The Reg4-CD44ICD pathway and tumor aggression in early-stage colon adenocarcinoma.

Abstract

234 Background: Reg4, or regenerating islet-derived protein 4, is highly expressed in gastrointestinal malignancies and acts as a mitogenic factor. Our prior work suggests a direct invitro interaction between Reg4 and CD44, a non-kinase transmembrane glycoprotein over-expressed in cancer stem cells. Reg4 is hypothesized to activate CD44 proteolytic cleavage to release intra-cytoplasmic domain of CD44 (CD44ICD). The goal of this study was to demonstrate clinical validity of the Reg4-CD44/CD44ICD pathway in stage II/III colon cancer and its association with disease recurrence and clinical parameters of aggression. Methods: We constructed a tissue microarray (TMA) consisting of 94 stage II/III matched colon adenocarcinoma patients, 23 of which had recurrent disease after potentially curative treatment. The TMA was immunohistochemically stained for Reg4, CD44, and CD44ICD proteins with specific antibodies. Spearman’s correlation, Chi-square tests, and Kaplan-Meier analyses were used to identify protein associations with tumor characteristics, recurrence and overall survival. Results: Expression of Reg4, CD44, and CD44ICD was associated with tumor location, size, tumor grade, mismatch repair (MMR) status, and disease recurrence. TMA data analysis supported our proposed mechanism of Reg4-mediated release of CD44ICD into the cytoplasm. A significant correlation was observed between Reg4 and CD44 (r2= 0.23, P = 0.028), CD44 and CD44ICD (r2= 0.36, p = 0.0004), and Reg4 and CD44ICD (r2= 0.45, p = < 0.0001). Reg4 expression was associated with larger tumor size (r2= 0.23, p = 0.026) . No association was observed between Reg4, CD44, or CD44ICD expression with disease recurrence. In patients who recurred, Reg4-positive patients had a median survival of 4 years vs. 7 years for Reg4-negative patients (p = 0.04). Our lab has performed in vitroexperiments with CD44ICD interfering peptides blocking the pro-proliferative effect of Reg4. Conclusions: These results suggest that Reg4-positive tumors, while not at higher risk of recurrence, are associated with decreased survival in established recurrent colon adenocarcinoma. Inhibition of the Reg4-CD44/CD44ICD pathway may be a future therapeutic target.