EGFR and MET receptor tyrosine kinase-altered microRNA expression induces tumorigenesis and gefitinib resistance in lung cancers

Abstract

The involvement of the MET oncogene in de novo and acquired resistance of non-small cell lung cancers (NSCLCs) to tyrosine kinase inhibitors (TKIs) has previously been reported, but the precise mechanism by which MET overexpression contributes to TKI-resistant NSCLC remains unclear. MicroRNAs (miRNAs) negatively regulate gene expression, and their dysregulation has been implicated in tumorigenesis. To understand their role in TKI-resistant NSCLCs, we examined changes in miRNA that are mediated by tyrosine kinase receptors. Here we report that miR-30b, miR-30c, miR-221 and miR-222 are modulated by both epidermal growth factor (EGF) and MET receptors, whereas miR-103 and miR-203 are controlled only by MET. We showed that these miRNAs have important roles in gefitinib-induced apoptosis and epithelial-mesenchymal transition of NSCLC cells in vitro and in vivo by inhibiting the expression of the genes encoding BCL2-like 11 (BIM), apoptotic peptidase activating factor 1 (APAF-1), protein kinase C ɛ (PKC-ɛ) and sarcoma viral oncogene homolog (SRC). These findings suggest that modulation of specific miRNAs may provide a therapeutic approach for the treatment of NSCLCs.

Figure 1. MiR-221-222, 30b-c, 103, 203 target APAF-1, BIM, PKC-ε and SRC

(a) EGFR and MET proteins and mRNAs down-regulation after EGFR and MET silencing. (b) Unsupervised hierarchical clustering based on miRNA expression profiles in shControl versus shEGFR and shMET-Calu-1 cells at a p value <0.05. (c) Intersection of shEGFR and shMET regulated microRNAs. (d) Northern blots showing miR-221,-222, 103, -203, -30b, and -30c deregulation after MET knockdown. SnRNA U6, loading control. (e) Decreased luciferase activity indicated direct miR-PKC-ε, SRC, APAF-1 and BIM 3′ UTR interactions (Fig. 1e) and target gene repression was rescued by mutations or deletions in the complementary seed sites. In the case of SRC only the site 1595-1601 is implicated in the binding with miR-203; deletion of the site 1706-1712 did not rescue luciferase activity (See also Supplementary Fig. 2). Relative repression of firefly luciferase expression was standardized to a transfection control. (f) Inverse correlation between miR-103,-203, 221-222 and -30b-c and target proteins in a panel of NSCLC cells. Correlation coefficients of -0.91 (miR-203/SRC), -0.92 (miR-221/APAF-1), -0.90 (miR-222/APAF-1), -0.55 (miR-30b/BIM), -0.91 (miR-30c/BIM), -0.87 (miR-103/PKC-ε), P<0.05. (g) MiR-221&222, miR-30b-c overexpression decreased endogenous levels of APAF-1 and BIM. (h) MiR-103 and miR-203 overexpression decreased endogenous levels of PKC-ε and SRC. (i) Anti-miR-221-222 and anti-miR-30b-c increased APAF-1 and BIM expression. (j) MET knockdown induced APAF-1 and BIM upregulation and SRC and PKC-ε down-regulation. Results are representative of at least, three independent experiments. Error bars depict ± s.d. * P< 0.001, ** P< 0.05 by two tailed student's t test.

Figure 2. MiR-103-, 222-, 203-, 30c-MET co-expression analyses

(a) 110 lung cancer tissues were analyzed for miR-103, -222, -203, -30c expression by ISH and then for MET by IHC. Upper row, from the left miR-103 signal (blue), MET (red) and the mixed signal in which fluorescent yellow is indicative of miR and protein co-expression; note the lack of miR-103 in presence of MET expression. In the serial section of the same cancer (row below) one sees miR-222, the MET image and the co-expression of miR-222 and MET. Many cancer cells positive for miR-222 also express MET (yellow). The arrows in the left panel (third row) depict benign stromal cells that express miR-203 (blue) and do not express MET. Next panels represent MET (red) and the mixed signal. The arrow in the fourth row depicts cancer cells positive for miR-30c; next panels depict MET signal and the co-expression of miR-30c (yellow). Right panels show the RGB image of the ISH/lHC reaction. (b) Box plots showing miR-30b-c, -221-222 expression in 40 lung cancer patients. Real time PCR was used to classify tumors into two groups: EGFR-MET low and EGFR-MET high by round function with the cut-off at 0.5 (2^(-DeltaCt)). (See also Supplementary Fig. 5). *P<0.0001 by Student's t test. (c) XY Scatter plots showing inverse correlation between MET-103, MET-203. P<0.0001. (d) MET and EGFR immunostaining on 40 lung tumor tissues. One representative case of 17 metastatic tumors expressing both MET and EGFR is shown. Large arrow = tumor cells, small arrow = stroma. Scale bar, 100 μm.

