GPCRs profiling and identification of GPR110 as a potential new target in HER2+ breast cancer

Abstract

Purpose: G protein-coupled receptors (GPCRs) represent the largest family of druggable targets in human genome. Although several GPCRs can cross-talk with the human epidermal growth factor receptors (HERs), the expression and function of most GPCRs remain unknown in HER2+ breast cancer (BC). In this study, we aimed to evaluate gene expression of GPCRs in tumorigenic or anti-HER2 drug-resistant cells and to understand the potential role of candidate GPCRs in HER2+ BC.

Methods: Gene expression of 352 GPCRs was profiled in Aldeflur+ tumorigenic versus Aldeflur- population and anti-HER2 therapy-resistant derivatives versus parental cells of HER2+ BT474 cells. The GPCR candidates were confirmed in 7 additional HER2+ BC cell line models and publicly available patient dataset. Anchorage-dependent and anchorage-independent cell growth, mammosphere formation, and migration/invasion were evaluated upon GPR110 knockdown by siRNA in BT474 and SKBR3 parental and lapatinib+ trastuzumab-resistant (LTR) cells.

Results: Adhesion and class A GPCRs were overexpressed in Aldeflur+ and anti-HER2 therapy-resistant population of BT474 cells, respectively. GPR110 was the only GPCR overexpressed in Aldeflur+ and anti-HER2 therapy-resistant population in BT474, SKBR3, HCC1569, MDA-MB-361, AU565, and/or HCC202 cells and in HER2+ BC subtype in patient tumors. Using BT474 and SKBR3 parental and LTR cells, we found that GPR110 knockdown significantly reduced anchorage-dependent/independent cell growth as well as migration/invasion of parental and LTR cells and mammosphere formation in LTR derivatives and not in parental cells.

Conclusion: Our data suggest a potential role of GPR110 in tumorigenicity and in tumor cell dissemination in HER2+ BC.

Keywords: Breast cancer; Drug resistance; Drug targets; GPR110; HER2; Tumorigenesis.

Figure 1.. Distribution of 352 GPCRs with high, intermediate, low, and very low expression in Aldeflur+ tumorigenic versus Aldeflur- population and in parental cells and their anti-HER2 drug resistant derivatives of BT474 cells.

GPCR gene expression was determined using the 384-well Human GPCR TaqMan Array cards in (A) Aldeflur+ cell versus Aldeflur- cell population and (B) anti-HER2 drug resistant derivatives compared to parental cells of BT474 cells (N=2–4). Aldeflur+ cells were separated using Aldeflur assay followed by FACS sorting. Trastuzumab-resistant (TR), lapatinib-resistant (LR), and lapatinib + trastuzumab-resistant (LTR) cells were generated by long-term culture of the parental cells in their original media with increasing concentrations of respective drugs. GPCR expression was considered high, moderate, low, or very low if the Ct values were <25.0, 25.0 – 29.9, 30.0 – 39.9, and ≥ 40, respectively. Although most GPCRs had very low or low expression, a few GPCRs had moderate or high expression in various BT474 cell line model of tumorigenic cells and anti-HER2 drug resistance.

Figure 2.. Candidate GPCR selection in Aldeflur+ vs. Aldeflur- cells and anti-HER2 resistant cells:

Eleven GPCRs (ADORA1, EDG2, EFNRA, GPR87, MTNR1A, GCCR, BAI3, EMR2, GPR116, GPR124, GPR110) were upregulated in Aldeflur+ tumorigenic cells compared to Aldeflur- cell population of BT474 cells. In addition, 10 GPCRs (CCBP2, CCR9, EBI2, F2RL1, GALR2, GPR1, GPR24, LGR4, OXER1 AND GPR110) were over-expressed in the LTR and LR or TR derivatives compared to the parental BT474 cells. GPR110 was the only GPCR overexpressed in both tumorigenic as well as resistant derivatives of BT474 cells, and hence was selected as a candidate GPCR for further investigation.

Figure 3.. Relative gene expression of GPR110 in Aldeflur+ tumorigenic cells versus Aldeflur- cells population of various HER2+ BC cell line models.

Aldeflur+ cells were separated using Aldeflur assay followed by FACS sorting. GPR110 gene expression was determined using real time quantitative PCR using Taqman gene expression assays (Life Technologies). The level of the different mRNAs was normalized to the level of the housekeeping genes (PPIA). A comparative C_T analysis was utilized to determine fold change in RNA expression based on the ΔΔC_T approach (N=3). GPR110 expression was significantly higher in Aldeflur+ compared to Aldeflur- populations of (A) BT474, (B) SKBR3, (C) HCC1569, and (D) MDA-MB-361 cell lines, but not in (E) AU565 and (F) HCC1954 cells. * indicates P < 0.05 (student’s t-test).

Figure 4.. Relative gene expression of GPR110 in anti-HER2 drug-resistant derivatives versus parental cells of various HER2+ BC cell line models.

