tapi-2 and Colorectal-Neoplasms

Epithelial Cell Adhesion Molecule (EpCAM), or CD326, is a trans-membrane glycoprotein expressed by multiple normal epithelia as well as carcinoma. Human hepatic stem cells and bile duct epithelium of the liver are EpCAM positive. In tumor cell lines, its intracellular domain can be released after cleavage of the extracellular domain. Within the cell nucleus, it induces cell proliferation, but cleavage depends on cell contact. Fragments of various lengths have been described in tumor cells. Despite its described important role in proliferation in tumor cells, there is not much known about the expression and role of EpCAM fragments in primary human liver cells. Here, we demonstrate that EpCAM protein fragments and function are considerable different between tumor cells, normal fetal and adult liver cells. Contrary to previously reported findings in tumor cells, gene knockdown or treatment with an inhibitor of the cleavage enzyme ADAM17 (TACE) rather increased cell numbers in primary human fetal liver-derived EpCAM-positive cells. EpCAM fragment sizes were not affected by treatment with inhibitor. Knockdown of EPCAM gene expression by siRNA in sorted cells did not significantly affect proliferation-associated genes or cell numbers. The intracellular domain could not be detected within cell nuclei of fetal and adult liver cells. In conclusion, signaling through the intracellular domain of EpCAM appears to be a mechanism that induces proliferation specifically in tumorigenic cells but not in normal primary EpCAM-positive liver cells.

Topics: ADAM17 Protein; Adult Stem Cells; Cell Proliferation; Colorectal Neoplasms; Epithelial Cell Adhesion Molecule; Fetal Stem Cells; Gene Expression Regulation; Glycosylation; HT29 Cells; Humans; Hydroxamic Acids; Liver; Neoplastic Stem Cells; Peptide Fragments; Primary Cell Culture; Protein Domains; Signal Transduction

Evidence is accumulating for the role of cancer stem cells (CSCs) in mediating chemoresistance in patients with metastatic colorectal cancer (mCRC). A disintegrin and metalloproteinase domain 17 (ADAM17; also known as tumor necrosis factor-α-converting enzyme [TACE]) was shown to be overexpressed and to mediate cell proliferation and chemoresistance in CRC cells. However, its role in mediating the CSC phenotype in CRC has not been well-characterized. The objective of the present study was to determine whether ADAM17 regulates the CSC phenotype in CRC and to elucidate the downstream signaling mechanism that mediates cancer stemness. We treated established CRC cell lines and a newly established human CRC cell line HCP-1 with ADAM17-specific small interfering RNA (siRNA) or the synthetic peptide inhibitor TAPI-2. The effects of ADAM17 inhibition on the CSC phenotype and chemosensitivity to 5-fluorouracil (5-FU) in CRC cells were examined. siRNA knockdown and TAPI-2 decreased the protein levels of cleaved Notch1 (Notch1 intracellular domain) and HES-1 in CRC cells. A decrease in the CSC phenotype was determined by sphere formation and ALDEFLUOR assays. Moreover, TAPI-2 sensitized CRC cells to 5-FU by decreasing cell viability and the median lethal dose of 5-FU and increasing apoptosis. We also showed the cleavage and release of soluble Jagged-1 and -2 by ADAM17 in CRC cells. Our studies have elucidated a role of ADAM17 in regulating the CSC phenotype and chemoresistance in CRC cells. The use of drugs that inhibit ADAM17 activity might increase the therapeutic benefit to patients with mCRC and, potentially, those with other solid malignancies.

Topics: ADAM Proteins; ADAM17 Protein; Apoptosis; Cell Line, Tumor; Cell Proliferation; Colorectal Neoplasms; Fluorouracil; Gene Expression Regulation, Neoplastic; Humans; Hydroxamic Acids; Neoplastic Stem Cells; Receptor, Notch1; Signal Transduction