1. Abstract P3-14-06: Evaluation of the therapeutic efficacy of a Rad6 small molecule inhibitor in triple negative breast cancer cells
- Author
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Y Zhang, Malathy P.V. Shekhar, Andrew D. Westwell, Guangzhao Mao, Jing Li, Brittany Haynes, and Sarah Petit
- Subjects
Cancer Research ,Oncology ,business.industry ,Immunology ,Cancer research ,Medicine ,business ,Small molecule ,Triple-negative breast cancer - Abstract
Triple negative breast cancers (TNBCs) lack estrogen and progesterone receptors and Her2/neu amplification, and are hence not treatable with therapies targeting these molecules. TNBCs have upregulated DNA damage response mechanisms, including the Rad6 postreplication repair (PRR) pathway, that potentially contribute to chemoresistance. Rad6 is a major component of the PRR pathway and its ubiquitin conjugating (UBC) activity is critical for its function. Rad6 expression is low in normal breast cells and tissues but the Rad6 homolog Rad6B is overexpressed in invasive, metastatic and chemoresistant BrCas. Constitutive overexpression of Rad6B in MCF10A cells induces resistance to cisplatin and doxorubicin. TCGA analysis of TNBC patient data showed an association between high Rad6B expression (but not Rad6A) and decreased overall survival. We recently reported the development of a novel Rad6-selective small molecule inhibitor (SMI#9) that inhibits Rad6 UBC activity, migration, and induces apoptosis in TNBC cells but has no effect on MCF10A cells. Since SMI#9 has limited aqueous solubility, in this study we synthesized a modified analog of SMI#9 to enable conjugation via a hydrolyzable ester bond to gold nanoparticle (GNP) and to improve delivery. GNP tethered SMI#9 (SMI#9-GNP) was characterized for purity, ligand conjugation and size by thermogravimetric analysis, atomic force microscopy, transmission electron microscopy, UV-Vis spectroscopy and zeta sizer, and for cellular uptake and drug release by FTIR and mass spectrometry. We compared the activities of SMI#9-GNP and free SMI#9 for cytotoxicity and intracellular localization in mesenchymal (MDA-MB-231 and SUM1315) and basal (MDA-MB-468 and HCC1937) subtypes of TNBC, and in MCF10A cells. Whereas free SMI#9 was cytotoxic to all TNBC cells, SMI#9-GNP demonstrated as good or better cytotoxicity than free SMI#9 only in mesenchymal TNBC cells. MCF10A cells were unaffected by both free and SMI#9-GNP. Consistent with cellular sensitivities, SMI#9-GNP is efficiently endocytosed and processed in lysosomes in mesenchymal TNBC cells, while uptake into basal TNBC cells is compromised by cell microenvironment induced SMI#9-GNP aggregation. SMI#9-GNP treatment induces mitochondrial dysfunction, and stabilization and hyperactivation of PARP-1 that was commensurate with autophagy (indicated by LC3-I to LC3-II conversion). Rad6 loss and PARP-1 hyperactivation are associated with mitochondrial dysfunction, and since inhibition of Rad6 induces both mitochondrial dysfunction and PARP-1 activation this implicates a potential novel role for Rad6 in linking these processes. In summary, our data show that SMI#9-GNP is a suitable delivery vehicle and that the SMI#9 released from GNP conjugate functions similarly as free SMI#9. Our data also illustrate how cell microenvironment induced changes in the physical properties of GNP-drug conjugates can have important implications in the application of nanoparticles in cancer therapy. Supported by NIH R21 CA178117. Citation Format: Haynes B, Zhang Y, Li J, Petit S, Westwell A, Mao G, Shekhar M. Evaluation of the therapeutic efficacy of a Rad6 small molecule inhibitor in triple negative breast cancer cells. [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 P3-14-06.
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- 2016