Article Figures & Data
Figures
- Figure 1.
HGF induces gefitinib resistance of lung adenocarcinoma cells with EGFR-activating mutations. A, HGF induced gefitinib resistance of PC-9 and HCC827 cells with EGFR-activating mutation. Tumor cells were incubated with increasing concentrations of gefitinib and/or HGF, and cell growth was determined after 72 h of treatment by MTT assay. B, pretreatment of HGF with anti-HGF antibody abrogated HGF-induced resistance of HCC827 cells to gefitinib. HGF (20 ng/mL) was pretreated with control IgG (2 μg/mL) or anti-HGF antibody (2 μg/mL) at 37°C for 1 h. The resultant solutions were added to the cultures of tumor cells with or without gefitinib (300 nmol/L). Cell growth was determined in the same way as in A. *, P < 0.01 (one-way ANOVA). C, HGF was most potent in induction of gefitinib resistance of PC-9 and HCC827 cells. PC-9 and HCC827 cells were incubated with or without gefitinib and/or 50 ng/mL of HGF, EGF, TGF-α, or IGF-I. Cell growth was determined after 72 h of treatment. The percentage of growth is shown relative to untreated controls.
- Figure 2.
HGF induces gefitinib resistance of lung adenocarcinoma cells with EGFR-activating mutations by restoring phosphorylation of Akt, but not EGFR or ErbB3. A, the phosphorylation of Akt, but not EGFR or ErbB3, was restored by HGF. Tumor cells were treated with or without gefitinib (300 nmol/L) and/or HGF (20 ng/mL) for 1 h. Cells were lysed, and the indicated proteins were detected by immunoblotting. B, cell extracts were immunoprecipitated with an antibody to MET or p85. The precipitated proteins were determined by immunoblotting with the indicated antibodies.
- Figure 3.
Specific down-regulation of MET, but not ErbB3, reversed gefitinib resistance and phosphorylation of Akt induced by HGF. A, siRNA specific for MET and ErbB3 down-regulated mRNA expression of MET and ErbB3, respectively. Control, MET-specific, or ErbB3-specific siRNAs were introduced into PC-9 and HCC827 cells. mRNA was extracted 24 h later and RT-PCR was done. B, siRNA specific for MET, but not for ErbB3, reversed gefitinib resistance by HGF. The growth of cells from A with or without gefitinib (300 nmol/L) and/or HGF (20 ng/mL) was measured with a Cell Counting Kit-8. *, P < 0.05 (one-way ANOVA). C, down-regulation of MET, but not ErbB3, by specific-siRNA inhibited restored Akt-phosphorylation by HGF in cells treated with gefitinib. Control, MET-specific, or ErbB3-specific siRNAs were introduced into PC-9 cells. After 48 h, the cells were treated with or without gefitinib (300 nmol/L) and/or HGF (20 ng/mL) for 1 h, and then cell extracts were prepared and immunoblotted with the indicated antibodies.
- Figure 4.
High immunoreactivity for HGF in cancer cells of intrinsic resistant tumors and those with acquired resistance without T790M second mutation or MET amplification. Immunohistochemical HGF staining of tumor obtained before gefitinib treatment from patient 4 who showed partial response (A), tumor obtained from patient 4 after acquired resistance by T790M second mutation (B), tumor obtained before gefitinib treatment from patient 1 who showed intrinsic resistance (C), and tumor obtained from patient 7 after acquired resistance without T790M second mutation or MET amplification (D). Magnification, ×200.
- Figure 5.
HGF derived from tumor cells induces gefitinib resistance of lung adenocarcinoma cells with EGFR-activating mutation. A, HGF production by PC-9, HCC827, mock-transfected PC-9 (PC-9/mock), and HGF gene–transfected PC-9 (PC-9/HGF) cells. The cells were incubated in medium for 48 h and culture supernatants were harvested. The level of HGF in the supernatants was determined by ELISA. B, HGF gene transfection into PC-9 cells resulted in resistance to gefitinib. Human HGF cDNA was introduced into PC-9 cells (PC-9/HGF). Vector alone served as a mock control (PC-9/mock). The cells were incubated with or without gefitinib (300 nmol/L) in the presence or absence of HGF (20 ng/mL). Cell growth was measured with a Cell Counting Kit-8. C, anti-HGF antibody abrogated the resistance of HGF gene–transfected PC-9 cells to gefitinib. PC-9/mock and PC-9/HGF cells were cultured in the presence or absence of control IgG (2 μg/mL) or anti-HGF antibody (2 μg/mL) with or without gefitinib (300 nmol/L). The growth of cells was measured with an MTT assay. *, P < 0.001 (one-way ANOVA).
Tables
- Table 1.
Summary of tissue samples from patients with lung adenocarcinoma with EGFR-activating mutations
Patient Specimen Type BOR EGFR mutation T790M MET amplification HGF MET Intrinsic resistance Pretreatment specimens 1 Pre-gefitinib Primary lung cancer PD L858R N N High 2+ 2 Pre-gefitinib Primary lung cancer SD L858R N N High 3+ 3 Pre-gefitinib Lymph node PD L858R N N High 3+ Acquired resistance Paired specimens 4 Pre-gefitinib Primary lung cancer PR Ex19Del N Low 3+ Post-gefitinib Y N Low 1+ 5 Pre-gefitinib Primary lung cancer PR Ex19Del N Low 3+ Post-gefitinib Y N Low 3+ 6 Pre-gefitinib Primary lung cancer PR Ex19Del N Low 3+ Post-gefitinib Y N Low 2+ Posttreatment specimens 7 Post-gefitinib Lymph node PR L858R N N High 3+ 8 Post-gefitinib Lung metastasis PR L858R N N Low 2+ Post-gefitinib Lung metastasis PR L858R Y N Low 2+ Responder Pretreatment specimens 9 Pre-gefitinib Primary lung cancer PR Ex19Del N NE Low 3+ 10 Pre-gefitinib Lymph node PR L858R N N Low 1+ 11 Pre-gefitinib Primary lung cancer PR Ex19Del N NE Low 2+ 12 Pre-gefitinib Primary lung cancer PR Ex19Del N NE Low 1+ 13 Pre-gefitinib Lymph node PR L858R N N Low 3+ 14 Pre-gefitinib Primary lung cancer PR L858R N NE Low 3+ 15 Pre-gefitinib Primary lung cancer PR L858R N NE Low 3+ 16 Pre-gefitinib Lymph node PR Ex19Del N N High 3+ -
Abbreviations: BOR, best overall response to gefitinib treatment; PR, partial response; SD, stable disease; PD, progressive disease; L858R, L858R point mutation in exon 21; Ex19Del, in-frame deletion in exon 19; N, no; Y, yes; NE, not evaluated.
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Supplementary Data, Yano, et al.
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Volume 68, Issue 22
