KLF4-dependent, PPARγ-induced expression of GPA33 in colon cancer cell lines

The glycoprotein A33 (GPA33) is a colon cancer antigen. Phase I trials with I-131 and I-125 monoclonal antibody A33 in colon carcinoma patients showed excellent localization to colorectal cancer and some evidence of tumor response. Using DNA microarrays, we have identified the GPA33 gene as a target of PPAR gamma in HT29-Cl.16E colon cancer cells. Treatment of HT29-Cl.16E, Caco2, SW1116 and LS174T colon cancer cells with the PPAR gamma agonist GW7845 induced a 2- to 6-fold increase in GPA33 mRNA as determined by real-time PCR. This induction was also found in HT29-Cl.16E cells treated with rosiglitazone and ciglitazone and was prevented by cotreatment with the PPAR gamma antagonist GW9662, indicating that this regulation was PPAR gamma dependent. No canonical PPAR responsive element was found in the GPA33 promoter. We therefore analyzed the expression of transcription factors involved in GPA33 expression. CDX1, CDX2 and KLF5 expression was not modified by PPAR gamma activation. By contrast, a significant increase in KLF4 was seen, both at mRNA and protein levels. Furthermore, chromatin immunoprecipitation studies demonstrated that an increased amount of KLF4 protein was bound to the GPA33 promoter in cells treated with rosiglitazone. Finally, downregulation of KLF4 expression by siRNA reduced rosiglitazone-induced GPA33 expression. This indicates that PPAR gamma activation induces KLF4 expression, which in turn increases GPA33 expression. We also demonstrate that PPAR gamma activation leads to increased (p21(WAF1/Cip1) and keratin 19) or decreased (cyclin D1) expression of known KLF4 targets, suggesting that KLF4 is a nodal player in a network of PPAR gamma-regulated genes. (C) 2009 UICC