All cell lines were cultured in RPMI 1640 medium containing l-glutamine (Thermo Fisher Scientific, cat. Cell lines were authenticated by the ATCC or DSMZ before distribution via morphology, karyotyping, and PCR-based approaches. Human UC cell lines (5637, HT-1197, HT-1376, TCCSUP, and T24) were purchased from the American Type Culture Collection (ATCC), with the exception of Cal29 (DSMZ, cat. Moreover, we demonstrate a functional link between SHH- and PPARγ-dependent cell functions, thus providing insights into the tumorigenic nature and molecular mechanism involved in PPARγ-dependent UC tumorigenesis. Importantly, our data demonstrated for the first time direct transcriptional regulation of the oncogene Sonic Hedgehog ( SHH) by PPARγ. In this study, our objective was to further elucidate cell-intrinsic roles of PPARγ signaling in UC and identify candidate target genes responsible for UC progression based on genome-wide approaches. However, the direct molecular mechanisms by which PPARγ promotes tumor growth have not been described, nor have the downstream mediators of PPARγ–RXR transcriptional activity been defined. Indeed, multiple recent studies link PPARγ activity to UC, describing its ability to promote disease progression in both cell-autonomous ( 5, 14–16) and non–cell-autonomous manners ( 17). Nevertheless, these observations have led our group and others to speculate that PPARγ might be an important factor in UC development and progression in a subset of patients. Of note, rosiglitazone treatment alone did not induce UC tumors, suggesting a potential synergistic relationship between PPARγ activation and carcinogen exposure in UC development in vivo. In a preclinical study employing a carcinogen-induced model of bladder cancer, rosiglitazone treatment increased incidence and size of UC in rats in a dose-dependent manner ( 13). Long-term use of certain thiazolidinediones, synthetic agonists of PPARγ, Pioglitazone, but not rosiglitazone, used to manage blood glucose levels in type 2 diabetics, has been associated with increased risk of bladder cancer development ( 10–12). Collectively, our data indicate that PPARγ promotes UC progression in a subset of patients, at least in part, through cell-autonomous mechanisms linked to SHH signaling. Finally, we demonstrate the PPARγ dependency of UC tumors in vivo by genetic and pharmacologic PPARγ inhibition in subcutaneous xenografts. Similar to PPARγ, genetic inhibition of SHH reduces proliferation and motility. Through genome-wide approaches including chromatin immunoprecipitation sequencing and RNA sequencing, we define a novel set of PPARγ-regulated genes in UC, including Sonic Hedgehog ( SHH). In vitro assays revealed for the first time that treatment of UC cells with PPARγ inverse agonist or PPARG knockout by CRISPR-Cas9 reduces proliferation, migration, and invasion of multiple established UC cell lines, most strongly in those characterized by PPARG genomic amplification or activating mutations of RXRA, the obligate heterodimer of PPARγ. Here, we report robust expression and nuclear accumulation of PPARγ in 47% of samples of patients with UC, exceeding mRNA expression patterns published by The Cancer Genome Atlas. Although PPARγ has been recently demonstrated to play non–cell-autonomous roles in promoting bladder urothelial carcinoma (UC) progression, underlying mechanisms of the cell-intrinsic oncogenic activity remain unknown. The role of PPAR gamma (PPARγ) has been well characterized in the developmental process of adipogenesis, yet its aberrant expression patterns and functions in cancer subtypes are less understood.
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