Breast cancer (BrCa) is the second leading cause of cancer mortality in women in the U.S., and triple negative breast cancers (TNBCs) comprise ~15-20% of these cancers. Treatment of TNBC poses a clinical challenge because they are not treatable with therapies targeting estrogen receptor and Her2/neu as they lack expression of estrogen, progesterone, and Her2/neu receptors. TNBCs are associated with a shorter time to recurrence and death. Approximately 70% of BrCas in individuals carrying a germline BRCA1 mutation are triple negative, and the incidence of BRCA1 mutations in TNBC ranges from 16-42%. BRCA1-associated BrCas have aberrant DNA repair, and since TNBCs share several histologic features with BRCA1-related BrCa, DNA repair pathways are thought to play a significant role in TNBC development and therapy response. In the U.S., there has been a shift to treating TNBCs with platinum (Pt)-based therapies. Pt compounds cause DNA crosslink breaks (ICLs). Repair of ICLs require activities of BRCA/Fanconi anemia (FA) network and postreplication DNA repair (PRR) pathways. PRR pathway confers tolerance to DNA damage by enabling cells to complete DNA replication in the face of damage in order to avoid mitotic catastrophe, and can be error-free or error-prone. The mechanisms contributing to progression and therapy resistance in TNBC are not well understood and no key targets useful for prevention and treatment have thus far been identified. In this application, we propose that targeting Rad6, a principal component of the PRR pathway, will be beneficial to TNBCs treated with Pt by preventing acquisition of resistance and overcoming Pt resistance. The Rad6 gene encodes an ubiquitin (Ub) conjugating enzyme, and its catalytic activity is essential for PRR function. Rad6B is overexpressed in BrCa. Constitutive overexpression of Rad6B in normal breast cells induces aneuploidy and cisplatin (CDDP) resistance, whereas Rad6B suppression confers CDDP sensitivity. This relationship between Rad6 expression and CDDP sensitivity is directly related to PRR activity. Whether the PRR activity is error-prone or error-free is dependent upon whether PCNA is mono- or poly-ubiquitinated, respectively, by Rad6. Rad6 also regulates FA pathway activation by promoting FancD2 ubiquitination, a critical event for ICL repair. We have identified a small molecule inhibitor (SMI) of Rad6 that targets its Ub conjugating activity. Treatment of MDA-MB-231 TNBC cells with Rad6 SMI attenuates CDDP-induced PCNA and FancD2 ubiquitination, and enhances CDDP sensitivity. We hypothesize that Rad6 is a major player in stimulation of Pt-induced FA/BRCA repair pathway. We propose that inhibiting Rad6 will sensitize TNBCs to Pt therapy by inactivating PRR and consequent disruption of PRR crosstalk with the FA pathway. We will test this hypothesis with the following two specific aims: (1) Determine the functional role of Rad6 in ICL repair in BRCA1 wild type and BRCA1 mutant TNBC cells. (2) Determine the therapeutic utility of Rad6 intervention in treatment of BRCA1 wild type and BRCA1 mutant TNBC cells using in vivo and a novel in vitro three-dimensional culture platform.
Treatment of triple negative breast cancer poses a major clinical challenge because they are not treatable with estrogen receptor or Her2/neu targeted therapies as they lack estrogen, progesterone and Her2/neu receptors. Triple negative breast cancers are associated with early and high relapse rates and poor survival rates. Intensive efforts are made to identify and develop new therapeutic targets and strategies for addressing this clinical problem. Our proposed studies will: (1) provide mechanistic and preclinical insights into the role of Rad6 in repairing platinum therapy induced DNA damage lesions, (2) test the therapeutic efficacy of a novel small molecule inhibitor of Rad6, and (3) validate Rad6 as a novel druggable target for treating triple negative breast cancers.
|Rosner, Karli; Mehregan, Darius R; Kirou, Evangelia et al. (2014) Melanoma Development and Progression Are Associated with Rad6 Upregulation and ? -Catenin Relocation to the Cell Membrane. J Skin Cancer 2014:439205|
|Rosner, Karli; Adsule, Shreelekha; Haynes, Brittany et al. (2014) Rad6 is a Potential Early Marker of Melanoma Development. Transl Oncol :|