Biological sex is an independent risk factor of cancer. Men are three to five times more likely than women to develop bladder cancer even when known risk factors are taken into consideration. Development of sex in mammals is often viewed as a two-step process. The first step is sex determination, of which the XX and XY sex chromosome complements trigger gonad differentiation to form the ovary and testis, respectively. After that, sex hormones secreted by gonads initiate sexually dimorphic differentiation of nongonadal tissues. However, this model has been challenged by recent findings revealing an independent contribution of sex chromosomes to sexual dimorphism. In this chapter, we discuss how the sex chromosomes and sex hormones together cause gender disparities in bladder cancer. We propose a concept of epigenetic sex – epigenetic differences between males and females – and suggest that the sex epigenome is a previously unknown biasing factor contributing to gender disparities in bladder cancer.
- Bladder Cancer
- Gender Disparities
- Sex Hormones
- Sex Chromosomes
- Sex Epigenome
Bladder cancer (BC) originates primarily from the urothelium – the inner lining of bladder lumen. Men are disproportionally affected by the disease. Males are 3–5 times more likely than females to BC . This difference persists even after adjusting other known risk factors [2, 3, 4, 5], suggesting that male sex is an independent risk factor of BC.
Typical males have one copy each of the X and Y chromosomes (XY) while females have two copies of the X chromosome (XX). For XY individuals, sex-determining region Y (SRY) gene on the Y chromosome triggers gonadal differentiation to form testes, which secrete androgens and promote male primary and secondary sex characteristics. Females with XX chromosome complement have ovarian development and estrogen secretion leading to female primary and secondary sex characteristics .
Strong evidence exists that androgens acting through androgen receptor, promote bladder tumorigenesis . Female gonadal hormones acting through estrogen receptors also influence BC risk albeit playing a minor role when compared to androgens . While sex hormones clearly play important roles in gender disparities in BC, potential role of the sex chromosomes is not nearly as apparent. Because the sex chromosomes (
2. The impact of biological sex on bladder cancer development
2.1 The role of androgen
A retrospective study revealed that male patients who received α-reductase inhibitors before the diagnosis of BC had better survival and was positively correlated with duration of administration . Similarly, prostate cancer patients who received androgen deprivation therapy (ADT) had a lower incidence of BC compared with patients that did not . Paradoxically, a majority of expression analyses of androgen receptor (AR) in BC patients showed a negative correlation between AR expression and the aggressiveness of BC [12, 13, 14, 15, 16, 17]. Offering a possible explanation underlying this observation is our finding that reduced AR expression may lead to upregulation of cancer stem cell related genes such as CD44 . Clearly the role of androgens and AR in BC is complex and likely in molecular and cellular context-dependent manner.
Animal models of BC support the role of androgens/AR in modulating bladder tumorigenesis. Male mice were much more vulnerable than female mice to BC induced by N-butyl-N-(4- hydroxybutyl) nitrosamine (BBN), a bladder-specific carcinogen . This sex difference was blunted by castration. Similarly, Okajima and colleagues have observed male-biased responses to BBN-induced BC in rats. Moreover, administration of Diethylstilbestrol (DES), a nonsteroidal estrogen, suppressed bladder carcinogenesis in male rats. In contrast, testosterone supplementation increased the incidence of BC in female rats . Miyamoto
2.2 The role of estrogens and their receptors
Postmenopausal women had a higher risk of BC and early menopause enhanced this risk, suggesting that female sex hormones protect women from BC development [29, 30, 31]. Classical estrogen receptors (ERs) include estrogen receptor α (ERα) and estrogen receptor β (ERβ) . Clinically, inconsistent results existed between the expression of ERα and ERβ and the grades and stages of BC patients although more reports supported that expression of ERα favors a better while ERβ is associated with a worse prognosis [14, 32, 33, 34, 35, 36, 37, 38]. Experimentally, both whole body ERα KO or urothelium-specific ERα KO increased the incidence of BBN-induced BC in female mice. Disruption of ERα decreased the expression of Inositol polyphosphate-4-phosphatase, type II (INPP4B), resulting in a higher activation of AKT . On the contrary, whole body deletion of ERβ impeded BBN-induced BC in female mice . Moreover, knockdown (KD) of ERβ suppressed transformation of normal bladder cells and growth of BC cells partly through reducing expression of mini-chromosome maintenance complex component 5 (MCM5) because reintroduction of MCM5 into BC cells blunted ERβ KD phenotype . Interestingly, tamoxifen treatment conferred a chemoprevention in female mice against BBN-induced BC . Since tamoxifen is a selective estrogen-receptor modulator with mixed estrogenic and antiestrogenic activity depending on targeted tissues, it would be interesting to see which ERs, ERα or ERβ, is activated or inhibited and whether any of these receptors plays a more dominant role in the BBN-induced bladder carcinogenesis.
