TRIM32, a gene initially detected by binding to HIV tat transcription factor, was found upregulated at the mRNA and protein level in independent chemically initiated cell lines of a clonal in vitro/in vivo mouse epidermal carcinogenesis model. These initiated cells give rise to lineage-specific tumorigenic fates of papilloma or squamous cell carcinoma when transplanted to athymic nu/nu mice, and tumorigenic cells continue to express Trim32, suggesting that it offers an advantage to malignant cells. In normal tissues TRIM32 is expressed at high levels only in brain. TRIM32 (human, Trim32 mouse) belongs to the tripartite motif (TRIM) family of proteins that contain zinc finger RING and B-box domains followed by a coiled-coil domain, and to date comprise 37 members. Several members have been implicated in human diseases, including cancer. While little is known about TRIM protein biological and molecular functions, proteins containing RING domains including TRIM family member PML have been found to be E3-ubiquitin ligases. A role for Trim32 in cancer is supported by our finding that transduced Trim32 cDNA causes in vitro initiation of non-transformed epidermal keratinocytes. Transplantation of these cells to athymic nu/nu mice resulted in an epidermal phenotype of keratinizing cysts and a thickened epidermal lesion resembling Bowen's disease, a human squamous cell carcinoma in situ found on sun-exposed skin. The same cells are resistant to the synergistic effects of TNFalpha and UVB light-induced apoptosis in vitro. Furthermore, analysis of mouse sporadic skin tumors revealed that Trim32 protein expression was elevated in all UVB-induced tumors and a subset of chemically induced tumors analyzed so far and analysis of human head and neck squamous cell carcinomas revealed 5/7 with at least 2 fold elevated TRIM32 mRNA. Our underlying hypothesis is that the TRIM32 gene encodes an E3-ubiquitin ligase that regulates TNFalpha cellular signaling to foster an initiated state in epidermis, favoring survival rather than differentiation or apoptosis. The proposed studies are directed toward determining specific mechanisms of action of TRIM32 in vivo responsible for its role in cancer development.
The aims of this proposal are to: 1) Establish the role of Trim32 in tumorigenesis and apoptosis using skin-grafted keratinocytes and Trim32 transgenic mice, 2) Determine the mechanism of Trim32 inhibition of TNFalpha/UVB-induced apoptosis, 3) Test Trim32 activity as an E3-ubiquitin ligase, and 4) Establish the relevance of TRIM32 to human squamous cell tumors. Trim32 protein activities in initiation and malignancy may provide a new molecular identifier of initiated epithelium and leads for reversing survival mechanisms in precancers and malignant cells.
