Abstract
Cholangiocarcinoma (CCA) is a terminal diagnosis, with 5-year survival rates averaging less than 10%. Thus, developing therapeutic options for cholangiocarcinoma patients is essential. FGFR4 is a heavily N-glycosylated receptor tyrosine kinase that signals through Akt, ERK and STAT3 pathways, and whose overexpression in cancer has previously been observed. The activation and signaling through FGFR4 involve the intracellular tyrosine kinase domain that is activated upon ligand binding the extracellular receptor region. Examination of expression in the TCGA database and our studies in CCA cells demonstrated a role for FGFR4 in promoting tumor cell survival and proliferation. Further, we have observed the presence of a proteolytic cleavage product of FGFR4, that is comprised of the intracellular kinase domain, which we call R4-ICD. Our data and previous studies on recombinant protein demonstrated that the intracellular kinase domain in the absence of the transmembrane and ligand-binding domains is constitutively active. HuCCT-1 cells (human CCA cell line lacking endogenous FGFR4 expression) stably transfected to express R4-ICD had increased proliferation and pro-survival phenotypes when compared to the parental cell line. Of 14 frozen CCA human tumor samples, all showed expression of R4-ICD. Compared to normal liver tissue, R4-ICD expression was greater in 65% (9 of 14) of tumor samples than in normal liver. In addition, total FGFR4 expression was lower in normal cholangiocytes than human CCA samples. Full length FGFR4 expression was observed in 12 tumor samples (85%). Of note, full-length FGFR4 expression levels were far weaker than R4-ICD expression in the 10 samples that showed both signals. Because R4-ICD is produced from full length receptor, we interpret the predominance and higher prevalence of R4-ICD to reflect efficient processing to a stable intracellular kinase fragment. We demonstrated at least two glycoforms of FGFR4, terminal and core, in cell lines, and these were also observed in human tumors. We hypothesized that glycosylation may regulate R4-ICD proteolytic production. Live CCA cells in culture were treated with PNGase F to remove extracellular N-linked sugars from cell surface proteins, including FGFR4. Deglycosylation of FGFR4 was confirmed by immunoblotting. Upon deglycosylation, R4-ICD levels rapidly increased. In summary, CCA cells in culture and in tumors were demonstrated to have high levels of R4-ICD, a constitutively active kinase that promoted tumor cell survival and proliferation. The stable cleavage product of FGFR4, R4-ICD, was increased on receptor deglycosylation. Whether aberrant FGFR4 glycosylation contributes to R4-ICD predominance in tumor samples or to tumor aggressiveness remains to be tested. Further understanding of R4-ICD formation will allow us to better target CCA, possibly by reducing proteolytic processing in conjunction with kinase inhibition. Citation Format: Andrew J. Phillips, Ashley M. Mohr, Mary Anne Phillippi, Justin L. Mott, Keith R. Johnson. N terminal glycosylation regulates FGFR4 cleavage in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 856.
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