XBP1 increases transactivation of somatic mutants of ESR1 and loss of XBP1 reverses endocrine resistance conferred by gain-of-function Y537S ESR1 mutation

Abstract

Somatic mutations of the estrogen receptor 1 gene (ESR1) is an emerging mechanism of acquired resistance to endocrine therapy in hormonal breast cancers. Hotspot point mutations in the ligand- binding domain of estrogen receptor α (ER) and genomic rearrangements producing ESR1 fusion genes are the two major types of mutations that are associated with endocrine resistance. The crosstalk between X-box binding protein 1 (XBP1), a key transcription factor of the unfolded protein response (UPR) and ER signalling creates a positive feedback loop that results in increased expression of XBP1 in ER-positive breast cancer. Here we report that XBP1 co-operated with point mutants (Y537S, D538G) and fusion mutants (ESR1-YAP1, ESR1-DAB2) of ESR1 to increase their transcriptional activity. XBP1 was required for optimal expression of estrogen-regulated genes, and up to 40% of XBP1-dependent genes were estrogen-responsive genes. Knockdown of XBP1 in genome-edited MCF7 cells expressing Y537S mutant reduced their growth, re-sensitized them to anti-estrogens and attenuated the constitutive and estrogen-stimulated expression of estrogen-regulated genes. Our study provides a rationale for overcoming endocrine resistance in breast cancers expressing ESR1 mutation by combining the XBP1 targeting agents with anti-estrogen agents.

Keywords: Biochemistry; Cancer research; Cell biology; ER-Positive breast cancer; ESR1 mutations; Endocrine resistance; Molecular biology; Transcriptomics; XBP1.

Figure 1

Ligand-independent transcriptional activity of ESR1 mutants. (A) Upper panel, HEK293T cells were transfected with ERE-LUC and indicated plasmids, 24h post-transfection, reporter assays were performed. Normalised LUC activity is presented as mean ± SD (n = 3). Lower panel, 24h post-transfection cell lysates were analysed by western blotting using antibodies against ER and β-actin. B) Upper panel, MCF7 cells were transfected as in A, 24h post-transfection reporter assay was performed. Normalised LUC activity is shown as mean ± SD (n = 3). Lower panel, 24h of post-transfection with indicated plasmids, cell lysates were analysed by western blotting using antibodies against ER and β-actin. ∗p < 0.05, two-tailed unpaired t-test. ERE, estrogen response element; LUC, luciferase; ER, estrogen receptor alpha. Uncropped full-length pictures of Western blotting membranes presented in the Supplementary Figure 1.

Figure 2

XBP1s enhances the transactivation function of ESR1 mutants. (A) MCF7 cells were transfected with ERE-LUC and indicated ER expressing plasmid, both in the absence and presence of XBP1s. The LUC activity of lysate without exogenous ER and spliced XBP1 was set as 1. Normalised LUC activity is presented as mean ± SD (n = 3). (B) MCF7 cells were transfected as in A, cell lysates were analysed by western blotting using antibodies against ER and β-actin. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 two-tailed unpaired t-test. Uncropped full-length pictures of Western blotting membranes presented in the Supplementary Figure 2.

Figure 3

Estrogen-responsive genes induced by point mutants of ER require XBP1. (A) Venn diagram displaying overlapping genes between XBP1-regulated genes and estrogen- responsive genes. XBP1 shRNA: genes downregulated in XBP1 shRNA expressing cells; E2 Early: genes regulated at 4h of E2 stimulation, E2 Late; genes regulated at 24h of E2 stimulation. (B) After 2 days of synchronization in phenol red free DMEM containing 3% CSS, MCF7 control (CTL) and XBP1 knockout (XBP1 KO) cells were either treated with (Veh) or estrogen (E2) for 24h. Relative expression of the indicated genes is shown (n = 3). (C) MCF7 WT, MCF7 Y537S and MCF7 D538G cells were grown in steroid free conditions in phenol red free DMEM containing 5% CSS. Relative expression of the indicated genes is shown (n = 3). (D) After 3 days of synchronization in phenol red free DMEM containing 5% CSS, MCF7 WT, MCF7 Y537S and MCF7 D538G cells were either treated (Veh) or estrogen (E2) for 24h. Relative expression of the indicated genes is shown (n = 3). ∗p < 0.05, ∗∗P < 0.01, two-tailed unpaired t-test, N.S not significant.

Figure 4

Depletion of XBP1 attenuates cell growth and sensitizes MCF7-Y537S cells to tamoxifen. (A) MCF7-Y537S CTL and XBP1 shRNA cells were treated with BFA (2 μg/ml) for 8h to induce the expression of XBP1s. Whole cell lysates were analysed by immunoblotting using antibodies against XBP1s and β-actin. (B) Cells were treated as in A, qRT-PCR was performed for the indicated genes. Mean of two independent experiments performed in triplicates are shown. (C) Indicated sub-clones of MCF7 cells were plated in 6-well plate (500 cells/well) and grown for 14 days. Representative image of colonies stained with crystal violet are shown (n = 3). (D) Quantification of number of colonies as determined by Image J is shown (n = 3). (E) Proliferation of indicated sub-clones of MCF7 cells was assessed by MTS assay. Data represents here as mean ± SD of three independent experiments performed in six replicates. (F) Indicated sub-clones of MCF7 cells were treated with tamoxifen (1 μM) and MTS assay was performed. Fold change in the absorbance at 490 nm are shown, with value of untreated samples set at 1. Data represents here as mean ± SD of three independent experiments performed in six replicates.∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 from two-tailed unpaired t-test. Uncropped full-length pictures of Western blotting membranes presented in the Supplementary Figure 3.

Figure 5

Depletion of XBP1 reduces the expression of estrogen-responsive genes in MCF7-Y537S cells. Indicated sub-clones of MCF7 cells were synchronized for 3 days in phenol red free DMEM containing 5% CSS. Cells were either treated with (Veh) or estrogen (E2) for 24h. Relative expression of the GREB1 and PGR was evaluated by RT-PCR. A representative of two experiments performed in triplicates is shown. ∗p < 0.05, ∗∗P < 0.01, two-tailed unpaired t-test.