AR and UPR gene expression are correlated in CRPC

We previously showed that androgen receptor (AR) activated the IRE1α-XBP1s signaling in LNCaP and VCaP cells that model the androgen-sensitive state of PCa11. To assess whether this applies to CRPC, we used two established CRPC models, 22Rv1 and C4-2B lines, both of which are responsive to, but not dependent on, androgens. Androgens significantly upregulated IRE1α and XBP1s expression, as well as that of XBP1s target P58IPK in both cell lines (Supplementary Fig. 1a). Consistently, there was a significant positive correlation between AR gene expression signature and IRE1 arm gene expression in four independent gene expression datasets from patients with both primary and metastatic PCa, including CRPC (Supplementary Fig. 1b). These data suggest that androgens are important for IRE1α-XBP1s arm activation in all phases of PCa.

Discovery and characterization of an optimized specific IRE1α inhibitor−MKC8866

MKC8866 (see Fig. 1a for chemical structure) was optimized and refined from a family of IRE1α-specific endoribonuclease inhibitors obtained from a chemical library screen14,15,16. Previous characterization of these compounds, including structural analyses, confirmed its specificity on IRE1α RNase activity15. Similar to its earlier versions, MKC8866 potently inhibited the RNase activity of human IRE1α in vitro with an IC 50 of 0.29 μM (Supplementary Fig. 2a). In MM1 myeloma cells, MKC8866 strongly inhibited DTT-induced XBP1s expression with an EC 50 of 0.52 μM (Supplementary Fig. 2b). Dose titration experiments in unstressed RPMI 8226 cells corroborated these data with an IC 50 of 0.14 μM (Supplementary Fig. 2c).

Fig. 1 Functional characterization of MKC8866 on IRE1α RNase activity. a Chemical structure of MKC8866. b LNCaP cells were cultured in regular growth medium and treated with 30 nM TG and the indicated doses of MKC8866 for 24 h. Protein expression was determined by Western analysis. XBP1s Western blot intensity was quantified and normalized to GAPDH from three different experiments, and used to calculate the IC 50 . c LNCaP cells were either left untreated (UT), or treated with vehicle (DMSO) or MKC8866 with indicated doses. Cell viability was measured after 3 days. d LNCaP cells were transfected with either empty vector (pCDNA3) or the pCDNA3-Flag-XBP1s plasmid, and treated with vehicle (DMSO) or 10 μM MKC8866. Cell viability was measured after 3 days. e Mice (n = 3) were orally dosed with indicated amounts of MKC8866 and its plasma concentration was profiled post treatment. f Mice (n = 3) were intraperitoneally injected with 1 mg kg−1 tunicamycin (Tm) for 4 h and orally gavaged with the indicated dosage of MKC8866 for 2 h before harvesting its liver. Quantified average inhibition ratio is shown below the corresponding treatment. g Mice (n = 5) were dosed with 1 mg kg−1 tunicamycin (Tm) and 4 mg kg−1 MKC8866 for the indicated time length before harvesting its kidney. Quantified average inhibition ratio is shown below the corresponding treatment. *P < 0.05, Student t-test, error bars denote SD Full size image

In LNCaP PCa cells, MKC8866 suppressed XBP1s expression in a dose-dependent manner under conditions of mild ER stress (30 nM thapsigargin, TG) with an IC 50 of 0.38 μM (Fig. 1b). The expression of two XBP1s target genes, BiP and P58IPK, were similarly inhibited, whereas phospho-IRE1α or total IRE1α, or phospho-eIF2α, levels were not affected (Fig. 1b). Similar results were obtained in three independent PCa cell lines (VCaP, 22Rv1, and C4-2B) modeling different stages of PCa (Supplementary Fig. 2d). MKC8866 suppressed the viability of all four cell lines in a dose-dependent manner under normal conditions, with the most robust effect in LNCaP cells (Fig. 1c and Supplementary Fig. 2e). The efficacy of MKC8866 was further increased in C4-2B cells under TG-induced mild ER stress (Supplementary Fig. 2f). Ectopically expressed XBP1s rescued the inhibitory effect of MKC8866 (Fig. 1d). The levels of ectopically expressed flag-XBP1s was a few-fold higher compared with endogenous XPB1s; whereas endogenous XBP1s expression was inhibited by MKC8866, that of flag-XBP1s was not affected (Supplementary Fig. 2g). Furthermore, MKC8866 potently impaired XBP1s levels induced either by androgen treatment (Supplementary Fig. 2h), or glucose deprivation (Supplementary Fig. 2i). In addition, MKC8866 had moderate oral bioavailability (30%) in mice, with maximum concentrations in blood observed at 4 h after oral administration (Fig. 1e); it also suppressed the tunicamycin (Tm)-induced XBP1 splicing in mouse liver (Fig. 1f), as well as in mouse kidney (Fig. 1g). These data suggest that MKC8866 effectively represses IRE1α-mediated XBP1 splicing in PCa cells and has favorable pharmacokinetic and pharmacodynamic properties.

