Additive clinical impact of epidermal growth factor receptor and podocalyxin-like protein expression in pancreatic and periampullary adenocarcinomas

Abstract

The outcome of periampullary adenocarcinomas remains poor with few treatment options. Podocalyxin-like protein (PODXL) is an anti-adhesive protein, the high expression of which has been shown to confer a poor prognosis in numerous malignancies. A correlation and adverse prognostic synergy between PODXL and the epidermal growth factor receptor (EGFR) has been observed in colorectal cancer. Here, we investigated whether this also applies to periampullary adenocarcinomas. We analyzed the immunohistochemical expression of PODXL and EGFR in tissue microarrays with tumors from two patient cohorts; (Cohort 1, n = 175) and (Cohort 2, n = 189). The effect of TGF-β-induced expression and siRNA-mediated knockdown of PODXL and EGFR, were investigated in pancreatic cancer cells (PANC-1) in vitro. We found a correlation between PODXL and EGFR in these cancers, and a synergistic adverse effect on survival. Furthermore, silencing PODXL in pancreatic cancer cells resulted in the down-regulation of EGFR, but not vice versa. Consequently, these findings suggest a functional link between PODXL and EGFR, and the potential combined utility as biomarkers possibly improving patient stratification. Further studies examining the mechanistic basis underlying these observations may open new avenues of targeted treatment options for subsets of patients affected by these particularly aggressive cancers.

Figure 1

Correlations between the immunohistochemical expression of PODXL and EGFR. Bar charts visualizing the correlations between low vs. high (0 vs. 1) PODXL and low vs. high (0 vs. 1) EGFR. The top row shows the correlations for Cohort 1, for the entire cohort and divided into morphological subtypes. The bottom row shows the correlations for Cohort 2.

Figure 2

Overall survival according to PODXL and EGFR expressions in Cohort 1. Kaplan-Meier estimates for the overall survival according to the combined strata of low and high PODXL and EGFR expression in Cohort 1, shown for the entire cohort, for PB-type and I-type tumors. The p-value was based on a pairwise comparison with PODXL-/EGFR- and PODXL+/EGFR+.

Figure 3

Overall survival according to PODXL and EGFR expressions in Cohort 2. Kaplan-Meier estimates for the overall survival in pancreatic ductal adenocarcinoma according to the combined strata of low and high expression of PODXL and EGFR in Cohort 2. The p-value was based on a pairwise comparison with PODXL-/EGFR- and PODXL+/EGFR+.

Figure 4

Effects of TGF-β incubation and siRNA-mediated silencing of PODXL and EGFR in pancreatic cancer cells. (A) 3D organotypic model of the PANC-1 cell line on gel sections, as visualized in an H&E stain (top row), without (left column) and after (right column) incubation with TGF-β. As shown in the second row, the expression of PODXL increases upon incubation with TGF-β, particularly along the invasive front (inserts show the immunocytochemistry on cell pellets). The third row shows that the expression of EGFR does not markedly change upon incubation with TGF-β. The fourth row shows the EGFR protein expression in siPODXL silenced PANC-1 cells and the bottom panel shows PODXL protein expression in siEGFR silenced PANC-1 cells. (B) qPCR demonstrating the mRNA levels of PODXL and EGFR in siPODXL and siEGFR PANC-1 cell line. (C) qPCR demonstrating the mRNA levels of PODXL and EGFR in TGF-β incubated PANC-1 cell line. (D) qPCR demonstrating mRNA levels of PODXL and EGFR where PANC-1 cells were incubated with TGF-β following siRNA transfection.