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Role of tumor suppressor p33ING2 inhuman cutaneous melanoma Lu, Fuqu 2006

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R O L E O F T U M O R SUPPRESSOR P33ING2 IN H U M A N C U T A N E O U S MELANOMA by FUQU L U B . M . , Harbin Medical University, 2000  A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T OF T H E REQUIREMENTS FOR THE D E G R E E OF M A S T E R OF S C I E N C E in T H E F A C U L T Y OF G R A D U A T E STUDIES (Experimental Medicine)  T H E U N I V E R S I T Y OF BRITISH C O L U M B I A  December 2006 © Fuqu L u , 2006  ABSTRACT  P33JNG2 was cloned through a homology search with p33INGlb,  the founding member o f ING  family proteins in 1998. There have been several studies indicating that p331NG2 is a tumor suppressor candidate since it is involved in the regulation o f transcription, apoptosis and senescence. People in our lab already revealed that p33ING2 plays an essential role in cellular stress response against U V irradiation ( U V R ) either by enhancing nucleotide excision repair (NER) or promoting apoptosis in melanoma cell line. So far, reduced I N G family proteins have been reported in different tumor types including the loss o f nuclear expression o f p 3 3 I N G l b in melanoma. Since p33ING2 is highly homologous to p331NGlb, we hypothesized that aberrant expression o f p331NG2 may play a role in melanoma tumorigenesis. To test this hypothesis, we used tissue microarray ( T M A ) and immunohistochemistry to evaluate the expression pattern o f p33ING2 in different stages o f melanocyte lesions. We found that nuclear 1NG2 expression is significantly reduced in melanomas compared with dysplastic nevi. Moreover, there is no correlation between TNG2 nuclear expression and patient clinicopathological parameters or between 1NG2 nuclear expression and patient's 5-year survival in primary melanomas and metastatic melanomas. We then investigated i f gene mutation is the reason for reduced ING2 nuclear expression in melanomas. Our results showed that no mutation is found in melanoma cell lines or melanoma tissue samples. At last, we restored the expression o f p33TNG2 in melanomas by establishing the 1NG2 stable cell line, and c D N A microarray analysis was performed to identify the most abundantly induced or suppressed genes by overexpressed I N G 2 or U V R . We found that U V R can enhance expressions o f two relatively new genes BTG2 and PLK3 while stably expression o f p33.ING2 significantly decreased R A R R E S 1 (TIG 1) expression.  ii  TABLE OF  CONTENTS  ABSTRACT  ii  T A B L E OF CONTENTS  iii  LIST OF T A B L E S  v  LIST OF F I G U R E S  vi  LIST OF A B B R E V I A T I O N S  vii  ACKNOWLEDGEMENTS CHAPTER 1.1  viii  1. I N T R O D U C T I O N  1  Human Melanoma  1  1.1.1 M e l a n o m a Incidence and M o r t a l i t y R a t e  1  1.1.2 R i s k Factors  1.1.3 C h a r a c t e r i s t i c s o f D i f f e r e n t Stages o f M e l a n o c y t e L e s i o n s 1.1.4 M o l e c u l a r C h a n g e s in M e l a n o m a  1.2  2  3  1.1.5 M e l a n o m a Treatment and P r o g n o s i s  5  1.2.1 I N G 1  7  Inhibitor o f G r o w t h (ING) F a m i l y  6  1.2.2 P 3 3 I N G 2  10  1.2.2.1 G e n e and Protein Structures  1.2.2.2 E x p r e s s i o n Profiles  1.2.2.3 F u n c t i o n s o f P 3 3 I N G 2  1.3  2  1.2.2.4 M o d e s o f P 3 3 I N G 2 A c t i o n  10  ,  Objective  C H A P T E R 2. M A T E R I A L S A N D M E T H O D S  11  12  14  14  16  2.1  T M A Construction  16  2.2  Immunohistochemistry o f T M A  16  2.3  Evaluation o f Immunostaining  17  2.4  Statistical A n a l y s i s  18  2.5  C e l l L i n e s and C e l l C u l t u r e  18  2.6  N o r m a l and M e l a n o m a T i s s ues  1.9  2.7  U V Irradiation  19  2.8  P l a s m i d s , T r a n s f e c t i o n and I N G 2 Stable C l o n e G e n e r a t i o n  19  2.9  Western Blot Analysis  20  2.10  S R B Cell Survival Assay  20  2.11  D N A and R N A E x t r a c t i o n  21  2.12  Polymerase Chain Reaction  21  2.13  D N A Sequencing  22  2.14  c D N A Microarray  22  in  2.15  Q u a n t i t a t i v e Real T i m e P C R  23  C H A P T E R 3. N U C L E A R I N G 2 E X P R E S S I O N IS R E D U C E D I N  HUMAN  CUTANEOUS MELANOMAS  24  3.1  R a t i o n a l e and H y p o t h e s i s  24  3.2  Results  25  3.2.1 C l i n i c o p a t h o l o g i c a l F i n d i n g s  25  3.2.2 R e d u c e d I"NG2 N u c l e a r E x p r e s s i o n i n H u m a n M e l a n o m a s  25  3.2.3 C o r r e l a t i o n between I N G 2 N u c l e a r E x p r e s s i o n and C l i n i c o p a t h o l o g i c a l Parameters or 5-year Patient S u r v i v a l  3.3  Discussion  26  32  C H A P T E R 4. M U T A T I O N A L S T A T U S O F P33ING2 I N M E L A N O M A S  36  4.1  36  4.2  R a t i o n a l e and H y p o t h e s i s Results  37  4.2.1 A l t e r a t i o n s o f P 3 3 I N G 2 G e n e in M e l a n o m a C e l l L i n e  37  4.2.2 A l t e r a t i o n s o f the N L S and P H D D o m a i n o f P 3 3 I N G 2 G e n e in Melanoma  4.3  Discussion  40  C H A P T E R 5. I D E N T I F I C A T I O N O F G E N E S T R A N S C R I P T I O N A L L Y B Y U V I R R A D I A T I O N O R P33ING2 5.1  5.2  5.3  37  R a t i o n a l e and H y p o t h e s i s  REGULATED 43 43  Results  5.2.1 G r o w t h I n h i b i t i o n o f P 3 3 I N G 2  45  45  5.2.2 G e n e E x p r e s s i o n A n a l y s i s b y c D N A M i c r o a r r a y  45  5.2.3 R e a l time P C R A n a l y s i s  46  Discussion  53  C H A P T E R 6. G E N E R A L C O N C L U S I O N S  57  6.1  Summary  57  6.2  Future D i r e c t i o n s  58  REFERENCES  60  iv  LIST O F T A B L E S Table 3.1  C l i n i c o p a t h o l o g i c a l parameters o f 122 cases o f m e l a n o m a s  27  Table 4.1  JNG2 alterations in n o r m a l melanocyte and m e l a n o m a c e l l lines  38  Table 5.1  T o p 10 genes i n d u c e d b y U V i rradi ati on in M M R U  49  Table 5.2  T o p 10 genes suppressed b y U V irradiation in M M R U  50  Table 5.3  T o p 10 genes i n d u c e d b y p 3 3 1 N G 2  51  Table 5.4  T o p 10 genes suppressed b y p 3 3 I N G 2  52  v  LIST O F FIGURES  F i g u r e 1.1  Structural features o f the TNG family proteins  Figure 3.1  Representative images o f 1NG2 immunohistochemical staining in human  15  melanocytic lesions  28  Figure 3 . 2  I N G 2 nuclear expressions at different stages o f melanocytic lesions  29  Figure 3 . 3  N o correlation was found between ING2 nuclear expression and tumor thickness or tumor ulceration of primary melanomas  30  Figure 3 . 4  1NG2 nuclear expression and 5-year patient survival  31  F i g u r e 4.1  Representative sequence chromatograms o f ING2 mutations in Sk-mel-110 melanoma cell line  39  F i g u r e 5.1  I N G 2 is overexpressed in stable clones  47  F i g u r e 5.2  Cell survival rate by S R B assay o f UVB-irradiated M M R U cells and ING2 stable cell line  48  vi  LIST OF  ABBREVIATIONS  AJCC  A m e r i c a n J o i n t C o m m i t t e e on C a n c e r  CDK4  C y c l i n - d e p e n d e n t kinase 4  BCC  Basal cell carcinoma  CDKN2A  C y c l i n - d e p e n d e n t kinase i n h i b i t o r 2 A  C ECM  T h e c y c l e n u m b e r o f threshold  T  HAT  HDAC ING LOH MDM2 MMP-7  NER NLS  E x t r a c e l l u l a r matrix  H i stone acetyltransferase Histone.deacetylase Inhibitor o f g r o w t h Loss o f heterozygosity M o u s e d o u b l e minute 2  M a t r i x metalloproteinase 7 N u c l e o t i d e e x c i s i o n repair  N u c l e a r l o c a l i z a t i o n signal  NMuMG PBS PCNA  M o u s e m a m m a r y epithelial c e l l  PHD PIP  Plant h o m e o d o m a i n P C N A - i n t e r a c t i n g protein d o m a i n  PTEN Rb  RGP SRB  TCA TIG1 TMA  Phosphate-buffered saline  P r o l i f e r a t i n g c e l l u l a r nuclear antigen  Phosphatase and tensin h o m o l o g u e  R e t i n o b l a s t o m a protein R a d i a l g r o w t h phase p r i m a r y m e l a n o m a Sulforhodamine B  Trichloroacetate Tazarotene i n d u c e d gene 1 Tissue microarray  TSG  T u m o r suppressor gene  VGP  V e r t i c a l g r o w t h phase p r i m a r y m e l a n o m a  UVR  U l t r a v i o l e t irradiation  vu  ACKNOWLEDGEMENTS  I w o u l d l i k e to express m y sincere thanks to m y supervisor D r . G a n g L i , for p r o v i d i n g me the opportunity to study in y o u r laboratory and set up m y first step in research. T o D r . B i l l S a l h , Dr. Y o u w e n Z h o u , D r . K e v i n J M c E l w e e , and D r . Torsten N i e l s e n , I w a n t to thank y o u for b e i n g m y committee members and f o r y o u r suggestions on m y study. I w o u l d l i k e to thank D r . M a g d a l e n a M a r t i n k a and D e r e k L. D a i for the technical assistance in tissue m i c r o a r r a y s t a i n i n g evaluation and statistical a n a l y s i s ; D r . C o l l e e n N e l s o n and A n n e Haegert for the assistance in  cDNA  m i c r o a r r a y studies; M i n w a n S u and Jun L i for assistance in tissue s a m p l e c o l l e c t i o n and D N A sequencing.  I w o u l d also l i k e to thank the m e m b e r s o f L i lab and m y f a m i l y , y o u r encouragement, support and help are greatly appreciated.  vin  C H A P T E R 1. I N T R O D U C T I O N  1.1.  Human Melanoma  Human cutaneous  malignant melanoma originates from the melanocytes o f the skin or  melanocyte lesions including common acquired nevus, dysplastic nevus, congenital nevus and cellular blue nevus (Balch et al, 2001). Although it only accounts for 4 percent o f all dermatologic cancers, melanoma is responsible for 80 percent o f deaths from skin cancer. Only 14 percent o f patients with metastatic melanoma survive for 5 years (Cancer facts & figures, 2003. Atlanta: American Cancer Society, 2003). The intractability o f melanoma promotes the need to learn more about the changes that facilitate melanoma genesis, progression and metastasis.  1.1.1  Melanoma Incidence and Mortality Rate  Once considered as an uncommon disease, the incidence o f malignant melanoma has doubled within the last 10 years in the United States (Balch et al, 2001). It is estimated that the chance o f an American developing melanoma during his/her lifetime leaped from 1 i n 1500 in 1960, to 1 in 68 in 2000, and is projected to increase to 1 in 50 by the year 2010 (Rigel, 2002; Dunlap et al, 2004). Primary melanoma usually progresses from the nonmetastatic radial growth phase to the vertical growth phase, in which the malignant cells invade the dermis and develop the ability to metastasize (Elder, 1999). The large majority o f patients with early stages o f melanoma can be cured by surgical removal while 50% o f patients with distant metastasis die within the first 6 months after the diagnosis (Balch et al, 2001; Schoenlaub et al, 2001).  1.1.2 Risk Factors The strongest risk factors for melanoma are family history o f melanoma, previous melanoma and multiple benign or atypical nevi. Additional factors include immunosuppression and ultraviolet radiation exposure. Each risk factor reflects a genetic predisposition or an environmental stressor that favors melanomagenesis. For example, the mutation o f cyclin-dependent kinase inhibitor 2 A ( C D K N 2 A ) was observed in 33/36 cases o f melanoma in nine families and a few rare kindreds have mutations in cyclin-dependent kinase 4 ( C D K 4 ) (Hussussian et al, 2004; Zuo et al, 1996). U V R is the major environmental risk factor for melanoma. It is well documented that U V R promotes the malignant change i n the skin by having direct mutagenic effects on D N A , by stimulating the cellular components to produce growth factors and by reducing cutaneous immune defenses (Gilchrest et al, 1999; Thompson et al, 2005). O n the other hand, the tanning response is a defensive mechanism in which melanocytes produce melanin and deliver it to neighboring keratinocytes. Melanin then forms a protective cap over the outer part o f keratinocyte nuclei where it absorbs and dissipates ultraviolet energy (Gilchrest et al, 1999; Ortonne, 2002).  