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

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

Norihiko Sasaki  1 , Seiichi Shinji  2   3 , Yuuki Shichi  3 , Toshiyuki Ishiwata  3 , Tomio Arai  4 , Takeshi Yamada  2 , Goro Takahashi  2 , Ryo Ohta  2 , Hiromichi Sonoda  2 , Akihisa Matsuda  2 , Takuma Iwai  2 , Kohki Takeda  2 , Kazuhide Yonaga  2 , Koji Ueda  2 , Sho Kuriyama  2 , Toshimitsu Miyasaka  2 , Hiroshi Yoshida  2
Affiliations

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

Norihiko Sasaki et al. Biochem Biophys Rep. .

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.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1
Fig. 1
TGF-β1 signaling in NEC cell line SS-2. (A) Immunoblotting for TGFβR-I, TGFβR-II and β-actin (loading control) in SS-2, DLD-1, and HT-29 cells. (B) Immunoblotting for p-Smad2, p-Smad3 and Smad2/3 in SS-2 cells cultured with or without indicated concentration of TGF-β1 for 2 d. (C) The histogram shows mean densitometric readings for the proteins normalized to Smad2/3. NEC: neuroendocrine carcinoma; TGF- β: transforming growth factor-beta.
Fig. 2
Fig. 2
EMT of NEC cell line SS-2. (A) Representative phase contrast images (upper) and SEM analysis (lower) of SS-2 cells cultured with or without under 10 ng/mL TGF-β1 for 2. Original magnification, ×100; inset, ×200; scale bars 10 μm. (B) qRT-PCR of EMT markers in SS-2 cells cultured with or without 10 ng/mL TGF-β1 for 2 d. Results are presented as the mean ± SD of three independent experiments; *p < 0.05, **p < 0.01. (C) Immunocytochemical staining in SS-2 cells cultured with or without 10 ng/mL TGF-β1 for 2 d. Representative images are shown (E-cadherin, red; DAPI, blue). scale bars 20 μm. (D) Matrigel invasion assays performed in SS-2 cells cultured with or without 10 ng/mL TGF-β1 for 2 d. Representative results from measurements of 12 fields are shown. **p < 0.01. EMT: epithelial–mesenchymal transition; NEC: neuroendocrine carcinoma; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; SEM: Scanning electron microscopic; TGF- β: transforming growth factor-beta. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3
Fig. 3
ECM degradation activity and adhesion ability in NEC cell line SS-2. (A) qRT-PCR of MT1-MMP and MMP2 in SS-2 cells cultured with or without 10 ng/mL TGF-β1 for 2 d. Results are presented as the mean ± SD of three independent experiments; *p < 0.05. (B) Gelatin zymography was performed using culture supernatants from SS-2 cells that were cultured with or without 10 ng/mL TGF-β1 for 2 d. Relative band intensity is shown. (C) Adhesion assays in SS-2 cells cultured with or without 10 ng/mL TGF-β1 for 2 d. Each cell type exhibited negligible adhesion to BSA. **P < 0.01. ECM: extracellular membrane; MMP: matrix metalloproteinase; NEC: neuroendocrine carcinoma; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; TGF- β: transforming growth factor-beta.
Fig. 4
Fig. 4
α2-integrin dependent adhesion and invasion in NEC cell line SS-2. (A) qRT-PCR of integrins in SS-2 cells cultured with or without 10 ng/mL TGF-β1 for 2 d. Results are presented as the mean ± SD of three independent experiments; **P < 0.01. (B) FACS of β1-integrin and α2-integrin levels in SS-2 cells cultured with or without 10 ng/mL TGF-β1 for 2 d. Controls are indicated by thin lines with gray color. MFIs relative to non-treated control cells are shown on the right side. Results are presented as means ± SD from three independent experiments. *P < 0.05, **P < 0.01. (C) Immunoblotting for α2-integrin and β-actin (loading control) in SS-2 and colon cancer cells cultured with or without 10 ng/mL TGF-β1 for 2 d. (D) Adhesion assays in SS-2 cells cultured with or without 10 ng/mL TGF-β1 for 2 d. Cells were treated with or without anti-α2-integrin antibody before assays. **P < 0.01. (E) Matrigel invasion assays performed in SS-2 cells cultured with or without 10 ng/mL TGF-β1 for 2 d. Cells were treated with or without anti-α2-integrin antibody before assays. Representative results from measurements of 12 fields are shown. **p < 0.01. FACS: fluorescence activated cell sorter; MFI: mean fluorescence intensity; NEC: neuroendocrine carcinoma; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; TGF- β: transforming growth factor-beta.
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