| Background and ObjectiveLung cancer is the leading cause of cancer-related death worldwide. Non-small cell lung cancer (NSCLC) accounts for more than85%of all lung cancer,most patients with NSCLC are in a later stage at the time of diagnosis,the overall5-year relative survival rate for lung cancer is about15.6%In the recent years, the greatest advance in some cancer therapy is that molecular targeted drugs take place of traditional cytotoxic drugs. Gefitinib and erlotinib,as the representatives of the epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), are used for the first-line therapy of advanced NSCLC patients with EGFR mutations.Although EGFR-TKIs treatment shows good response rates in NSCLC patients with EGFR gene mutations, drug resistance eventually emerges and this limits the mean duration of response to8-10months.Therefore,how to overcome the acquired resistance to EGFR-TKIs is one of the most urgent problems in the treatment of advanced NSCLC.It is reported that the major mechanisms of acquired resistance to EGFR-TKIs include secondary mutation of the target gene and activation of EGFR-bypass signals in the tumors. In recent years,it is observed threonine-to-methionine mutations in codon790(T790M) in exon20of the EGFR gene and MET gene amplification account for about60%of acquired resistance,the drugs is being developed to overcome resistance against the above two mechanisms in the clinical research.However,there is still40%remain unknown.The unclearly mechanisms may include the activation of unexpected EGFR tyrosine kinase receptor signal pathway,the abnormal activation of moleculars in EGFR downstream signaling pathway,the absence of protein tyrosine phosphatase(PTEN)gene, EML4-ALK fusion gene and EMT,etc.The process of epithelial-mesenchymal transitions (EMT) is associated with down-regulation of epithelial hallmarks such as E-cadherin,catenin,etc,and up-regulationof mesenchymal marks such as vimentin,fibronectin,etc.EMT,which is basic physiological and pathological phenomena,not only plays crucial roles in embryonic development and organ formation of multi-cellular organisms, chronic inflammation and the development of many chronic diseases but also takes part in the process of tumor development,tumor cells in situ invasion and metastasis’12"151. Insulin-like growth factor I receptor(IGF-1R) is a transmembrane tetraploid on the cell surface with activity of tyrosine kinase. Intracellular tyrosine kinase is activated with the combination of IGF-1and IGF-2with IGF-1R to promote the phosphorylation of tyrosine residues of various proteins,and then two major pathways including PI3K/AKT and ERK/MAPK will be activated to promote mitosis and cell growth.Previous studies show that there is overexpression of IGF-1R in tumor cells and its abnormal expression is closely related to the appearance of malignant tumors.Recent studies show that NSCLC cells with wild-type EGFR gene take acquired resistance to EGFR-TKIs accompanied by EMT and up-regulation of IGF-1R protein.The results suggest that EMT and IGF-1R may be closely related to NSCLC cells acquired resistance to EGFR-TKIs,and the resistant mechanism is still for further study.It is observed in previous research that the NSCLC cells developed EMT with the up-regulation of p-AKT and down-regulation of p-ERK and without the activation of EGFR during acquired resistance to EGFR-TKIs.The results indicate that the development of EMT may be mediated by the activation of PI3K/AKT signal pathway.Studies also show that the activation of IGF-1R and its downstream signal pathways not only stimulates the EGFR-bypass signal pathway,but also plays a great role in the development of EMT.Therefore,We speculate that the NSCLC cells acquire resistance to EGFR-TKIs can be induced by EMT which medicated by the activation of IGF-1R and its downstream PI3K/AKT signal pathway. Athough previous studies have shown that EMT developed in the EGFR-wild NSCLC cells acquired resistance to EGFR-TKIs,it is not certain that whether the same phenomenon developed in the NSCLC cells with EGFR gene mutation and whether the change is universal in the wild-type EGFR NSCLC cells.Since previous studies have observed the overexpression of IGF-1R protein in the acquired resistance to EGFR-TKIs,the chang in the IGF-1R signal traduction pathway is required for further research.Both EGFR mutant and wild-type NSCLC cells were used in this study,and the two cells were