Characterization of N-terminal Myc ubiquitylation and the novel oncogene CUL7

Transcription

1 Characterization of N-terminal Myc ubiquitylation and the novel oncogene CUL7 by Sam Sulgi Kim A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Medical Biophysics University of Toronto Copyright by Sam Sulgi Kim 2012

2 Characterization of N-terminal Myc ubiquitylation and the novel oncogene CUL7 Abstract Sam Sulgi Kim Doctor of Philosophy Department of Medical Biophysics University of Toronto 2012 Myc is an oncogene that is commonly deregulated in human cancers. Mechanistic studies reveal that Myc is a transcription factor that interacts with a protein partner called Max. Heterodimerization and the formation of the Myc:Max complex enables Myc:Max to bind to the E-box and subsequently regulate the activation and repression of Myc target genes. Since regulation of its target genes are essential for Myc to drive transformation, the Myc and Max interaction has been targeted in mouse model studies to determine whether the oncogenic activity of Myc can be inhibited. Surprisingly, these studies reveal that targeting Myc is not only possible but a powerful way to suppress tumour growth. Since a better understanding of how Myc carries out its biological functions makes the possibility of targeting Myc a reality, it is essential to investigate and study the mechanisms of how Myc promotes tumourigenesis. In the first part of this thesis, we investigate the idea that the N-terminal end of Myc may be post-translationally modified and this modification may dictate Myc activities. Indeed we report here that the N-terminal end of Myc can be ubiquitylated as well as acetylated, and that the loss of these modifications results in a decrease in Myc activities. Furthermore, we characterize Mdm2 as a potential E3 ubiquitin ligase that may ubiquitylate the N-terminal end of Myc. In the second part of this thesis, we investigate CUL7 as a novel oncogene that may inhibit Myc-potentiated apoptosis and cooperate with Myc in transformation. Indeed, CUL7 is a novel p53 interacting protein that inhibits Myc potentiated apoptosis through the inhibition of ii

3 p53. We have also characterized CUL7 to be overexpressed in primary human lung cancers, and a higher level of CUL7 expression associates with short-term survival of lung cancer patients. Through the better understanding of the enzymes that post-translationally modify the N- terminal end of Myc and proteins, such as CUL7, that can cooperate with Myc to drive tumourigenesis, we may begin to devise ways to target and control deregulated Myc in cancer cells. iii

4 Acknowledgments I thank both the past and present Penn lab members for all their suggestions and help during my study. I would like to especially thank Jamie Clendening, Paul Boutros, Amanda Wasylishen, and Lindsey Lustig for being my best friends and going extra distances to help me. I am also extremely grateful to my mentor Dr. Linda Penn for her amazing support and teaching. She is my role model who inspired me to be creative, persistent, and strong. I will always remember her generosity and kindness in helping everyone. Lastly, I thank my lovely wife Emily for supporting me throughout my study. She was there for me in both good and bad days, and my study would not have been possible without her support and help. iv

5 Table of Contents Contents Acknowledgments... iv Table of Contents... v List of Figures... ix List of Abbreviations... xi Chapter 1 Introduction Life, death, and immortality Overview Life Death Myc Overview Structure of Myc Myc NTD Myc CTD Myc-dependent proliferation Myc-dependent apoptosis P Overview P53-mediated cell cycle arrest P53-mediated apoptosis P53 summary Mdm2 and p53 ubiquitylation... 9 v

6 4.1 Overview of ubiquitylation Mdm Parc CUL ARF ARF-independent, p53-dependent pathway of Myc potentiated apoptosis P53-independent role of Mdm Myc ubiquitylation Targeting Myc for the treatment of cancer Hypotheses Abstract Introduction Materials & methods Cell culture Constructs Retroviral production and infection Immunoblot analysis Cell death assay Colony growth in soft agar Co-immunoprecipitation Intracellular ubiquitylation assay Results and discussion Chapter 3 Characterization of CUL7 as a novel oncogene Abstract Introduction Materials & methods vi

7 17.1 Cell culture Constructs Retroviral production and infection Functional screen Immunoblot analysis Cell death assay Colony growth in soft agar Co-immunoprecipitation CUL7 knockdown with RNAi Purification of GST-p In vitro translation and GST pulldown assay Generation of p53 fusion proteins and use in CUL7 (1-375) pulldown assay Bioinformatics Results and Discussion Identification of an inhibitor of Myc-potentiated apoptosis CUL7 blocks both c-myc and N-Myc-potentiated apoptosis CUL7 interacts with p53 both in vitro and in vivo CUL7 cooperates with Myc in transformation Chapter 4 Discussion and Future Directions Myc and Mdm CUL7 and p Concluding remarks References or Bibliography Copyright Acknowledgements vii

8 List of Tables Table 1: Biological comparison of WT Myc to 6KR-N viii

9 List of Figures Figure 1. 6KR-N mutant is significantly less transforming than wild-type Myc Figure 2. 6KR-N Myc mutant promotes anchorage independent growth similar to WT Myc in Rat1A cells. Figure 3. Full length 6KR-N Myc mutant stability is not significantly different from full length WT Myc in HO15.19 Rat Myc null cells. Figure 4. HO15.19 Rat Myc null cells expressing full length 6KR-N Myc mutant proliferate slightly slower than but not significantly different than cells expressing full length WT Myc. Figure 5. HO15.19 Rat Myc null cells expressing full length 6KR-N Myc mutant is defective in potentiating cell death than cells expressing full length WT Myc. Figure 6. Lysine 52, 148, and 157 of Myc is ubiquitylated in 293T cells. Figure 7. K51 and K52 of Myc are required for Myc to allow SHEP cells to grow in a anchorage independent manner. Figure 8. N-Myc upregulates endogenous nuclear and cytoplasmic Mdm2. Figure 9. Myc interacts with Mdm2. Figure 10. Ectopic expression of Mdm2 promotes endogenous Myc ubiquitylation. Figure 11. K51 and K52 of Myc is required for Mdm2 to ubiquitylates soluble N-terminal Myc peptide (1-167). Figure 12. Working Model of how Mdm2 regulates Myc. Figure 13. A functional screen identifies CUL7 as an inhibitor of Myc-induced apoptosis Figure 14. CUL7 inhibits Myc potentiated apoptosis and cooperates with Myc in transformation. ix

