Pparg Drives Luminal Differentiation and Luminal Tumor Formation in the Urothelium

The urothelium is a crucial stratified epithelial barrier that protects the urinary tract. It consists of basal cells in the lower layers and intermediate and superficial cells in the luminal layer. These urothelial cells can be identified by their distinct gene expression patterns. Superficial cells are terminally differentiated, binucleated, post-mitotic cells that are responsible for the barrier function of the urothelium via the production of uroplakin proteins. Intermediate cells act as the progenitor cells for superficial cells during development, homeostasis, and after acute injury. Basal cells consist of two populations, K14-basal cells and K5-basal cells. K14- basal cells have been shown to be progenitors that can repopulate the urothelium after chronic injury and are the cells of origin that produce bladder cancer. Bladder cancer can be classified as basal subtype or luminal subtype. The basal subtype is generally immune infiltrated, aggressive, and invasive with a poor prognosis. The luminal subtype is generally immune poor, less aggressive, and non-invasive with a better prognosis compared to basal tumors. Pparg is a nuclear hormone receptor that has been described as a master regulator of adipogenesis and cellular differentiation that also carries out important anti-inflammatory functions (in part by antagonizing the NFKB pathway). Pparg is downregulated in basal subtype muscle invasive bladder cancer and amplified in luminal subtype bladder cancer. In vivo we find that Pparg is a master regulator of cell specification during urothelial development, homeostasis, regeneration, and cancer. When Pparg is ablated in the entire urothelium, Pparg KO mutants lack mature superficial cells and undergo squamous differentiation, with an expansion of the K14-basal cell population. These Pparg KO mutants also display persistent inflammation and squamous metaplasia after injury by urinary tract infection (UTI), due to unregulated NFKB signaling. However, the squamous differentiation in the Pparg KO mutants did not progress to bladder cancer. Constitutive activation of Pparg in basal cells using a novel VP16;Pparg transgenic mouse line crossed to an Krt5CreERT2 driver induces basal cells to undergo a luminal differentiation program towards post-mitotic S-cells during homeostasis. Not surprisingly, these cells did not progress to form bladder cancer on their own. Interestingly, expression of VP16;Pparg in basal cells only drives tumor formation when the basal cells are in an “activated state,” induced by 1 month of BBN treatment. In a BBN mouse model which produces basal subtype bladder cancer in wild type animals, expression of the VP16;Pparg transgene in activated basal cells drives the formation of luminal tumors with papillary morphology, suggesting that this transcription factor is a master regulator of urothelial luminal differentiation, as has been suggested from previous in vitro studies. Like their human counterparts, these VP16;Pparg luminal tumors are immune cold. Additionally, these VP16;Pparg luminal tumors have different domains; a top domain that is “luminal,” and a bottom domain that is “basal”, suggesting the luminal tumors produced by activation of Pparg are not homogenous and undergo a phenotypic shift that mimics what has previously been reported in patient-derived organoids. Understanding the molecular mechanism that drives luminal bladder cancer provides critical information in bettering our approach in diagnosing and treating MIBCs.