Abstract 3573: Novel G protein-coupled receptor targets in HER2+ breast cancer

Abstract

Abstract Background: G protein-coupled receptors (GPCRs) are excellent drug targets. However, not many GPCRs have been identified as potential drug targets in breast cancer (BC). This may be due to two factors: (1) The primary mode of GPCR regulation is at the level of signaling rather than gene expression, which is the common method for large-scale target identification. (2) GPCR expression in BC stem cells or drug-resistant cells could have been overlooked because these cells exist in small fractions. In this study, we aimed to identify novel GPCR targets based on large panel functional screening and differential gene expression in HER2+ BC stem cells and drug resistant cells. Methods: (I) For the functional screening, each of the 370 GPCR genes was knocked down using targeted Silencer® Select siRNA library comprising of 3 individual siRNAs per target in BT474 and SKBR3 cell lines (N = 3). Candidate GPCRs were selected based on at least 20% alteration in cell growth by at least 2 out of 3 siRNAs. (II) For the expression screening, gene expression of 343 GPCRs was measured in ALDH+ BC stem cells vs. ALDH- cells as well as in anti-HER2 drug-resistant derivatives vs. parental cells of BT474 cells using Taqman RT-PCR arrays (N = 2-4). Melatonin MT1 receptor (MTNR1A) gene knockdown and pharmacological modulation were employed to functionally validate its role in anchorage-dependent (MTT assay) and -independent (soft agar colony formation assay) cell growth. Results: Gene knockdown of 11 GPCRs (ADORA, ADRA1D, CCR8, GNRHR, GPR26, GPR113, GPR120, LPAR3, MC5R, MTNR1A, NTSR2) altered cell growth of HER2+ BC cells by at least 20%. The MT1 receptor was selected as a candidate GPCR for this study because (1) its knockdown resulted in at least 20% inhibition of cell growth in both BT474 and SKBR3 cells, and (2) its expression was increased in ALDH+ vs. ALDH- as well as in drug-resistant cells vs. parental BT474 cells. MTNR1A knockdown resulted in 25-35% inhibition in the anchorage-dependent cell growth of both BT474 and SKBR3 cells. Furthermore, MTNR1A knockdown caused a marked (3-fold) decrease in the number of colonies in soft agar assay. MT1 receptor antagonist luzindole reduced the anchorage-dependent cell growth of BT474 cells in a concentration-dependent manner (IC50 = 20-55 nM). However, the efficacy of luzindole was only ∼50% of lapatinib efficacy in reducing cell growth. Luzindole (1μM) also significantly reduced (by 2-fold) anchorage-independent cell growth of BT474 cells. Neither MTNR1A knockdown nor luzindole significantly improved the efficacy of lapatinib (1nM) in inhibiting cell growth of BT474 cells. Conclusion: In summary, functional knockdown screening and differential gene expression of GPCRs is a powerful tool for identifying GPCRs as candidate targets in BC. Using the combination of these two approaches, we have discovered that melatonin MT1 receptor as a potential drug target in HER2+ BC cells. The place in therapy for MT1 receptor antagonists, however, still remains to be determined. Citation Format: Raksha Bhat, Puja Yadav, Pavel Christiny, Rachel Schiff, Meghana V. Trivedi. Novel G protein-coupled receptor targets in HER2+ breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3573. doi:10.1158/1538-7445.AM2015-3573