Loss of p53 enhances the function of the endoplasmic reticulum through activation of the IRE1α/XBP1 pathway

Abstract

Altered regulation of ER stress response has been implicated in a variety of human diseases, such as cancer and metabolic diseases. Excessive ER function contributes to malignant phenotypes, such as chemoresistance and metastasis. Here we report that the tumor suppressor p53 regulates ER function in response to stress. We found that loss of p53 function activates the IRE1α/XBP1 pathway to enhance protein folding and secretion through upregulation of IRE1α and subsequent activation of its target XBP1. We also show that wild-type p53 interacts with synoviolin (SYVN1)/HRD1/DER3, a transmembrane E3 ubiquitin ligase localized to ER during ER stress and removes unfolded proteins by reversing transport to the cytosol from the ER, and its interaction stimulates IRE1α degradation. Moreover, IRE1α inhibitor suppressed protein secretion, induced cell death in p53-deficient cells, and strongly suppressed the formation of tumors by p53-deficient human tumor cells in vivo compared with those that expressed wild-type p53. Therefore, our data imply that the IRE1α/XBP1 pathway serves as a target for therapy of chemoresistant tumors that express mutant p53.

Keywords: ER function; IRE1α/XBP1 pathway; p53 target genes; tumor suppressor p53.

Figure 1. ER stress response in p53-deficient or knockdown cells

A. HCT116 p53^+/+ or HCT116 p53^−/− cells, B. MEF p53^+/+ or MEF p53^−/− cells, and C. U2OS shLuc or U2OS shp53 cells were incubated with Tm (0.5 μg/mL) or BFA (1 μg/mL) for the times indicated. Cell lysates were analyzed using western blotting with the indicated antibodies. The blot was cut based on the size of proteins or stripped. Total RNAs were extracted and subjected to RT-PCR analysis using specific primer sets for XBP1(U) and XBP1(S). Cell lysates were analyzed using western blotting with indicated antibodies.

Figure 2. IRE1α expression is regulated by p53

A. Western blot analysis of the expression of endogenous IRE1α in 23 human cancer cell lines. Cell lines were grouped according to expression of wild-type or mutant p53 as indicated. (A well between wt-p53 and mutant-p53 cell lines was cut, from the gel as indicated by a black line, due to the controversial p53 status of the cell line). Right panel: The intensities of the IRE1α bands (left panel) are expressed relative to those of β-actin. Values shown are the mean ± standard deviation (s.d.). The P value was calculated using two-way ANOVA. B. Downregulation of p53 expression induces increased expression of IRE1α. HCT116 p53^+/+ and U2OS cells were transfected with shLuc, shp53 (753), or shp53 (814), and selected using puromycin. Whole cell lysates of a pool of transfectants were analyzed using western blotting with the indicated antibodies. C. Overexpression of wild-type p53 inhibits IRE1α expression in mutant-p53 cell lines. Cell lysates, prepared 48 h after transfection with wild-type p53, were analyzed for the expression of indicated proteins. D. Mutant p53 proteins do not inhibit IRE1α expression. Cell lysates were prepared from cells transfected with p53-G245S, p53-R248W, p53-249S, and p53-R273H expression vectors or from cells that constitutively expressed wild-type p53 and were analyzed for the expression of the indicated proteins.

Figure 3. p53 stimulates IRE1α protein degradation

A. The level of p53 did not affect the expression of IRE1α mRNA. Total RNAs were extracted and subjected to qRT-PCR analysis using specific primer sets for IRE1α, p53, and GAPDH, and the data were normalized to those of GAPDH. Data shown are the mean ± s.d. (triplicates measured at the same time). B. p53 promotes IRE1α degradation. The indicated cells were treated with cycloheximide (25 μg/mL) for 1 h and incubated further for the indicated times. Cell lysates were analyzed for the expression of the indicated proteins. The blot was cut based on the size of the proteins of interest (left panel). The intensities of the IRE1α bands were determined (one of the gels is shown in left panel) and are expressed relative to those of β-actin (right panel). Values shown are the mean ± s.d. of three independent experiments.

