Patient Mutation Directed shRNA Screen Uncovers Novel Bladder Tumor Growth Suppressors

Abstract

Next generation sequencing (NGS) of human bladder cancer has revealed many gene alterations compared with normal tissue, with most being predicted to be “loss of function”. However, given the high number of alterations, evaluating the functional impact of each is impractical. Here we develop and use a high-throughput, in vivo strategy to determine which alterations are loss of function in tumor growth suppressors. Genes reported as altered by NGS in bladder cancer patients were bioinformatically processed by MutationTaster and MutationAssessor, with 283 predicted as loss of function. A shRNA lentiviral library targeting these genes was transduced into T24 cells, a non-tumorigenic human bladder cancer cell line, followed by injection into mice. Tumors that arose were sequenced and the dominant shRNA constructs were found to target IQGAP1, SAMD9L, PCIF1, MED1 and KATNAL1 genes. In vitro validation experiments revealed that shRNA molecules directed at IQGAP1 showed the most profound increase in anchorage-independent growth of T24 cells. The clinical relevance of IQGAP1 as a tumor growth suppressor is supported by the finding that its expression is lower in bladder cancer compared with benign patient urothelium in multiple independent datasets. Lower IQGAP1 protein expression associated with higher tumor grade and decreased patient survival. Finally, depletion of IQGAP1 leads to increased TGFBR2 with TGFβ signaling, explaining in part how reduced IQGAP1 promotes tumor growth. These findings suggest IQGAP1 is a bladder tumor growth suppressor that works via modulating TGFβ signaling and is a potentially clinically useful biomarker. Implications This study used gene mutation information from patient-derived bladder tumor specimens to inform the development of a screen used to identify novel tumor growth suppressors. This included identification of the protein IQGAP1 as a potent bladder cancer growth suppressor.

Cap-specific terminal N 6 -methylation of RNA by an RNA polymerase II–associated methyltransferase

“…This finding might partly explain why CAPAM KO strains are sensitive to oxidative stress. An RNA interference-based genetic screen identified CAPAM/PCIF1 as a putative tumor suppressor in a bladder cancer model (45), indicating that it might be involved in cell proliferation under certain conditions. Further studies seem necessary to unveil the physiological role of this gene.…”

TGF-β and microRNA Interplay in Genitourinary Cancers

“…On the other hand, multiple studies indicated that TGF-β1 and its receptors stimulate the progression of bladder cancer cells. Loss of the IQGAP1 tumor suppressor leads to increased expression of TGFBR2 and activated the TGF-β1 signaling pathway, thereby stimulating growth of human bladder cancer cells [193]. TGF-β1-stimulated migration and invasion of bladder cancer cells arise mediated by Src and FAK kinase [194] as well as transgelin (TAGLN), an actin-binding protein that stimulates colony formation, migration, and invasion, as well as epithelial-mesenchymal transition [195].…”

Cantharidin alters the expression of genes associated with the NKG2D-associated immune response in TSGH-8301 human bladder carcinoma cells

“…A previous study demonstrated that CTD affects gene expression in the human lung cancer NCI-H460 cell line ( 32 ). Additionally, a recent study identified, using next generation sequencing, that human bladder cancer has numerous gene alterations compared with normal tissue, with most predicted to be loss-of-function mutations; however, evaluating the functional impact of each genetic alteration is impractical ( 39 ). The present study, to the best of our knowledge, is the first to demonstrate the effects of CTD on gene expression in human bladder TSGH-8301 cancer cells.…”