Semaphorin 7A promotes EGFR-TKI resistance in EGFR mutant lung adenocarcinoma cells
Yuhei Kinehara, Izumi Nagatomo, Shohei Koyama, Daisuke Ito, Satoshi Nojima, Ryota Kurebayashi, Yoshimitsu Nakanishi, Yasuhiko Suga, Yu Nishijima-Futami, Akio Osa, Takeshi Nakatani, Yasuhiro Kato, Masayuki Nishide, Yoshitomo Hayama, Masayoshi Higashiguchi, Osamu Morimura, Kotaro Miyake, Sujin Kang, Toshiyuki Minami, Haruhiko Hirata, Kota Iwahori, Takayuki Takimoto, Hyota Takamatsu, Yoshito Takeda, Naoki Hosen, Shigenori Hoshino, Yasushi Shintani, Meinoshin Okumura, Toru Kumagai, Kazumi Nishino, Fumio Imamura, Shin-ichi Nakatsuka, Takashi Kijima, Hiroshi Kida, Atsushi Kumanogoh
Yuhei Kinehara, Izumi Nagatomo, Shohei Koyama, Daisuke Ito, Satoshi Nojima, Ryota Kurebayashi, Yoshimitsu Nakanishi, Yasuhiko Suga, Yu Nishijima-Futami, Akio Osa, Takeshi Nakatani, Yasuhiro Kato, Masayuki Nishide, Yoshitomo Hayama, Masayoshi Higashiguchi, Osamu Morimura, Kotaro Miyake, Sujin Kang, Toshiyuki Minami, Haruhiko Hirata, Kota Iwahori, Takayuki Takimoto, Hyota Takamatsu, Yoshito Takeda, Naoki Hosen, Shigenori Hoshino, Yasushi Shintani, Meinoshin Okumura, Toru Kumagai, Kazumi Nishino, Fumio Imamura, Shin-ichi Nakatsuka, Takashi Kijima, Hiroshi Kida, Atsushi Kumanogoh
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Research Article Oncology Pulmonology

Semaphorin 7A promotes EGFR-TKI resistance in EGFR mutant lung adenocarcinoma cells

Abstract

Although responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs) are initially positive, 30%–40% of patients with EGFR-mutant tumors do not respond well to EGFR-TKIs, and most lung cancer patients harboring EGFR mutations experience relapse with resistance. Therefore, it is necessary to identify not only the mechanisms underlying EGFR-TKI resistance, but also potentially novel therapeutic targets and/or predictive biomarkers for EGFR-mutant lung adenocarcinoma. We found that the GPI-anchored protein semaphorin 7A (SEMA7A) is highly induced by the EGFR pathway, via mTOR signaling, and that expression levels of SEMA7A in human lung adenocarcinoma specimens were correlated with mTOR activation. Investigations using cell culture and animal models demonstrated that loss or overexpression of SEMA7A made cells less or more resistant to EGFR-TKIs, respectively. The resistance was due to the inhibition of apoptosis by aberrant activation of ERK. The ERK signal was suppressed by knockdown of integrin β1 (ITGB1). Furthermore, in patients with EGFR mutant tumors, higher SEMA7A expression in clinical samples predicted poorer response to EGFR-TKI treatment. Collectively, these data show that the SEMA7A–ITGB1 axis plays pivotal roles in EGFR-TKI resistance mediated by ERK activation and apoptosis inhibition. Moreover, our results reveal the potential utility of SEMA7A not only as a predictive biomarker, but also as a potentially novel therapeutic target in EGFR-mutant lung adenocarcinoma.

Authors

Yuhei Kinehara, Izumi Nagatomo, Shohei Koyama, Daisuke Ito, Satoshi Nojima, Ryota Kurebayashi, Yoshimitsu Nakanishi, Yasuhiko Suga, Yu Nishijima-Futami, Akio Osa, Takeshi Nakatani, Yasuhiro Kato, Masayuki Nishide, Yoshitomo Hayama, Masayoshi Higashiguchi, Osamu Morimura, Kotaro Miyake, Sujin Kang, Toshiyuki Minami, Haruhiko Hirata, Kota Iwahori, Takayuki Takimoto, Hyota Takamatsu, Yoshito Takeda, Naoki Hosen, Shigenori Hoshino, Yasushi Shintani, Meinoshin Okumura, Toru Kumagai, Kazumi Nishino, Fumio Imamura, Shin-ichi Nakatsuka, Takashi Kijima, Hiroshi Kida, Atsushi Kumanogoh

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Figure 6

The SEMA7A–ITGB1 axis regulates Erk phosphorylation under erlotinib treatment.

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The SEMA7A–ITGB1 axis regulates Erk phosphorylation under erlotinib trea...
(A and B) ITGB1 expression in SEMA7A WT or KO HCC827 cells generated by sh-Ctrl or sh-ITGB1 RNA, tested by FCM and qPCR. Data (means ± SE) are representative of 3 independent experiments. MFI, median fluorescence intensity; iso, isotype control. Gray area indicates the isotype control. The 2-sample t test was used to detect significance. ***P < 0.001. (C) Representative immunoblots showing p-EGFR and p-Erk in HCC827 cells 24 hours after treatment with erlotinib. After long-term treatment, the p-Erk level was maintained in SEMA7A WT cells, despite a reduction in the level of p-EGFR, which was suppressed by knockdown of ITGB1. (D) SEMA7A WT and KO HCC827 cells were treated with low-dose erlotinib for several weeks. Treated cells were passaged and cultured on 96-well plates for 24 hours, exposed to erlotinib and U0124 (5 μM) or U0126 (5 μM) for 72 hours, and then subjected to modified MTT assay. Data (means ± SE) are representative of 3 independent experiments. To evaluate significance, ANOVA followed by Tukey–Kramer multiple comparison test was used. *P < 0.05. (E) PFS for patients with EGFR mutation for whom EGFR-TKI was used as the first-line treatment. The P value for the difference between the 2 curves was determined by the log-rank test. IHC scores were defined as follows: 0, 0%–1% positive tumor cells; 1, 1%–20%; 2, 21%–50%; 3, 51%–100%. Based on SEMA7A IHC, patients with scores of 0 or 1 were classified into the low-SEMA7A group, and those with scores of 2 or 3 were classified into the high-SEMA7A group.