Degradation.…………........ 23 ING Family Proteins…………………………………………………………….... 24 1.4.1 Gene Location and Splicing Variants……………………………………… 25 1.4.2 Structure of ING Family ………………………………………………....... 25 1.4.3 ING Proteins and Chromatin Regulation…………………………………... 30 1.4.4 Biological Functions of ING Proteins………………………………………31 1.4.5 ING Family Proteins and Cancer…………………………………………... 35 1.4.6 Novel Tumour Suppressor ING3……………………………………….... .. 36 Objectives….…………………………………………………………………….... 37  CHAPTER 2 MATERIALS AND METHODS ………………………………………... 39 2.1 2.2 2.3 2.4 2.5  Construction of Tissue Microarray (TMA) …………………………………….. 39 Immunohistochemistry ………………………………………………………… .. 40 Evaluation of TMA Immunostaining ………………………………………….... 40 Statistical Analysis of TMA Immunostaining ………………………………….. 41 Cell Lines and Cell Culture………………………………………………………. 41 iv  2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 2.15 2.16 2.17 2.18 2.19 2.20 2.21 2.22 2.23  Plasmids and Transfection……………………………………………………….. 42 siRNA and Transfection………………………………………………………….. 43 Antibodies…………………………………………………….…………………… 43 Generation of ING3 Stable Clones………………………………………………. 44 UV Irradiation…………………………………………………….………………. 44 Light Microscopy ………………………………………………….……………... 45 Cell Survival Assay…………………………………………………….…………. 45 Flow Cytometry…………………………………………………….…………… .. 45 Measurement of Caspase-8 Activity……………………………………………... 46 Hoechst Staining………………………………………………………………….. 47 Western Blot Analysis……………………………………………………………. 47 Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) ………………. 48 Immunoprecipitation……………………………………………………………... 49 Immunofluorescent Staining……………………………………………………... 49 Isolation of Proteasomes…………………………………………………………..50 In Vitro Degradation Assay…………………………………………………….. .. 50 In Vivo Ubiquitination Assay…………………………………………………….. 51 Statistical Analysis for in vitro Studies………………………………………….. 51  CHAPTER 3 DEREGULATION OF ING3 IN MELANOMA……………………….. 52 3.1 3.2  3.3  Rationale and Hypothesis………………………………………………………… 52 Results……………………………………………………………………………... 52 3.2.1 Correlation between ING3 Staining and Melanoma Progression………….. 52 3.2.2 Reduced Nuclear ING3 Staining Correlates with Tumour Location………. 56 3.2.3 Reduced Nuclear ING3 Staining Correlates with 5-year Patient Survival… 61 3.2.4 Nuclear ING3 Staining Independently Predicts 5-year Patient Survival…... 63 Discussion …………………………………….……………………………………65  CHAPTER 4 ING3 ENHANCES UV-INDUCED APOPTOSIS OF MELANOMA CELLS…………………….…………………………….... 69 4.1 4.2  4.3  Rationale and Hypothesis…………………….…………………………………... 69 Results…………………….……………………………………………………….. 69 4.2.1 ING3 Is Induced by DNA Damaging Agents…………….……………….. 69 4.2.2 ING3 Promotes UV-induced Apoptosis……………….…………………... 72 4.2.3 ING3 Mediates UV-induced Apoptosis Independently of p53……………..79 4.2.4 ING3 Mediates UV-induced Apoptosis via Death Receptor Pathway…….. 82 4.2.5 ING3 Induces Fas Expression………….………………….………….……. 83 Discussion…………………….…………………………………………………….87  CHAPTER 5 RAPID DEGRADATION OF ING3 REGULATES ITS TUMOUR SUPPRESSIVE ROLES IN MELANOMA CELLS………………….. ...91 5.1 5.2  Rationale and Hypothesis…………………….…………………………………...91 Results…………………….……………………………………………………... ...91 5.2.1 ING3 Is Rapidly Degraded in Melanoma Cells….………………………. ...91 5.2.2 ING3 Is Stabilized upon Proteasome Inhibitor Treatment…..……………...95 5.2.3 ING3 Is Degraded by the Ubiquitin-proteasome System….………………..99 5.2.4 Lysine Residue 96 Is Required for ING3 Ubiquitination and Degradation.102 5.2.5 SCFSkp2 E3 ligase Complex Mediates ING3 Ubiquitination and Degradation….………………….……………….……105 v  5.3  5.2.6 K96R Mutation Enhances the Tumour Suppressive Roles of ING3 .……..111 Discussion ………………….……………………………………………………..113  CHAPTER 6 GENERAL CONCLUSIONS ..……………….…………………………117 References………………………….……………………………………….…………….120 Appendix...……………………………………………………………………………….. 145 List of Publications…………………….…………………………………………145  vi  LIST OF TABLES Table 3.1 Table 3.2 Table 3.3  Clinicopathologic features of 114 primary melanomas…….……………… 58 Nuclear ING3 staining and clinicopathologic characteristics of primary melanomas……………….……………….………………………………... 59 Multivariate Cox regression analysis of ING3 nuclear staining in 114 cases of primary melanoma…….……….……………………………..........64  vii  LIST OF FIGURES Figure 1.1 Figure 1.2 Figure 1.3 Figure 1.4 Figure 1.5 Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7 Figure 4.8 Figure 4.9 Figure 4.10 Figure 4.11 Figure 5.1 Figure 5.2 Figure 5.3 Figure 5.4 Figure 5.5 Figure 5.6 Figure 5.7 Figure 5.8 Figure 5.9 Figure 5.10 Figure 5.11 Figure 5.12  Stages of melanoma progression….………………….……………….……...7 Overview of apoptotic pathways in cells………………….……………….. 17 The ubiquitin-mediated proteasome degradation system….………………. 21 Structure and signaling features of ING proteins….………………….….…29 Alignment of ING3 proteins in different species……………….….............. 38 Representative images of ING3 immunohistochemical staining in human melanocytic lesions ……………….…....……………….….……………… 54 Correlation between ING3 staining and melanoma progression…….…….. 55 Correlation between ING3 staining and the location of melanomas...…..… 60 Correlation between the nuclear ING3 staining and 5-year survival in patients with primary melanomas..….…....……………….….…………. 62 ING3 was induced by DNA damage agents in melanoma cells………….... 71 ING3 was stably overexpressed in melanoma cells……………….….......... 73 ING3 promoted UV-induced cell death in melanoma cells………………... 74 ING3 enhances UV-induced apoptosis in melanoma cells……………….... 76 ING3 enhances the UV-triggered activation of caspases in melanoma cells.77 Knockdown of ING3 inhibited UV-induced apoptosis in melanoma cells... 78 ING3 promoted UV-induced apoptosis independent of p53 in MMRU cells..….…....…………….…...…………….…...……………... 80 ING3 promoted UV-induced apoptosis in p53 wt and null HCT116 cells… 81 Blockage of caspase-8 activation abolished ING3-mediated apoptosis in melanoma cells……………….…...…………….…...…………….…..........84 Blockage of Fas activation abolished ING3-mediated apoptosis in melanoma cells…………….…...…………….…....……………….….... 85 ING3 induced Fas expression in melanoma cells………….………….….... 86 Aberrant ING3 expression in melanoma cells…………………….…....... ...93 Rapid degradation of ING3 in melanoma cells……………….….................94 ING3 accumulates in the presence of proteasome inhibitors…………….....96 ING3 is stabilized in the presence of proteasome inhibitors……………......98 ING3 is degraded by the ubiquitin-dependent pathway…………...…........101 96-111 residues are crucial for ING3 ubiquitination and degradation…….103 K96 residue is essential for ING3 ubiquitination and degradation………. .104 ING3 interacts with SCF E3 ligase complex……………….…................. .106 Cul1 is required for ING3 ubiquitination and degradation....…................. .107 ING3 interacts with the F-box protein Skp2......................………………. .109 Skp2 mediates the ubiquitination and degradation of ING3.....………….. .110 Blockage of ING3 degradation stimulates its tumour suppressive functions……………….…....…………….…...…………….…...………. .112  viii  ABBREVIATIONS ACTH α-MSH Apaf-1 APC Bax Bid BH Caspase CDK CHX DN-caspase-8 DTIC FACS FADD Fas-L GAPDH HAT HDAC HNSCC IAP ING IP LOH LZL MAPK MC1R MITF MMP NCR NER NF-kB NLS NuA4 PAGE PARP PBS PCNA PCR PHD PI p-NA PVDF RGP RING RT-PCR SCF SDS siRNA  Adrenocorticotropic hormone α-melanocyte stimulating hormone Apoptotic protease activating factor-1 Anaphase promoting complex Bcl-2-associated X protein Bcl-2 interacting domain Bcl-2 homology Cysteinyl aspartate-specific protease Cyclin-dependent kinase Cycloheximide Dominant negative caspase-8 Dacarbazine Fluorescence-activated cell sorting Fas-associating death domain Fas ligand Glyceraldehyde 3-phosphate dehydrogenase Histone acetytransferase Histone deacetylase Human head and neck squamous cell carcinoma Inhibitor of apoptosis Inhibitor of growth Immunoprecipitation Loss of heterozygosity leucine-zipper like Mitogen-activated protein kinase Melanocortin-1 receptor Microphthalmia transcription factor Matrix metalloproteinase Novel chromatin regulatory Nucleotide excision repair Nuclear factor kappa B Nuclear localization signal Nucleosomal acetyltransferase of histone H4 Polyacrylamide gel electrophoresis Poly(ADP-ribose) polymerase Phosphate buffered saline Proliferating cell nuclear antigen Polymerase chain reaction Plant homeodomain Propidium iodide para-Nitroaniline Polyvinylidene difluoride Radial growth phase Really interesting new gene Reverse transcriptase-polymerase chain reaction Skp1-Cul1-F-box protein Sodium dodecyl sulphate Small interfering ribonucleic acid  ix  Skp2 Smac SRB Tip60 TMA TNF TRADD TRAIL Ub UV VGP WB WCE WT  S-phase kinase-associated protein 2 Second mitochondria-derived activator of caspases Sulforhodamine B Tat-interactive protein 60 Tissue microarray Tumor necrosis factor TNF receptor-associated death domain protein TNF-related apoptosis-inducing ligand Ubiquitin Ultraviolet Vertical growth phase Western blot Whole cell extract Wild-type  x  ACKNOWLEDGEMENTS  I would like to graciously thank my supervisor, Dr. Gang Li, who accepted and supported me for my Ph.D. study at UBC. Throughout these years Dr. Li has shown me the way of doing quality research. I would also like to thank my thesis committee members Drs. Baljinder Salh, Michael Cox and William Jia for their constructive inputs, encouragement and kind support in scholarship applications. I would also like to thank Dr. David Huntsman, Nikita Makretsov and Hamid Masoudi for the construction of tissue microarray and thank Dr. Magdalena Martinka for the evaluation of tissue microarray staining. I thank all the previous and current members from Dr. Li’s lab. I owe many thanks to their help and friendship. I also thank my friends and colleagues from different labs at Jack Bell Research Centre, Vancouver General Hospital for their assistance. I am grateful to the following agencies which provided me with scholarships and awards: Roman M. Babicki Fellowship in Medical Research from UBC Faculty of Medicine, PhD Student Travel Award and Terry Fox Foundation PhD Studentship from National Cancer Institute of Canada, and Rising Star Award from Vancouver Coastal Health Research Institute. I also thank UBC Experimental Medicine Program and Department of Dermatology and Skin Science for their continuous supports of my studies. Lastly, I would like to express my thanks to my family for their love, understanding and encouragement over the years.  xi  DEDICATION  To My Parents  xii  CHAPTER 1 GENERAL INTRODUCTION 1.1 Cutaneous Melanoma 1.1.1  The Biology of Melanocytes Melanocytes, originating from the neural crest cells of the neuroectoderm  primordium, are melanin-producing cells found in human skin, retina of eyes, and the mucosal epithelia of inner ears and meninges. In human skin, dendritic cells migrate to the basal layer of the epidermis and differentiate into functional melanocytes that make dendritic contacts primarily with keratinocytes at a ratio of about 1:36 (Scott and Haake, 1991). Thus melanocytes constitute 2-4% of the total epidermal cell population (Vancoillie et al., 1999). Proliferation of melanocytes is normally suppressed by gap junctions with keratinocytes that are mediated by the adhesion molecule E-cadherin expressed on both melanocytes and keratinocytes (Hsu et al., 2000). Switching from E-cadherin to N-cadherin releases melanocytes from growth suppression, allowing them to proliferate and selfaggregate to form nevi, confers increased resistance of melanocytes to apoptosis, and also permits the migration of melanocytes among N-cadherin-expressing dermal fibroblasts (Li et al., 2001). The more rapidly proliferating keratinocytes retain melanin granules delivered from melanocytes as they terminally differentiate to establish the keratinized epithelium of the skin, a physical barrier for epidermal melanocytes. Melanocytes produce and export light absorbing melanins, which are packaged into specialized organelles called melanosomes. Melanin production regulates several functions in humans include photoprotection, trapping reactive oxygen species, sequestering metal ions, and binding certain drugs and organic chemicals (Riley, 1992; Riley, 1997). In addition  1  to carotenoids and haemoglobin, melanin is the main contributor to pigmentation. The variation in the number, size, composition and distribution of melanosomes determine the pigmentation difference, whereas melanocyte numbers typically remain constant (Tolleson, 2005). There are two major types of melanin: red/yellow pheomelanin and brown/black eumelanin, depending on the genotype of melanocortin-1 receptor (MC1R) gene (Rees, 2003). The MC1R gene encodes a transmembrane G-protein coupled receptor, which is activated by α-melanocyte stimulating hormone (α-MSH) or adrenocorticotropic hormone (ACTH) (Barsh et al., 2000; D'Orazio et al., 2006) and inhibited by agouti (Suzuki et al., 1997). Effects of α-MSH/ACTH on melanogenesis are mediated via microphthalmia transcription factor (MITF), the transcriptional factor controlling the expression of several pigmentation enzymes including tyrosinase, the rate-limiting enzyme in melanogenesis (Levy et al., 2006). During the differentiation of neural crest-derived melanocytes, activation of the c-Kit receptor tyrosine kinase initiates signaling towards the activation of mitogenactivated protein kinase (MAPK) ERK1/2, resulting in the phosphorylation and activation of MITF (Hemesath et al., 1998). In addition, the potassium-dependent sodium-calcium exchanger protein NCKX5 (Na-Ca-K-Exchanger-5) has also been shown to regulate melanogenesis (Ginger et al., 2008; Lamason et al., 2005). After ultraviolet (UV) irradiation, the activated p38 MAPK stimulates the expression of α-MSH released from keratinocytes or melanocytes by phosphorylating both MITF and upstream transcription factor (USF-1) to stimulate the promoter activity of tyrosinase leading to the production of melanin (Corre et al., 2004; D'Orazio et al., 2006). Meanwhile, inhibition of p38 MAPK activity blocks α-MSH-induced melanogenesis (Smalley and Eisen, 2000), suggesting that p38 MAPK plays crucial roles in both the upregulation of α-  2  MSH and its downstream signalling.. UV also inhibits the expression of bone morphogenetic protein (BMP) receptor-1b, thereby preventing its inhibitory role on tyrosinase mRNA transcription (Yaar et al., 2006). In addition, UV radiation-triggered thymidine breaks in DNA itself may induce tyrosinase and pigment production by upregulating the expression of p53 tumour suppressor (Hadshiew et al., 2001; Park and Gilchrest, 1999). Transfer of the melanin to keratinocytes ultimately forms a cap over the nucleus of keratinocytes protecting chromatin from exposure to hazardous UV light. 1.1.2  Epidemiology of Melanoma Cutaneous malignant melanoma, arising from the melanocytes in the epidermis, is a  life-threatening skin cancer. The incidence of melanoma has been rising rapidly, by as much as 3-8% yearly since the 1960s, and has doubled in the last decade (Dauda and Shehu, 2005; Thompson et al., 2005). Data from Canadian Cancer Statistics reveal the diagnosis of 4600 new cases of melanoma and the death of 900 individuals in 2007. Although it accounts for only 6% of all dermatologic cancers, it is responsible for 80% of deaths from skin cancer (National Cancer Institute of Canada: Canadian Cancer Statistics 2007, Toronto, Canada, 2007). In addition, melanoma is one of the most common causes of cancer deaths in young adults (Lens and Dawes, 2004). Despite the fact that the strongest risk factors for melanoma are a family history of melanoma and previous melanoma, the incidence of melanoma is strongly affected by race and geographic location (Thompson et al., 2005). It is a disease predominantly of populations with lighter pigmentation such as Caucasians. Among Caucasians, people with red hair, pale skin and a tendency to freckle are three times more likely to develop melanoma than others (Bliss et al., 1995; Thompson et al., 2005). In addition, the atypical nevi are potential precursor lesions. Although only 10-20% of primary  3  melanomas are associated with nevi, 5-30% of the lentigo maligna lesions, benign pigmented lesions occurring on heavily sun-exposed skin, progress to invasive lentigo maliga melanoma (Houghton and Polsky, 2002). The evidence of a causative link with sun exposure is compelling, with severe episodic sunburn in early life correlating best with melanoma risk (Oliveria et al., 2006; Thompson et al., 2005). 1.1.3  UV Exposure and Melanoma Accumulated epidemiological studies support the notion that exposure to UV  radiation in sunlight is the primary environmental factor in melanoma development (Gilchrest et al., 1999; Oliveria et al., 2006). However, the pattern of sun exposure also seems very important since sites that are intermittently exposed behave differently from those that are continually exposed (Oliveria et al., 2006; Rees, 2008). Although the association between melanoma risk and the intermittent sun exposure during vacation or recreation is still controversial, a positive correlation between early age sunburn and melanoma risk is well recognized now (Oliveria et al., 2006). The UV spectrum can be separated into ranges from the high-energy UVC (wavelengths below 280 nm) to the mid-range of UVB (280-320 nm) and to the weakenergy UVA (320-400 nm).UVB is overwhelmingly responsible for inducing the DNA lesions and its carcinogenesis aspect has been extensively investigated in the past (Cooper and Bowden, 2007; Ehrhart et al., 2003). However, UVA is the most abundant UV source in sunlight since the ozone layer of the outer space of the earth absorbs wavelengths up to about 310 nm thus preventing UVC and the majority of UVB from reaching the earth’s surface. Despite the fact that the carcinogenetic effects of UVA are supported by more studies (Bachelor and Bowden, 2004; de Gruijl, 2002), the residual UVB can still have  4  deleterious effects on macromolecules of the skin especially as a consequence of the continuing diminution of the protective ozone (a rarified layer which extends from 12 to 50 km in the stratosphere) (Bjorn and McKenzie, 2007). UV radiation promotes malignant change in the skin by causing direct mutagenic DNA lesions, stimulating cellular constituents of the skin to produce growth factors, reducing cutaneous immunological mechanisms of cancer surveillance, and promoting reactive oxygen species of melanin that cause DNA damage and suppress apoptosis (Meyskens et al., 2004). UV radiation-induced DNA lesions result in accumulated abnormalities in genetic pathways within the melanocytes, which promote cell proliferation and prevent apoptosis (Satyamoorthy and Herlyn, 2002). Failure to repair the UV-induced DNA lesions leads to the accumulation of genetic mutations and eventually contributes to the malignant transformation of melanocytes as observed in patients with xeroderma pigmentosum, a family of diseases characterized by grossly deficient repair of DNA photoproducts induced by UV radiation (Webb, 2008). 1.1.4  Staging and Subtypes of Melanoma The current standard classification and clinical and pathological staging of  melanoma was adopted in 2002 by the American Joint Committee on Cancer. This essentially takes into consideration of prognostic indicators such as primary tumour thickeness (Breslow thickness), primary tumour ulceration, number of lymph nodes involved and anatomic site of metastases (Kim et al., 2002; Thompson et al., 2005). The Clark model of melanoma progression describes the histologic changes that accompany the progression from normal melanocytes to malignant melanoma (Miller and Mihm, 2006). The first event is a proliferation of normal melanocytes to form the benign nevi (Fig. 1.1).  5  Such lesions have an increased number of nested melanocytes along the basal layer. Next is the development of dysplastic nevi characterized by the random and discontiguous cytologic atypia. During the radial growth phase (RGP) melanoma cells are confined to the epidermis, whereas cells invade the dermis and form an expansible nodule during the vertical growth phase (VGP). The VGP is associated with increased malignancy because of the potential for metastasis to other areas of the skin and other organs. The subtypes of melanoma are distinguished by clinical and pathologic growth patterns: superficial spreading, lentigo maligna, nodular, and acral lentiginous. Superficial spreading melanoma represents approximately 70% of melanomas and is the most common type occurring in people with fair skin complexion. Lentigo maligna melanoma is the least common subtype of melanoma (accounting for 4-15% of all melanomas) and occurs almost exclusively on the sun-exposed skin of the head and neck. Nodular melanoma represents 15% of all melanomas, occurs mostly on the trunk, head, and neck, and grows rapidly. Acral lentiginous melanoma accounts for 10% of all melanomas. The incidence of acral lentiginous melanoma is similar in people with widely different skin colors. So far, melanoma diagnosis has been based on pathology. A recent study demonstrates that genome-wide alterations in DNA copy number together with analysis of individual somatic mutation can be used to distinguish between the different melanoma subtypes with 70% accuracy (Curtin et al., 2005), suggesting that the different melanomas develop along distinct genetic pathways.  