Supplementary Figure S1. BRCA1 mutation confirmation and RAD51 gene expression in SUM149 cells. Supplementary Figure S2. RAD51 foci is associated with CSC population of BRCA1-mutant TNBC cells and effect of long-term PARP inhibition on RAD51. Supplementary Figure S3: Knockdown of RAD51 prevents foci formation in SUM149 and SUM159 cells. Supplementary Figure S4. The effect of RAD51 KD on TNBC viable cell count, CSCs, and cell cycle. Supplementary Figure S5. RAD51 KD sensitizes TNBC CSCs to PARPi. Supplementary Figure S6. Efficiency of RAD51 shRNA knockdown with additional shRNA vectors. Supplementary Figure S7. Knocking down RAD51 with two additional shRNA vectors also sensitizes CSCs to PARPi. Supplementary Figure S8. "Physiologic" RAD51 knockdown decreases cancer stem cells in SUM149 cells.
ARTICLE ABSTRACT
Introduction: PARP inhibitors have shown promising results in early studies for treatment of breast cancer susceptibility gene (BRCA)–deficient breast cancers; however, resistance ultimately develops. Furthermore, the benefit of PARP inhibitors (PARPi) in triple-negative breast cancers (TNBC) remains unknown. Recent evidence indicates that in TNBCs, cells that display “cancer stem cell” properties are resistant to conventional treatments, mediate tumor metastasis, and contribute to recurrence. The sensitivity of breast cancer stem cells (CSC) to PARPi is unknown.Experimental Design: We determined the sensitivity of breast CSCs to PARP inhibition in BRCA1-mutant and -wild-type TNBC cell lines and tumor xenografts. We also investigated the role of RAD51 in mediating CSC resistance to PARPi in these in vitro and in vivo models.Results: We demonstrated that the CSCs in BRCA1-mutant TNBCs were resistant to PARP inhibition, and that these cells had both elevated RAD51 protein levels and activity. Downregulation of RAD51 by shRNA sensitized CSCs to PARP inhibition and reduced tumor growth. BRCA1–wild-type cells were relatively resistant to PARP inhibition alone, but reduction of RAD51 sensitized both CSC and bulk cells in these tumors to PARPi treatment.Conclusions: Our data suggest that in both BRCA1-mutant and BRCA1–wild-type TNBCs, CSCs are relatively resistant to PARP inhibition. This resistance is mediated by RAD51, suggesting that strategies aimed at targeting RAD51 may increase the therapeutic efficacy of PARPi. Clin Cancer Res; 23(2); 514–22. ©2016 AACR.