2.3 The role of sex chromosomes in driving gender disparities
A potential role of the sex chromosomes in gender disparities in BC was implicated initially by cancer epidemiological findings of Turner and Klinefelter patients. Turner syndrome is a genetic disorder of female X0 patients who lost one copy of the X chromosome. Conversely, Klinefelter syndrome has two or more copies of the X chromosome among the affected male patients. Turner patients displayed an increased risk of BC ; and Klinefelter patients had an overall reduced rate of solid tumors . These observations suggest that an extra copy of X chromosome is tightly associated with low BC risk in both sexes. However, because the sex chromosomes are tightly coupled with their respective sex/gonadal hormones, the confounding effect of sex chromosomes cannot be excluded. As a result, the sex hormone–independent roles of the sex chromosomes have largely been overlooked.
To overcome the aforementioned challenges of studying independent roles of the sex-biasing factors, De Vries
By taking advantage of the FCG mice, we showed that, independent of the sex hormones, the sex chromosomes had a sex-biasing effect on BC development . We further showed that regardless of gonadal sex XY mice had 2.55 times of higher chance of developing BBN-induced BC than XX mice, demonstrating an independent role of the sex chromosomes. This study has also confirmed the sex-biasing role of androgens and further revealed that having testes was 4.71 times more likely than having ovaries to develop BC. More strikingly, wild type male mice with the XY chromosome complement and testes were 12.39 times more likely than wild type female mice with the XX chromosome complement and ovaries. This is unexpected because it is close to the product of 2.55 and 4.71 instead of the sum. Such finding suggests that both the sex chromosomes and the sex hormones have independent and dependent sex-biasing effects on BC. Moreover, the sex chromosomes interact with the sex hormones to amplify the difference (Figure 1). The underlying mechanism of interaction between these sex-biasing factors is yet to be defined.
Because the Y chromosome is frequently lost in BC cells and its loss has been associated with a higher cancer risk [47, 48, 49], it is less likely that the Y chromosome explains the male dominance in BC. A more reasonable possibility is that copy number difference of the X chromosome may render females better protected than males. To understand the potential tumor suppressing role of the X chromosome, we have examined the X chromosome-linked genes that escape X chromosome inactivation (XCI) (Figure 1a) , hence, genes that are expressed in higher levels in XX than in XY urothelial cells. By comparing gene expression levels in the bladder urothelium of FCG mice, we have identified Lysine Demethylase 6A (KDM6A) as a top candidate of X-linked tumor suppressors .
2.4 The sex epigenome
Through the intrinsic histone demethylase activity, KDM6A regulates downstream gene transcription by antagonizing Polycomb Repressive Complex 2 (PRC2)-dependent epigenetic gene silencing program [60, 61, 62, 63]. Specifically, KDM6A catalyzes removal of the methyl groups from histone H3 lysine 27 trimethylation (H3K27me3), making H3K27 available for acetylation (H3K27ac). H3K27me3 and H3K27ac are closely associated with transcription repression and activation, respectively. In a demethylase-independent manner, KDM6A functions in the COMPASS-like protein complex that harbors MLL3/KMT2C and MLL4/KMT2D lysine methyltransferases . KMT2C and KMT2D catalyze formation of H3K4 monomethylation (H3K4me1), which is tightly linked to active transcription enhancers [65, 66, 67]. COMPASS and PRC2 display an antagonistic relationship in regulating downstream gene expression [68, 69]. Therefore,
Whole-genome transcriptome analysis of
A new concept of sex epigenome begins to emerge. In addition to gonadal hormones, the copy number difference of the X chromosome between males and females contributes to sex differences in BC - an extra copy of the X chromosome confers a better protection of females. Moreover, there is a cooperative interaction between the sex hormones and chromosomes during BC development. The tumor suppressing effect of the X chromosome is largely mediated by
This work was supported by the NIH/National Cancer Institute (1R01DK110477, 1R01HL136921, and 5R21CA249701 to X.L.).
Conflict of interest
The authors declare no conflict of interest.