The Interplay Between p120ctn and EGFR Causes Esophageal Squamous Cell Carcinoma Invasion Through NFkB

Four out of five patients diagnosed with esophageal squamous cell carcinoma (ESCC) will die within five years. This is primarily a result of the aggressive invasive potential of the disease. While the individual roles of oncogenes and tumor suppressors in cancer are widely studied, our research is focused on the interplay between these types of genes in the invasive process. Specifically, EGFR and p120‐catenin (p120ctn) are commonly dysregulated genes that are indicative of poor prognosis in ESCC. Our data show that 63% of ESCC patients harbor both EGFR overexpression and p120ctn inactivation, thus emphasizing the relevance of this gene combination in human ESCC pathogenesis. We hypothesized that EGFR and p120ctn cooperate to drive the invasive phenotype of ESCC. By studying these genes individually and together, we have demonstrated that in our unique 3D organotypic culture model, only when EGFR overexpression is combined with p120ctn inactivation do the cells transform and invade – as opposed to either event alone. This cell type phenotypically and molecular mimics human ESCC. Global gene analysis of our 3D cultures identified approximately 900 genes unique to EGFR overexpression and p120ctn inactivation. Interestingly, “Cancer” is the most overrepresented gene ontology functional category in these data. By subcategorizing the cancer genes, “Metastasis” was identified as the most enriched subcategory. This strongly points to the fact that there is a robust and unique relationship between EGFR and p120ctn that is creating a more aggressive and invasive ESCC cell type. From these gene expression data, we have identified a putative invasion pathway downstream of EGFR and p120ctn that involves NFkB as a central regulator. Interestingly, we found that NFkB is hyperactivated in cells with EGFR overexpression and p120ctn inactivation than with either EGFR or p120ctn alone. Inhibition of this NFkB hyperactivation results in complete loss of invasion, suggesting that NFkB signaling is necessary for invasion in this aggressive cell type. We have identified RhoA GTPase and Rho‐kinase (ROCK) as upstream regulators of NFkB. We found that active RhoA levels are dramatically increased in cells with EGFR overexpression and p120ctn inactivation. Inhibition of Rho with C3 exotransferase or inhibition of ROCK activity downstream of RhoA, results in a decrease in NFkB activation levels. These data suggest that RhoA and ROCK are playing a role in the invasion pathway downstream of EGFR and p120ctn. Downstream of NFkB we have identified that Twist2 (which is a novel NFkB target) and uPAR expression are increased when NFkB is hyperactivated. Inhibition of NFkB activation results in a decrease in both Twist2 and uPAR expression when EGFR is overexpressed and p120ctn is inactivated together. These data suggest that EMT and MMP activation may be mechanisms by which NFkB effects invasion in ESCC. In conclusion, we have demonstrated that EGFR and p120ctn are working synergistically together to create a highly invasive phenotype in ESCC. We believe this cooperation is not only important in the aggressive mechanisms of ESCC but will be broadly applicable to many cancer types since p120ctn and EGFR are frequently involved in other cancers. Support or Funding Information This work was supported by a Research Scholar Grant, RSG‐16‐219‐01‐TBG, from the American Cancer Society, and by a Penn State Cancer Institute Research Pilot Grant (CURE Funds).