Alteration In Mir-21/PTEN Expression Modulates Gefitinib Resistance In Non-small Cell Lung Cancer

Non-small cell lung cancer(NSCLC)is the leading cause of cancer related death worldwide.Unfortunately,in spite of advances in early detection of cancer,the majority of patients with NSCLC are diagnosed with advanced-stage disease,resulting in poor prognosis with a median survival of only 10-12 months.The development of drugs that target the epidermal growth factor receptor(EGFR),such as EGFR-TKIs(gefitinib and erlotinib)has improved the efficacy of NSCLC therapy.Indeed,the presence of activating EGFR exon mutations plays a key role in predicting the therapeutic efficacy of EGFR-TKIs.These activating EGFR mutations often significantly correlate with an adenocarcinoma-associated histology,smoking status,gender,and ethnicity.Thus,EGFR-TKIs have been recommended as the first line therapy for NSCLC patients with EGFR mutations.Unfortunately,their clinical efficacy is limited by the development of acquired resistance:most patients who initially respond would subsequently experience disease progression with continuous use of TKI for about 9-11 months;about 50%of patients who initially respond to EGFR-TKI acquire resistance to EGFR-TKI,while EGFR T790M mutation in exon20 accounts for 50%of this acquired resistance;another 20%of TKI acquired resistance results from MET amplification.It is still unclear how the remaining 30%of patients develop acquired resistance.MicroRNA(miRNA),known to intrinsically suppress mRNA by pairing with the 3’-untranslated region(UTR)of the mRNA was shown to negatively modulate expression of targeted genes.As genes regulators,miRNA regulate about one third of genes and play important roles in cellular functions including proliferation,growth,differentiation and apoptosis.In recent years,the crucial role of miRNAs in carcinogenesis have been demonstrated.Moreover,some miRNAs function as tumor suppressors in vitro.Therefore,over-expression of such miRNAs may suppress the target proteins which function as carcinogenic factors.In contrast to short interfering RNA,miRNAs is believed to regulate the same pathway at multiple levels.MiR-21 is an important oncogenic miRNA,closely related to tumor growth and metastasis.Indeed,expression of miR-21 was shown to be associated with poor prognosis and chemo-sensitivity in colon adenocarcinoma.In addition,anti-miR-21 suppressed cell growth of breast cancer through down-regulation of the antiapoptotic factor,B-cell lymphoma 2(Bcl-2).These data,taken together,support an important role of altered miR-21 expression during tumor development.So we investigated whether miR-21 modulates TKI sensitivity in NSCLC patients as well.In this article,we assessed 47 pairs of cancer tumor specimens and adjacent normal tissues by qRT-PCR and found a significantly higher expression of miR-21 in 37/47(78.7%)in tumor tissues in comparison with adjacent normal tissues.Since Pten is one of the important targets of miR-21,we investigated the relationship between expression of miR-21 and Pten in human NSCLC specimens both by immunoblot and immunohistochemistry(IHC).We found that Pten protein levels were significantly reduced in 34/47(72.3%)tumor tissues compared with adjacent normal ones.In addition,scatter plot analysis showed an inverse correlation between miR-21 expression levels and IHC scores of Pten signals.Kaplan-Meier survival analysis showed that patients with high miR-21 expression levels had a shorter disease free survival(DFS)compared with patients with low miR-21 expression.To further explore the relation between miR-21/PTEN expression alteration and TKI sensitivity,we assessed miR-21 expression levels using ISH assays and Pten expression levels by IHC on paraffin embedded samples from 46 NSCLC patients treated with TKI(gefitinib or erlotinib)as treatment and followed up their clinical response and survival status.As expected,miR-21 expression levels in PD patients were significantly higher than those in PR and SD groups(.Higher miR-21 expression levels also indicated shorter overall survival in TKI treated patients.In addition,Pten expression levels in PD patients was significantly lower than in PR and SD patient groups,and higher Pten expression correlated with longer overall survival in TKI treated patients.In order to test whether the effect of high miR-21/low Pten expression on modulation of TKI sensitivity in vitro,we selected pc-9,a TKI sensitive cell line,and the gefitinib resistant cell line PC-9/GR We also tested the miR-21 and PTEN protein expression level.The resulted revealed that PC-9/GR cells showed high miR-21/low Pten expression comparing with pc-9 cells.Therefore,we hypothesized that miR-21/Pten expression alteration plays an important role in modulating gefitinib sensitivity in pc-9 cells and pc-9/GR cells.We transfected pc-9 cells with miR-21 mimics and then co-transfected Pten plasmid to observe whether Pten could rescue miR-21 induce biological change in pc-9 cells.As expected,miR-21 did reduce gefitinib sensitivity in pc-9 cells and over-expression Pten could rescue this gefitinib sensitivity change.We found that miR-21 transfected pc-9 cells were more invasive showed anti-apoptosis ability than the NC group and over-expression Pten could rescue this invasive ability assessed by trans-well assay and flow cytometry.To better understand the molecular mechanisms involved in miR-21 induced gefitinib resistance in pc-9 cells,western-blot assays were used to assess the expression of related proteins.In pc-9 cells,elevated expression of miR-21 resulted in repression of Pten expression and activation of p-AKT and p-ERK in comparison with the NC group,but did not change total protein expression of AKT and EKR.Over-expression of Pten expression could rescue up-regulation of miR-21 induced activation of p-AKT and p-ERK.To further confirm the function of miR-21 on gefitinib sensitivity regulation in pc-9/GR cells,pc-9/GR cells were transfected with miR-21 inhibitor to decrease miR-21 expression and then co-transfected these cells with Pten siRNA.CCK-8 assay data showed that miR-21 knockdown significantly decreased gefitinib sensitivity of pc-9/GR and reduced Pten expression level could restore gefitinib sensitivity of pc-9/GR.Transwell assay and flow cytometry data also showed that miR-21 knockdown reduced the invasive ability and increased gefitinib induced apoptosis ability of pc-9/GR cells but it could be rescued by down-regulation of Pten.Finally,western-blot data showed that knockdown of miRNA 21 in pc-9/GR cells increased Pten expression and inactivated p-AKT and p-ERK relatively to NC group,but did not affect total protein expression levels in AKT and EKR.Down-regulation of Pten expression could rescue knockdown miR-21 induced inactivation of p-AKT and p-ERK.To analyze the role of miR-21 in modulating gefitinib sensitivity in vivo,we stably transfected pc-9/GR cells with GFP lentivirus constructs containing full-length anti-miR-21 or anti-Ctr and then injected into the posterior flank of nude mice.We found that knockdown of miR-21 in pc-9/GR cells resulted in sharp inhibition of tumor growth and increased sensitivity to gefitinib in nude mice.In this study,we propose alteration of miR-21/Pten expression as a novel mechanism for TKI resistance in NSCLC cancer.Our findings provide a new basis for using miR 21/Pten-based therapeutic strategies to reverse gefitinib resistance in NSCLC.