Research On The Role And Mechanism Of SHP2 In Regulating Cisplatin Resistance In Small Cell Lung Cancer

Background and Objectives:Lung Cancer is one of the most common causes of cancer-related death.Small Cell Lung Cancer(SCLC)is the most malicious lung cancer,accounts for about 20% of lung cancer,which grows fast,migrates early and has poor prognosis.Even after regular chemotherapy,the median survival time(MST)of patients with SCLC is only about 8 to 13 months.Poor treatment effect caused by cisplatin resistance is an important factor leading to the poor survival rate of patients with SCLC.Therefore,the research on the specific mechanisms of SCLC drug resistance is of great guiding significance for improving prognosis of disease.Cisplatin(CDDP)is an important anti-cancer drug.Most patients with SCLC adopt cisplatin-based combination therapy.However,the malignant cells that exposed to cisplatin may activate its adaptive response to resist cisplatin’s antiproliferative/cytotoxic effect,eventually produce cisplatin resistance and cause patient’s poor response to cisplatin-based chemotherapy.PI3K/AKT is an important signal pathway,whose signaling axis can provide powerful pro-survival signals.Therefore,AKT pathway may play an important role in regulating cisplatin resistance in tumor.However,the functional mechanism and its significance in cisplatin resistance are still unclear.Src homology 2-containing protein tyrosine phosphatase 2(SHP2)is a non-receptor type protein-tyrosine-phosphatase(PTP)and widely expressed in human tissues.It plays an important role in the process of various cellular events,such as cell proliferation,survival and migration.Mutation or abnormal expression of SHP2 is closely related to hematologic malignant tumor and other tumors.SHP2 can connect PI3 K and P85 subunit and related activation-promoting factor,leading to the activation of PI3 K.SHP2 can also activate PI3K/AKT signal pathway via its PTP catalytic activity.However,the researches on the effect of SHP2 in activating PI3K/AKT mainly focus on tumorigenesis and metastasis,its role in tumor drug resistance has not been fully recognized.Our previous study found the high expression of SHP2 in chemotherapy-resistant lung cancer cells,suggesting that it may participate in the chemotherapy-resistant process.CA916798 is a protein related to drug resistance,which is found in human lung cancer cell line SPC-A-1/CDDP through suppression subtractive hybridization(SSH)and no homology was found with known proteins.There are multiple serine/threonine kinase and tyrosine kinase activation sites in CA916798 structure,indicating that it may be activated by relevant kinases and participate in signal transmission process.Our previous study indicates that CA916798 promotes cisplatin resistance in lung cancer,and could be regulated by PI3K/AKT pathway.Hypothesis is made according to above studies: SHP2 regulates the expression of CA916798 by regulating PI3K/AKT pathway,and causes SCLC’s resistance to cisplatin.This study is carried out to verify the hypothesis.Materials and Methods:This research is divided into 3 parts: in vitro experiment,in vivo test and clinical analysis.1.Cisplatin-sensitive SCLC cell lines and drug-resistant cell line derived from cisplatin-induced sensitive cells were taken.2.Plasmid with SHP2 expression was transfected into sensitive cell,SHP2 RNA interference plasmid was transfected into drug-resistant cell,and cell lines with stable high/low expression of SHP2 were screened.3.While intervening SHP2,plasmid with AKT expression was transfected or AKT RNA interference therapy was made.At different levels of AKT expression,SHP2-induced cisplatin resistance in SCLC was observed.4.CCK8 method was adopted to test various cell lines’ sensitivity to cisplatin.5.Co-immunoprecipitation method was adopted to detect the expressions of proteins combined with SHP2.6.Western-blotting method was used to detect the protein expressions.7.RT-PCR method was adopted to detect the m RNA expression level of related proteins.8.Nude mice were injected subcutaneously with cells,in order to establish xenograft model in nude mice.9.Cisplatin or physiological saline was intraperitoneally injected into the transplanted tumor of nude mice,cell volume of each group was observed,in order to determine cell’s sensitivity to cisplatin treatment in each group.10.Qualified clinical cases were selected,data were collected,and tumor tissues were treated with HE and immunohistochemical staining,the correlation between SHP2 and CA916798 protein expression was tested,and the survival time of patients with different levels of protein expression after chemotherapy were compared.Results:1.SHP2 promotes cisplatin resistance.