The objective of this proposal is to elucidate the role of androgen-response gene calreticulin in prostate cancer progression. Calreticulin is an evolutionarily conserved protein with demonstrated functions in cell adhesion and intracellular Ca++ homeostasis. We showed that calreticulin is abundantly expressed and regulated by androgen in prostatic epithelial cells. Further analysis showed that calreticulin indeed regulates cell adhesion and intracellular Ca++ homeostasis in cultured prostate cancer cells. Our recent preliminary data indicate that calreticulin overexpression markedly suppressed anchorage-independent growth of prostate cancer cells in soft agar and that calreticulin expression is down-regulated in clinical prostate cancer specimens. Our research hypothesis is that escape from androgen-dependent growth restriction, via calreticulin down-regulation, is an essential step in prostate cancer progression.
Four specific aims are proposed. 1. Test the hypothesis that calreticulin down-regulation is more frequent in high Gleason grade prostate tumors. Calreticulin expression will be determined by a semiquantitative immunohistochemistry. 2. Determine the functional domains and motifs of calreticulin in inhibiting anchorage-independent growth. Deletion and substitution mutants will be generated to map domain(s) essential for inhibiting anchorage-independent growth of prostate cancer cells. The impact of calreticulin mutants on the growth of PC3 prostate cancer cells in soft agar will be tested. 3. Determine if down-regulation of calreticulin enhances the anchorage-independent growth of prostate cancer cells. Calreticulin down-regulation will be achieved by antisense RNA expression and its effect on anchorage-independent growth examined. 4. Study the role of calreticulin in prostate tumor growth and metastasis in vivo in xenograft tumors. The effect of calreticulin overexpression or down-regulation on the growth and metastasis of subcutaneous and orthotopic xenograft prostate tumors will be studied in nude mice.
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