6  Fig. 1.1 Stages of melanoma progression. Five distinct stages have been proposed in the evolution of melanoma: common acquired benign nevus, dysplastic nevi, radial-growth phase melanoma, vertical-growth phase melanoma and metastatic melanoma.  7  1.1.5 Molecular Progression of Melanoma Numerous molecular events have been associated with the initiation and progression of melanoma. Alterations in the cyclin dependent kinase inhibitor 2A (CDKN2A) gene is almost uniformly present in melanoma. CDKN2A encodes two proteins, p16INK4A and p14ARF. The p16INK4A protein regulates the Rb1 pathway and p14ARF regulates the p53 pathway, both acting as brakes on the cell cycle (Giacinti and Giordano, 2006; Lara and Paramio, 2007). About 50% of melanomas have a deletion of the CDKN2A gene and 9% contain inactivating point mutations (Bennett, 2008). Hyper-methylation of the p16 promoter has been reported to occur in 20% to 75% of melanomas (Marini et al., 2006). Therefore, genetic or epigenetic changes resulting in a functional loss of p16INK4A and p14ARF ultimately lead to uninhibited cellular growth and proliferation. In addition, melanoma appears to rely on downregulated p14ARF levels to suppress the function of p53 since the p53 gene is rarely mutated in melanoma (Petitjean et al., 2007). Significant inverse correlation is also observed between p16INK4A or p14ARF expression and melanoma progression, as well as poor prognosis (Dobrowolski et al., 2002; Piepkorn, 2000; Sanki et al., 2007), implying that there is a remarkablely close relationship between the alternations in CDKN2A gene and melanoma progression. RAS genes (NRAS, HRAS, and KRAS) are among the most frequently mutated genes in human cancers. Human melanomas carry RAS mutations almost exclusively in NRAS, and 12% of metastatic melanomas carry NRAS alterations (Haluska et al., 2007). RAS controls cellular proliferation through the BRAF-MAPK pathway and controls cell apoptosis through the phosphatidylinositol 3-kinase (PI3K)-phosphatase and tensin homolog (PTEN)-Akt pathway (Rajalingam et al., 2007). Mutations of BRAF are detected in 66% of malignant  8  melanomas (Davies et al., 2002) that are associated with melanoma progression and poor patient outcome (DeLuca et al., 2008). Loss of PTEN is found in 30% of melanomas and this results in activated Akt, which antagonizes apoptosis and correlates with melanoma progression and a poorer 5-year survival (Dai et al., 2005; Wu et al., 2003). Further studies also suggest that concurrent BRAF/PTEN mutations may function like NRAS mutation in melanoma development (Haluska et al., 2006). Therefore, the alternations in major components of the NRAS-BRAF/PTEN pathways have significant effects on melanoma progression. Invasion and spread of melanoma are related to alterations in cell adhesion molecules. Progression from the radial-growth phase to the vertical-growth phase of melanoma is marked by the loss of E-cadherin and the expression of N-cadherin and αVβ3 integrin (Danen et al., 1996; Danen et al., 1994; Miller and Mihm, 2006). The integrin induces expression of matrix metalloproteinase (MMP) 2 to degrade the collagen in basement membrane, rendering N-cadherin-expressing melanoma cells to invade into adjacent tissues. In addition, transcriptional factors such as AP-2, CREB and ATF-1, growth factor receptor C-KIT and the angiogenic factor interleukin-8 (IL-8) may play an important role in melanoma development and progression (Leslie and Bar-Eli, 2005; Poser and Bosserhoff, 2004; Sekulic et al., 2008). Although many efforts have been made to elucidate the mechanisms of melanoma development, discoveries reported to date are not likely to account fully for the molecular pathogenesis of melanoma.  9  1.1.6  Treatment of Melanoma Current treatment strategies for melanoma are generally dictated by whether the  disease is local or whether metastases have occurred. Most primary melanomas are curable by surgical resection followed by adjuvant therapy. However, the 10-year survival expectancy dramatically drops below 3% once distant metastases have occurred (Lens and Dawes, 2004). Radiation therapy is usually used as primary and adjuvant therapy for localized or locally advanced melanoma if surgery is not an option (Berk, 2008). Because melanoma is notoriously radioresistant, radiotherapy is rarely used on malignant tumours (Jenrette, 1996). In the past few decades, many chemotherapeutic regimens have been tried without significantly increasing overall survival rates of melanoma patients with metastases. Dacarbazine (DTIC), a methylating drug, is the only currently used cytotoxic drug approved by the U.S. Food and Drug Administration for the treatment of metastatic melanoma. However, the response rates with single-agent DTIC did not exceed 12% (Gogas et al., 2007). Temozolomide (TMZ), an oral congener of DTIC, has similar efficacy as DTIC in the treatment of a variety of solid tumors, especially in brain malignancies (Gogas et al., 2007; Middleton et al., 2000). Other single agents include platinum analogues like cisplatin or carboplatin, nitrosoureas, and microtubule toxins such as the vinca alkaloid, vindesine have shown modest activity as single agents in patients with metastatic melanoma (Gogas et al., 2007). To improve response rates, many combination regimens have been evaluated in clinical trials. However, 2-agent regimens show little superiority to single-agent DTIC (Atallah and Flaherty, 2005; Chapman et al., 1999; Rosenberg et al., 1999). Another approved regimen in use for metastatic melanoma is immunotherapy using cytokines, particularly interferon-α (INFα) and interleukin-2 (IL-2) (Tarhini and Agarwala,  10  2006). However, their efficacy is still a matter of debate and toxicity is a problem. Pegylated interferon also seems to be at least equally efficacious as interferon in the treatment of metastatic melanoma with lower acute toxicity (Bukowski, 2003). A recent study showed that pegylated interferon α2b can improve recurrence-free survival in patients with resected stage III (lymph-node metastatic) melanoma and may be very relevant for the treatment of the N1 subset, whose metastatic melanoma was discovered on sentinel-node biopsy rather than as a clinically palpable node (N2/3) (Eggermont et al., 2008). Vaccines utilizing the melanoma-associated antigens as well as humanized monoclonal antibodies targeting these markers have been developed, however, clinical responses to melanoma vaccines are still poor and currently there is no melanoma vaccine with a proven efficacy (Lens, 2008; Terando et al., 2007). The intrinsic resistance of melanoma to conventional chemotherapies highlights the necessity of developing novel strategies to defeat metastatic melanomas, leading many studies to evaluate new approaches such as protein kinase inhibitors (e.g. sorafenib), agents that act on cytotoxic T-lymphocyte antigen 4 (CTLA-4) or on apoptotic mechanisms (e.g. oblimersen sodium), and antiangiogenic agents (e.g. bevacizumab, SU5416, MEDI-522, PI88) (Gray-Schopfer et al., 2007). The multi-kinase inhibitor sorafenib targets BRAF, CRAF and the VEGF and PDGF receptor tyrosine kinases (Wilhelm et al., 2004). Monotherapy with sorafenib shows only modest activity against melanoma; its combination with carboplatin and paclitaxel are more encouraging (Flaherty, 2006). Many other protein kinase inhibitors have also been developed recently, such as MEK inhibitors PD0325901 and AZD6244 (Solit et al., 2006) and mTOR inhibitors CCI-779 or RAD001 (Dancey, 2006).  11  1.2 Apoptosis 1.2.1  Apoptosis versus Necrosis Cell death can occur by either of two major distinct mechanisms, necrosis or  apoptosis. Necrosis represents a passive consequence of mechanical damage or exposure of cells to toxins. It is characterized by rapid swelling of the dying cell, rupture of the plasma membrane, and release of cytoplasmic contents to the cell environment (Hetz et al., 2005). It affects groups of contiguous cells and usually causes significant imflammatory response. Apoptosis, also termed programmed cell death, is a mode of cell death induced by physiological stimuli such as lack of growth factors and changes in hormonal environment. Apoptosis plays a pivotal role in development, cancer, normal ageing and in neurological disorders such as Alzheimer’s disease, amyotrophic lateral sclerosis and Parkinson’s disease (Thompson, 1995). Cells undergoing apoptosis is an active participant in its own demise and exhibit a characteristic pattern of morphologic changes, including cell shrinkage, nuclear condensation and DNA fragmentation of the nucleus and bubbling of the plasma membrane (Ziegler and Groscurth, 2004). It affects individual cells and does not trigger imflammatory response. A critical stage of apoptosis involves the acquisition of surface changes by dying cells that eventually results in the recognition and the uptake of these cells by phagocytes (Hart et al., 1996). In particular, cells undergoing apoptosis break up the phospholipid asymmetry of their plasma membrane and expose phosphatidylserine from the inner layer of their membrane to the outer layer (Schlegel et al., 2000). This phosphatidylserine exposure occurs in the early phases of apoptosis during which the cell membrane remains intact and represents a hallmark in early apoptotic cells, therefore providing a target for the detection of apoptotic cells using dyes such as Annexin V (van  12  Engeland et al., 1998). Since melanoma is resistant to most forms of treatment that usually elicits tumour shrink by either suppressing tumour cell proliferation or inducing its apoptosis (Soengas and Lowe, 2003), understanding of the apoptotic signaling pathway will help us design the novel strategies for the treatment of melanoma. Autophagic cell death, another type of programmed cell death, has also been reported, which is characterized by the formation of large vacuoles that eliminate organelles in a specific sequence prior to the nucleus being destroyed (Bursch et al., 2000). 1.2.2  Apoptosis Pathways Apoptosis is initiated and promoted by a wide variety of intra- and extra-cellular  stimuli via different mechanisms including two major routes that involve either the mitochondria activation (the intrinsic pathway) or the activation of death receptors (the extrinsic pathway) (Fig. 1.2). Both pathways converge to induce the activation of the caspase (cysteinyl aspartate–specific protease) family. Subsequently, cleavage of key cellular substrates ensues, leading to cell demise (Danial, 2007). The intrinsic pathway of apoptosis functions in response to various types of intracellular stress including growth factor withdrawal, DNA damage, unfolding stresses in the endoplasmic reticulum and death receptor stimulation. The Bcl-2 family of proteins governs the intrinsic apoptotic pathway and controls the release of cytochrome c from the mitochondria, which forms apoptosome with apoptotic protease activating factor-1 (Apaf-1) and pro-caspase-9 to trigger the activation of caspase cascade (Adams and Cory, 1998). The Bcl-2 family proteins are classified on the basis of structural similarity to the Bcl-2 homology (BH) domains (BH1, BH2, BH3 and BH4), and a transmembrane domain: prosurvival proteins, whose members are most structurally similar to Bcl-2, such as Mcl-1 and  13  Bcl-XL; pro-apoptotic proteins, Bax, Bak and Bok, which are structurally similar to Bcl-2 and antagonize their pro-survival functions; and the pro-apoptotic ‘BH3-only’ proteins (Danial, 2007). The BH3-only pro-apoptotic molecules, including Bad, Bid, Bim, Noxa, Bik, Hrk and Puma, show sequence homology only within a single α-helical segment, the BH3 domain, which is also known as the minimal death domain required for binding to multidomain Bcl-2 family members (Wang et al., 1996). BH3-only molecules are upstream sentinels that selectively respond to proximal death and survival signals, and require Bax/Bak to induce death, whereas the principal function of antiapoptotic Bcl-2, Bcl-XL and Mcl-1 is to inhibit Bax and Bak (Kim et al., 2006). Although melanoma is very resistant to apoptosis, the level of Bcl-2 in melanoma is usually downregulated in comparison to benign nevi and the level of Bax is increased in melanoma (Tang et al., 1998). Recent studies demonstrate that both Mcl-1 and Bcl-XL are overexpressed in melanoma and correlates with melanoma tumor progression (Zhuang et al., 2007), implying that these two anti-apoptotic factors may be key players in overriding the apoptotic signals generated by increased Bax and decreased Bcl-2 expression. The extrinsic pathway is activated upon the activation of death receptors, which are cell surface receptors belonging to the tumour necrosis factor (TNF) superfamily, which trigger apoptosis upon ligand binding (Hussein et al., 2003; Jin and El-Deiry, 2005). The best characterized death receptors are Fas, tumour necrosis factor receptor (TNF-R), TNFrelated apoptosis-inducing ligand receptor (TRAIL-R), TNF receptor apoptosis-mediating protein (TRAMP or DR3), TRAIL-R1 (DR4) and TRAIL-R2 (DR5). Upon ligand binding, monomeric receptors trimerize and form receptor clusters, thereby establishing an intracellular activated death domain to recruit adaptor proteins such as Fas-associated  14  protein with death domain (FADD), TNF receptor-associated protein with death domain (TRADD) and receptor-interacting protein (RIP) to the death receptors. The death domaincontaining adaptor proteins also interact with pro-caspase-8 or pro-caspase-10 to form a complex at the receptor called the death inducing signalling complex (DISC). Once assembled, DISC induces the activation of caspase-8, which in turn precipitates the activation of downstream effector caspase cascades. Death receptor signalling is also regulated by cellular FLICE-like inhibitory protein (c-FLIP), an endogenous inhibitor that interacts with FADD to antagonise apoptosis (Irmler et al., 1997). Furthermore, the intrinsic and extrinsic pathways of apoptosis are connected by the BH3-only Bcl-2 interacting domain (Bid) protein (Luo et al., 1998). The full-length Bid is cleaved by death receptoractivated caspase-8 and translocates to the mitochondria to activate the intrinsic pathway (Luo et al., 1998) (Li et al., 1998b). Activation of apoptosis can be blocked by a family of proteins termed inhibitors of apoptosis (IAPs), which are characterized by the presence of one or more repeats of highly conserved 70 amino acids domain called the baculoviral IAP repeat (BIR) (Hunter et al., 2007). Eight homologues have been described in the past decades including neuronal apoptosis inhibitory protein (NAIP), c-IAP1, c-IAP2, Drosophila IAP1 (DIAP1), X-linked inhibitor of apoptosis protein (XIAP), livin, survivin and apollon. They interfere with the activation of caspases through their conserved BIR domains (Salvesen and Renatus, 2002). In addition, c-IAP1, c-IAP2 and XIAP contain a highly conserved RING domain which function as an E3 ubiquitin ligase to regulate apoptosis by controlling the degradation of caspases (Morizane et al., 2005). The antiapoptotic effect of IAPs is modulated by other mitochondrial proteins such as second mitochondrial-derived activator of caspase  15  (Smac/DIABLO), which is released along with cytochrome c at the initiation of apoptosis in response to apoptotic stimuli (Ceballos-Cancino et al., 2007; Creagh et al., 2004; Fu et al., 2003; McNeish et al., 2005; Vucic et al., 2000). Consistent with the resistance of melanoma to apoptosis, the expression of IAPs including survivin and livin (also called melanoma IAP) is significantly increased in melanomas (Emanuel et al., 2008; Kluger et al., 2007; Vucic et al., 2000), whereas that of Smac/DIABLO is reduced in melanomas. Inhibition of survivin, XIAP or livin dramatically improves the chemosensitivity of melanoma cells (ChawlaSarkar et al., 2004; Schmollinger et al., 2003).  16  Fig. 1.2 Overview of apoptotic pathways in cells. Apoptosis may involve either deathreceptor-mediated or mitochondria-mediated apoptotic events. Ligand-activated death receptors recruit adaptor molecules such as FADD, which subsequently activates caspase-8 and caspase-3 and induces DNA fragmentation, an apoptotic hallmark. Meanwhile, DNA damaging agents lead to p53-mediated mitochondria activation and trigger cytochrome c release. The released cytochrome c binds to Apaf-1 together with pro-caspase-9 and activates caspase-9. The caspase-9 cleaves pro-caspase-3 and activates caspase-3. These two pathways converge through caspase-8-mediated Bid truncation. In addition, caspase activation can be inhibited by IAPs, which is usually antagonized by Smac released from activated mitochondria in apoptosis.  17  1.2.3 UV-induced Apoptosis Ultraviolet-B (UVB) radiation triggers a variety of biological effects such as the induction of apoptosis. UVB-induced apoptosis provides a well controlled scavenging mechanism protecting cells from malignant transformation. UVB radiation mainly targets the genomic DNA, leading to the formation of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproduct, which are usually removed by nucleotide excision repair (NER) to prevent the unwanted mutations (Murphy et al., 2001). Insufficient repair of DNA correlates with increased apoptotic rate, thereby implying a role for photolesions in triggering apoptosis. This is also supported by the observation that photoreaction resulted in a pronounced reduction of DNA lesions which correlated with a significant inhibition of UVB-induced apoptosis (Yarosh et al., 1994). UVB radiation can also modulate the activation of apoptosis by affecting the activation of death receptors. The expression of Fas is upregulated upon UVB irradiation in epidermal cells (Bang et al., 2002). The role of UV radiation on Fas ligand (Fas-L) expression is cell type restricted. In lymphocytes UV upregulates Fas-L expression via the activation of nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1) (Kasibhatla et al., 1998), while in keratinocytes it downregulates Fas-L expression (Ouhtit et al., 2000). Furthermore, UVB is also able to induce ligand-independent clustering of Fas and TNF-R and lead to the activation of the extrinsic apoptotic pathway (Bang et al., 2002; Elyassaki and Wu, 2006; Kulms et al., 1999; Rehemtulla et al., 1997). In addition, the contributions of both UVB-induced DNA lesions and UVB-induced death receptor activation to UVBtriggered apoptosis are additive and independent (Kulms and Schwarz, 2002), suggesting that UVB-induced apoptosis can be triggered through different mechanisms.  18  The tumour suppressor p53 plays a central role in UV radiation-induced signaling cascade. Upon UVB irradiation, the ataxia telangiectasia-mutated and Rad3-related (ATR) protein and checkpoint1 (Chk1) protein is subsequently activated, which phosphorylated p53, leading to its stabilization and activation (Lu et al., 2008). p53 plays a key role in UVBinduced apoptosis by modulating the activation of mitochondria. Several pro-apoptotic Bcl-2 family proteins are transcriptionally regulated by p53 such as Bax, Noxa, Puma, Bid and Pig3 through the p53-responsive elements located in the promoter or intron region of these genes (Haupt et al., 2003). UVB-triggered p53 activation leads to the upregulation of these pro-apoptotic proteins and results in the activation of the intrinsic apoptotic pathway. In addition, p53 can modulate the expression or subcellular distribution of Fas, thereby playing a vital role in UVB-induced apoptosis. P53 can activate the Fas gene by binding to its transcriptional activation site and promoter region (Muller et al., 1998) and promotes the redistribution of cytoplasmic Fas to the cell surface through transportation from the Golgi complex upon DNA damage (Bennett et al., 1998). Moreover, TRAIL-R is also a p53regulated death receptor in response to DNA damage (Wu et al., 1997). 1.3 Protein Degradation 1.3.1 Pathways of Protein Degradation Fundamental cellular activities are largely maintained by coordinated protein synthesis and degradation. Breakdown of this balance usually results in aberrant protein levels and their associated cellular functions, which eventually contribute to the development of many diseases including cancer and central nervous system diseases (Bordoli et al., 2001; Kim et al., 2006; Zetter and Mangold, 2005). Selective inhibition of the activities of disease-specific components of the degradation pathway has arisen as a  19  promising approach for innovative anticancer therapies (Burger and Seth, 2004; Goldberg, 2007; Montano et al., 1994; Nalepa et al., 2006). At least two major pathways can degrade cellular proteins, the proteasome pathway and the lysosomal pathway. In mammalian cells, about 90% of cellular proteins are believed to be degraded by the proteasome pathway, while the breakdown of the extracellular and membrane proteins is by the lysosome-endosomal pathway (Tanaka et al., 2004). Proteasomes are multicatalytic complexes that constitute the major proteolytic activity in all eukaryotic cells and include two major types, 26S and 20S proteasomes. The 20S proteasome is a barrel-shaped complex consisting of four stacked rings, each composed of seven related subunits (Hershko and Ciechanover, 1998). The outer rings are formed by noncatalytic α subunits, whereas catalytic β subunits occupy the inner two rings. The 20S proteasome is an ATP-independent protease that cleaves peptides and digests several unfolded proteins. The 26S proteasome is formed by an ATP-dependent association of the 20S core particle with one or two ATPase regulatory complexes termed PA700 (or the 19S particle) at one or both ends of the 20S proteasome barrel, respectively (Fig. 1.3A) (Walz et al., 1998). The 26S proteasome is an ATP-dependent protease that degrades mainly ubiquitinated proteins that are misfolded and/or predominantly involved in the regulation of cell differentiation, cell cycle control, stress response, and antigen presentation.  