Abstract P4-06-02: Identification of novel G-protein coupled receptor targets in HER2-positive breast cancer.

Background: HER2 is a member of human epidermal growth factor receptor (HER) superfamily and HER2-overexpressing (HER2+) breast cancer (BC) is an aggressive tumor. Despite the clinical success of anti-HER2 therapies, de novo and acquired drug resistance occur in many patients. Identification of novel drug targets to overcome anti-HER2 therapy resistance is an unmet need. Since G-protein coupled receptors (GPCRs) are known to cross-talk with the HER superfamily, it is possible that some GPCRs may signal to modulate the HER2 pathway. GPCRs are considered excellent drug targets due to their plasma membrane localization, unique ligand-binding pocket, and availability of high throughput assays for drug screening. The expression and function of the majority of GPCRs are largely unknown in HER2+ BC. The goal of this study was to identify novel GPCR targets in HER2+ BC, in the context of anti-HER2 therapy resistance. Methods: We examined the differential GPCRs expression in BC stem cells, suggested to be involved in resistance, as well as in anti-HER2 treatment-resistant BT474 cell line model of HER2+ BC. BC stem cells were identified as aldehyde dehydrogenase-positive (ALDH+) cells using the Aldefluor assay. Brightly fluorescent ALDH+ cells were separated from ALDH- cells using FACS Aria II cell sorter. Anti-HER2 resistant derivatives of BT474 cells were established by long-term exposure to increasing drug concentration of trastuzumab (T), lapatinib (L), or their combination (T+L). RNA was isolated and subjected to profiling using TaqMan real time RT-PCR GPCR 384-well microarray to quantify the expression of mRNA encoding 343 GPCRs from 50 different subfamilies. Only overexpressed GPCRs were considered of interest as drug targets, and the overexpression of GPCRs was verified by RT-PCR and western blotting for the selected targets. Publically available TCGA dataset for mRNA expression was also interrogated to determine differential expression of selected GPCRs in HER2+ vs. other subtypes of BC. Results: Nine GPCRs [BAI3, EDNRA, GPR110, GPR116, GPR124, MTNR1A, EDG2, EMR2, GCGR] were upregulated in ALDH+ compared to ALDH- BT474 cells. In addition, 11 GPCRs [CCBP2, CCR9, F2RL1, GALR2, GPR1, GPR24, GPR87, GPR110, GPR183, LGR4, OXER1] were over-expressed in the resistant derivatives (T, L, and T+L) compared to the parental BT474 cells. Out of these, 13 belong to Class A and 6 to Class B, designated by The International Union of Basic and Clinical Pharmacology (IUPHAR). GPR110 was the only GPCR common to BC stem cells as well as resistant derivatives of BT474 cells. In TCGA dataset, GPR110 expression was significantly higher in HER2+ and basal subtypes of BC compared to ER+ luminal A and B subtypes. GPR110 as well as other differentially expressed GPCRs are currently being investigated as potential targets by determining the effects of their downregulation or exogenous over-expression on the growth and drug-sensitivity of these BC cells. Conclusions: We report for the first time the differential expression of a large panel of GPCRs in the BC stem cell population and in anti-HER2 resistant derivatives of the BT474 cell line model of HER2+ BC. Results of the functional studies will guide novel strategies to improve the treatment for HER2+ BC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-02.