1.1.3  Characteristics of Different Stages of Melanocytic Lesions  Normal nevi are the first phenotypic changes in melanocytes which derive from the proliferation o f structurally normal melanocytes. Clinically, these nevi present as flat or slightly raised lesions with either uniform coloration or a regular pattern o f dot-like pigment i n a tan or dark brown background. Atypical nevi, also referred to as dysplastic nevi, are moles that develop from aberrant growth o f preexisting benign nevi or in a new location. Clinically, such lesions may be  asymmetric, have irregular borders, contain multiple colors or have increasing diameters. The clinical importance o f dysplastic nevi lies in their association with an increased risk o f malignant melanoma which is supported by cohort and case-control studies (Hussein, 2005). The characteristic o f the radial growth phase ( R G P ) primary melanoma is that the malignant cells grow only within or i n close proximity to the epidermis and they do not have competence for metastasis (Hsu et al, 1998). Clinically, they sometimes present as raised lesions. These lesions no longer display random atypia and instead show cytomorphologic cancer throughout the lesion (Urso, 2004). Lesions that progress to the vertical growth phase ( V G P ) acquire the ability to invade the dermis. The melanoma cells i n V G P are capable o f growth in soft agar, and have the capacity to form tumor nodules when implanted to nude mice. The conversion o f primary melanomas from R G P to V G P is the most critical step i n melanoma progression and ultimately in disease outcome (Erhard et al, 1997; H s u et al, 1998). The final step melanoma metastasis is the spread o f malignant cells to other parts o f skin and other organs. These cells can grow in soft agar and when implanted i n nude mice can form tumor nodules and metastasize. A t this stage, cells usually spread to nearby lymph node and distant metastases typically are found in the skin, liver, lung, bone, and brain.  1.1.4  Molecular Changes in Melanoma  The histological changes o f melanocytic lesions relate to particular gene mutations and these mutations further affect molecular signaling and are responsible for the progression from normal melanocyte to melanoma (Miller and M i h m , 2006). P53, a central sensor linking D N A damage to apoptosis, plays an essential role in tumor suppression and chemosensitivity in many tumor  types ( L i et al, 1998; Raffo et al, 2000; Fridman and Lowe, 2003). However, mutational analysis reveals that the p53 gene is altered i n only approximately 11% o f melanomas (Hussein, 2004). The low mutation rate of p53 suggests that other genes may play important roles i n pathogenesis of melanoma.  N - R A S , B R A F , and M A P K G r o w t h F a c t o r Signaling Pathway Activation o f this pathway is due to the somatic mutations o f NRAS, which occur i n up to 30% cases o f cutaneous malignant melanoma or BRAF, which are found in 59% melanoma patients (Omholt et al, 2002; Omholt et al, 2003). Most o f NRAS mutations are at codon 61 (Q61R and Q61K) and result i n the expression o f p 2 1 R A S oncoprotein, which remains constitutively G T P bound and active (Malumbres and Barbacid, 2003); A l l the B R A F mutations were found within the kinase domain, with a single substitution (T to A ) o f glutamate for valine at codon 600 (V600E) being responsible for 90% o f the observed mutations (Davies et al, 2002). NRAS and BRAF mutations, which occur exclusively o f each other, cause constitutively active expression o f the serine-threonine kinase in the E R K - M A P K pathway. Similar frequency o f BRAF mutations in benign nevi, primary and metastatic melanoma suggests that nevi must acquire additional molecular changes to free themselves o f growth constraint and become malignant. For instance, i n zebrafish, melanocyte specific expression o f mutant B R A F protein leads to proliferation o f melanocytes, analogous to human nevi. However, the combination o f BRAF mutation and p53 inactivation causes melanocytes to transform and become melanoma (Patton et al, 2005).  CDKN2A Cyclin-dependent kinase inhibitor ( C D K N ) 2 A is located on chromosome 9p21. Germline mutations o f CDKN2A gene have been reported in numerous melanoma-prone families or melanoma cases selected because o f young age or multiple primary tumors (Kamb et al, 1994; Ruas and Peters, 1998). Alternative splicing o f various exons within CDKN2A yields two distinct tumor suppressor proteins, I N K 4 A ( p l 6  I N K 4 A  ) and alternate reading frame ( A R F ) .  INK4A  contributes to cell cycle arrest at the G l - S check point by inhibiting cyclin-dependent kinases 4 and 6. I N K 4 A suppresses the proliferation o f cells with damaged D N A or activated oncogenes and also acts when cells are old or crowded (Sharpless and Chin, 2003). I N K 4 A deficient mice appear normal but are more susceptible to carcinogens and easily develop various tumors (Serrano et al, 1996). The INK4A" "mice develop spontaneous cutaneous melanomas when /  combined with melanocyte-specific expression o f activated H-ras after a short latency and with high penetrance (Chin et al, 1997). A R F participates i n the core regulatory process that controls the levels o f p53 by binding to mouse double minute 2 ( M D M 2 ) protein, sequestering it from p53 and therefore causing p53 to accumulate. P53 can arrest the cells at G 2 - M phase, allowing for repairing o f damaged D N A or inducing apoptosis (Harris and Levine, 2005). In animals, A R F deficient shortens the time required to develop melanoma after exposure to U V light. When both CDKN2A product (pl6  1.1.5  I N K 4 A  and A R F ) are deficient, the latent time is even shorter (Recio et al, 2002).  Melanoma Treatment and Prognosis  To date, the success o f systemic therapy for melanoma remains unsatisfactory. Melanoma is associated with a very high mortality rate, especially for advanced disease. Treatment options  and follow up intervals for melanoma patients vary with the depth o f primary lesion and stage. Surgical excision is the primary therapeutic approach for > melanoma, which is almost 100% effective when treating an early tumor. Chemotherapy and adjuvant therapy including biologic response modifiers and vaccines, provide supplemental treatment for more advanced tumors (Rigel and Carucci, 2000). In general, the prognosis for the melanoma depends on the stage at diagnosis. For patients with American Joint Committee on Cancer ( A J C C ) , the overall survival is 70% for A J C C stages I and II, 30% for A J C C stage III patients and only 10% for stage I V patients (Balch et al, 2001). For certain stage primary melanoma, the dominant predictors for prognosis are lesion thickness, ulceration and lymph node involvement. Factors such as age, sex, anatomic location and satellite/in transit lesions are important. Moreover, for the metastatic melanoma, the most important prognostic factors include sites o f metastasis and the presence o f increased serum lactic dehydrogenase (Homsi et al, 2005).  1.2  I n h i b i t o r of G r o w t h ( I N G ) F a m i l y  The I N G tumor suppressor family consists o f five members including I N G 1 , p33ING2, p47ING3, p29ING4 and p28ING5. The I N G 1 gene, due to alternative splicing o f its m R N A product, encodes three isoforms p 4 7 I N G l a , p 3 3 I N G l b , p24DSfGlc (Garkavtsev et al, 1999; Gunduz et al, 2000). It is not clear whether the other I N G family members also contain multiple splicing isoforms, but structure analysis shows they all share a nuclear localization signal ( N L S ) and a plant homeodomain ( P H D ) type zinc finger which indicate I N G family members may have similar biochemical functions (Fig. 1.1) (Gong et al, 2005). N L S domain indicates the nuclear localization o f I N G family proteins and mutations o f this domain may disrupt interactions  between I N G intracellular trafficking proteins resulting in a loss o f nuclear I N G expression and affects their normal functions. In addition, P H D domains o f I N G 1 and I N G 2 can bind to phosphoinositides both i n vitro and i n vivo and this interaction is crucial for I N G 2 to activate p53 and p53 dependent apoptotic pathways (Gozani et al, 2003). O n the other hand, proteins with P H D domain are intimately associated with chromatin remodeling and subsequent transcriptional regulation o f specific genes. A recent article published i n Nature reported that the I N G P H D domains are specific and highly robust binding modules for d i - and tri-methylated lysine 4 o f histone H 3 (H3K4me2 and H3K4me3) and this association is important for I N G 2 mediated cellular responses to genotoxic insults (Shi et al, 2006). I N G proteins have been found to be differentially contributed to other biologic activities including cell cycle arrest, apoptosis, D N A repair, senescence, anti-angiogenesis (Campos et al, 2004; Shi and Gozani, 2005). The mechanisms underlying these functions are partly due to the ability o f I N G proteins associated with different histone acetyltransferases ( H A T s ) and histone deacetylase ( H D A C ) to posttranslationally modify the core histones and the p53 tumor suppressor (Kuzmichev et al, 2002; Vieyra et al, 2002; Doyon et al, 2006).  1.2.1  ING1  I N G 1 , the founding member o f the I N G tumor suppressor family, was discovered in 1996 through suppressive subtractive hybridization between normal human mammary epithelial cells and breast cancer cell line, and further in vivo selection o f genetic suppressor elements (Garkavtsev et al, 1996). Genetic mapping disclosed that ING1 gene is located on the long arm of chromosome 13 in which genetic deletions have been reported in a variety o f human tumors (Garkavtsev etal, 1997).  A m o n g all three isoforms o f I N G 1 , p 3 3 I N G l b is the predominant form expressed in normal tissues, with various expression levels in different types o f tissues (Saito et al, 2000). ING1 expression screening showed that down-regulation o f ING1 is a frequent event in different tumor types including breast (Garkavtsev et al, 1996; Toyama et al, 1999), gastric (Oki et al, 1999), esophageal (Chen et al, 2001), blood (Ito et al, 2002), lung (Kameyama et al, 2003) and brain (Tallen et al, 2003). The mechanisms resulting in decreased I N G expression are unknown and currently under active investigation. Lines o f evidence suggest that ING1 can influence cell cycle progression and are actively involved in cellular checkpoints. One candidate mechanism is the cooperation o f p 3 3 I N G l b with p53 tumor suppressor to enhance the transcription o f cyclin dependent kinase inhibitor ( C D K I ) p21 (Garkavtsev et al, 1998). P21 affects the G l cellular checkpoint by binding and inactivating c y c l i n - C D K complexes, therefore leading to reduced phosphorylation o f retinoblastoma protein (Rb), transcriptional E 2 F sequestration and G l - S cell cycle arrest (Harper et al, 1993). Moreover, c D N A microarray analysis relates p33INGb induced G 2 - M arrest to cyclin B l (Takahashi et al, 2002), which accumulates during G 2 - M phase o f the cell cycle and associates with cdc2 for mitotic initiation (Elledge, 1996). The study is performed using mouse mammary epithelial cells ( N M u M G ) which are transformed with antisense ING1 and presented 14 upregulated genes including cyclin Bl,  proto-oncogene  DEK,  and osteopontin while overexpressed ING1 in  N M u M G cells resulted i n down-regulation o f cyclin Bl, DEK, and osteopontin (). Consistent with the c D N A microarray finding, later studies revealed that overexpression o f p 3 3 I N G l b was able to enhance adriamycin-induced G 2 arrest i n the H1299 non-small cell lung carcinoma cell line (Tsang et al, 2003).  