10 Figure 15. CUL7 and p53 interact in vivo and in vitro. Figure 16. CUL7 collaborates with Myc in transformation and is overexpressed and associated with poor prognosis in lung cancer. x

11 List of Abbreviations ARF Alternative reading frame of the INK4a locus Bax Bcl2-associated X protein Bcl2 B-cell lymphoma 2 BET Bromodomain and extra terminal BH3 Bcl2 homology 3 domain bhlh-lz Basic region - helix loop helix- leucine zipper Bid BH3-interacting domain death BR Basic region CDK Cyclin dependent kinase ChIP Chromatin immunoprecipitation CHX Cycloheximide CPH Conserved in Cul7, Parc, and Herc2 domain CTD Carboxy terminal domain CUL7 Cullin 7 DMEM H21 Dulbecco's Modified Eagle's Medium DNA Deoxyribonucleic acid E1 Ubiquitin activating enzyme E2 Ubiquitin conjugating enzyme E3 Ubiquitin ligase FBS Fetal bovine serum GADD45 Growth arrest and DNA damage inducible gene 45 GFP Green fluorescent protein GSK3 Glycogen synthase kinase hr Hours IP Immunoprecipitate K Lysine Max Myc-associated protein X MBI Myc box I MBII Myc box II Mdm2 Mouse double minute 2 MEF Mouse embryo fibroblast Myc Myelocytomastosis protein NTD Nmino terminal domain PBS Phosphate buffered saline PCR Polymerase chain reaction PUMA p53 upregulated modulator of apoptosis R Arginine RPMI 1640 Roswell Park Memorial Institute 1640 medium TRRAP Transformation/transcription domain-associated protein xi

12 Ub Ubiquitin xii

13 1 Chapter 1 Introduction 1 Life, death, and immortality 1.1 Overview The cell is described as the unit of life. Every individual cell is a living entity that displays key characteristic behaviours such as growth, reproduction, and in most cases death. Whether an organism is a single cell such as a bacterium or yeast, or is multi-cellular, such as a worm, fly, or human, it is composed of a living cell or cells which perform the basic but critical functions that define life. Since the cell is a living organism, it must somehow decide when to grow and proliferate, and importantly, when to die. How and what determines whether a cell lives or dies is a fascinating question that many biologists have asked and sought to answer through scientific experimentation. As it turns out there is not one simple answer for this question, but numerous answers that reflect the complexity and diversity of cell biology. It first became apparent that cellular behaviours such as proliferation and death were all highly regulated, as scientists learned that they could manipulate the behaviour of cells by applying or removing mitogens to induce cell proliferation or cell death, respectively. Secondly, scientists discovered that the decisions made by a cell depend on molecular events happening internally, as scientists could alter either the expression or activity of the proteins within, and thereby control or change the way in which the cells behaved. Thirdly, scientists realized that nature also introduces alterations to proteins via mutation, which can result in either healthy or abnormal changes to the way a given cell behaves. A profound example of what can happen when cells behave abnormally is human cancer. In cancer, cells acquire genetic lesions which lead to the alteration of protein expression or activity, permitting the cell to evade death and transform into cells that proliferate in an uncontrolled manner. Since all cancer cells derive from normal cells, it is imperative that scientists understand the molecular mechanisms that regulate life and death, and importantly, how these molecular programs can been altered in cancer cells to allow them to become immortal. One key molecule that profoundly governs the life and death of the cell is c-myc (Myc). The work in this thesis aims to better understand how Myc regulates these two key cellular programs.

14 2 1.2 Life Prior to 1961, it was commonly believed that cells had the ability to proliferate indefinitely. By allowing human cells to grow in flasks in tissue culture conditions, Leonard Hayflick and Paul Moorhead were the first to observe that primary cells are only capable of replicating a finite number of times as the cells ceased to divide after 50 replications (1). Thanks to decades of research, it is now understood that when cells proliferate, they undergo specific phases that make up the cell cycle termed G1, S, G2, and M (2). G1 phase is gap period where cells grow in size and prepare for DNA synthesis. S phase is where the DNA replicates in the nucleus (2). G2 phase describes the gap period from the end of DNA replication to the beginning of M phase where cells divide into two identical daughter cells (2). Mitosis, M phase, is the period where the nuclear envelope temporarily disappears, chromosomes line up in the middle of the cell and microtubule structures attach themselves to the kinetichores in order to separate each chromosome into chromatids that are destined for each daughter cell (2). During the cell cycle, proteins called Cyclins and Cyclin Dependent Kinases (abbreviated as CDKs) are activated in a highly regulated manner to dictate how and when the cell enters each phase. For example, Cdk4 and Cdk6 form a complex with Cyclin D in G1 phase (3). These Cdk4-Cyclin D and Cdk6-Cyclin D complexes then phosphorylate a protein called Retinoblastoma (Rb) (3). Rb is bound to a protein called E2F from late M phase to the middle of G1 phase. Once the Cdk4-Cyclin D and Cdk6-Cyclin D complexes phosphorylate Rb, it is released from E2F permitting E2F to induce target genes required for DNA synthesis and S phase to proceed (3). There are many different types of cyclins and CDKs. For example, cyclins A, B, D, and E as well as Cdk 2, 4, 5, 6 and Cdc2. Cdk2, Cdk4, Cdk5 and Cdk6 form complexes with cyclin D at the beginning of G1 phase to the end of G1 phase. Cdk2 and cyclin E form a complex from mid G1 to the beginning of S phase (2). Lastly, the Cdc2-Cyclin A and Cdc2- Cyclin B complexes are formed from the end of S phase to the middle of mitosis (2). Therefore, the progression of cell cycle and proliferation is determined by sequential activation of different CDK and cyclin complexes and by proteins that can regulate their expression.

