Mechanisms of Cancer Initiation by TRIM32

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PROJECT SUMMARY Trim32 is an E3-ubiquitin ligase and a member of a family of scaffold proteins involved in both cancer and human development. We made the novel association of Trim32 activation with cancer through carcinogenesis studies in mouse keratinocyte models and have preliminarily confirmed this association in human squamous cell carcinoma (SCC). We have identified Piasy as a substrate protein targeted for degradation by Trim32 ubiquitylation, providing the first evidence for how Piasy is regulated in cells. Others have established that Piasy is a pro-apoptotic E3-SUMO ligase involved in regulation of tumor suppressor p53 and of NFkB cellular survival pathways. Piasy's role in cancer is evident as it is targeted for inactivation by the high-risk HPV-E6 protein implicated in human cervical carcinoma, and its downregulation is prognostic for conversion from myelodysplastic syndrome (MDS) to later stages and chronic myeloid leukemia (CML). Piasy is a pro- apoptotic factor, inducing apoptosis in CML cells, human fibroblasts and mouse keratinocytes. We show that Trim32 protects keratinocytes from apoptosis induced by UVB and synergized by TNFa. Moreover, we find an inverse correlation between Piasy and Trim32 expression levels in human SCC. These data suggest a direct role of Trim32 activation and Piasy degradation in epithelial carcinogenesis and tumor progression. We propose to define Trim32/Piasy interactions in intracellular signaling, carcinogenesis and human SCC according to the following aims: 1) define the dynamic regulation of Piasy subcellular localization by the Trim32 E3 ligase scaffold protein and the role of these interactions in survival pathways in epidermal keratinocytes; 2) examine the role of Trim32 and Piasy in initiation, promotion and malignant progression in keratinocytes during epidermal carcinogenesis; 3) explore the predictive value of Trim32/Piasy status in human skin and head and neck SCC prognosis as a basis for future strategies for molecular targeting of Trim32/Piasy interactions or downstream effectors in cancer. The expression of Trim32 and Piasy beyond epithelia, such as in hematologic, muscle and neurological tissues and the presence of Trim32 inactivating mutations in two human hereditary syndromes imply broader significance of understanding Trim32 and Piasy regulation in human disease. PROJECT NARRATIVE Trim32 and Piasy: Two enzymes with opposing roles in cancer development Trim32 is a ¿scaffold¿ protein that controls the timing and location of other proteins that must interact appropriately to cause normal tissue development and that, if abnormal, can lead to cancer. We have shown that Trim32 adds a small molecule ¿ubiquitin¿ and destroys Piasy (an enzyme that is needed for tumor suppression and cell survival mediated by important gene regulators p53, NFB, STAT and Smad/TGFbeta), providing the first evidence for how Piasy is regulated in cells. Understanding how the very earliest changes detectable in cancer, such as Trim32 activation, extend abnormal cell survival and promote further changes (activation of cancer genes and inactivation of tumor suppressor genes) provides a basis for novel selective, less toxic molecular-targeted prevention and therapy of skin cancers and perhaps cancers from other tissues where Trim32 and Piasy are expressed, such as chronic myelogenous leukemia, muscle and brain tumors.