Shp2 regulates migratory behavior and response to EGFR-TKIs through ERK1/2 pathway activation in non-small cell lung cancer cells

Abstract

In the clinical treatment of lung cancer, therapy failure is mainly caused by cancer metastasis and drug resistance. Here, we investigated whether the tyrosine phosphatase Shp2 is involved in the development of metastasis and drug resistance in non-small cell lung cancer (NSCLC). Shp2 was overexpressed in a subset of lung cancer tissues, and Shp2 knockdown in lung cancer cells inhibited cell proliferation and migration, downregulated c-Myc and fibronectin expression, and upregulated E-cadherin expression. In H1975 cells, which carry double mutations (L858R + T790M) in epidermal growth factor receptor (EGFR) that confers resistance toward the tyrosine kinase inhibitor gefitinib, Shp2 knockdown increased cellular sensitivity to gefitinib; conversely, in H292 cells, which express wild-type EGFR and are sensitive to gefitinib, Shp2 overexpression increased cellular resistance to gefitinib. Moreover, by overexpressing Shp2 or using U0126, a small-molecule inhibitor of extracellular signal-regulated kinase 1/2 (ERK1/2), we demonstrated that Shp2 inhibited E-cadherin expression and enhanced the expression of fibronectin and c-Myc through activation of the ERK1/2 pathway. Our findings reveal that Shp2 is overexpressed in clinical samples of NSCLC and that Shp2 knockdown reduces the proliferation and migration of lung cancer cells, and further suggest that co-inhibition of EGFR and Shp2 is an effective approach for overcoming EGFR T790M mutation acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). Thus, we propose that Shp2 could serve as a new biomarker in the treatment of NSCLC.

Keywords: ERK1/2; Shp2; epidermal growth factor receptor; non-small cell lung cancer; tyrosine kinase inhibitors.

Figure 1. Shp2 expression is increased in non-small cell lung cancer (NSCLC)

Anti-Shp2 antibody staining of (A) normal lung tissue and (B) NSCLC tissue. The IHC semi-quantitative score was derived based on two criteria: the antibody staining intensity was multiplied by the percentage of tumor cells stained. IHC scores for each set of specimens were averaged (N = 23) and statistically analyzed (C).

Figure 2. Transfection of Shp2 siRNA and Shp2^WT cDNA decreased and increased cellular Shp2 expression, respectively

Equal amounts of lysates prepared from cells transfected with control/Shp2 siRNAs (A) or vector/Shp2^WT (B) were immunoblotted with anti-Shp2 and anti-actin antibodies (N = 3).

Figure 3. Shp2 knockdown inhibits tumor growth and enhances cellular response to gefitinib

(A), (B) Shp2 knockdown decreased the proliferation of H1975 cells (A) and H292 cells (B). Lung cancer cells were transfected with Shp2 or control siRNA for 72 h in RPMI 1640 medium containing 0.5% FBS. Cell proliferation was measured using the CCK8 assay at 72 h after transfection, and the results are expressed as means ± SD (N = 4); *P < 0.05 versus control. (C), (D) Shp2 knockdown enhanced gefitinib sensitivity in NSCLC cells. (E), (F) Shp2 overexpression decreased gefitinib sensitivity in NSCLC cells. H1975 and H292 cells transfected with Shp2/control siRNAs or Shp2^WT/vector were treated with 0–10 μM gefitinib for 3 days, and then cell proliferation was measured using the CCK8 assay. The cell-viability percentage was calculated by adjusting the DMSO control group to 100%. Points and error bars represent the mean and SD values from 3 independent experiments. Black points: control siRNAs/vector; Gray points: Shp2-siRNAs/Shp2^WT.

Figure 4. Shp2 knockdown inhibits the migration of lung cancer cells

Cells transfected with Shp2/control siRNAs were used in wound-healing assays (A) and Transwell-migration assays (B). In the wound-healing assay, cell images were captured immediately after wounding and 24 h later. In Transwell-migration assays, equal numbers cells were cultured on Transwell membranes for 24 h, and then the migrated cells were counted. *P < 0.05 versus controls.

Figure 5. Shp2 expression enhances c-Myc expression and EMT

Shp2 siRNA transfection reduced c-Myc and fibronectin expression, enhanced E-cadherin expression, and decreased ERK1/2 activation (A). Shp2 overexpression reduced E-cadherin expression, enhanced fibronectin and c-Myc expression, and induced ERK1/2 activation (B). Equal amounts of lysates from cells transfected with control/Shp2 siRNAs or vector/Shp2^WT were immunoblotted with the indicated antibodies (N = 3).

Figure 6. Shp2 promotes c-Myc expression and EMT through Ras/MAPK signaling

(A) Comparison of gefitinib sensitivity of H292 cells transfected with vector or Shp2^WT and treated with DMSO or U0126; cell survival was measured using the CCK8 assay. (B) U0126 treatment abrogated Shp2^WT-induced upregulation of c-Myc and fibronectin expression and downregulation of E-cadherin expression in H292 cells. Equal amounts of lysates from cells transfected with vector/Shp2^WT were immunoblotted with the indicated antibodies. (C), (D) U0126 inhibits H292 cell migration; cells treated with DMSO or U0126 were used in wound-healing assays (C) and Transwell-migration assays (D). *P < 0.05 versus controls.