15 3 1.3 Death Apoptosis is a programmed cell suicide program that can be triggered when the cell is damaged (eg. DNA damage) or under negative growth conditions (eg. low mitogen levels) (4). The end result of apoptosis is the complete breakdown of the cytoplasmic and nuclear skeletons, fragmentation of the cell, and degradation of the chromosomes (5). In vivo the cellular remnants of apoptosis are cleared by a non-inflammatory process. Apoptosis has two main pathways: death receptor and mitochondrial. The death receptor pathway involves cell surface receptors such as Fas binding to death ligands like FasL to initiate apoptosis (6). Once the Fas ligand has bound to this receptor, the common adaptor protein Fas- Associated death domain (FADD) binds to Fas (6). FADD interaction with Fas then creates the death-inducing-signaling complex (DISC) which attracts and activates caspase-8 (7). Caspase-8 then activates caspase 3 to begin the breakdown of the cell (6). The mitochondrial pathway begins by proapoptotic signals such as DNA damage activating an apoptosis regulator such as p53. p53 can induce apoptosis by increasing the levels of the pro-apoptotic molecules Bax and Puma at the mitochondria (8, 9). Bax and Puma are members of the Bcl-2 family of proteins (10). Members of the Bcl-2 family share regions of homology called Bcl-2 homology (BH) domains and can be divided into three groups. First, proapoptotic members that have multiple BH domains (BH1-3) such as Bax and Bak can form homo-multimers. These multimers then form large permeable pores in the mitochrondrial membrane and allow cytochrome c to be released from the intermembrane space (10). Second, members that have BH4 domains such as Bcl-2, Bcl-XL, and Mcl-1, are anti-apoptotic and are dedicated to protecting cells from apoptosis by binding to Bax or Bak and inhibiting multimer formation. Third, BH3 domain only containing proteins such as Bid and Puma, function as proapoptotic molecules by disrupting the binding of anti-apoptotic molecules to Bax and Bak, thereby allowing Bax and Bak to form multimers. P53, through its transcription-dependent functions increases the level of Bax and Puma thereby favouring pore formation at the mitochondria (8, 9). Cytochrome c release from the mitochondria subsequently activates caspase-9, which in turn activates downstream effector caspases to breakdown the cell (6). It has also been recently reported that p53 can function as a proapoptotic molecule when localized at the mitochondria in a transcription-independent manner (11-14). Apoptosis is an important mechanism in preventing cancer since it eliminates genetically damaged cells from the population before they can undergo transformation.

16 4 Therefore, for cancers to develop, evasion of apoptosis is necessary, and this trait is a hallmark of most cancers (5). 2 Myc 2.1 Overview c-myc is a potent oncogene, that is tightly regulated in normal human cells, but quite often deregulated in human cancer. c-myc is the cellular homologue to v-myc which was initially discovered within acute transforming virus MC29, a virus that was isolated from a chicken myelocytoma (15). c-myc is a member of a large gene family which include N-, L-, S-, and B- Myc (16). Out of all the family members of Myc, N-Myc was the first one discovered to be amplified in neuroblastoma patients (17). L-Myc was then discovered to be overexpressed in small cell lung carcinoma (18). c-myc, hereafter referred to as Myc, was discovered shortly after to be involved in numerous human cancers. For example, in all cases of Burkitt s lymphoma, Myc is deregulated through chromosomal translocation of immunoglobulin gene promoters on chromosome 14, 2, or 22, to the Myc gene locus on chromosome 8 (19, 20). Amplification of Myc is also associated with poor prognosis in breast cancer (21), and many other cancers. Although Myc family members are frequently deregulated in human cancers, their individual functions are not identical. Knock-out of different Myc family members in mouse results in different phenotypes. For instance, c-myc null mice die between E (22), while N-myc null mice die between E (23, 24), and L-Myc null mice remain viable (25). These knock-out mouse phenotype results indicate that there are differences between c-, N-, and L-Myc. However, c- and N-Myc function may be relatively similar as embryonic lethality of the c-myc null mouse can be rescued by an N-Myc knock-in (26). 2.2 Structure of Myc Myc protein is broadly considered to have two major domains: the N-terminal and C-terminal domains. The full-length human c-myc is comprised of 439 amino acids. Residues1-262 comprise the N-terminal domain (NTD), and amino acids are considered the C-terminal domain (CTD).