IRE1α targeting inhibits PCa cell growth in vitro and in vivo

We next investigated the potential effect of MKC8866 on PCa cell growth under various culture conditions. MKC8866 significantly reduced colony formation in all four PCa cell lines tested under either anchorage-dependent or anchorage-independent conditions (Supplementary Fig. 3a). Prostatosphere growth of these cell lines, an indication of stemness, was also markedly inhibited in the presence of MKC8866, which was consistent with IRE1α or XBP1 knockdown experiments (Supplementary Fig. 3b), suggesting that IRE1α may be involved in mechanisms of tumor initiation in PCa.

Based on these favorable in vitro results, we next evaluated the efficacy of MKC8866 in vivo. Consistent with the in vitro findings, MKC8866 strongly inhibited xenografted tumor growth in all PCa cell lines tested (Fig. 2a). XBP1s expression was significantly lower in MKC8866-treated tumors compared to controls, confirming that MKC8866 was active in mice harboring the tumors and that IRE1α activity was appropriately inhibited in vivo (Supplementary Fig. 4a). In addition, there was a decrease in PCNA expression and an increase in cleaved Caspase-3 levels, indicating that MKC8866 treatment resulted in decreased proliferation and increased apoptosis, respectively (Supplementary Fig. 4b). Removal of MKC8866 during the course of the treatment resulted in rebounding of XBP1s levels and enhanced tumor growth (Fig. 2b and Supplementary Fig. 4c), indicating the importance of sustained MKC8866 application for its growth inhibitory effects. These results show that pharmacological targeting of IRE1α exerts potent antitumor effects in preclinical mouse models of PCa.

Fig. 2 Therapeutic efficacy of MKC8866 in preclinical mouse models of PCa. a Nude mice bearing LNCaP, VCaP, 22Rv1, or C4-2B xenografts were treated orally with either vehicle or 300 mg kg−1 MKC8866 daily and tumor growth was recorded. b Nude mice bearing VCaP or 22Rv1 tumors were treated with either vehicle or 300 mg kg−1 MKC8866 daily. At day 7 (indicated by the orange arrow), the MKC8866-treated mice were randomly divided into two groups, one of which continuously received MKC8866 of the same dosage while the other group was treated with vehicle for the rest of the experiment. c Nude mice bearing VCaP xenografts were orally treated with either vehicle, 200 mg kg−1 MKC8866 daily, or 300 mg kg−1 MKC8866 every other day. Tumor growth was recorded weekly. d Nude mice bearing VCaP xenografts were orally treated either with vehicle, MKC8866 (300 mg kg−1 every two days), enzalutamide (ENZA, 30 mg kg−1 every two days), or MKC8866+ENZA. Tumor weight was recorded at the end of the experiment. e As in d, but the treatments were vehicle, MKC8866 (300 mg kg−1 every two days), abiraterone acetate (AA, 20 mg kg−1 every two days), or a combination of both drugs. f As in d, but the treatments were vehicle, or MKC8866 (300 mg kg−1 every two days), or cabazitaxel (CABA, 5 mg kg−1) twice a week intraperitoneally, or a combination of both drugs. *P < 0.05, all P values were calculated by Student’s t-test, error bars denote the SEM Full size image

MKC8866 synergizes with clinical PCa drugs in vitro and in vivo

The striking effect of MKC8866 as monotherapy on PCa tumor growth in vivo suggests that it can potentially synergize with drugs that are currently in clinical use for PCa. To assess this, LNCaP cells were treated with sub-optimal doses of MKC8866 along with antiandrogens, including abiraterone acetate and enzalutamide, two drugs that are used in the management of CRPC, as well as taxanes, including docetaxel, cabazitaxel, and paclitaxel. There was additive inhibition on LNCaP cell viability when MKC8866 was combined with either abiraterone acetate, enzalutamide, paclitaxel, or docetaxel, while clear synergy was seen in combination with cabazitaxel (Supplementary Fig. 5a). In CRPC cell lines 22Rv1 and C4-2B, MKC8866 also showed additive effects with abiraterone acetate in vitro, but failed to re-sensitize these cells to enzalutamide (Supplementary Fig. 5b and 5c). These results suggested that there may be enhanced efficacy of currently used PCa drugs when administered in combination with MKC8866 in vivo.

