MOLECULAR CHANGES DURING PROSTATE CARCINOMA PROGRESSION

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The transition from prostatic intrepithelial neoplasia (PIN) to invasive carcinoma is characterized by a loss of two major regulatory proteins, beta4 integrin and its ligand, laminin 5. We hypothesize that the mechanism for the loss of laminin 5 is the presence of a post-transcriptional defect. The failure of laminin 5 assembly causes instability of beta4 which is lost due to degradation. The loss of these regulatory proteins destabilizes the normal signaling pathway of the cells contributing to prostate tumor progression. We will examine this hypothesis by using the LNCaP cell line which, like the primary prostate carcinomas, expresses the three laminin subchain mRNAs but fails to assemble the fully functioning heterotrimeric laminin 5 protein. In Aim 1 we will examine the three laminin 5 subchain mRNAs for mutations by sequencing fragments of the cDNA products produced by RT-PCR. The mRNAs will also be tested for their in vitro translational capacity. In Aim 2 we will test the effect the purified laminin 5 on the synthesis, degradation, and cytoplasmic membrane stability of beta4 integrin. We will also study the ability of purified laminin 5 to cause signal transduction. These experiments will utilize pulse labeling or surface biotinylation followed by immunoprecipitation, and in the cause of signal transduction, Western blotting with anti-phosphotyrosine, Sh2, and GrB2 specific antibodies. The functional effect of laminin 5 on prostrate cell growth, adhesion, migration, and tumorigenicity will also be studied in Aim 3. These studies will be carried out using quantitative assays of adhesion and migration established in our laboratory. In Aim 4 we will test the clinical relevance of the persistence of alpha 6 and alpha 3 integrins in the absence of beta4.