Long non-coding RNA CASC9 promotes gefitinib resistance in NSCLC by epigenetic repression of DUSP1

Abstract

Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, has greatly affected clinical outcomes in non-small cell lung cancer (NSCLC) patients. The long noncoding RNAs (lncRNAs) are known to regulate tumorigenesis and cancer progression, but their contributions to NSCLC gefitinib resistance remain poorly understood. In this study, by analyzing the differentially expressed lncRNAs in gefitinib-resistant cells and gefitinib-sensitive cells in the National Institute of Health GEO dataset, we found that lncRNA CASC9 expression was upregulated, and this was also verified in resistant tissues. Gain and loss of function studies showed that CASC9 inhibition restored gefitinib sensitivity both in vitro and in vivo, whereas CASC9 overexpression promoted gefitinib resistance. Mechanistically, CASC9 repressed the tumor suppressor DUSP1 by recruiting histone methyltransferase EZH2, thereby increasing the resistance to gefitinib. Furthermore, ectopic expression of DUSP1 increased gefitinib sensitivity by inactivating the ERK pathway. Our results highlight the essential role of CASC9 in gefitinib resistance, suggesting that the CASC9/EZH2/DUSP1 axis might be a novel target for overcoming EGFR-TKI resistance in NSCLC.

Fig. 1. Relative CASC9 expression in gefitinib-resistant tissues and cell lines.

a Data mining of altered CASC9 expression in microarray gene profiles (GSE34228). b IC50 values of gefitinib in PC9/GR and their respective parental PC9 cells was examined by CCK8 assay. c Observation the morphological differences of PC9/GR and PC9 cells by microscope. d The expression of p-EGFR, p-AKT, p-ERK, total EGFR, AKT, ERK, and GAPDH of PC9 and PC9/GR cells treated with 5 μM gefitinib were measured by western blot. e CASC9 was detected in BT group and AR group by qRT-PCR. The levels of CASC9 in AR tissues are significantly higher than that in BT tissues. The ΔCt value was determined by subtracting the GAPDH Ct value from the CASC9 Ct value. A smaller ΔCt value indicates higher expression. f CASC9 expression of PC9, HCC827, H1975 and PC9/GR cells was evaluated by qRT-PCR. *P < 0.05, **P < 0.01.

Fig. 2. Downregulation/Upregulation of CASC9 increases/reduces the sensitivity of PC9/GR and PC9 cells to gefitinib.

a, b qRT-PCR analysis of CASC9 expression in PC9 and PC9/GR cells overexpressing or depleted of CASC9. c, d CCK8 assays were measure the IC50 ability of CASC9-depleted or -overexpressing PC9/GR and PC9 after various concentration of gefitinib treatment for 72 h. e, f CCK8 assays were performed to determine the proliferation of CASC9-depleted or -overexpressing PC9/GR and PC9 cells treated with gefitinib. g, h Colony formation assays were used to evaluate the colony formation capacity of CASC9-depleted or -overexpressing PC9/GR and PC9 cells treated with gefitinib. *P < 0.05, **P < 0.01.

Fig. 3. Downregulation of CASC9 reduces acquired gefitinib resistance in vivo.

a FACS analysis of the effect of CASC9 down-regulation on PC9/GR cells apoptosis treated with gefitinib. b The expression of p-EGFR, p-AKT, p-ERK, total EGFR, AKT, ERK, and GAPDH of CASC9-depleted or -overexpressing PC9/GR and PC9 cells for 48 h were examined by western blot. c PC9/GR/sh-CASC9 or PC9/GR/Empty vector cells were injected into nude mice (n = 6). 9 days later, the mice were treated with normal saline or gefitinib (25.0 mg/kg) by the method of gavage. d Tumor volume vs time growth curves were measured every 3 days. e Tumor weight was measured after at 18 days after inoculation. f qRT-PCR analysis of relative expression of CASC9 in xenograft tumors. g Representative tumor sections derived from cells expressing PC9/GR/sh-CASC9 or PC9/GR/Empty vector treated with normal saline or gefitinib were subjected to H&E, Ki67 staining and FISH. *P < 0.05, **P < 0.01.

Fig. 4. CASC9 represses DUSP1 expression by binding to EZH2.

a Hierarchically clustered heatmap of differentially expressed genes in PC9/GR cells after transfection of CASC9 or control siRNAs. b Nine representative genes mRNA levels in PC9/GR cells depleted of CASC9. c Western blot analysis of DUSP1 expression in PC9/GR cells transfected with CASC9 siRNA and CASC9-ASO. d qRT-PCR analysis was performed to determine the subcellular localization of CASC9 in PC9/GR cells. e RIP assays were performed in PC9/GR cells to show CASC9 co-immunoprecipitation with EZH2, LSD1, SUZ12 and Ago2. f qRT-PCR analysis of EZH2 expression in PC9 and PC9/GR cells. g IC50 values of gefitinib in EZH2-depleted PC9/GR cells was examined by CCK8 assay. h Colony formation assays were used to evaluate the colony formation capacity of PC9/GR cells depleted of EZH2 treated with gefitinib. i FACS analysis of the effect of EZH2 down-regulation on PC9/GR cells apoptosis treated with gefitinib. j, k qRT-PCR and western blot analysis of EZH2 and DUSP1 mRNA and proteins expression in PC9/GR cells transfected with siRNA-NC or si-EZH2. l ChIP-qPCR assay showing EZH2 occupancy on the DUSP1 promoters was reduced by CASC9 knockdown. *P < 0.05, **P < 0.01.

Fig. 5. Upregulation of DUSP1 increases the sensitivity of PC9/GR cells to gefitinib and regulates ERK pathway.

a qRT-PCR analysis of DUSP1 expression in PC9 and PC9/GR cells. b qRT-PCR analysis of DUSP1 mRNA levels in PC9/GR cells overexpressing DUSP1. c CCK8 assays were performed to determine the IC50 ability of DUSP1-overexpressing PC9/GR cells. d, e CCK8 and colony formation assays were performed to determine the proliferation of DUSP1-overexpressing PC9/GR cells treated with gefitinib. f FACS analysis of PC9/GR cells apoptosis overexpressing DUSP1 treated with gefitinib. g Western blot analysis of ERK and p-ERK proteins expression in PC9/GR cells transfected with DUSP1 vector. *P < 0.05, **P < 0.01.

Fig. 6. Downregulation of DUSP1 could rescue the effect of si-CASC9 on the sensitive of PC9/GR cells to gefitinib and regulate ERK signaling pathway.

a qRT-PCR analysis detection of DUSP1 mRNA expression after co-transfection of PC9/GR cells with si-DUSP1 and si-CASC9. b ERK, p-ERK and DUSP1 protein levels were detected after co-transfection of PC9/GR cells with si-DUSP1 and si-CASC9. c CCK8 assays were performed to determine the IC50 ability of PC9/GR cells after co-transfection with si-DUSP1 and si-CASC9. d, e Colony formation assays were performed to determine the proliferation of PC9/GR cells after co-transfection with si-DUSP1 and si-CASC9. f Proposed model which medicated by CASC9 in gefitinib resistance of NSCLC. *P < 0.05, **P < 0.01.