Figure 3. Gefitinib downregulates miR-221-222 and 30b-c

(a) Calu-1, A549, PC9 and Hcc827 cells were treated with increasing concentrations of gefitinib. Cell viability, relative to untreated controls, was measured after 24h. Each data point represents the mean ± s.d. of five wells. (b) qRT-PCR showing miR-30b-c and -221-222 down-regulation only in PC9 and Hcc827 gefitinib-sensitive cells and not in Calu-1 and A549-resistant cells after treatment with 5 or 10 μM gefitinib. (c) PC9, Calu-1 and Hcc827 cells were treated for 24h with 5 or 10 μM gefitinib. An increase of BIM and APAF-1 expression and a decrease of ERKs phosphorylation were observed only in the Hcc827 and PC9 gefitinib-sensitive cells but not in the Calu-1 -resistant cells. β-actin was used as loading control. (d) qRT-PCR showing that miR-30b-c and -221-222 expression did not decrease in PC9 GR and HCC827GR cells (with acquired gefitinib resistance) exposed to 10 μM gefitinib for 24h. All quantitative data were generated from a minimum of three replicates. Error bars depict ± s.d. Two tailed student's t test was used to determine P values. *P<0.001, **P<0.05.

Figure 4. MiR-30b-c, 221-222, 103, 203 regulates gefitinib sensitivity

(a) Parental and resistant HCC827 GR and PC9GR and Calu-1 cells treated with increasing concentrations of gefitinib. Each data point represents the mean ± s.d of 6 wells. (b) Western blot showing an increase in PARP cleaved fragment after BIM and APAF-1 over-expression and gefitinib treatment (15μM) in A549 cells. (c) BIM and APAF-1 silencing in HCC827 and PC9 cells reduces the response to gefitinib. (d) Overexpression of miR-30b/c- and -221/222-insensitive BIM and APAF-1 cDNAs, induces gefitinib sensitivity in A549 cells. (e-f) MiR-103, -203 overexpression and miR-30c, -222 silencing increase gefitinib sensitivity in vivo. (e) Growth curve of engrafted tumors and (f) comparison of engrafted tumors in nude mice injected with A549 cells stable infected with anti-miR-control (ctr), anti-30c, anti-221 and with miR-103 and -203 or an empty virus as control. The images show average-sized tumors from among five of each category. In a, c, d and e error bars indicate ± s.d. *P < 0.001, **P < 0.05 by two tailed student's t test.

Figure 5. MiR-103, 203 inhibit migration and proliferation of NSCLC

(a)Representative pictures of cells that migrated through the filter and stained with crystal violet. Scale bar, 40μm. The results are means ± s.d, n=3. *P < 0.001. (b) Representative photographs of scratched areas of confluent monolayer of A549 cells transfected with miR-103, -203 or ctr miR at 0h and 24h after wounding with a pipet tip. Scale bar, 500 μm. Significance values of *P< 0.00001 and **P<0.001 relative to miR scrambled transfected cells. (c) Flow cytometric distributions of Calu-1 and A549 cells transfected with control miR, miR-103, miR-203, control siRNA, PKC-ε and SRC siRNAs. The effect of miR-203 on cell cycle is slightly stronger than that of miR-103, as assessed by the ratio between Go-G1 and S phases. (A549 G0-G1/S NT= 3.1±0.2; Src miR= 3.6±0.2; 103= 4.8±0.25; 203= 6.4±0.2; scr siRNA= 2.9±0.3; siPKC-ε= 4.8±0.1; siSrc= 5.7±0.25. Calu-1 G0-G1/S NT= 2.8± 0.34; Scr miR= 3.1± 0.21; 103= 4.3±0.25; 203= 6.3±0.25; Scr siRNA= 2.5±0.21; siPKC-ε= 4.6±0.26; siSrc= 5.2±0.32). All quantitative values show mean ± s.d, n=5. Two tailed student's t test was used to determine P values between G0-G1/S ratio. *P<0.00001 versus scr miR. **P<0.005 versus scr miR.

Figure 6. c-Met knockdown induces MET through miR-103 and -203

(a) Morphological changes of Calu-1 cells after MET knockdown. Scale bar indicates 20μm. (b) Immunofluorescence: Snail, mesenchymal intermediary filament vimentin and N-cadherin in Calu-1 shControl and Calu-1 shMET. Snail expression is strong and nuclear in Calu-1 ShCtr and is weaker and cytoplasmic in Calu-1 ShMET. Scale bar, 20μm. (c) Western blots showing the downregulation of mesenchymal proteins as fibronectin, vimentin and Snail and the upregulation of E-cadherin after MET knockdown in Calu-1 cells. Loading control, GAPDH. (d) qRT-PCR showing the expression of epithelial and mesenchymal markers in Calu-1 ShCtr and Calu-1 shMET. (e) Immunofluorescence: fibronectin, Snail and vimentin expression decreases after miR-103 or -203 overexpression in Calu-1 cells. Scale bar indicates 20μm. (f) Immunofluorescence: E-cadherin increased signal after miR-103 or -203 enforced expression in Calu-1 cells. Scale bar, 40μm. (g) Immunoblot showing the down-regulation of mesenchymal markers after miR-103 or -203 overexpression. (h) A model is reported in which MET inhibition elicits upregulation of miR-103 and -203, which in turn, downregulating PKC-ε, Dicer and SRC, induce gefitinib-sensitivity and mesenchymal-epithelial transition (MET). MET knockdown induces also miR-30b/c, -221/222 and -21 downregulation and consequent gefitinib-sensitivity through BIM, APAF-1 and PTEN upregulation. EGFR knockdown decreases miR-221/222 and -30b/c expression levels. In red are the up-regulated and in green the down-regulated miRs after EGFR and MET silencing. Results are representative of at least four independent experiments. P values were obtained by two-tailed student's t test. Error bars represent standard deviation.