Target sequencing and CRISPR/Cas editing reveal simultaneous loss of UTX and UTY in urothelial bladder cancer

Abstract

UTX is a histone demethylase gene located on the X chromosome and is a frequently mutated gene in urothelial bladder cancer (UBC). UTY is a paralog of UTX located on the Y chromosome. We performed target capture sequencing on 128 genes in 40 non-metastatic UBC patients. UTX was the most frequently mutated gene (30%, 12/40). Of the genetic alterations identified, 75% were truncating mutations. UTY copy number loss was detected in 8 male patients (22.8%, 8/35). Of the 9 male patients with UTX mutations, 6 also had copy number loss (66.7%). To evaluate the functional roles of UTX and UTY in tumor progression, we designed UTX and UTY single knockout and UTX-UTY double knockout experiments using a CRISPR/Cas9 lentiviral system, and compared the proliferative capacities of two UBC cell lines in vitro. Single UTX or UTY knockout increased cell proliferation as compared to UTX-UTY wild-type cells. UTX-UTY double knockout cells exhibited greater proliferation than single knockout cells. These findings suggest both UTX and UTY function as dose-dependent suppressors of UBC development. While UTX escapes X chromosome inactivation in females, UTY may function as a male homologue of UTX, which could compensate for dosage imbalances.

Genomic Subtypes of Non-invasive Bladder Cancer with Distinct Metabolic Profile and Female Gender Bias in KDM6A Mutation Frequency

“…None is apparent in MIBC (Gui et al, 2011; Guo et al, 2013; Cancer Genome Atlas Research Network, 2014a). Relatively few NIBC have been screened for mutations, but in a series of exome-sequenced recurrent NMIBC there appears to be a skew towards female patients (Lamy et al, 2016) and a similar skew is apparent in a study of tumors of all grades and stages (Ahn et al, 2016). However, a recent study that included 51 stage Ta samples from patients in Singapore and Taiwan showed no gender bias (Ler et al, 2017).…”

Association between loss of Y chromosome and poor prognosis in male head and neck squamous cell carcinoma

“…Loss of UTY is linked to increased cell proliferation in urothelial bladder cell lines. 28 Its X-chromosome homolog (UTX, also known as KDM6A) is a demethylase of lysine 27 on histone 3 (H3K27), which is linked to dysregulated squamous cell differentiation in HNSCC cell lines 29 ; UTY may share this enzymatic activity, albeit at a reduced level. 30 Loss of UTY has also recently been implicated in both myeloid malignancies and pancreatic cancer via demethylase-independent mechanisms.…”

The histone demethylase UTX/KDM6A in cancer: Progress and puzzles

“…By comparison, fewer than 5% of the cases of the four most common cancers (breast, lung, prostate, colorectal) contain genomic alterations affecting KDM6A. 30,31 Functions of UTX in Cancer Mutations are concentrated in the TPR motifs and in the JmjC domain and are thought to disturb the interaction of UTX with the histone 3 substrate or other proteins, or its enzymatic function.…”

“…Notably, UTY deletions are difficult to call in genome-wide studies, because of its location on the Y-chromosome, and are usually reported as being much more prevalent in dedicated investigations. 30,31…”