Knockdown of UTX/KDM6A Enriches Precursor Cell Populations in Urothelial Cell Cultures and Cell Lines

Abstract

The histone demethylase UTX (gene: KDM6A) directs cell and tissue differentiation during development. Deleterious mutations in KDM6A occur in many human cancers, most frequently in urothelial carcinoma. The consequences of these mutations are poorly understood; plausibly, they may disturb urothelial differentiation. We therefore investigated the effects of UTX siRNA-mediated knockdown in two in vitro models of urothelial differentiation; namely, primary cultures of urothelial epithelial cells treated with troglitazone and PD153035 and the immortalized urothelial cell line HBLAK treated with high calcium and serum. In both models, efficient UTX knockdown did not block morphological and biochemical differentiation. An apparent delay was due to a cytotoxic effect on the cell cultures before the initiation of differentiation, which induced apoptosis partly in a p53-dependent manner. As a consequence, slowly cycling, smaller, KRT14 high precursor cells in the HBLAK cell line were enriched at the expense of more differentiated, larger, proliferating KRT14 low cells. UTX knockdown induced apoptosis and enriched KRT14 high cells in the BFTC-905 papillary urothelial carcinoma cell line as well. Our findings suggest an explanation for the frequent occurrence of KDM6A mutations across all stages and molecular subtypes of urothelial carcinoma, whereby loss of UTX function does not primarily impede later stages of urothelial differentiation, but favors the expansion of precursor populations to provide a reservoir of potential tumor-initiating cells.

Alterations of Chromatin Regulators in the Pathogenesis of Urinary Bladder Urothelial Carcinoma

“…Rather, chromatin regulator mutations may enhance the self-renewal ability of urothelial precursor cells, favoring their clonal expansion. In support of this idea, we observed that knockdown of KDM6A in primary urothelial cell cultures and the normal urothelial cell line HBLAK, which comprise basal cells and KRT14-expressing stem cells, increased the proportion of cells expressing KRT14 [ 148 ]. Mutations inactivating KDM6A in normal urothelium may therefore augment the capacity of urothelial KRT14-expressing precursor cells for self-renewal, allowing them to gradually colonize the tissue at the expense of non-mutant urothelial precursors.…”

“…Accordingly, the KDM6A paralog KDM6B/JMJD3 is known to contribute to differentiation of cultured primary keratinocytes [ 147 ]. In primary urothelial and HBLAK cells, knockdown of KDM6A affected the balance between KRT14-positive stem cells and KRT5-positive basal cells but had only limited effects on further differentiation of luminal cells [ 148 ].…”

“…This issue clearly calls for deeper investigation. The findings so far suggest that restoration of KDM6A function does affect proliferation of UC cells but usually only over time [ 132 , 148 , 181 , 182 ]. These delayed effects could be explained by decreased self-renewal ability of tumor stem cells.…”

KDM6 Demethylases and Their Roles in Human Cancers

“…KDM6A mutations are commonly lost in all stages and subtypes of urothelial cancer (UC), which is classified into non-muscle-invasive and muscle-invasive tumors (62)(63)(64)(65). Papillary UC is driven by loss of mutation of KDM6A in growth signaling pathway, especially activating FGFR3 and PIK3CA (119).…”