In addition to its involvement in cell cycle regulation, ING1 is w e l l documented to be able to induce apoptosis. Initial evidence o f this function came from the observation o f prominent expression o f ING1 in regressing tail o f Xenopus tadpoles while absence from growing hind limbs as well as the induced expression o f I N G 1 in serum-starved P I 9 teratocarcinoma cells (Helbing et al, 1997; Wagner et al, 2001). Cooperation with p53 was quickly recognized since ING1 expressing fibroblasts had little effect on cell survival in the absence o f p53, while both ING1 and p53 were required to suppress colony formation (Garkavtsev et al, 1998). Moreover, people i n our lab reported that overexpressed ING1 was able to increase UV-induced apoptosis in p53 wild-type M M R U melanoma cell line but not in p53 mutant M E W O cell line and we further found overexpression o f ING1 enhances endogenous bax expression and alter the mitochondrial membrane potential (Cheung and L i , 2002). In addition, one study demonstrated that the ectopic expression o f p 3 3 I N G l b rather than p 4 7 I N G l a sensitized early passage o f fibroblast to apoptosis (Vieyra et al, 2002). This difference reflects the different N-terminal sequence o f I N G proteins since p 3 3 I N G l , but not p 4 7 I N G l a , contains a PCNA-interacting protein (PIP) domain, which permits p 3 3 I N G l b to physically interact with P C N A following U V exposure and induce apoptosis (Scott et al, 2001). Previous studies have shown that ING1 physically binds to p53 and shares similar functions with p53. People i n our lab therefore hypothesized that I N G 1 , like p53, may play an important role in D N A repair. They found that U V irradiation could induce cell type specific p3 3 I N G l b expression in a time- and dose-dependent manner (Cheung et al, 2000) and overexpression  o f I N G 1 enhanced  nucleotide  excision repair  ( N E R ) o f UVC-damaged  exogenous plasmid D N A and U V B - d a m a g e d genomic D N A . A s expected, p 3 3 I N G l b mediated enhancement o f D N A repair was also dependent on the existence o f functional p53. They further  demonstrated that there is a weak association between p 3 3 I N G l b and G A D D 4 5 , but not other repair proteins like X P A and X P B (Cheung et al, 2001). Several other studies also clarified the involvement o f p 3 3 I N G l b i n D N A repair machinery, however, the mechanism underlying p 3 3 I N G l b enhanced D N A repair is still not very clear.  1.2.2  P33ING2  P33ING2, also known as I N G 1 L ( " I N G l - l i k e molecule"), was first isolated by Shimada and his colleagues through a homology search from a private Otsuka c D N A data base (Shimada et al, 1998). P33ING2 has been shown to negatively regulate cell growth, enhance nucleotide excision repair upon U V R as well as regulate the onset o f replicative senescence. A l l these functions require the presence o f functional p53 (Chin et al, 2005; Pedeux et al, 2005; Wang J et al, 2006).  1.2.2.1  Gene and Protein Structures  The full-length p33ING2  c D N A contains an 840bp length open reading frame, encoding a 280  amino acid protein with a 32.8 k D a molecular weight. The gene product o f p33ING2 shows 58.9% identity with p 3 3 I N G l b , while nucleotide identities between the two genes are 60% (Shimada et al, 1998). Fluorescence in situ hybridization and radiation-hybrid analysis assigned p33ING2 chromosome 4 (Shimada et al, 1998).  to  Besides the common P H D zinc finger motif and N L S  domain, I N G 2 also contains a unique leucine zipper domain which is thought to mediate hydrophobic protein-protein interaction (Feng et al, 2002). I N G 2 is mainly localized in the nucleus with 74% i n the chromatin/nuclear matrix and 9% i n the nucleoplasm i n HT1080 fibrosarcoma cells (Gozani et al, 2003).  -10-  1.2.2.2  Expression Profiles  Northern blot analysis revealed ubiquitous expression o f p33ING2 i n various normal tissues with highest expression detected in testis. Subsequent expression screening o f 20 colon tumors showed higher p33ING2 expression level in all colon cancers compared with the normal tissues acquired at the same surgical sites. The importance and involved mechanism o f elevated p33ING2 expression in colon cancers was still unknown but the p53 abnormalities were found to occur frequently in this type o f cancer (Shimada et al, 1998). Nagashima et al also detected p33ING2 expression in 12 different types o f human cell lines. The level o f p33ING2 was highly variable among the cell lines with no visible expression in 5 o f the 12 cell lines and the expression status did not correlate with the mutational status o f p53 (Nagashima et al, 2001). A recent study detected the expression o f p33ING2 in lung cancers and found decreased I N G 2 expression i n 6 o f 7 lung cancer cell lines with mutant p53 which suggests I N G 2 gene may be an independent tumor suppressor candidate on p53 in this type o f cancer. In addition, they detected p33ING2 mutations i n 30 human lung cancer cell lines and 31 primary lung cancer tumors but failed to detect any mutation (Okano et al, 2006). Furthermore, studies on loss o f heterozygosity ( L O H ) in sporadic basal cell carcinomas ( B C C ) reported a high frequency o f L O H (30%) on chromosome 4q32-35, which is mapped to p33ING2 and SAP30, both o f which are believed to be involved in chromatin remodeling and gene regulation (Sironi et al, 2004). The importance o f these reported abnormal expressions o f p33ING2 i n cancer cells is not clear. However, since p 3 3 I N G 2 is capable o f activating both the B a x and p 2 1  W a f l  promoters, loss  of p33ING2 may impair the proper regulation o f cell cycle and apoptosis and lead to cellular transformation and tumorigenesis (Nagashima et al, 2001).  1.2.2.3  Functions of P 3 3 I N G 2  The first evidence supports p33ING2  as a tumor suppressor gene is based on the finding that  overexpressed p33ING2 can strongly inhibit colony formation i n w i l d type p53 containing colorectal carcinoma R K O cells, but not as completely as in R K O E 6 cells which contain inactive p53. The tumor suppression function o f p33ING2 is further supported b y the finding that its expression is specifically induced by D N A damage agents including etoposide  and  neocarzinostatin, but not by y irradiation, doxorubicin, bleomycin or cw-platinum. The molecular mechanism underlying this function is due to the ability o f p33ING2 to increase the acetylation of p53, therefore stabilizes p53 and further enhances the promoter activities o f p 2 1  W a f l  and Bax  (Nagashima et al, 2001). Consistent with this, people in our lab also found that overexpression o f p33ING2 induces more apoptosis in UVB-irradiated and non-irradiated melanoma M M R U cells and the enhancement o f apoptosis requires the existence o f functional p53. Furthermore, we found  overexpressed  p 3 3 I N G 2 significantly downregulates  antiapoptotic  molecule  Bcl-2  expression, resulting in an increased Bax/Bcl-2 ratio. Moreover, p33ING2 can promote the translocation o f B a x to mitochondria, alter the mitochondrial membrane potential and initiate mitochondrion-mediated apoptotic pathway (Chin et al, 2005). The severity o f D N A damage decides whether the cells w i l l go through D N A repair or apoptosis. Our previous study has shown that overexpressed p 3 3 I N G l b increases nucleotide excision repair ( N E R ) o f UV-damaged D N A in a p53 dependent manner (Cheung et al, 2001). The structural and functional similarities between p 3 3 I N G l b and p 3 3 I N G 2 prompted us to analyze the role o f p 3 3 I N G 2 in N E R . W e found that p33ING2 significantly enhances N E R in melanoma cell line i n a p53 dependent manner by rapidly inducing the acetylation o f histone H 4 , chromatin relaxation and facilitating the recruitment o f the photolesion-recognition protein X P A  - 12-  to the D N A damage site (Wang J et al, 2006). The recruitment o f X P A to the D N A damage site is considered to be a rate-limiting process for N E R (Thoma et al, 2003). Cellular senescence, a process originally described by Hayflick i n 1965, prevents normal human fibroblasts from growing indefinitely in culture (Hayflick, 1965). The fact that p53 acetylation, which is required for its full activity, increases in replicative senescence and the capability o f p33ING2 to increase p53 acetylation prompted people to investigate  the  involvement o f p33ING2 i n replicative senescence (Pearson et al, 2000; Nagashima et al, 2001). Their studies demonstrated that expression levels o f I N G 2 and p53 lysine 382 acetylation are elevated during replicative senescence  in human fibroblasts. Theoretically, p33ING2 can  enhance the binding o f p53 to histone acetyltransferase p300 and acts as a cofactor for p300mediated p53 acetylation. In addition, p33ING2 also regulates the onset o f replicative senescence since overexpressed I N G 2 in young fibroblasts can induce premature senescence in a p53dependent manner while down-regulation o f p33ING2 decreases p53 acetylation and delays the onset o f replicative senescence (Pedeux et al, 2005). Gozani and colleagues first reported that I N G 2 functions as a nuclear phophoinositide (PtdlnsP) receptor through its P H D domain by performing a library expression screening using PtdlnsP-affmity resins and found p33ING2 can bind to both the PtdIns(3)P and PtdIns(5)P. Furthermore, they demonstrated that the I N G 2 P H D finger interacts with PtdIns(5)P in vivo and this interaction regulates the ability o f p33ING2 to activate p53 and p53-dependent apoptosis. Moreover, the P H D finger is found to be a general PtdlnsP bindng molecule (Gozoni et al, 2003).  -13 -  1.2.2.4 M o d e s of P 3 3 I N G 2 A c t i o n Except for promoting p53 posttranslational modification that activates the p53 tumor suppressor protein, I N G 2 is a stable component o f a S i n 3 A - H D A C complex (Doyon et al, 2006) and it can also binds to histone acetyl transferase p300. Recent studies found that the I N G P H D domains are specific binding modules for d i - or tri-methylated lysine 4 o f histone H 3 . P33ING2 especially binds with high affinity to the trimethylated species. The interaction is pivotal to stabilize the S i n 3 A - H D A C complex at the promoters o f proliferation genes, leading to active gene repression in response to D N A damage (Shi et al, 2006).  1.3  Objective  The primary objective o f this study is to understand the role o f tumor suppressor p33ING2 in melanoma. W e , therefore,  evaluated the expression o f p33ING2 i n different  melanocytic lesions and correlated the expression pattern  stages o f  to various clinicopathological  parameters and 5-year patient survival. Moreover, we also evaluated the mutational status o f p33ING2 gene using the c D N A from melanoma cell lines and genomic D N A from formalinfixed, paraffin-embedded normal tissues as well as melanoma tissues. A t last, c D N A microarray was performed to analyze the transcriptional changes in M M R U melanoma cell line treated with U V R or stably transfected p33ING2.  - 14-  Figure 1.1 Structure features of the I N G family proteins  P33INGlb  ING2  ING3  NLS  PIP  Leucine zipper  NLS  NLS  PHD  PHD  PHD  ING4  NLS  NLS  ING5  - 15 -  PHD  PHD  NLS  NLS  C H A P T E R 2. M A T E R I A L S A N D M E T H O D S  2.1  T M A Construction  Formalin-fixed, paraffin-embedded tissue blocks containing normal nevi, dysplastic nevi, primary melanomas and metastatic melanomas were used i n this study. Sample collection was approved by the University o f British Columbia and was performed i n accordance with the Declaration o f Helsinki Guidelines. A l l the tissue samples were from 1990 to 1997 archives o f the Department  o f Pathology, Vancouver General Hospital.  For each case,  the  most  representative lesion area was selected and marked on haematoxylin-eosin-stained slides. Taking into account the limitations o f the representative areas o f the tumor, duplicate 0.6-mm-diameter tissue cores were taken from each biopsy and the T M A s were assembled using a tissue array instrument (Beecher Instruments, Silver Spring, M D , U S A ) . Multiple 4-um sections were cut with a Leica microtome and then transferred to adhesive-coated slides using routine histology procedures. One section from each T M A was routinely stained with haematoxylin and eosin. The remaining sections were stored at room temperature for immunohistochemistry staining.  