20  Fig. 1.3 The ubiquitin-mediated proteasome degradation system. (A) Structure of the 26S proteasome. Ubiquitinated substrates are recognized, deubiquitinated and unfolded by the 19S subunit. The unfolded substrates will then be cleavaged by the 20S core subunit. (B) The ubiquitination process is mediated by three major enzymes: E1 activating enzyme, E2 conjugating enzyme and E3 ligases. The HECT-domain E3 ligases transfer Ub moiety by forming a thioester bond between the E3 ligases and Ub. Ub is then transferred to the substrates. The RING-domain E3 ligases interact with Ub-conjugated E2 which transfers Ub directly to the substrates. (C) A schematic presentation of the RING-domain E3 ligases-SCF (Skp1-Cul1-F-box) complex. Substrates are recognized through the carboxyl-terminus of Fbox proteins and the Ub is transferred to the substrates from E2. The interaction between the F-box proteins and the substrates requires the phosphorylation of substrates.  21  1.3.2  Ubiquitin-dependent Proteasome Degradation Proteins targeted for degradation by the 26S proteasome are covalently attached to a  chain consisted of small molecule ubiquitin (Ub) moieties, a process called ubiquitination (Welchman et al., 2005). Ub itself is a 76 amino acid protein containing seven lysine residues that can be used as an acceptor for the attachment of other Ub molecules, allowing the formation of polyubiquitin chains (Xu and Peng, 2006). Ubiquitination requires three major enzymes: an E1 activating enzyme, an E2 conjugating enzyme and an E3 ubiquitin ligase (Pickart, 2000). Upon the activation by E1 enzyme, Ub moieties are subsequently conjugated to E2 enzyme and transferred to the E3 ligase-recognized substrates. The substrate protein is then polyubiquitinated and destined for destruction by the 26S proteasome (Fig. 1.3B) (Nandi et al., 2006; von Mikecz, 2006). 19S subunits of the 26S proteasome are responsible for recognition of ubiquitinated substrates and cleavage of the ubiquitin chains prior to translocation into the proteasome core (Walz et al., 1998). Opposite to the ubiquitination, Ub is removed from the substrates or the free polyUb chains by a large number of deubiquitinating enzymes, a process that is called deubiquitination that counteracts the effect of ubiquitination or recycles the Ub moieties (Kim et al., 2003; Wilkinson, 2000). The selectivity of the ubiquitin-proteasome pathway for a particular substrate relies on E3 ligases. Two types of E3 ligases, the homology to E6-AP carboxylterminal (HECT) domain E3s, and the really interesting new gene (RING) finger motifcontaining E3s, utilize different mechanisms to transfer the Ub adducts to substrates (Fig. 1.3B). RING-domain E3 ligases transfer Ub directly onto substrates from the E2 conjugating enzymes, whereas HECT-domain E3s bind to Ub first and then target it to the substrates next. The activity of E3 ligases is tightly regulated through variable mechanisms, which may  22  include the subcellular relocalization of the E3 enzymes, the binding of E3 ligases to accessory proteins, and the posttranslational modification of substrates or a component of the ubiquitin conjugating machinery (Baer and Ludwig, 2002; Kawai et al., 2007; Lin et al., 2006; Woelk et al., 2007). 1.3.3  SCFSkp2 E3 ligase Complex-Mediated Protein Degradation Two types of E3 ligases that regulate cell cycle progression have been well  characterised: the SCF (Skp1-Cul1-F-box) E3 ligase complex responsible for regulatory events at the G1-S transition, and the anaphase promoting complex (APC) that acts at the end of mitosis and controls passage back into the G1 phase of the cell cycle. Both of them belongs to the superfamily of the RING-domain E3 ligases (Pray et al., 2002). The SCF E3 ligase complex contains four major components: Skp1, Cul1, Roc1 and F-box protein (Ang and Wade Harper, 2005; Schwechheimer and Calderon Villalobos, 2004). Cul1 acts as a scaffold protein within the complex. The carboxyl-terminus of Cul1 interacts with Roc1 to recruit the Ub-conjugated E2 enzyme, whereas its amino-terminus interacts with Skp1 (Fig. 1.3C). Skp1 binds to the F-box motif of an F-box protein that interacts with the substrates directly and determines the specificity of SCF E3 ligase complex. The F-box motif consists of approximately 50 amino acid residues located at the amino-terminus of an F-box protein. The F-box proteins bind to phosphorylated substrates through their carboxyl-terminus and are categorized into three major families based on the motifs at the carboxyl-terminus: the FBW family containing F-box proteins with Trp-Asp (WD) repeats, the FBL family containing F-box proteins with leucine-rich repeats (LRR), and the FBX family consisting of F-box proteins with other protein-protein interaction domains at their carboxyl-termini (Ho et al., 2006; Jin et al., 2004).  23  S-phase kinase-associated protein 2 (Skp2), an F-box protein, has been implicated in targeting many cellular proteins for degradation including cyclin-dependent kinase (CDK) inhibitors such as p27, p21 and p57, TOB1 (transducer of Erbb2), RASSF1 (Ras association domain family 1) and FOXO1 (forkhead box-containing transcription factor 1) (Frescas and Pagano, 2008). In general, Skp2 recognizes substrates for ubiquitination through the phosphorylated consensus sequence in their target proteins. Upregulation of Skp2 has been shown in several types of cancers including colorectal cancer, breast cancer, gastric cancer, prostate cancer, lung cancer, sarcoma, ovarian cancer, brain cancer and leukemia (Hershko and Ciechanover, 1998). Increased Skp2 expression correlates with a poor patient prognosis in those cancers and may be an attractive target for the development of novel cancerintervention strategies (Chiappetta et al., 2007; Hershko and Ciechanover, 1998; Masuda et al., 2002; Tanami et al., 2004; Yang et al., 2002; Yokoi et al., 2004). In melanoma, the nuclear Skp2 level is inversely correlated with the expression of p27 (Li et al., 2004; Woenckhaus et al., 2005). Suppression of Skp2 by RNA interference significantly inhibits melanoma cell growth and invasion (Katagiri et al., 2006; Sumimoto et al., 2006). However, the prognostic value of Skp2 is controversial as one study indicates that increased nuclear Skp2 but not cytoplasmic Skp2 correlates with a poorer patient survival, whereas a separate study reports an opposite result (Li et al., 2004; Woenckhaus et al., 2005). Future studies are demanded to understand the prognostic role of Skp2 in patients with melanomas. 1.4 ING Family Proteins The evolutionarily conserved inhibitor of growth (ING) family of proteins are widely present in eukaryotic proteomes (He et al., 2005a). The best-characterized members include the five human members (ING1-5) and three S. cerevisiae members (Yng1, Yng2, and  24  Pho23). The human ING tumor suppressors ING1-5 were discovered during the past decade (Garkavtsev et al., 1996; Nagashima et al., 2003; Shimada et al., 1998; Shiseki et al., 2003). Studies from multiple groups impy that ING proteins can modulate cell proliferation, apoptosis, cellular senescence, contact inhibition, DNA damage repair and angiogenesis (Campos et al., 2004; Russell et al., 2006; Soliman and Riabowol, 2007). 1.4.1  Gene Location and Splicing Variants The five human ING family genes are located on different chromosomes: ING1  (13q34), ING2 (4q35), ING3 (7q31.3), ING4 (12p13.3), and ING5 (2q37.3). Among them, ING1, ING2, ING4, and ING5 are in regions very close to the terminus of their respective chromosome, thereby having the potential to be affected by telomere erosion. In contrast, ING3 is located in the middle of the long arm of chromosome 7, suggesting that ING3 may be functionally distinct from other ING proteins. Splicing variants have been reported for several members. Alternative splicing of the first exon of ING1 give rise to several ING transcripts which encode four different ING1 protein isoforms (He et al., 2005a). ING3 utilizes differential incorporation of an internal exon to produce mRNA variants, encoding p47ING3 (ING3) and p11ING3, respectively (Kawaji et al., 2002; Nagashima et al., 2003). Four ING4 mRNA variants are generated from the use of closely juxtaposed splicing sites that results in the differential incorporation of a 12-nucleotide coding region within a nuclear localization signal (Ho et al., 2006; Unoki et al., 2006). 1.4.2  Structure of ING Family Proteins All the ING proteins share a highly conserved plant homeodomain (PHD) at their  carboxyl-terminus (C-terminus), and contain a centrally localized nuclear localization sequence (NLS) as well as a conserved novel chromatin regulatory (NCR) region (Fig. 25  1.4A) (He et al., 2005a). The PHD finger, discovered in 1993 as a Cys4-His-Cys3 motif in the homeodomain protein HAT3 in Arabidopsis thaliana (Schindler et al., 1993), is known to be involved in protein-protein interactions and is commonly found in nuclear chromatinbinding proteins to regulate chromatin organization or gene expression (Aasland et al., 1995; Bordoli et al., 2001; Capili et al., 2001; Pascual et al., 2000). The PHD finger is a Zn(2+)binding domain and resembles the metal binding RING domain (Cys3-His-Cys4) (Bienz, 2006), which usually binds to E2 ligases to mediate the ubiquitination of substrate proteins (Ho et al., 2006). Although there is no evidence for PHD fingers functioning as ubiquitin ligases (Scheel and Hofmann, 2003), recent studies indicate that PHD domain can act as a small ubiquitin-like modifier (SUMO) E3 ligase for the adjacent bromodomain (GarciaDominguez et al., 2008; Peng and Wysocka, 2008). In addition, a small conserved proteininteracting motif (PIM) is found along with PHD region of ING1 and ING2, which consists of acidic, basic and aromatic residues, and can bind to phosphatidylinositol monophosphates (Gozani et al., 2003). The NLS is a strong basic region and can be classified into two major groups (Dingwall and Laskey, 1991; Garcia-Bustos et al., 1991; Makkerh et al., 1996). The first is a single type containing 3-5 basic amino acids with the weak consensus Lys-Arg/Lys-XArg/Lys; the other is a bipartite type NLS containing two clusters of basic regions of 3-4 residues, each separated by approximately 10 amino acids. One NLS motif is predicted residing upstream of the PHD fingers of ING proteins (He et al., 2005a). Three potential nucleolar targeting signals (NTS) including RRQR, KEKK and KKKK are also present within this NLS motif of ING1 and ING2 proteins, which has been shown to target ING1 to the nucleolus after UV damage (Scott et al., 2001a). A recent report indicates that the  26  deletion of three NTS-containing NLS motif (residues 133-199) was able to prevent nucleolar translocation of ING1b, but was not sufficient to abolish the nuclear localization of ING1b (Russell et al., 2008). However, deletion of both the NLS motif and a C-terminus NLS-like motif (residues 265-279) completely disrupted the nuclear import of ING1b (Russell et al., 2008), thereby pointing out the significance of basic acid-enriched Cterminus NLS motif in regulating the subcellular localization of ING proteins. Both ING1 and ING2 contain a conserved REASP motif between NLS and PHD finger, which closely matches the canonical, phosphorylation-dependent 14-3-3 binding site RSXpSXP or RX(Y/F)XpSXP (X denotes any amino acid residues except cysteine) (Rittinger et al., 1999). It was shown that the S199 residue within the motif REASP of ING1b mediates its interaction with 14-3-3 protein (Gong et al., 2006). Using the online tool MOTIFSCAN program (http://scansite.mit.edu/motifscan_seq.phtml), one potential 14-3-3 protein binding signal is also predicted for ING3 protein (RTSS247LKAS), which likely matches another variation of the 14-3-3 binding motif, the Ser-rich motif as postulated as RX1–2SX2–3S (X denotes any amino acid and at least one of the Ser is phosphorylated) (Liu et al., 1997). The conserved NCR region of ING proteins was firstly identified by Kawaji et al (Kawaji et al., 2002) and subdivided into three submotifs: Q-E-L-G-D-E/D-K-I/L/M-QI/L, K-E-F/Y-S/G-D-D-K-V-Q-L and L-E-D-A-D-E-K-V-Q-L that are specific for ING1/ING2, ING4/ING5, and ING3 subfamilies, respectively (He et al., 2005a). This region has been proposed to be involved in the binding of HAT, HDAC, MYC and other cell cycle related proteins (Helbing et al., 1997). Accordingly, it has been shown that ING1b is recruited to the Sin3/HDAC complexes by interacting with SAP30 through the N-terminus region including the NCR domain (Kuzmichev et al., 2002).  27  Several other motifs are also found in ING proteins. ING1b contains a specific amino-terminus proliferating cell nuclear antigen (PCNA)-interacting protein (PIP) domain, through which it competes with p21 in mediating the cellular response to UV irradiation (Scott et al., 2001b), and a partial bromodomain that is known to regulate protein-protein interactions influencing chromatin remodelling and transcriptional activation. The aminoterminus of ING2 contains a leucine-zipper like (LZL) motif that consists of leucine residues spanning every seven amino acids to form a hydrophobic patch. The LZL of ING2 is required for ING2-p53 interaction and has been shown to be crucial for ING2-mediated DNA repair and apoptosis in response to UV irradiation (Wang et al., 2006b). In addition, ING3 contains two major insertions between NCR and NLS region: one is located between residues 110-212 and the other spans from residues 233 through 287 (He et al., 2005a). These distinct motifs indicate that ING family proteins may also be involved in different cellular events in addition to their shared biological functions.  28  Fig. 1.4 Structure and signaling features of ING proteins. (A) The structural features of ING proteins. All ING proteins shared a conserved plant homeodomain (PHD), a nuclear localization signal (NLS) and a novel chromatin regulatory (NCR) region. The aminoterminus of ING1b and ING2 also contain a proliferating cell nuclear antigen (PCNA)interacting protein (PIP) domain and a leucine-zipper like (LZL) domain, respectively. Both ING1b and ING2 also have a small conserved protein-interacting motif (PIM) in their carboxyl-terminus. (B) Schematic representation of potential ING-mediated signaling mechanisms. By binding to the methylated histone tails via their PHD regions, ING proteins recruite their associated HAT or HDAC complexes to modify the state of chromatin acetylation and change the accessibility of chromatin, which plays a crucial role in the regulation of different biological events such as transcription, apoptosis, DNA repair and senescence.  29  1.4.3 ING Proteins and Chromatin Regulation Eukaryotic DNA is packaged and compacted by histones (H2A, H2B, H3 and H4) into a higher order structure of chromatin called nucleosomes. The level of compaction of chromatin determines the accessibility of chromatin to transcriptional factors to initiate gene transcription or repairing of DNA. The conserved PHD finger of ING proteins binds to histones in a methylation-sensitive manner (Champagne et al., 2008; Palacios et al., 2006; Pena et al., 2006; Shi et al., 2006; Taverna et al., 2006) suggesting that ING proteins may contribute to the regulation of chromatin structure and the deciphering of the epigenetic histone code hypothesis, which predicts that distinct modifications of the histone tails would be recognized by different chromatin-associated proteins thereby initiating downstream pathways (Fig. 1.4B) (Jenuwein and Allis, 2001). A decade of study reveals that ING family proteins are crucial components of distinct histone acetyl-transferases (HATs) and histone deacetylases (HDACs) complexes, and appear to facilitate the association and enzymatic activity of their associated HATs or HDACs with the substrates (Soliman and Riabowol, 2007). The involvement of ING proteins in histone acetylation was first noted in yeast (Choy and Kron, 2002; Loewith et al., 2000). Saccharomyces cerevisae Yng2 was found to be a component of the nucleosomal acetyltransferase of histone 4 (NuA4) complex, a yeast multi-subunit complex composed of the MYST Esa1 protein, which is involved in the acetylation of histones H2A and H4 (Choy and Kron, 2002; Nourani et al., 2001; Nourani et al., 2003). ING1b was found to coprecipitate with various HATs, including transactivation–domain associated protein (TRRAP), p300, PCAF and CBP which contains HAT activity for H2A, H2B, H3 and H4 (Skowyra et al., 2001; Vieyra et al., 2002). Both ING1b and ING2 are also present in human  30  Sin3-HDAC and the SWI/SNF complexes (Doyon et al., 2006; Doyon et al., 2004; Kuzmichev et al., 2002; Xin et al., 2004). The ING1b-Sin3-HDAC complex is important for histone deacetylation and methylation of histone H3 at Lys9 in heterochromatin and is therefore important for the maintenance of gene silencing at heterochromatin (Pena et al., 2006; Shi et al., 2006; Shi et al., 2007), but the functional significance of the interaction of ING proteins with SWI/SNF is unknown. In addition, ING4 is associated with the HBO1 HAT that is required for the majority of histone H4 acetylation in vivo, whereas ING5 is involved in two distinct complexes containing HBO1 or nucleosomal H3-specific MOZ/MORF HATs (Doyon et al., 2006). Therefore, it is well accepted that ING proteins execute their biological functions by facilitating the assembly and subnuclear localization of their associated HATs and HDACs. 1.4.4  Biological Functions of ING Proteins Abundant evidence indicates that ING proteins can regulate cell cycle checkpoints,  apoptosis, nucleotide excision repair (NER), senescence, cell migration and angiogenesis (Campos et al., 2004; Russell et al., 2006). Most of these biological functions of ING proteins require functional p53 (Campos et al., 2004), the crucial gate keeper of genomic integrity. p21 is a potent mediator of the G1 cellular checkpoint. It binds and inactivates cyclin-cyclin-dependent kinase (Cdk) complexes, resulting in hypophosphorylation of retinoblastoma protein (Rb), E2F sequestration and cell cycle arrest at the G1/S transition (Abukhdeir and Park, 2008). ING proteins, except ING1a, inhibit the colony formation, induce cell cycle arrest and negatively regulate cell growth by enhancing the promoter activity of the p53-target gene p21,(Kataoka et al., 2003; Nagashima et al., 2001; Nagashima  31  et al., 2003). It is also found that cyclin B1, a key G2 cell phase cyclin, is also a downstream target of ING1b and ING1c (Garkavtsev and Riabowol, 1997; Nakamura et al., 2002; Porter and Donoghue, 2003; Takahashi et al., 2002). Coexpression of p53 and ING1b significantly reduces the mRNA level of cyclin B1 (Takahashi et al., 2002). Cyclin E is also involved in the G1/S transition and is used as a marker for cell proliferation. Although no direct evidence links ING proteins to cyclin E, a recent report indicates that cyclin E tends to express more in hepatocellular carcinomas with low ING1b expression (Ohgi et al., 2002). In addition, ING1b-mediated cell cycle regulation involves both the amino-terminus and carboxyl-terminus of ING1b and requires functional Ras signalling (Goeman et al., 2005). ING2 interacts with the Smad-interacting transcriptional modulator SnoN and Smad2, thereby inducing transcription and cell cycle arrest in epithelial cells (Sarker et al., 2008). ING4 also inhibits human lung adenocarcinoma cell growth both in vitro and in vivo by inducing p27, suppressing cyclinD1, Skp2, and Cox2, and inactivating the Wnt-1/betacatenin pathway (Li et al., 2008c). Replicative senescence acts as an anti-tumour mechanism by preventing cancer development due to the ageing-related accumulation of genetic mutations in somatic tissues (Hornsby, 2005). Senescence, marked by terminal cell cycle arrest, limits the replicative capacity of cells, thus preventing the proliferation of cells that are at different stages of malignancy (Dimri, 2005). The expression levels of both ING1a and ING2 are dramatically induced during replicative senescence (Garkavtsev and Riabowol, 1997; Pedeux et al., 2005; Soliman et al., 2008). Overexpression of ING1a or ING2 induces a senescent phenotype in primary fibroblasts, whereas knockdown of ING1 or ING2 delays onset of senescence and results in a moderate extension of the replicative lifespan of diploid human fibroblasts  32  (Garkavtsev and Riabowol, 1997; Goeman et al., 2005; Pedeux et al., 2005). Furthermore, ING2 is shown to promote binding of p53 and p300 and enhanced p300-dependent acetylation of p53, thereby playing a positive role in encouraging p53-mediated replicative senescence (Pedeux et al., 2005). ING family proteins appear to also play a crucial role in apoptosis. Overexpression of ING1b enhances