Tumor-suppressor genes that escape from X-inactivation contribute to cancer sex bias

Abstract

There is a striking and unexplained male predominance across many cancer types. A subset of X-chromosome genes can escape X-inactivation, which would protect females from complete functional loss by a single mutation. To identify putative 'escape from X-inactivation tumor-suppressor' (EXITS) genes, we examined somatic alterations from >4,100 cancers across 21 tumor types for sex bias. Six of 783 non-pseudoautosomal region (PAR) X-chromosome genes (ATRX, CNKSR2, DDX3X, KDM5C, KDM6A, and MAGEC3) harbored loss-of-function mutations more frequently in males (based on a false discovery rate < 0.1), in comparison to zero of 18,055 autosomal and PAR genes (Fisher's exact P < 0.0001). Male-biased mutations in genes that escape X-inactivation were observed in combined analysis across many cancers and in several individual tumor types, suggesting a generalized phenomenon. We conclude that biallelic expression of EXITS genes in females explains a portion of the reduced cancer incidence in females as compared to males across a variety of tumor types.

Figure 1. Escape from X-inactivation Tumor Suppressor (EXITS) genes

(a) SEER data of annual incidence rates over time for the indicated cancer types in males (blue), females (green), or all patients (black). (b-c) The EXITS hypothesis: (b) “Traditional” tumor suppressor genes (TSGs) on chrX are represented in the top row for females and males. A single deleterious mutation in a TSG is equally likely to occur in male and female cancers because males have only one chrX, and females have one active chrX (Xa, pink) and one inactive chrX (Xi, purple). (c) On the bottom row is a model for EXITS gene behavior. In females, there are two active alleles of EXITS genes and therefore females are protected from complete gene loss after a single alteration. Complete inactivation of an EXITS gene may require biallelic mutations, or mutation with loss of the other chrX. In males, one mutation could inactivate the only allele of an EXITS gene that has no functional Y homolog, and therefore males would be more likely to develop cancers associated with mutations in those TSGs. Alternatively, because some genes that escape X inactivation have chrY homologs with redundant function, cancers with mutations in those genes would be more likely to occur in males who also have somatic loss of chrY.

Figure 2. Genes with higher frequencies of somatic loss-of-function (LOF) alterations in male cancers

Permutation testing for genes on chrX across all cancer datasets is shown. The log₂ M:F ratio of events is plotted for each gene against the significance (P) value. The size and color of each circle represent the number of (a) LOF mutations, or (b) LOF mutation/CN loss events in that gene. Genes with significantly higher (FDR<0.1) frequencies of mutation in male cancers are identified. Disease-specific permutation testing of LOF mutations in (c) lower-grade glioma and (d) clear cell kidney cancer is shown.

Figure 3. EXITS gene alterations are associated with male cancers

(a) Calculation of the number of tumor/normal pairs needed for 80% power to detect 4-fold male-biased LOF mutations with FDR<0.1 (i.e., 4x more prevalent in male than female tumors). The x-axis represents the fraction of all mutations on chrX occurring in males in the cohort (a function of the M:F ratio of disease incidence and overall mutation rate in males and females). Lines represent the percentage of cancers in a given tumor type that harbor a specific mutation (blue, 2%; red, 5%; yellow, 10%; purple, 20%). Each of the 21 tumor types we analyzed is plotted to show the power we had to detect a male-biased mutation based on the fraction of mutations on chrX in males and number of tumors/normal pairs in the dataset. BLCA, bladder carcinoma; CLL, chronic lymphocytic leukemia; CRC, colorectal carcinoma; DLBCL, diffuse large B cell lymphoma; ESO, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous carcinoma; KIRC, clear cell kidney cancer; KIRP, papillary kidney cancer; LAML, acute myeloid leukemia; LGG, lower-grade glioma; LUAD, lung adenocarcinoma; LUSC, lung squamous carcinoma; MED, medulloblastoma; MEL, melanoma; MM, multiple myeloma; NB, neuroblastoma; PAAD, pancreatic ductal adenocarcinoma; RHAB, rhabdoid tumor; STAD, stomach adenocarcinoma; THCA, thyroid carcinoma. (b) RNA-seq expression levels (log₂) for DDX3X, KDM5C, and KDM6A in head and neck squamous carcinoma (HNSC) and clear cell kidney cancer (KIRC) in the TCGA datasets, separated by patient sex (data visualization from www.cbioportal.org). Each dot represents one tumor; blue symbols have no mutation in the gene, and red have a mutation of the indicated type (P<0.0001 for all female-male expression comparisons by K-S test, either including or excluding mutated cases, see also Supplementary Figure 8). (c) M:F ratio of LOF mutations in the EXITS genes identified in Table 1, all other chrX escape (n=56), or chrX non-escape genes (data compared by t-test; bar represents median;‘+’, mean; box, interquartile range; whiskers,10–90%ile). (d) M:F ratio of LOF mutations in the EXITS genes that have functional Y homologs (DDX3X, KDM5C, and KDM6A), all other chrX genes with predicted functional Y homologs (n=14), or chrX genes without a Y homolog (data compared by t-test; plotted as in (c)).