In search for a potentially suitable dose of MKC8866 for an in vivo combination experiment, we reduced its concentration (200 mg kg−1) or frequency of its administration (every two days) in a pilot experiment. Daily treatment with the lower dose still strongly inhibited tumor growth, whereas the normal dose every other day was less effective and was thus chosen for further combinatorial tests in vivo (Fig. 2c).

There was strong synergy in tumor growth inhibition when MKC8866 was co-administered with enzalutamide (Fig. 2d). Hematoxylin and eosin (H&E) staining of the tumors revealed that combinatorial treatment had the lowest cell content concomitant with highest necrosis levels among the different treatment regimens demonstrating increased efficacy, which was accompanied by loss of nuclear PCNA expression and marked elevation in caspase-3 staining (Supplementary Fig. 6a–c). Western analysis confirmed reduced expression of XBP1s in tumors treated with both drugs compared to those treated with either vehicle or a single drug, while no significant differences were observed on ATF6α cleavage or eIF2α phosphorylation, indicating that the other canonical UPR arms were not affected (Supplementary Fig. 6d). Co-administration of MKC8866 with abiraterone acetate and cabazitaxel also synergistically inhibited tumor growth (Fig. 2e, f). None of the treatments significantly affected body weight of mice (Supplementary Fig. 6e), suggesting that there were no noteworthy toxicities. Taken together, these data demonstrate that in preclinical models MKC8866 synergizes with some of the central PCa drug regimens that are currently used in the clinic.

XBP1s is required for activation of the c-MYC transcriptional program

MKC8866 specifically inhibits the RNase activity of IRE1α thus decreasing XBP1s levels, which should thus be responsible for the phenotypic effects that we observe in PCa cells. To gain insight into this process and XBP1s-regulated genes, we performed RNA-seq analysis in LNCaP cells upon either XBP1 siRNA-mediated knockdown (siXBP1) or MKC8866-mediated IRE1α inhibition. Differential gene expression analysis of the RNA-seq data was highly concordant amongst experimental replicates for both siXBP1-treated and MKC8866-treated cells (Supplementary Fig. 7a and 7b). Both approaches robustly depleted XBP1s expression, indicated by a significant decrease in the mRNA levels of the spliced compared to the unspliced XBP1 isoforms (siXBP1: P < 2.2e−16, MKC8866: P < 2.2e−16, negative binomial generalized linear model) (Fig. 3a). This was further confirmed by qPCR analysis of XBP1s mRNA expression, as well as expression of its target genes RAMP4, EDEM1, and P58IPK (Fig. 3b). Interestingly, scatter-plot analysis showed that the down-regulated genes upon XBP1 knockdown and MKC8866 treatment showed high concordance, whereas no strong correlation was observed for the upregulated genes (compare lower left quadrant with other quadrants, Fig. 3c), indicating that XBP1s primarily functions as a transcriptional activator in PCa cells. Consistently, there was significant overlap (26 genes) between the top 100 down-regulated genes (ranked by P value) in each treatment (P < 2.2e−16, binomial test), whereas the overlap (three genes) was much smaller between the top 100 upregulated genes (P < 0.05, binomial test). This suggested that the down-regulated genes by IRE1α inhibition in PCa cells are largely mediated by XBP1s. We also validated the RNA-seq data by individual qPCR analysis of top 10 genes where their expression was inhibited upon both MKC8866 treatment and XBP1 knockdown (Supplementary Fig. 7c).