serum starvation-elicited apoptosis (Helbing et al., 1997). Coexpression of ING1b and p53 synergistically induce apoptosis in cancer cells derived from gliomas, and esophageal carcinomas (Shimada et al., 2002; Shinoura et al., 1999). In response to UV irradiation, ING1b binds to PCNA through its amino-terminus located PCNA-interacting protein (PIP) motif and contributes to UV-induced apoptosis (Scott et al., 2001b). Further studies demonstrate that ING1b may play a crucial role in the p53-mediated intrinsic apoptotic pathway by incorporating with p53 to activate the expression of the pro-apoptotic protein Bax upon UV irradiation and subsequentially modify the mitochondrial membrane potential in MMRU melanoma cells (Cheung and Li, 2002). In addition, both ING2 and ING3 induce apoptosis in RKO cells in a p53-dependent manner (Nagashima et al., 2001; Nagashima et al., 2003). Overexpression of ING2 also enhances UV-induced apoptosis in MMRU cells, which tends to be mediated by its LZL domain (Chin et al., 2005; Wang et al., 2006b). ING1b and ING2 are also involved in NER in a p53-dependent manner (Cheung et al., 2001; Kuo et al., 2007; Wang et al., 2006a), suggesting that ING proteins and p53 may regulate NER in a cooperative manner. Although they do not colocalize with UV-induced lesions, ING1b and ING2 enhance rapid histone H4 acetylation, chromatin relaxation, and the recruitment of XPA to photolesions after UV irradiation (Kuo et al., 2007; Wang et al.,  33  2006b), indicating the involvement of ING proteins and global histone acetylation and chromatin remodelling in DNA repair. Furthermore, the LZL domain of ING2 is important for ING2-p53 interaction and the proper functions of ING2 in NER (Wang et al., 2006b). Furthermore, certain ING proteins have also been shown to modulate cell migration, invasion and angiogensis. Although ING1b has no effect in melanoma cell migration and angiogenesis (Cheung and Li, 2002), overexpression of ING4 significantly inhibits stress fiber formation, cell migration and invasion in melanoma cells (Li et al., 2008b). It represses breast cancer cell colony formation efficiency in soft agar (Kim et al., 2004) and inhibits the activities of Rho A GTPase and MMP-2 and MMP-9 in melanoma cells (Li et al., 2008b). Other studies also demonstrate that ING4_v1 (originally identified as ING4) play an important role in suppressing cell spreading and cell migration by interacting with a novel binding partner, liprin alpha1, in the cytoplasm (Shen et al., 2007). Other variants of ING4 have different roles in cell migration. The variant 2 of ING4 (ING4_v2) loses a cell spreading suppressive effect but can still suppress cell migration, whereas the variant 4 of ING4 (ING4_v4) loses both cell spreading and migration suppression (Unoki et al., 2006). Furthermore, ING4 physically interacts with the p65 subunit of NF-κB and negatively regulates glioma angiogenesis by repressing the promoter activities of NF-κB-responsive genes (Garkavtsev et al., 2004; Nozell et al., 2008). It also reduces the expression of the proangiogenic molecules IL-8 and osteopontin (Colla et al., 2007), and suppresses the activity of hypoxia-inducible factor-1α (HIF-1α) in multiple myeloma cells (Colla et al., 2007; Ozer and Bruick, 2005). Many functions of ING proteins are enhanced by the functional wild-type p53 (Russell et al., 2006). It has been proposed that ING proteins facilitate p53-dependent  34  transcription through mechanisms including: a) opening the promoter chromatin of p53targeted genes, b) facilitating p53 acetylation mediated by ING-associated HATs, and c) inhibiting p53 deacetylase SIRT1 (Binda et al., 2008; Shi and Gozani, 2005). However, accumulated results show that ING proteins also regulate biological function independent of p53. ING1b is shown to impair cell proliferation in p53-null H1299 cells (Tsang et al., 2003). Results from knockout mouse studies reveal that ING1 also regulates apoptosis in a p53-independent manner as loss of ING1 induces Bax expression and increases DNA damage-induced apoptosis in primary cells and mice irrespective of p53 status (Coles et al., 2007). ING1b can also induce HSP70 expression and therefore mediates tumor necrosis factor alpha (TNF-alpha)-stimulated apoptosis in primary fibroblasts (Feng et al., 2006). 1.4.5  ING Family Proteins and Cancer ING family proteins are rarely mutated but frequently deregulated in human cancers  including basal cell carcinoma, bladder cancer, brain cancer, breast cancer, colorectal carcinoma, gastrointestinal cancer, head and neck squamous cell carcinoma, ovarian cancer, lymphoma, and melanoma (Campos et al., 2004; Gunduz et al., 2005; Lu et al., 2006; Nouman et al., 2002a; Okano et al., 2006; Takahashi et al., 2004). Recent studies demonstrate that the distorted expression levels of ING proteins are associated with cancer progression. Downregulation of ING1 mRNA correlates with the progression of astrocytomas and bladder cancers (Sanchez-Carbayo et al., 2003; Vieyra et al., 2003). However, increased ING1 expression correlates with cellular chemoresistance in cancer cell lines and correlates with a poorer patient survival in bladder cancer (Tallen et al., 2003). Downregulation of ING1 tumour suppressor sensitizes glioblastoma cells to cisplatininduced cell death (Tallen et al., 2003). Reduced ING2 expression correlates with the  35  progression of hepatocellular carcinoma and can predict the outcome of patients with hepatocellular carcinoma (Zhang et al., 2008). Reduced ING4 expression correlates with the progression of glioma, whereas the restoration of ING4 expression inhibits glioma tumour growth and angiogenesis in nude mouse model (Ozer and Bruick, 2005). Reduced ING4 expression also correlates with melanoma progression, predicts the outcome of patients with primary melanoma and is associated with the enhanced tumour invasive potential (Li et al., 2008b). Therefore, loss of ING expression may be involved in malignancy progression, but high ING expression may constrain cancer chemotherapy as ING proteins are able to enhance DNA repair rate. 1.4.6  Novel Tumour Suppressor ING3 The human ING3 gene is mapped to 7q31.3 and consists of 12 exons, which  produces two mRNA variants and encodes p47ING3 (ING3) and p11ING3, respectively (Kawaji et al., 2002; Nagashima et al., 2003). Alignment of ING3 protein in different species including human, mouse, rat, chicken, dog, frog and horse reveals that ING3 is highly conserved across the genome (Fig. 1.5). The yeast homologue of ING3, yng2, also exhibits a high degree of homologue with the first and last 100 residues of yng2 matching the equivalent regions in human ING3 (Doyon et al., 2004). ING3 protein has been shown to be ubiquitously expressed in normal human tissues and to modulate cell cycle control and apoptosis in RKO cells by regulating p53-mediated transcription of p21 and bax (Nagashima et al., 2003). ING3 is predominantly present in the NuA4 multisubunit complex and required for the HAT activity of Tip60 (Doyon et al., 2006; Doyon et al., 2004). The yeast homologue yng2 is also an important component of the yeast NuA4 complex and abrogation  36  of yng2 function results in genome-wide deficiency of nucleosomal histone H4 acetylation and an increased sensitization to DNA damage in S phase (Choy and Kron, 2002). The ING3 gene is rarely mutated in human head and neck squamous cell carcinomas (HNSCCs). However, 50% of primary HNSCCs exhibit decreased or no expression of ING3 mRNA (Gunduz et al., 2002) (Gunduz et al., 2008). 63% of tongue and larynx tumors with decreased ING3 expression showed a tendency of higher mortality (Gunduz et al., 2002). Although most clinicopathological variables were not correlated to ING3 downregulation, the 5-year survival rate of HNSCCs patients with normal to high ING3 expression is 60% compared with 35% in the patients with low ING3 expression (Gunduz et al., 2008). In addition, ING3 mRNA level may be an independent prognostic factor in patients with HNSCCs under a multivariate Cox regression model (Gunduz et al., 2008; Gunduz et al., 2002). 1.5 Objectives The objectives of this study are to evaluate the prognostic significance of the novel tumour suppressor ING3 in melanoma and its tumour suppressive roles in melanoma cells. Since understanding the regulatory pathway of tumour suppressors can contribute to the development of novel strategies for the prevention and treatment of cancer, this study also aims to investigate the possible mechanism leading to ING3 deregulation in melanoma.  37  Fig. 1.5 Alignment of ING3 proteins in different species. ING3 protein sequences in species including human, mouse, rat, chicken, dog, frog and horse were aligned with the Clustalx Software.  38  CHAPTER 2 MATERIALS AND METHODS 2.1 Construction of Tissue Microarray Formalin-fixed, paraffin-embedded tissues from 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas were used for the construction of a tissue microarray (TMA). All specimens were obtained from the 1990 to 1998 archives of the Department of Pathology, Vancouver General Hospital. The use of human skin tissues was approved by the medical ethics committee of the University of British Columbia and was performed in accordance with the Declaration of Helsinki guidelines. The most representative tumor areas were carefully selected and marked on the hematoxylin and eosin-stained slides. The TMA slides were assembled using a tissue-array instrument (Beecher Instruments, Silver Spring, MD) consisting of thin-walled stainless steel punches and stylets used to empty and transfer needle content. The assembly was held in an X-Y position guide equipped with semiautomatic micrometers, with a 1-mm increment between individual samples and 3-mm punch depth stop device. Briefly, the instrument was used to create holes in a recipient block with defined array cores. A solid stylet, which closely fit the needle, was used to transfer the tissue cores into the recipient block. Duplicate or triplicate 0.6-mm diameter tissue cores from each donor block were used to avoid the limitations of the representative areas of the tumor. Multiple 4-µm sections were cut with a Leica microtome. Sections were transferred to adhesive-coated slides using routine histology procedures.  39  2.2 Immunohistochemistry TMA slides were dewaxed at 55°C for 30 min and washed with xylene. Tissues were rehydrated by a series of washes in 100%, 95%, and 80% ethanol, and distilled water. Antigen retrieval was performed by heating the samples at 95°C for 30 min in 10 mM sodium citrate (pH 6.0). Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 20 min. After blocking with universal blocking serum for 30 min, slides were incubated with a polyclonal rabbit anti-ING3 antibody (Protein Tech Group, Chicago, IL) at 4°C overnight. The sections were then incubated with biotin-labeled secondary antibody and streptavidin-peroxidase for 30 min each, followed by developing with 3, 3'diaminobenzidine substrate and counterstained with hematoxylin. Slides were finally dehydrated and sealed with coverslips. As negative controls the ING3 antibody was omitted during primary antibody incubation. 2.3 Evaluation of TMA Immunostaining The evaluation of ING3 nuclear and cytoplasmic staining was made blindly by two independent observers (including one dermatopathologist) simultaneously, and a consensus score was reached for each core. The nuclear or cytoplasmic ING3 staining was scored into four grades according to their staining intensity: 0, 1, 2, and 3. Percentages of nuclear or cytoplasmic ING3-positive cells were also scored into four categories: 0 (0%), 1 (1-33%), 2 (34-66%), and 3 (67-100%). The sum of the intensity and percentage scores was used as the final ING3 nuclear or cytoplasmic staining score (Dai et al., 2005), defined as follows: 0-2, negative or weak; 3-4, moderate; and 5-6, strong. The sum of scores from nuclear and cytoplasmic ING3 staining was used as the final ING3 overall staining score, defined as follows: 0-4, negative or weak; 5-7, moderate; and 8-12, strong. Eighty percent of the  40  biopsies had uniform staining between different cores. In the 20% of cases with a discrepancy between different cores, the higher score was taken as the final score. The reason for differential ING3 staining in some biopsies could be that melanoma is a heterogeneous tumor, so different areas in the tumor may represent different stages of tumor progression. 2.4 Statistical Analysis of TMA Immunostaining Statistical analysis of ING3 staining was performed with SPSS 11.5 software. The chi-square test was used to compare the quantitative differences of overall, nuclear or cytoplasmic ING3 expression in different stages of melanoma progression. The Spearman test was used to analyze the correlation between nuclear and cytoplasmic ING3 staining and the correlation between ING3 staining and the clinicopathologic parameters including age, gender, tumor thickness, ulceration, and tumor location. The Kaplan-Meier method and logrank test were used to evaluate the correlations between nuclear ING3 staining and patient survival. Cox regression analysis was performed under a multivariate model including patient age, gender, tumor thickness, ulceration, tumor location and ING3 nuclear staining. P values less than 0.05 were considered statistically significant. 2.5 Cell Lines and Cell Culture The MMRU, MMAN and MMLH melanoma cell lines were kind gifts from Dr. H.R. Byers (Boston University School of Medicine, Boston, MA, U.S.A.). The MEWO, Sk-mel3, Sk-mel-24, Sk-mel-93, and Sk-mel-110 cell lines were kindly provided by Dr A.P.Albino (Memorial Sloan-Kettering Cancer Center, New York, U.S.A.). Sk-mel-5 and Sk-mel-28 cell lines were obtained from the Tissue Bank at the National Institutes of Health, U.S.A. KZ-13 cells were kindly provided by Dr H Silver (British Columbia Cancer Research 41  Center, Vancouver, BC, Canada). HCT116 p53 wild-type and HEK293T cell lines were purchased from the American Type Culture Collection.  HCT116 p53-/- cell line was  granted by Dr. B. Vogelstein (Johns Hopkins University, Baltimore, MD, U.S.A.). All the melanoma cell lines and HEK293T cell line were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Invitrogen, Burlington, ON, Canada) in the presence of 100 units/ml penicillin, 100 µg/ml streptomycin and 25 µg/ml amphotericin B. HCT116 cells were maintained in McCoy's 5A medium (Invitrogen) supplemented with 10% fetal bovine serum. Normal human epidermal melanocytes were purchased from PromoCell (Heidelberg, Germany) and cultured in melanocyte basal medium supplemented with 0.4% bovine pituitary extract, 50 ng/ml amphotericin B, 1.0 ng/ml basic fibroblast growth factor, 50 µg/ml gentamicin, 5.0 µg/ml hydrocortisone, 5.0 µg/ml bovine insulin, and 10 ng/ml phorbol 12-myristate 13-acetate (PromoCell). All cell lines were maintained in a 5% CO2 atmosphere at 37°C. 2.6 Plasmids and Transfection pcDNA3 vector was purchased from Invitrogen. pcDNA3-ING3 was a kind gift of Dr. M. Gunduz at Okayama University (Gunduz et al., 2002). The cDNA of ING3 was obtained from pcDNA3-ING3 with polymerase chain reaction (PCR) and sub-cloned into p3×FLAG vector (Sigma) between BglII and XbaI restriction sites to generate the FlagING3. Plasmids expressing ING3 truncated proteins, K96R or K103/105R point mutants were generated with PCR from Flag-ING3 using site-specific primers and sub-cloned into the p3×FLAG vector. All the constructs were sequenced across the newly created junctions to confirm no reading frame shift induced by polymerase chain reaction.  42  HA-tagged pcDNA3-Ubiquitin plasmid (HA-Ub) was a kind gift of Dr. R. Zhang from the University of Alabama at Birmingham (Zhang et al., 2008). HA-tagged vector, HA-Cul1 and HA-Roc1 plasmids were kindly provided by Dr. M. Pagano at New York University (Busino et al., 2007). HA-βTRCP, Myc-tagged vector and Myc-Skp2 plasmid were generously granted by Dr. S. Sun at Pennsylvania State University (Fong and Sun, 2002) and Dr. J. Hsieh from Washington University at St. Louis (Liu et al., 2007). Dominant negative caspase-8 plasmid (DN-Caspase-8) was a gift of Dr. K. Arul at Massachusetts Institute of Technology (Cambridge, MA, U.S.A.) (Keiler et al., 1996). Plasmids were transfected into cells at 50-70% confluency using the Effectene transfection kit (Qiagen, Mississauga, ON, Canada) according to manufacturer’s procedures. 2.7 siRNA and Transfection The  sequences  of  ING3  siRNA  (Ambion,  Austin,  TX,  U.S.A.)  were  GCUGAUAAUGCUGGAAUU AUU (sense) and UAAUUCCAGCAUUAUCAG CUU (antisense).  The  sequences  of  p53  siRNA  (Qiagen)  were  GCAUGAACCGGAGGCCCAUdTdT (sense) and AUGGGCCUCCGGUUCAUGCdTdT (antisense). The Fas siRNA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). The Cul1 siRNA oligomers were synthesized by Dharmacon (Lafayette, CO, U.S.A.) and the sequence was: 5’-CGAAGAGUUCAGGUUUACC-3’ (Chaturvedi et al., 2005). At 50-60% confluency, cells were transfected with siRNA using SiLentFect reagent (Bio-Rad, Hercules, CA, U.S.A.) according to the manufacturer’s instruction. 2.8 Antibodies Normal mouse and goat serum IgG was purchased from Santa Cruz biotechnology. The primary antibodies included rabbit anti-ING3 (Proteintech Group Inc, Chicago, IL. 43  U.S.A.), goat anti-ING3 (Abcam, Cambridge, MA, U.S.A.), rabbit anti: -Bax, Bcl-2, caspase-3, caspase-8, caspase-9 (Cell Signaling, Beverly, MA, U.S.A.), rabbit anti-Bid (BD Pharmingen, Mississauga, ON, Canada), rabbit anti-PARP (Oncogene, Cambridge, MA, U.S.A.), mouse anti: -p53, Bad, Noxa, Fas, Fas-L, -PSMC4, -α4, -Chk1, -Cul1, Skp2, actin (Santa Cruz Biotechnology), mouse anti: -HA and -Myc (Epitope Biotech, Vancouver, BC, Canada), mouse anti-Flag (Applied Biological Materials Inc., Richmond, BC, Canada) antibodies. 2.9 Generation of ING3 Stable Clones MMRU cells in 35-mm dishes were transfected with pcDNA3 or pcDNA3-ING3 and incubated at 37°C for 24 h. Cells were then reseeded into 100-mm dishes and selected in culture medium supplemented with 800 µg/ml G418 (Sigma, St. Louis, MO, U.S.A.) for two weeks. Single clones were then isolated and expanded in the culture medium containing 200 µg/ml G418. The expression levels of ING3 in isolated clones were examined by Western blot and reverse transcriptase-PCR assays. The clones with at least twofold higher expression of ING3 than that of parental MMRU cells were kept and used for in vitro studies. 2.10 UV Irradiation A bank of four FS40 sunlamps (Westinghouse, Bloomfield, NJ) was used for UVB irradiation. The intensity of the UV light was measured by an IL 700 radiometer fitted with a WN 320 filter and an A127 quartz diffuser (International Light, Inc., Newburyport, MA). Cells were rinsed with phosphate buffered saline (PBS) and the Petri dish cover was left on to filter possible UVC emissions during the irradiation. Immediately after irradiation, cells were washed with phosphate-buffered saline, re-fed with complete media and returned to 44  37°C. Non-irradiated controls were kept from light exposure but subjected to all other manipulations. 2.11 Light Microscopy The morphology of MMRU cells and MMRU cells stably transfected with pcDNA3ING3 were subjected to microscopic assessment 24 hours after seeding, using an inverted microscope (Zeiss, Chester, VA). Images were taken using a cooled mono 12-bit E× camera (Q-imaging, Burnaby, BC, Canada). 2.12 Cell Survival Assay Cells in 24-well plates at 80% confluency were irradiated with UVB. Twenty-four hours after UV irradiation, cell survival was determined with the sulforhodamine B (SRB) (Sigma) assay as described previously (Li et al., 1998a). Briefly, after the medium was removed, cells were fixed with 500 µl of 10% trichloroacetic acid for 1 h at 4°C. Cells were then washed with tap water, air-dried and stained with 500 µl of 0.4% SRB (dissolved in 1% acetic acid) for 30 min at room temperature. The cells were then destained with 1% acetic acid, and air-dried. For quantification, the cells were incubated with 500 µl of 10 mM Tris (pH 10.5) on a shaker for 20 min to dissolve the bound dye followed by colorimetric determination at 550 nm for 100 µl aliquots. 2.13 Flow Cytometry Cells (1×106) were collected by trypsinization and pelleted by centrifugation at 2000× g for 2 min. After washing twice with cold PBS, cell pellets were resuspended in 1 ml of hypotonic fluorochrome buffer [0.1% Triton X-100, 0.1% sodium citrate] containing 50 µg/ml propidium iodide (PI) (Sigma) and 25 µg/ml RNase A. The samples were then  45  incubated at 4°C for 30 min in the dark, and then analyzed by flow cytometry to determine the percentage of subdiploid DNA. Samples were run on a Coulter EPICS XL-MCL flow cytometer (Beckman Coulter, Mississauga, ON, Canada) and analyzed with EXPO32 ADC analysis software. About 10,000 cells of each sample were analyzed each time. To examine the apoptotic cells after UV irradiation, both floating and adherent cells were collected for the flow cytometry analysis. Cells in Sub-G1 phase were considered apoptotic. To differentiate the cells in apoptosis from necrosis, cells were collected as abovementioned and incubated with 100 µl binding buffer [10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2] containing 5 µl annexin V-FLUOS and 5 µl PI solution (Roche Diagnostics GmbH) for 30 min in the dark. Viable cells are nonfluorescent; cells in the metabolically active stages of apoptosis are stained with annexin (green fluorescence) but not with PI (red fluorescence);  necrotic cells are stained with PI staining; cells undergoing late stage  apoptosis bind both annexin and PI. 2.14 Measurement of Caspase-8 Activity Caspase-8 activity was determined using ApoAlert Caspase-8 colorimetric assay kits (Clontech, Palto Alto, CA, U.S.A.). Briefly, cells (2×106) were treated with or without 600 J/m2 UVB. After incubation for 5 h cells were harvested and lysed. Cell lysate was incubated in the provided reaction buffer containing 200 µM Ile-Glu-Thr-Asp-p-nitroaniline (IETD-NA) and 10 mM dithiothreitol (DTT) at 37°C for 1h. Liberated p-NA was monitored colorimetrically by absorbance at 405 nm. The protease activity of Caspase-8 was determined by comparing the absorbance of p-NA in UV-irradiated cells with those in nonirradiated cells.  