Epithelial cell adhesion molecule (EpCAM) regulates HGFR signaling to promote colon cancer progression and metastasis

(2023) 21:530 Lee et al. Journal of Translational Medicine https://doi.org/10.1186/s12967-023-04390-2 Journal of Translational Medicine Open Access RESEARCH Epithelial cell adhesion molecule (EpCAM) regulates HGFR signaling to promote colon cancer progression and metastasis Chi‑Chiu Lee1, Chia‑Jui Yu1, Sushree Shankar Panda1, Kai‑Chi Chen1, Kang‑Hao Liang1,2, Wan‑Chen Huang1, Yu‑Shiuan Wang1, Pei‑Chin Ho2 and Han‑Chung Wu1,2* Abstract Background Epithelial cell adhesion molecule (EpCAM) is known to highly expression and promotes cancer progres‑ sion in many cancer types, including colorectal cancer. While metastasis is one of the main causes of cancer treatment failure, the involvement of EpCAM signaling in metastatic processes is unclear. We propose the potential crosstalk of EpCAM signaling with the HGFR signaling in order to govern metastatic activity in colorectal cancer. Methods Immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), and fluorescence resonance energy transfer (FRET) was conducted to explore the extracellular domain of EpCAM (EpEX) and HGFR interaction. Western blotting was taken to determine the expression of proteins in colorectal cancer (CRC) cell lines. The functions of EpEX in CRC were investigated by proliferation, migration, and invasion analysis. The combined therapy was vali‑ dated via a tail vein injection method for the metastasis and orthotopic colon cancer models. Results This study demonstrates that the EpEX binds to HGFR and induces downstream signaling in colon cancer cells. Moreover, EpEX and HGF cooperatively mediate HGFR signaling. Furthermore, EpEX enhances the epithelialto-mesenchymal transition and metastatic potential of colon cancer cells by activating ERK and FAK-AKT signaling pathways, and it further stabilizes active β-catenin and Snail proteins by decreasing GSK3β activity. Finally, we show that the combined treatment of an anti-EpCAM neutralizing antibody (EpAb2-6) and an HGFR inhibitor (crizotinib) significantly inhibits tumor progression and prolongs survival in metastatic and orthotopic animal models of colon cancer. Conclusion Our findings illuminate the molecular mechanisms underlying EpCAM signaling promotion of colon cancer metastasis, further suggesting that the combination of EpAb2-6 and crizotinib may be an effective strategy for treating cancer patients with high EpCAM expression. Keywords EpCAM, EpEX, HGFR, Cancer progression, Epithelial-to-mesenchymal transition, Invasion, Colorectal cancer *Correspondence: Han‑Chung Wu 1 Institute of Cellular and Organismic Biology, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 11529, Taiwan 2 Biomedical Translation Research Center (BioTReC), Academia Sinica, Taipei 11529, Taiwan Background EpCAM is a type I transmembrane protein with 314 amino acids and an observed molecular weight of 39–42 kDa. It contains an extracellular domain (EpEX, 265 amino acids), a single transmembrane domain, and a short intracellular domain (EpICD, 26 amino acids). The domains are cleaved and released via regulated intramembrane proteolysis (RIP) by a disintegrin and © The Author(s) 2023. 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The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Lee et al. Journal of Translational Medicine (2023) 21:530 metalloprotease ADAM17 (also called tumor necrosis factor-α converting enzyme, TACE) and a multi-subunit protease complex, γ-secretase [1]. RIP can trigger EpCAM-mediated signal transduction through the shedding of EpEX by ADAM17 and EpICD by γ-secretase complex [1, 2] and play important roles in tumor initiation and progression [3–6]. Although, EpCAM is absent or weakly expressed in the vast majority of healthy epithelial squamous cells, it is strongly expressed in squamous cell carcinomas [7]. Furthermore, the expression of EpCAM in squamous carcinomas is correlated with increased cellular proliferation and decreased differentiation [8]. Our group previously developed a neutralizing antibody against EpCAM, EpAb2-6, which has strong potential for use as a colorectal carcinoma (CRC) treatment [5, 9, 10]. Despite its promise as a therapeutic target in CRC, the mechanisms through which EpCAM contributes to tumorigenesis and metastasis are still not completely known. HGFR (hepatocyte growth factor receptor), also called c-Met, is a high affinity receptor tyrosine kinase (RTK) that is activated by hepatocyte growth factor (HGF, also known as Scatter Factor) and encoded by the MET gene [11]. The tyrosine kinase domain of HGFR contains two tyrosine residues at positions 1234 and 1235, and the phosphorylation of these two sites is essential for activating the HGFR receptor [12]. Many reports have demonstrated critical roles for HGFR in tumorigenesis, cell growth, survival, and metastasis [13, 14]. Hepatocyte growth factor (HGF), the ligand of HGFR, is a pleiotropic cytokine mainly produced by mesenchymal cells, including fibroblasts and macrophages. The activation of HGFR can induce through HGF binding to HGFR resulting in HGFR homodimerization, or HGFR dimerizes with different receptor pathways [15]. HGFR activation induces tumor progression via signaling cascades that mainly affect the tumor angiogenesis, growth, motility, and metastasis in many cancer cells, including colorectal cancer (CRC) [16, 17]. HGFR overexpression has been reported in CRC, which has been demonstrated to be critically attributable to CRC stemness and poor prognosis [18, 19]. Clarifying the mechanism of regulating HGFR signaling in CRC is important for finding effective therapy for CRC. Epithelial–mesenchymal transition (EMT) is associated with tumorigenic process and metastasis [20]. Cancer cells that undergo EMT exhibit enhanced cell motility and invasion through the induction of mesenchymal properties and loss of epithelial cell adhesion. Indicators of EMT include increased expression of mesenchymal markers, such as Vimentin, Snail, and Slug, along with decreased expression of epithelial markers (...truncated)