RRM2 and CDC6 are novel effectors of XBP1-mediated endocrine resistance and predictive markers of tamoxifen sensitivity

Abstract

Background: The molecular mechanisms that determine endocrine resistance in estrogen receptor α (ER)-positive breast cancer patients are poorly understood. A positive feedback loop created by the intersection between estrogen signalling and unfolded protein response (UPR) leads to increased expression of x-box binding protein 1 (XBP1) and estrogen receptor α in ER-positive breast cancer. The endocrine-resistant breast cancers have elevated expression of XBP1 where it drives endocrine resistance by controlling the expression of its target genes. Despite the in-depth understanding about the biological functions of XBP1 in ER-positive breast cancer not much is known about the effectors of endocrine resistance downstream of XBP1. Methods and Results: We generated sub-clones of MCF7 cells with deletion of XBP1 using CRISPR-Cas9 for this study. Deletion of XBP1 compromised the upregulation of UPR-target genes during conditions of endoplasmic reticulum (EnR) stress and sensitized cells to EnR stress-induced cell death. Loss of XBP1 in MCF7 cells decreased cell growth, attenuated the induction of estrogen-responsive genes and sensitized them to anti-estrogen agents. The expression of cell cycle associated genes RRM2, CDC6, and TOP2A was significantly reduced upon XBP1 deletion/inhibition in several ER-positive breast cancer cells. Expression of RRM2, CDC6, and TOP2A was upregulated upon estrogen stimulation and elevated expression was observed in cells harbouring point-mutants (Y537S, D538G) of ESR1 in the steroid free conditions. Ectopic expression of RRM2 and CDC6 increased cell growth and reversed the hypersensitivity of XBP1 KO cells towards tamoxifen. Importantly, increased expression of XBP1-gene signature was associated with poor overall and relapse free survival, and reduced efficacy of tamoxifen treatment in ER-positive breast cancer. Conclusions: Our results suggest that expression of RRM2, CDC6 and TOP2A in ER-positive breast cancer is regulated by concerted effort of both ER and XBP1. RRM2 and CDC6 contribute to endocrine resistance downstream of XBP1 in ER-positive breast cancer. XBP1-gene signature was associated with poor outcome and response to tamoxifen in ER-positive breast cancer. Taken together these results reveal mechanistic insights about the role of XBP1 in endocrine resistant breast cancer.