Natalya N. Pavlova

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.

Activation of Multiple Proto-oncogenic Tyrosine Kinases in Breast Cancer via Loss of the PTPN12 Phosphatase

Among breast cancers, triple-negative breast cancer (TNBC) is the most poorly understood and is refractory to current targeted therapies. Using a genetic screen, we identify the PTPN12 tyrosine phosphatase as a tumor suppressor in TNBC. PTPN12 potently suppresses mammary epithelial cell proliferation and transformation. PTPN12 is frequently compromised in human TNBCs, and we identify an upstream tumor-suppressor network that posttranscriptionally controls PTPN12. PTPN12 suppresses transformation by interacting with and inhibiting multiple oncogenic tyrosine kinases, including HER2 and EGFR. The tumorigenic and metastatic potential of PTPN12-deficient TNBC cells is severely impaired upon restoration of PTPN12 function or combined inhibition of PTPN12-regulated tyrosine kinases, suggesting that TNBCs are dependent on the proto-oncogenic tyrosine kinases constrained by PTPN12. Collectively, these data identify PTPN12 as a commonly inactivated tumor suppressor and provide a rationale for combinatorially targeting proto-oncogenic tyrosine kinases in TNBC and other cancers based on their profile of tyrosine-phosphatase activity.

The pro-apoptotic kinase Mst1 and its caspase cleavage products are direct inhibitors of Akt1

Akt kinases mediate cell growth and survival. Here, we report that a pro‐apoptotic kinase, Mst1/STK4, is a physiological Akt1 interaction partner. Mst1 was identified as a component of an Akt1 multiprotein complex isolated from lipid raft‐enriched fractions of LNCaP human prostate cancer cells. Endogenous Mst1, along with its paralog, Mst2, acted as inhibitors of endogenous Akt1. Surprisingly, mature Mst1 as well as both of its caspase cleavage products, which localize to distinct subcellular compartments and are not structurally homologous, complexed with and inhibited Akt1. cRNAs encoding full‐length Mst1, and N‐ and C‐terminal caspase Mst1 cleavage products, reverted an early lethal phenotype in zebrafish development induced by expression of membrane‐targeted Akt1. Mst1 and Akt1 localized to identical subcellular sites in human prostate tumors. Mst1 levels declined with progression from clinically localized to hormone refractory disease, coinciding with an increase in Akt activation with transition from hormone naïve to hormone‐resistant metastases. These results position Mst1/2 within a novel branch of the phosphoinositide 3‐kinase/Akt pathway and suggest an important role in cancer progression.

CDYL Bridges REST and Histone Methyltransferases for Gene Repression and Suppression of Cellular Transformation

The neuronal gene repressor REST/NRSF recruits corepressors, including CoREST, to modify histones and repress transcription. REST also functions as a tumor suppressor, but the mechanism remains unclear. We identified chromodomain on Y-like (CDYL) as a REST corepressor that physically bridges REST and the histone methylase G9a to repress transcription. Importantly, RNAi knockdown of REST, CDYL, and G9a, but not CoREST, induced oncogenic transformation of immortalized primary human cells and derepression of the proto-oncogene TrkC. Significantly, transgenic expression of TrkC also induced transformation. This implicates CDYL-G9a, but not CoREST, in REST suppression of transformation, possibly by oncogene repression. CDYL knockdown also augments transformation in a cell culture model of cervical cancer, where loss of heterozygosity of the CDYL locus occurs. These findings demonstrate molecular strategies by which REST carries out distinct biological functions via different corepressors and provide critical insights into the role of histone-modifying complexes in regulating cellular transformation.

A role for PVRL4-driven cell–cell interactions in tumorigenesis

During all stages of tumor progression, cancer cells are subjected to inappropriate extracellular matrix environments and must undergo adaptive changes in order to evade growth constraints associated with the loss of matrix attachment. A gain of function screen for genes that enable proliferation independently of matrix anchorage identified a cell adhesion molecule PVRL4 (poliovirus-receptor-like 4), also known as Nectin-4. PVRL4 promotes anchorage-independence by driving cell-to-cell attachment and matrix-independent integrin β4/SHP-2/c-Src activation. Solid tumors frequently have copy number gains of the PVRL4 locus and some have focal amplifications. We demonstrate that the transformation of breast cancer cells is dependent on PVRL4. Furthermore, growth of orthotopically implanted tumors in vivo is inhibited by blocking PVRL4-driven cell-to-cell attachment with monoclonal antibodies, demonstrating a novel strategy for targeted therapy of cancer. DOI: http://dx.doi.org/10.7554/eLife.00358.001

