X chromosome protects against bladder cancer in females via a KDM6A-dependent epigenetic mechanism

Abstract

Men are much more likely than women to develop bladder cancer (BCa), but the underlying cause of this gender disparity remains poorly defined. Using sex-reversed mice, we show that the sex chromosome complement is an independent cause and, moreover, amplifies the biasing effects of sex hormones. We also show that the X-linked lysine demethylase 6A (KDM6A) is a sexually dimorphic gene. Wild-type but not catalytically dead KDM6A confers sustained tumor suppressor activity in vitro. Knockout of mouse Kdm6a reduces expression of Cdkn1a and Perp, canonical gene targets of the tumor suppressor p53. Consistently, loss of Kdm6a increases BCa risk in female mice, and mutations or reduced expression of human KDM6A predicts poor prognosis of female BCa patients. Collectively, the study reveals that the X chromosome protects against BCa among females via a KDM6A-dependent epigenetic mechanism and further suggests that KDM6A is a prototypical sex-biasing tumor suppressor with both demethylase-dependent and demethylase-independent activities.

Fig. 1. Sex chromosomes play an important and independent role in sex difference in BCa.

(A) Schematic diagram of the sex-reversed or FCG mice. (B) Outline of the BBN-induced BCa regimen. BBN (0.1%) is supplied to mice (7 to 8 weeks old) in drinking water for 14 weeks. Mice are monitored daily for death or moribundity. (C and D) Kaplan-Meier analysis of overall survival of the FCG mice after BBN exposure (C) and P values are shown in (D) (log-rank test). Mice that survive the 40-week regimen are considered as censored.

Fig. 2. KDM6A functions as a demethylase-dependent and demethylase-independent tumor suppressor of BCa.

(A) Venn diagram of the DEGs identified by RNA-seq analysis of bladder urothelium. (B) Quantitative Kdm6a expression levels [quantitative reverse transcription polymerase chain reaction (qRT-PCR)] in bladder urothelium. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is used as an internal control. n = 3, Student’s t test. (C and D) Cell proliferation rate (C) and anchorage-independent growth in soft agar assay (D) are measured and compared between MB49 BCa cells that express either wild-type (WT) or the catalytically dead (Mut) KDM6A. Mock, parental cells with vector control; *P < 0.05, Student’s t test. (E and F) Schematics of the doxycycline (Dox)–inducible strategy (E) to express transiently the myc-tagged wild-type (WT) or mutant (Mut) KDM6A (myc-KDM6A) in UM-UC-13 cells. Cell lysates were immunoblotted using Myc and GAPDH-specific antibodies (F). (G and H) Cell proliferation assays. D0, without Dox; D4, transient Dox induction for 4 days; D7, persistent Dox induction for 7 days; *P < 0.05, Student’s t test.

Fig. 3. Knockout of Kdm6a significantly increases BCa risk among female mice.

(A) Kaplan-Meier analysis of overall survival of control and Kdm6a cKO mice under the BBN-induced BCa regimen. ^aP = 0.0048, XXF Kdm6a cKO versus XXF WT; ^bP = 0.0025, XXF Kdm6a cKO versus XYM WT; ^cP = 0.0261, XXF Kdm6a cKO versus XYM Kdm6a cKO; ^dP = 0.2732, XYM Kdm6a cKO versus XYM WT; log-rank test. (B) Volcano plot of the DEGs of bladder urothelium between wild-type and homozygous Kdm6a cKO females (n = 2; P_adj < 0.05). (C) qRT-PCR analysis of the candidate Kdm6a gene targets Cdnk1a and Perp in bladder urothelium of Kdm6a cKO mice. (D and E) qRT-PCR analysis of endogenous CDKN1A (D) and PERP (E) gene expression in UM-UC-13 cells that express either wild-type or catalytically dead KDM6A. *P < 0.05; ns, not significant, Student’s t test. (F and G) Chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) assays of UM-UC-13 cells shown in (D) and (E) using H3K27me3- and H3K4me4-specific antibodies. Location of the qPCR primer relative to the transcription start site (TSS) is indicated. *P < 0.01, Student’s t test. (H and I) qRT-PCR analysis of Cdnk1a (H) and Perp (I) gene expression in bladder urothelium of Eed cKO mice. GAPDH is used as an internal control. n = 3, Student’s t test.

Fig. 4. Mutations and reduced expression of human KDM6A predict poor prognosis of female patients with BCa.

(A and B) Outlier box plot of KDM6A gene expression in BCa samples from males and females (A) and from different cancer stages (B). Student’s t test. (C and D) KDM6A mutations (C) and decreased expression (D) correlate tightly to poor disease-free survival of female but not of male patients with BCa. Kaplan-Meier analysis, log-rank test.