Fig. 3 IRE1/XBP1s pathway is required for multiple hallmark pathways in PCa. LNCaP cells were either left untreated or subjected to siRNA-mediated XBP1 knockdown. In parallel, cells were either treated with vehicle or MKC8866. Total RNA was isolated and used in NextGen sequencing. a The presence of the XBP1s junction is measured in the spliced RNA-seq reads and represented as a ratio of total spliced reads. The box represents the interquartile range, the horizontal line in box is the median, and the whiskers represent 1.5 times interquartile range. b The expression levels of XBP1s, XBP1u, and three XBP1s target genes were determined by qPCR. *P < 0.05, Student t-test, error bars denote SD. c Scatter plot representing the concordance of gene expression data obtained upon XBP1 knockdown and MKC8866 treatment. d Hallmark pathways enriched under both MKC8866-treated and XBP1 knockdown cells. Graph displays category scores as −log 10 (P value) from Fisher’s exact test. e Hallmark pathways MYC TARGETS V1 and V2 both enriched by GSEA on combined data of siXBP1 and MKC8866 treatment Full size image

Hallmark pathway enrichment analysis of the RNA-seq data showed that MKC8866 treatment and XBP1 knockdown affected similar pathways (Fig. 3d). As expected, UPR and Protein Secretion pathways were both strongly correlated with XBP1 gene expression profile (Supplementary Fig. 8a), mirroring the established central role of IRE1α-XBP1s signaling in these processes4,17. Interestingly, among the most highly enriched pathways by both treatments was signaling by c-MYC (ranked first in siXBP1 and second in MKC8866 by P value) (Fig. 3d), an oncoprotein which is very frequently deregulated in cancer12 and has been centrally implicated in PCa13. XBP1-induced gene expression changes were positively associated with two different hallmark signatures for MYC TARGETS, V1 and V2 (Fig. 3e). Furthermore, Kyoto Encyclopedia of Genes and Genome (KEGG) analysis demonstrated that the ribosome pathway ranks at the top in both conditions (Supplementary Fig. 8b and 8c), whereas gene set enrichment analysis (GSEA) revealed XBP1-deregulated genes were positively enriched in KEGG ribosome and protein export (Supplementary Fig. 8d). Together, these data indicate that XBP1 not only affects fundamental aspects of ER biology in PCa cells, consistent with previous findings in other tissues, but also impacts oncogenic c-MYC signaling.

XBP1s directly activates c-MYC expression

To explore whether XBP1s expression may indeed be linked to c-MYC expression in PCa, we analyzed published gene expression datasets for human PCa. Strikingly, there was a positive and strong correlation between XBP1s and c-MYC target gene expression in 9 out of 11 available cohorts (Fig. 4a, b, and Supplementary Fig. 9a). To assess whether this correlation extends to the protein level, serial sections of human prostatectomy samples were examined by immunohistochemistry for XBP1s and c-MYC. As shown in Fig. 4c, there was clear colocalization of XBP1s and c-MYC expression in human PCa specimens. Furthermore, XBP1s and c-MYC expression was significantly correlated in a tissue microarray (TMA) consisting of 260 human PCa specimens11,18 (Fig. 4d). These results suggest that XBP1s expression is functionally linked to c-MYC signaling in human PCa.

Fig. 4 XBP1s activates c-MYC expression and activity in human PCa. a The plots show significant positive correlation between XBP1s and c-MYC target gene expression in five independent PCa datasets. b The bar graph shows the P values of the correlation between XBP1s and c-MYC target gene expression in nine different human PCa cohorts, using the Pearson correlation test. c Consecutive sections of human PCa prostatectomy samples were immunostained for XBP1s and c-MYC. Representative images from one patient at two different magnifications are shown. Scale bars, 100 μΜ (upper panel) and 30 μΜ (lower panel). d Tissue microarrays containing 260 tumor samples stained with either a XBP1s or c-MYC-specific antiserum were scored. Dots represent values of individual samples; thick horizontal lines represent the median; box represents the upper and lower quartile; whiskers represent 1.5 times interquartile range. The P value indicates the significance of correlation between XBP1s and c-MYC scores by ANOVA. e LNCaP or VCaP cells were treated with either siXBP1 or MKC8866, and c-MYC levels were determined. f LNCaP cells were transfected with either empty vector or flag-XBP1s expression vector. XBP1s and c-MYC levels were then determined after 2 days. g LNCaP c-MYC inducible cells were treated with either siXBP1 or MKC8866 and then grown with or without doxycycline (Dox). Cell viability was measured after 3 days. h Same procedure was performed as in g, colony formation (CF) was measured after 2 weeks while prostatospheres (PS) after 1 week. i LNCaP cells were transfected with 1 μg of pGL3-MYC luciferase reporter plasmid plus either empty vector (pCDNA3) or the pCDNA3-Flag-XBP1s plasmid. Luciferase activity was determined after 48 h. Results are from a representative experiment in triplicate. * Indicates statistical differences in LUC values compared to vector-transfected cells (P < 0.05) by Student's t-test. j LNCaP cells were transfected with either empty pCDNA3 vector (Ctrl) or the pCDNA3-Flag-XBP1s plasmid. After 48 h, ChIP assay was performed using Flag antibody. The data are representative of two experiments in duplicate. Two responsive sites (#1 and #2) as well as one non-responsive site (#3) are presented. *P < 0.05, Student's t-test, error bars denote the SD Full size image

