Investigation of Transcription Factors Regulating Urothelial Differentiation and Signalling Pathways involved in Urothelial Cancer Cells

Background: Normal human urothelial (NHU) cells can be induced to differentiate by inhibition of the EGFR pathway and activation of PPARγ, which is considered a master transcription factor (TF) for urothelium. Activation of PPARγ results in up-regulation of downstream TFs, amongst which ELF3 was identified as the most up-regulated by microarray. Different phenotypes of NHU cells (proliferating or differentiated) assessed by expression of differentiation markers were found to resemble different types of urothelial cancer (UC) cell lines. The hypothesis is that the differentiation status in UC cell lines reflects cancer evolution pathways, which are selected to be either retained or lost, and define regulatory pathways that UC cells use for growth. Aim: To understand the role of TFs in regulating urothelial differentiation and pathways regulating proliferation in different UC cell lines. Results: Knockdown of ELF3 in NHU cells affected the expression of ELF3. Functional study also showed an essential role of ELF3 in maintaining an effective barrier. Evidence suggested that PPARγ was coincidentally knocked down in some cells. Over-expression of ELF3 demonstrated increased ELF3 transcript, but had no effect on phenotype. A UC cell line (5637) with low expression of differentiation markers resembled non-differentiated NHU cells. When maintained in medium without serum, 5637 cells relied on PPARγ and GSK3β pathways for growth. By contrast, UMUC9 cells, which resembled differentiated NHU cells, could not proliferate when serum or other growth factors were removed. NHU cells or UC cells maintained in different media (± serum) demonstrated different expressing patterns of PPARγ transcripts and protein isoforms. Conclusions: ELF3 was important in regulating differentiation in NHU cells and may affect the expression of other TFs, but was not alone sufficient to initiate the differentiation programme. The phenotype and signalling pathways associated with proliferation in 5637 and UMUC9 cells may have implications for different subtypes of Muscle Invasive Bladder Cancer (MIBC).