Concurrent alterations in TERT, KDM6A, and the BRCA pathway in bladder cancer

Abstract

Purpose: Genetic analysis of bladder cancer has revealed a number of frequently altered genes, including frequent alterations of the telomerase (TERT) gene promoter, although few altered genes have been functionally evaluated. Our objective is to characterize alterations observed by exome sequencing and sequencing of the TERT promoter, and to examine the functional relevance of histone lysine (K)-specific demethylase 6A (KDM6A/UTX), a frequently mutated histone demethylase, in bladder cancer.

Experimental design: We analyzed bladder cancer samples from 54 U.S. patients by exome and targeted sequencing and confirmed somatic variants using normal tissue from the same patient. We examined the biologic function of KDM6A using in vivo and in vitro assays.

Results: We observed frequent somatic alterations in BRCA1 associated protein-1 (BAP1) in 15% of tumors, including deleterious alterations to the deubiquitinase active site and the nuclear localization signal. BAP1 mutations contribute to a high frequency of tumors with breast cancer (BRCA) DNA repair pathway alterations and were significantly associated with papillary histologic features in tumors. BAP1 and KDM6A mutations significantly co-occurred in tumors. Somatic variants altering the TERT promoter were found in 69% of tumors but were not correlated with alterations in other bladder cancer genes. We examined the function of KDM6A, altered in 24% of tumors, and show depletion in human bladder cancer cells, enhanced in vitro proliferation, in vivo tumor growth, and cell migration.

Conclusions: This study is the first to identify frequent BAP1 and BRCA pathway alterations in bladder cancer, show TERT promoter alterations are independent of other bladder cancer gene alterations, and show KDM6A loss is a driver of the bladder cancer phenotype.

Conflict of interest statement

Conflicts of Interest: The authors have no conflicts to disclose.

©2014 American Association for Cancer Research.

Figures

Figure 1. Altered BC genes

A, 41 BC genes with somatic nonsynonymous and SJ alterations. Top histogram, NGS predicted variants by nucleotide change for non-muscle invasive (NMI, stage Ta, T1) and muscle invasive (MI, stage T2-T4) tumors. Central panel, genes (left) and CRM-DA functions (teal color, see text); asterisk, a proven or likely oncogene; black dot, tumor has ≥1 somatic mutation; histogram, right side, percent of tumors with somatic gene mutation; bold or hatched box, CNV; bottom, tumor IDs (1-14). B, Somatic alterations in five CRM-DA BC genes (top) in 40 validation tumors (left side), annotated as in panel A.

Figure 2. Chromatin remodeling genes frequently altered in BC

A, Locations of somatic alterations on annotated proteins. BAP1: ubiquitin C terminal hydrolase domain (UCH, purple); HCF-1 binding motif (orange line); BRCA1 binding region (BRCA1 Domain, red); UCH37-like domain (ULD, gray); NLS, aa 717-722 (green); active site residues (vertical arrows). Adapted from (21, 51). ARID1A: LXXLL motif (black line); AT-interaction domain (ARID, red); HIC1 binding domain (yellow); Pfam homology domain (orange); B/C box (green); acetylation site (+); region omitted in isoform B, aa 1366-1583 (left bracket); GR binding domain (Glucocorticoid Receptor, right bracket). KDM6A: alanine rich region (green); 8 TPR repeats (black lines); Jumonji C domain (JmjC, red); iron (ˆ) and zinc (ˆ) binding sites. STAG2: STAG domain (STAG, green); SCD domain (SCD, red); serine rich region (black line). In all proteins, recurrent phosphorylation sites are shown (*). B, A ribbon diagram of BAP1 residues 5-291 (UniProtKB Q92560) is based on the crystal structure of homologous UCHL3-UbVME (19). Active site residues, H169 (proton donor, green) and cysteine 91 (nucleophile, yellow) are shown (inset at higher magnification). C, Alignment of NLS residues in BAP1 orthologues. Residue numbers are shown for human BAP1 (top), p.R718 mutation (asterisk). Adapted from (20). D, Papillary features in H&E stained tumor sections from bladder tumors with somatic BAP1 mutations. Top: tumor 48, a low grade papillary urothelial carcinoma; bottom: tumor 9, a low grade urothelial carcinoma, inverted papillary type.

Figure 3. TERT promoter variants in BC

A, The proximal TERT locus in the UCSC Genome Browser with HG19 genomic coordinates (Hg19), TERT isoforms (TERT transcripts), TFBS from ENCODE ChIP-seq, the amplicon location (Amplicon BLAT), and a CpG-rich region (CpG Island, green). B, The TERT promoter amplicon (5′, top left, to 3′, bottom right) with germline variants (above) and somatic variants (below) the genomic reference sequence (solid line). Variant annotation is based on the TERT coding strand (not shown). Frequencies of variants (n=54, in parentheses), novel variants (asterisk), and variants in new TFBS (blue, details in Fig. 3C) are indicated. C, TFBS in the TERT promoter (bold horizontal line, center) are shown relative to the transcription start site (TSS) and the protein-coding start codon (ATG): ◆, Sp1; ○, E-box; and ✶, Ets TFBS. New TFBS created by novel (blue) and previously identified variants (red) are shown in enlarged regions.

Figure 4. Biological and co-mutational connections of KDM6A to a network of genes with somatic mutations in BC

A, The third most significant network (Fisher's Exact Test, p<10-42) constructed by IPA from genes with confirmed somatic mutations in BC, including KDM6A and RB1 (highlighted in blue). The number of independently identified mutations is listed above each respective gene. Evidence of direct (solid lines) or indirect (dashed lines) relationships between gene products is shown. Colored genes indicate decreased (green) or increased (red) gene expression in tumors compared to normal bladder samples in at least two out of three patient cohorts (FDR < 5%). B, The network was created using the co-mutation rate and the frequency of mutation to plot the mutations in 3D space. The size of each gene (node) is representative of the times it was mutated in different tumors. The connection lines (edges) in the network indicate whether the genes were found mutated together in the same tumor, with the length and thickness of each line shorter and wider with an increasing co-mutation rate. The nodes and edges of our model have been filtered to show only those genes found mutated in five or more tumors and edges between genes that were mutated together more than once. Node color corresponds to a gene's relative centrality score, with genes closer to the center of the network red and genes farther away turning to yellow then green at the periphery.

Figure 5. Relationship between a KDM6A signature score and BC

The KDM6A signature score is plotted for normal and tumor samples in A, CNUH (n = 197), B, Lindgren (n=156), and C, MSKCC (n=129) cohorts. AUC, area under receiving operator characteristics curve, with AUC>0.50 corresponding to a signature score higher in tumor than normal samples. P-values were calculated by the Wilcoxon rank-sum test.

Figure 6. KDM6A loss drives the BC phenotype

KDM6A WT MGHU3 cells were treated with short hairpin RNA (shRNA) targeting KDM6A (shKDM6A) or a scrambled shRNA (shCTL). These were compared to KDM6A-mutant T24T cells transiently re-expressing FLAG-tagged KDM6A (FLAG-KDM6A) or empty FLAG vector (FLAG). A, Relative KDM6A mRNA expression by quantitative PCR. B, Anchorage independent growth assay (n=6 wells/line). C, Transwell cell migration (n=4 wells/line). D, Monolayer growth of cells (left) assessed by CYquant fluorescence assay (n=4 wells/line) and subcutaneous tumor growth (right, n=20 mice/line). Results are shown as the mean +/- SEM. P-values were calculated using a Student's t-test on the final day of the assay. Assay details are described or referenced in the Materials and Methods.