Regulation of human PAX2 in cancer cells by Transforming Growth Factor-beta (TGF-β)
The PAired boX (PAX) family is a group of related genes that are highly conserved throughout evolution and are known to play important roles in mammalian development. So far nine members of the PAX gene family (PAX1-9) have been identified in mice and humans. Their expression patterns are restricted to developing structures such as the central nervous system, the vertebral column, the eye and the kidney. This study was based on the regulation of the PAX2 gene, which plays a central role in kidney development. PAX2 gene expression is frequently observed in cancer and is often required for cancer cell survival. Expression of PAX2 has been observed in carcinomas of kidney, prostate, breast, and ovary and in Wilms’ tumor, whereas PAX2 expression is down-regulated in normal adult tissues in which these cancers originate. However, the mechanisms through which PAX2 expression continues to be expressed in cancer remain poorly explored. An increasing number of studies relate patterns of PAX gene expression to signaling by members of the TGF-β superfamily. A number of studies suggest that TGF-β functions as a tumor suppressor, consistent with its ability to arrest cell growth and/or induce apoptosis in normal tissues or differentiated tumors. TGF-β1 expression and function is implicated in extracellular matrix regulation and it is known to be a negative regulator of renal cell carcinoma. The present study was performed to define the mechanism by which PAX2 expression is regulated by TGF-β1 in cancer cells. The results obtained in this study demonstrated a repressive effect of TGF-β1 on PAX2 mRNA levels as well as protein expression in renal cancer cell (RCC) lines - 786-O and A498. In contrast, the bladder cancer (EJ) and colon cancer (RKO) cells did not show any response to TGF-β1. Transient transfections of PAX2 promoter reporter constructs also showed that the presence of exogenous TGF-β1 repressed PAX2 promoter activity in HEK293 (embryonic kidney transformed with adenovirus 5 DNA) cells and TK10 (RCC) cells. In contrast TGF-β1 did not repress PAX2 promoter activity in K1 (thyroid cancer) cells and Hs578T (breast cancer) cells thereby implicating a cell-lineage specific response to TGF-β1. Further, to investigate the mechanism of PAX2 regulation by TGF-β1 an inhibitory SMAD (SMAD7) was overexpressed in 786-O cells to block the TGF-β signaling pathway. The overexpression of SMAD7 released the repressive effect of TGF-β1 on PAX2 protein and PAX2 mRNA levels in 786-O cells. Chromatin immunoprecipitation (ChIP) of HEK293 cells and 786-O cells with and without TGF-β1 treatment and using an anti-Smad2/3 antibody indicated that TGF-β1 signaling leads to the direct binding of SMAD2/3 to the PAX2 promoter. Further, the binding of SMAD to the predicted binding sites in the PAX2 promoter was abrogated in the presence of an inhibitory SMAD (SMAD7) in both cell lines. This study therefore concludes that TGF-β1 regulates PAX2 in RCC at the transcriptional level through the direct binding of SMAD2/3 at one or more putative binding sites on the PAX2 promoter. Therefore, the dissection of developmental signaling pathways, such as the TGF-β signaling pathway, may provide an insight into the factors controlling the expression of key regulatory genes, such as PAX2 during tumorigenesis. Overall this study has provided novel evidence that TGF-β/SMAD signaling may be at least in part involved in the regulation of PAX2 and that this regulation is mediated by direct binding of SMAD to a putative binding site/sites in the PAX2 promoter. Moreover, the repressive effect of TGF-β1 on PAX2 observed in this study suggests that in renal cancers involving overexpression of PAX2 there may be decreased or disrupted TGF-β activity. Thus in such clinical scenarios, a chemopreventive strategy to restore or to increase TGF-β signaling could be used to repress PAX2 expression in cancer cells.
Advisor: Eccles, Michael; Horsfield, Julia
Degree Name: Doctor of Philosophy
Degree Discipline: Pathology
Publisher: University of Otago
Keywords: PAX2; RCC; TGF-beta; SMAD2/3
Research Type: Thesis