Study On The Inhibitory Effects Of FGFR Inhibitor PRN1371 Combined With Gefitinib On EGFR-TKI Resistance In Non-small Cell Lung Cancer And Its Mechanism

Part One The application of MPRP method in the detection of EGFR-TKI resistanceWith the continuous development and application of targeted drugs,it is particularly desirable to find a non-invasive diagnostic approach to screen patients for precision treatment.Specifically,detection of multiple cancer-related mutations is very important for targeted therapy and prediction of drug resistance.developing a multiplex,sensitive and convenient method to detect tumor-related mutations is an urgent need for clinical precision treatment.In this study,we presented the MPRP method to combine padlock RCA assay and real-time PCR technology to detect multiplex tumor-related mutations.The padlock probes were specifically linked to form a DNA ring in the presence of Hi Fi Taq DNA ligase which can distinguish single base variation,when the padlock probe is completely complementary to the target DNA.The RCA reaction was followed to amplify the circularized DNA ring,and finally the product was detected for mutant discrimination by multiplex real time PCR.In order to explore the application of MPRP technology in the detection of tumor related mutations,the plasma of 8 patients with non-small cell lung cancer was collected and the drug-resistant mutations were detected by MPRP test and digital PCR.The coincident results between these two methods indicated that the MPRP assay can provide a specific,sensitive,and convenient method for multiplex detection of cancer-related mutations.MPRP is an innovative strategy to combine padlock RCA with multiplex real-time PCR for the non-invasive detection of lung cancer-related mutations,which appears to be very promising in tumor molecular genotyping,clinical medication,drug resistance research,etc.Part Two The investigation of the molecular mechanism of EGFR-TKI resistance in primary tumor cellsTargeted drugs mainly inhibit the growth of tumor cells by blocking the signaling pathway and promoting apoptosis,and the drug resistance of tumor cells is closely related to apoptosis.In this study,we intend to explore the relationship between the signaling pathway and drug resistance,so as to provide a new way to explain the mechanism of EGFR-TKI resistance.Two NSCLC patients’ primary tumor cells,PC-9/GR and three TKI sensitive cell lines were analyzed by microarray to achieve the diffierential gene expression which related to different signaling pathways.Combined with the results of database and subsequent verification,it was found that the expression of FGFR1 was significantly up-regulated in TKI resistant cells,indicating that the increase of FGFR1 expression level may be closely related to TKI resistance.In order to study whether the increased expression of FGFR1 is directly related to the resistance of EGFR-TKI,we overexpressed FGFR1 in TKI sensitive cell line PC-9,and detected cell proliferation and apoptosis to assess whether the up-regulation of FGFR1 leads to the resistance of NSCLC cells to TKI.The results showed that over expression of FGFR1 increased the cell growth.Meanwhile,FGFR1 overexpression significantly inhibited the apoptosis of PC-9 cells and promoted the development of TKI resistance.FGFR1-si RNA was transfected into PC-9/GR cells and significantly inhibited the growth of PC-9/GR cells induced by gefitinib.At the same time,FGFR1 may play an important role in the inhibition of cell proliferation,and may be able to effectively induce cell apoptosis of TKI resistant cells and resistant to EGFR-TKI by activating Akt signaling pathway.Part Three The inhibitory effects of FGFR inhibitor PRN1371 combined with gefitinib on the growth of EGFR-TKI resistance cellsOver-expression of FGFR1 activated Akt signaling pathway,which promoted the proliferation of cancer cells and result in EGFR-TKI resistance.However,it is not clear whether FGFR1 inhibitor can enhance the sensitivity of gefitinib to TKI resistant cells.In order to verify the drug effect of FGFR1 inhibitor,we combined with gefitinib and FGFR1 inhibitor to observe the apoptosis and proliferation of PC-9,PC-9GR and primary tumor cells,so as to understand the mechanism of TKI resistance.The results showed that PRN1371,a FGFR1 inhibitor,could significantly inhibit the expression of FGFR1 m RNA in PC-9/GR resistant cells.The combination of gefitinib and PRN1371 significantly inhibited the cell viability,invasion and migration in PC-9/GR resistant cells.Therefore,the combination of gefitinib and PRN1371 can synergistically inhibit the proliferation of gefitinib resistant cells in NSCLC,which may be a therapeutic strategy to inhibit TKI resistance.