Characterization of PHGDH expression in bladder cancer: potential targeting therapy with gemcitabine/cisplatin and the contribution of promoter DNA hypomethylation

Abstract

d-3-Phosphoglycerate dehydrogenase (PHGDH) conducts an important step in the synthesis of serine. Importantly, the PHGDH gene is often amplified in certain cancers. Our previous studies revealed that PHGDH gene amplification was associated with poor overall survival in clear cell renal cell carcinoma (ccRCC) and that metabolic reprogramming of serine synthesis through PHGDH recruitment allowed ccRCC cells to survive in unfavorable environments. There have been no investigations of the role of PHGDH expression in bladder cancer (BC). In this investigation, we examined the clinical importance of PHDGH in BC. Furthermore, we asked whether PHGDH expression could be exploited for BC therapy. Finally, we investigated the regulatory mechanisms that modulated the expression of PHGDH. Using data from The Cancer Genome Atlas, we found that patients with high-grade BC had significantly higher PHGDH expression levels than did those with low-grade BC. In addition, patients with high PHGDH expression did not survive as long as those with low expression. PHGDH downregulation by si-RNAs or an inhibitor in BC cell lines significantly inhibited proliferative ability and induced apoptosis. Furthermore, combined treatment using a PHGDH inhibitor and gemcitabine/cisplatin achieved synergistic tumor suppression compared to use of a single agent both in vitro as well as in vivo. Mechanistic analyses of PHGDH regulation showed that PHGDH expression might be associated with DNA copy number and hypomethylation in BC. These findings suggest novel therapeutic strategies could be used in BC. Finally, our data enhance our understanding of the role of PHGDH in BC.

Keywords: GC therapy; PHGDH; apoptosis; bladder cancer; methylation.

Fig. 1

Clinical significance of PHGDH expression in BC according to TCGA data. (A) Left: the expression levels of PHGDH mRNA in normal human bladders and BCs. Right: the significant positive correlation between PHGDH expression and pathological grade (P < 0.0001). In BLCA, G1 was for ‘Low grade’ and G3 for ‘High grade’. The Mann–Whitney U‐test was performed to assess the statistical relationship, and error bars are represented as mean ± SD. (B) Overall survival (left) and disease‐free survival periods (right) were significantly shortened in patients with high PHGDH expression defined as Z‐score > 0 compared with those in patients with low PHGDH expression defined as Z‐score ≤ 0 (P = 0.0032 and P = 0.0218, respectively). The Kaplan–Meier method and log‐rank test were performed to assess the statistical relationship.

Fig. 2

PHGDH inhibition by si‐RNA and inhibitor. (A) Immunoblotting analysis showed that PHGDH expression was elevated in all BC cells compared to breast cancer cells. PHGDH expression values normalized by β‐actin were indicated. (B) Cell proliferation after treatment with PHGDH si‐RNA (*P < 0.01, **P < 0.001). The Bonferroni‐adjusted Mann–Whitney U‐test was performed to assess the statistical relationship, and error bars are represented as mean ± SD. (C) Apoptosis levels after treatment with PHGDH si‐RNA (*P < 0.02, **P < 0.001). The representative quadrant figures of apoptosis assay determined by flow cytometry are shown. Early apoptotic cells can be seen in the bottom right quadrant and late are in the upper right (lower). The Bonferroni‐adjusted Mann–Whitney U‐test was performed to assess the statistical relationship, and error bars are represented as mean ± SD. (D) Decreased Ki‐67 and increased cleaved caspase3 levels in si‐PHGDH‐transfected BC cells. PHGDH expression values normalized by β‐actin are indicated. Each experiment was carried out in triplicate.

Fig. 3

PHGDH inhibition by a PHGDH inhibitor. (A) Cell proliferation after treatment with a PHGDH inhibitor (CBR‐5884) (*P < 0.01, **P < 0.001). The Bonferroni‐adjusted Mann–Whitney U‐test was performed to assess the statistical relationship, and error bars are represented as mean ± SD. (B) Apoptosis after treatment with CBR‐5884 (*P < 0.0001). The representative quadrant figures of apoptosis assay determined by flow cytometry are shown. Early apoptotic cells can be seen in the bottom right quadrant and late are in the upper right (right). Each experiment was carried out in triplicate. The Bonferroni‐adjusted Mann–Whitney U‐test was performed to assess the statistical relationship, and error bars are represented as mean ± SD.

