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Published on: July 22, 2020
KLF4-dependent, PPARgamma-induced expression of GPA33 in colon cancer cell lines
Julie Rageul1, Stéphanie Mottier, Anne Jarry
1Faculté de Médecine, CNRS UMR 6061, Université Rennes 1, IFR140, Rennes, France.
Abstract:
The glycoprotein A33 (GPA33) is a colon cancer antigen. Phase I trials with 131I and 125I 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 PPARgamma in HT29-Cl.16E colon cancer cells. Treatment of HT29-Cl.16E, Caco2, SW1116 and LS174T colon cancer cells with the PPARgamma 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 PPARgamma antagonist GW9662, indicating that this regulation was PPARgamma dependent. No canonical PPAR responsive element was found in the GPA33 promoter. We therefore analyzed the expression of transcription factors involved in GPA33 expression. CDXl, CDX2 and KLF5 expression was not modified by PPARgamma 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 PPARgamma activation induces KLF4 expression, which in turn increases GPA33 expression. We also demonstrate that PPARgamma activation leads to increased (p21WAF1/Cip1 and keratin 19) or decreased (cyclin D1) expression of known KLF4 targets, suggesting that KLF4 is a nodal player in a network of PPARgamma-regulated genes.
Insights
Peroxisome proliferator-activated receptor gamma (PPARγ) activation increases glycoprotein A33 (GPA33) expression in colon cancer cells. This occurs via induction of KLF4, a transcription factor that binds to the GPA33 promoter, highlighting a new therapeutic target.
Area of Science:
- Molecular Biology
- Oncology
- Gene Regulation
Background:
- Glycoprotein A33 (GPA33) is a colon cancer antigen targeted by monoclonal antibody therapy.
- Previous studies showed promising localization and some tumor response in clinical trials.
Purpose of the Study:
- To investigate the molecular mechanisms regulating GPA33 expression in colon cancer cells.
- To identify potential therapeutic targets for enhancing GPA33-mediated cancer therapy.
Main Methods:
- Utilized DNA microarrays to identify GPA33 as a target of PPARγ.
- Treated various colon cancer cell lines with PPARγ agonists (GW7845, rosiglitazone, ciglitazone) and an antagonist (GW9662).
- Assessed GPA33 mRNA levels via real-time PCR and analyzed transcription factor expression (KLF4, CDX1, CDX2, KLF5) using RT-PCR and Western blot.
- Performed chromatin immunoprecipitation (ChIP) assays to study KLF4 binding to the GPA33 promoter.
- Investigated the role of KLF4 using siRNA-mediated gene silencing.
Main Results:
- PPARγ activation significantly increased GPA33 mRNA levels (2- to 6-fold) in multiple colon cancer cell lines, a process dependent on PPARγ.
- PPARγ activation led to a substantial increase in both mRNA and protein levels of KLF4.
- ChIP assays confirmed increased binding of KLF4 to the GPA33 promoter upon PPARγ activation.
- Downregulation of KLF4 expression abolished the induction of GPA33 by rosiglitazone.
- PPARγ activation modulated the expression of known KLF4 target genes, including p21WAF1/Cip1, keratin 19, and cyclin D1.
Conclusions:
- PPARγ activation induces GPA33 expression in colon cancer cells through the upregulation of the transcription factor KLF4.
- KLF4 acts as a key mediator, binding to the GPA33 promoter and driving its expression.
- This pathway reveals a novel regulatory network involving PPARγ, KLF4, and GPA33, offering potential new strategies for colon cancer treatment.
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