Anthony C. Liang

A Genetic Screen for Candidate Tumor Suppressors Identifies REST

Tumorigenesis is a multistep process characterized by a myriad of genetic and epigenetic alterations. Identifying the causal perturbations that confer malignant transformation is a central goal in cancer biology. Here we report an RNAi-based genetic screen for genes that suppress transformation of human mammary epithelial cells. We identified genes previously implicated in proliferative control and epithelial cell function including two established tumor suppressors, TGFBR2 and PTEN. In addition, we uncovered a previously unrecognized tumor suppressor role for REST/NRSF, a transcriptional repressor of neuronal gene expression. Array-CGH analysis identified REST as a frequent target of deletion in colorectal cancer. Furthermore, we detect a frameshift mutation of the REST gene in colorectal cancer cells that encodes a dominantly acting truncation capable of transforming epithelial cells. Cells lacking REST exhibit increased PI(3)K signaling and are dependent upon this pathway for their transformed phenotype. These results implicate REST as a human tumor suppressor and provide a novel approach to identifying candidate genes that suppress the development of human cancer.

Cancer Proliferation Gene Discovery Through Functional Genomics

Retroviral short hairpin RNA (shRNA)–mediated genetic screens in mammalian cells are powerful tools for discovering loss-of-function phenotypes. We describe a highly parallel multiplex methodology for screening large pools of shRNAs using half-hairpin barcodes for microarray deconvolution. We carried out dropout screens for shRNAs that affect cell proliferation and viability in cancer cells and normal cells. We identified many shRNAs to be antiproliferative that target core cellular processes, such as the cell cycle and protein translation, in all cells examined. Moreover, we identified genes that are selectively required for proliferation and survival in different cell lines. Our platform enables rapid and cost-effective genome-wide screens to identify cancer proliferation and survival genes for target discovery. Such efforts are complementary to the Cancer Genome Atlas and provide an alternative functional view of cancer cells.

SCFbeta-TRCP controls oncogenic transformation and neural differentiation through REST degradation.

The RE1-silencing transcription factor (REST, also known as NRSF) is a master repressor of neuronal gene expression and neuronal programmes in non-neuronal lineages. Recently, REST was identified as a human tumour suppressor in epithelial tissues, suggesting that its regulation may have important physiological and pathological consequences. However, the pathways controlling REST have yet to be elucidated. Here we show that REST is regulated by ubiquitin-mediated proteolysis, and use an RNA interference (RNAi) screen to identify a Skp1-Cul1-F-box protein complex containing the F-box protein β-TRCP (SCFβ-TRCP) as an E3 ubiquitin ligase responsible for REST degradation. β-TRCP binds and ubiquitinates REST and controls its stability through a conserved phospho-degron. During neural differentiation, REST is degraded in a β-TRCP-dependent manner. β-TRCP is required for proper neural differentiation only in the presence of REST, indicating that β-TRCP facilitates this process through degradation of REST. Conversely, failure to degrade REST attenuates differentiation. Furthermore, we find that β-TRCP overexpression, which is common in human epithelial cancers, causes oncogenic transformation of human mammary epithelial cells and that this pathogenic function requires REST degradation. Thus, REST is a key target in β-TRCP-driven transformation and the β-TRCP–REST axis is a new regulatory pathway controlling neurogenesis.

A SUMOylation-Dependent Transcriptional Subprogram Is Required for Myc-Driven Tumorigenesis

Taking the Myc Despite nearly 30 years of research into the mechanisms by which Myc oncogene dysregulation contributes to tumorigenesis, there are still no effective therapies that inhibit Myc activity. Kessler et al. (p. 348, published online 8 December; see the Perspective by Evan) searched for gene products that support Myc-driven tumorigenesis. One pharmacologically tractable target that emerged from the screen was the SUMO-activating enzyme complex SAE1/2, which catalyzes a posttranslational modification (SUMOylation) that alters protein behavior and function. SUMOylation was found to control the Myc transcriptional response, and its inhibition caused mitotic defects and apoptosis in Myc-dependent breast cancer cells. An RNA interference screen identifies a “druggable” enzyme whose inhibition halts tumor cell growth. Myc is an oncogenic transcription factor frequently dysregulated in human cancer. To identify pathways supporting the Myc oncogenic program, we used a genome-wide RNA interference screen to search for Myc–synthetic lethal genes and uncovered a role for the SUMO-activating enzyme (SAE1/2). Loss of SAE1/2 enzymatic activity drives synthetic lethality with Myc. Inactivation of SAE2 leads to mitotic catastrophe and cell death upon Myc hyperactivation. Mechanistically, SAE2 inhibition switches a transcriptional subprogram of Myc from activated to repressed. A subset of these SUMOylation-dependent Myc switchers (SMS genes) is required for mitotic spindle function and to support the Myc oncogenic program. SAE2 is required for growth of Myc-dependent tumors in mice, and gene expression analyses of Myc-high human breast cancers suggest that low SAE1 and SAE2 abundance in the tumors correlates with longer metastasis-free survival of the patients. Thus, inhibition of SUMOylation may merit investigation as a possible therapy for Myc-driven human cancers.