2.2  Immunohistochemistry of T M A  The T M A slides were deparaffmed by heating at 55°C for 30 m i n following by three washes with xylene, 5 m i n each. Tissues were then rehydrated in a series o f 5 m i n washes i n 100, 90 and 70% ethanol and rinsed with phosphate-buffered saline (PBS). Antigen retrieval was performed by heating the samples at 95°C for 30 m i n in 10 m M sodium citrate (pH 6.0). Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 20 m i n and nonspecific binding was blocked by universal blocking serum ( D A K O Diagnostics, Mississauga, Ontario,  -16-  Canada) for 30 min. The primary polyclonal rabbit anti-ING2 antibody Ping2, a kind gift o f Dr. C C . Harris ( N I H , Bethesda, M D , U S A ) , was diluted 1: 250 and incubated at 4 ° C overnight. After three washes, 2 m i n each with P B S , the sections were incubated with a biotinylated goat anti-rabbit secondary antibody for 30 min (Santa Cruz Biotechnology, Santa Cruz, C A , U S A ) . After three washes with P B S for 2 m i n each, horseradish peroxidase-streptavidin (Santa Cruz Biotechnology) was added to the section for 30 min, followed b y another three washes, 2 min each with P B S . The samples were developed with 3,3'-diaminobenzidine substrate (Vector Laboratories, Burlington, Ontario, Canada) for 7 min and counterstained with haemotoxylin. Then the slides were dehydrated following a standard procedure and sealed with coverslips. Negative controls were performed by omitting I N G 2 antibody during the primary antibody incubation.  2.3  Evaluation of Immunostaining  I N G 2 staining intensity was evaluated blinded by three independent observers (including one dermatopathologist) simultaneously, and a consensus score was reached for each core. In all, 11 normal nevi and 57 dysplastic nevi were evaluated for I N G 2 staining, and informative tumor staining and complete clinicopathological information were obtained in 79 primary melanoma cases and 43 metastatic melanoma cases. The staining intensity was scored as negative (0), weak (1), moderate (2) and strong (3). For I N G 2 staining intensity, 87% o f the biopsies have uniform staining between different cores. In the 13% cases with a discrepancy between duplicated cores, the average score was obtained. In addition, the percentage o f cells showing staining in the nucleus was assessed by counting a minimal 400 cells per tissue core and the average percentage of duplicate biopsy cores was calculated. The multiplication o f the average intensity score (0-3)  - 17-  and the average percentage (0-100%) was used as the final staining score for statistical analysis. The range for the final score was from 0 to 3.  2.4  Statistical Analysis  Nuclear I N G 2 expression among different melanocyte lesions as well as its correlation with clinicopathological parameters o f the melanoma patients, including age, gender, tumor thickness, location, histological subtype and tumor ulceration status was evaluated by Mann-Whitney test or Kruskal-Wallis test. Survival curves were plotted according to Kaplan-Meier method and the comparison o f survival curves was performed with the log-rank test. The Mann-Whitney and Kruskal-Wallis tests were performed by using GraphPad Prism and the survival analysis was performed with SPSS 11.5 (SPSS, Chicago, I L , U S A ) . A P-value <0.05 was considered significant.  2.5  Cell Lines and Cell Culture  Ten melanoma cell lines were used for this study. The M M A N , M M R U and P M W K cell lines were kind gifts from D r H . R . Byers, Boston University, School o f Medicine, Boston, M A ) . The M E W O , Sk-mel-3, Sk-mel-24, Sk-mel-93, Sk-mel-110, K Z - 2 , K Z - 2 8 cell lines were kind gifts from Dr. A . P . A l b i n o (Memorial Sloan Kettering Cancer Center, N e w Y o r k , U . S . A . ) . Normal melanoma cell line was purchased from Clonetics (Walkersville, M D , U . S . A . ) . A l l melanoma cell lines were maintained in Dulbecco's modified Eagle's medium (Invitrogen), supplied with 10% fetal bovine serum, 100 units/ml o f penicillin, 100 ug / m l o f streptomycin in a 5% C O 2 atmosphere at 37°C. N o r m a l melanocytes  were cultured i n melanocyte  (Clonetics) at 37°C i n a 5% C O 2 atmosphere.  - 18-  growth medium  2.6  Normal and Melanoma Tissues  Most normal tissue and tumor tissue (n=15) sections (12-um) were obtained from formalin-fixed, paraffin embedded blocks from the 1990-1998 archives o f the Department o f Pathology, Vancouver General Hospital. The tissues were dissected under a microscope and the paraffin was removed through three washes, 5 m i n each o f xylene followed by 100% ethanol dehydration prior to D N A extraction. In addition, normal tissue samples N M - 3 C , N M N - 2 B and tumor tissue samples M M - 4 , M M - 3 A and M M - 2 B were kind gifts from D r . Y o u w e n Zhou and all these samples were stored in liquid nitrogen before use. Fresh melanoma tissue sample S-9650069 was also obtained from the Department o f Pathology, Vancouver General Hospital.  2.7  U V Irradiation  M e d i u m was removed and the cells (at 80% confluency) were rinsed with P B S and exposed to U V B (280-320nm) using a bank o f four unfiltered FS40 sunlamps (Westinghouse, Bloomfield, NJ). The Petri-dish cover was left on to filter possible U V C emissions from the U V B bulb. Medium was replaced and cells were incubated in a 5% C O 2 incubator at 37°C for desired time periods after U V B irradiation. The intensity o f the U V light was measured by the I L 700 radiometer fitted with a W N 320 filter and an A 1 2 7 quartz diffuser (International Light, Newburyport, M A ) .  2.8  Plasmids, Transfection and P33ING2 Stable Clone Generation  The p c D N A 3 - I N G 2 plasmid (kind gift from D r . C C . Harris, National Cancer Institute, National Institutes o f Health, Bethesda, M D ) was transfected into the M M R U cells using Effectene reagent (Qiagen, Mississauga, O N , Canada). For the I N G 2 stable clone generation, transfected  - 19-  M M R U cells were incubated at 37°C for 48h followed by a 14-day selection in the culture medium supplemented with 1 mg/ml o f G418 (Sigma). Single clones were then picked up and maintained in the culture medium containing 500 ug/ml o f G418.  2.9  Western B l o t A n a l y s i s  C e l l pellets were lysed and extracted in 50 ul o f triple detergent buffer (50 m M T r i s - H C l [pH8.0], 150 m M N a C l , 0.02 % N a N , 0.1% S D S , 1% Nonidet P-40, 0.5% sodium deoxycholate) 3  containing freshly added protease inhibitors (100 ug / m l o f phenylmethylsulfonyl fluoride, 1 ug/ml o f aprotinin, 1 ug/ml o f leupeptin, 1 ug/ml o f pepstatin A ) . The protein concentration was determined by a Bradford assay, and Western blot was performed as previously described (Chin et al, 2005). The primary antisera included anti-p33ING2 rat monoclonal antibody (a kind gift from Dr. O. Gozani and D r . J. Yuan, Harvard Medical School, Boston, M A ) and anti-/?-actin mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, C A ) .  2.10  S R B C e l l S u r v i v a l Assay  M M R U cell line and I N G 2 stable cell line 17 were grown in 24-well plates at 50% confluency. They were irradiated with U V B at 300 J/m and 600 J/m 24h after seeding the cells. Cell 2  2  survival was determined with the sulforhodamine B ( S R B ) (Sigma) assay 24 hours after U V R . S R B is a pink dye with two sulfonic groups that bind to basic amino acid residues in cells fixed with trichloroacetate ( T C A ) . S R B provides a sensitive index o f cellular protein content. Briefly, the medium was removed and the cells were fixed with 500 ul o f 10% T C A for 2 h at 4 ° C after treatment. The cells were then washed five times with tap water and the excess water was removed by flicking. The cells were air-dried and then stained with 500 ul o f 0.4% S R B  -20-  (dissolved in 1% acetic acid) for 30 m i n at room temperature, washed four times with 1% acetic acid, and air-dried. The cells were then incubated with 500 ul o f 10 m M Tris ( P H 10.5) on a shaker for 20 m i n to solubilize the bound dye. Spectrophotometric readings were then taken at 550nm for 100 ul aliquots.  2.11  D N A and R N A Extraction  Total R N A was prepared by T R I z o l extraction (Invitrogen) and reverse transcribed into c D N A with the Superscript First-Strand Synthesis Systerm (Invitrogen) according to the manufacture's protocol. For the genomic D N A extraction, the tissues was incubated with lysis buffer (10 m M Tris.CI, 0.1 M N a C l , 1 m M E D T A ) containing freshly added proteinase K (20mg/ml) (Invitrogen) and followed by phenol chloroform extraction using standard protocols.  2.12  Polymerase Chain Reaction  Polymerase chain reaction ( P C R ) was performed to amplify the whole p33ING2 coding sequence from c D N A o f melanoma cell lines and normal melanocyte using following primers: forward 5'CGCGGATCGGCAGGATGTTA-3'  and reverse 5' - T G G A T G G C C T T T A C T A C C T C - 3 ' ;  primers used to amplify the N L S and P H D domain o f p33ING2  The  from genomic D N A o f the  normal and melanoma tissues are: forward 5 ' - A A A A T C G G G C A A G A C A A A T G - 3 ' and reverse 5'-TGTGGATGGCCTTT ACT ACCTC-3'.  Hotstart  P C R was  performed  with  Taq D N A  polymerase reaction system (Qiagen). Amplification was carried out as follows: (i) initial denaturation at 9 6 ° C for 3 m i n ; (ii) denaturation at 96°C for 1 min; (iii) annealing at 55°C for 1 min; (iv) polymerization at 72°C for 2 min; (v) repeats o f step i i - i v for 35 cycles; (vi)fmal  -21 -  polymerization at 72°C for 10 min. Samples were then electrophoresed on 2% agarose gels containing 0.5 pg / m l o f ethidium bromide and visualized under U V irradiation.  2.13  D N A Sequencing  P C R products were purified using a Qiagen P C R Purification K i t , re-amplified at following conditions using forward and reverse primers respectively: (i) 96°C 1 m i n ; (ii) 96°C 10 sec; (iii) 50°C 5 sec; (iv) 60°C 4 m i n ; (v) repeats o f step ii-iv for 25 cycles and then re-purified using 100% ethanol and 3 M sodium acetate ( P H 5.2) for direct sequencing. Sequencing was performed using a B i g D y e Terminator K i t ( A B I ) and analyzed on an A B I P R I S M 310 Genetic Analyzer. Sequence chromatograms were scrutinized by eye to confirm the results.  2.14  c D N A microarray  The whole c D N A microarray analysis was done by cooperating with the Microarray Facility o f The Prostate Center at Vancouver General Hospital. R N A s were extracted from M M R U and I N G 2 stable cell lines which treated with or without 100 J / m U V R using the standard Trizol 2  method. The quality and quantity o f R N A was measured using the Agilent 2100 bioanalyzer and R N A 6000 N A N O kit (Agilent Technologies, Palo Alto, C A ) . Human Operon v.2.1 ( 2 I K ) glass arrays were produced (based on human 70 mers from Operon, Huntsville, A L ) . Total R N A from test samples and universal human reference  R N A (Stratagene,  Cedar Creek, T X ) were  differentially labeled with Cy5 and Cy3 respectively with the 3 D N A array detection 350 kit (Genisphere, Hatfield, P A ) and cohybridized to c D N A microarrays as previous described method (Kojima et al, 2006). Briefly, reverse transcription incorporates a specific sequence present at the 5' end o f the R T primer. Then the c D N A was hybridized to the array overnight at 42°C. After  -22-  stringent washing, the fluorescent  3DNA  reagent, which includes a "capture  sequence"  complementary to the sequence at the 5' end o f the R T primer, was hybridized to the c D N A (47°C for 2-3 h). Following further washing, the arrays were immediately scanned on a Scan Array Express Scanner (PerkinElmer, Boston, M A ) . Image analysis and quantification were conducted with commercial software (Imagene 6.0 software:Biodiscovery Inc, E l Segundo, C A ) . After grid assignment, the adjusted intensity for each gene was calculated b y subtracting the background median from the signal median. This value was then used as the input for the Genespring 7.2 program (Silicon Genetics, Redwood City, C A , U S A ) , which allows multiple further comparisons using data from different experiments. List o f differentially expressed genes with greater than 2 fold expression were generated and unpaired t-tests were subsequently performed.  