Despite recent advances in clinical management, the molecular complexity of triple-negative breast cancers (TNBC) and therapy-associated side effects often limit effectiveness of many therapies. Development of new and improved strategies for TNBC treatment remains urgent. We previously discovered an apoptosis regulatory protein CARP-1/CCAR1. CARP-1 functions to transduce cell growth as well as chemotherapy (adriamycin, etoposide, or Iressa)-dependent inhibitory signaling. CARP-1 expression correlates inversely with breast cancer tumor grades. A yeast-two-hybrid screen together with co-immuno-precipitation analyses revealed that CARP-1 binds with the anaphase-promoting complex (APC/C) E3 ubiquitin ligase subunit APC-2, cytoskeletal scaffold filamin C , and apoptosis-transducing DEDD2 proteins. High-throughput screening of a chemical library yielded inhibitors of CARP-1:APC-2 binding termed CARP-1 functional mimetics (CFMs). Lead compound CFM-4 binds CARP-1, stimulates CARP-1 expression and apoptosis. CFM-4 inhibits TNBC cell growth in vitro and in vivo. CFM-4 also attenuates growth of tamoxifen (TAM) or adriamycin (ADR)-resistant breast cancer cells without inhibiting growth of the non-tumorigenic and immortalized mammary epithelial MCF-10A cells. Our on-going studies further revealed that (1) CARP-1 is a part of filamin/c-jun N-terminal kinase (JNK) proteome, and JNK2 regulates apoptosis by ADR or CFM-4, (2) CFM-4.16, a novel CFM-4 analog, enhances ADR inhibition of TNBC cells, (3) CFM-4 and its analogs function in part by elevating CARP- 1 and DEDD2 levels, and promote apoptosis by activating JNKs and caspases-8/9/3, and attenuate TNBC cell migration and invasion, and (4) CARP-1 depletion blocks breast cancer cell growth inhibition by CFMs or ADR. Hypothesis: CARP-1, a peri-nuclear phospho-protein, is a key regulator of apoptosis by ADR or CFMs, and that modulation CARP-1 and its signaling by these agents is a novel mechanism for treating TNBCs and other breast cancers. Objectives: Elucidation of mechanisms of CARP-1-dependent breast cancer growth inhibition, and exploitation of this knowledge to develop anti-breast cancer agents are our long-term goals. Loss of p53 promotes development of aggressive breast cancers and development of ADR-resistant TNBCs remains a formidable problem in clinic. The facts that CARP-1 is a co-activator of p53 while CFMs that bind and elevate CARP-1 also inhibit ADR-resistant breast cancer cells, apoptosis-stimulating functions of CARP-1 could compensate for loss of p53, especially in TNBCs and their drug-resistant variants that lack functional p53. Together with our brief findings listed above, the current preliminary studies provide a strong rationale to test our hypothesis by: (1) Investigating molecular mechanism(s) of filamin-CARP-1 binding, and determining the extent to which JNK/CARP-1-dependent apoptosis regulates TNBC growth for optimal efficacy of ADR or CFMs. (2) Elucidating mechanism(s) CARP-1 interaction with DEDD2, and determining the extent to which this interaction regulates activation of caspases-8/10, and contributes to apoptosis by CFMs or ADR. (3) Determining whether CARP-1-dependent JNK/Caspase-8 signaling regulates TNBC growth in vivo following administration of nano-lipid formulations of CFM analogs alone or in combination with ADR. Potential Impact on Veterans Health Care: The proposed studies will yield valuable knowledge to facilitate development of novel and effective strategies to combat breast cancer that will benefit female VA workforce, contribute to Veterans Health Care and further the mission of the VA.
We are proposing to continue our studies investigating 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 regulates chemotherapy (adriamycin or iressa)-dependent growth inhibition of human breast cancer (HBC) cells. We recently identified novel, small molecule inhibitors (SMIs) of HBC cell growth that function in part by inducing CARP-1 and apoptosis. These CARP-1 functional mimetic (CFM) compounds inhibit growth of Adriamycin-resistant HBC cells without suppressing growth of immortalized, mammary epithelial MCF-10A cells. Loss of CARP-1 prevents apoptosis by Adriamycin or CFMs. CFM-4 analog, CFM- 4.16 enhances Adriamycin-dependent inhibition of triple-negative breast cancer (TNBC) cells. Thus, knowledge of the mechanisms of HBC growth suppression by CFM analogs and CARP-1 could be exploited to enhance Adriamycin efficacy and effectively target Adriamycin-resistant breast cancers in the clinic.
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