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. 2010 Jan;101(1):167-72.
doi: 10.1111/j.1349-7006.2009.01368.x. Epub 2009 Sep 14.

Effects of Src inhibitors on cell growth and epidermal growth factor receptor and MET signaling in gefitinib-resistant non-small cell lung cancer cells with acquired MET amplification

Takeshi Yoshida  1 , Isamu Okamoto, Wataru Okamoto, Erina Hatashita, Yuki Yamada, Kiyoko Kuwata, Kazuto Nishio, Masahiro Fukuoka, Pasi A Jänne, Kazuhiko Nakagawa
Affiliations

Effects of Src inhibitors on cell growth and epidermal growth factor receptor and MET signaling in gefitinib-resistant non-small cell lung cancer cells with acquired MET amplification

Takeshi Yoshida et al. Cancer Sci. 2010 Jan.

Abstract

The efficacy of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors such as gefitinib and erlotinib in non-small cell lung cancer (NSCLC) is often limited by the emergence of drug resistance conferred either by a secondary T790M mutation of EGFR or by acquired amplification of the MET gene. We now show that the extent of activation of the tyrosine kinase Src is markedly increased in gefitinib-resistant NSCLC (HCC827 GR) cells with MET amplification compared with that in the gefitinib-sensitive parental (HCC827) cells. In contrast, the extent of Src activation did not differ between gefitinib-resistant NSCLC (PC9/ZD) cells harboring the T790M mutation of EGFR and the corresponding gefitinib-sensitive parental (PC9) cells. This activation of Src in HCC827 GR cells was largely abolished by the MET-TKI PHA-665752 but was only partially inhibited by gefitinib, suggesting that Src activation is more dependent on MET signaling than on EGFR signaling in gefitinib-resistant NSCLC cells with MET amplification. Src inhibitors blocked Akt and Erk signaling pathways, resulting in both suppression of cell growth and induction of apoptosis, in HCC827 GR cells as effectively as did the combination of gefitinib and PHA-665752. Furthermore, Src inhibitor dasatinib inhibited tumor growth in HCC827 GR xenografts to a significantly greater extent than did treatment with gefitinib alone. These results provide a rationale for clinical targeting of Src in gefitinib-resistant NSCLC with MET amplification.

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Figures

Figure 1
Figure 1
Activation of Src in non‐small cell lung cancer cells with or without MET amplification. (A) HCC827 cells, their gefitinib‐resistant clones with MET amplification (HCC827 GR5 and GR6), PC9 cells, and their gefitinib‐resistant clone with a secondary T790M mutation of epidermal growth factor receptor (PC9/ZD) were incubated for 24 h in medium containing 10% serum. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to phosphorylated (p‐) or total forms of MET, ErbB3, and Src as well as with those to β‐actin (loading control). (B) H1299 and H460 cells without MET amplification, and H1838, EBC‐1, and H820 cells with MET amplification were incubated for 24 h in medium containing 10% serum. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to phosphorylated (p‐) or total forms of MET and Src as well as with those to β‐actin (loading control).
Figure 2
Figure 2
Effects of various inhibitors on epidermal growth factor receptor (EGFR) and MET signaling in gefitinib‐resistant non‐small cell lung cancer cells with MET amplification. (A) HCC827 cells and a gefitinib‐resistant clone with MET amplification (HCC827 GR5) were incubated for 12 h in the absence (control) or presence of gefitinib alone (1 μm), PHA‐665752 alone (1 μm), gefitinib and PHA‐665752 combined, PP1 (10 μm), or dasatinib (500 nm) in medium containing 10% serum. Cell lysates were then subjected to immunoblot analysis with antibodies to phosphorylated (p‐) or total forms of EGFR, MET, ErbB3, Src, Akt, and Erk. (B) HCC827 and HCC827 GR5 cells were incubated for 24 h in medium containing 10% serum, lysed, and subjected to immunoprecipitation (IP) with an antibody to Src. The resulting precipitates were subjected to immunoblot analysis with antibodies to EGFR, MET, ErbB3, and Src.
Figure 3
Figure 3
Effects of dasatinib on growth and apoptosis in gefitinib‐resistant non‐small cell lung cancer cells with MET amplification. (A) HCC827 cells or (B) HCC827 GR5 cells were treated for 72 h with increasing concentrations of gefitinib alone, PHA‐665752 alone, gefitinib and PHA‐665752 in combination, or dasatinib alone in medium containing 10% serum, after which cell viability was assessed. Data are means of triplicates from a representative experiment and are expressed as a percentage of the value for untreated cells. (C) HCC827 and HCC827 GR5 cells were incubated for 72 h with gefitinib (1 μm) alone, PHA‐665752 (1 μm) alone, gefitinib plus PHA‐665752, or dasatinib (1 μm) in medium containing 10% serum. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to poly(ADP‐ribose) polymerase (PARP) and to β‐actin. The positions of intact PARP (116 kDa) and the 85‐kDa cleavage fragment (c‐PARP) are shown.
Figure 4
Figure 4
Effects of dasatinib on the growth of gefitinib‐resistant non‐small cell lung cancer cells with MET amplification in vivo. Nude mice with tumor xenografts established by s.c. implantation of HCC827 GR5 cells were treated daily for 28 days with vehicle (control), gefitinib (50 mg/kg), or dasatinib (15 mg/kg) by oral gavage. Tumor volume was determined at the indicated times after the onset of treatment. Points indicate the mean of values from five mice per group; bars indicate SE. *P < 0.05 for dasatinib versus control or gefitinib alone (Student’s t‐test).
Figure 5
Figure 5
Models for signaling pathways in gefitinib‐sensitive non‐small cell lung cancer (NSCLC) cells (A) and gefitinib‐resistant NSCLC cells with acquired MET amplification (B). Src functions downstream of both epidermal growth factor receptor (EGFR) and MET as well as upstream of Akt and Erk signaling pathways and EGFR. However, the dependency of Src signaling is shifted from EGFR to MET and MET associates with ErbB3 after the acquisition of MET amplification. EGFR mediates, at least in part, activation of MET in gefitinib‐sensitive NSCLC cells, whereas EGFR and MET function independently of each other in gefitinib‐resistant NSCLC cells with acquired MET amplification. Pathways targeted by gefitinib or PHA‐665752 are indicated, and the relative activities of signaling pathways are denoted by the width of the arrows.
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