Abstract A62: PARP-1 regulates NF-κB-mediated IL-8 expression in HER2 positive breast cancer

Abstract

Abstract Background: We have previously reported that HER2+ breast tumors are sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi) independent of a basal or induced homologous recombination (HR) repair deficiency. In addition to its DNA repair functions, PARP-1 acts as a coactivator for NF-κB, a tumor-promoting signaling pathway. In this study we investigated the effect of PARP inhibition on trastuzumab resistant HER2+ breast cancer cells as well as the role of PARP-1 in NF-κB-mediated transcription. Methods: Two trastuzumab resistant (TR) HER2+ breast cancer cell lines (BT-474 and UACC-812 TR) were used in these studies. Cell survival was determined by colony formation assays. PARP-1 knockdown was performed using siRNA and results were compared to cells treated with scrambled (SCR) siRNA. Cell proliferation after PARP-1 knockdown was measured using a coulter counter. Gene expression levels of NF-κB regulated genes were measured via the nCounter Gene Expression Assay and qRT-PCR. We also investigated gene expression levels in PARP-1 knockdown cells stimulated with human recombinant TNF-α, a ligand used to induce NF-κB activation. To validate these results, a chromatin immunoprecipitation (ChIP) assay was utilized to detect NF-κB (p65) at the IL-8 promoter in trastuzumab resistant HER2+ breast cancer cells. IL-8 protein expression levels were also determined via an ELISA assay. Results: HER2+ trastuzumab resistant breast cancer cells retained sensitivity to increasing concentrations of PARPi (ABT-888) as compared to their parental counterparts (survival fraction of more than 70% at 10 μM). Cell proliferation was also reduced by 40% after PARP-1 knockdown. NanoString analysis revealed that PARP-1 knockdown had variable effects on NF-κB-regulated genes, including IL-8, BRCA2, NFKBIZ, VEGFC, PIM1, and FASLG. Interestingly, PARP-1 knockdown had the greatest effect on IL-8 and strongly inhibited its gene expression levels by 90% compared to cells treated with SCR siRNA. These results were also validated by qRT-PCR analysis. PARP-1 knockdown also significantly inhibited TNF-α induced IL-8 gene expression (30%) as compared to cells treated with SCR siRNA. Further, there was an increased amount of p65 detected at the IL-8 promoter after TNF-α stimulation, which was then decreased by 30% after PARP-1 knockdown. IL-8 protein expression was also inhibited by 40% in TNF-α treated PARP-1 knockdown cells. Conclusions: Trastuzumab resistant HER2+ breast cancer cells remain sensitive to PARP inhibition. Further, PARP-1 regulates the expression of IL-8, an NF-κB regulated gene. These results suggest that inhibition of the interaction between PARP-1 and the NF-κB signaling pathway may be a potential mechanism behind the sensitivity to PARPi in HER2+ trastuzumab resistant breast cancer cells. Citation Format: Monicka E. Wielgos, Rajani Rajbhandari, Susan Nozell, C. Kent Osborne, Rachel Schiff, Eddy S. Yang. PARP-1 regulates NF-κB-mediated IL-8 expression in HER2 positive breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A62.