CCL2 promotes EGFR-TKIs resistance in non-small cell lung cancer via the AKT-EMT pathway

Abstract

Acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs) represents a primary cause of treatment failure in non-small cell lung cancer (NSCLC) patients. Chemokine (C-C motif) ligand 2 (CCL2) is recently found to play a pivotal role in determining anti-cancer treatment response. However, the role and mechanism of CCL2 in the development of EGFR-TKIs resistance have not been fully elucidated. In the present study, we focus on the function of CCL2 in the development of acquired resistance to EGFR-TKIs in NSCLC cells. Our results show that CCL2 is aberrantly upregulated in EGFR-TKIs-resistant NSCLC cells and that CCL2 overexpression significantly diminishes sensitivity to EGFR-TKIs. Conversely, CCL2 suppression by CCL2 synthesis inhibitor, bindarit, or CCL2 knockdown can reverse this resistance. CCL2 upregulation can also lead to enhanced migration and increased expressions of epithelial-mesenchymal transition (EMT) markers in EGFR-TKI-resistant NSCLC cells, which could also be rescued by CCL2 knockdown or inhibition. Furthermore, our findings suggest that CCL2-dependent EGFR-TKIs resistance involves the AKT-EMT signaling pathway; inhibition of this pathway effectively attenuates CCL2-induced cell migration and EMT marker expression. In summary, CCL2 promotes the development of acquired EGFR-TKIs resistance and EMT while activating AKT signaling in NSCLC. These insights suggest a promising avenue for the development of CCL2-targeted therapies that prevent EGFR-TKIs resistance in NSCLC.

Keywords: CCL2; EGFR-TKIs resistance; epithelial-mesenchymal transition; non-small cell lung cancer.

Figure 1

CCL2 is upregulated in NSCLC cells with acquired resistance to EGFR-TKIs (A) Clustered heatmap of the upregulated ( P-value<0.05 and log2FC≥1) and downregulated genes ( P-value<0.05 and log2FC<1) between gefitinib-resistant (gefi-R) and gefitinib-sensitive (gefi-S) NSCLC cells. (B) KEGG pathway analysis of gefitinib-resistant and gefitinib-sensitive NSCLC samples in the GSE122005 dataset. (C) Volcano plot showing genes that were differentially expressed between gefitinib-resistant and gefitinib-sensitive NSCLC cells. (D,E) Cells were treated with different concentrations of gefitinib or osimertinib for 72 h. CCK-8 assays were used to detect the IC 50 values of gefitinib in HCC827 and HCC827-GR cells (D) and the IC 50 values of osimertinib in HCC827 and HCC827-OR cells (E). (F) qRT-PCR was used to assess the mRNA expression of CCL2 in HCC827, HCC827-GR, and HCC827-OR cells. (G) ELISA was performed to detect the protein expressions of CCL2 in HCC827, HCC827-GR, and HCC827-OR cells. Data are shown as the mean±SEM of three independent experiments (*** P<0.001).

Figure 2

CCL2 upregulation induces resistance to EGFR-TKIs in NSCLC cells (A,B) The viability of HCC827 cells treated with gefitinib (A) and osimertinib (B) was determined by a CCK-8 assay after pretreatment with rhCCL2 (100 ng/mL) or PBS (control) for 120 h. (C‒E) Validation of CCL2 overexpression in HCC827 cells at the mRNA (D) and protein (C,E) levels. (F,G) The viability of gefitinib-(F) and osimertinib-(G) treated HCC827 cells after CCL2 overexpression was determined by CCK-8 assay. Data are shown as the mean±SEM of three independent experiments (** P<0.01, *** P<0.001).

