ARRB1 cooperates with estrogen receptor to drive endocrine resistance in ER-positive breast cancer

Background: β-Arrestin1(ARRB1) is a member of arrestins protein family and act as a scaffold to regulate proteins through G-protein-coupled receptors signaling (1). Recent studies show that it plays an important role in different cancers. ARRB1 enhances progression of breast and prostate cancer via regulation of HIF1A activity (2, 3), promotes lung cancer by increasing E2F1 activity (4). ARRB1 associates with androgen receptor and augments its activity in prostate cancer (5) . However, a tumour-suppressor role of ARRB1 has also been observed. Overexpression of ARRB1 inhibited the growth of human neuroblastoma cells through regulating p27 transcription (6) and triple-negative breast cancer (7, 8). These reports suggest that ARRB1 exerts diverse roles in cancers. Breast cancer is the most common cancer diagnosed in women. Around 70% of all breast cancers are estrogen receptor α (ER) positive(9). Despite advances in treatment of ER-positive breast cancers, the development of resistance remains a major challenge necessitating the targeting novel mechanisms of endocrine resistance to improve clinical outcome (10). Indeed, Razavi et al., 2018 reported that 60% of endocrine-resistant metastatic breast cancers lacked known somatic drivers of resistance suggesting that there are yet unidentified factors that contribute to endocrine resistance (11). Here we have evaluated the role of ARRB1 in ER positive breast cancer. Methods: ARRB1 overexpressed stable clone is generated through a lentiviral expression plasmid. ARRB1 knockout stable clone is generated using the CRISPR-Cas9 gene knockout strategy. These clones are validated using western blot. Colony formation assay and MTS assay are used to evaluate cell viability and cell proliferation. Luciferase reporter assay is used to evaluate the effect of ARRB1 on estrogen receptor. qRT-PCR and Western blot are used to identify the mRNA and protein level. CBioPortal is used to analyze genetic status. KM plotter is used for survival analysis. Results: The expression of ARRB1 is increased in ER-positive breast cancer compared to ER-negative breast cancer. Depletion of ARRB1 decreases colony formation of ER-positive breast cancer cells. While overexpression of ARRB1 in MCF7 cells increases cell growth and amplifies the induction of estrogen-responsive genes. What’s more, overexpress ARRB1 increases E2-dependent growth in MCF7 cells while absence of ARRB1 reduces E2-dependent growth in T47D cells. Further, ARRB1 co-expression enhances the transactivation function of ERα WT and ER point mutation but not ESR1 fusion mutation. In contrast, estrogen downregulates mRNA and protein of ARRB1. In addition, KM Plotter analysis shows that increased expression of ARRB1 is associated with poor outcome in ER-positive breast cancer. Combined with Spiperone treatment, which downregulates ARRB1, increases the effect of Tamoxifen on viability of MCF7 cells.Conclusions: Our results suggest that ARRB1 functionally collaborates with ER to drive endocrine resistance and targeting of ARRB1 will provide a novel approach to overcome endocrine resistance.

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