The prognostic and therapeutic value of XBP1 signalling in ER-positive breast cancer

Breast cancer is a heterogeneous disease with distinct subtypes having specific molecular features. Approximately, 75% of breast cancers are estrogen receptor α (ER)-positive. Endocrine therapies have been used as a standard treatment option to treat ER-positive breast cancers in all stages of the disease. However, one-third of patients previously treated with endocrine therapies gain resistance over the time of treatment, leading to disease progression and death. Several molecular mechanisms of endocrine resistance were identified, including the presence of ER mutations, altered activity of co-regulator proteins, constitutive activation of growth factor receptors and the activation of unfolded protein response (UPR). UPR is considered as an adaptive cellular signalling, which is activated upon the accumulation of unfolded/misfolded proteins in the lumen of endoplasmic reticulum. UPR induction can play an essential role in allowing cancer cells to survive and proliferate during hypoxia, nutrient deprivation, and environmental stress and thus drive therapy resistance in several tumours. IRE1-XBP1 signalling is one of the branches of UPR which has been linked with several cancers including breast cancer. Activated IRE1 catalyses an unconventional cytoplasmic mRNA-splicing reaction, and generates active transcription factor, spliced XBP1 (XBP1s). XBP1s is a multitasking transcription factor which is a key component of the UPR. Recent studies indicate a crucial role for the IRE1/XBP1 pathway in several aspects of ER-positive breast cancer. XBP1 physically interacts with ER and potentiates ER-dependent transcriptional activity in a ligand-independent manner. XBP1s expression can be upregulated following estradiol (E2) treatment in ER-positive human breast cancer cell lines. Thus, XBP1 and ER generate a positive feedback regulatory loop that leads to increased expression of XBP1 and ER in hormonal breast cancer. We hypothesize that XBP1 is a key signalling node during estrogen stimulation with important regulatory role in resistance to endocrine therapy in ER-positive breast cancer. The current thesis aimed (i) to determine the transcriptional network of XBP1s in the context of ER-positive breast cancer and (ii) to evaluate the functional interaction between XBP1 and gain-of-function mutants of ESR1 in ER-positive breast cancer. The first section of results describes the generation of XBP1 knockout sub-clones in breast cancer cells and the effect of XBP1 deficiency on UPR-mediated cell death. The second section of results presents the identification of XBP1-regulated targets and their prognostic significance in ER-positive breast cancer, and the last section describes the co-operation between XBP1s and ESR1 mutants and the effect of XBP1 depletion in ESR1 point mutant breast cancer cells. XBP1 deletion in MCF7 cells showed compromised induction of UPR-responsive genes and increased sensitivity to UPR-mediated cell death. Deletion of XBP1 in MCF7 cells showed decreased cell growth and proliferation compared to control MCF7 cells, also, depletion of XBP1 in MCF7 cells demonstrated compromised E2-mediated cell growth. Notably, we observed the requirement of XBP1 for the optimal basal and estrogen-stimulated expression of ER-target genes, including GREB1, PGR, H19, PDZK1, and EGR. XBP1 deficiency in MCF7 cells enhanced sensitivity towards anti-estrogens, including tamoxifen and fulvestrant. The expression of cell cycle-associated genes RRM2, CDC6, and TOP2A was found to be significantly reduced upon XBP1 deletion/inhibition in ER-positive breast cancer cells, including MCF7, T47D, and BT474 cells. We found that RRM2, CDC6, and TOP2A are estrogen-responsive genes and demonstrated their increased expression in MCF7 cells harbouring ESR1-Y537S point mutation cultured in steroid free conditions. We demonstrated that overexpression of RRM2 and CDC6 increased cell growth and conferred anti-estrogen resistance in MCF7 cells. Importantly, XBP1 gene-signature analysis (RRM2, CDC6, and TOP2A) revealed that higher expression of XBP1-gene signature is significantly associated with poor clinical outcome in ER-positive breast cancer. Our results identified a XBP1-dependent gene signature which could serve as potential marker to predict clinical outcome in ER-positive breast cancer patients. Multiple genomic alterations at the ESR1 locus is an established mechanism to drive endocrine therapy resistance in ER-positive breast cancer. Hotspot point mutations clustering in the ligand binding domain (LBD) of ESR1 have been found in treatment-refractory, metastatic ER-positive breast cancer patients. Two most prevalent ESR1 point mutations (Y537S and D538G) show constitutive, ligand-independent transcriptional activity, and partial resistance to endocrine agents. Besides, several other ESR1 fusion mutations have also been identified including ESR1-DAB2 and ESR1-YAP1, which are implicated in advanced, treatment refractory breast cancer patients. Resistance conferred by both types of mutations is a significant challenge to treat metastatic ER-positive breast cancer, and there is an urgent need to identify new effective treatments, in order to best treat metastatic, ER-positive breast cancers harbouring ESR1 mutations. We hypothesized that XBP1 functionally collaborates with ESR1 mutants to drive endocrine resistance and, co-targeting of XBP1 and estrogen signalling may provide a novel approach to overcome endocrine resistance. Luciferase reporter analysis demonstrated estrogen independent transcriptional activity of mutant ER constructs (ER Y537S, ER D538G, ESR1-DAB2, and ESR1-YAP1) in both MCF7 cells and 293T cells. We observed the co-operation of XBP1s with point mutants (Y537S, D538G) and fusion mutants (ESR1-DAB2, ESR1-YAP1) of ESR1, where co-expression of XBP1s enhanced ERE-transcriptional activity in MCF7 cells. We identified estrogen-responsive genes that were regulated by XBP1 from the overlap of genes downregulated in T47D XBP1-shRNA cells (GSE49953) and estrogen-regulated genes. We found a total of 442 differentially regulated genes in T47D XBP1-shRNA cells. Meta-analysis revealed that almost 40% of genes that were differentially regulated by XBP1 inhibition were estrogen responsive. Later, we confirmed ligand-independent stimulation of estrogen-induced genes including GREB1, PGR, and H19 in genome-edited MCF7 Y537S and MCF7 D538G cells. Concurrently, the expression of XBP1 was also found to be induced in a ligand-independent manner. Interestingly, knockdown of XBP1 in genome-edited MCF7 Y537S cells showed compromised activation of estrogen-stimulated genes. Knockdown of XBP1 in genome-edited MCF7 Y537S cells demonstrated attenuation of cell growth and proliferation, re-sensitization to anti-estrogens. Together, our study provides a rationale for overcoming endocrine resistance in breast cancers expressing ESR1 mutation by combining the XBP1 targeting agents with anti-estrogens.

Funder

Irish Research Council

Publisher

NUI Galway

Rights

Attribution-NonCommercial-NoDerivs 3.0 Ireland

cbn

Collections

University of Galway Theses (PhD Theses)