The Role Of IGF-1R In The Acquired Resistance Of EGFR Wild-type Non-small Cell Lung Cancer To EGFR-TKIs And Associated Mechanisms

Background and ObjectiveLung cancer is a disease with the highest morbidity and mortality in malignant tumors worldwide,which has become the leading cause of death in cancer. Non-small cell lung cancer (NSCLC) accounts for more than80%of all lung cancer,most patients with NSCLC are in a later stage at the time of diagnosis, the overall5-year survival rate for lung cancer is only about15%The epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), in which gefitinib is one of the representatives, are used in the first-, second-,or third-line therapy of advanced NSCLC patients with EGFR mutations. Efficiency ratio of EGFR-TKIs for EGFR wild-type NSCLC also reaches5%-6%However, the emergence of acquired resistance has blocked further application of EGFR-TKIs, which leads to a median effective time of8-10months. Therefore, to find out the mechanism of acquired resistance is important for clinical therapy.It is now considered that the major mechanisms of acquired resistance of EGFR mutant NSCLC to EGFR-TKIs include the secondary mutation of EGFR gene and the amplification of MET gene. This two mechanisms account for about60%of acquired resistance. The drugs(such as HKI-272,ERK-569,CI1033, AZD9291and BIBW2992,etc.) aimed at the above two mechanisms have already been applied to clinical practice. However,40%of acquired resistant mechanism of EGFR mutant NSCLC and the resistant mechanism of EGFR wild-type NSCLC remain unknown. The unclear mechanisms may include the activation of unexpected EGFR tyrosine kinase receptor signal pathway, the abnormal activation of moleculars in EGFR downstream signaling pathway, EML4-ALK fusion gene, the absence of protein tyrosine phosphatase gene (PTEN) and EMT,etc. Among them, the activation of signaling pathway mediated by IGF-1R and EMT are particularly noteworthy.As a transmembrane tyrosine protein receptor, IGF-1R plays an important role in the regulation of cell division, cell differentiation and cell proliferation. It is widely expressed in various types of cell surface. Intracellular tyrosine kinase is activated with the combination of IGF-1R and its ligand to promote the phosphorylation of tyrosine residues of various proteins,and then the two major pathways including PI3K/AKT and ERK/MAPK will be activated to promote mitosis and cell growth. It has been reported that IGF-1R expresses in some types of malignant tumors and its abnormal expression/high expression plays an important role in sustained growth of NSCLC and some other malignant tumors. Epithelial-mesenchymal transitions (EMT) characteristic with down-regulation of epithelial markers (such as E-cadherin) and up-regulation of mesenchymal markers (such as Vimentiri).is a biological process, which is usually associated with the activation of some signaling pathways. EMT, in which a number of factors involved in this complex pathological reaction, not only plays crucial roles in embryonic development, chronic inflammation, tissue reconstruction and the development of many chronic diseases but also takes part in the process of tumor development,tumor cells in situ invasion and metastasis. What is more, EMT is closely related to the prognosis of NSCLC and EGFR-TKIs sensitivity.Recent studies show that the activation of IGF-1R and EMT in tumor cells are closely related to the EGFR-TKIs-resistance in some types of NSCLC cells. It has also been confirmed in our prophase research that H460cells take acquired resistance to EGFR-TKIs accompanied by EMT and up-regulation of IGF-1R protein, which suggests that EMT and IGF-1R may play a great role in the acquired resistance of EGFR wild-type NSCLC cells to EGFR-TKIs. Rho and his colleagues discovered that the NSCLC cells developed EMT with the up-regulation of p-AKT and down-regulation of p-ERK 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 activation of IGF-1R and its downstream PI3K/AKT or ERK/MAPK signal pathways may cause the acquire resistance of EGFR wild-type NSCLC cells to EGFR-TKIs by inducing EMT. However, the causal relationship between IGF-1R activation and EMT,as well as the detailed resistant mechanism mediated by IGF-1R, is still unknown. Although previous studies have confirmed that application of IGF-1R-TKIs and IGF-1R gene knockout may reverse the drug resistance of EGFR wild-type NSCLC to erlotinib, the molecular mechanism of above reversal effect has not been elucidated both home and abroad.Therefore, the purpose of this study is to observe the effects of IGF-1R gene on the acquired resistance to EGFR-TKIs and cell EMT by constructing lentiviral vectors of RNA interference targeting IGF-1R gene to inhibit the expression of IGF-1R in EGFR wild-type NSCLC cells, and then further investigate the molecular mechanism of reversal effect on the EGFR-TKIs-resistance to find out new targets and new methods to improve the curative effect of EGFR-TKIs.Methods1.EGFR wild-type human lung adenocarcinoma H460and A549cells were used to creat erlotinib-resistant(named as H460/ER) and gefitinib-resistant cells cells (named