17 Myc NTD The Myc NTD encodes a tri-partite transactivation domain (TAD), which is composed of glutamine rich (amino acids 1-41), proline rich (amino acids ), and acidic (amino acids ) regions (27). Within the TAD, there are two regions of Myc that are highly conserved between Myc family members, called Myc box I (MBI) and Myc box II (MBII) (28). MBI encompasses amino acids of Myc (28). The residue threonine 58 (T58) is frequently mutated in Burkitt s lymphoma and resides in MBI (29). Using a threonine 58 to alanine Myc mutant (T58A), which has been observed in Burkitt s lymphoma, it was reported that compared to wild type Myc, the T58A mutant demonstrates an increase in stability and transforming potential (29). This increase in stability is attributed to the alanine mutation interfering with a critical phosphorylation event, which would otherwise occur at T58 by glycogen synthase kinase 3 (GSK3) leading to Myc ubiquitylation and degradation (30, 31). The increase in transformation is reported to be due to a defect in the T58A Myc mutant to induce apoptosis through a loss of function in inducing the proapoptotic molecule Bim (32). Therefore, MBI plays a significant role in regulating Myc stability and Myc potentiated apoptosis. MBII is comprised of amino acids MBII is a region of Myc that is required for Myc to interact with many proteins such as Skp2 (33, 34), TIP48 (35), and TRRAP (36). Deletion of MBII ( ΜΒΙΙ) blocks the recruitment of TRRAP to the cyclin D2 promoter and inhibits Myc from inducing its expression (37), suggesting that MBII is a critical region for Mycprotein interactions that are necessary for target gene expression. The ΜΒΙΙ when expressed in immortalized rat fibroblast cells or human breast MCF10A cells, it fails to form colonies in soft agar (29, 38). MBII, when expressed in Rat-derived Myc null cells causes a slower rate of proliferation compared to wild type Myc expressing cells, and fails to potentiate apoptosis upon serum withdrawal (38). Together, these findings suggesting that MBII is required for Myc to fully promote proliferation and potentiate apoptosis Myc CTD The C-terminal domain of Myc contains a basic αhelix-connecting loop- αhelix leucine zipper (bhlh-lz) comprised of amino acids The Myc bhlh-lz allows Myc to interact with Myc-associated protein X (Max) (39). It is this heterodimerization and the formation of the Myc:Max complex that enables site-specific binding to the CA(C/T)GTG DNA E-box and

18 6 subsequent regulation of Myc target genes (39, 40). Myc BHLH-LZ deletion studies indicate that this region is essential for Myc to drive cell proliferation, apoptosis, and transformation (41-43). These important findings have contributed to our understanding of Myc s role as a transcription factor and function in driving different biological programs in the cell. 2.3 Myc-dependent proliferation One of the many biological functions that Myc regulates is cell proliferation (28). The first evidence for this role of Myc emerged in 1987, when expression of Myc in T lymphocytes was down regulated through microinjection of antisense Myc and inhibited cells from entering S- phase even in the presence of mitogen (44). In 1989, the converse experiment was conducted where Myc expression was upregulated through activation of inducible Myc, and this activated Myc was shown to be sufficient to allow quiescent fibroblast cells enter into S-phase even in the absence of mitogen (45). In 1997, the first Myc null cells were generated (46). These cells were defective in proliferation as they were completing the cell cycle at a rate 3 times slower than cells with endogenous wild type Myc (46). It was reported that this slow growth phenotype stems from the lengthening of G1 and G2 phase (46, 47). This result was consistent with two additional studies where Myc was conditionally knocked out using Cre-recombinase in mouse embryonic fibroblasts (MEFs) (48) and B-cells (49). In both cases, cells arrested at G0/G1. Mechanistically, Myc has been shown to up regulate a plethora of target genes, such as cyclin D2, cdc25a, CDK4, and E2F1, which are known to play critical roles in promoting cell cycle progression (50-53). Myc has also been shown to down regulate various cell cycle inhibitors, such as p15, p21, and p27 (54-56). Through both the up- and down- regulation of key cell cycle regulators, Myc plays an important role in the control of cellular proliferation. 2.4 Myc-dependent apoptosis Although Myc promotes proliferation, when deregulated, Myc can also sensitize cells to undergo apoptosis under anti-proliferative conditions such as mitogen withdrawal (57) or DNA damage (4). The mechanism by which Myc potentiates apoptosis is not well characterized. One group has suggested that deregulated Myc can potentiate apoptosis by inhibiting the growth arrest function of p53, while leaving its apoptotic functions intact (55). Myc can also increase the expression of Fas ligand (FasL) which binds to the Fas receptor and initiates death receptor mediated apoptosis (58). Furthermore, Myc has been shown to reduce the expression of the

19 7 anti-apoptotic proteins Bcl2 and Bcl-XL in hematopoietic cells (59). Previously, our lab has reported that Myc is required for full activation of the pro-apoptotic molecule, Bax (4). Under apoptotic conditions, Bax translocates from the cytoplasm to the mitochondria where it oligomerizes to form multimers (10). Bax oligomerization results in the irreversible induction of apoptosis and this critical activation step is dependent upon Myc (4). Although, it is not yet clear as to how Myc promotes Bax oligomerization, the current model is that Myc regulates specific target genes that deregulate Bax. Another way that Myc potentiates apoptosis is through the induction of its target gene Bim (32). The mutated T58A Myc that is often observed in Burkitt s lymphoma, demonstrates a loss of function by decreasing the transactivation of this key apoptotic molecule. The most well characterized mechanism in which Myc to potentiates apoptosis however, is through activation of the ARF-Mdm2-p53 pathway (60). 3 P Overview P53 is a tumour suppressor initially discovered through its interaction with the SV40 Large T- Antigen protein and named for its molecular mass of kilo daltons (61, 62). P53 was initially thought to promote cell proliferation since it could cooperate with Ras to transform rat embryonic fibroblasts (63-65). However, later studies revealed that the previous work had been performed with a mutant version of p53, and that the wild-type protein actually had potent tumour suppressing activities (66, 67). P53 knock-out mice were then produced by disrupting both alleles through insertional mutagenesis and homologous recombination (68). The p53 null mice were viable suggesting that p53 is not required for normal development, however the mice were highly susceptible to spontaneous tumour formation, mostly lymphoma and tissue sarcomas at 3 to 5 months of age (68). Mutant p53 (273H) transgenic mice were also generated, and these mice too were susceptible to tumour formation, further supporting the role of p53 as a tumour suppressor (69, 70). In humans, p53 mutations are found in about 50% of all cancers (71). The majority of these mutations were missense mutations and frequently occurred at amino acids 175, 245, 248, 249, 273, and 282 (72, 73). These residues are within the central region of p53 which is required for p53 to bind DNA, suggesting the importance of p53 to function as a transcription factor in order to suppress tumour formation (74). Interestingly, recent reports

