Irwin H. Gelman - US grants

DESCRIPTION: (provided by applicant) A major enigma in the field of cell biology is how signaling proteins with seemingly antithetical functions, such as protein kinase (PKA) and PKC, are regulated temporally and spatially during mitoqenic stimulation. Scaffolding proteins are thought to facilitate the efficiency and specificity of substrate phosphorylation and also coordinate the actions of kinases and phosphatases in a spatiotemporal manner. The long-term goal of this research is to understand how SSeCKS, a plasma membrane-and cytoskeleton-associated PKC substrate that binds PKC, PKA, calmodulin and cyclins, helps regulate signaling crosstalk and G1-S progression in untransformed fibroblasts and epithelial cells, compared to prostate cancer cells, in which SSeCKS expression is downregulated severely. Our working hypothesis is that SSeCKS (and its orthologue, Gravin/AKAP25O) prevents PKC/PKA crosstalk: in quiescent cells, SSeCKS binds to and inhibits PKC activity whereas during G1 S progression, mitogen-activated phosphorylation of SSeCKS selectively antagonizes PKC binding; PKA, whose binding is phosphorylation insensitive, is then sequestered to "inactive" sites. With mounting data that SSeCKS has metastasis suppressor activity in prostate cancer, we envision that dissection of the PKC/PKA/SSeCKS control network will further understanding of signaling and cytoskeletal controls during mitogenesis and oncogenesis. In Aim #1, we will use site-directed mutagenesis and in vitro binding assays to identify the SSeCKS domains that bind PKC and PKA and that mediate multimerization. In Aim #2, we will examine how SSeCKS binding affects the in vitro kinase activity of PKC and PKA on specific substrates. We will also determine how the binding sites regulate PKC-and PKA-mediated G1-S signaling and cytoskeletal pathways in vivo in untransformed NIH3T3 and prostate epithelial cells, and in SSeCKS-deficient human LNCaP prostate cancer cells. We will analyze how SSeCKS affects the mitogen-and integrin-mediated cellular localization of PKC and PKA. Aim #3 will investigate how PKC-induced phosphorylation modulates SSeCKS in vivo scaffolding activity and involvement in G1-S regulation. To this end, we will map the in vivo PKC phosphorylation sites on SSeCKS under growth-promoting and oncogenic conditions using a combination of peptide microsequencing and MALDITOF mass spectrometry. The role of PKC-induced phosphorylation on SSeCKS' scaffolding/regulatory functions will be accessed using SSeCKS phospho-specific antibodies and cell lines expressing SSeCKS PKC-phosphorylation-site mutants. Given that many G1-S regulatory functions are undermined in oncogenesis, the results of the proposed research on SSeCKS should broaden our understanding of spatial and temporal controls governing cell cycle progression in normal and cancer cells.

DESCRIPTION (provided by applicant): A major enigma in the field cell biology is how signaling proteins with seemingly antithetical functions, such as 1 protein kinase (PK) A and PKC, are regulated temporally and spatially during mitogenic stimulation. Scaffolding proteins are thought to facilitate the efficiency and specificity of substrate phosphorylation and also coordinate the actions of kinases and phosphatases in a spatiotemporal manner. The long-term goal of this research is to understand how SSeCKS, a plasma membrane- and cytoskeleton-associated PKC substrate that binds PKC, PKA, calmodulin and cyclins, helps regulate signaling crosstalk and G1-S progression in untransformed fibroblasts and epithelial cells, compared to prostate cancer cells, in which SSeCKS expression is downregulated severely. Our working hypothesis is that SSeCKS (and its orthologue, GravinfAKAP25O) prevents PKCIPKA crosstalk: in quiescent cells, SSeCKS binds to and inhibits PKC activity whereas during G1-S progression, mitogen-activated phosphorylation of SSeCKS selectively antagonizes PKC binding; PKA, whose binding is phosphorylation insensitive, is then sequestered to "inactive" sites. With mounting data that SSeCKS has metastasis suppressor activity in prostate cancer, we envision that dissection of the PKC/PKA/SSeCKS control network will further understanding of signaling and cytoskeletal controls during mitogenesis and oncogenesis. In Aim #1, we will use site-directed mutagenesis and in vitro binding assays to identify the SSeCKS domains that bind PKC and PKA and that mediate multimerization. In Aim #2, we will examine how SSeCKS binding affects the in vitro kinase activity of PKC and PKA on specific substrates. We will also determine how the binding sites regulate PKC- and PKA-mediated G1-S signaling and cytoskeletal pathways in vivo in untransformed NIH3T3 and prostate epithelial cells, and in SSeCKS-deficient human LNCaP prostate cancer cells. We will analyze how SSeCKS affects the mitogen- and integrin-mediated cellular localization of PKC and PKA. Aim #3 will investigate how PKC-induced phosphorylation modulates SSeCKS in vivo scaffolding activity and involvement in G1-S regulation. To this end, we will map the in vivo PKC phosphorylation sites on SSeCKS under growth-promoting and oncogenic conditions using a combination of peptide microsequencing and MALDI-TOF mass spectrometry. The role of PKC-induced phosphorylation on SSeCKS scaffolding/regulatory functions will be accessed using SSeCKS phospho-specific antibodies and cell lines expressing SSeCKS PKC-phosphorylation-site mutants. Given that many G1-S regulatory functions are undermined in oncogenesis, the results of the proposed research on SSeCKS should broaden our understanding of spatial and temporal controls governing cell cycle progression in normal and cancer cells.

