Abstract #2223: FOXC2 and FOXL1 contribute to metastatic potential of non-small cell lung cancer by downregulation of p120-catenin.
Authors: Fariborz Mortazavi, Jiabin An, Steven Dubinett, Matthew Rettig
Journal: Cancer Research (2009)

Introduction: The p120-catenin protein (p120ctn) is a regulator of E-cadherin and also RhoGTPase and Rac therefore playing a major role in cell adhesion and motility. p120ctn is down regulated at the protein and mRNA levels in a number of malignancies including non-small cell lung cancer (NSCLC). Here we investigated transcriptional regulation of p120ctn in NSCLC. Methods: We cloned a 1400 bp amplicon of chromosome 11 from position -1082 to +320 relative to the transcription start site into a firefly luciferase reporter vector. In addition, we prepared multiple serial deletion constructs in order to pinpoint the  cis -acting elements involved in regulation of  p120ctn.  We transfected NSCLC cell lines A549, H157, Rh2 and H358 as well as immortalized normal human respiratory epithelial cell line (BEAS-2B) with the above mentioned constructs. Results: Our experiments show that  p120ctn  promoter activity and p120ctn mRNA message are reduced in lung cancer cell lines compared to non-cancerous immortalized lung epithelial cells. This finding suggests that differential transcriptional regulation of  p120ctn  in malignant versus non-malignant lung epithelial cells contributes to heightened metastatic potential of malignant lung epithelium. Serial deletion analysis of  p120ctn  promoter identified a region between positions +267 and +282 that potentially mediates transcriptional repression of  p120ctn.  Computer based sequence analysis of this region predicted putative binding sites for the transcription factors FOXC1 and FOXL1. Both FOXC1  and FOXL1 are mesenchymal forkhead related proteins, which are involved in specifying mesenchymal cell fate during embryogenesis. FOXC2 is known to play a role in breast cancer metastasis. Since FOXC1 is not expressed in our non-small cell lung cancer cell lines we focused on FOXC2 and FOXL1 in the transcriptional regulation of the  p120ctn  promoter. Silencing both FOXC2 and FOXL1 in NSCLC cell lines with siRNA resulted in 2-2.5-fold enhancement of  p120ctn  promoter activity and mRNA message. Consistent with this finding, stable downregulation of FOXL1 by shRNA expressed from a lentivirus resulted in increased p120ctn mRNA levels. In addition, these lentivirally transduced cells show decreased cell invasion compared to control in Matrigel invasion assay. Experiments for creating stable NSCLC cell lines with low levels of FOXC2 are currently underway. Electromobility shift assay (EMSA) showed binding of nuclear extract from A549 cells with DNA sequence between positions +267 and +282 relative to transcription initiation site of  p120ctn , which supports the notion that certain FOX family proteins are mediating transcriptional repression of  p120ctn.    Conclusion: These findings suggest that FOXL1 and FOXC2 transcription factors control cell adhesion and motility through transcriptional repression of  p120ctn.     Citation Information:  In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2223.