20 8 indicate that the central region of p53 is also necessary for interaction with Bak, and Bcl-XL in the cytoplasm, suggesting that missense mutations in p53 may inhibit both transcriptiondependant and -independent roles for p53 (13, 75). In humans, p53 mutation is also detected in the germ-line. These individuals are born with one mutated copy of the p53 gene and develop Li- Fraumeni syndrome (LFS) (76). LFS is an autosomal dominant syndrome characterized by increased occurrence and earlier onset of various cancers such as brain, osteosarcoma, breast carcinoma, soft tissue sarcoma, and leukemia. Individuals with LFS lose the one remaining wild type p53 alleles during the development of disease (77-79). 3.2 P53-mediated cell cycle arrest There are two main mechanisms of action through which p53 suppresses tumour formation. The first mechanism is by inducing cell cycle arrest. When cells acquire DNA damage, it is imperative that such lesions are repaired before the cell enters S phase so as to avoid replicating the mutated DNA and passing it on to daughter cells in mitosis. Several p53 target genes are responsible for inducing cell cycle arrest. P53 has been described to induce cell cycle arrest in G1 through the induction of p21 (80). p21 acts as a strong inhibitor of several cyclin-cdk complexes, including cyclin A-CDK2, cyclin D-CDK4 and cyclin E-CDK2 (81). By inhibiting the cyclin-cdk complexes that are required for the G1 to S transition, cells are arrested upon p21 induction by p53. p53 can also induce GADD45 and to induce cell cycle arrest at the G2 checkpoint (82, 83). p53 has also been reported to directly repress Myc by binding to the Myc promoter (84). Since Myc promotes cell proliferation through the activation of cyclins and the repression of p21, p53 must down-regulate Myc in order to induce cell cycle arrest. There are also reports that suggest that p53 can induce cell cycle arrest through induction of microrna mir34 (85). Elevated expression of mir34 leads to cell cycle arrest at both G1 and G P53-mediated apoptosis The second mechanism by which p53 exerts its tumour suppressing function is by inducing apoptosis. P53 can induce apoptosis in both a transcription -dependent and -independent manner. With respect to the former, p53 can function as a transcription factor to induce many proapoptotic target genes such as PUMA, Bax and Noxa (9, 86, 87). PUMA, Bax, and Noxa are located at the mitochondria, and regulate permeabilization of the outer membrane and subsequently the release of cytochrome c. P53 can also induce apoptosis by translocating to the

21 9 cytoplasm where it can engage Bax, Bak, or Bcl-XL (11, 13, 75). The current understanding of cytoplasmic p53 is that it can directly activate either Bax or Bak depending on the cell system used. In an in vitro cell-free system where purified mitochondria are present, purified p53 from UV-irradiated MCF7 cells was added together with either purified Bax, Bim, or Bid. In this cell system only Bax was able to function together with p53 to promote cytochrome c release (11). By contrast, it was Bak and not Bax that p53 was able to activate in H1299 cells, a p53 null lung line (13). P53 was also shown to bind Bcl-XL through its DNA binding domain, and this interaction was important for inducing apoptosis (75). 3.4 P53 summary It is evident that when activated, p53 is programmed to induce either cell cycle arrest or cell death. What is not yet clear is what dictates whether p53 will induce cell cycle arrest or apoptosis. One report indicates that Myc may play a role in mediating p53-dependent apoptosis, by repressing the p53 target gene p21, but leaving Puma expression intact (55). This finding is consistent with earlier observations that rat fibroblasts undergo p53-dependent cell cycle arrest upon cytotoxic stress, unless the cells harbour deregulated Myc or E1A, which causes the cells to undergo apoptosis (88, 89). 4 Mdm2 and p53 ubiquitylation 4.1 Overview of ubiquitylation Since active p53 results in either cell cycle arrest or apoptosis, it is essential for proliferating cells to keep p53 expression low. One of the ways in which cells regulate p53 is through ubiquitylation: a process that requires multiple enzymes (90). First, ubiquitin, a protein comprised of 76 amino acids, must be activated in an ATP-dependent reaction that allows a thioester linkage to form between the active cysteine residue of E1 and the carboxy-terminal glycine of ubiquitin. E1 enzymes are therefore called ubiquitin activating enzymes. The ubiquitin is then transferred to an active cysteine residue on a ubiquitin conjugating enzyme (E2), where the activated ubiquitin forms a new thioester linkage. There are over 40 human E2s that have been identified to date; the E2 that usually mediates p53 ubiquitylation is UbcH5b/c (91). Once the E2 is linked to ubiquitin, the E2:ubiquitin complex then interacts with an E3 ubiquitin ligase such as Mdm2 which is responsible for substrate specificity and binds to proteins