DESCRIPTION (provided by applicant): Molecular analysis of chromosomal abnormalities has allowed the identification of many genes directly involved in the pathogenesis of lymphoid malignancies. However, in non-Hodgkin's lymphoma (NHL), the development of the full neoplastic phenotype depends on the acquisition of multiple genetic events including concurrent activation of synergistic "dominant" oncogenes and loss of tumor suppressor gene functions. NHLs often present complex karyotypes that may prevent complete analysis. To overcome this difficulty, many transformed NHL cell lines have been established. Others and we have identified chromosome 12q24 as a recurrent breakpoint in mature lymphoid malignancies of both T- and B-cell lineage and have recently mapped the gone encoding SMRT to 12q24.3. We observed that SMRT is altered in all transformed NHL cell lines/patients tested at the genomic, transcript and protein levels. We propose that SMRT plays an important role in NHL. This implies that SMRT acts as a tumor suppressor gene. To test this hypothesis, we propose the following Specific Aims: 1-To perform a retrospective study of a large series of transformed and non-transformed patient samples by FISH, LOH and immunohistochemistry, to expand upon our observation of transformed lymphoma phenotype and deletion of one SMRT allele. 2- To study the mechanisms by which SMRT regulates apoptosis and cell survival in SMRT-deficient NHL cells. For this purpose, we will study variation of expression of a set of genes involved in cell survival and of the different caspases (and their activation) upon SMRT-restoration-driven apoptosis. The ultimate goal of this aim is to identify future investigative area for potential (new) therapeutic approaches to induce apoptosis in SMRT-deficient NHL cells. 3- To establish a cause: effect relationship between SMRT down-regulation and the NHL transformation process. For this purpose, we will use an antisense strategy to down-regulate SMRT in different normal and low-grade transgenic mice hematopoietic environment. Confirmation of the role of SMRT in NHL transformation and in apoptosis could potentially allow development of new diagnostic and prognostic tools as well as new therapeutic strategies.

