The objective of this revised proposal is to elucidate the anti- apoptotic mechanism of clusterin in prostate cancer. Clusterin has many names, including sulfated glycoprotein-2 (SGP-2), apolipoprotein J (Apo-J), complement lysis inhibitor (CLI), testosterone repressed prostate message-2 (TRPM-2), etc. It is a ubiquitous secretory protein with multiple proposed functions. Our data demonstrate that the apoptotic effects of TNF-alpha (tumor necrosis factor-alpha) and TGF-beta (transforming growth factor-beta) on prostate cancer cells can be blocked by extra- cellular levels of clusterin. Furthermore, the level of clusterin expression correlates with the degree of clinical aggressiveness in human prostate cancer. These exciting observations offer new insights in tumor biology and add a new function for clusterin. Our hypothesis states that clusterin is a natural anti-apoptotic mediator in cancer cells and that its expression can be manipulated.
Five specific aims are proposed.
Aim 1 will study the interaction between clusterin and TNF-alpha. Clusterin prevents TNF-alpha induced apoptosis. We shall determine if clusterin action is mediated through extra- or intra-cellular components of prostate cancer cells. PC3 and LNCaP cells will be used as targets.
Aim 2 will study the interaction between clusterin and TGF-beta. Clusterin also prevents TGF-beta apoptosis. Unlike TNF-alpha, TGF-beta not only induces apoptosis but also inhibits proliferation in target cells. PC3 and TSU-Pr1 cells will be used as targets.
Aim 3 will study the binding characteristics of clusterin in prostate cancer cells. We propose to test if there is a high affinity, low capacity binding site for clusterin in prostate cancer cells. The presence of such a site would suggest the presence of specific receptors and the possibility of an intra-cellular involvement.
Aim 4 will determine the functional domains of clusterin. Our hypothesis favors the need of specific structural domains for clusterin function. We propose to use a PCR program to construct expression vectors of putative domains of clusterin and transfect them into cancer cells.
Aim 5 will study the in vivo effect of clusterin. We propose to use subcutaneous as well as orthotopic xenograft tumors in nude mice as the testing system. Expression constructs will be used to regulate clusterin expression, which will be correlated with the status of tumor progression.