22 10 such as p53. The E2 binds to the either a RING (Really Interesting New Gene) or HECT (Homologous to E6-associated protein Carboxyl Terminus) domain found within the E3 ligase (92, 93). Mdm2 contains a RING domain where E2s such as UbcH5b can attach (94). The ubiquitin:e2 attaches to the E3:substrate forming a complex of ubiquitin:e2:e3:substrate, permitting the E2 to transfer the ubiquitin to lysines that are available on the substrate (90). Several types of ubiquitylation events are known to occur. If the E3 only allows the E2 to add a single ubiquitin to the substrate, this addition is termed mono-ubiquitylation. Similarly, multiple mono-ubiquitylation is when several mono-ubiquitylation events occur on independent lysines of the same substrate. Lastly, when the E3 allows the E2 to transfer ubiquitin onto already ubiquitylated lysines, branches are formed and this is referred to as poly-ubiquitylation (90). There are two predominant forms of poly-ubiquitylation have been characterized. The lysine 48 (K48) linkage involves binding of the carboxy-terminus of a glycine residue on one ubiquitin molecule to the lysine 48 of another ubiquitin that is already attached to the substrate. K48 poly-ubiquitylation tends to form long chains and is known to target the substrate for degradation by the 26S proteasome (95). The other common form of poly-ubiquitylation generates substrates with the lysine 63 (K63)-linked chains of ubiquitin. In contrast to targeting proteins for degradation, it signals activation of signal transduction cascades that promote DNA repair (96). 4.2 Mdm2 As previously mentioned, Mdm2 is an E3 ubiquitin ligase for p53. Mdm2 binds to the amino terminus of p53 and can signal either mono- or multiple mono-ubiquitylation at lysines 101, 292, 305, 319, 357, 370, 372, 373, 381, 382, and 386 (97). Ubiquitylated p53 then translocates from the nucleus to the cytoplasm where it is thought to undergo K48 dependent poly-ubiquitylation and degradation (98-100). The mechanism as to how Mdm2 mediated mono-ubiquitylated p53 progresses to poly-ubiquitylation is still an open question. One report indicates that it is the amount of Mdm2 available in the cell that dictates whether Mdm2 mono- or poly-ubiquitylates p53 (101). It was argued that at low expression levels, Mdm2 can only mono-ubiquitylate p53, whereas at much higher levels of expression, Mdm2 poly-ubiquitylates p53 (101). Proteins such as p300, which lack E3 ligase activity on their own, have also been suggested to promote poly-

23 11 ubiquitylation of p53 when Mdm2 is present (102). Similar to p300, the protein YY-1 does not have native E3 ligase activity, but, in the presence of Mdm2, was able to promote polyubiquitylation of p53 (103). It is not yet clear how p300 and YY-1, both nuclear proteins, could target mono-ubiquitylated p53 for poly-ubiquitylation when this process has been shown to occur in the cytoplasm (100). This suggests that there may be further mechanisms of regulation. 4.3 Parc Another protein named Parc, recently renamed Cul9, has been shown to be localized in the cytoplasm, and interact with p53 (104). However, Parc does not ubiquitylate p53, rather it serves to anchor p53 in the cytoplasm (104). To that end, knock down of Parc expression results in translocation of p53 back to the nucleus, triggering p53-dependent apoptosis (104). Cell culture based evidence suggested that Parc was inhibiting p53 and may function as an oncogene in neuroblastoma where p53 is frequently found anchored in the cytoplasm (104). However, Parc null mice were generated and surprisingly, they were viable and free of any detectable defect (105). This result was surprising because Mdm2 null mice are embryonic lethal due to induction of massive apoptosis; a phenotype that can be rescued if p53 is is concurrently knocked out (106). This suggests that Mdm2 is a key negative regulator of p53 that keeps p53 expression low during mouse development. If Parc functions as a key negative regulator of p53 during mouse development like Mdm2, we would have expected ablation of Parc to lead to massive induction of apoptosis. When Parc null and wild type expressing MEFs were subjected to DNA damage, both induced p53 to a similar level, and had no difference in the level of apoptosis (105). Therefore is is believed that Parc is not required during normal mouse development, and does not negatively regulate p53 in this context. 4.4 CUL7 Shortly after the identification of Parc, CUL7 was discovered by Dias et al. as a cullin protein with E3 ubiquitin ligase activity and the ability to form the Skp1-Cullin7-Fbx29 complex (107). However, CUL7 was in fact a rediscovery, as it had initially been described by by Kohrman et al, as a novel SV40 Large T antigen binding protein termed p185 for its molecular mass (108). Additionally, Tsai et al also discovered CUL7 in mouse prior to Dias et al and named it p193,

24 12 hypothesizing that it may be a pro-apoptotic molecule since it contained a BH3 domain (109). BH3 domains are frequently observed in pro-apoptotic mitochondrial proteins such as Puma, Bim, and Bid and mediate inhibitory interactions with the BH1 and BH2 domains of antiapoptotic molecules such as Bcl-2, Bcl-XL, and Mcl-1. Although three labs have independently discovered and studied CUL7, the function of CUL7 remains a mystery. Ali et al. suggested that CUL7 may be a tumour suppressor because when they generated an SV40 Large T antigen mutant that could not bind to CUL7 and used this mutant to transform MEFs, it was less transforming than wild type SV40 Large T antigen (110). The authors argue that for SV40 Large T antigen to be a fully transforming protein it required its interaction with CUL7. CUL7 and Parc have highly similar sequences and both contain a CPH (conserved in CUL7, Parc, and Herc2) domain at the N-terminus (111). Therefore, since the N-terminal end of Parc interacts with p53, it is possible that, by extension, CUL7 also interacts with p53. Lastly, Mdm2 is an important negative regulator of p53, but not the only one. There are other E3 ligases that have been characterized to target p53 for ubiquitylation and subsequent degradation. PIRH2, HectH9/ARF-BP1, and COP1 have all been reported to negatively regulate p53 ( ). Additional study of PIRH2 revealed that PIRH2 targets p53 for degradation when p53 is activated upon DNA damage (115). p53 is phosphorylated at serines 15 and 37 upon DNA damage, which leads to the inhibition of Mdm2-p53 interaction (116). The function of PIRH2 is then to regulate such phosphorylated p53 that Mdm2 can no longer regulate. This is an interesting finding, since this may explain why multiple E3 ligases are required to recognize and regulate multiple post-translationally modified forms of a protein. 5 ARF ARF (Alternative Reading Frame of the INK4a locus) is a tumour suppressor. ARF was named because its transcript is formed from the alternative reading frame of the Ink4a locus (117). This locus encodes two proteins, p16 and ARF. p16 is also a tumour suppressor and functions by binding to CDK4 and CDK6 to inhibit cell cycle progression (118). Since the Ink4a locus encodes two tumour-suppressing proteins, it is often silenced in cancers. Studies of ARF revealed that ARF inhibits Mdm2, the key negative regulator of p53. There are two ways in which ARF can inhibit Mdm2. First, ARF can directly bind to Mdm2 and prevent Mdm2 from ubiquitylating p53 for proteasomal degradation (119). Second, ARF can sequester Mdm2 in the nucleolus and prevent it from interacting with p53 (120). It has been demonstrated that ARF is