2.15  Quantitative Real Time P C R  The same R N A samples that had served for the microarray analysis were used here. The primer sequences  for  ATF3  are  5' - C A G G T C T C T G C C T C G G A A G T - 3 '  CAAAGGGCGTCAGGTTAGCA-3' (forward)  and  (reverse);  TIG1 are  5'-CGTCCCTCACCTTCCTGAAG-3'  GCTCTTTTCC AGCCTTCCTT-3'  (forward)  and  5'  (forward)  and  5'-  5'-GCCGCGCGTCCATTAAT-3' (reverse)  and  P-actin  are  5'-  CGGATGTCAACGTACCACTT-3'  (reverse) . A l l the P C R reactions were performed in duplicated i n a total o f 25 pi reaction mix including Platinum S Y B R Green q P C R S u p e r M i x - U D G with R o x (Invitrogen), primers and a portion o f each c D N A . P C R was carried out at 50°C for 2 min, then 95°C for 10 min, followed by 40 cycles at 95 °C for 15 sec and 60°C for 1 min. The cycle number o f threshold (CT) was recorded for each reaction and the d value o f A T F - 3 was normalized to that o f (3-actin.  -23 -  C H A P T E R 3. N U C L E A R ING2 E X P R E S S I O N IS R E D U C E D IN HUMAN CUTANEOUS MELANOMAS  3.1  Rationale and Hypothesis  Cutaneous malignant melanoma is highly resistant to conventional radio- and chemo-therapy. Dacarbazine (DTIC), the only F D A approved drug for treatment o f melanoma, yields a response rate o f only 16% (Atallah and Flaherty, 2005). Therefore, better understanding o f the molecular mechanism o f melanoma progression and chemoresistance  is needed for designing novel  treatment regimes. P53, a central sensor linking D N A damage to apoptosis, plays an essential role in tumor suppression and chemosensitivity i n many tumor types ( L i et al, 1998; Raffo et al, 2000; Fridman et al, 2003). However, mutational analysis reveals that the p53 gene is altered in only approximately 11% o f melanomas (Hussein, 2004). The low mutation rate oip53 suggests that other tumor suppressor genes may play important roles in pathogenesis o f melanoma. Recent studies suggest that I N G family proteins function as tumor suppressors. Five members o f I N G proteins have so far been identified and they all share a conserved P H D domain in the C-terminus (Campos et al, 2004). P 3 3 I N G l b is the founding member which has been shown to be able to induce cell cycle arrest, enhance D N A repair and promote apoptosis after D N A damage events (Garkavtsev et al, 1998; Cheung et al, 2001; Cheung and L i , 2002). I N G 2 is cloned through a homology search with p 3 3 I N G l b and is found to be located to human chromosome 4 (Shimada et al, 1998). Downregulated expressions o f I N G proteins have been reported in several tumor types including the loss o f nuclear expression o f p 3 3 I N G l b in melanoma. A s I N G 2 exhibits 58.9% homology with p 3 3 I N G l b , we first want to know i f aberrant expression o f I N G 2 is involved in melanomagenesis.  -24-  3.2  Results  3.2.1  Clinicopathological Findings  The clinicopathological features o f the melanomas for this study are summarized in Table 3.1. In primary melanomas, 79 cases (50 male and 29 female) were available for the evaluation o f I N G 2 staining. The median age o f the patients was 58 y ranging from age 25 to 92. There were 12 cases o f R G P and 67 cases o f V G P . For the thickness o f these primary melanomas, 23 were <1.0 mm, 27 were 1.01-2.0 m m , 15 were 2.01-4.0 mm, and 14 were >4 mm. For the tumor subtype, superficial spreading melanoma accounted for 36 cases, lentigo maligna melanoma 13 cases, acrolentigous melanoma 2 cases, nodular melanoma 13 cases, and the remaining 15 cases were unspecified. The majority o f the melanomas located i n sun-protected sites (65 cases, trunk, arm, leg and feet) while 14 located in sun-exposed sites (head and neck). Tumor ulceration was found in 16 patients. Forty-three out o f 50 metastatic melanoma cases were available for I N G 2 staining. There are 29 male and 14 female, with age ranging from 27 to 89 (median 59 y). Clinically, dysplastic nevi can be identified as mild, moderate, or severe. While mildly and moderately dysplastic nevi can be closely observed, severely dysplastic nevi should certainly be surgically removed. In this study, information on 53 o f 57 dysplastic nevi are available for sub-categorization: 22 mild, 20 moderate, and 11 severe.  3.2.2  Reduced ING2 Nuclear Expression in Human Melanomas  W e examined I N G 2 nuclear expression in normal nevi, dysplastic nevi, primary melanomas and metastatic melanomas b y immunohistochemistry (Figure 3.1). There is no significant difference in ING2 nuclear expression between normal nevi and dysplastic nevi (P>0.05, Mann-Whitney test). N o difference in I N G 2 expression was found among mildly, moderately, or severely  -25 -  dysplastic nevi (data not shown). However, reduced I N G 2 expression was observed in R G P and V G P primary melanomas as w e l l as metastatic melanomas compared with dysplastic nevi (P=0.029, P=0.0001 and.P=0.0286 respectively, Mann-Whitney test). There are no significant differences in I N G 2 nuclear expression among R G P , V G P , and metastatic melanomas (P>0.05, Kruskal-Wallis test) (Figure 3.2).  3.2.3  Correlation between ING2 Nuclear Expression and Clinicopathological Parameters  or 5-year Patient Survival Tumor thickness and ulceration are well-known indicators for melanoma prognosis. However, no correlation was found between I N G 2 nuclear staining and these parameters (P>0.05 for both) (Figure 3.3). In addition, no association was found between I N G 2 nuclear expressions and other clinicopathological parameters including age, gender, subtype and location o f tumors (data not shown). To investigate whether I N G 2 expression was correlated with patient survival, KaplanMeier survival curves were plotted to see i f there was a relationship between I N G 2 nuclear expression and five-year patient survival in primary melanomas or metastatic melanomas. W e defined the staining as strong i f the score is between 1.51 and 3.0 or weak i f the score is <1.5. Our results showed that I N G 2 nuclear expression did not significantly correlate with both 5-year overall and disease-specific patient survival in primary and metastatic melanomas (.P>0.05, logrank test) (Figure 3.4).  -26-  Table 3.1 Clinicopathological parameters of 122 cases of melanomas No. of Patient  %  Primary melanomas Age 38  48  41  52  50 29  63 37  <1 1.01-2 2.01-4 >4 Ulceration Absent Present Tumor subtype Superficial spreading melanoma Lentigo maligna melanoma Acrolentigous melanoma Nodular melanoma Unspecified Tumor growth phase Radial growth phase Vertical growth phase Site Sun-protected Sun-exposed Metastatic melanomas Age  23  29  27 15 14  34 20 17  63 16  80 20  36 13 2 13 15  46 16 3 16 19  12 67  15 85  65 14  82 18  <59 >59 Gender Male Female  22  51  21  49  29 14  67 33  <58 >58 Gender Male Female Tumor thickness (mm)  a  Sun-protected sites: trunk, arm, leg and feet. Sun-exposed sites: head and neck.  -27-  F i g u r e 3.1 Representative images o f I N G 2 immunohistochemical staining in human melanocyte lesions. Strong I N G 2 expression in adjacent normal epidermis ( A ) , normal nevi ( B ) , dysplastic nevi ( C ) , and weak I N G 2 staining in primary melanoma ( D ) and metastatic melanoma ( E ) . Arrows indicate strong staining in melanocyte. Magnification, X 4 0 0 .  -28 -  Figure 3.2 I N G 2 nuclear expression at different stages o f melanocyte lesions. There are less I N G 2 nuclear expression i n R G P , V G P and metastatic melanomas compared with dysplastic nevi (P=0.029, 0.0001 and 0.0286, respectively, Mann-Whitney test).  3-1  *****  en  5  2  cs as o =3  C  CM (3  N N  —f— DN  RGP  -29-  VGP  MM  Figure 3.3 N o correlation was found between I N G 2 nuclear expression and tumor thickness (P>0.05, Kruskal-Wallis test) ( A ) or tumor ulceration (P>0.05, Mann-Whitney test) (B) o f primary melanomas.  A  3-  c "c  •5 2 • tn co J£ o  c <M  O  1 •  1-2  ;1  2-4  >4  Tumour thickness (mm) B  3-H  "c  "5  2  « 3  e 1  <3  Absent  Present Ulceration  -30-  Figure 3.4 I N G 2 nuclear expression and 5-year patient survival. I N G 2 nuclear expression is not correlated with 5-year overall ( A , C ) and disease-specific survival ( B , D ) i n primary melanoma patients ( A , B ) or metastatic melanoma patients ( C , D ) .  0-1.5  B 100  -1.51-3 90 CO  >  w E o  80  70 P= 0.1556 60  —i  10  (  20  1  30  1  40  1  50  1  60  Time (months)  Time (months)  i  10 Time (months)  -31 -  1  1  r  20 30 40 Time (months)  1  1  50  60  3.3  Discussion  The main purpose o f this study is to investigate i f the novel tumor suppressor I N G 2 is aberrantly expressed  in  human  cutaneous  melanomas.  Using  tissue  microarray  technology  and  immunohistochemistry, we for the first time demonstrated that I N G 2 nuclear expression is reduced in human melanomas compared to dysplastic nevi (Fig.3. 2). Although a number o f studies indicated that I N G 2 possesses tumor suppressive functions, such as inducing growth arrest, senescence, apoptosis, and enhancing D N A repair (Nagashima et al, 2001; Gozani et al, 2003; Pedeux et al, 2005; C h i n et al, 2005; Wang J et al, 2006), there is limited information on I N G 2 expression level i n human cancers. There is only one recent report by Okano et al (2006) showing that I N G 2 expression is reduced in 6 o f 7 lung cancer cell lines. However, these authors did not detect any ING2 mutation i n 31 human lung cancer cell lines and 30 lung cancer biopsies. Although the reason for reduced I N G 2 expression in human melanomas is unclear, we cannot rule out mutation o f the ING2 gene i n melanoma because different tumor types may have different mechanisms for gene inactivation. For example, reduced I N G 1 expression was found in 73% o f non-small cell lung cancer biopsies (Kameyama et al, 2003) and 44% o f breast cancer primaries (Toyama et al, 1999), while no missense mutation was found in these lung cancer biopsies and only 1 missense mutation was found in 377 breast cancer carcinomas (0.27%). O n the other hand, aberrant I N G l expression was associated with a much higher mutation rate o f the ING1  gene in human primary melanomas  (Campos et al, 2004). In addition, loss o f  heterozygosity o f the region 4q32 in the long arm o f chromosome 4, which includes I N G 2 and S A P 3 0 , was found in 20% o f basal cell carcinomas (Sironi et al, 2004), suggesting that genetic alteration o f the I N G 2 gene does occur i n human tumors. Future studies on I N G 2 expression and  -32-  gene mutation in different cancer types w i l l provide further evidence on the important role o f this tumor suppressor in the pathogenesis o f human cancers. Similar levels o f I N G 2 nuclear expression in melanomas regardless o f the growth phases ( R G P vs V G P ) (Fig. 3.2), tumor thickness or ulceration (Fig. 3.3) suggest that reduced I N G 2 expression may be involved i n the initiation, rather than progression o f melanoma. W e have recently shown that I N G 2 plays an essential role for maintaining genomic stability upon U V irradiation. I N G 2 acts as a D N A damage sensor for nucleotide excision repair, as physiological level o f I N G 2 is required for rapid induction o f histone H 4 acetylation, chromatin relaxation, and the recruitment o f repair recognition factor X P A to the photolesion sites (Wang J et al, 2006). ING2 can also enhance the removal o f D N A damage by triggering the apoptosis process when the D N A damage is too severe. W e have found that overexpression o f I N G 2 significantly enhances UV-induced apoptosis by downregulating the expression o f B c l - 2 , promoting the translocation o f the B a x protein to the mitochondria, resulting i n the mitochondrial membrane potential change, release o f