The molecular complexity of breast cancers and therapy-associated side effects often limit effectiveness of many therapies, warranting the development of new agents for specific molecular targets while minimizing the off-target effects. Our goal is to develop novel, safer, and effective human breast cancer (HBC) therapies by exploiting functions of an apoptosis regulatory protein CCAR1/CARP-1. CARP-1 functions to transduce cell growth as well as chemotherapy (adriamycin, etoposide, or Iressa)-dependent inhibitory signaling. CARP-1 expression correlates inversely with HBC tumor grades. A yeast-two-hybrid (Y2H) screen revealed that CARP- 1 binds with the E3 ubiquitin ligase subunit anaphase promoting complex (APC)-2 protein. Following mapping of the interacting epitopes of CARP-1 and APC-2, we developed a fluorescence polarization assay (FPA) and screened a chemical library to identify small molecule inhibitors (SMIs) of CARP-1:APC-2 binding. The lead SMI termed CARP-1 Functional Mimetic (CFM)-4, inhibits HBC growth in vitro and in vivo. CFM-4 also attenuates growth of tamoxifen (TAM) or adriamycin (ADR)-resistant HBC cells but does not suppress growth of the non-tumorigenic and immortalized MCF-10A cells. CFM-4 binds with CARP-1, leads to increased CARP- 1 levels, activates pro-apoptotic p38 mitogen-activated protein kinase (MAPK), caspases-9 and 3, and apoptosis. CFM-4 induces loss of mitotic cyclin B1 and Cdc20 proteins, possibly through caspase-dependent but ubiquitin proteasome pathway (UPP)-independent mechanism(s). Hypothesis: CARP-1, a peri-nuclear phospho-protein, functions to regulate HBC cell growth and apoptosis, and is a key regulator of ADR signaling. We further hypothesize that CFM-4 stimulation of CARP-1 levels will provide a novel mechanism for treating breast and other cancers. Objectives: Our long-term objective is to elucidate mechanisms of CARP-1-dependent HBC growth inhibition, and to exploit this knowledge to develop agents for targeting of HBC. The rationale for this objective is based on the facts that loss of tumor suppressor p53 promotes development of aggressive HBCs and that resistance to ADR continues to be a problem. Since CARP-1 regulates HBC growth inhibition by ADR and is a co- activator of p53, the CFMs that bind and elevate CARP-1, activate pro-apoptotic p38 MAPK and caspases, and suppress Cdc20 and cyclin B1 levels have potential to target HBCs including those that have mutant p53 as well as have resistance to ADR or TAM. We will test our hypothesis by pursuing the following objectives. (1) To investigate molecular mechanism(s) of p38 activation, and the extent to which CARP-1 binding with p38 regulates HBC growth in the presence of CFM-4 or ADR. (2) To elucidate mechanism(s) of CARP-1-dependent activation of caspases-9 and -3, and determine the extent to which activated caspases target Cyclin B1 in the presence of CFM-4. (3) To conduct pharmacokinetic (PK) profiling and to demonstrate the anti-tumor activities of CFM(s) in mouse xenograft models of HBC. Potential Impact on Veterans Health Care: This investigation will facilitate development of strategies to prevent/combat breast cancer that will benefit women in the VA workforce, contribute to Veterans Health Care and further the mission of the VA.
We are proposing to study molecular mechanisms of human breast cancer growth suppression by a cellular phospho-protein CARP-1/CCAR1 that was previously identified and characterized by us. CARP-1 functions to regulate chemotherapy (adriamycin or iressa)-dependent growth inhibition of human breast cancer (HBC) cells. We have performed additional studies and identified novel, small molecule inhibitors (SMIs) of HBC cell growth that function in part by inducing CARP-1 and HBC cell death/apoptosis. Thus, knowledge of the mechanisms of HBC growth suppression by CARP-1 could be exploited to identify and develop anti-HBC SMIs for their potential utility as therapeutics against breast and possibly other cancers.
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