Abstract 1113: The role of BRCA1 in taxane resistance

Abstract

Abstract The taxanes are among the most effective chemotherapeutic agents used for the treatment of solid tumors, yet eventually most tumors become resistant to taxane chemotherapy. Mechanisms responsible for clinical resistance to taxanes have not been fully elucidated. Breast cancer susceptibility gene 1 (BRCA1) is a tumor suppressor gene with DNA damage repair activity, whose expression has been clearly correlated with taxane sensitivity in many solid tumors including non-small cell lung cancer (NSCLC). However, the molecular mechanism underlying the relationship between BRCA1 expression and taxane activity remains unclear. In addition to its DNA damage repair activity, BRCA1 was shown to ubiquitinate γ-tubulin and affect centrosomal nucleation of microtubules. Here, we test the hypothesis that reduced BRCA1 expression will enhance microtubule nucleation (due to centrosome hyperactivity) which in turn, will produce hyper-acetylated microtubules (since this post-translational modification is specific for and accumulates on the microtubule polymer) making them non-responsive to taxanes. To this end, we developed a lentiviral shRNA-knockdown system of BRCA1 in a NSCLC model cell line, A549. Real-time PCR and Western Blot analyses confirmed 80% BRCA1 knockdown in the A549-BRCA1-KD cell line. Microtubule regrowth experiments following nocodazole treatment with subsequent washout showed an increase in centrosomally-nucleated microtubules (i.e. centrosomal hyperactivity) in the A549-BRCA1-KD cells compared to controls. These experiments also showed that the large majority of the microtubules that regrew were acetylated. Western Blot analysis showed that A549-BRCA1-KD cells indeed contain increased Ac-Tubulin content compared to controls. Interestingly, analysis of 155 NSCL patient samples from the randomized Neoadjuvant/Adjuvant Taxol Carboplatin Hope (NATCH) trial revealed that patients with high basal Ac-Tubulin in the adjuvant arm had a significant shorter time to progression suggesting that Ac-Tubulin content is a negative predictor for taxane efficacy. To assess taxane sensitivity in the A549-BRCA1-KD cell line, we treated cells for 72 hrs with paclitaxel and used two different cell viability assays, Cell-Titer Glo and Sulforhodamine B. Both showed A549-BRCA1-KD cells have a modest resistance (2-3 fold) to paclitaxel treatment, recapitulating the clinical data. Currently, we are correlating BRCA1 expression levels with Ac-Tubulin content in the NATCH patients as well as developing an siRNA screen to assess signaling pathways that modulate taxane sensitivity in the BRCA1-KD model. Elucidation of how lung cancer cells become resistant to taxanes will provide us with insight into how to potentially reverse these resistance mechanisms and enhance therapeutic efficacy. (Supported by NCI 5P01CA116676 and T32CA062948) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1113.