Abstract A19: CCR5 contributes to breast cancer stem cell expansion by enhancing DNA damage repair

Abstract

Abstract Tumors are highly heterogeneous with a few cells capable of generating a new tumor referred to as cancer stem cells. Cancer stem cells contribute to metastasis and therapy resistance (chemotherapy, radiation). Therapy resistance in some circumstances is attributable to induction of the DNA repair machinery. Herein the role of endogenous CCR5 in cancer stem cells was assessed in the SUM159 basal breast cancer cell line. By FACS analysis, 1-10% of the SUM159 cells were CCR5+. CCR5+ cells formed more mammospheres, a surrogate measurement of progenitor cell expansion. CCR5+ SUM159 cells preferentially gave rise to tumors when injected subcutaneously. The tumors formed by CCR5+ cells were >40 times larger. When CCR5 was overexpressed in CCR5- cells and injected subcutaneously to the mice, the tumors were ten-thousand times larger than vector-control cells. In order to characterize the functional pathways regulated by CCR5, total RNA was prepared from both SUM159 CCR5+ and CCR5- cells isolated by FACS sorting. Microarray analysis showed that 321 genes were differently regulated in CCR5+ cells vs CCR5- cells. GOTERM_BP_FAT analysis revealed 18 Gene Ontology pathways were regulated by CCR5 including “DNA repair” and “response to DNA damage stimulus”. Genes elated to DNA repair (FANCB, LIG3, POLE and CRY1) were confirmed by qtPCR. Histone H2AX phosphorylation at Serine 139 (p-γH2AX) serves to recruit proteins that sense or signal the presence of DNA damage and can be used as surrogate marker of DNA damage/repair. SUM159 cells stably transfected with CCR5 or control vector were compared for the DNA damage/repair response. γ-radiation of SUM159 cells induced p-γH2AX, however CCR5-overexpressing cells showed reduced p-γH2AX at 24 hours. Treatment of SUM159 cells with the DNA intercalating anthracycline doxorubicin induced γH2AX phosphorylation, and CCR5-overexpressing cells showed less p-γH2AX than its control with 24 hours treatment. The DNA repair reporter, DR-GFP is used to measure homology-directed DNA repair (HDR). In order to measure HDR activity CCR5+ SUM159 cells, the cells were co-transfected with the plasmid encoding I-SceI and the I-SceI based DNA repair reporter DR-GFP and stained with APC labeled anti-CCR5 antibody. GFP+ cells, generated by HDR of I-SceI induced double-strand DNA, were sorted by FACS into CCR5- and CCR5+ populations. The percentage of DR-GFP+ cells was increased in CCR5+ or CCR5-overexpressing cells compared with CCR5- or vector control cells. Together these studies demonstrate that CCR5+ breast cancer cells possess cancer stem cell-like properties and have increased DNA damage repair activity. CCR5 promote stem cell expansion and breast cancer tumor formation through enhancing DNA damage repair. CCR5 antagonists were approved by the FDA for HIV treatment. These results suggest potential clinical value of these compounds for breast cancer metastasis management. Citation Format: Xuanmao Jiao, Marco Velasco, Zhiping Li, Shaohua Xu, Massimo Cristofanilli, Hallgeir Rui, Richard G. Pestell. CCR5 contributes to breast cancer stem cell expansion by enhancing DNA damage repair. [abstract]. In: Proceedings of the Fourth AACR International Conference on Frontiers in Basic Cancer Research; 2015 Oct 23-26; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2016;76(3 Suppl):Abstract nr A19.