Figure 3

CCL2 suppression reverses EGFR-TKIs resistance in vitro (A,B) In the presence or absence of bindarit (300 nM), HCC827 cells were pretreated with rhCCL2 (100 ng/mL) for 120 h, and the sensitivities to gefitinib (A) and osimertinib (B) were tested by CCK-8 assay. (C,D) Viability of HCC827-GR cells treated with gefitinib (C) and HCC827-OR cells treated with osimertinib (D) pretreated with or without bindarit (300 nM) was assayed by CCK-8 assay. (E,F) Validation of CCL2 knockdown in HCC827-GR cells and HCC827-OR cells at the mRNA (E) and protein levels (F). (G,H) The viability of HCC827-GR cells treated with gefitinib (G) and HCC827-OR cells treated with osimertinib (H) was determined by CCK-8 assay after CCL2 knockdown. Data are shown as the mean±SEM of three independent experiments (* P<0.05, ** P<0.01, *** P<0.001).

Figure 4

NSCLC cells with acquired EGFR-TKIs resistance exhibit enhanced migration and EMT (A) Western blot analysis of E-cadherin, Vimentin, N-cadherin, and ZEB1 levels in HCC827, HCC827-GR, and HCC827-OR cells. (B,C) Immunofluorescence assays of E-cadherin (B) and vimentin (C) in HCC827, HCC827-GR, and HCC827-OR cells (original magnification: 630×, scale bar: 25 μm). (D) Transwell assays were performed to detect the migration of HCC827, HCC827-GR, and HCC827-OR cells (scale bar: 5 mm). (E,F) Differences in the proliferation of HCC827 and EGFR-TKIs-resistant HCC827 cells (HCC827-GR cells in B, HCC827-OR cells in C) were determined by CCK-8 assay at 24 h, 48 h, 72 h and 96 h. Data are shown as the mean±SEM of three independent experiments (ns P>0.05, *** P<0.001).

Figure 5

CCL2 upregulation promotes EMT and migration in NSCLC cells (A) Volcano plot showing genes differentially expressed between HCC827-Empty and HCC827-CCL2 cells. (B) GO and KEGG pathway analyses of the differentially enriched pathways. (C) Transwell assays were performed to detect the migration of HCC827-Empty and HCC827-CCL2 cells (scale bar: 100 μm). (D,E) The migration ability of HCC827-GR cells (D) and HCC827-OR cells (E) after CCL2 knockdown was detected by Transwell assay (scale bar: 100 μm). (F) Western blot analysis of ZEB1, E-cadherin, N-cadherin, and Vimentin levels in HCC827-Empty and HCC827-CCL2 cells. (G,H) Immunofluorescence assays of E-cadherin (G) and vimentin (H) in HCC827-Empty and HCC827-CCL2 cells (original magnification: 630×, scale bar: 25 μm). (I) Western blot analysis of E-cadherin, N-cadherin and vimentin levels in HCC827-GR and HCC827-OR cells after CCL2 knockdown. Data are shown as the mean±SEM of three independent experiments (* P<0.05, *** P<0.001).

Figure 6

CCL2 promotes acquired EGFR-TKIs resistance through AKT-EMT signaling (A) Western blot analysis of p-AKT and AKT levels in HCC827, HCC827-GR and HCC827-OR cells. (B) Western blot analysis of p-AKT and AKT in levels HCC827 cells pretreated with rhCCL2 or rhCCL2 combined with bindarit. (C) Western blot analysis of p-AKT and AKT levels in HCC827-Empty and HCC827-CCL2 cells. (D) Western blot analysis of p-AKT and AKT levels in HCC827-GR and HCC827-OR cells after CCL2 knockdown. (E) The migration ability of HCC827-CCL2 cells treated with LY2940002 (10 μM) was detected by a transwell assay (scale bar: 100 μm). (F) Western blot analysis of p-AKT, AKT, E-cadherin, Vimentin, N-cadherin, and ZEB1 levels in HCC827-CCL2 cells treated with LY2940002 (10 μM). (G) Schematic diagram of CCL2-mediated resistance to EGFR-TKIs by activating the AKT signaling pathway and promoting EMT. Data are shown as the mean±SEM of three independent experiments (* P<0.05, ** P<0.01, *** P<0.001).