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RUNX3 is a candidate tumor suppressor gene localized in 1p36, a region commonly inactivated by deletion and methylation in various human tumors. To elucidate the role of RUNX3 in transforming growth factor (TGF)-β signaling in biliary tract cancer, we transfected Mz-ChA-2 cells, which do not express RUNX3 but have intact TGF-β type II receptor and SMAD4 genes, with the RUNX3 expression plasmid pcDNA3.1/RUNX3 or with the vector pcDNA3.1 as a control. Four Mz-ChA-2/RUNX3 clones and one control clone were obtained. Although TGF-β1 only slightly inhibited growth of the control cells, growth inhibition and TGF-β-dependent G1 arrest were significantly enhanced in the RUNX3-transfected clones. None of the clones, however, exhibited apoptosis. The slightly increased TGF-β1-induced p21 expression in the control clone was strongly enhanced in the RUNX3-transfected clones, and was accompanied by augmented decreases in the expression of cyclins D1 and E. When RUNX3 small interfering RNA was added, TGF-β-dependent induction of p21 was reduced in the RUNX3-transfected clones. Xenografts of the clones in nude mice demonstrated that tumorigenicity was significantly decreased in the RUNX3-transfected clones in inverse proportion to the expression levels of RUNX3. Based on these results, RUNX3 is involved in TGF-β-induced expression of p21 and the resulting induction of TGF-β-dependent G1 arrest. (Cancer Sci 2007; 98: 838–843)
Although carcinomas of the biliary tract are relatively rare, their incidence is increasing in Japan.(1) Most carcinomas of the biliary tract are unresectable when diagnosed, and their prognosis is poor because of high resistance to chemoradiation therapies.(2) Therefore, elucidating the biological characteristics of biliary tract cancer cells is necessary to establish better treatment strategies and thereby improve prognosis.
The RUNX3 gene, which is located on chromosome 1 at 1p36.1, encodes a protein that belongs to the runt domain family of transcription factors that act as master regulators of gene expression in major developmental pathways.(3–5) Recent studies have revealed that RUNX3 is frequently inactivated in various carcinomas, including gastric,(5) lung,(6,7) hepatocellular,(8) breast,(9) colon,(10) pancreatic and biliary tract,(11) prostate(12) and laryngeal carcinomas.(9) Interestingly, in primary cultures of RUNX3-null gastric epithelial cells, the cells are less sensitive to transforming growth factor (TGF)-β-dependent growth inhibition and apoptosis.(5) In addition, the RUNX3 protein has been found to bind the Smad2 and Smad3 proteins.(13) These data suggest a possible role for RUNX3 in transducing TGF-β signaling.
TGF-β initiates its signal by bringing together type I and type II serine-threonine kinase receptors that form complexes with the ligand on the cell surface. This process results in the phosphorylation of the type I receptor by the type II receptor.(14) The type I receptor then phosphorylates and activates members of the Smad family of tumor suppressors (i.e. R-Smads), which includes Smad2 and Smad3.(14,15) The activated R-Smads form oligomers with the unique co-Smad, Smad4, and rapidly translocate to the nucleus to regulate expression of target genes. It is well known that TGF-β stimulation generally induces inhibition of cell growth, which involves various mechanisms, such as downregulation of c-myc and cyclin-dependent kinase (CDK)-2/CDK-4 activity by modulating the functions of p15INK4B,(16,17) p21Waf1/Cip1,(18,19) and/or p27Kip1.(20,21) Among these factors, p21 plays a central role in the inhibition of CDK and in control of the cell cycle.
Forkhead transcription factor FOXO is a negative regulator of the PI3K/Akt pathway, which is activated by growth and survival cytokines such as insulin-like growth factor-1 and platelet-derived growth factor.(22) Among the FOXO family of molecules, FOXO3A directly binds and activates the p21 promoter, which has forkhead-binding elements adjacent to the SMAD-binding element, in cooperation with SMAD3 and SMAD4.(23) Moreover, Akt directly phosphorylates Smad3 to control TGF-β-dependent apoptosis or cell cycle arrest.(24) Altogether, the PI3K/Akt/FOXO pathway may have important cross-communication for TGF-β signaling.(25)
To elucidate the role of RUNX3 in the TGF-β signaling pathway, we established and characterized stable transfectants of Mz-ChA-2 cells that constitutively expressed RUNX3. We demonstrate how the restoration of RUNX3 expression in Mz-ChA-2 biliary tract cancer cells led to enhancement of TGF-β-induced upregulation of both p21 and FOXO3A and downregulation of cyclin D1 while significantly enhancing G1 arrest. We also show how RUNX3 is involved in the TGF-β-dependent expression of p21 and cell cycle arrest, most likely through upregulation of p21.
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RUNX3 has been implicated as a tumor suppressor gene.(5) The gastric epithelium of Runx3 knockout mice exhibits reduced TGF-β-dependent apoptosis, as well as cell-growth inhibition, suggesting that RUNX3 exerts its tumor suppressor activity in a region downstream of the TGF-β signaling pathway.(26,31) Although previous studies demonstrated that overexpression of RUNX3 enhanced TGF-β-dependent cell cycle arrest and apoptosis,(5,29,30,32) the precise role of RUNX3 in the TGF-β signaling pathway remains unknown. In our study, we demonstrated that restoration of RUNX3 in the Mz-ChA-2 cells significantly enhanced TGF-β-induced cell growth inhibition and TGF-β signaling. Furthermore, our study also showed that tumorigenicity of the Mz-ChA-2 cells, when injected into nude mice, was reduced in proportion to the expression levels of RUNX3. These data strongly suggest that RUNX3 plays an important role in inhibiting cellular growth by participating in the TGF-β signaling pathway.