Construction of a rationally designed antibody platform for sequencing-assisted selection

Antibody discovery platforms have become an important source of both therapeutic biomolecules and research reagents. Massively parallel DNA sequencing can be used to assist antibody selection by comprehensively monitoring libraries during selection, thus greatly expanding the power of these systems. We have therefore constructed a rationally designed, fully defined single-chain variable fragment (scFv) library and analysis platform optimized for analysis with short-read deep sequencing. Sequence-defined oligonucleotide libraries encoding three complementarity-determining regions (L3 from the light chain, H2 and H3 from the heavy chain) were synthesized on a programmable microarray and combinatorially cloned into a single scFv framework for molecular display. Our unique complementarity-determining region sequence design optimizes for protein binding by utilizing a hidden Markov model that was trained on all antibody-antigen cocrystal structures in the Protein Data Bank. The resultant ∼1012-member library was produced in ribosome-display format, and comprehensively analyzed over four rounds of antigen selections by multiplex paired-end Illumina sequencing. The hidden Markov model scFv library generated multiple binders against an emerging cancer antigen and is the basis for a next-generation antibody production platform.

Protein interaction discovery using parallel analysis of translated ORFs (PLATO)

Identifying physical interactions between proteins and other molecules is a critical aspect of biological analysis. Here we describe PLATO, an in vitro method for mapping such interactions by affinity enrichment of a library of full-length open reading frames displayed on ribosomes, followed by massively parallel analysis using DNA sequencing. We demonstrate the broad utility of the method for human proteins by identifying known and previously unidentified interacting partners of LYN kinase, patient autoantibodies, and the small-molecules gefitinib and dasatinib.

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 IA28: Haploinsufficiency in cancer

Breast cancer is a collection of diseases with distinct clinical behaviors and underlying genetic causes. We have searched genetically for genes that have cancer relevant phenotypes including genes that alter cellular proliferation, cellular transformation, cell survival, cellular senescence, and genes that are essential for the proliferation of cancer cells. We have approached this by the generation of libraries of shRNAs for loss of function experiments and libraries of ORFs for gain of function experiments. We will discuss these technologies and how they can be applied to the functional dissection of genes important for breast cancer. Using these new technologies we have identified new oncogenes and tumor suppressors. We find that tumor cells selectively delete negative growth regulators and suggest that the deletion of clusters of these genes may drive tumorigenesis by haploinsufficiency. These recurrent deletions also avoid deletion of one copy of many essential genes through haploinsufficiency. We put forward the Cancer Gene Island model that postulates that tumors select for hemizygous loss of islands of gene enriched in negative regulators of proliferation and depleted in essential genes to promote tumor cell proliferation through cumulative haploinsufficiency. Citation Format: Teresa Davoli, Nicole L. Solimini, Natalya N. Pavlova, Qikai Xu, Kristen Mengwasser, Laura M. Sack, Anthony C. Liang, Michael R. Schlabach, Ji Luo, Anna E. Burrows, Anthony N. Anselmo, Mamie Z. Li, Stephen J. Elledge. Haploinsufficiency in cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr IA28.

Abstract IA8: Genetic approaches to cancer

Breast cancer is a collection of diseases with distinct clinical behaviors and underlying genetic causes. We have searched genetically for genes that have cancer relevant phenotypes including genes that alter cellular proliferation, cellular transformation, cell survival, cellular senescence, and genes that are essential for the proliferation of cancer cells. We have approached this by the generation of libraries of shRNAs for loss of function experiments and libraries of ORFs for gain of function experiments. We will discuss these technologies and how they can be applied to the functional dissection of genes important for breast cancer. Citation Format: Nicole L. Solimini, Andrea L. Bredemeyer, Mamie Z. Li, Rameen Beroukhim, Matthew Meyerson, Stephen J. Elledge, Natalya N. Pavlova, Qikai Xu, Craig H. Mermel, Anthony C. Liang, Michael R. Schlabach, Ji Luo, Anna E. Burrows, Anthony N. Anselmo. Genetic approaches to cancer [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr IA8.