46  2.15 Hoechst Staining Cells grown on coverslips in 6-well plates were irradiated with UVB at 600 J/m2. Twenty-four hours later, cells were fixed with fixation buffer (2% formaldehyde, 0.5% Triton X-100 in PBS, pH 7.2) and incubated for 45 min at room temperature. The fixed cells were washed with 0.1% Triton X-100/PBS for 5 min and then stained with a mixture of 2.5 µg/ml Hoechst 33258 (Sigma), PBS/0.1% Triton X-100 at room temperature for 5 min. Coverslips were washed twice with PBS before being mounted onto slides with mounting media (Fisher Scientific, Nepean, ON, Canada). The slides were visualized under a fluorescent microscope (Zeiss, Chester, VA, U.S.A.) for apoptotic bodies. Images were taken with a cooled mono 12-bit Retiga-Ex camera. 2.16 Western Blot Analysis Cells were washed with cold PBS, harvested by scraping on ice and pelleted by centrifugation at 2,000 g for 2 min. Cells pellets were lysed in 80 µl of triple detergent buffer (50mM Tris-Cl, (PH 8.0), 150 mM NaCl, 0.02% NaN3, 0.1% SDS, 1% NP-40, 0.5% sodium  deoxycholate)  containing  freshly  added  protease  inhibitors  (100µg/ml  phenylmethylsulfonyl fluoride, 1µg/ml aprotinin, 1µg/ml leupeptin, 1µg/ml pepstatin A). The samples were then sonicated, incubated on ice for 30 min, and centrifuged at 12,000 × g for 30 min at 4°C. The supernatants were collected and a Bradford assay was performed to determine the protein concentration. Proteins (50 µg/lane) were separated on 12% SDSpolyacrylamide gels and blotted onto polyvinylidene difluoride (PVDF) membranes (BioRad, Mississauga, ON, Canada). The PVDF was blocked with 5% skim milk for 1 h at room temperature before incubating with primary antisera prepared in 5% bovine serum albumin for 1 h at room temperature. Blots were washed three times in PBST (PBS containing 0.04%  47  Tween-20) for 5 min each and then incubated with horseradish peroxidase (HRP)conjugated secondary antisera (Jackson ImmunoResearch Laboratory, West Grove, PA, U.S.A.) for 1 h at room temperature. The signals were detected with enhanced chemiluminescence detection kit (Amersham Bioscience, Baie d’Urfe, Quebec, Canada). Alternatively, blots were incubated with secondary antibodies labelled with the near-infrared fluorescent dyes IRDye800 or IRDye 680 (LI-COR Biosciences, Lincoln, NE, U.S.A.) for 1 h at room temperature, followed by scanning on the Odyssey Infrared Imaging System to visualize proteins (LI-COR Biosciences). The protein expression levels were quantitated with the Quantity One software (Bio-Rad) or ImageJ software (National Institutes of Health, U.S.A.). The fold-induction or reduction was corrected for differences in the actin loading control. 2.17 Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Total RNA from melanoma cells and normal human melanocytes was extracted by TRIzol reagent (Invitrogen). The RNA concentrations were measured with a spectrometer. 2 µg of total RNA was reverse-transcribed into cDNA with the SuperScript First-Strand Synthesis System (Invitrogen) according to the manufacturer's protocol. Hotstart PCR system was performed with the Taq DNA polymerase reaction system (Qiagen). The sequences of human ING3 primers were 5'-CAGCCTCTTCTAACAATGCCTA-3' (sense) and 5'-CTTCATCAAACAAAAGGACCAC-3' (antisense). The primers for human glyceraldehyde-3-phosphate CTCATGACCACAGTCCATG  dehydrogenase CCATC-3'  (GAPDH)  were  5'-  (sense)  and  5'-  CTGCTTCACCACCTTCTTGATGTC-3' (antisense) that served as an input control. Amplification was carried out for as follows: i) denaturation at 95°C for 1 min, ii) annealing  48  at 55°C for 1 min and iii) polymerization at 72°C for 1min. The reaction was repeated for 32 cycles for ING3 and 25 cycles for GAPDH. PCR products were electrophoresed on 1% agarose gel stained with ethidium bromide (10mg/ml). The gel was viewed under UV light. 2.18 Immunoprecipitation Cells were lysed in NP-40 lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1% NP-40). Pre-cleared lysates (~300 µg of proteins) were incubated for 2 h at 4°C with 2 µg of monoclonal anti-Flag, HA, Cul1, or Skp2 antibodies and then precipitated with 40 µl of fresh Protein-G agarose beads (GE Healthcare Bioscience, Uppsala, Sweden) at 50% slurry overnight at 4°C. Mouse IgG (Santa Cruz Biotechnology) was used as a negative control. The beads were then pelleted by centrifugation at 10,000 × g for 30 sec, washed thrice with IP washing buffer (10 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA) and boiled in 2 × Tris–glycine SDS sample buffer for Western blot analysis. 2.19 Immunofluorescent Staining Cells were seeded on coverslips and transfected with wild-type (WT) or K96R mutant Flag-ING3 for 24 h. Cells were fixed with 2 ml of fixation solution (2% paraformaldehyde, 0.5% Triton X-100 in PBS) for 30 min at 4oC. Cover-slips were then washed three times for 5 min each with PBS and then blocked 1 h in 1% BSA at 4°C. Cells were then gently inverted in mouse anti-Flag antibody at a dilution of 1:200 for 1 h at room temperature. Cover-slips were washed three times for 5 min each with PBS and incubated at room temperature for 1 h with Cy5-conjugated goat anti-mouse (Jackson ImmunoResearch) at a dilution of 1:500. DNA was stained with 1 µg/ml Hoechst 33258 for 1 min. Cover-slip were then washed three times and mounted with Permount mounting media (Fischer 49  Scientific). Pictures were captured using a CCD camera under a Zeiss Axioplan 2 microscope (ZEISS, Jena, Germany). 2.20 Isolation of Proteasomes MMRU cells were harvested and lysed in 50 mM Tris-HCl buffer [pH 7.4] containing 10 mM NaCl, 5mM MgCl2, 0.5% NP-40, 1 mM DTT, 1 mM PMSF and a protease inhibitor cocktail. Pelleted nuclei were lysed in 20 mM HEPES buffer [pH 7.9] containing 0.35 M NaCl, 1 mM EDTA, 20% Glycerol, 1 mM DTT, 1 mM PMSF and a protease inhibitor cocktail for nuclear protein extraction. The nuclear extracts were then used for the isolation of proteasomes as previously described (Garate et al., 2008). Briefly, 26S proteasomes were precipitated in the presence of ammonium sulfate added to 38% saturation, while 20S proteasomes were precipitated in the presence of ammonium sulfate added to 70% saturation. The precipitated fractions were loaded on a Superose 6 column (GE Healthcare), previously calibrated with a HMW gel filtration calibration kit (GE Healthcare). Fractions were collected for Western Blot analysis of the 26S proteasome marker PSMC4 and the 20S proteasome marker α4. 2.21 In Vitro Degradation Assay The 26S containing fraction from Section 2.20 was then electrophoresed through a native 4.5% polyacrylamide gel, where the 26S proteasome complex was identified by overlay staining using the fluorogenic substrate Suc-LLVY-AMC (Biomol Int., Plymouth Meeting, PA) as described (Vieyra et al., 2003). The band containing 26S proteasome was excised and eluted from the gel. For in vitro degradation assay, 50 µg of nuclear extracts were incubated at 37°C in the presence or the absence of 50 ng of isolated 26S proteasome for different time periods. 50  2.22 In Vivo Ubiquitination Assay In vivo ubiquitination was assayed as described (Campos et al., 2004). Briefly, cells were treated with 2 µM MG132 (Calbiochem, La Jolla, CA, U.S.A.) for 6 h after transfection with HA-Ub plasmid or cotransfection together with Flag-ING3 plasmid for 24 h. Cells were then harvested and lysed in modified RIPA buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, EDTA 1 mM) containing a protease inhibitor cocktail and 10 mM N-ethylmaleimide (NEM, Sigma) to inhibit ubiquitin hydrolases. Mouse anti-HA monoclonal antibody was used to precipitate HA-tagged ubiquitinated proteins that were then denatured and resolved on 8% SDS-PAGE for the analysis of ING3 or Flag-ING3. 2.23 Statistical Analysis for In Vitro Studies The data were presented as the mean ± SD. Statistical analyses were performed using student-t test and p values < 0.05 were considered significant.  51  CHAPTER 3 DEREGULATION OF ING3 IN MELANOMA  3.1 Rationale and Hypothesis ING3 expression is significantly reduced in human head and neck squamous cell carcinomas (HNSCCs) and higher mortality has been observed in cases with decreased ING3 expression (Gunduz et al., 2002). Although a comparative genomic hybridization (CGH) analysis found that the chromosome 7q31-q34 region, which contains the ING3 gene, is frequently amplified in melanoma cells (Tanami et al., 2004), the expression level of ING3 and the cytogenetic characteristics of the ING3 gene in malignant melanoma biopsies has never been examined. In this study we hypothesize that the expression of ING3 is distorted in melanomas which may be associated with melanoma development. We used tissue microarray (TMA) technology and immunohistochemistry to evaluate ING3 staining in different stages of human melanocytic lesions and analyzed the correlation between ING3 staining and clinicopathologic parameters and patient survival. 3.2 Results 3.2.1 Correlation between ING3 Staining and Melanoma Progression Various levels of ING3 staining were observed in dysplastic nevi and melanoma biopsies (Fig. 3.1). Strong overall ING3 staining was obtained in 71%, 71% and 52% of the biopsies in dysplastic nevi, primary melanomas, and melanoma metastases, respectively (Fig. 3.2A). The difference of overall ING3 staining (negative-to-moderate vs. strong staining) between metastatic melanoma and dysplastic nevi or primary melanoma was significant (P < 0.05, χ2 test). Strong nuclear ING3 staining was recorded in 60%, 33% and  52  10% of the biopsies in dysplastic nevi, primary melanomas, and melanoma metastases, respectively (Fig. 3.2B). Significant differences for nuclear ING3 staining (negative-tomoderate vs. strong staining) were observed between dysplastic nevi and primary melanomas (P < 0.001, χ2 test), between dysplastic nevi and melanoma metastases (P < 0.001, χ2 test), and between primary melanomas and melanoma metastases (P < 0.001, χ2 test). In addition, strong cytoplasmic ING3 staining was also recorded in 24%, 39% and 62% of the biopsies in dysplastic nevi, primary melanoma, and melanoma metastases, respectively (Fig. 3.2C). Significant differences for the cytoplasmic ING3 staining (negative-to-moderate vs. strong staining) were observed between dysplastic nevi and primary melanomas (P < 0.05, χ2 test), between primary melanomas and melanoma metastases (P < 0.05, χ2 test) and between dysplastic nevi and melanoma metastases (P < 0.001, χ2 test). To investigate if the reduced nuclear ING3 staining was due to a subcellular compartment shift to the cytoplasm, we evaluated ING3 staining in the cytoplasm. In addition, reduced nuclear ING3 staining was positively associated with increased cytoplasmic ING3 staining in either primary melanomas or metastatic melanomas (Fig. 3.2D; P = 0.001 and 0.042, respectively, Spearman test).  53  Fig. 3.1 Representative images of ING3 immunohistochemical staining in human melanocytic lesions. A, Strong nuclear ING3 staining in dysplastic nevi; B, Moderate nuclear ING3 staining in primary melanoma; C, Weak nuclear ING3 Staining and strong cytoplasmic ING3 staining; D, Weak overall ING3 staining in metastatic melanoma.  54  Fig. 3.2 Correlation between ING3 staining and melanoma progression. A-C, Overall (A), nuclear (B) and cytoplasmic (C) ING3 staining in dysplastic nevi (DN), primary melanoma (PM) and metastatic melanoma (MM). D, Reduced nuclear ING3 staining is correlated with increased cytoplasmic ING3 staining in primary and metastatic melanoma. Asterisks indicate statistical significance (* p < 0.05, ** p < 0.01, *** p<0.001)  55  3.2.2 Reduced Nuclear ING3 Staining Correlates with Tumour Location The clinicopathologic features of 114 primary melanomas are shown in Table 3.1. There were 64 men and 50 women, with the age ranging from 21 to 93 years (median, 56 years). For melanoma staging, we used Breslow thickness as our criteria for evaluating the expression of ING3: 76 tumours were less than 2.0 mm thick (defined as low-risk primary melanoma), and 38 were thicker than 2.0 mm (defined as high-risk primary melanoma). Nineteen melanomas were located at sun-exposed sites (head and neck), and 95 melanomas were located at sun-protected sites (other locations including trunk, arm, leg and feet). Ulceration was observed in 16 cases of melanoma. To assess whether reduced nuclear ING3 staining correlates with the transition of melanoma from RGP to VGP, we examined the correlation between ING3 staining and tumour thickness in 114 primary melanomas. Thirty-six percent (27/76) and thirty-two percent (12/38) of tumours had strong ING3 nuclear staining in low-risk and high-risk primary melanomas, respectively. The difference was not statistically significant (P > 0.05). In addition, no significant difference was observed between nuclear ING3 staining and other clinicopathologic parameters including tumour subtype, ulceration, patient’s age or gender (Table 3.2). Since UV radiation is the main environmental factor for melanoma formation, we also compared the ING3 staining in tumours occurring at sun-exposed with that at sunprotected sites. Reduced nuclear ING3 staining (negative-to-moderate vs. strong staining) significantly correlated with the location of primary melanomas (Fig. 3.3A; P < 0.05, Spearman test). Thirty-seven percent (35/95) of tumours at sun-protected sites had strong ING3 nuclear staining, whereas strong nuclear ING3 staining was only observed in 10%  56  (2/19) of tumours at sun-exposed sites (head and neck). However, increased cytoplasmic ING3 staining did not significantly correlate with the location of primary melanomas (Fig. 3.3B; P > 0.05, Spearman test), although the strong ING3 cytoplasmic staining increased from 38% in primary melanomas occurred at sun-protected sites to 55% in that at sunexposed sites.  57  Table 3.1. Clinicopathologic features of 114 primary melanomas  Age, years ≤56 >56 Gender Male Female Tumour thickness, mm <2.0 ≥2.0 Ulceration Absent Present Tumour subtypea SSM LMM Others Siteb Sun-exposed Sun-protected  No. of Patients  %  56 58  49 51  64 50  56 44  76 38  67 33  98 16  86 14  52 17 45  46 15 39  19 95  17 83  a  SSM, superficial spreading melanoma; LMM, lentigo malignant melanoma; Others include  desmoplastic melanoma, acrolentigous melanoma and nodular melanoma. b  Sun-protected sites: trunk, arm, leg and feet; Sun-exposed sites: head and neck.  58  Table 3.2 Nuclear ING3 staining and clinicopathologic characteristics of primary melanomas Nuclear ING3 staining Neg-to-mod Strong Age, years ≤56 >56 Gender Male Female Tumour thickness, mm <2.0 ≥2.0 Ulceration Absent Present Tumour subtypea SMM LMM others Tumour sitec Sun-protected Sun-exposed  Total  P valuea  39 37  17 21  56 58  >0.05  38 38  26 12  64 50  >0.05  49 26  27 12  76 38  >0.05  65 11  33 5  98 16  >0.05  37 9 30  15 8 15  52 17 45  60 9  35 10  95 19  >0.05  <0.05  a  Spearman test for cases with strong nuclear ING3 staining versus the cases with negative-  to-moderate (neg-to-mod) nuclear ING3 staining. b  SSM, superficial spreading melanoma; LMM, lentigo malignant melanoma; Others  including desmoplastic melanoma, acrolentigous melanoma and nodular melanoma. c  Sun-protected sites: trunk, arm, leg and feet; Sun-exposed sites: head and neck  59  Fig. 3.3 Correlation between ING3 staining and the location of melanomas. A, Reduced nuclear ING3 staining is correlated with sun-exposed tumour sites in primary melanomas (P < 0.05). B, Increased cytoplasmic ING3 staining did not significantly correlate with tumour location in primary melanomas (P > 0.05). Asterisks indicate statistical significance (* p < 0.05)  60  3.2.3 Reduced Nuclear ING3 Staining Correlates with 5-year Patient Survival To evaluate whether the reduced nuclear ING3 staining in human primary melanomas correlates with a worse prognosis, Kaplan-Meier survival curves were constructed using overall or disease-specific 5-year survival to evaluate the biopsies with strong nuclear ING3 staining to those with negative-to-moderate nuclear ING3 staining. The overall 5-year survival rate dropped from 87% in patients with the strong nuclear ING3 staining to 74% in patients showing negative-to-moderate nuclear ING3 staining, but they were not significantly correlated (P = 0.119, log-rank test; Fig. 3.4A). On the other hand, the disease-specific 5-year survival rate dropped from 97% in patients showing strong nuclear ING3 staining to 82% in those with negative-to-moderate nuclear ING3 staining, and this correlation between reduced ING3 nuclear staining and a poorer 5-year disease-specific survival was statistically significant (P < 0.05, log-rank test; Fig. 3.4C). Since the majority of death cases occurred in the high-risk group (tumour thickness ≥ 2.0 mm) and only 1 of 76 patients in the low-risk group (tumour thickness < 2.0 mm) died, we analyzed the correlation between reduced nuclear ING3 staining and patient survival in high-risk primary melanomas. We found that the overall survival rate was 67% in patients with strong nuclear ING3 staining compared with 35% in patients with negative-to-moderate nuclear ING3 staining although this correlation only showed a border line significance (P = 0.0601, logrank test; Fig. 3.4B). In addition, the disease-specific survival rate was 93% in patients with strong nuclear ING3 staining compared with 44% in patients with negative-to-moderate nuclear ING3 staining, and this correlation was statistically significant (P = 0.004, log-rank test; Fig. 3.4D).  61  Fig. 3.4 Correlation between nuclear ING3 staining and 5-year survival in patients with primary melanomas. (A, B) Patients with strong nuclear ING3 staining have a better 5-year overall survival than those with negative-to-moderate ING3 staining in 114 primary melanomas (PM) (A) and 38 high-risk PM (B) although they are not statistically significant. (C, D) Patients with strong nuclear ING3 staining have a significantly better 5-year diseasespecific survival than those with negative-to-moderate ING3 staining in PM (C) and 38 high-risk PM (D).  62  3.2.4 Nuclear ING3 Staining Independently Predicts 5-year Patient Survival We then examined whether nuclear ING3 is an independent prognostic marker for melanoma. We performed a multivariate Cox regression analysis including patient age, gender, tumour thickness, ulceration and location, and ING3 nuclear staining for 114 primary melanomas. Besides the tumour thickness being an independent prognostic marker for disease-specific survival, the nuclear ING3 level showed significance in predicting patient outcome independently of other clinicopathologic parameters for disease-specific survival (relative risk, 9.31; 95% CI, 1.13 to 76.5; P < 0.05; Table 3.3). In high-risk primary melanoma, nuclear ING3 staining showed a boarder-line significance in predicting the patient outcome as an independent factor (relative risk, 8.34; 95% CI, 0.959 to 72.6; P = 0.055; Table 3.3), while tumour thickness did not show significance in predicting patient outcome independently of other clinicopathologic parameters for disease-specific survival (P = 0.151; Table 3.3).  63  Table 3.3 Multivariate Cox regression analysis of ING3 nuclear staining in 114 cases of primary melanoma.  Variable Gender Age Thickness Site Ulceration ING3  All primary melanomas Relative Risk 95% CI .471 .151-1.47 .648 .159-2.64 54.8 6.43-470 .910 .304-2.72 .708 .218-2.30 9.31 1.13-76.5  P .194 .545 .000 .865 .566 .038  High-risk melanomas Relative Risk 95% CI .477 .145-1.56 .430 .097-1.92 .391 .108-1.41 1.30 .397-4.28 .769 .216-2.74 8.34 .959-72.6  P .222 .268 .151 .662 .685 .055  Coding of variables: ING3 nuclear staining was coded as 0 (negative-to-moderate staining) and 1 (strong staining); Gender was coded as 0 (male) and 1 (female); Age was coded as 0 ( 56 years) and 1 (< 56 years); Thickness was coded as 0 (≥2.0 mm) and 1 (< 2.0 mm) in all primary melanomas or 0 (≥ 4.0 mm) and 1 (< 4.0 mm) in high-risk melanomas (thickness ≥ 2.0 mm); Ulceration was coded as 0 (absent) and 1 (present); Site was coded as 0 (head and neck) and 1 (other sites).  64  3.3 Discussion Acquired resistance to apoptosis is a major hallmark of cancers (Hanahan and Weinberg, 2000), which allows cancer cells to escape the apoptotic death induced by anticancer agents and enables the establishment of tumours. Human cutaneous malignant melanoma is a particularly aggressive type of cancer in this regard. Its metastatic form is very resistant to conventional radiotherapy and chemotherapy (Lens and Eisen, 2003; Soengas and Lowe, 2003; Tsao and Sober, 2005). In this study, using tissue microarray and immunohistochemistry technology, we, for the first time, demonstrated that ING3 expression is deregulated in melanomas compared with dysplastic nevi. This finding is consistent with the report by Gunduz et al showing that ING3 is reduced in HNSCCs (Gunduz et al., 2002). However, different mechanisms may be involved for the reduction of ING3 expression in HNSCC and melanoma. 