β-Arrestin1 act as a rheostat for ERα and TP53 signalling in ER-positive breast cancer and modulate response to therapy

Breast cancer is the most common cancer in women worldwide, with 2.3 million new cases in 2022. Globally, it was estimated that more than 666,000 women died from breast cancer in 2022. Approximately 70% of breast cancer cases are hormonal positive cancer characterized by expression of the oestrogen receptor α (ERα). Endocrine therapy (ET) targeting the ERα is the main treatment for patients with hormone receptor-positive breast cancer. Resistance to ET is a major problem in ER positive breast cancer, with recurrence and metastasis occurring in up to 40% of patients. Drugs that can reverse endocrine resistance when used in combination with endocrine therapy are urgently needed, and targeting gene synthetic lethality provide a new approach to it. β-Arrestin1 (ARRB1) is a member of the arrestins protein family and acts as a scaffold for regulating proteins through G protein-coupled receptor signalling. Recent studies have shown that it plays an important role in different cancers and is a key regulator of cancer development and progression. Cytoplasmic ARRB1 promotes liver and colorectal cancer by activating AKT signalling pathway. Nuclear ARRB1 enhances the progression of breast and prostate cancer via regulation of the HIF1A activity; promotes lung cancer by increasing E2F1 activity. Nuclear ARRB1 also associated with androgen receptor and augments its activity in prostate cancer. However, the tumour-suppressor role of ARRB1 has also been observed. Overexpression of ARRB1 inhibited the growth of human neuroblastoma cells through regulating p27 transcription in the nucleus and inhibited the progression of T-cell acute lymphoblastic leukaemia cells and triple-negative breast cancer (TNBC) cells via ARRB1 binding partners in the cytoplasm. These reports suggest that ARRB1 plays a diverse, context-dependent role in cancer. Surprisingly, not much is known about the role of ARRB1 in ER-positive breast cancer. Earlier work in our group has shown significant downregulation of ARRB1 mRNA and protein during conditions of EnR stress in multiple ER-positive breast cancer cell lines. PERK signalling was required for the downregulation of ARRB1. Further, we showed ARRB1 expression was upregulated by TP53. Here we have studied the crosstalk of ARRB1 with ER signalling, UPR pathway and TP53, the main aims of the study are to: 1) Evaluate the functional interactions between ARRB1 and ER in breast cancer. 2) Investigate the role of ARRB1 in regulating the response to endocrine therapy and cell fate during EnR stress. 3) Determine the role of ARRB1 in regulating expression and function of TP53 in ER-positive breast cancer. Result 1: Functional interactions between ARRB1 and ER in breast cancer. We found that ARRB1 enhances transactivation activity of ERα through physical interaction with ERα. In addition, ARRB1 enhances the transactivation activity of ER WT and point mutants (ER Y537S and ER D538G) but has no effect on the activity of fusion mutants ESR1 DAB2 and ESR1 YAP1) of ERα, it suggests that the binding site is in the ligand-binding domain of ERα. We demonstrated that ARRB1 positively regulates the growth and proliferation of ER-positive breast cancer, promotes E2-dependent growth. We further reveal that ARRB1 increases the expression of ER target genes like PGR, GREB1 and EGR, but has no effect on ERα protein expression. Results 2: Role of ARRB1 in regulating the response to endocrine therapy and cell fate during EnR stress. ARRB1 overexpression provided resistance against tamoxifen and fulvestrant while knock-down of ARRB1 had opposite effect. We found that ARRB1 inhibits the activation of the PERK pathway during conditions of UPR but has no effect on IRE1-XBP1 or ATF6 pathway. ARRB1 expression protected cells from UPR-induced cell death. From a list of clinically approved drugs with relatively higher bioavailability we found that Spiperone treatment remarkably reduced ARRB1 expression. Spiperone-mediated reduction of ARRB1 expression is linked to the ability of Spiperone to activate UPR. Further, we demonstrated that targeted downregulation of ARRB1 with Spiperone, could enhance the sensitivity towards tamoxifen. Results 3: Role of ARRB1 in regulating expression and function of TP53 in ER-positive breast cancer. Next, we evaluated the role of ARRB1 on TP53 response pathway. We observed increased expression and function of TP53 after treatment with DNA damage reagents (5FU, cisplatin & Doxorubicin) in ARRB1 overexpressing cells. Moreover, ARRB1 knockdown cells showed decreased expression and function of TP53 after treatment with DNA damage reagents (5FU, cisplatin & Doxorubicin). Further ARRB1 co-expression enhances the transactivation of TP53 in reporter assays. Furthermore, ARRB1 enhanced 5FU-induced cell death and compromised the recovery and growth of cells after a short-term exposure to 5FU. We show that ARRB1 is at interface of ER signalling, UPR pathway and TP53 pathway in breast cancer. The results revealed the complex role of ARRB1 in ER-positive breast cancer cells. On the one hand, it contributes to endocrine resistance in ER positive breast cancer cells, and on the other hand, it increases the sensitivity towards DNA-damage inducing agents (5FU). The results presented in thesis suggest that reduction of ARRB1 can sensitize the cells to endocrine therapy. The patients with increased ARRB1 and wild type TP53 will respond better to 5FU treatment.

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University of Galway

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CC BY-NC-ND

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University of Galway Theses (PhD Theses)