Investigation On Molecular Mechanism Of Quickly Acquired Resistance To EGFR-TKIs Mediated By FGF-2Signaling Pathway

Objective:Quickly acquired resistance to EGFR-TKIs is induced in PC-9cell lines (one cell line of NSCLC harboring19exon del in EGFR gene) by silence or activation of endogenous FGF2as well as co-incubation of Gefitinib and exogenous FGF2, so that the molecular mechanism of quickly acquired resistance to EGFR-TKIs mediated by FGF-2signaling pathway can be further clarified.Methods:1. FGF2gene targeting shRNA lentiviral vectors and recombinant plasmid human-FGF2-PLJM1lentiviral vectors based on PCR-based accurate synthesis were constructed in human lung cancer cell line PC-9. Quantitative real-time PCR was performed to detect mRNA levels of FGF2gene, and western blot was performed to detect the expression levels of FGF2protein, thus cell line of FGF2silencing (PC-9-FGF2-KD) or FGF2overexpression (PC-9-FGF2-OE) can be selected;2. Induction and identification of quickly acquired resistance to EGFR-TKIs in PC-9cell line. PC-9cell line with FGF2silencing or over-expression was incubated with gefitinib alone or in combination with exogenous FGF2to screen a quickly acquired resistance cell line. Biological indicators were detected in drug-resistant NSCLC cell lines to further confirm the drug-resistance activity. Biological indicators include:(1) Proliferation of cells via MTT assay;(2) Apoptosis and cell cycle via Annexin V/PI kit and PI single staining method;(3) Anchorage-independent growth via soft agar method;(4) Migration and invasion of cells via Transwell determination;(5) T790M point mutation and cMet amplification via Real-time PCR.3. Whole genome Affymetrix3’IVT microarray chip was applied to detect the expression of downstream genes in PC-9-NC+FGF2, PC-9-FGF2-KD+FGF2, PC-9-FGF2-OE+FGF2cells as well as in PC-9cells.Results:1. FGF2gene silencing ox overexprssion of PC-9cells were constructed by using lentiviral vector-mediated gene transfer method, which is further confirmed by Western blot detection on FGF2’s expression. Stable cell lines of PC-9with FGF2gene silencing (PC-9-FGF2-KD) or overexprssion (PC-9-FGF2-OE) were successfully screened;2. Cell viability was increased in cell line of PC-9with FGF2gene overexpression (PC-9-FGF2-OE) and under the condition of exogenous FGF2, which indicated that both overexpression of endogeneous FGF2gene and incubation with exogenous FGF2help to induce quickly acquired resistance to EGFR-TKIs in PC-9cells. Cellular biology assays further demonstrated notable EGFR-TKIs resistance behaviors such as a decrease of apoptosis, an increase of proliferation and an enhancement of invasion as well as migration ability in PC-9-FGF2-OE cells incubated with exogenous FGF2. Thus, the result further confirmed that PC-9-FGF2-OE with exogenous FGF2is a stable NSCLC cell model of quickly acquired resistance to EGFR-TKIs mediated by FGF-2signaling pathway;3. Microarray test found that PI3K-AKT pathway, MAPK pathway, ErbB pathway and VEGF pathway were significantly upregulated in PC-9-FGF2-OE+FGF2cells when compared with PC-9-NC+FGF2cells. PI3K-AKT signaling pathway might play an important role in FGF2-mediated fast acquired drug resistance to EGFR-TKIs.Conclusion:1. Cell model of FGF2-mediated quickly acquired resistance to EGFR-TKIs in NSCLC cell line was successfully constructed, which laid basis for further investigations on molecular mechanisms of FGF2-mediated quickly acquired resistance to EGFR-TKIs;2. PI3K-AKT signaling pathway may play an important role in pathogenesis of fast acquired drug resistance to EGFR-TKIs mediated by FGF2.