Recurrent Hemizygous Deletions in Cancers May Optimize Proliferative Potential

Cancer Gene Islands Human tumors are riddled with genomic alterations that rearrange, remove, amplify, or otherwise disrupt a wide spectrum of genes, and a key challenge is identifying which of these alterations are causally involved in tumorigenesis. The role of recurrent hemizygous focal deletions is especially puzzling because these deletions preferentially affect certain chromosomal regions and result in the loss of one copy of a whole cluster of adjacent genes. Solimini et al. (p. 104, published online 24 May; see the Perspective by Greenman) found that these deletions span genomic regions that are enriched in genes that negatively regulate cell proliferation. The cumulative reduction in dosage and tumor suppressive function of the genes within these “cancer gene islands” may represent a critical factor driving tumor growth. The genomes of cancer cells have preferentially lost genes that inhibit cell growth. Tumors exhibit numerous recurrent hemizygous focal deletions that contain no known tumor suppressors and are poorly understood. To investigate whether these regions contribute to tumorigenesis, we searched genetically for genes with cancer-relevant properties within these hemizygous deletions. We identified STOP and GO genes, which negatively and positively regulate proliferation, respectively. STOP genes include many known tumor suppressors, whereas GO genes are enriched for essential genes. Analysis of their chromosomal distribution revealed that recurring deletions preferentially overrepresent STOP genes and underrepresent GO genes. We propose a hypothesis called the cancer gene island model, whereby gene islands encompassing high densities of STOP genes and low densities of GO genes are hemizygously deleted to maximize proliferative fitness through cumulative haploinsufficiencies. Because hundreds to thousands of genes are hemizygously deleted per tumor, this mechanism may help to drive tumorigenesis across many cancer types.

A functional genomic screen identifies a role for TAO1 kinase in spindle-checkpoint signalling

Defects in chromosome–microtubule attachment trigger spindle-checkpoint activation and delay mitotic progression. How microtubule attachment is sensed and integrated into the steps of checkpoint-signal amplification is poorly understood. In a functional genomic screen targeting human kinases and phosphatases, we identified a microtubule affinity-regulating kinase kinase, TAO1 (also known as MARKK) as an important regulator of mitotic progression, required for both chromosome congression and checkpoint-induced anaphase delay. TAO1 interacts with the checkpoint kinase BubR1 and promotes enrichment of the checkpoint protein Mad2 at sites of defective attachment, providing evidence for a regulatory step that precedes the proposed Mad2–Mad1 dependent checkpoint-signal amplification step. We propose that the dual functions of TAO1 in regulating microtubule dynamics and checkpoint signalling may help to coordinate the establishment and monitoring of correct congression of chromosomes, thereby protecting genomic stability in human cells.

A Systematic Analysis of Factors Localized to Damaged Chromatin Reveals PARP-Dependent Recruitment of Transcription Factors.

Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the amyotrophic lateral sclerosis (ALS) candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a poly-(ADP-ribose) polymerase (PARP)-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors; > 70% of randomly tested transcription factors localized to sites of DNA damage, and of these, ∼90% were PARP dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding-domain dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins.

STOP gene Phactr4 is a tumor suppressor

Significance Cell proliferation control is central to tumor suppression. We previously identified hundreds of suppressors of tumorigenesis and/or proliferation (STOP) genes that restrain normal cell proliferation. Here we show that one such STOP gene, phosphatase and actin regulator 4 (PHACTR4), acts to prevent tumorigenesis. Phactr4 suppresses proliferation and transformation in vitro and tumorigenesis in vivo. PHACTR4 is significantly mutated or downregulated in several cancers, and reintroduction of Phactr4 limits proliferation and tumor growth in cells from these cancers. Thus, our studies provide strong evidence that PHACTR4 is a tumor suppressor. Cancer develops through genetic and epigenetic alterations that allow unrestrained proliferation and increased survival. Using a genetic RNAi screen, we previously identified hundreds of suppressors of tumorigenesis and/or proliferation (STOP) genes that restrain normal cell proliferation. Our STOP gene set was significantly enriched for known and putative tumor suppressor genes. Here, we report a tumor-suppressive role for one STOP gene, phosphatase and actin regulator 4 (PHACTR4). Phactr4 is one of four members of the largely uncharacterized Phactr family of protein phosphatase 1 (PP1)- and actin-binding proteins. Our work suggests that Phactr4 restrains normal cell proliferation and transformation. Depletion of Phactr4 with multiple shRNAs leads to increased proliferation and soft agar colony formation. Phactr4 acts, in part, through an Rb-dependent pathway, because Rb phosphorylation is maintained upon growth factor withdrawal in Phactr4-depleted cells. Examination of tumor copy number analysis and sequencing revealed that PHACTR4 is significantly deleted and mutant in many tumor subtypes. Furthermore, cancer cell lines with reduced Phactr4 expression exhibit tumor suppressor hypersensitivity upon Phactr4 complementation, leading to reduced proliferation, transformation, and tumor formation. Thus, Phactr4 acts as a tumor suppressor that is deleted and mutant in several cancers.

