Research ArticleCANCER

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

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Science Advances  13 Jun 2018:
Vol. 4, no. 6, eaar5598
DOI: 10.1126/sciadv.aar5598
  • 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; Padj < 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.

  • Table 1 Independent and combined biasing effects of the sex chromosomes and gonadal hormones in BCa.

    The HR is determined using the Cox proportional hazard model. Chro, sex chromosomes; n, mouse numbers; O, ovary; T, testis.

    nHRP95% CI
    Chro (XY vs. XX)(52, 44)2.5490.00021.55–4.28
    Gonad (T vs. O)(49, 47)4.714<0.00012.77–8.28
    Combination(29, 24)12.39<0.00015.54–31.63

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/6/eaar5598/DC1

    fig. S1. Schematic diagram of the BBN-induced mouse BCa model.

    fig. S2. KDM6A functions as a demethylase-dependent and demethylase-independent tumor suppressor of BCa.

    fig. S3. Schematic diagram and results of the BBN-induced BCa regimen of urothelium-specific Kdm6a cKO mice.

    fig. S4. KDM6A expression levels in cancer samples are higher among female than male patients.

    fig. S5. Kaplan-Meier analysis of overall survival of cancer patients stratified by KDM6A expression levels.

    fig. S6. Candidate XCI escape genes and their mutation rates in human BCa.

    fig. S7. Expression of the candidate XCI escape genes in BCa tissues from female and male patients.

    fig. S8. Kaplan-Meier analysis of overall survival of cancer patients stratified by expression levels of the candidate XCI escape genes.

    fig. S9. A model of molecular basis underlying the sex differences in cancer.

    table S1. BCa HR of mice with different sex chromosome complement (XX versus XY) or Kdm6a status (wt versus mut).

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Schematic diagram of the BBN-induced mouse BCa model.
    • fig. S2. KDM6A functions as a demethylase-dependent and demethylase-independent tumor suppressor of BCa.
    • fig. S3. Schematic diagram and results of the BBN-induced BCa regimen of urothelium-specific Kdm6a cKO mice.
    • fig. S4. KDM6A expression levels in cancer samples are higher among female than male patients.
    • fig. S5. Kaplan-Meier analysis of overall survival of cancer patients stratified by KDM6A expression levels.
    • fig. S6. Candidate XCI escape genes and their mutation rates in human BCa.
    • fig. S7. Expression of the candidate XCI escape genes in BCa tissues from female and male patients.
    • fig. S8. Kaplan-Meier analysis of overall survival of cancer patients stratified by expression levels of the candidate XCI escape genes.
    • fig. S9. A model of molecular basis underlying the sex differences in cancer.
    • table S1. BCa HR of mice with different sex chromosome complement (XX versus XY) or Kdm6a status (wt versus mut).

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