cytochrome c and activation o f caspases-9 and -3 (Chin et al, 2005). Since U V radiation is the main environment factor for melanoma formation, reduced I N G 2 expression would impair the removal o f UV-damaged D N A , leading to gene mutation and malignant cell transformation. Our data that no significant correlation between I N G 2 expression and melanoma thickness supports a multiple genetic change model during melanoma pathogenesis. M a n y different molecules in the apoptosis and survival pathways have been shown to be involved in melanoma initiation and progression. It appears that defects in the apoptosis pathway mostly contribute to the initiation step i n melanoma pathogenesis, while activated survival pathway often correlates with tumor progression. For example, the expression o f pro-apoptotic factor Apaf-1, a p53  -33 -  downstream effector, which links the release o f cytochrome c to the activation o f caspase-9, is reduced in human cutaneous melanomas compared with normal nevi (Dai et al, 2004). The expression o f another p53 downstream effector P U M A is significantly reduced i n melanoma compared to dysplastic nevi (Karst et al, 2005). Furthermore, the expression o f proapototic protein X A F 1 which blocks the XIAP-mediated inhibition o f caspase-3 (Liston et al, 2001) is also reduced in human melanomas (Ng et al, 2004). However, the expression o f Apaf-1, P U M A and X A F 1 expression did not correlate with tumor thickness. O n the other hand, increased expression o f integrin-linked kinase and phospho-Akt in the PI3 kinase survival pathway has been observed i n melanoma and correlated with tumor thickness and 5-year patient survival (Dai et al, 2003; 2005). Furthermore, reduced expression o f P T E N , the negative regulator o f the PI3 kinase, was also found to be reduced in melanoma and significantly associated with tumor thickness (Goel et al, 2006). However, some factors are involved in more than one step in melanoma pathogenesis. For instance, although P U M A expression does not correlate with tumor thickness i n primary melanoma, it is further reduced i n metastatic melanoma and weaker P U M A expression is correlated with a poorer 5-year patient survival (Karst et al, 2005). Based on the complexity o f the apoptotic and survival pathways which govern the fate o f a cell, additional studies on the timing o f the gene inactivation/overexpression i n these pathways from the same set o f tumor biopsies and the interdependence among these events w i l l provide a more complete picture o f the molecular changes during melanoma pathogenesis. Our data that reduced I N G 2 expression does not significantly correlate with 5-year patient survival (Fig. 3.4) is consistent with the findings that I N G 2 reduction is an early event in the R G P and does not correlate with tumor progression (Fig. 3.2, 3.3). O n the other hand, although it is not statistically significant, we observed that patients with weaker I N G 2 expression have a  -34-  trend towards better prognosis for both overall and disease-specific 5-year patient survival in primary melanomas (Figure 3.4). Interestingly, decreased expression o f p 3 3 I N G l b was also associated with better prognosis in childhood acute lymphoblastic leukemia patients as well as in invasive bladder cancer (Nouman et al, 2002; Sanchez et al, 2003). Although the mechanism underlying these phenomena is unclear, we propose that the enhancement o f D N A repair by I N G 2 (Wang J et al, 2006) may account for a better trend o f 5-year patient survival in weaker I N G 2 expression group because enhanced D N A repair have been shown to reduce the cytotoxicity o f anticancer drugs (Siddik et al, 2003). Future studies on the correlation between I N G 2 expression and patient survival in a large set o f melanoma biopsies from patients with and without chemotherapy w i l l provide a clearer answer. In summary, we found that I N G 2 nuclear expression is reduced in human cutaneous melanomas compared to normal or dysplastic nevi. These data, together with our previous findings that I N G 2 plays essential roles in cellular stress responses (Chin et al, 2005; Wang J et al, 2006), suggest the importance o f this tumor suppressor in the pathogenesis o f melanoma.  -35 -  C H A P T E R 4. M U T A T I O N A L S T A T U S O F P33ING2 I N M E L A N O M A S  4.1  Rationale a n d Hypothesis  Melanomagenesis is a multistep progress which requires the activation o f oncogenes and the inactivation o f the tumor suppressor genes. The molecular abnormalities attributable to the progression from dysplastic nevi to malignant melanoma affect cell growth, D N A repair, and susceptibility to cell death. For instance, in 25 to 50 percent o f cases o f non-familial melanoma, the tumor suppressor gene, phosphatase and tensin homologue (PTEN),  is inactivated by  mutation ( L i et al, 1997; Steck et al, 1997). In murine models o f melanoma, mutation o f either CDKN2A  or PTEN alone fails to cause melanoma, but when combined with each other or with  mutations i n other genes, melanomas arise ( Y o u et al, 2002). U V R is the primary environmental cause for melanoma formation. Our group has shown that p33ING2 is capable o f regulating melanoma cellular stress response to U V R either by enhancing nucleotide excision repair or promoting apoptosis (Chin et al, 2005; Wang J et al, 2006). We also found that reduced p33ING2 is important for the progression from the dysplastic nevi to malignant melanoma ( L u et al, 2006). Considering the importance o f p33ING2 in melanoma, we therefore, investigated the mutational status o f the p33ING2 cutaneous melanomas.  -36-  gene in human  4.2  Results  4.2.1  Alterations of P33ING2 Gene in Melanoma Cell Line  W e searched for mutations i n the whole coding regions o f the p33ING2  i n ten melanoma cell  lines by direct sequencing o f R T - P C R products. W e also sequenced a normal melanocyte cell line to determine whether the variant was tumor specific. W e found except for normal melanocytes, K Z - 2 and Sk-mel-110 melanoma cell lines, all other eight melanoma cell lines contained the same C - » T mutation which located i n the codon 13 i n the leucine zipper domain. However, the mutation did not change the amino acid (Ala—>Ala). The c D N A from normal melanocytes did not have this mutation indicating it is tumor specific. Surprisingly, one melanoma cell line Sk-mel-110 contains another twelve mutations including three mutations residing in the P H D domain o f p33ING2. A l l the mutated nucleotides located at the third nucleotide o f the codon and thus did not change the coded amino acid (Table 4.1; Figure 4.1). U V signature C - » T transitions were found at codon 121, 142, 165. The other changes are transitions A—»G; G—>A; T—»C and transversions C—>A; A—>C. A l l o f the point mutations were confirmed by sequencing from both ends.  4.2.2  Alterations of the N L S and P H D Domain of P33ING2 Gene in Melanoma  Taking into account o f the limited numbers o f the melanoma cell lines, we further examined the mutational status o f p33ING2 gene in twelve melanoma biopsies and eight normal tissue biopsies. The primers we used amplified around 400 base pairs including both the N L S and P H D domain of p33ING2. W e found that genomic D N A from both the normal tissues and melanoma tissues did not show any mutations.  -37-  Table 4.1 ING2 alterations in normal melanocyte and melanoma cell lines Cell Line Normal Melanocyte  No  KZ-2  No  KZ-28 MEWO MMAN MMRU PMWK Sk-mel-3 Sk-mel-28 Sk-mel-93 Sk-mel-110 Sk-mel-110 Sk-mel-110 Sk-mel-110 Sk-mel-110 Sk-mel-110 Sk-mel-110 Sk-mel-110 Sk-mel-110 Sk-mel-110 Sk-mel-110 Sk-mel-110  Silent Silent Silent Silent Silent Silent Silent Silent Silent Silent Silent Silent Silent Silent Silent Silent Silent Silent Silent Silent  Mutation Type  mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation mutation  GCC-GCT GCC-GCT GCC-GCT GCC-GCT GCC-GCT GCC-GCT GCC-GCT GCC-GCT CAC-CAT CGA-CGG TCC-TCT AGG-AGA CAC-CAT ATT-ATC AAA-AAG AAT-AAC TGT-TGC CTT-CTC GAT-GAC ACA-ACC  -38-  Location  Codon Codon Codon Codon Codon Codon Codon Codon Codon Codon Codon Codon Codon Codon Codon Codon Codon Codon Codon Codon  13, L E L domain 13, L E L domain 13, L E L domain 13, L E L domain 13, L E L domain 13, L E L domain 13, L E L domain 13, L E L domain 121 133 142 154 N L S domain 165 N L S domain 170 N L S domain 188 N L S domain 210 242 P H D domain 245 P H D domain 261 P H D domain 265  F i g 4.1 Representative sequence chromatograms o f ING2 mutations in Sk-mel-110 melanoma cell line. Arrows indicate the locations o f the mutation.  10 8330 8340 8350 83G0 'T G T C C A A T T f T A A T G G T T T C f f C T T T T C A T G C G T T T C A C T C f r C C T A T  8370 838C A A A C T A A A G G G G A A A"  TGTCCRRTTG RRTGGTTTCRC TTTTCRTGCGTTTCRCTCRCCTRT RRRC G RRRGGGC R R R 1  A —pro  m  GCAJTGG G A G A C A A T G A G  m  TED AAAACCffTGG  ACAAAAiTTAC  TGAAA  G C RG GG G RG RC RR T G RGRRRRC CRT GGRC Rflfl AG T RC T G flflfl  -39-  4.3  Discussion  P33ING2 is a tumor suppressor candidate. It can negatively regulate cell growth in a p53dependent manner through induction o f G l cell cycle arrest and apoptosis (Nagashima et al, 2001). It also interacts with phosphoinositides, Ptdlns(3)P and Ptdlns(5)P through its P H D zinc finger, which plays a crucial role in D N A damage-initiated stress signaling (Gozani et al, 2003). Moreover, significantly reduced p33ING2 expression has been reported in melanomas compared to dysplastic nevi ( L u et al, 2006). To further reveal the role o f p33ING2 i n melanoma formation, in this study we screened for p33ING2  mutations i n 10 melanoma cell lines and 12 melanoma  biopsies. We did not find any missense mutations in this gene which is consistent with a previous finding that no mutation o f ING2 was detected i n 30 human lung cancer cell lines and 31 primary lung cancer tumors (Okano et al, 2006). W e observed same C —»T synonymous variant which is located i n codon 13 o f the L E L domain in eight o f t e n melanoma cell lines (Table 4.1). Okano et al also showed same locus variant existing in six o f thirty-one lung cancer tissues (Okano et al, 2006). However, these authors reported a T to C change which is incorrect i n our opinion. The sequence from PubMed (NM_001564) shows cytosine instead o f thymidine in this location suggesting twenty-five instead o f six lung cancer tissues contain this variant in the study by Okano et al. The L E L domain o f p33ING2 is important for interacting with other L E L domain containing proteins and hydrophobic interactions and a previous study from our group also indicates that the L E L domain is important for the proper functions o f p33ING2 i n D N A repair, apoptosis and chromatin remodeling after U V irradiation (Wang Y et al, 2006). However, we believe this variant is most probably a common polymorphism in melanoma as it is a synonymous variant and C - » T mutation is a U V signature mutation. Especially, the mutation rate at this location is  -40-  quite high (80%). Previous studies indicated that within coding sequences the silent or synonymous polymorphisms are much more common than are changes that result in amino acid substitution, presumably because many amino acid changes interfere with normal function o f the protein and are eliminated by natural selection. However, different alternative codons for the same amino acid may differ i n speed corresponding to different  and accuracy o f transcription, and the  alternative codons may have different  mRNA  accuracy and speed o f  translation. Although Sk-mel-110 melanoma cell line does not contain the silent mutation in codon 13, it contains another twelve synonymous variants i n the whole coding sequence o f p33JNG2. The specialty o f Sk-mel-110 melanoma cell line has been reported before since it has five missense mutations o f p53 although the mutation of p53 is rare i n melanoma (Albino et al, 1994; Hussein, 2004). Besides the melanoma cell lines, we also analyzed the mutational status o f p33ING2 using