IRE1α-XBP1s axis regulates SREBP1-dependent MRP1 expression to promote chemoresistance in non-small cell lung cancer cells

Abstract

Background: Inositol-requiring enzyme 1 (IRE1) is an endoplasmic reticulum (ER)-resident transmembrane protein that senses ER stress and mediates an essential arm of the unfolded protein response (UPR). IRE1 reduces ER stress by upregulating the expression of multiple ER chaperones through activation of X-box-binding protein 1 (XBP1). Emerging lines of evidence have revealed that IRE1-XBP1 axis serves as a multipurpose signal transducer during oncogenic transformation and cancer development. In this study, we explore how IRE1-XBP1 signaling promotes chemoresistance in lung cancer.

Methods: The expression patterns of UPR components and MRP1 were examined by Western blot. qRT-PCR was employed to determine RNA expression. The promoter activity was determined by luciferase reporter assay. Chemoresistant cancer cells were analyzed by viability, apoptosis. CUT & Tag (Cleavage under targets and tagmentation)-qPCR analysis was used for analysis of DNA-protein interaction.

Results: Here we show that activation of IRE1α-XBP1 pathway leads to an increase in MDR-related protein 1 (MRP1) expression, which facilitates drug extrusion and confers resistance to cytotoxic chemotherapy. At the molecular level, XBP1-induced c-Myc is necessary for SREBP1 expression, and SREBP1 binds to the MRP1 promoter to directly regulate its transcription.

Conclusions: We conclude that IRE1α-XBP1 had important role in chemoresistance and appears to be a novel prognostic marker for lung cancer.

Keywords: IRE1α; MRP1; SREBP1; XBP1; chemoresistance.

FIGURE 1

Endoplasmic Reticulum Stress Orchestrates Chemotherapy Resistance Through the IRE1α‐XBP1‐MRP1 Axis. (a) The A549 and H1299 cells were treated with etoposide (40 μM) for different time point (left panel) and various concentrations (right panel) in the presence or absence of TG (30 nM). MTT assay was used to detect cell viability (*p < 0.05, **p < 0.01, ***p < 0.001 for difference from control cells). (b) The A549 and H1299 cells were pretreated with TG (30 nM) for 2 h and then were treated with etoposide (20, 40, and 60 μM) for 48 h. Cell viability was assessed using the MTT assay. (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 for difference from control cells). (c) H1299 and A549 cells were treated with etoposide (40 μM) in the presence or absence of TG (30 nM). Flow cytometry analysis revealed that TG could induce resistance to etoposide in NSCLC. The bars represent the mean ± S.D. of triplicates (***p < 0.001, ****p < 0.0001 for difference from control cells, ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, ^#### p < 0.0001 for difference from etoposide‐treated cells by ANOVA with Dunnett's correction for multiple comparisons, ns means no statistical difference). (d) The correlation between ABCC1 with UPR related gene ERN1 (IRE1) expression from GEPIA data (GEPIA‐LUAD). (e) The results of the TCGA database statistical analysis reveal that the MRP1 protein encoded by ABCC1 is highly expressed in cancer cells. (f) The results of the GEPIA database reveal a correlation between the expression of MRP1 and the prognosis of lung cancer cell. (g) Western blot revealed that treating H1299 and A549 cells with TG (15, 30 nM) for 48 h resulted in a dose‐dependent enhancement of MRP1 protein expression. (h) The cells were transfected with IRE1α and XBP1s cDNA for 48 h. Western blot analysis demonstrated an increase in the expression level of MRP1 upon overexpression of IRE1α and XBP1s. (i) Cells treated with TG (30 nM) for 12 h or untreated subjected to subcellular fractionation. Equivalent celly lysates from cytoplasmic and nuclear fraction were analyzed by Western blot for subcellular localization of XBP1s. (j) After transfecting cells with XBP1s cDNA, Western blot analysis revealed the expression level of XBP1s and MRP1. (k) Western blot analyzed the expression levels of Bip/GRP78, phosphorylated IRE1α, XBP1s, ATF6, and MRP1 after treatment A549 and H1299 cells with 4‐PBA (10 mM) in the presence or absence of TG (30 nM). (l–n) The A549 and H1299 cells were first transfected with siRNA of IRE1α, XBP1s or MRP1. At 48 h post‐transfection, cells were treated with etoposide for 24 h in the presence and absence of TG followed by MTT assay (****p < 0.001 for difference from control cells, ^#### p < 0.0001 for the difference from etoposide‐treated cells, ^$$$$ p < 0.001 for difference from etoposide and TG‐treated cells by ANOVA with Dunnett's correction for multiple comparisons).

