Abstract 1662: Therapeutic relevance of the Rad6/translesion synthesis pathway in BRCA1-related triple-negative breast cancer cells

Abstract

Abstract Triple negative breast cancers (TNBCs) comprise ∼15-20% of all breast cancers (BrCas), and their treatment is challenging because they are not treatable with ER, PgR or Her2/neu-targeted therapies. Since TNBCs and BRCA-mutated BrCas share many histopathologic features including aberrant DNA repair pathways, the concept of targeting DNA repair defects with agents such as platinum (Pt) compounds is applied for treatment of TNBC patients. Pt drugs induce DNA replication stalling interstrand DNA crosslinks (ICLs), the repair of which require concerted activities of nucleotide excision repair, Fanconi anemia (FA)/BRCA homologous recombination repair (HRR) and translesion synthesis (TLS) pathways. The therapeutic efficacies of these drugs are often limited by the cancer cell's enhanced ability to repair/tolerate these toxic DNA lesions. The TLS pathway a.k.a DNA damage tolerance pathway is critical for cell survival in the face of DNA damage. TLS is a crucial initial step in ICL repair as it synthesizes DNA across the lesion thus preparing the damaged DNA template for repair by the FA network and HR pathway, processes critical for ICL repair. The Rad6 gene is a principal component of the TLS pathway and previous work in our lab has shown its important role in BrCa development/progression and acquisition of cisplatin (CDDP) resistance. To understand the involvements of Rad6/TLS and FA/BRCA in CDDP-induced DNA damage repair, we evaluated CDDP sensitivities, CDDP-induced DNA damage responses and stalled replication restarts in wt-BRCA1 (MDA-MB-231, MDA-MB-468) and mut-BRCA1 (HCC1937, SUM1315) TNBCs. Data from MTT assays showed no correlation between CDDP sensitivity and BRCA1 status; however, pretreatment with our recently developed Rad6 small molecule inhibitor (SMI) enhanced CDDP sensitivity of all TNBC cell lines regardless of their BRCA1 status. Consistent with these data, immunoblot analysis showed that treatment with the Rad6 SMI attenuated the CDDP-induced PCNA ubiquitinations (a hall mark of Rad6/TLS activity), as well as the steady-state levels of FancD2 (a surrogate marker of FA pathway activation), pol eta (TLS polymerase), and Rad51 (critical for HRR) in both wt-BRCA1 and mut-BRCA1 TNBC cells. IdU/CldU labeling assays showed that Rad6 inhibition mitigated the restart of CDDP-induced stalled replication forks. Whereas Rad6 is expressed weakly in normal breast tissues and overexpressed in BrCas, immunohistochemical analysis showed strong nuclear staining for Rad6 in the normal ducts of mut-BRCA1 clinical breast tissues implicating an important role for Rad6 in mut-BRCA1 BrCas. Our data suggest an important role for the Rad6/TLS pathway in processing Pt-induced ICLs in both wt-BRCA1 and mut-BRCA1 TNBCs and the potential therapeutic value of inhibiting this pathway in TNBCs. Supported by NIH CA178117-01. Note: This abstract was not presented at the meeting. Citation Format: Brittany Haynes, Hend Kothayer, Andrew Westwell, Malathy Shekhar. Therapeutic relevance of the Rad6/translesion synthesis pathway in BRCA1-related triple-negative breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1662. doi:10.1158/1538-7445.AM2015-1662