melanoma biopsies. W e focused our studies on the P H D domain o f p33ING2 since it is the most widely studied domain and it is pivotal for the normal function o f p33ING2 in different experimental models (Gozani et al, 2003; Shi et al, 2006). However, we failed to detect any mutations o f p33ING2 P H D domain in the tissue samples. The limited tissues presented here are due to the difficulty o f obtaining high quality D N A from the formalin-fixed, paraffin-embedded tissues. Although some literatures suggest that high-temperature heating may improve D N A extraction from archival formalin-fixed, paraffin-embedded tissues (Coombs et al, 1999; Shi et al, 2002), we found the best method is still the traditional phenol-chloroform extraction followed by ethanol precipitation method. W e also found that the genomic D N A quality to a larger degree depends on the size o f the tissues and the presence o f melanin or not. Normally, it is easier to  -41 -  obtain good quantity and quality o f D N A from larger tissue biopsies while the presence o f melanin w i l l affect the P C R reaction and lead to failure o f the experiment. W e also applied Sephadex 25 i n our experiments, but it did not help get rid o f the melanin from the D N A samples. In summary, our results suggest that p33ING2, like other I N G family members, is rarely mutated in human cancers. Other mechanisms may be responsible for the reduced p33ING2 expression in melanoma. First, since p33INGlb  gene and flanking regions are highly G C rich  and p33ING2 shows 58.9% identity with p 3 3 I N G l b (Shimada et al, 1998; Gunduz et al, 2000), we postulate that epigenetic modification o f p33ING2 promoter which can cause reduced expression o f corresponding protein may be the reason for the loss o f p33ING2 expression in melanoma. Second, abnormal subcellular translocation o f p 3 3 I N G 2 may happen  during  melanomagenesis. Aberrant cytoplasmic expression o f p 3 3 I N G l b has been reported to be associated with malignancy i n melanocyte lesions and the failure o f induction o f cyclin dependent kinase inhibitor p 2 1  W A F 1  upon D N A damage stress (Nouman et al, 2002; Gong et al,  2006). In addition, we recently found that the nuclear p29ING4 is translocated to cytoplasm in melanoma (unpublished data). These results indicate that translocation o f I N G family proteins is likely a common phenomenon in melanoma and more studies are required to clarify this possibility for p33ING2 i n the future.  -42-  C H A P T E R 5. I D E N T I F I C A T I O N O F G E N E S T R A N S C R I P T I O N A L L Y R E G U L A T E D B Y U V I R R A D I A T I O N O R P33ING2  5.1  Rational and Hypothesis  U V R is the main environmental factor causing melanoma. Studies o f U V R responses at the molecular level have advanced most quickly using primary melanocyte cultures and more readily melanoma cells. For instance, using normal melanocytes, it was found that U V R exposure induces a G l arrest in melanocytes that is at least partially attributable to both p53 (Barker et al, 1995) and p l 6 I N K 4 a (Pavey et al, 1999). Although much o f the understanding o f U V R responses in melanocytes are based on single-gene/pathway approaches, Valery et al undertook a 9,000-transcript human c D N A microarray approach to study U V R i n melanocytes at the transcriptional level. They successfully identified 198 out o f 9,000 genes which were shown to be altered (>1.9-fold). Altogether, 117 clones and 81 clones were suppressed and induced by U V R , respectively (Valery et al, 2001). On the other hand, deregulated apoptosis is believed to be another key factor contributing to melanomagenesis. I N G family proteins, as candidate tumor suppressors, are aberrantly expressed in different tumor types including melanoma (Nouman et al, 2002; Kameyama et al, 2003; Garkavtsev et al, 2004; L u et al, 2006). B y using c D N A microarray analysis in mouse mammary epithelial cells ( N M u M G ) after antisense ING1 -induced transformation, a Japanese group analyzed expression profiles o f 2304 genes and their studies provided evidence that overexpression o f antisense ING1 stimulated expression o f 14 genes including cyclin B l and proto-oncogene DEK (Takahashi et al, 2002).  -43 -  W e already found that I N G 2 nuclear expression is significantly reduced in melanomas compared with dysplastic nevi ( L u et al, 2006). In this study, we restored I N G 2 expression in melanomas by establishing I N G 2 stable clones and further identified the genes transcriptionally regulated by overexpessed I N G 2 or U V R using the M M R U experimental model.  -44-  melanoma cell line as an  5.2  Results  5.2.1  Growth Inhibition of P33ING2  We first generated stable clones overexpressing p33ING2 in M M R U melanoma cells. Figure 5.1A showed that compared with the parental M M R U cells, I N G 2 stable clones (SC) have much higher I N G 2 expression and S C I 6 and S C I 7 showed the highest expressions among all the clones. Consistent with previous finding (Wang Y and L i , 2006), we also noticed that the cells stably expressed p 3 3 I N G 2 are slightly bigger and less dendritic compared with the parental M M R U cells (Fig. 5.1B).We next showed that I N G 2 SC17 had reduced cell survival after various doses o f U V B irradiation (Fig. 5.2). W e thus chose S C I 7 for the following studies.  5.2.2  Gene Expression Analysis by cDNA Microarray  The target samples we used in this study include parental M M R U cells and I N G 2 S C I 7 , both were treated with or without U V B irradiation. The U V dose we used was 100 J / m and the cells 2  were harvested 6 hours after irradiation. The c D N A s derived from the target samples were fluorescently labeled with Cye5 which would emit red fluorescence and the c D N A s from human universal reference R N A  were labeled with Cye3 which would emit green color. Then, the Cyt5  labeled target c D N A and Cyt3 labeled reference c D N A were applied simultaneously on the same microarray and the two fluorescent images were scanned with a fluorescence laser-scanning device. Each signal was normalized so that the Cyt5:Cyt3 intensity ratio o f the housekeeping gene signal like ji-actin was 1. The red and green fluorescent signals indicated genes whose expression levels were relatively higher in target samples or in human universal reference R N A while yellow signals indicate genes with equal expression. First, we compared the gene expression differences between M M R U cells and M M R U cells which are treated with U V B  -45 -  irradiation. The final score is acquired by comparing the two Cyt5:Cyt3 ratio deriving from the untreated and treated M M R U cells. W e considered genes that exhibited a final score >2 as significantly different genes. Overall, 361 genes were shown to be altered with 143 genes and 218 genes induced and suppressed by U V R , respectively. The top 10 induced genes are listed in Table 5.1. A m o n g them, genes ATF-3 and CDKN1A  (p2I CIP) are well known to be able to be  induced by U V R . Table 5.2 listed top 10 genes suppressed by U V R . Except Smad 3, a member of Smad family and a key factor in wound healing and fibrogenesis, all the other genes are rarely studied. Similarly, we also evaluated gene expression changes between M M R U parental cells and I N G 2 stable transfected cells. W e found that 17 genes are suppressed by overexpressed p33ING2 while 57 genes are induced. The top 10 induced and suppressed genes are listed in Table 5.3 and Table 5.4, respectively. W e found that stably overexpressed p33ING2 significantly induced the expressions o f genes like MMP-7 and NDRG-1 while reduced expression o f genes including RARRES1  5.2.3  (TIG1).  R e a l T i m e P C R Analysis  To examine the reliability o f the expression changes detected by the profiling analysis using the c D N A microarray, the real time P C R analysis with the same R N A samples that had served for the microarray analysis was performed to detect the most abundantly changes genes ATF-3 and RARRES1.  The real time P C R results are consistent with the c D N A microarray analysis,  although the induction of ATF-3 detected by real P C R is more dramatic (34.58 fold) than by using c D N A microarray analysis (18.03).  -46-  F i g 5.1 I N G 2 was overexpressed in stable clones. Overexpressed p 3 3 I N G 2 was detected in stable clones by western blot ( A ) and microscopic images were taken from M M R U and I N G 2 stable clone (B).  -47-  Fig 5.2 Cell survival rate by S R B assay o f UVB-irradiated M M R U cells and I N G 2 stable cell line. Columns, mean from duplicates; bars, S D .  -48 -  Table 5.1  Gene  Top 10 genes induced by U V irradiation in M M R U  Fold Induction  Description  PubMed Accession Number  ATF-3  18.03  HEXJM1  11.59  N M 001674, N M 004024 NM_006460  GPR3  11.2  NM_005281  IL6  11.15  NM_000600  BTG2  8.63  NM_006763  PLK3  6.65  NM_004073  DLL1  6.39  NM_005618  FOSB CDKN1A (P21CIP)  6.25 5.95  N M 006732 NM_078467  CXCL2  5.48  NM_002089  Cyclic-AMP-dependent transcription factor ATF-3 (Activating transcription factor 3 HMBA-inducible; likely ortholog of mouse cardiac lineage protein 1; menage a quatre 1; hexamethylene bisacetamide-inducible protein Probable G protein-coupled receptor GPR3 ( A C C A orphan receptor). Interleukin-6 precursor (IL-6) (B-cell stimulatory factor 2) (BSF-2) (Interferon beta-2) (Hybridoma growth factor) ( C T L differentiation factor) (CDF) BTG2 protein (NGF-inducible anti-proliferative protein PC3) Serine/threonine-protein kinase PLK3 (EC 2.7.1.37) (Polo-like kinase 3) (PLK-3) (Cytokine-inducible serine/threonine-protein kinase) (FGF- inducible kinase) (Proliferation-related kinase) Delta-like protein 1 precursor (Drosophila Delta homolog 1) (Delta 1) (H-Delta-1) Protein fosB (G0/G1 switch regulatory protein 3). Cyclin-dependent kinase inhibitor 1 (p21) (CDKinteracting protein 1) (Melanoma differentiation associated protein 6) (MDA-6) Macrophage inflammatory protein-2-alpha precursor (MIP2-alpha) (CXCL2) (Growth regulated protein beta)  -49-  Table 5.2 Top 10 genes suppressed by U V irradiation in M M R U  Gene LHFPL2 PUM2 THRAP2  Fold Suppression 7.14 6.76 6.26  PubMed Accession Number N M 005779 N M 015317 NM_015335  PUM1 NAV3  5.31 5.17  N M 014676 NM_014903  BTEB1  4.95  NM_001206  ASXL2  4.90  NM_018263  SASH1 AMOTL1  4.59 4.58  N M 015278 NM_130847  SMAD3  4.31  NM_005902  -50-  Description Lipoma H M G I C fusion partner-like 2 Pumilio homolog 2 (Pumilio2) Thyroid hormone receptor associated protein 2; protein similar to TRAP240 Pumilio homolog 1 (Pumilio 1) (HsPUM). Neuron navigator 3; pore membrane and/or filament interacting like protein 1; steerin 3 Transcription factor BTEB1 (Basic transcription element binding protein 1) (BTE-binding protein 1) (GC box binding protein 1) (Krueppel-like factor 9) Additional sex combs like 2; polycomb group protein ASXH2 S A M and SH3 domains containing protein 1 Angiomotin like 1; junction-enriched and associated protein M A D , mothers against decapentaplegic homolog 3; mad protein homolog; mad homolog JV15-2; S M A and MAD-related protein  Table 5.3 T o p 10 genes induced by p 3 3 I N G 2  Gene ILIA MMP7 GPR51  Fold Induction 6.94 6.13 5.46  PubMed Accession Number N M 000575 N M 002423 NM_005458  PAPPA DLL1 PLAC8 IGFBP3  4.74 3.97 3.83 3.61  N M 002581 N M 005618 N M 016619 NM_000598  NDRG1  3.52  NM_006096  RAMP1 EFNB2  3.41 2.98  N M 005855 N M 004093  -51 -  Description Interleukin-1 alpha precursor (IL-1 alpha) Matrilysin precursor Gamma-am inobutyric acid type B receptor, subunit 2 precursor Pappalysin-1 precursor Delta-like protein 1 precursor Placenta-specific gene 8 protein (CI5 protein) Insulin-like growth factor binding protein 3 precursor (IGFBP-3) (IBP- 3) (IGF-binding protein 3). NDRG1 protein (N-myc downstream regulated gene 1 protein) (Differentiation-related gene 1 protein) (DRG1) (Reducing agents and tunicamycin-responsive protein) (RTP) (Nickelspecific induction protein Cap43) Receptor activity-modifying protein 1 precursor Ephrin-B2 precursor  