TGF-β1 increases cellular invasion of colorectal neuroendocrine carcinoma cell line through partial epithelial-mesenchymal transition

Norihiko Sasaki; Seiichi Shinji; Yuuki Shichi; Toshiyuki Ishiwata; Tomio Arai; Takeshi Yamada; Goro Takahashi; Ryo Ohta; Hiromichi Sonoda; Akihisa Matsuda; Takuma Iwai; Kohki Takeda; Kazuhide Yonaga; Koji Ueda; Sho Kuriyama; Toshimitsu Miyasaka; Hiroshi Yoshida

doi:10.1016/j.bbrep.2022.101239

TGF-β1 increases cellular invasion of colorectal neuroendocrine carcinoma cell line through partial epithelial-mesenchymal transition

Biochem Biophys Rep. 2022 Mar 1:30:101239. doi: 10.1016/j.bbrep.2022.101239. eCollection 2022 Jul.

Authors

Norihiko Sasaki¹, Seiichi Shinji^{2

3}, Yuuki Shichi³, Toshiyuki Ishiwata³, Tomio Arai⁴, Takeshi Yamada², Goro Takahashi², Ryo Ohta², Hiromichi Sonoda², Akihisa Matsuda², Takuma Iwai², Kohki Takeda², Kazuhide Yonaga², Koji Ueda², Sho Kuriyama², Toshimitsu Miyasaka², Hiroshi Yoshida²

Affiliations

¹ Research Team for Geriatric Medicine (Vascular Medicine), Tokyo Metropolitan Institute of Gerontology, Tokyo, 173-0015, Japan.
² Department of Gastrointestinal and Hepato-Biliary-Pancreatic Surgery, Nippon Medical School, Tokyo, 113-8603, Japan.
³ Division of Aging and Carcinogenesis, Research Team for Geriatric Pathology, Tokyo Metropolitan Institute of Gerontology, Tokyo, 173-0015, Japan.
⁴ Department of Pathology, Tokyo Metropolitan Geriatric Hospital and Institute of Gerontology, Tokyo, 173-0015, Japan.

Abstract

Epithelial-mesenchymal transition (EMT) plays a pivotal role in cancer progression and metastasis in many types of malignancies, including colorectal cancer. Although the importance of EMT is also considered in colorectal neuroendocrine carcinoma (NEC), its regulatory mechanisms have not been elucidated. We recently established a human colorectal NEC cell line, SS-2. In this study, we aimed to clarify whether these cells were sensitive to transforming growth factor beta 1 (TGF-β1) and whether EMT could be induced through TGF-β1/Smad signaling, with the corresponding NEC cell-specific changes in invasiveness. In SS-2 cells, activation of TGF-β1 signaling, as indicated by phosphorylation of Smad2/3, was dose-dependent, demonstrating that SS-2 cells were responsive to TGF-β1. Analysis of EMT markers showed that mRNA levels changed with TGF-β1 treatment and that E-cadherin, an EMT marker, was expressed in cell-cell junctions even after TGF-β1 treatment. Invasion assays showed that TGF-β1-treated SS-2 cells invaded more rapidly than non-treated cells, and these cells demonstrated increased metalloproteinase activity and cell adhesion. Among integrins involved in cell-to-matrix adhesion, α2-integrin was exclusively upregulated in TGF-β1-treated SS-2 cells, but not in other colon cancer cell lines, and adhesion and invasion were inhibited by an anti-α2-integrin blocking antibody. Our findings suggest that α2-integrin may represent a novel therapeutic target for the metastasis of colorectal NEC cells.

Keywords: Adhesion; BSA, bovine serum albumin; CSC, cancer stem cell; EMT; EMT, epithelial-to-mesenchymal transition; FACS, fluorescence activated cell sorter; Invasion; MFI, mean fluorescence intensity; MMP, matrix metalloproteinase; NEC, neuroendocrine carcinoma; NENs, neuroendocrine neoplasms; Neuroendocrine carcinoma; SD, standard deviation; SEM, scanning electron microscopic; TGF, transforming growth factor-beta; TGF-β1; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; α2-integrin.