FIGURE 2

Reversal of MRP1 Enhances Cellular Sensitivity to Drugs. (a) After treating cells with TG (30 nM) for 12 h, cells were either left untreated or treated with the ABC transporter inhibitor Verapamil (10 μM). Representative histogram profiles of untreated cells (control), cells treated with 30 nM TG, 10 μM Verapamil, and the combined treatment of TG and Verapamil. The quantitative results are presented in the right panel, with data represented as the mean ± S.D. of triplicate measurements. Statistical analysis was performed using ANOVA with Dunnett's correction for multiple comparisons, indicating ****p < 0.0001 for the difference from control cells and ^#### p < 0.0001 for the difference from TG‐treated cells. (b) H1299 and A549 cells were first transfected with MRP1 siRNA, At 48 h post‐transfection, cells were treated with Diaminodichloroplatinum (DDP, 30 μM) or Doxorubicin (30 μM) in the presence or absence of TG (30 nM). Flow cytometry analysis revealed MRP1 siRNA rescued increased cell apoptosis in TG‐treated cells depletion of MRP1 by siRNA rescued increased cell apoptosis in TG‐treated cells (**p < 0.01 for difference from control cells, ^## p < 0.01 for the difference from DDP or Doxorubicin‐treated cells, ^$$ p < 0.01 for difference from DDP or Doxorubicin and TG‐treated cells by ANOVA with Dunnett's correction for multiple comparisons). (c) The A549 and H1299 cells were treated with DDP or Doxorubicin for different time point (Left panel) and various concentrations (Right panel) in the presence or absence of TG (30 nM). MTT assay was used to detect cell viability (*p < 0.05, **p < 0.01, ***p < 0.001 for difference from control cells). (d) After transfecting with MRP1 siRNA for 48 h, cells were treated with DDP (30 μM) or Doxorubicin (30 μM) for 36 h in the presence or absence of TG (30 nM). MTT assay was used to detect cell viability. (****p < 0.001 for difference from control cells, ^#### p < 0.0001 for the difference from DDP or Doxorubicin‐treated cells, ^$$$$ p < 0.001 for difference from DDP or Doxorubicin and TG‐treated cells by ANOVA with Dunnett's correction for multiple comparisons). (e) The A549 and H1299 cells were first transfected with MRP1 siRNA. At 48 h post‐transfection, cells were treated with etoposide, DDP, and Doxorubicin in the presence and absence of TG followed by Western blot analysis of Cleaved‐PARP.

