Repression of transcription factor AP-2 alpha by PPARγ reveals a novel transcriptional circuit in basal-squamous bladder cancer

The discovery of bladder cancer transcriptional subtypes provides an opportunity to identify high risk patients, and tailor disease management. Recent studies suggest tumor heterogeneity contributes to regional differences in molecular subtype within the tumor, as well as during progression and following treatment. Nonetheless, the transcriptional drivers of the aggressive basal-squamous subtype remain unidentified. As PPAR gamma has been repeatedly implicated in the luminal subtype of bladder cancer, we hypothesized inactivation of this transcriptional master regulator during progression results in increased expression of basal-squamous specific transcription factors (TFs) which act to drive aggressive behavior. We initiated a pharmacologic and RNA-seq-based screen to identify PPAR gamma-repressed, basal-squamous specific TFs. Hierarchical clustering of RNA-seq data following treatment of three human bladder cancer cells with a PPAR gamma agonist identified a number of TFs regulated by PPAR gamma activation, several of which are implicated in urothelial and squamous differentiation. One PPAR gamma-repressed TF implicated in squamous differentiation identified is Transcription Factor Activating Protein 2 alpha (TFAP2A). We show TFAP2A and its paralog TFAP2C are overexpressed in basal-squamous bladder cancer and in squamous areas of cystectomy samples, and that overexpression is associated with increased lymph node metastasis and distant recurrence, respectively. Biochemical analysis confirmed the ability of PPAR gamma activation to repress TFAP2A, while PPAR gamma antagonist and PPAR gamma siRNA knockdown studies indicate the requirement of a functional receptor. In vivo tissue recombination studies show TFAP2A and TFAP2C promote tumor growth in line with the aggressive nature of basal-squamous bladder cancer. Our findings suggest PPAR gamma inactivation, as well as TFAP2A and TFAP2C overexpression cooperate with other TFs to promote the basal-squamous transition during tumor progression.