as A549/GR), respectively. The EGFR and K-RAS gene mutation were examined by qPCR-HRM. 2.GV248plasmid was used to construct both siRNA expression vector targeting to IGF-1R gene and its negtive control vector. The vectors were identified by PCR and gene sequencing.The identified vectors was given to transfect293T cells and then been screened for effective targets by Western Blot.The effective vector was chosen to be packaged with293T cells and the virus titer was determinated. The virus acquired was transfected into targeting cells H460/ER and A549/GR. Real-time quantitative RT-PCR and Western Blot detected the inhibited effeciency of IGF-1R in H460/ER and A549/GR cells.3.After IGF-1R was inhibited, 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 expressions of p-IGF-1R, EGFR (epidermal growth factor receptor), ERK (extracellular signal regulated kinase), AKT (protein kinase B),E-Cadherin and Vimentin were determined by Western blotting.4.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.Results1. The sensitivities of H460/ER and A549/GR cells to EGFR-TKIs were both decreased.The four cell lines grew multiply indefinitely with cultured by ordinary medium. The proliferative effects of H460and H460/ER cells on Erlotinib are both concentration dependent. Compared with H460cells, the sensitivity to EGFR-TKIs of H460/ER was significantly decreased. The IC50values of H460/ER and H460cells were52.66±2.79and12.22±0.55μmol/L (P<0.05). The proliferative effects of A549and A549/GR cells on Gefitinib are also concentration dependent. Compared with H460cells, the sensitivity to EGFR-TKIs of A549/GR was significantly decreased. The IC50values of A549/GR and A549cells were41.84±3.70μmol/L and9.73±1.29μmol/L (P<0.05) 2.EGFR and K-ras gene states were determinated in the cells.EGFR gene exon19, exon20, exon21and K-RAS gene exon3were all wild-type, while EGFR gene exon18and K-RAS exon2were not determinated in A549/GR cells and no gene mutation in H460/ER cells.3.The lentiviral vector of RNAi targeting IGF-1R gene was constructed and it inhibited the expression of IGF-1R in H460/ER cell.Three lentiviral vectors of RNAi targeting IGF-1R gene (named IGF-1R-siRNA-1,IGF-1R-siRNA-2,IGF-1R-siRNA-3) and their negtive control vector IGF-1R-siRNA-NC were successfully constructed. The effective vector IGF-1R-siRNA-3was identified and packaged. IGF-1R-siRNA-3had significant inhibitory effect on H460/ER cells, and the inhibition rate of mRNA and protein were67.7±2.6%and86.6±1.6%respectively (P<0.05),while it had no effect on A549/GR cells.4.The effect of IGF-1R silence on the acquied resistance of H460/ER cells to EGFR-TKIs.The two cell lines grew multiply indefinitely with cultured by ordinary medium. The proliferative effects of siControl H460/ER and si1GF-lR H460/ER cells on Erlotinib are both concentration dependent. Compared with siControl H460/ER cells, the sensitivity to EGFR-TKIs of siIGF-1R H460/ER was significantly increased. The IC50values of siIGF-1R H460/ER and siControl H460/ER cells were6.29±1.56and65.34±3.30μmol/L (P<0.05)5.IGF-1R downstream siganling pathways was inhibited after IGF-1R gene was knocked down.The result shows that the expressions of IGF-1R and its phosphorylation in siIGF-1R H460/ER cells were significantly decreased (P<0.05) than those in siControl H460/ER cells, accompanied by the down-regulation of AKT and ERK phosphorylation. No significant difference was found in the expressions of EGFR and its phosphorylation between siControl H460/ER and siIGF-1R H460/ER cells(P>0.05).6. The effect of IGF-1R silence on EMT in H460/ER cells.(1) Morphological changes happened in the cells. Compared with siControl H460/ER cells, silGF-1R H460/ER cells displayed epithelial status with pebble shaped and less pseudopods among the cells.(2) The capability of invasion and migration was weakened in the siIGF-1R H460/ER cells.As shown in the wound-healing assay, the distance of scratches edge of siIGF-1R H460/ER cells was significantly extended:compared with siControl H460/ER cells, the distance of scratches edge in the siIGF-1R H460/ER cells extended61%(P <0.05).It was observed in the tanswell assay that there were less cells invaded through the membrane-coated commercial Matrigel in the siIGF-lR H460/ER cells: the number of invading cells was69%of siControl H460/ER cells. These phenomena demonstrated that the capability of invasion and migration was weakened in the cells in which the IGF-1R gene was knocked down.(3) The result determined by Western Blot shows that the expressions of Vimentin and Snail were decreased in the silGF-1R H460/ER cells (P<0.05) and the expression of E-cadherin was increased significantly (P<0.05).ConclusionIGF-1R silence can block IGF-1R downstream signaling pathways PI3K/AKT and ERK/MAPK and reverse the acquired resistance of EGFR wild-type non-small cell lung cancer to EGFR-TKIs as well as EMT, which indicates that IGF-1R may play an important role in the acquired resistance of EGFR wild-type non-small cell lung cancer to EGFR-TKIs and EMT should be the mechanism of the acquired resistance mediated by IGF-1R to EGFR-TKIs in EGFR wild-type non-small cell lung cancer.