One potential mechanism for these observations is that c-MYC expression is regulated by the IRE1α-XBP1s axis. In support of this hypothesis, c-MYC protein expression was inhibited by XBP1 knockdown as well as MKC8866 treatment, in both LNCaP and VCaP cells, whereas levels of its heterodimer partner MAX and cofactor TRRAP were not significantly affected (Fig. 4e). Consistently, there was a significant decrease in c-MYC mRNA expression, as well as its target gene expression, represented by SHMT1, PCNA, MTHFD1, CDC25A, ATF4, and PPAT, whereas MAX and TRRAP mRNA levels were unchanged upon XBP1 knockdown or MKC8866 treatment (Supplementary Fig. 9b). In contrast, ectopic expression of XBP1s dose-dependently increased c-MYC protein expression in LNCaP (Fig. 4f) and VCaP cells (Supplementary Fig. 9c), which led to an increased resistance to the bromodomain inhibitor JQ1 (Supplementary Fig. 9d). In keeping with this finding, c-MYC immunohistochemical staining intensity was significantly weaker in xenograft tumor sections from MKC8866-treated mice compared with untreated mice (Supplementary Fig. 9e). Furthermore, XBP1-knockdown or MKC8866-mediated inhibition of LNCaP cell viability, colony formation, as well as prostatosphere growth were rescued, at least in part, upon inducible expression of c-MYC (Fig. 4g, h). Similar results were obtained upon c-MYC ectopic expression (Supplementary Fig. 9f and 9g). In addition, ectopic expression of flag-XBP1s activated a c-MYC promoter-driven luciferase reporter in a dose-dependent manner in LNCaP (Fig. 4i) and 293T cells (Supplementary Fig. 9h). TG-induced mild stress also activated the c-MYC reporter activity in 293T cells, while stronger stress did not, likely due to the translation inhibition by PERK-eIF2α activation (Supplementary Fig. 9i). Finally, chromatin immunoprecipitation (ChIP) analysis showed that XBP1s associated with two response elements in the c-MYC gene 5ʹ flanking region (Fig. 4j); consistently, the deletion of these elements in the c-MYC promoter significantly impaired the c-MYC reporter activation in 293T cells (Supplementary Fig. 9j), indicating that XBP1s directly regulates c-MYC transcription. Together, these data suggest that the IRE1α-XBP1s axis is required for c-MYC expression and function in PCa cells.

XBP1 gene expression signature is strongly associated with PCa prognosis

Integrated analysis of gene expression profiles upon XBP1 knockdown and MKC8866 treatment identified 733 genes which were significantly downregulated by XBP1 knockdown or MKC8866 (Supplementary Data 1). Next, we evaluated the possibility that the expression of these genes could serve as potential prognostic biomarkers for PCa. To assess this, a subset of significantly differentially expressed IRE1α-XBP1s genes was tested as disease-free survival predictors in three independent cohorts of PCa patients. To avoid cohort-dependent effects, the data was quantile normalized. Eventually, a five-gene combination, defined as the XBP1 signature, was identified using an unbiased prediction and feature selection algorithm for high-dimensional survival regression19. This five-gene signature, including ANLN, CSNK1G3, RRM2, SLC35A2, and UBAC2, demonstrated remarkable predictive power on disease-free survival in the TCGA (logrank test, P = 8 × 10−5), MSKCC (logrank test, P = 7.8 × 10−4), and GSE94767 datasets (logrank test, P = 2.6 × 10−4) (Fig. 5a–c). Further analyses showed significant correlation between elevated XBP1 signature gene expression and shorter biochemical recurrence (BCR)-free survival in the TCGA (logrank test, P = 1.9 × 10−2), GSE70768 (logrank test, P = 5.9 × 10−3), and GSE70769 datasets (logrank test, P = 5.5 × 10−4) (Fig. 5d–f). Taken together, these data show that XBP1 pathway activation correlates with poor survival in PCa patients and a gene signature derived from XBP1-regulated genes may have clinical utility.