Table 5.4 T o p 10 genes suppressed by I N G 2  Genes RARRES 1 RPS4Y1 LASS4 IGFBP5  Fold Suppresstion 15.54  PubMed Accession Number NM_002888  6.46 2.91 2.64  N M 001008 N M 024552 NM_000599  RPL39L HAMP  2.60 2.36  N M 052969 NM_021175  TBX2  2.27  NM_005994  CTGF  2.14  NM_001901  MICA APOB  2.13 2.03  N M 000247 N M 000384  -52-  Description Retinoic acid receptor responder protein 1 40S ribosomal protein S4, Y isoform (PR02646) LAG1 longevity assurance homolog 4 Insulin-like growth factor binding protein 5 precursor (IGFBP-5) (IBP- 5) (IGF-binding protein 5) 60S ribosomal protein L39-like (L39-2). Hepcidin precursor (Liver-expressed antimicrobial peptide) (LEAP-1) T-box transcription factor T B X 2 (T-box protein 2) Connective tissue growth factor precursor (Hypertrophic chondrocyte- specific protein 24). M H C class I chain-related gene A protein Apolipoprotein B-100 precursor (Apo B-100)  5.3  Discussion  With the availability o f much higher density microarrays, a more thorough annotation of the human genome and a stronger technical and technological platform to analyze expression data, we undertook a global view o f UV-response or ING2-response gene expressions to generate novel hypothesis regarding the effects o f either U V R or I N G 2 on melanoma cells. In our analysis, we focused on the most significant gene expression changes modulated by U V R or overexpression o f p33ING2. The relatively low dose o f U V B irradiation (100 J/m ) 2  allows us to identify genes more related to D N A repair rather than apoptosis. However, the use of a 6-hour time point o f analysis limited our analysis to enrich for early-gene responses. A T F - 3 and C D K N 1 A (P21CIP1) are well known to be able to work as stress inducible factors (Yang et al, 2006). In addition, A T F - 3 has also been shown to participate i n axonal growth and neural development (Pearson et al, 2003). It has long been observed that melanocytes w i l l form dendrites in the direction o f light when exposed to U V R (Yamashita et al, 2005). Therefore, it is possible that melanocytes coordinate critical development genes from its  neuroectodermal  lineage in order to regulate dendrite outgrowth in a targeted orientation. The lack o f induction o f p53 R N A by U V R is consistent with the known UVR-mediated post-transcriptional stabilization of p53 protein rather than an effect on p53 transcript levels. Moreover, we also found that U V R can enhance the expression o f GADD45A,  another well known U V R induced gene, by 3.82 fold  compared with control. A t the same time, we revealed the induction o f two new genes by U V irradiation including BTG2 and PIK3. PIK3 is reported to prime phosphorylation o f Chk2 and mediate its full activation i n response to D N A damage (Bahassi et al, 2006). The identification of U V R induced B T G 2 expression is worthy o f mention. B T G 2 belongs to a newly identified family o f structurally related genes whose other known human members are B T G 1 , B T G 3 and  -53 -  Tob. However, BTG2 is the only p53 transciptional target gene (Cortes et al, 2000; Boiko et al, 2006). In addition, B T G expression was found to be significantly reduced i n a large proportion of human kidney and breast carcinomas and it works as a tumor suppressor that links both the p53 and Rb pathways i n human tumorigenesis (Boiko et al, 2006). The study on the role of B T G 2 in melanoma is still lacking and we anticipate that B T G 2 may have similar functions as I N G family proteins i n cancer. Although it is not the most abundantly induced gene, we did find increased p33ING2 c D N A expression (2.49 fold) in I N G 2 stable cell line when comparing with the parental M M R U cell. It further supports the successful establishment o f our stable clone. A m o n g the top 10 genes induced by p33ING2, one gene is MMP-7. This finding is surprising since p33ING2 is believed to work as a tumor suppressor while overexpressed M M P - 7 has been found in different types o f tumors (Shiomi et al, 2003; Lee et al, 2006). There is substantial evidence that overexpression of M M P - 7 correlates with a more aggressive phenotype o f tumor cells and poorer prognosis (Shiomi et al, 2003). Moreover, M M P s not only degrade extracellular matrix ( E C M ) , but degrade n o n - E C M proteins including insulin-like growth factor binding protein (IGFBP) resulting in increased bioavailability o f insulin-like growth factors and enhancing cellular proliferation (Ii et al, 2006). However, we found the induction instead o f suppression o f I G F B P 3 in I N G 2 stable cell line (Table 5.3) suggesting the existence o f other genes which can compete with M M P - 7 on the regulation o f I G F B P 3 . O n the other hand, we also observed the upregulated NDRG1  gene which is necessary for p53 mediated apoptosis and overexpression o f this gene,  leads to the inhibition o f growth in colon cancer cells as well as suppression o f metastasis in prostate, colon and breast cancer cell lines (Stein et al, 2004; Maruyama et al, 2006). The contrary functions o f M M P - 7 and N D R G 1 indicate the complexity o f transcriptional changes  -54-  resulting from overexpressed I N G 2 in melanoma and we thus hypothesize the function o f p33ING2 in melanoma invasion and metastasis depends on the balance o f these molecules. Overexpression o f p33ING2  most significantly suppressed the expression o f RARRES1  gene. R A R R E S 1 also called tazarotene induced gene 1 (TIG1) and it is a retinoid acid response gene. So far all the published literature focused on clarifying the mechanism o f TIG1 inactivation in different tumor types and their results support that hypermethylation o f TIG1 gene promoter contributing to the inactivation o f it (Zhang et al, 2004; Shutoh et al, 2005). Although I N G 2 can recognize H3 lysine methylation and activate gene suppression (Shi et al, 2006), no correlation between I N G 2 and gene methylation has been reported. Therefore, it would be interesting to examine the expression levels o f TIG1 gene i n melanoma i n future studies and investigate i f epigenetic modification w i l l lead to gene inactivation and whether p33ING2 plays an important role in this process. P33ING2 plays a crucial role in D N A repair (Wang J et al, 2006), we therefore further compared the gene expression patterns between U V treated M M R U cell line and U V treated LNG2 stable clone. However, the difference between the two cell lines doesn't seem to be affected  by U V R . The most abundantly modified genes are still MMP-7,  RARRESl(TIGl)(data  NDRG1  and  not shown). A s reported previously, the importance o f p331NG2 on D N A  repair depends on its involvement in U V induced rapid H 4 acetylation and the recruitment of damage recognition protein xeroderma pigmentosum group A protein (Wang J et al, 2006) which could not be reflected by our c D N A microarray analysis. We propose other studies like C H I P maybe more powerful to identify the molecular changes contributing to p33ING2 mediated D N A repair.  -55 -  In summary, the application o f c D N A microarray in this study is a effective approach to identify novel genes which are involved in the same signaling pathway as ING2 or U V R in melanoma cells.  -56-  C H A P T E R 6. G E N E R A L C O N C L U S I O N S  6.1  Summary  P33ING2, as a tumor suppressor candidate, has been shown to share similar biological functions with its homologue p 3 3 I N G l b i n cell cycle arrest, apoptosis, D N A repair and senescence. A l l the functions require the presence o f functional p53. In addition, both p 3 3 I N G l b and p33ING2 are stable components  o f S i n 3 - H D A C indicating the involvement o f them in transcriptional  regulation o f genes. Several studies have provided evidence that aberrant expressions o f p3 3 I N G l b were found i n different tumor types including the loss o f p3 3 I N G l b in melanoma. Considering the highly homology between p 3 3 I N G l b and p33ING2, i n this thesis, we started with investigating the expression patterns o f p33ING2 in different melanocytic lesions using tissue microarray and immunohistochemistry. We found significantly reduced I N G 2 nuclear expressions in primary melanomas and metastatic melanomas compared with dysplastic nevi. However, there was no difference o f I N G 2 nuclear expression among R G P melanomas, V G P melanomas and metastatic Furthermore,  there was  no  correlation between  ING2  nuclear  expression  melanomas. and  patient  clinicopathological parameters or between I N G 2 nuclear expression and 5-year patient survival. The above results indicate that the loss o f nuclear expression o f p33ING2 may only be involved in the initiation o f melanoma rather than the progression o f melanoma. Loss o f heterozygosity ( L O H ) , mutation and epigenetic modification are three main mechanisms causing decreased tumor suppressor gene expressions i n tumor samples. W e therefore investigated the mutational status o f p33ING2 tissue samples. However, no mutation o f p33ING2  -57-  in melanoma cell lines and melanoma  was found in both the melanoma cell lines  and tissue samples suggesting that mutation is not the reason causing reduced I N G 2 expression in melanomas. Inactivation o f p33ING2 may be due to transcriptional or post-transcriptional modification. We also investigated the transcriptional changes caused by U V R or stably transfected p33ING2 by performing c D N A microarray analysis. We not only confirmed a subset of U V induced genes like ATF-3 and P21, but also unveiled some new genes including BTG2 and PLK3. The effect o f overexpressed p33ING2 on M M R U is quite complicated because it activates both the tumor suppressor genes and oncogenes. The simultaneous expressions o f these ostensibly antagonistic processes may be due to the unknown adjunctive functions o f these genes and more studies are required to find the real functions o f p33ING2 in melanoma. Taken together, we have elucidated in this thesis the expression and mutational status o f p33ING2 in melanoma. W e also described the transcriptional changes caused by overexpressed p33ING2. Together with further exploration, our knowledge o f this gene may lead to more effective prevention and treatment for human melanoma.  6.2  F u t u r e Directions  In this thesis, our studies showed that mutation o f p33ING2  gene is not the mechanism  contributing to the significantly reduced nuclear expression o f p33ING2 in melanomas. Since the p331NGlb  gene and flanking regions are highly G C rich and p33ING2 shows 58.9% identity  with p 3 3 I N G l b , we anticipate that epigenetic modification o f p33ING2 promoter which can cause reduced expression o f corresponding protein may be the reason for the loss o f p33ING2 expression in melanoma.  -58-  Using I N G 2 stable clones and c D N A microarrays, we identified that the expression o f R A R R E S 1 (TIG1) is negatively regulated by overexpressed p33ING2. This finding is interesting since the inactivation o f T I G 1 has been reported i n different types o f tumors and it is believed to act as a tumor suppressor gene. To confirm this result, s i R N A can be used to knock down the expression of p33ING2 in M M R U cell line and see i f we can restore the expression o f T I G 1. In addition, similar studies are needed to be done using normal melanocyte and different origins o f melanoma cell lines to exclude the possibility o f cell type specific regulation o f p33ING2 on TIG1. After all these studies, we believe the confirmation o f the relationship between p33ING2 and TIG1 w i l l generate new hypothesis about the role o f p33ING2 in melanomas.  -59-  REFERENCES  Albino A P , V i d a l M J , M c N u t t N S , Shea C R , Prieto V G , Nanus D M , Palmer J M , Hayward N K . Mutation and expression o f the p53 gene i n human malignant melanoma. Melanoma Res 1994, 4:35-45.  Atallah E , Flaherty L . Treatment o f metastatic malignant melanoma. Curr Treat Options Oncol 2005,6:185-193.  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