Abstract P3-06-12: Are specific interactions of proteasome with Hsp72 responsible for resistance of triple negative breast cancer cells to proteasome inhibitors?

Proteasome, the essential giant protease, is the hub of the ubiquitin-proteasome pathway critical for maintaining intracellular proteostasis. Inhibitors of the proteasome cause overload of cells with non-degraded protein debris and activate proapoptotic signaling. The effects are especially dramatic in fast metabolizing cancer cells; hence the proteasome inhibitors are used as anti-cancer drugs. There are two FDA-approved drugs directly blocking the proteasome activity, bortezomib and carfilzomib, and several others are in trials. So far, the high effectiveness of these drugs is limited to multiple myeloma and other hematological malignancies. Unfortunately, breast and other solid cancers are much more resistant to apoptosis triggered by attenuation of the ubiquitin-proteasome pathway than majority of blood cancers. The relatively low effectiveness of proteasome inhibitors has been noted even for cancers that have been identified in genome-wide screens as addicted to the proteasome activity, such as triple negative breast cancers. The source of the resistance, noted in cell culture, animal studies and clinical trials, remains unknown. Here we propose that, at least in part, the resistance can be tracked down to protein-protein interactions between the drug target, the proteasome, and a cytosolic heat shock protein, Hsp72 (inducible Hsp70). Indeed, high levels of Hsp72 have been observed in many cancers, including breast, and generally correlate with poor prognosis. The intracellular roles of the ubiquitous chaperone are undoubtedly very diverse, however we postulate a novel molecular action: direct protection of the proteasome from competitive inhibitors of its enzymatic activity. The hypothesis has been inspired by our recent work exploring the very effective ubiquitin-proteasome pathway in the naked mole rats. These small African rodents do not develop cancers and maintain unusually long lifespan and health-span. Interestingly, the proteasome in naked mole rats is exceptionally resistant to inhibition. We discovered that the resistance is bestowed by a cytosolic protein factor containing Hsp72 and Hsp40 chaperones (Biochim. Biophys. Acta-MBD 1842; 2060-2072; 2014). We partially purified the factor and tested its in vitro effectiveness with human proteasomes. The molecular mechanism beneficial for naked mole rats may be also protective for human cancer cells. We used pifithrin μ, a specific small molecule inhibitor blocking the interactions of Hsp72 with other proteins, to test the hypothesis. We prepared lysates from cultured triple negative breast cancer cells MDA-MB-231. Such lysates are rich in proteasome activity and preserve interactions of the proteasomes with natural protein ligands. Interestingly, upon the treatment with pifithrin μ proteasomes in lysates maintained their high activity while becoming significantly more sensitive to inhibition with bortezomib. The studies are in progress to assess the identity of a putative chaperone-based proteasome resistance factor in breast cancer cells. Citation Format: Gaczynska M, Osmulski PA. Are specific interactions of proteasome with Hsp72 responsible for resistance of triple negative breast cancer cells to proteasome inhibitors?. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-06-12.