50% of the HNSCC cases showed reduced or absent ING3 mRNA and 48% of cases had allelic deletion in 7q31 region (Gunduz et al., 2002). However, reduced nuclear ING3 levels in melanomas was positively correlated with increased cytoplasmic ING3 levels indicating that in melanoma, translocation of ING3 from the nucleus to cytoplasmic compartment (Fig. 3.3). By analogy, the mRNA level of ING1b, a homologue of ING3, is decreased in 61% of human exocrine pancreatic carcinoma (Yu et al., 2004), 42% of non-small cell lung cancer (Kameyama et al., 2003), and 44% of primary breast cancers (Toyama et al., 1999), while the translocation of ING1b from nucleus to cytoplasm was observed in invasive breast cancer (Nouman et al., 2003), childhood acute lymphoblastic leukaemia (Nouman et al., 2002b), and melanoma (Nouman et al., 2002a), which may be caused by the sequestration of ING1b by 14-3-3 proteins in the cytoplasm (Gong et al., 2006). The mechanism controlling ING3 nuclear-to-cytoplasmic translocation  65  remains to be determined. In addition, the overall ING3 staining was also significantly reduced in advanced melanomas compared with dysplastic nevi and primary melanomas (Fig. 3.2A), suggesting that mechanisms other than ING3 nuclear-to-cytoplasm translocation play an important role in the deregulation of ING3 in advanced melanomas. Future studies such as analyzing the mRNA level of ING3, mutational status of ING3 gene and protein degradation will help understand the possible mechanisms leading to deregulation of ING3 in melanomas. Although the nuclear ING3 staining was significantly reduced in melanomas compared to dysplastic nevi, only 60% (35/58) of the dysplastic nevi showed strong nuclear ING3 staining and 24% (14/58) of them had strong ING3 staining in the cytoplasm. This suggests that the ING3 nuclear-to-cytoplasm translocation may be an early event in melanocytic neoplasia. In support of this idea, reduced nuclear ING3 staining was positively correlated with the tumour location at sun-exposed sites (Fig. 3.3A), indicating a crucial role of UV radiation in regulating nuclear ING3 levels. However, although increased strong cytoplasmic ING3 staining was observed in tumours at sun-exposed sites, the correlation between cytoplasmic ING3 localization and sun exposure was not significant (Fig. 3.3B). This may be explained by the fact that the sample volume of melanomas occurred at sunexposed sites was much smaller compared with that occurred at sun-protected sites in this study (19 vs. 95). Another possible explanation for this discrepancy is that the nuclear-tocytoplasmic translocation of ING3 may be only partially responsible for reduced nuclear ING3 staining after UV irradiation since 32% (6/19) of tumours at sun-exposed sites with reduced nuclear staining did not show strong cytoplasmic ING3 staining.  66  Defects in the apoptosis pathway including Apaf-1, Puma and XAF1mostly affect the initiation step in melanoma pathogenesis (Dai et al., 2004), while activated survival pathways by increased integrin-linked kinase and phospho-Akt levels or reduced PTEN expression often correlates with tumour progression (Dai et al., 2003; Dai et al., 2005). In this study, reduced nuclear ING3 levels correlated with the progression from primary melanoma to metastatic melanoma, indicating that ING3 plays a role not only in the initiation of melanoma, but also in melanoma progression. In agreement with the correlation between reduced ING3 expression and a poorer patient survival in HNSCCs (Gunduz et al., 2002), reduced nuclear ING3 levels significantly correlated with a poorer 5-year diseasespecific survival of patients with primary melanoma (Fig. 3.4C-D) and is likely an independent factor predicting patient outcome (Table 3.3). With TMA and immunohistochemistry, our lab have recently found that both nuclear ING2 levels and overall ING4 expression were significantly reduced in human melanomas compared with dysplastic nevi (Li et al., 2008a; Lu et al., 2006). Although there was no correlation between nuclear ING2 levels and clinicopathological parameters, ING4 staining is inversely associated with melanoma thickness, ulceration (P = 0.034 and 0.002, respectively, χ2 test) as well as poor overall and disease-specific 5-year survival of primary melanoma patients (P = 0.0002 and 0.001, respectively, χ2 test) (Li et al., 2008a). Cox regression analysis also revealed that the reduced ING4 level is an independent factor for the poor prognosis of patients with primary melanomas. In an earlier report, Nouman et al also demonstrated that nuclear ING1b levels were significantly decreased and shifted to the cytoplasm in melanocytic lesions (Nouman et al., 2002a). These results, together with our current data, suggest that ING family proteins play a crucial role in the development of  67  melanoma. This is also supported by our in vitro studies showing that both ING1b and ING2 significantly enhance the repair of UV-damaged DNA and promote apoptosis in melanoma cells in a p53-dependent pathway (Cheung and Li, 2002; Cheung et al., 2001; Chin et al., 2005; Wang et al., 2006a). Overexpression of ING4 significantly inhibits stress fiber formation, cell migration and invasion in melanoma cells by inhibiting the activities of Rho A GTPase and MMP-2 and MMP-9 in melanoma cells (Li et al., 2008a). The biological roles of ING3 in melanoma cells remain to be explored. ING proteins have been known to cooperate with p53 and modulate its downstream signalling pathways (Campos et al., 2004). Considering that the mutation of p53 gene is rare in melanoma (Albino et al., 1994; Montano et al., 1994; Ragnarsson-Olding et al., 2002), the aberrant expression of ING family tumour suppressors may be an important step during melanoma development and progression. Future studies on the mutational status of ING proteins and their cooperative roles in melanoma tissues will provide new insights on the mechanisms of aberrant expression of ING proteins and biological functions in melanoma tumourigenesis. In conclusion, the data presented in this report demonstrated that nuclear ING3 staining is significantly reduced with human melanoma progression. Reduced nuclear ING3 levels correlated with increased cytoplasmic ING3 levels. Strikingly, reduced nuclear ING3 levels also correlated with a worse 5-year disease-specific survival of primary melanoma patients and may be an independent prognostic factor for primary melanomas. These data indicate that ING3 may play an important role in both melanoma initiation and progression and serve as a promising prognostic marker and therapeutic target for malignant melanoma.  68  CHAPTER 4 ING3 ENHANCES UV-INDUCED APOPTOSIS OF MELANOMA CELLS  4.1 Rationale and Hypothesis ING3 can inhibit the growth of RKO cells and NIH3T3 cells, and stimulate apoptosis of RKO cells by upregulating Bax expression in a p53-dependent manner (Doyon et al., 2004; Nagashima et al., 2003). In melanoma, we have demonstrated that ING3 expression is deregulated in melanoma biopsies (Wang et al., 2007). However, its cellular role in melanoma development is not clear. UV radiation is the major environmental factor for melanoma development. The expression of ING family proteins ING1b and ING2 are able to be induced by DNA damaging agents including UV irradiation in some cell types (Cheung and Li, 2002; Nagashima et al., 2003; Scott et al., 2001a). In response to UV irradiation, both ING1b and ING2 promote UV-induced apoptosis in a p53-dependent manner (Cheung and Li, 2002; Chin et al., 2005; Scott et al., 2001b), and enhance p53mediated repair of UV-damaged DNA (Cheung et al., 2001; Wang et al., 2006a). However, the role of ING3 in cellular response to UV irradiation is not clear. In this study, we hypothesized that ING3 may enhance UV-induced apoptosis in melanoma cells. 4.2 Results 4.2.1 ING3 Is Induced by DNA Damaging Agents To test our hypothesis that ING3 may play an important role in the cellular response to UV irradiation, we analyzed ING3 expression level after UVB irradiation in melanoma cells. After UV irradiation, we found that the level of p53 steadily increased within 24 h (Fig. 4.1A). ING3 protein levels were rapidly induced in MMRU melanoma cells as early as 69  0.5 h after UVB irradiation, reached the peak at 6 h (4.6-fold compared to un-irradiated MMRU cells) and then returned to normal levels 24 h later (Fig. 4.1A). Using RT-PCR, we also found that this induction was at the mRNA level (Fig. 4.1B). To assess whether UVmediated induction of ING3 is a common event rather than a cell line specific observation, we further examined ING3 expression after UV irradiation in other melanoma cell lines. Despite the differences in basal expression level of ING3 among melanoma cell lines, we detected the induction of ING3 in all five cell lines (MEWO, MMAN, Sk-mel-3, Sk-mel-5 and Sk-mel-24) albeit to very different degrees (Fig. 4.1C). To evaluate if ING3 can be induced by other DNA damage stimuli, we treated MMRU cells with genotoxic chemicals including cisplatin, etoposide, doxorubicin and camptothecin. Results showed that ING3 was induced by all treatments except cisplatin (Fig. 4.1D).  70  Fig. 4.1 ING3 was induced by DNA damage agents in melanoma cells. MMRU cells were irradiated with 600 J/m2 UVB and cells were harvested at indicated time points for protein or RNA extraction. Western Blot (A) and RT-PCR (B) were used to analyze the protein and mRNA levels of ING3, respectively. The expression level was quantitated with Quantity One software and the fold of induction was listed below the blots; (C) Several melanoma cell lines were irradiated with 600 J/m2 UVB and harvested for western blot analysis 6 h after irradiation; (D) MMRU cells were treated with 50 µM cisplatin, 50 µM etoposide, 2 µM doxorubicin and 2 µM camptothecin for 6h before being harvested for western blot analysis. GAPDH and actin were used as loading control for PCR and western blot analysis, respectively.  71  4.2.2 ING3 Promotes UV-induced Apoptosis MMRU cells stably expressing ING3 were generated and the expression of ING3 was analyzed in these stable clones by both Western blot (Fig. 4.2A) and RT-PCR (Fig. 4.2B). The ING3 stable clones (F5, F6 and F9) had a three to five fold higher ING3 expression compared with their parental MMRU cells. Under the microscope, it was observed that the shape of most ING3 stable clone cells looked rounder compared with their parental MMRU cells (Fig. 4.2C). To investigate if ING3 plays a role in cellular stress response to UV radiation in melanoma cells, we irradiated MMRU cells and ING3 stable clone F5 cells with 300 or 600 J/m2 UVB radiation. The results of SRB cell survival assays showed that fewer ING3 stable clone F5 cells survived in comparison to their parental MMRU cells after UV irradiation (Fig. 4.3A). Fluorescence-activated cell sorting (FACS) analysis of cells stained with propidium iodide (PI) indicated similar levels of cell death (sub-G1 popullation) in non-irradiated parental MMRU cells and ING3 stable clones (F5, F6, and F9) (Fig. 4.3B, P > 0.05). However, all three ING3 stable clones were more sensitive to UVB radiation compared with parental MMRU cells (Fig. 4.3B). In particular, the rate of cell death induced by UV irradiation was two-folds higher in the F5 stable clone cells compared with that of the parental MMRU cells (P < 0.001, Fig. 4.3B).  72  Fig. 4.2 ING3 was stably overexpressed in MMRU cells. MMRU cells were transfected with pcDNA3-ING3, incubated at 37°C for 48 h followed by a 14-day selection in the culture medium containing 800 µg/ml G418. Single clones were then picked and maintained in the culture medium containing 100 µg/ml G418 for ING3 expression detection by western blot (A) or RT-PCR (B). GAPDH and actin were used as loading control for PCR and western blot analysis, respectively; (C) Microscopic images were taken for MMRU and stable clones. The magnification is 200×.  73  Fig. 4.3 ING3 promoted UV-induced cell death in melanoma cells. (A) MMRU and F5 cells were irradiated with 300 or 600 J/m2 UVB and the cell survival was determined by SRB assay 24 h after UV irradiation. The bottom panel shows the quantitation of cell survival. (B) MMRU and ING3 stable clone cells (F5, F6 and F9) were irradiated with 600 J/m2 UVB. Cells were then harvested, fixed, stained by PI for FACS analysis. Cells in sub-G1 phase were regarded as dead cells. Asterisks indicate statistical significance (* p < 0.05, ** p < 0.01, *** p<0.001)  74  To distinguish if ING3-enhanced cell death is due to apoptosis or necrosis, MMRU cells and ING3 stable clone F5 cells were subjected to Hoechst staining. Apoptotic cells showed significant condensed chromosome and fragmented nuclei that were indicated by bright spots in Hoechst staining. As shown in Fig. 4.4A-B, significant differences between the apoptotic rate of MMRU cells and that of ING3 stable clone F5 cells appeared as early as 8 h after irradiation with 600 J/m2 UVB and the dramatic difference was observed after 16 h (32% vs. 60%, P < 0.001). An early event in apoptosis is the flipping of phosphatidylserine of the plasma membrane from the inside surface to the outside surface (Reutelingsperger and van Heerde, 1997). To further confirm that ING3-enhanced cell death was not due to necrosis, we analyzed UV-irradiated MMRU and F5 cells with Annexin-VFLUOS and PI double staining because Annexin V binds specifically to phosphatidylserine and FLUOS-conjugated Annexin V can be used as a fluorescent probe to label apoptotic cells. Results from double staining demonstrated that F5 cells were significantly hypersensitive to 600 J/m2 UVB-induced apoptosis compared with MMRU cells (Fig. 4.4CD; 41% vs. 20%, P < 0.001). When we examined the activation of caspases, more cleaved forms of caspase-9, caspase-3 and their target poly ADP ribose polymerase (PARP) were detected in ING3 stable clone F5 cells after UV irradiation (Fig. 4.5). Therefore, it is concluded that stable overexpression of ING3 in MMRU cells significantly enhanced UVinduced apoptosis. To determine the physiological role of ING3 in MMRU cells, we knocked down ING3 expression by ING3 siRNA (Fig. 4.6A). FACS analysis with PI staining showed that UV-induced apoptosis in MMRU cells were significantly reduced after the knockdown of ING3 (Fig. 4.6B).  75  Fig. 4.4 ING3 enhances UV-induced apoptosis in melanoma cells. (A, B) MMRU and F5 cells were irradiated with 600J/m2 UVB and cultured for 8 and 16 h before fixation and staining with 2.5µg/ml Hoechst 33342. The cells that are condensed, fragmented and bright were regarded as apoptotic cells. (C, D) Sixteen hours after being irradiated with 600 J/m2 UVB, MMRU and F5 cells were stained by both Annexin-V-FLUOS and PI for FACS analysis. Quadrants: lower left, viable cells; lower right, apoptotic cells; upper left, cell debris; upper right, necrotic cells. (* p<0.05, ** p<0.01, *** p<0.001).  76  Fig. 4.5 ING3 enhances the UV-triggered activation of caspases in melanoma cells. MMRU and F5 cells were exposed to 600 J/m2 UVB. Twenty-four hours later, floating and adherent cells were collected separately for Western blot analysis of caspase-3, caspase-9 and PARP. ‘Fl’ and ‘Ad’ stand for floating and adherent cells, respectively. Actin was used as loading control.  77  Fig. 4.6 Knockdown of ING3 inhibited UV-induced apoptosis in melanoma cells. MMRU cells were transfected with scrambled siRNA (ctrl siRNA) or ING3 siRNA, and harvested for Western blot analysis of the knockdown of ING3 (A) or irradiated with 600 J/m2 UVB. Twenty-four hours later, cells will be processed for PI staining and FACS analysis (B). Asterisks indicate statistical significance (** p < 0.01).  78  4.2.3 ING3 Mediates UV-induced Apoptosis Independently of p53 Since both ING1b and ING2, the homologs of ING3, can promote UV-induced apoptosis in a p53-dependent manner (Cheung and Li, 2002; Scott et al., 2001b), it would be necessary to demonstrate whether ING3 cooperated with p53 to boost UV-triggered apoptosis in melanoma cells. The p53 siRNA was employed to knockdown p53 expression in ING3 stable clone F5 cells, which harbors wild-type p53. Western blot analysis showed that the protein levels of p53 and its target Bax were dramatically reduced in both MMRU and F5 cells after siRNA treatment (Fig. 4.7A, bottom panel). However, knockdown of p53 in ING3-overexpressing F5 cells did not bring down the apoptosis rate to the same level in their parental MMRU cells after UVB irradiation (Fig. 4.7A, top panel), suggesting that ING3-mediated apoptosis is p53-independent. Accordingly, ING3 overexpression did not significantly affect the expression of p53-targeting mitochondrial proteins Bax, Bcl-2, Bad and Noxa (Fig. 4.7B), indicating that ING3 did not directly activate the mitochondrial apoptosis pathway. In addition, ING3 promoted UV-induced apoptosis in either p53 wild type or p53-null HCT116 cells (Fig. 4.8), concluding that p53 function is not required for ING3-enhanced apoptosis in response to UV irradiation.  79  Fig. 4.7 ING3 promoted UV-induced apoptosis independent of p53 in mealnoma cells. (A) MMRU and F5 cells were transfected with either control or p53 siRNA, cultured for 24 h and irradiated by 600 J/m2 UVB. After another 24 h, cells were harvested for PI staining FACS analysis (upper panel). The bottom panel was the western blot analysis of p53 knockdown efficiency; (B) MMRU and F5 cells were collected for western blot analysis of p53-targeting mitochondria proteins 24 h after 600 J/m2 UVB irradiation. ‘Fl’ and ‘Ad’ stand for floating and adherent cells, respectively. Actin was used as loading control. Asterisks indicate statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001).  80  Fig. 4.8 ING3 promoted UV-induced apoptosis in p53 wt and null HCT116 cells. HCT116 p53 +/+ or HCT116 p53 -/- cells were transfected with pcDNA3 or pcDNA3-ING3 for 24 h and irradiated with 600 J/m2 UVB. Cells were fixed for PI staining FACS analysis 24 h later. The bottom panel was the western analysis of overexpression of ING3 in HCT116 cells. Actin was used as loading control. Asterisks indicate statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001).  81  4.2.4 ING3 Mediates UV-induced Apoptosis via Death Receptor Pathway Since ING3 does not directly modify the expression of mitochondria proteins in response to UV radiation, we then examined the role of ING3 in the death receptor pathway. Western blot analysis showed that more caspase-8 and its target BH3 only protein Bid were cleaved in F5 cells upon UV irradiation (Fig. 4.9A). In addition, the colorimetric assay suggested that the activity of caspase-8 was significantly higher in F5 cells compared with MMRU cells in response to UV irradiation (Fig. 4.9B). Transfection with a dominant negative caspase-8 (DN-caspase-8) vector resulted in reduced caspase-8 activity in both MMRU and F5 cells. No significant difference was observed for the activity of caspase-8 between that of MMRU and F5 cells after UV irradiation (Fig. 4.9B). Accordingly, inhibition of caspase-8 completely abolished ING3-mediated apoptosis (Fig. 4.9C). Furthermore, when the activation of caspase-8 was blocked, we did not observe a significant difference in caspase-9 activation between F5 and MMRU cells in response to UV irradiation (Fig. 4.9D), confirming that caspase-9 was partially activated by caspase-8 via the cleavage of Bid in F5 cells. These data support the notion that ING3 may mediate UVinduced apoptosis by activating the death receptor pathway. The Fas death receptor is activated by binding to Fas ligands (Fas-L). Fas death receptors then recruit Fas-associated death domain protein (FADD) to the plasma membrane, which in turn activates pro-caspase-8, leading to the activation of a cascade of caspases (Nagata, 1999; Peter and Krammer, 1998). Studies have shown that the Fas death receptor pathway is activated by UV radiation and contributes to UV-induced apoptosis (Gozani et al., 2003; Leverkus et al., 1997). To examine whether the Fas death receptor pathway was crucial for ING3-mediated apoptosis under UV stress, MMRU and F5 cells  82  were pre-incubated at 4oC for 30 min, which was previously shown to block Fas aggregation (Bush et al., 2001; Kulms et al., 1999). Upon UV irradiation, no significant difference in apoptosis rate was observed between MMRU and F5 cells after blocking the Fas aggregation (Fig. 4.10A). Meanwhile, transfection of F5 cells with specific Fas siRNA resulted in over 90% knockdown efficiency, and caused a significant, but not complete, reduction in the apoptosis of F5 cells after UV irradiation (Fig. 4.10B). 4.2.5 ING3 Induces Fas Expression To detect how ING3 activated the Fas death receptor pathway, we investigated whether ING3 could modulate Fas or Fas-L expression. Our data indicated that ING3 induced Fas protein expression in both non-stressed and UVB irradiated cells (Fig. 4.11A). The induction of Fas protein by ING3 was observed to be maximal at 8 h after 600 J/m2 UVB irradiation. Moreover, the induction of Fas by ING3 was at the transcriptional level (Fig. 4.11B). ING3 did not have obvious effects on Fas-L expression (Fig. 4.11A), but UV radiation upregulated the expression of Fas-L in both MMRU and F5 melanoma cells (Fig. 4.11B).  