Chromosomal and genomic abnormalities affecting chromosome 16q have frequently been reported in cytogenetic and allelotypic studies of various epithelial tumors. We recently cloned and described WWOX, a gene spanning a genomic region of more than 1 MB located in ch. 16q23.3-24.1. This specific region is the most frequent target for LOH affecting 16q in breast, ovarian, prostate and other tumors. Others and we demonstrated that this is the very same region as that of the common chromosomal fragile site 16D (FRA16D). Translocation breakpoints common in multiple myeloma are also found within the WWOX genomic region. We determined that one of the WWOX alleles is inactivated very early in breast carcinogenesis and the vast majority of primary breast tumors and breast cancer lines display this abnormality. Homozygous deletions affecting WWOX exons were detected in ovarian carcinoma, and mutations affecting WWOX were reported in esophageal carcinoma. Recently, the occurrence of aberrantly and other cancer lines spliced forms affecting WWOX mRNA have also been reported to occur in multiple tumor types. In summary, evidence from various sources indicate that WWOX is a gene commonly affected at the genomic and transcriptional level in various tumor types. Recently, we demonstrated that ectopic WWOX expression strongly inhibits anchorage-independent growth of breast cancer cell lines, and induces a dramatic inhibition of tumorigenicity of the most aggressive breast cancer cell lines. We speculate that the normal cellular function of WWOX may be affected by various different mechanisms. Our hypothesis is that WWOX is an important cancer gene that can behave as a suppressor of tumor growth and that abnormalities affecting this gene at the genomic and transcriptional level are relevant in carcinogenesis. Thus, we propose in Aim 1: To generate mouse strains containing WWOX disrupted alleles. We will A) generate a conditionally targeted WWOX mutant allelic series utilizing the cre-lox and tip-fit recombinase system and B) generate mice expressing a WWOX-LacZ fusion protein product of disruption of the endogenous WWOX alleles. This truncated WWOX form will be similar to that produce in various tumor types.
Aim 2 : To identify WWOX protein domains important for tumor inhibition and cellular localization. To this end mutant WWOX forms will be created affecting the WW domains and critical portions of the oxidoreductase enzymatic domain.
Aim 3 : To identify cellular proteins that interact with WWOX. Various candidate in vitro WWOX binding proteins containing the matching PPXP motifs have been already identified and cloned. Various approaches will be followed to define the true in vivo interacting proteins.
Aim 4 : To determine patterns and levels of WWOX protein expression in breast and ovarian carcinomas assessing its relevance as a prognostic tool.
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