used to created gefitinib-resistant cells and erlotinib-resistant cells, respectively.We observed the change of related molecules in EMT and IGF-1R signal pathway during the acquired resistance of NSCLC cells to EGFR-TKIs to investigate the other possible acquired resistance mechanisms.Methods1.Human lung adenocarcinoma PC-9cells and H460cells were used to created gefitinib-resistant cells (named as PC-9/ZD) and erlotinib-resistant cells (named as H460/ER), respectively. MTT assay was used to measure the cell proliferation. Wound-healing assay and transwell assay were used to determine the invasive and migratory capabilities of cells. The protein and mRNA expressions of E-Cadherin, Vimentin, EGFR (epidermal growth factor receptor), ERK (extracellular signal regulated kinase), AKT (protein kinase B) and IGF-1R were determined by Western blotting and reverse transcription PCR (RT-PCR), respectively.2.The results analyzed by SPSS13.0were expressed as means±S.D.s.Two groups were analyzed by t-test and Multiple groups were analyzed by one-way ANOVA followed by LSD multiple comparison test.Results 1.EGFR and K-ras gene status in the cellsEGFR gene exon19mutation was found in PC-9cells and no gene mutation in H460cells.2.Both PC-9/ZD and H460/ER cells were acquired as the resistant cells to EGFR-TKIs.The four cell lines grew multiply indefinitely with cultured by ordinary medium,EGFR wild-type cells and mutant cells grew into logarithmic growth phase in two and three days,respectively. Gefitinib (0.01～10μmol/L) inhibits the growth of both PC-9and PC-9/ZD cells in dose dependent manner,so does erlotinib (1～100μmol/L) to H460and H460/ER cells. Compared with PC-9and H460cells, the sensitivity to EGFR-TKIs of PC-9/ZD and H460/ER was significantly decreased, respectively (P<0.05). Both PC-9/ZD and H460/ER cells were acquired as the resistant cells to EGFR-TKIs.3.Morphological changes in the resistant cellsCompared with PC-9and H460cells,PC-9/ZD and H460/ER cells displayed mesenchymal states with spindle shaped and looser contacts among the cells.4.The capability of invasion and migration was enhanced in the resistant cellsAs shown in the wound-healing assay, regardless of EGFR status,the distance of scratches edge was significantly short in resistant cells:compared with PC-9and H460cells, the distance of scratches edge in the PC-9/ZD and H460/ER shorten66%and44%,respectively (P<0.05).It is observed in the tanswell assay that there were more cells invaded through the membrane-coated commercial Matrigel in the resistant cells:compared with PC-9and H460cells,the number of invading cells increased1.82and3.34times,respectively (P<0.05). These phenomena demonstrated that the capability of invasion and migration was enhanced in the cells acquired resistance to EGFR-TKIs.5.The acquired resistance to EGFR-TKIs induced characteristics that were consistent with EMT The results determined by Western blotting and RT-PCR show that the expression of vimentin was increased in both PC-9/ZD and H460/ER cells (P<0.05) and the expression of E-cadherin was decreased in PC-9/ZD cells (P<0.05).Moreover, the expression of vimentin increased time-dependently when H460acquired resistance to erlotinib. The expression of E-cadherin was not found in the wild-type cells.6.The level of IGF-1R was enhanced in the resistant cellsThe data show that the expression of IGF-1R and its phosphorylation in both PC-9/ZD and H460/ER cells were significantly increased (P<0.05) than those in PC-9and H460cells, accompanied by the up-regulation of AKT and ERK phosphorylation (P<0.05). No significant difference was found in the expression of EGFR and its phosphorylation between PC-9and PC-9/ZD (P<0.05), while the phosphorylation of EGFR was suppressed in H460/ER cells (P<0.05).The results detected by RT-PCR determined that there was no significantly difference in the mRNA level of related-molecular between parental and resistant cells (P>0.05)Conclusion1.The EGFR gene of H460and PC-9cells was wide-type and the19exon mutation, respectively. Both PC-9/ZD and H460/ER cells were acquired as the resistant cells to EGFR-TKIs.2.Regardless of EGFR status, human lung adenocarcinoma cells acquire the resistance to EGFR-TKIs accompanied by EMT and activation of IGF-1R signal transduction. EMT and IGF-1R signal transduction may play a great role in the NSCLC cells acquire the resistance to EGFR-TKIs. |
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