Trastuzumab-resistant (TR), lapatinib-resistant (LR), and lapatinib + trastuzumab-resistant (LTR) cells were generated by long-term culture of the parental cells in their original media with increasing concentrations of respective drugs. GPR110 gene expression was determined using real time quantitative PCR using Taqman gene expression assays (Life Technologies). The level of the different mRNAs was normalized to the level of the housekeeping gene (PPIA). A comparative C_T analysis was utilized to determine fold change in RNA expression based on the ΔΔC_T approach (N=3). GPR110 expression was significantly elevated in the TR, LR, and LTR derivatives compared to parental cells of (A) BT474, (B) SKBR3, and (C) UACC812; and in TR and LR derivatives of (D) AU565 and (E) HCC202 cells, but not in (F) HC1954 cells. * indicates P < 0.05 (One-way ANOVA followed by Dunnett’s posthoc test).

Figure 5.. GPR110 expression in publicly available datasets with different BC subtypes.

Publicly available TCGA dataset [25] was used to determine the expression of GPR110 mRNA in different subtypes of BC. Box and Whisker plot was used for showing differential GPR110 expression in basal, HER2+, luminal A, and luminal B subtypes of BC in human patients. As indicated, gene expression of GPR110 was significantly higher in HER2+ and basal like BC compared to luminal A and B subtypes. * indicates P < 0.05, t-test on log-transformed data. Box plots represent 5%, 25%, 50%, 75%, and 95%. Log2 FPKM expression values are normalized to standard deviations from the median.

Figure 6.. Concentration-response curve of lapatinib in affecting anchorage-dependent cell growth of HER2+ BC cells in presence or absence of GPR110 knockdown by siRNA.

Parental and LTR derivatives of (A) BT474 and (B) SKBR3 cells were reverse transfected with non-targeting (si-control, solid line) or with 2 independent GPR110-targeting (si-GPR110, dotted lines) siRNAs. Parental (C) BT474 and (D) SKBR3 cells were also treated with vehicle or various concentrations of lapatinib (1 nM to 10 μM) for 72 hours. MTT assay was conducted using the kit from ATCC to assess cell growth and viability (N=3). The data was normalized to vehicle (100%) and was plotted using the lapatinib concentration ([lapatinib]) in log M for concentration curve. The data was fitted using the 3-parameter logistic equation: Y = Bottom + (Top-Bottom)/(1+10^((X-LogIC50))). The growth of parental and LTR derivatives of BT474 and SKBR3 cells was significantly inhibited by GPR110-targeting siRNAs. But, knockdown of GPR110 did not affect potency of lapatinib in BT474 and SKBR3 parental cells. * indicates P < 0.05, Repeated Measures ANOVA followed by Dunnett’s post-hoc test.

Figure 7.. GPR110 knockdown inhibits anchorage-independent cell growth as measured by soft agar assay better in LTR derivatives compared to parental cells in HER2+ BC cell lines.

Parental and LTR derivatives of (A) BT474 and (B) SKBR3 cells were reverse transfected with non-targeting (si-control, white bar) or with 2 independent GPR110-targeting (si-GPR110, lined bars) siRNAs. After 24 hours, the cells were subjected to soft agar assay to assess anchorage-independent growth (N=3–4). Number of colonies (at least 50 μm in size) was counted on day 10 with Gelcount™ (Oxford Optromix, Germany). Knocking down GPR110 by two independent siRNAs resulted in a significant decrease in the number of colonies in both parental and LTR derivatives of BT474 and SKBR3 models. However, the decrease was more pronounced in the LTR cells compared to parental BT474 and SKBR3 cells. * indicates P < 0.05, Repeated Measures ANOVA followed by Dunnett’s post-hoc test.

Figure 8.. GPR110 knockdown inhibits mammosphere formation in LTR derivatives but not in parental BT474 cells.

BT474 parental cells and their LTR derivatives were reverse transfected with non-targeting (si-control, white bar) or with independent GPR110-targeting (si-GPR110, lined bars) siRNAs. After 24 hours, the cells were subjected to mammosphere assay using the Mammocult media from stem cell technologies in 24-well ultra low attachment plates (N=3). Numbers of (A) first- and (B) second-generation mammospheres of at least 50 μm in size were counted on day 7 with Gelcount™ (Oxford Optromix, Germany) after plating. BT474 LTR derivatives formed more mammospheres compared to parental cells. GPR110 knockdown by siRNAs resulted in a significant inhibition of first and second generation mammosphere formation in BT474 LTR derivatives, but not in parental cells. * indicates P < 0.05, Repeated Measures ANOVA followed by Dunnett’s posthoc test.

Figure 9.. GPR110 knockdown inhibits migration and invasion better in LTR derivatives compared to parental cells of SKBR3 cell line.

SKBR3 parental cells and their LTR derivatives were reverse transfected with non-targeting (si-control, white bar) or with 2 independent GPR110-targeting (si-GPR110, lined bars) siRNAs. After 24 hours, the cells were subjected to migration (A) and invasion (B) assays using 8μm cell culture inserts (N=3). GPR110 knockdown resulted in significant inhibition of migration and invasion of SKBR3 parental as well as LTR cells. * indicates P < 0.05, Repeated Measures ANOVA followed by Dunnett’s posthoc test.