Search

Supplemental Figure Legends

The Golgi apparatus is responsible for transporting, processing, and sorting numerous proteins in the cell, including cell surface-expressed receptor tyrosine kinases (RTK). The small-molecule compound M-COPA [2-methylcoprophilinamide (AMF-26)] disrupts the Golgi apparatus by inhibiting the activation of Arf1, resulting in suppression of tumor growth. Here, we report an evaluation of M-COPA activity against RTK-addicted cancers, focusing specifically on human gastric cancer (GC) cells with or without MET amplification. As expected, the MET-addicted cell line MKN45 exhibited a better response to M-COPA than cell lines without MET amplification. Upon M-COPA treatment, cell surface expression of MET was downregulated with a concurrent accumulation of its precursor form. M-COPA also reduced levels of the phosphorylated form of MET along with the downstream signaling molecules Akt and S6. Similar results were obtained in additional GC cell lines with amplification of MET or the FGF receptor FGFR2. MKN45 murine xenograft experiments demonstrated the antitumor activity of M-COPA in vivo. Taken together, our results offer an initial preclinical proof of concept for the use of M-COPA as a candidate treatment option for MET-addicted GC, with broader implications for targeting the Golgi apparatus as a novel cancer therapeutic approach. Cancer Res; 76(13); 3895–903. ©2016 AACR.

The antibodies used in this study.

Supplemental Materials and Methods for "Authentication of cell lines"

Dose-dependent effects on cell surface expression and processing of MET protein, and phosphorylation status of downstream signaling molecules.

The effect of M-COPA on cell surface expression of BCRP1 and P-glycoprotein (MDR1).

Effect of M-COPA on cell surface expression of EGFR in MET-unamplified GC cell lines.

Effect of M-COPA on cell surface expression and phosphorylation status of HER3 in MKN-45 cells.

The Golgi apparatus plays an essential role in the transport, processing, and sorting of cell surface proteins. We previously demonstrated that a Golgi disrupting agent, 2-methylcoprophilinamide (M-COPA), exhibited antitumor effect in human cancer cells in vitro and in vivo; however, in vivo antitumor efficacy of M-COPA did not simply reflect its in vitro efficacy, probably due to the lack of microenvironmental features of tumor tissues in the monolayer culture conditions. To bridge the gap between in vitro and in vivo efficacies, we exploited 3-dimensional (3D) spheroid culture models, which can recapitulate physiological characteristics of tumor tissues; i.e., nutrient/oxygen gradient, cell-cell interaction and cell-extracellular matrix (ECM) interaction, if cancer cells synthesize their own ECM molecules. Interestingly, human gastric cancer MKN1 cells displayed hypersensitivity to M-COPA under the 3D culture conditions compared to those in the monolayer culture conditions. Especially, we found that M-COPA caused loss of spheroid integrity. We previously demonstrated that M-COPA exerted antitumor effects via inhibiting intracellular transport of receptor tyrosine kinases onto the cell surface. Therefore, we examined the involvement of cell adhesion molecules in the antitumor effect of M-COPA. As a result, we found that specific integrins, which serve as cell surface receptors responsible for cell-ECM adhesion, were overexpressed and their downstream signaling molecules such as Src, Akt and MEK/ERK were highly phosphorylated in the cells under the 3D culture conditions compared to those under the monolayer culture conditions. Upon exposure to M-COPA, cell surface expression of these integrins was efficiently decreased and the downstream signaling molecules were dephosphorylated along with loss of spheroid integrity. Moreover, expression knockdown or neutralizing antibody of the integrins clearly suppressed sphere formation of MKN1 cells. These results suggest that M-COPA exerted in vivo antitumor effects via targeting specific integrins that mediate cell-matrix adhesion and its downstream intracellular signaling pathway observed in 3D spheroid culture models. Citation Format: Yoshimi Ohashi, Kazuma Takeuchi, Mutsumi Okamura, Akinobu Akatsuka, Isamu Shiina, Kentaro Yoshimatsu, Shingo Dan. Potential antitumor effects of a Golgi disrupting agent, M-COPA, via targeting cell-extracellular matrix interaction under the spheroid culture conditions [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 1299.