25 13 usually sensitive to improper oncogene activation and expression. ARF expression is rapidly increased upon Myc, E2F1, Ras, E1A, or v-abl over-expression (118). Although there is no evidence to date that Myc can bind to the Ink4a locus, upon Myc over-expression, ARF is activated (60). As to how Myc can elevate ARF expression is not clear. One possibility may lie in E2F1. Myc has been reported to induce E2F1 in primary cells, and another report suggested that E2F1 could directly induce ARF (52, 121). Therefore, Myc potentiates apoptosis through the activation of ARF. When ARF is activated, it interacts with Mdm2 and inhibits Mdm2 from ubiquitylating p53. Given that ARF inhibits Mdm2, ARF expression leads to the activation of p53 (60). Activated p53 then upregulates other key apoptotic molecules, such as PUMA, Bax, Fas, PIDD, and Killer DR5, to induce cellular apoptosis. This pathway has been called the Myc- ARF-Mdm2-p53 axis. 6 ARF-independent, p53-dependent pathway of Myc potentiated apoptosis Although Myc can potentiate apoptosis through Myc-ARF-Mdm2-p53 axis, there is yet another axis through which Myc can potentiate apoptosis. The Myc-L11-Mdm2-p53 axis has been discovered in recent years, where Myc can directly induce the L11 ribosomal protein (122). L11 is a ribosomal protein that is usually induced upon ribosomal stress caused by changes in nutrient availability or when cells are subjected to Actinomycin D treatment (123). Myc-induced L11 protein then binds to Mdm2 and inhibits Mdm2 from suppressing p53 levels, therefore promoting p53 activation and cell death (123). 7 P53-independent role of Mdm2 Although, Mdm2 is key negative regulator of p53, recent findings indicate there may be a p53- independent role of Mdm2 in promoting tumourigenesis. First, in Eµ-Myc transgenic mouse studies, it was reported that one third of lymphomas with p53 mutations also had Mdm2 overexpression (124). Since p53 is already mutated, Mdm2 over-expression would be redundant if Mdm2 was only promoting tumourigenesis through inhibition of p53. Second, p53-/- mice overexpressing Mdm2 developed phenotypically different tumours compared to p53-/- mice with endogenous Mdm2 expression (125). This result suggested that over-expression of Mdm2 may have an additional function in promoting tumourigenesis even in the absence of p53. Third,

26 14 Mdm2 splice variants that do not have the N-terminal domain required for binding to p53 were also able to promote tumourigenesis in vivo (126). This result suggested that Mdm2 s interaction with p53 is not entirely required for Mdm2 to function as an oncogene. Lastly, there has been a study where Mdm2 and p53 status was investigated in human sarcomas (127). It was found that a subset of these tumours had both over-expression of Mdm2 and mutated p53. Interestingly, patients whose tumours that had Mdm2 over-expression and mutated p53 showed decreased survival when compared to patients tumours with either Mdm2 overexpression or p53 mutation alone. Based on these findings, it appears that Mdm2 must have additional functions and roles in promoting tumourigenesis that are independent of p53. 8 Myc ubiquitylation Just like p53 expression is mainly regulated by Mdm2 mediated ubiquitylation and subsequent degradation, Myc expression is also regulated by E3 ligase mediated ubiquitylation and subsequent degradation. The most well characterized E3 ligase that ubiquitylates Myc to regulate its expression is SCF Fbw7 (128, 129). For SCF Fbw7 to recognize Myc, a combination of dephosphorylation at S62 by PP2A and phosphorylation at T58 by GSK3 is required (30). These events allow for the binding of Fbw7 to Myc and this leads to the subsequent ubiquitylation and degradation of Myc. Another E3 ligase that has been characterized to ubiquitylate Myc is SCF Skp2 (33, 34). Skp2 interacts with Myc through the MBII and bhlh-lz domains (34). Ectopic expression of Skp2, like ectopic expression of Fbw7, leads to the destabilization of Myc and a decrease in Myc expression. However, unlike Fbw7, expression of Skp2 seems to also play a role in regulating Myc transcription. The expression of a Skp2 dominant negative, inhibited Myc from activating its target genes, cyclin D2 and cad (34). From this result, it has been suggested that Skp2 may have dual functions, where it ubiquitylates Myc to allow Myc to become transcriptionally active, but this active ubiquitylated Myc is programmed to undergo rapid proteasomal degradation. Additional E3 ligases that have been recently discovered to ubiquitylate Myc are PIRH2 and HectH9 (115, 130). As mentioned previously both of these E3 ligases ubiquitylate p53 and down regulate p53. However, PIRH2 seems to also poly-ubiquitylate and down-regulate Myc (115). HectH9 has also been characterized to ubiquitylate Myc, but HectH9-mediated Myc ubiquitylation did not result in Myc down-regulation through degradation (130). It was reported

