Abstract P6-04-05: GPR110 overexpression increases tumorigenic potential of HER2+ breast cancer cells

Abstract

Abstract Human epidermal growth factor receptor-2-overexpressing (HER2+) breast cancer is an aggressive tumor. Despite the clinical success of anti-HER2 drugs such as lapatinib (L) and trastuzumab (T), intrinsic and acquired drug resistance occurs in many patients. Identification of novel drug targets in HER2+ breast cancer is an unmet clinical need. In this context, G-protein coupled receptors (GPCRs) may be excellent drug targets because they cross-talk with the HER family members. However, the expression and function of the majority of GPCRs are unknown in HER2+ breast cancer. In a preliminary study, we examined the differential gene expression of GPCRs in anti-HER2 treatment-resistant derivatives as well as in the tumorigenic cell population, suggested to be involved in resistance, of a BT474 cell line model of HER2+ breast cancer. Anti-HER2 resistant derivatives of BT474 cells were established by long-term exposure of parental cells to increasing concentrations of L, T, or their combination (L+T). Tumorigenic cells were identified as aldehyde dehydrogenase-positive (ALDH+) cells using the Aldefluor assay. RNA was profiled using TaqMan real time RT-PCR GPCR 384-well microarray to quantify the expression of mRNA encoding 343 GPCRs. The publically available TCGA dataset was interrogated to determine differential mRNA expression of selected GPCRs in HER2+ and other subtypes of breast cancer. To determine the functional role of GPR110, BT474 cells were infected with lentiviral GPR110 construct (GPR110-OE) or empty vector (EV), and stable pools were obtained. Anchorage-dependent cell growth was evaluated using MTT cell proliferation assay over 8 days. Tumorigenic potential was determined by calculating the% of ALDH+ cells using Aldefluor assay and by evaluating the anchorage-independent cell growth using soft agar assay over 14 days. The influence of GPR110 overexpression on HER signaling pathway was investigated by measuring the levels of phosphorylated (active) and total protein levels of HER1 and HER2 using immunoblotting. GPR110 was the only GPCR overexpressed in resistant derivatives versus parental cells as well as in ALDH+ versus ALDH- cells of BT474 cells. In TCGA dataset, GPR110 expression was significantly higher in HER2+ and basal subtypes of breast cancer compared to ER+ luminal A and B subtypes. Overexpression of GPR110 in BT474 cells (9-fold in GPR110-OE vs. EV cells) resulted in a marked 5-fold increase in the number of colonies when grown in soft agar compared to EV cells even though anchorage-dependent cell growth was not significantly different between EV and GPR110-OE cells. In addition, GPR110-OE cells had a significantly higher% of ALDH+ population compared to EV cells. Phosphorylated (but not total) HER1 and HER2 protein levels were significantly higher in GPR110-OE cells compared to EV cells, suggesting hyperactive HER signaling with GPR110 overexpression. Experiments with L and T treatment will reveal the role of GPR110 in drug efficacy and acquired resistance. In summary, we show for the first time a pro-tumorigenic role of GPR110 in HER2+ breast cancer. Therefore, GPR110 may be a novel pharmacological target in HER2+ breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-05.