Fig. 4

PHGDH overexpression in PHGDH‐downregulated cells. (A) Immunoblotting analysis showed that PHGDH expression was significantly elevated in UMUC cells (P = 0.0495). The Mann–Whitney U‐test was performed to assess the statistical relationship, and error bars are represented as mean ± SD. (B) Cell proliferation of parental and PHGDH‐overexpressing cells (P = 0.0039). The Mann–Whitney U‐test was performed to assess the statistical relationship, and error bars are represented as mean ± SD. (C) Representative image of colony formation by parental and PHGDH‐overexpressing UMUC cells (magnification, ×1). The graph showed the ratio of the number of colonies by parental and PHGDH‐overexpressing cell (P = 0.0209). Each experiment was carried out in triplicate. The Mann–Whitney U‐test was performed to assess the statistical relationship, and error bars are represented as mean ± SD.

Fig. 5

PHGDH inhibition promoted gemcitabine‐ and cisplatin‐induced antitumor effects. (A) Cell proliferation after cisplatin (CDDP) (left) or gemcitabine (GEM) (right) treatment in the absence or presence of a PHGDH inhibitor (NCT‐503) (*P < 0.01, **P < 0.001). The Bonferroni‐adjusted Mann–Whitney U‐test was performed to assess the statistical relationship, and error bars are represented as mean ± SD. (B) Time course of tumor volumes formed by subcutaneously injected BOY cells into nude mice. Four groups were examined: (a) vehicle, (b) GC (GEM 150 mg·kg⁻¹, i.p., days 7 and 14. CDDP 6 mg·kg⁻¹, i.p., days 6 and 13), (c) NCT‐503 (40 mg·kg⁻¹, 5 times a week), or (d) a combination of GC and NCT‐503 (*P < 0.01) (n = 4 for vehicle or NCT‐503 group, n = 6 for GC or combination group). The Bonferroni‐adjusted Mann–Whitney U‐test was performed to assess the statistical relationship, and error bars are represented as mean ± SD. (C) Detection of apoptotic cells by TUNEL in tumor xenografts. Bright cells indicate apoptotic cells. Each experiment was carried out in triplicate.

Fig. 6

PHGDH expression was positively correlated with PSAT1, PSPH, and SHMT1 expression in cancer cells. (A) Flowchart of the strategy for identification of enriched metabolic pathways in the PHGDH high group. (B) Pathway analysis by Enrichr was performed using the HumanCyc Database. Fisher's exact test, followed by the Benjamini–Hochberg procedure was performed to assess the statistical relationship. (C) Spearman's rank test demonstrated that PHGDH and PSAT1, PSPH or SHMT1 mRNA expression levels were positively correlated (each P < 0.0001). (D) Expression of PSAT1, PSPH, or SHMT1 mRNA was positively correlated with PHGDH mRNAs expression in BC and breast cancer cell lines. Each value indicates the relative ratio compared to PHGDH mRNA expression in BOY cells. (E) Positive correlations between the expression of PHGDH and PSAT1, PSPH or SHMT1 mRNAs were observed in other cancer cell lines (left). Each value indicates the correlation coefficient of the heat map (right). Spearman's rank test was performed to assess the statistical relationship.

Fig. 7

Relationship between PHGDH mRNA expression and DNA methylation or copy number. (A) Spearman's rank test indicated a negative correlation between PHGDH mRNA expression and DNA methylation (left), and a positive correlation between PHGDH mRNA expression and its copy number (right). (B) Correlation between PHGDH, PSAT1, PSPH or SHMT1 mRNA expression and PHGDH copy number, methylation, or mRNA expression. Spearman's rank test was performed to assess the statistical relationship. (C) PHGDH expression was decreased by SAM in BC cells. PHGDH expression values are normalized by β‐actin as indicated. Experiments were carried out in triplicate. The Bonferroni‐adjusted Mann–Whitney U‐test was performed to assess the statistical relationship, and error bars are represented as mean ± SD. (D) Overall survival (left) and disease‐free survival periods (right) were shortened in patients with low methylation of PHGDH compared with those in patients with high methylation (0.0673 and P = 0.2438, respectively). The Kaplan–Meier method and log‐rank test were performed to assess the statistical relationship.