FIGURE 3

SREBP1 Mediated Regulation of MRP1 Expression Induced by Endoplasmic Reticulum Stress. (a) After treating cells with TG (30 nM), the mRNA levels of MRP1 were assessed by real‐time qPCR. (b) TG (30 nM) and exogenous XBP1 cDNA significantly increased the promoter activity of MRP1. (c) There are two putative binding sites for SREBP1 on the MRP1 promoter. (d) Following 48‐h transfection of SREBP1 cDNA, Western blot results revealed the expression of full‐length SREBP1, cleaved (active) SREBP1, and MRP1. (e) H1299 and A549 cells were transfected for 48‐h with SREBP1 siRNA or control siRNA. Western blot results revealed the expression of full‐length SREBP1, cleaved SREBP1 (active), and MRP1. (f) After transfection cells with SREBP1 siRNA, cells were further treated with TG (30 nM) for an additional 12 h. Western blot analysis revealed the expression of p‐IRE1α, XBP1s, SREBP‐P, SREBP‐N, and MRP1. (g) 48 h before Western blot experiments, cells were co‐transfected with IRE1α cDNA and SREBP1 siRNA. Western blot analysis revealed the expression of IRE1α, XBP1s, SREBP‐P, SREBP‐N, and MRP1. (h) Western blot analysis of cytosolic and nuclear extracts of SREBP1from TG (30 nM) or untreated cells. (i) H1299 cells were co‐transfected with MRP1 promoter reporter construct 1412 and SREBP1 cDNA plasmid. 48 h after transfection, luciferase activity was determined and normalized using the dual luciferase reporter system (***p < 0.001 for difference from control cells). (j) H1299 cells were co‐transfected with MRP1 promoter reporter construct 1412 and SREBP1 siRNA. Transfection of SREBP1 siRNA significantly reduced the promoter activity of MRP1. (k) The cells were co‐transfected with MRP1 promoter and SREBP1 siRNA in the presence and absence of TG (30 nM) (Left panel) or co‐transfection with IRE1α cDNA (Right panel). Luciferase activity was determined and normalized using the dual luciferase reporter system (**p < 0.01, ***p < 0.001 for difference from control cells, ^### p < 0.001 for the difference from TG or transfected with IRE1α cDNA cells by ANOVA with Dunnett's correction for multiple comparisons). (l) Schematic representation of two designated constructs of MRP1 promoter (1412‐Mut and 408). (m) Cells were co‐transfected either MRP1 promoter reporter construct 1412 or mutated MRP1 promoter reporter (1412‐Mut) with SREBP1 cDNA. After 48‐h transfection, luciferase activity was determined and normalized using the dual luciferase reporter system. (***p < 0.001 for difference of wild‐type MRP1 promoter, ^#### p < 0.0001 for difference of mutated MRP1 promoter). (n) The cells were either co‐transfected MRP1 promoter reporter construct 1412 or the short length MRP1 promoter construct 408 with SREBP1 cDNA. Luciferase activity was determined and normalized using the dual luciferase reporter system. (o) qRT‐PCR were taken to analyze the CUT&Tag results. qRT‐PCR products were run on a gel (Right panel). qRT‐PCR results were quantified and are indicated (Left panel) (***p < 0.001 for difference from IgG‐added cells). (p) After transfecting with SREBP1 siRNA alone or co‐transfected with IRE1α cDNA for 48 h, cells were treated with etoposide (40 μM) in the presence or absence of TG (30 nM). MTT assay was used to detect cell viability. (****p < 0.001 for difference from control cells, ^#### p < 0.0001 for the difference from etoposide‐treated cells, ^$$$$ p < 0.001 for difference from SREBP1 siRNA and TG‐treated cells by ANOVA with Dunnett's correction for multiple comparisons).

FIGURE 4

C‐Myc Orchestration of ER Stress‐Induced SREBP1 Activation in Lung Cancer Chemoresistance. (a) Western blot results revealed that transfection of c‐Myc cDNA increased the expression levels of SREBP1. (b) A549 cells were co‐transfected with the MRP1 promoter reporter construct and the c‐Myc cDNA plasmid. luciferase activity was determined and normalized using the dual luciferase reporter system (**p < 0.01, indicating differences relative to the untreated control group, assessed through ANOVA for multiple comparisons). (c) Following a 12‐h treatment with TG (15, 30 nM), Western blotting revealed the expression levels of c‐Myc. (d and e) 48 h after transfection cells with either IRE1α or XBP1s cDNA, Western blot was performed to show an elevation in the expression of IRE1α, XBP1s and c‐Myc. (f and g) Cell lysates were collected for Western blotting 48 h after co‐transfecting IRE1α‐ cDNA (f) or XBP1s‐ cDNA (g), and c‐Myc siRNA. Western blot results revealed the expression of IRE1α, XBP1s, c‐Myc, SREBP1 and MRP1. (h) The A549 and H1299 cells were first transfected with c‐Myc siRNA. After 48 h transfection, cells were treated with etoposide in the presence and absence of TG followed by MTT assay (****p < 0.001 for difference from control cells, ^#### p < 0.001 for the difference from etoposide‐treated cells, ^$$$$ p < 0.001 for difference from c‐Myc siRNA and TG‐treated cells by ANOVA with Dunnett's correction for multiple comparisons).