Figure 4. Synoviolin promotes IRE1α degradation in a wild-type p53-dependent manner

A. SYVN1 suppresses IRE1α protein expression in wild-type p53 cells. HCT116 p53^+/+ or HCT116 p53^−/− cells were transfected with siControl (−) or siSYVN1 (+) and cultured for 24 h. Cell lysates were analyzed using western blotting with indicated the antibodies (left panel). The intensities of the SYVN1 bands were quantified. The levels of SYVN1 are reported relative to those of β-actin (right panel). The blot was cut based on the size of proteins or stripped and reprobed. B. IRE1α and SYVN1 interaction is suppressed in p53-deficient cells. Proteins were cross-linked with DSP before protein extraction. Coimmunoprecipitation was performed with cell lysate using an IRE1α or an SYVN1 antibody. C. SYVN1 interacts with wild-type p53. H1299 cells transiently expressed wild-type p53, p53-R248S, or p53-R273H. Coimmunoprecipitation experiments were performed using the anti-p53 antibody. D. p53-SYVN1-IRE1α complex is observed by treatment with proteasome inhibitor. H1299 cells transiently expressing wild-type p53 (left panel) or HCT116 p53^+/+ (right panel) cells were treated with 50 μM MG132 for 3 h. Coimmunoprecipitation experiments were performed using the anti-p53 antibody.

Figure 5. p53 deficiency increases secretory the function of the ER through the IRE1α/XBP1 pathway

A. HCT116 p53^+/+ or HCT116 p53^−/− cells, MEF p53^+/+ or MEF p53^−/− cells, and U2OS shLuc or U2OS shp53 cells expressing secreted embryonic alkaline phosphatase (SEAP) were transduced with a pSEAP2 control vector and washed 24 h after transduction. The medium was then changed, and the cells were cultured for another 6 h. Culture media were analyzed for SEAP activity, and luminescence was normalized to cell number. The transfection efficiencies of HCT116 p53^+/+ and HCT116 p53^−/− cells were approximately 80% each (data not shown). B. Overexpression of wild-type p53 inhibited SEAP activity. SEAP activities of cells that constitutively expressed the indicated p53 molecules were analyzed using the same procedure described in (A). C. HCT116 p53^−/− cells that expressed SEAP were transfected with siControl, siATF6, siPERK, or siIRE1α, cultured for 24 h, and following a change of medium, the cells were cultured for another 6 h. Whole cell lysates were analyzed using western blotting with the indicated antibodies, and culture supernatants were analyzed for SEAP activity. Values shown are the mean ± s.d. of three different experiments simultaneously measured. The P value was calculated using two-way ANOVA.

Figure 6. The IRE1α inhibitor (STF-083010) suppresses the growth in vitro and in vivo of p53-deficient human cancer cells

A. Effects of an IRE1α inhibitor on cell viability and on Tm-induced cell death in p53-deficient cells. HCT116 p53^+/+ or HCT116 p53^−/− cells, MEF p53^+/+ or MEF p53^−/− cells, and U2OS shLuc or U2OS shp53 cells were treated with Tm (0.5 mg/mL), STF-083010 (50 μM), or both for 24 h. Cell viability was determined using an MTT assay. Values shown are the mean ± s.d. of three different experiments measured simultaneously. B. An IRE1α inhibitor selectively suppresses the growth of p53-deficient tumors in nude mice. HCT116 p53^+/+ and HCT116 p53^−/− cells were used to engraft nude mice, and 4 days after injecting the cells, DMSO or STF-083010 (40 mg/kg) was intraperitoneally administered once every 3 days. Tumor volume was measured on the indicated days. After 15 days, the weights of the tumors (left panel) were measured. Values shown are the mean ± standard error of the mean of eight mice from each group. The P value was calculated using two-way ANOVA.