Abstract 3091: A sumoylation-dependent transcriptional subprogram is required for Myc-driven tumorigenesis

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Myc is an oncogenic transcription factor frequently dysregulated in human malignancies. While the transcriptional programs and other functions of Myc have been intensively studied, there remains no effective strategy for inhibiting Myc in patients. To search for pathways that support the Myc oncogenic program, we employed a next-generation RNAi screen for Myc-synthetic lethal (MySL) genes. Using this strategy, we have identified several cellular processes required to tolerate oncogenic Myc. Key among these is the core sumoylation machinery, and we define the Sumo-activating enzyme (SAE) as a central component in this MySL network. Loss of SAE drives synthetic lethality with Myc, and the enzymatic activity of SAE is required to support the Myc oncogenic state. Inactivation of SAE leads to mitotic catastrophe and cell death selectively upon Myc hyper-activation. Mechanistically, depletion of SAE switches a subprogram of Myc transcriptional targets governing mitotic spindle function from activated to repressed, a subprogram we term Sumoylation-dependent Myc Switchers, or SMS genes. Notably, SMS genes are required to tolerate Myc hyper-activation, and SAE and the SMS program are required for Myc-dependent breast cancer cell survival in vitro and tumor growth and progression in vivo. Importantly, patient survival significantly correlates with levels of SAE and SMS gene expression in Myc-high tumors. Collectively, these studies reveal a mitotic vulnerability of Myc-driven cancers and demonstrate that inhibiting sumoylation can selectively impair mitosis and survival in an oncogenic Myc-dependent manner. We propose that drugs targeting SAE and its downstream SMS targets may have therapeutic benefits for patients with Myc-driven cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3091. doi:1538-7445.AM2012-3091

K63-linked polyubiquitin chains bind to DNA to facilitate DNA damage repair

Mutations in the DNA-interacting patch of ubiquitin sensitize cells to DNA-damaging agents. DNA-bound ubiquitin coordinates DNA repair Ubiquitylation is a posttranslational modification that reversibly alters protein stability, activity, interactions, or trafficking. The ubiquitylation of histones and various other proteins facilitates the response to DNA damage. However, Liu et al. discovered that ubiquitin also binds directly to DNA. In solution and in live cells, chains of ubiquitin specifically linked through Lys63 residues (referred to as K63-linked polyubiquitin chains) preferentially bound to the free ends of double-stranded DNA through a three-amino acid motif in ubiquitin that the authors call a “DNA-interacting patch” (DIP). These chains appeared to bind the broken ends of DNA and recruit repair proteins. Ubiquitins with mutations in the DIP were found in several types of tumors and, when expressed in cultured cells, impaired the cellular response to DNA-damaging agents, suggesting that these mutations might be exploited for therapeutic benefit in some cancer patients. Polyubiquitylation is canonically viewed as a posttranslational modification that governs protein stability or protein-protein interactions, in which distinct polyubiquitin linkages ultimately determine the fate of modified protein(s). We explored whether polyubiquitin chains have any nonprotein-related function. Using in vitro pull-down assays with synthetic materials, we found that polyubiquitin chains with the Lys63 (K63) linkage bound to DNA through a motif we called the “DNA-interacting patch” (DIP), which is composed of the adjacent residues Thr9, Lys11, and Glu34. Upon DNA damage, the binding of K63-linked polyubiquitin chains to DNA enhanced the recruitment of repair factors through their interaction with an Ile44 patch in ubiquitin to facilitate DNA repair. Furthermore, experimental or cancer patient–derived mutations within the DIP impaired the DNA binding capacity of ubiquitin and subsequently attenuated K63-linked polyubiquitin chain accumulation at sites of DNA damage, thereby resulting in defective DNA repair and increased cellular sensitivity to DNA-damaging agents. Our results therefore highlight a critical physiological role for K63-linked polyubiquitin chains in binding to DNA to facilitate DNA damage repair.