83  Fig. 4.9 Blockage of caspase-8 activation abolished ING3-mediated apoptosis in melanoma cells. (A) MMRU and F5 cells were collected for western blot analysis of caspase-8 and bid 24 h after 600 J/m2 UVB irradiation. ‘Fl’ and ‘Ad’ stand for floating and adherent cells, respectively; (B) MMRU and F5 cells were harvested for the determination of caspase-8 activity using colorimetric assay 24 h after 600 J/m2 UVB irradiation. The fold-induction of caspase-8 activity was normalized by the caspase-8 activity of MMRU cells; (C, D) MMRU and F5 cells transfected with either pcDNA3 or DN-caspase-8 plasmids were irradiated with 600 J/m2 UVB and harvested for PI staining FACS analysis or western blot analysis after another 24 h incubation. Asterisks indicate statistical significance (* p < 0.05, ** p < 0.01, *** p < 0.001).  84  Fig. 4.10 Blockage of Fas activation abolished ING3-mediated apoptosis in melanoma cells. (A) MMRU and F5 cells were pre-cultured at 4°C for 30 min before being irradiated with 600 J/m2 UVB. Twenty-four hour later, cells were harvested for PI staining FACS analysis; (B) MMRU and F5 cells were transfected with control or Fas siRNA for 24 h and then irradiated with 600 J/m2 UVB. Cells were harvested for PI staining FACS analysis 24 h after UV irradiation. The top panel was the western blot analysis of Fas knockdown efficiency in F5 cells. Asterisks indicate statistical significance ( ** p<0.01, *** p<0.001).  85  Fig. 4.11 ING3 induced Fas expression in melanoma cells. MMRU and F5 cells were irradiated with 600 J/m2 UVB, and harvested at indicated time for western blot (A) or RTPCR (B) analyses. GAPDH and actin were used as loading control for PCR and western blot analysis, respectively.  86  4.3 Discussion Like its homologues ING1b and ING2, the expression of ING3 was also rapidly induced after DNA damage. The rapid induction may be at the transcriptional level as the mRNA level of ING3 was also induced upon UVB irradiation. Although UVB irradiation can induce G1 or G2 cell cycle arrest (Ceruti et al., 2005; Gujuluva et al., 1994) and ING3 may be periodically expressed during the cell cycle, this induction is not likely due to the augmented cell cycle population because the induction of ING3 was rapid and transient, and ING3 expression returned to normal levels 24 h after UV irradiation. DNA damage caused by UV and other genotoxic stress activates checkpoint proteins ATM or ATR, which then modulate cell fate by cell cycle arrests, DNA repair or apoptosis through activating downstream mediators, transducers and effectors like p53, BRCA1 and Chk1/2 (Kastan and Bartek, 2004; Latonen and Laiho, 2005). Considering that ING3 is a key subunit of the NuA4 HAT complex (Doyon et al., 2004), inducible ING3 may play an important role in chromatin remodelling in response to UV irradiation. In this study we, for the first time, showed that stable overexpression of ING3 can strongly promote UV-induced apoptosis in melanoma cells. This increased apoptosis did not depend p53 function, although both ING1b and ING2, close family members of ING3, enhanced UV-induced apoptosis in a p53-dependent manner (Cheung et al., 2001; Chin et al., 2005). Since it was reported that ING3 can mediate p53-dependent growth arrest and apoptosis in RKO cells (Nagashima et al., 2003), it is likely that ING3 can modulate apoptosis in both p53-dependent and independent manners, which may depend on the cell type. The ING family proteins, ING1b, ING2, ING4 and ING5, all induce growth arrest and apoptosis in a p53-dependent manner. Among these ING proteins, ING1b, ING4, and ING5 can physically associate with p53,  87  whereas ING2 and ING3 do not (Shi and Gozani, 2005). Furthermore, ING1b, ING2, ING4 and ING5 also activate and stabilize p53 by stimulating the acetylation of p53 at residues K382 and/or K373 (Campos et al., 2004; Nagashima et al., 2001; Shiseki et al., 2003). Although ING1b also induced apoptosis in a p53-independent manner, it remains a possibility that this occurred through homologues of p53, p63α and p73α (Tsang et al., 2003). In this study, we did not detect any significant change of p53-responsive proteins after overexpressing ING3, which further supports the notion that ING3-mediated apoptosis after UV irradiation is p53-independent. This discrepancy of ING3 function from other family members is probably due to the fact that ING3 is evolutionarily distinct from other family members (He et al., 2005a). Both ING1 and ING2 contain a small protein-interacting motif (PIM) at C-terminal, which binds a defined subset of peptides and binds to phosphatidylinositol monophosphates (Feng et al., 2002; Gozani et al., 2003; Kaadige and Ayer, 2006). ING1b also contains a unique N-terminal PCNA-interacting protein (PIP) domain, through which ING1b promoted UV-induced apoptosis (Cheung and Li, 2002). Moreover, two distinct insertions of 102 and 54 amino acids are located between the NCR and NLS regions of ING3, respectively (Pedeux et al., 2005). Therefore, it will be interesting to clarify whether structural differences between ING3 and ING1b or ING2 are responsible for different pathways in UV-induced apoptosis. UV-induced apoptosis can be triggered through both both p53-mediated mitochondrial pathway and death receptor pathways (Kulms and Schwarz, 2002). In this study, both western blot analysis and caspase-8 activity assay indicated that caspase-8 was more activated in the ING3 stable clone F5 cells than in their parental MMRU cells in response to UV irradiation (Fig. 4.9A and 4.9B). Moreover, inhibition of caspase-8  88  activation blocked the cleavage of caspase-9 (Fig. 4.9D), confirming that ING3 does not directly affect the mitochondrial apoptosis pathway but through the crosstalk linked by the cleaved Bid protein. ING3-mediated apoptosis was abrogated when cells were transfected with DN-caspase-8 (Fig. 4.9C) or pre-incubated at 4°C (Fig. 4.10A), supporting the idea that death receptor pathways mediate ING3-triggered apoptosis. Regarding the observation that Fas siRNA treatment reduced ING3-mediated apoptosis significantly (but not completely) (Fig. 4.10B), the Fas death receptor pathway is likely involved in ING3-mediated apoptosis. However, the interuption of both caspase-8 activation and death receptors aggregation not only inhibit the Fas pathway, but affect all other death receptor signaling, including tumour necrosis factor (TNF) receptor-1, TNFα-related apoptosis inducing ligand (TRAIL) receptors and death receptor-3 (DR-3) signaling (Lavrik et al., 2005). Therefore, it remains possible that other death receptor pathways may also be involved in ING3-mediated apoptosis in response to UV radiation. Expression of Fas and Fas-L have both been shown to be markedly decreased or not expressed in melanomas (Chappell et al., 1999; Neuber and Eidam, 2006). Previous studies showed that ING2, a family member of ING3, was also able to upregulate Fas expression in melanoma cells (Chin et al., 2005). In the current study, we observed that ING3 stable clone F5 had higher Fas mRNA and protein levels in both basal and UV irradiated conditions (Fig. 4.11A and 4.11B). However, ING3 enhanced apoptosis only after cells were exposed to UV radiation, suggesting that ING3-induced Fas expression itself was insufficient, also requiring UV irradiation. UV radiation has been previously shown to upregulate Fas-L expression in lymphocytes (Kasibhatla et al., 1998). Therefore, the upregulated expression of Fas-L in melanoma cells after UV irradiation (Fig. 4.11B) may be crucial for the induction of ING3-  89  mediated apoptosis. In addition, UV radiation can induce the ligand-independent activation of Fas in melanoma cells (Elyassaki and Wu, 2006). Thus, it remains possible that UV radiation may also trigger the activation of ING3-mediated Fas expression independently of Fas-L in melanoma cells. Interestingly, p53 can activate the Fas gene by binding to its transcriptional activation site and its promoter region (Muller et al., 1998) and then promotes the redistribution of cytoplasmic Fas to cell surface through transportation from the Golgi complex upon DNA damage (Bennett et al., 1998). Since ING3 enhanced UV-induced apoptosis in a p53-independent manner, future studies to delineate how ING3 upregulates Fas expression in a p53-independent manner will help to understand the role of ING3 in UV response. Fas protein is a death receptor on cell membrane, whereas ING3 protein is mainly located in the nucleus. Similar to transcriptional factors, including NF-κB and Egr-1 (Thyss et al., 2005), the rapid induction and recovery of ING3 following UV irradiation suggested that ING3 may function as a transcription factor or co-factor in response to DNA damage. Although ING3 does not modulate p53 function, ING3 may possibly act as a transcription factor or cofactor to mediate apoptosis by modulating chromatin remodelling through the NuA4 HAT multisubunit complex (Doyon et al., 2004), which may help explain that loss of functional Tip60, the core HAT of NuA4 complex, failed to signal the existence of DNA damage to the apoptotic machinery (Ikura et al., 2000). The exact molecular mechanism of ING3-mediated chromatin-related transcriptional regulation remains to be revealed. In summary, we demonstrate that ING3 regulates UV-induced apoptosis through modulating Fas expression, thereby resulting in the activation of Fas/caspase8 pathway and the activation of caspase cascades via Bid cleavage.  90  CHAPTER 5 RAPID DEGRADATION OF ING3 REGULATES ITS TUMOUR SUPPRESSIVE ROLES IN MELANOMA CELLS 5.1 Rationale and Hypothesis We demonstrated in Chapter 3 that nuclear ING3 levels are remarkably reduced in malignant melanomas compared to dysplastic nevi, and reduced nuclear ING3 levels significantly correlate with a poorer disease-specific 5-year survival of patients with primary melanoma (Wang et al., 2007). In addition, overall ING3 is also significantly reduced in metastatic melanomas compared with dysplastic nevi and primary melanomas, suggesting that mechanisms other than the nucleus-to-cytoplasmic shift of ING3 contribute to its deregulation in advanced melanomas. In this study, we hypothesize that the overall expression of ING3 in metastatic melanoma cells is distorted in comparison to that in normal human melanocytes. We will also investigate the possible mechanisms leading to aberrant ING3 expression in melanoma cells and its effect on the tumour suppressive roles of ING3. 5.2 Results 5.2.1 ING3 Is Rapidly Degraded in Melanoma Cells To explore the mechanism responsible for reduced ING3 levels, we compared ING3 protein levels in nine metastatic melanoma cell lines with those in normal human melanocytes (Fig. 5.1A). Western blot analysis revealed that five melanoma cell lines (MMRU, MMLH, KZ-13, Sk-mel-3 and Sk-mel-93) had significantly lower ING3 expression in comparison to normal human melanocytes, whereas the other four melanoma cell lines (MMAN, MEWO, Sk-mel-5 and Sk-mel-110) showed ING3 levels similar to  91  melanocytes. To determine the mechanism resulting in low ING3 levels in MMRU, MMLH, KZ-13, Sk-mel-3 and Sk-mel-93 cells, we first checked the mRNA level of ING3 by RTPCR. No dramatic differences were observed for ING3 mRNA levels between these four cell lines (MMRU, MMLH, Sk-mel-3 and Sk-mel-93) and normal human melanocytes (Fig. 5.1B), but KZ-13 cells did have a significant lower ING3 mRNA level compared to melanocytes (Fig. 5.1B), implying that compromised mRNA expression is not the primary reason leading to the downregulation of ING3 in melanoma cells. Next, we examined the ING3 protein turnover rate in these cell lines by treating them with the protein synthesis inhibitor, cycloheximide, to inhibit de novo protein synthesis. The half-life of ING3 protein in either normal human melanocytes or KZ-13 cells was longer than 16 h, while it was shortened to about 5.0~6.6 h in MMRU, MMLH, Sk-mel-3 and Sk-mel-93 cells (Fig. 5.2). Therefore, rapid ING3 degradation may play an important role in the reduction of ING3 levels in melanomas.  92  Fig. 5.1 Aberrant ING3 expression in melanoma cells. (A) Whole cell extracts were obtained from normal human melanocytes and melanoma cell lines for western blot analysis of ING3; (B) RT-PCR analysis of ING3 mRNA level in melanocytes and melanoma cell lines. The line indicated the average expression level of ING3 in melanoma cells. * P<0.05, and **P< 0.01.  93  Fig. 5.2 Rapid degradation of ING3 in melanoma cells. (A) Melanocytes and melanoma cell lines were treated with 20 µg/ml cycloheximide (CHX) to block de novo protein synthesis. Cells were then harvested at indicated time and whole cell extracts obtained were subjected to western blot analysis of ING3. (B) Half-life of ING3 was determined by densitometric analysis of ING3 bands using the formula t1/2=-Ln2/S where S represents the slope from each linear regression.  94  5.2.2 ING3 Is Stabilized upon Proteasome Inhibitor Treatment Since the ubiquitin-proteasome degradation pathway accounts for the proteolysis of over 80% of cellular proteins, we examined if ING3 would be degraded by the proteasome complex by treating MMRU melanoma cells with proteasome inhibitors. p53, the wellcharacterized protein degraded by the ubiquitin-proteasome system, accumulated dramatically in the presence of proteasome inhibitors MG132 or lactacystin (Fig. 5.3A). The expression of ING3 was also significantly increased upon the treatment with proteasome inhibitor MG132 or lactacystin in both dose and time dependent manner (Fig. 5.3A-B). The level of ING3 was also significantly elevated in other melanoma cells including MMLH, Skmel-3 and Sk-mel-93 cells (Fig. 5.3C) and the transformed HEK293T cells (Fig. 5.3D), suggesting that the degradation of ING3 in these cell lines may be through the same pathway. The ectopic Flag-ING3 was also accumulated following the MG132 treatment in MMRU cells (Fig. 5.3E).  95  Fig. 5.3 ING3 accumulates in the presence of proteasome inhibitors. (A) MMRU cells were treated with MG132 or lactacystin at indicated doses for 6 h and harvested for Western blot analysis of ING3; (B) MMRU cells were treated with MG132 (2 µM) or lactacystin (2.5 µM) and harvested at indicated time for Western blot analysis of ING3; (C) Melanoma cells were treated with MG132 (2 µM) for 6 h and harvested for Western blot analysis of ING3; (D) HEK293T cells were treated with MG132 (2 µM) and harvested at indicated time for Western blot analysis of ING3; (E) MMRU cells were transfected with Flag-ING3 and treated with MG132 before being harvested for Western blot analysis of Flag-ING3.  96  To confirm that the increased expression of ING3 is a result of improved protein stability following the inhibition of proteasome activity, we examined the half-life of ING3 by treating MMRU cells with protein synthesis inhibitor cycloheximide. As expected, p53 was stabilized significantly in the presence of either MG132 or lactacystin (Fig. 5.4A). The half-life of ING3 was increased from 5.1 h in MMRU cells without any proteasome inhibitor treatment to 13.0 h and 9.6 h for MMRU cells in the presence of MG132 and lactacystin, respectively (Fig. 5.4A-B). Since we used ectopic Flag-tagged ING3 in a number of experiments to determine the degradation pathway for ING3, we also examined the half-life of Flag-ING3 in MMRU cells. The turnover rate of Flag-ING3 was estimated to be 4.2 h, which was increased to 15.1 h in the presence of MG132 (Fig. 5.4C-D). The fact that ectopic Flag-ING3 (4.2 h) has a shorter half-life than the endogenous ING3 (5.1 h) may be explained by the introduction of lysine residues inside Flag motif when we constructed the Flag-ING3 fusion protein.  97  Fig. 5.4 ING3 is stabilized in the presence of proteasome inhibitors. (A, B) MMRU cells pre-incubated with MG132 or lactacystin for 30 min were treated with CHX to block de novo protein synthesis in the presence of MG132 or lactacystin for indicated time. Cells were then processed for western blot analysis of ING3 and ING3 half-life estimation. (C, D) MMRU cells were transfected with Flag-ING3 plasmid and then treated with CHX for indicated time before being harvested for ING3 half-life determination.  98  5.2.3 ING3 Is Degraded by the Ubiquitin-proteasome System To distinguish if ING3 is degraded by the 20S or 26S proteasome, nuclear extracts from MMRU cells were precipitated with ammonium sulphate. Detection of 26S and 20S proteasome markers PSMC4 (subunit of 19S) and α4 (subunit of 20S) assured the enrichment of the crude preparations of 20S and 26S proteasomes (Fig. 5.5A). However, ING3 was predominantly associated to the 26S proteasome-rich fraction and its amount with this fraction was significantly increased in the presence of MG132. To confirm that the 26S proteasome is able to degrade ING3, we performed an in vitro degradation assay. Nuclear extracts from MMRU cells were incubated at 37°C for different time points in the presence or absence of isolated active 26S proteasomes. Samples were then blotted and probed with antibodies against ING3 or actin. Results showed that ING3, not actin, was rapidly degraded by the 26S proteasome (Fig. 5.5B). Since ubiquitination plays an essential role in protein degradation by the 26 proteasome,  we  examined  if  ING3  is  polyubiquitinated  using  a  two-step  immunoprecipitation (IP) assay to purify the ubiquitinated ING3. MMRU cells were cotransfected with HA-tagged ubiquitin (HA-Ub) and Flag-tagged ING3 (Flag-ING3) plasmids and processed for IP assay. We first pulled down Flag-ING3 containing immunocomplexes using mouse anti-Flag antibody, denatured the immunoprecipitates and then pulled down HA-tagged ING3 with mouse anti-HA antibody. The two-step purification assay gave a strong signal of ubiquitinated Flag-ING3 (Fig. 5.5C), confirming that ING3 itself is ubiquitinated. To examine the polyubiquitination of ING3, MMRU cells were transfected with HA-Ub and treated with proteasome inhibitor MG132 6 h before being harvested. Pulldown of ubiquitinated proteins by mouse anti-HA antibody indicated that  99  both the endogenous ING3 and ectopic Flag-ING3 protein were polyubiquitinated in MMRU cells and the ubiquitination level of ING3 was increased in the presence of MG132 (Fig. 5.5D). The parallel experiment to immunoprecipitate Flag-ING3 using mouse anti-Flag antibody also revealed that Flag-ING3 is polyubiquitinated (Fig. 5.5D). Taken together, all these data demonstrate that degradation of ING3 is mediated by the ubiquitin-dependent proteasome pathway.  100  Fig. 5.5 ING3 is degraded by the ubiquitin-dependent pathway. (A) 26S and 20S proteasome-containing fractions were isolated by ammonium sulphate precipitation from MMRU cells as described in Material and Methods for detection of ING3 by western blot. PSMC4 and α4 were used as specific markers for 26S and 20S proteasomes, respectively; (B) Nuclear extracts from MMRU cells were incubated at 37°C for different time points in the presence or absence of isolated active 26S proteasome. Samples were blotted and probed with antibodies against ING3 or actin; (C) Whole cell extracts obtained from MMRU cells co-transfected with HA-Ub and Flag-ING3 were subjected to IP by anti-Flag antibody (IP1). Captured proteins were then denatured and applied to IP by anti-HA antibody (IP2) for the detection of ubiquitinated Flag-ING3 by western blot; (D) MMRU cells transfected as indicated for 24 h were left untreated or treated with MG132 for 6 h. HA-tagged proteins were immunoprecitpitated (IP) by anti-HA or anti-Flag antibody for the detection of polyubiquitinated ING3 or Flag-ING3 by Western blot (WB).  101  5.2.4 Lysine Residue 96 Is Required for ING3 Ubiquitination and Degradation In the ubiquitin-proteasome system, an initial ubiquitin moiety is conjugated through its carboxyl-terminal glycine 76 residue to an α-NH2 group of an internal lysine residue of the substrate, which is usually located at the substrate protein’s N-terminus (Hershko and Ciechanover, 1998). To identify the lysine residues mediating ING3 protein ubiquitination and degradation, a series of Flag-tagged plasmids expressing amino-terminal-truncated ING3 proteins and one expressing carboxyl-terminal-truncated ING3 protein were constructed and transfected into HEK293T cells. Deletion of 1-95 amino acids did not affect MG132-mediated stabilization of ING3 protein, while deletion of 1-111 amino acids significant abolished MG132-triggered ING3 protein stabilization (Fig. 5.6A-B). In addition, ubiquitination of ING3 protein was also interrupted when amino acids 1-111, but not 1-95, was deleted (Fig. 5.6C), suggesting that amino acids 96-111 of ING3 plays a crucial role in ING3 ubiquitination and degradation. Since protein ubiquination usually occurs at lysine residues, we constructed the lysine-to-arginine point mutants for lysine residues K96, K103 and K105 (Fig. 5.7A), the three lysine residues located within amino acids 96-111 of ING3. The K96R mutant of ING3 did not affect the subcellular location of ING3 (Fig. 5.7B), but almost completely inhibited the ubiquitination of ING3 in HEK293T cells, while the K103/105R mutant of ING3 did not affect the ubiquitination of ING3 (Fig. 5.7C). In addition, the half-life of ING3 in HEK293T cells was elongated dramatically to 11.3 h from 4.0 h after the arginine substitution at K96 residue (Fig. 5.7D-E).  