27 15 that HectH9 poly-ubiquitylates the C-terminal end of Myc, and the nature of this ubiquitylation was of K63 linkage (130). This HectH9 mediated Myc ubiquitylation seems to also regulate Myc transcription. Moreover, a mutant Myc containing C-terminal lysines converted to arginines cannot be ubiquitylated by HectH9 and seems to be defective in up-regulating several Myc target genes. These results are interesting because two of the E3 ubiquitin ligases that ubiquitylate p53 seem to also ubiquitylate Myc. Since, Mdm2 has p53-independent function in promoting tumorigenesis, and Myc has been demonstrated to bind to the Mdm2 promoter (131), it would be interesting to determine if Mdm2 could also ubiquitylate Myc to regulate Myc expression and/or activities. 9 Targeting Myc for the treatment of cancer Because Myc is commonly deregulated in human cancers, many groups have explored the possibility of targeting this potent oncogene for patient treatment. There are three major approaches that have been taken to inhibit Myc activity in cancer: (1) direct downregulation of Myc protein expression; (2) targeting the activity of deregulated Myc; and (3) modulation of upstream pathways that regulate Myc expression and activity. With respect to approach 1, Jain et al. utilized a Doxycycline (Dox) inducible system to conditionally modulate Myc expression in osteogenic sarcoma cells (132). Interestingly, within as little as 24 hours of Myc inactivation, a rapid decrease in cellular proliferation was observed. Upon removal of Dox and therefore reactivation of Myc, massive apoptosis was induced which resulted in a decrease in osteosarcoma cell number. This data suggests that even reducing Myc protein expression in tumours for a short period of time could be beneficial as it promotes a decrease in tumour mass through the inhibition of cell proliferation and the induction of apoptosis. Using a different strategy to inhibit the oncogenic activity of Myc, its transcriptional programs were targeted using a dominant negative 92 amino acid protein called Omomyc. Omomyc consists of the bhlhzip dimerization domain of Myc with four amino acid substitutions that permit selective binding to certain Myc-interacting proteins but not others. In particular, expression of Omomyc has been shown to sequester Myc and prevent it from forming active transcription complexes with certain partner proteins, while leaving others, such as the Myc/Miz1 complex, intact. Omomyc lacks the transcription activation domain that is present in Myc, therefore, although Omomyc was shown to bind to Max, it acts as a dominant negative

28 16 inhibitor that is incapable of binding the E-box and activating Myc target genes (133). Transgenic mice with tetracycline-inducible Omomyc expression and K-Ras-mediated lung tumours demonstrated complete tumour regression in the presence of Omomyc. Significantly, withdrawal of tetracycline and therefore Omomyc expression, resulted in the complete recovery of normal cell tissues, while the mice remained tumour-free one year post-treatment (133). These studies provide the field with convincing evidence that it is possible to utilize our understanding of how Myc functions as a transcription factor to target its mechanism of action in cancer. A recent example of inhibiting the oncogneic activity of Myc by targeting upstream pathways is JQ1. JQ1 has been shown to inhibit BRD4. BRD4 is a member of the bromodomain and extra terminal (BET) family, which associates with regions of acetylated chromatin. In multiple myeloma cells, BRD4 binds to the Myc locus to regulate its expression. However, upon treatment with JQ1, Myc expression decreased resulting in the down regulation of Myc target gene transactivation (134). It was further elucidated that the down regulation of Myc through JQ1 treatment resulted in cell cycle arrest and senescence of multiple myeloma cells (134). This study demonstrates that indeed Myc can also be targeted indirectly by inhibiting upstream pathways that converge on Myc and promote its expression. Combined, the above described and additional work demonstrates that expression of Myc is crucial for tumour cell growth, and that the inhibition of Myc is a powerful way to suppress the neoplastic process. These studies also suggest that there are many ways in which we can target Myc, and interestingly, all of these different strategies are effective. Significantly, these proof-of-concept studies underscore the importance of understanding the regulation and deregulation of Myc as well the development of additional novel mechanisms - be it gene delivery, chemical or molecular inhibitors - to interfere with Myc activity and therefore, improve the current treatment of human cancer.

29 17 10 Hypotheses 1. Determine if the N-terminal six lysines of Myc regulate Myc biological programs such as proliferation, apoptosis, and transformation. Several key data are presented in the literature that suggest the N-terminal end of Myc is important for the regulation of Myc-dependent proliferation, apoptosis, and transformation. This is in part due to MBI and MBII of Myc are required for Myc to interact with its key co-factors to regulate its biological activities. I hypothesize that the N-terminal six lysines of Myc may be utilized for ubiquitylation and are required for Myc to fully promote Myc-dependent proliferation, apoptosis, and transformation. Therefore, I sought to mutate the N-terminal six lysines of Myc to abrogate ubiquitylation and determine the effect of these mutations on the function of Myc. 2. Investigate if CUL7 is an oncogene or tumor suppressor and determine its role in the regulation of p53 and Myc dependent apoptosis. A functional screening strategy identified CUL7 as an inhibitor of Myc-dependent apoptosis. Since Myc-potentiated apoptosis can be p53-dependent, and Parc, a highly similar protein to CUL7 has been characterized to bind p53 and inhibit p53 at the cytoplasm, I hypothesize that CUL7 may also antagonize Myc-potentiated apoptosis through the inhibition of p53. I sought to determine if CUL7 can cooperate with Myc in driving cellular transformation, and determine if CUL7 can interact with p53 both in cells and in vitro. Furthermore, I sought to determine if down-regulation of CUL7 in neuroblastoma cells would result in p53 activation.

30 18 Chapter 2 Characterization of N-terminal Myc ubiquitylation and functional analysis of a N-terminal lysine defective Myc mutant (6KR-N) Data attribution: Figures 1 A, B, C, D: Technical assistance was provided by Dr. Amanda Wasylishen. Figure 1 F, G, H: Experiments were designed and performed by Dr. Amanda Wasylishen. Mice injections and tumor measurements were done by Aleks Pandyra. Figure 3, 4: Technical assistance was provided by Dr. Angelina Stojanova. Figure 6: Technical assistance was provided by Manpreet Kalkat, and mass spectrometry analysis was performed by Michael Chan and Dr. Brian Raught Figure 11: Technical assistance was provided by Dr. Romi Ponzielli.