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TNBCs account for 15-20% of all breast and are unsuitable for common targeted therapies as they lack expression of estrogen, progesterone, and Her2/neu receptors. Cisplatin, a DNA damaging agent, is currently under clinical investigation for treatment of TNBCs and paclitaxel, a mitotic spindle poison, is a first-line treatment option for this disease. While both agents elicit therapeutic benefit BRCA1 mutation status, toxicity, and resistance are limiting factors. Rad6 functions as an E2 ubiquitin conjugating protein and its enzymatic activity is critical for its cellular function. Rad6 is known to play a critical role in the translesion synthesis (TLS) damage response pathway and we have shown constitutive Rad6 overexpression induces aneuploidy and multiple mitotic spindles. Our laboratory has recently reported the development of a novel Rad6 small molecular inhibitor, SMI#9, shown to inhibit Rad6 enzymatic activity and elicit cytotoxic affects in TNBC cells while having no effect on normal behaving MCF10A breast cells. In this dissertation, we show Rad6 is overexpressed in both BRCA1 wild type and BRCA1 mutant TNBCs. Data presented here show that treatment with SMI#9 enhances sensitivity and reverts resistance of TNBC cells to both cisplatin and paclitaxel, regardless of BRCA1 status. We show that SMI#9 mediated cisplatin sensitivity is associated with inhibition of the Rad6 TLS and attenuation of cisplatin-induced activation of FA network and HR repair pathway, implicating a central role for Rad6 in coordinating activities of these pathways. We also demonstrate that SMI#9 induced paclitaxel sensitivity corresponds with monopolar mitotic spindle formation and mitotic catastrophe, establishing a role for Rad6 in centrosome duplication/segregation and/or mitotic spindle nucleation. To overcome SMI#9 solubility limitations, we conjugated SMI#9 to gold nanoparticles. We present data to show similar efficacies and cellular responses in TNBC cells between unconjugated and gold nanoparticle conjugated SMI#9. These data implicate Rad6 as a potential regulator of TNBC drug response and provide support for the therapeutic benefit of SMI#9.
Haynes, Brittany, "Functional Analysis Of Rad6 In Brca1 Wild Type And Brca1 Mutant Triple Negative Breast Cancer Drug Response" (2017). Wayne State University Dissertations. 1807.
Available for download on Thursday, November 22, 2018