102  Fig. 5.6 96-111 residues are crucial for ING3 ubiquitination and degradation. (A) Illustration of wild-type (WT) and truncated ING3 proteins; (B) Plasmids expressing Flag-tagged WT or truncated ING3 were introduced into HEK293T cells for 24 h and then treated with MG132 for Western blot analysis; (C) HA-Ub was introduced into HEK293T cells together with either WT ING3 or amino acids 96-418 or 112-418 of ING3 for 24 h. Cells were treated with MG132 for 6 h and subjected to IP analysis of ubiquitinated ING3.  103  Fig. 5.7 K96 residue is essential for ING3 ubiquitination and degradation. (A) Illustration of residues 96-111 of ING3. Lysine residues were highlighted. (B) MMRU cells on cover-slips were transfected with either WT or K96R Flag-ING3 for 24 h. Cells were then fixed and stained with mouse anti-Flag antibody. Hoechst Staining was used to indicate the nuclei of cells. Magnification, ×630. (C) HA-Ub was introduced into HEK293T cells together with WT, K96R, or K103/105R of ING3 for 24 h followed by treatment with MG132 for 6 h. Cells were then processed for IP analysis of ubiquitinated ING3. (D) HEK293T cells were transfected with either WT or K96R ING3 for 24 h and then treated with CHX to inhibit de novo protein synthesis for indicated time before being harvested for the estimation of ING3 half-life.  104  5.2.5 SCFSkp2 E3 Ligase Complex Mediates ING3 Ubiquitination and Degradation To determine the E3 ligase targeting ING3 for ubiquitination and degradation by 26S proteasome, we first looked at the SCF complex since it comprises a large family of ubiquitin E3 ligases controlling the ubiquitination of many substrates including cell cycle regulatory proteins. To examine if ING3 is a target of SCF complex, we co-expressed FlagING3 and HA-tagged Cul1 (HA-Cul1) or HA-tagged Roc1 (HA-Roc1), two core subunitis of SCF complex, into HEK293T cells. Results from co-immunoprecipitation (Co-IP) assay showed that Flag-ING3 interacted with HA-Cul1 and HA-Roc1 in HEK293T cells (Fig. 5.8A). Although the ING3 antibody cannot be used for IP assay, pull-down of Flag-ING3 can precipitate endogenous Cul1 and pull-down of endogenous Cul1 can precipitate endogenous ING3 in MMRU cells (Fig. 5.8B-C), which support the idea of ING3-Cul1 interactions at physiological levels. To explore the role of Cul1 on ING3 degradation, we knocked down Cul1 expression and examined the half-life of ING3 with cycloheximide. Knockdown of Cul1 expression in MMRU cells significantly decreased the expression, and ubiquitination, of ING3 (Fig. 5.9AB). Knockdown of Cul1 also and elongated its half-life to 12.4 h compared with 4.7 h in MMRU cells treated with scrambled siRNA (Fig. 5.9C-D). Chk1 (checkpoint kinase 1), a well-known target of SCF complex, is also stabilized after repressing Cul1 (Fig. 5.9C-D).  105  Fig. 5.8 ING3 interacts with SCF E3 ligase complex. (A) HEK293T cells were cotransfected with Flag-ING3 and HA-Cul1 or HA-Roc1 for 24 h and the whole cell extracts were subjected to IP analysis by either anti-Flag or anti-HA antibodies. (B) MMRU cells were transfected with Flag-ING3 for 24 h followed by the treatment with MG132 for 6 h. The total cell protein was obtained and applied to IP assay with anti-Flag antibody and blotted with anti-Cul1 or anti-Flag antibodies. (C) MMRU cells were treated with MG132 for 6 h and the whole cell extracts were obtained for IP assay with anti-Cul1 antibody.  106  Fig. 5.9 Cul1 is required for ING3 ubiquitination and degradation. (A) MMRU cells were transfected with vector, HA-Cul1, scrambled control (Ctrl) siRNA or Cul1 siRNA for 48 h followed by Western blot analysis of ING3 expression. (B) MMRU cells were cotransfected with HA-Ub and either Ctrl or Cul1 siRNA for 48 h. After further incubation in the presence of MG132 cells were harvested and processed for IP analysis of polyubiquitinated ING3. (C, D) MMRU cells were treated with CHX to inhibit de novo protein synthesis after being transfected with Ctrl or Cul1 siRNA for 48 h. Cells were then harvested at indicated time and whole cell extracts obtained were subjected to Western blot analysis and ING3 half-life estimation.  107  Next, we attempted to determine the potential F-box proteins recognizing ING3. Skp2 and βTRCP are two important F-box proteins that behave as oncoproteins and direct many cell cycle regulatory proteins for degradation. We cotransfected Flag-ING3 and either Skp2 or βTRCP into HEK293T cells. Results from co-IP assay revealed that Flag-ING3 was bound to Myc-Skp2 but not HA-βTRCP (Fig. 5.10A-B). Pulldown of endogenous Skp2 was also able to precipitate endogenous ING3 (Fig. 5.10C) in MMRU cells. Treatment of MMRU cells with Skp2 siRNA reduced Skp2 expression by over 90% (Fig. 5.11A), reduced the ubiquitination of ING3 (Fig. 5.11A) and elongated the half-life of ING3 from 5.3 h in MMRU cells transfected with control siRNA to 8.7 h (Fig. 5.11C-D). In addition, knockdown of Skp2 in MMLH, Sk-mel-3 and Sk-mel-93 cells also significantly induced ING3 accumulation (Fig. 5.11B). Therefore, our data indicated that degradation of ING3 protein is under the tight control of SCFSkp2 E3 complex in melanoma cells and inhibition of the activity of SCFSkp2 complex will be able to restore its expression.  108  Fig. 5.10 ING3 interacts with the F-box protein Skp2. (A) HEK293T cells were cotransfected as indicated for 24 h followed by the treatment with MG132 for 6 h. WCE were obtained and applied to IP assay with anti-Flag antibody and blotted with anti-Myc or anti-Flag antibodies. (B) HEK293T cells were cotransfected as indicated for 24 h followed by the treatment with MG132 for 6 h. WCE were obtained and applied to IP assay with antiFlag antibody and blotted with anti-HA or anti-Flag antibodies. (C) MMRU cells were treated with MG132 for 6 h. The WCE were then obtained for IP assay with anti-Skp2 antibody followed by WB analysis of ING3.  109  Fig. 5.11 Skp2 mediates the ubiquitination and degradation of ING3. (A) MMRU cells were cotransfected with transfected with HA-Ub and either Ctrl or Cul1 siRNA and incubated for 48 h. After further incubation in the presence of MG132 for 6 h cells were harvested and processed for IP analysis of polyubiquitinated ING3. (B) Melanoma cells were transfected with Ctrl or Cul1 siRNA for 48 h and processed for Western blot analysis of ING3. (C, D) MMRU cells were treated with CHX to inhibit de novo protein synthesis after being transfected with Ctrl or Cul1 siRNA for 48 h. Cells were then harvested at the indicated times and whole cell extracts obtained were subjected to Western blot analysis and ING3 half-life estimation.  110  5.2.6 K96R Mutation Enhances the Tumour Suppressive Roles of ING3 ING3 has been shown to inhibit colony formation efficiency and modulate cell cycle control in both 3T3 and RKO cells (Doyon et al., 2004; Nagashima et al., 2003). Here, our data also indicated that 43.6% of MMRU cells were distributed at G1-phase following a transient transfection with empty vector, while it was increased to 51.5% and 58.2% after cells were transiently transfected with wild-type or K96R ING3 (Fig. 5.11A-C), respectively, confirming that ING3 plays a role in G1 phase cell cycle control. Since we have demonstrated that overexpression of ING3 significantly promotes UVB-induced apoptosis in MMRU cells (Wang et al 2006), we checked if stabilization of ING3 by K96R mutation would affect this sensitivity to UV irradiation. UVB at a dose of either 200 J/m2 or 600 J/m2 induced cell apoptosis at a rate of 14.6% and 27.5%, respectively (Fig. 5.11D-E). In the presence of wild type ING3, the apoptosis rates were increased to 22.6% and 38.2%, respectively, which were further elevated to 29.3% and 46.1% in cells overexpressing K96R ING3 mutant, respectively (Fig. 5.11D-E). These data suggest that interruption of ING3 degradation can stimulate its tumour suppressive roles.  111  Fig. 5.12 Blockage of ING3 degradation stimulates its tumour suppressive functions. (A-C) MMRU cells were transfected with vector, WT or K96R ING3 for 24 h. Cells were collected for Western blot analysis of ING3 expression (A) or cell cycle analysis by flow cytometry (B, C). (D, E) MMRU cells transfected with either vector, WT or K96R ING3 for 24 h were irradiated with UVB at indicated doses. After incubation for another 24 h cells were collected for analysis of cell cycle distribution. Cells at sub-G1 phase were considered apoptotic.  112  5.3 Discussion The ING3 tumour suppressor has been shown to modulate transcription, cell cycle control, and apoptosis. Recent studies by our group and Gunduz et al suggest that ING3 is deregulated and functions as a tumour suppressor in both melanomas and HNSCCs (Gunduz et al., 2008; Gunduz et al., 2002; Wang et al., 2007; Wang and Li, 2006). However, the mechanisms causing altered ING3 expression in melanomas appear different from those in HNSCCs. In HNSCCs, the ING3 gene is rarely mutated, but 50% of primary HNSCCs have decreased or no expression of ING3 mRNA due to loss of heterozygosity (Gunduz et al., 2008; Gunduz et al., 2002). In this work we find that rapid ING3 degradation plays an important role in the loss of ING3 levels in advanced melanomas. This provides another explanation for aberrant ING3 content in melanomas in addition to our previous finding that subcellular relocalization of ING3 causes its deregulation in both primary and metastatic melanomas. Meanwhile, ING1b has been shown to be shifted from the nucleus to the cytoplasm in melanocytic lesions (Nouman et al., 2002a), which may be mediated by 14-3-3 proteins (Gong et al., 2006). Both nuclear ING2 and overall ING4 expression are also downregulated in melanomas although only reduced ING4 levels were significantly associated with melanoma progression (Li et al., 2008b; Lu et al., 2006). The reduced ING4 level also correlated with tumour thickness, ulceration, and poorer 5-year patient survival with primary melanomas (Li et al., 2008b). These studies indicate that ING proteins are deregulated in melanomas and may play a crucial role in the development of melanoma. However, the reasons causing the aberrant expression of ING proteins, particularly ING2 and ING4, are largely unknown. Since ING genes are located on different chromosomes (He et al., 2005a), the aberrant expression of ING proteins are unlikely under a common genetic  113  control. The structural similarity of ING proteins, however, may provide alternative mechanisms by which deregulation of ING proteins in melanoma may be controlled by certain common proteins involved in their rapid protein turnover and/or subcellular relocalization. Future studies are needed to achieve a complete understanding of the aberrant expression of ING proteins in melanomas. It is worth noting that ING4 is also subjected to N-terminal ubiquitination and degraded through the ubiquitin-proteasome pathway (Ho et al., 2006). Thus it will be necessary to investigate if downregulation of ING4 is affected by enhanced degradation through the ubiquitin-proteasome system. Rapid ING3 degradation only occurs when primary melanomas become metastatic since overall ING3 is not reduced in primary melanomas compared with dysplastic nevi (Fig. 3.2). The underlying mechanism is not clear. Possible explanations may include increased E3 ligase level in melanoma metastases and/or any mutation or modification of ING3 favouring its recognition by E3 ligase. It was reported that mdm2-mediated p53 ubiquitination not only contributes to p53 degradation, but also controls the nucleus-tocytoplasm shift of p53, especially in the presence of a low level of mdm2 (Tanami et al., 2004). Considering that the expression of Skp2 is increased in melanomas and correlated with melanoma progression (Li et al., 2004; Woenckhaus et al., 2005), it is possible that the increased expression of Skp2 may mainly target ING3 to the cytoplasm in primary melanomas and subsequently enhance both subcellular translocation and degradation of ING3 in melanoma metastases. Further work is required to test this hypothesis. Furthermore, our study also reveals that the loss of ING3 expression in the metastatic melanoma cell line KZ-13 is a result of decreased ING3 mRNA expression (Fig. 5.1B), implying that the mechanisms leading to aberrant ING3 expression in melanomas are complicated.  114  Our current data strongly support the idea of a crucial role for the ubiquitinproteasome system in the proteolysis of ING3. The SCF E3 ligase complex plays an important role in controlling the level of proteins governing cell cycle progression (Ang and Wade Harper, 2005). Skp2 is well known as a critical S-phase promoting molecule by targeting the CDK inhibitor p27 for proteolysis (Frescas and Pagano, 2008). It also targets many other cellular proteins for degradation including cyclin-dependent kinase inhibitors p21, p27, p57, TOB1 (transducer of Erbb2), RASSF1 (Ras association domain family 1) and FOXO1 (forkhead box-containing transcription factor 1) (Frescas and Pagano, 2008). Upregulation of Skp2 has been shown in several types of cancers and may be an attractive target for the development of novel cancer-intervention strategies (Chiappetta et al., 2007; Hershko and Ciechanover, 1998; Masuda et al., 2002; Tanami et al., 2004; Yang et al., 2002; Yokoi et al., 2004). In melanoma Skp2 expression is significantly increased, which correlates with a poorer patient survival (Li et al., 2004; Woenckhaus et al., 2005). Suppression of Skp2 by RNA interference has been shown to inhibit melanoma growth effectively (Katagiri et al., 2006; Sumimoto et al., 2006). Consistent with the role of ING3 in cell cycle control (Nagashima et al., 2003), we find that degradation of ING3 protein is under the tight control of SCFSkp2 E3 complex. Since SCFSkp2 E3 ligase complex interacts with their substrates for ubiquitination through the phosphorylated consensus sequence in their target proteins (Frescas and Pagano, 2008), the ongoing work to identify the phosphomotifs of ING3 will improve our knowledge of the subtle mechanism(s) controlling ING3 protein turnover. In addition to the fact that the majority of ING3 is detected in the 26S proteasome fraction, ING3 is also detectable in the 20S proteasome (Fig. 5.5A), which may  115  be from the disassembled 26S proteasome, or suggest that degradation of ING3 may also take place in a proteasome pathway independently of ubiquitination. Interruption of ING3 degradation enhances its tumour suppressive roles in G1/S cell cycle transition and UV-induced apoptosis (Fig. 5.11). Interestingly, ING3 is able to enhance the promoter activation of the p21 gene (Nagashima et al., 2003), whereas p21 protein turnover during G1/S transition is also controlled by SCFSkp2 E3 ligase complex in non-stressed conditions (Bornstein et al., 2003; Li et al., 1998b; Wang et al., 2005). Therefore our current study may provide another explanation for how Skp2 affects p21 expression, by triggering the rapid turnover of ING3 in non-stressed conditions. Although overexpression of ING3 induced G1 cell cycle arrest in non-stressed MMRU cells (Fig. 5.11B), it promoted cell apoptosis upon UV irradiation instead of inducing G1 phase arrest (Fig. 5.11D), suggesting that the role of ING3 on p21 is overridden by other mechanisms including increased Fas expression (Wang and Li, 2006). Past studies have also demonstrated that UV can trigger rapid degradation of p21 through a Skp2-independent pathway (Bendjennat et al., 2003; Lee et al., 2006), and facilitate the induction of apoptosis (Bissonnette and Hunting, 1998; Rieber and Strasberg Rieber, 2000). Strategies based on the targeting of key components of ubiquitin-proteasome machinery have recently emerged as an effective way to kill tumour cells in several cancer types including hematologic malignancies and solid tumours (Chauhan et al., 2006; Cusack, 2003; Mitsiades et al., 2006; Zavrski et al., 2007). In the current study, the tumour suppressive role of ING3 in MMRU cells was enhanced when the degradation of ING3 was interrupted, implying that stabilization of ING3 protein by interfering with the ING3 degradation pathway may be a possible strategy in the treatment of malignant melanoma.  116  CHAPTER 6 GENERAL CONCLUSION ING proteins have been shown to be down-regulated in a broad variety of cancer types and may play an important role in cancer development and progression. Extensive studies have linked ING proteins to the regulation of cell growth, apoptosis, DNA repair, invasion and angiogenesis (Campos et al., 2004; Gong et al., 2005). In this study, we have established a fundamental role of ING3 in tumourigenesis of human cutaneous melanoma. Reduced nuclear ING3 level significantly correlated with melanoma progression and a poorer disease-specific 5-year survival of patients with primary melanoma (Wang et al., 2007), suggesting that ING3 may be a potentially valuable target for designing novel strategies for melanoma treatment. Our current studies reveal that the expression ING3 in melanomas is deregulated. Although the mechanisms underlying the aberrant ING3 expression in melanomas are not fully understood, our studies have demonstrated that i) reduced nuclear ING3 in primary and metastatic melanomas may be caused by a nucleus-to-cytoplasm relocalization; and ii) downregulation of ING3 in advanced melanomas is a result of rapid protein degradation. However, the molecular basis for the nucleus-to-cytoplasm shift of ING3 is not clear. Recent studies reported that 14-3-3 proteins can tether ING1b in the cytoplasm (Gong et al., 2006). It will be interesting to examine if ING3 is sequestered in cytosol through the same pathway. In addition, future mutational analysis of ING3 gene in melanomas will help explain the mechanisms leading to aberrant ING3 expression at both subcellular localization level and protein degradation level. Although ING3 has no obvious effect on melanoma cell apoptosis at basal level, it significantly enhances UV-induced apoptosis in melanoma cells in our study (Wang and Li,  117  2006). Apoptosis plays a fundamental role during the development and dysregulation of apoptosis significantly contributes to cancer initiation and progression (Hanahan and Weinberg, 2000; Thompson, 1995). Since UV radiation is the major environment factor of melanoma tumourigenesis, the inhibitory role of ING3 in UV-induced apoptosis in melanoma cells indicates that deregulation of ING3 contributes to melanoma development by evasion of apoptosis. We also find that ING3-enhanced apoptosis upon UV irradiation is independent of p53 function and is mediated by Fas/caspase-8 pathway. However, the mechanism for ING3-induced Fas expression remains to be explored. ING3 is an important component of NuA4 Tip60 HAT complex (Doyon et al., 2004), which is involved in ionizing radiation-induced apoptosis in Hela cells (Ikura et al., 2000) and UV-induced apoptosis in U2OS osteosarcoma cells (Tyteca et al., 2006). Interestingly both p53dependent and p53-independent pathways are involved in the function of Tip60 (tatinteractive protein 60) in cellular response to DNA damage (Eymin et al., 2006; He et al., 2005b). Therefore it is very important to investigate if the activity of NuA4 Tip60 HAT complex is required for the function of ING3 in UV-induced apoptosis and how ING3 may be involved in p53-independent function of Tip60 in cellular response to UV radiation. Furthermore, since p53 can activate the promoter activity of Fas gene (Muller et al., 1998) and promotes the redistribution of cytoplasmic Fas to the cell surface through transportation from the Golgi complex upon DNA damage (Bennett et al., 1998), it is also worthy to explore how ING3 upregulates Fas expression independently of p53 function. This thesis also demonstrates that degradation of ING3 in melanoma cells is mediated by ubiquitin-proteasome system. The ubiquination and degradation of ING3 is highly regulated by the SCFSkp2 E3 ubiquitin ligase complex. Since the F-box protein Skp2  118  recognizes substrates for ubiquitination through the phosphorylated consensus sequence in their target proteins (Frescas and Pagano, 2008), future studies are needed to provide the molecular basis for the interaction between ING3 and Skp2. Furthermore, two independent studies suggest that the increased expression of Skp2 correlates with melanoma progression (Li et al., 2004; Woenckhaus et al., 2005). In the future, it will be necessary to examine the expression of Skp2 in our established TMA system with immunohistochemistry and perform the correlation analysis to delineate the relationship between ING3 and Skp2 expression. Future studies may also be required to address why increased Skp2 in primary melanomas has no obvious affect on overall ING3 expression. The restoration of ING3 expression by either stable overexpression or interrupting its degradation significantly improves the sensitivity of melanoma cells to UV radiation and stimulates ING3-induced G1 cell cycle arrest in non-stressed melanoma cells. These observations not only highlight the role of ING3 in melanoma development, but support the potential value of ING3 in melanoma treatment. 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