DESCRIPTION (provided by applicant): Activation of protein kinase (PK) C has been considered cancer-promoting based on early data showing that phorbol esters could induce oncogenes is through the activation of so called classic and novel PKC isozymes. Although many human cancers exhibit increased levels of specific PKC isozymes there are conflicting data which show that specific isozymes can act as either promoters or suppressors of oncogenesis depending on tissue context. In human prostate cancer (CaP), PKC, levels increase with malignancy whereas PKC* levels are constant, yet only the overexpression of PKC, is sufficient to induce prostatic intraepithelial neoplasia (PIN). Therefore, characterization of the pro-oncogenic PKC-induced signaling pathways and mediators is paramount to our overall understanding of CaP disease progression. Our lab identified a novel PKC substrate, SSeCKS/Gravin/AKAP12 ("SSeCKS") that is also a scaffold for both PKC and PKA, capable of attenuating PKC kinase activity and altering its cellular compartmentalization, while regulating PKA through compartmentalization only. SSeCKS displays many of the hallmarks of a tumor suppressor in prostate cancer: it is severely downregulated in human CaP cell lines and tissues with Gleason sums =6, SSeCKS reexpression suppresses macroscopic CaP metastasis growth by inhibiting VEGF-induced neovascularization, genetic knockout (KO) in mice induces prostatic hyperplasia and focal dysplasia with evidence of epithelial cell senescence, and KO mouse fibroblasts (MEF) display premature senescence correlating with polyploidy and binucleation. Premature senescence seems Rb-dependent because it can be suppressed by HPV-16E7orpRb-siRNA but not byHPV-16 E6 or p53-siRNA. In keeping with SSeCKS'scaffolding function for PKC, KO-MEF have >2-fold higher total PKC activity than WT-MEF, with a >3-fold increase in PKC* activity. Based on preliminary data from expression microarray and biochemical experiments, we hypothesize the loss of SSeCKS induces hyperactive PKC*/,,which in turn induce i) cytokinesis defects through the activation of RhoA and LIMK, and through the suppression of the mitotic exit network kinase, WARTS, and ii) Rb-dependent senescence by increasing reactive oxygen species (ROS) mediators such as p47phox. We believe SSeCKS controls cytokinesis directly because a pool of SSeCKS enriches in the cleavage furrow during telophase where PKC* and , are known to normally regulate abscission by coordinating the timing of RhoA and LIMK activation, and thus, formation and contraction of the actomyosin abscission ring. Our overall aim is to use genetic, biochemical, and fluorescence and time-lapse microscopy techniques to dissect this novel SSeCKS-PKC pathway in WT vs. KO MEF and murine prostate epithelial cells (PrEC), and in human PrEC vs. CaP cell lines, using readouts of senescence/ polyploidy/ binucleation and cytokinesis completion (Aim 1), podosome formation and tumor invasiveness (Aim 2), and CaP formation, invasiveness and metastatic potential in transgenic mouse models (Aim 3). The experiments proposed are envisioned to elucidate the mechanism and pathways controlled by the SSeCKS/PKC scaffold complex and how dysregulation by the loss of SSeCKS contributes to CaP progression. PUBLIC HEALTH RELEVANCE: Our project focuses on SSeCKS, a protein that seems to suppress prostate cancer progression especially metastasis, by binding two opposing signaling proteins, PKA and PKC, and controlling their function by regulating when and where in the cell they are activated. We now propose to elaborate on pro-cancer pathways that PKC induces, yet which are normally regulated by SSeCKS. These studies will elucidate how the loss of SSeCKS expression in prostate cancer increases disease progression by allowing PKC to be hyperactivated and dysregulated.

PROJECT DESCRIPTION Synovial cell sarcoma (ScS) and Ewing's sarcoma (ES) are aggressive tumors with high mortality rates in children and adolescents. These cancers are marked by well-established fusion oncogene drivers, SYT- SSX1/2 for ScS and EWSR1-FLI1 for ES, yet because the fusion proteins encode transcriptional regulators and not enzymes, these drivers are poor drug targets and these cancers are considered undruggable. The low 3-year survival rate of these patients, often under 50%, is associated with high incidences of systemic metastatic disease which respond poorly to cytotoxic drugs. The use of histone deacetylate inhibitors (HDACi) promises to increase survival based on targeting the oncogenic genes induced by the fusion oncogene products. Yet, clinical trials with HDACi have had mixed results. Identification of shared drug-sensitive driver pathways that act alone or in concert with HDACi will undoubtedly improve the survival of ScS and CCSST patients. In Aim 1, we plan to identify testable ScS and ES drug sensitivities using a pipeline that combines shRNA/CRISPRi synthetic lethality screening with bioinformatics programs that identify essentiality pathways and cognate drug susceptibilities. We will develop a seamless work pipeline that incorporates i) deconvolution analysis to match barcode (shRNA) or sgRNA clone hits with gene identification, ii) removal of general essentiality genes, iii) optimized pathway identification from dropout gene lists, iv) identification of potential drug candidates and v) in silico prioritization of drug candidates based on existing pharmacogenomic databases. We will identify which essentiality pathways match those downregulated by HDACi, with the assumption that the drugs that target HDACi-independent essentiality pathways might synergize with HDACi against ScS and ES cells (Aim 1b). We will then test the ability of essentiality pathway drugs, alone or in combination with clinically-relevant HDACi, to inhibit ScS and ES growth in vitro and in vivo (Aim 1c). In Aim 2, we will then analyze the transcriptome of clinical ScS samples to determine whether they share expression of the druggable essentiality pathways identified in Aim 1. We will then attempt to develop patient organoid cultures in order to test their sensitivity to pathway essentiality drugs from Aim 1, alone or in combination with HDACi. Taken together, data from this project will allow us in future studies to develop preclinical therapeutic studies, with the goal of increasing ScS and ES patient survival. Our success in this proposal will validate the use of this screening/bioinformatics pipeline to identify drug sensitivities in other undruggable cancers, especially those targeting pediatric populations.