Several previous studies using gastric cancer cell lines have revealed that restoration of RUNX3 enhanced TGF-β-dependent apoptosis.(29,32–34) For example, Yamamura et al. demonstrated that RUNX3 directly activates the promoter activity of Bim, one of the proapoptotic Bcl family proteins, to enhance TGF-β-dependent apoptosis.(34) In contrast to their findings, we showed that restoration of RUNX3 in Mz-ChA-2 cells did not affect apoptosis but enhanced TGF-β-dependent G1 arrest. The reason for the discrepancy between their results and ours is unexplained at present. Although TGF-β did not elicit apoptosis in Mz-ChA-2 cells, it did enhance G1 arrest slightly, even in the RUNX3-deficient Mz-ChA-2 cells in our experiments. It is possible therefore that the TGF-β dependency of the apoptotic mechanism differs between the gastric cancer cell lines Yamamura et al. used and the biliary tract cancer cells we used, irrespective of the presence or absence of RUNX3. Another consideration is that the nuclei of Mz-ChA-2 cells stain strongly for p53 (data not shown), which suggests that the abnormal function of p53 is involved in the insensitivity of the Mz-ChA-2 cells to the apoptotic signal of TGF-β.
Our study suggested that the enhancement of TGF-β-induced G1 arrest by the restoration of RUNX3 expression would be associated with upregulation of p21 and downregulation of cyclins D1 and E. Our data are in accord with the recent report by Chi et al.,(35) which demonstrated that the restoration of RUNX3 enhanced TGF-β-induced p21 expression by direct activation of the p21 promoter by RUNX3 in cooperation with SMAD3 and SMAD4 in gastric cancer cell lines. Thus, it appears reasonable to consider that the enhancement of TGF-β-induced G1 arrest we observed is at least due in part to the enhanced expression of p21 by restoration of RUNX3.
TGF-β is also known to enhance p27.(36) Our study reconfirmed this finding, even in the RUNX3-deficient Mz-ChA-2 cells. Although a recent report suggested that RUNX3 restoration also enhanced TGF-β-induced upregulation of p27,(32) the TGF-β1-induced increase of p27 expression in Mz-ChA-2 cells in our study was not affected by RUNX3 restoration. Moreover, changes in c-myc expression have also been shown to be involved in TGF-β-dependent cell cycle regulation.(37,38) In our study, TGF-β elicited a slight decrease in c-myc expression in RUNX3-deficient Mz-ChA-2 cells. Similar to p27 expression, however, RUNX3 restoration did not influence TGF-β-induced decrease of c-myc expression. Thus, in our study, TGF-β1-induced increase of p27 expression and decrease of c-myc expression were not affected, but increase of p21 expression was strongly enhanced by restoration of RUNX3. Although the reasons why p27 as well as c-myc expression were not affected by restoration of RUNX3 in this study remains unknown, it is possible that RUNX3 has different roles in TGF-β-dependent p21 and p27 as well as c-myc expressions. It may be noted that TGF-β-dependent cell cycle arrest is observed in association with p21 and p27 increase and c-myc decrease even in RUNX3-deficient Mz-Ch-A2 cells in our study. Thus, it is likely that RUNX3 is not indispensable for TGF-β signaling, but is required for the potent inhibitory action of TGF-β on cell cycle arrest via enhancement of p21 expression.
Seoane et al. have shown that FOXO3A, a forkhead transcription factor, plays key roles in the TGF-β-dependent activation of p21 by partnering with Smad3 and Smad4.(23) It is noteworthy therefore that the induction of FOXO3A mRNA by TGF-β1 was enhanced in our study in proportion to the expression levels of RUNX3 in the RUNX3-restored Mz-ChA-2 clones. Because the FOXO3A promoter region has one complete RUNX-binding sequence (data not shown), it is reasonable to consider that RUNX3 might directly control the expression of FOXO3A and that it affects TGF-β-induced p21 expression through this control mechanism. Clearly, the importance of cross-communication between TGF-β signaling and the PI3K/Akt/FOXO pathway is suggested.
Interestingly, even in RUNX3-restored clones 3 and 4, in which induction of FOXO3A by TGF-β1 as well as expression levels of RUNX3 are low, tumorigenicity were significantly reduced. The reason why tumorigenicity is reduced in clones with low induction of FOXO3A and low expression of RUNX3 in this study remains unknown. However, the tumor suppressor function of RUNX3 appears to be strongly enhanced in vivo. This result is in accord with the recent report by Sakakura et al., which demonstrated that RUNX3-transfected gastric cancer cells could not make metastatic lesions in nude mice although there were no significant differences between growth of RUNX3-restored clones and mock-transfected gastric cancer cells in vitro.(29)
In summary, our data showed that RUNX3 plays important roles in TGF-β-induced p21 expression and cell cycle G1 arrest in human biliary cancer cell line Mz-ChA-2. Further elucidation of the role of RUNX3 as a tumor suppressor gene may contribute to the development of more optimal treatments for biliary tract carcinomas.