(1)Cisplatin-sensitive cell H446 and cisplatin-resistant H446/CDDP cells used in the experiment were identified,neuron-specific enolase(NSE)immunofluorescence indicates that both H446 and H446/CDDP have NSE expression,which confirms that the cells used in the experiment are cell lines of SCLC(2)IC50 value of H446 cell and H446/CDDP cell to cisplatin were 2.493 ug/ml and 15.239ug/ml,respectively.The drug resistance index of H446/CDDP to H446 is 6.1127.Compared with parental H446 cell,H446/CDDP has increased cisplatin resistance.(3)Western-blotting results indicated that,compared with H446 cells,the expression of SHP2 was increased in drug-resistant H446/CDDP cells(P <0.05)(4)SHP2 expression plasmid was transfected into H446 cells and screened,in order to establish H446-SHP2 cell lines with stable high expression of SHP2;SHP2 RNA interference plasmid was transfected into drug-resistant cells and screened,in order to establish H446/CDDP-shSHP2 cell lines with stable high expression of SHP2.Compared with H446 cells,the SHP2 expression increased in H446-SHP2;Compared with H446/CDDP cells,the SHP2 expression decreased in H446/CDDP-sh SHP2.This indicated that SHP2 expression intervention was successful.(5)The semi-quantitative measurement results indicated that each cell line’s SHP2 m RNA had the same trend with protein expression,but the difference is not statistically significant(P> 0.05)(6)The total phosphatase activity test results indicated that the total phosphatase activity of each cell line has the same trend with protein expression level,but the difference is not statistically significant.(7)IC50 value of H446 to cisplatin was 5.14ug/ml,while the IC50 value H446-CDDP to cisplatin was 10.414 ug/ml.The resistance index of H446-SHP2 to H446 was 2.03.IC50 value of H446/CDDP to cisplatin was 13.351 ug/ml,and which of H446/ CDDPsh SHP2 to cisplatin was 6.621ug/ml,the resistance index of H446/ CDDP-shSHP2 to H446 /CDDP was 0.4959.In cisplatin-sensitive cell lines,high expression of SHP2 could increase resistance to cisplatin;in cisplatin-resistant cell lines,partial knockout of SHP2 expression can resume sensitivity of drug-resistant cell lines to cisplatin.2.The mechanism that SHP2 promotes cisplatin resistance in SCLC(1)SHP2 combines with PI3 K,p85 subunit and CA916798 within the cells.When the total amount of SHP2 is roughly the same,SHP2 binds more p85 and CA916798 in H446/CDDP cells than in H446 cells.The amount of SHP2 combined with p85 and CA916798 also increases after cisplatin addition.(2)After SHP2 in H446 cells was high expressed,the expression of AKT,p AKT,pmTOR and CA916798 proteins also increases(P <0.05).In drug-resistant H446/CDDP cells,after partial knockout of SHP2 expression,the expression of AKT,pAKT,pm TOR,and CA916798 proteins decreases(P <0.05).These results suggest that the change of SHP2 expression can affect the activity of downstream AKT pathway and CA916798 expression.(3)Compared with transferred empty vector,after AKT RNA interference plasmids are transferred into H446 cells,the expression of AKT decreases;after AKT expression plasmids are transferred,AKT expression increases.This indicates that relevant plasmids can effectively intervene AKT expression.(4)Partial knockout of AKT expression in H446 cells with high expression of SHP2 can reduce the expression of CA916798(P <0.05).In the H446/CDDP cells with low expression of SHP2,high expression of AKT can restore the expression level of CA916798 to a relatively high level(P <0.05).This indicates that SHP2 can regulate the expression of CA916798.(5)IC50 values of H446 cells,H446-SHP2 cells and H446-SHP2-si AKT cell to cisplatin are 4.82ug/ml,10.902ug/ml and 6.236ug/ml,respectively.Partial knockout of AKT expression in H446 cells with high expression of SHP2 can increase drug-resistant cell’s sensitivity to cisplatin.IC50 values of H446/CDDP cells H446/CDDP-shSHP2-AKT are 11.032ug/ml and 9.344ug/ml.After partial knockout of SHP2,H446/CDDP cells exhibited high expression of AKT,which can restore the resistance of cells.to cisplatin.3.The influence of SHP2 on cisplatin resistance in SCLC in animal modelIn H446 group,the tumor volume was significantly smaller after cisplatin treatment than physiological saline treatment(P <0.05).In both H446-SHP2 group and H446/CDDP group,there were no significant difference of tumor volume between cisplatin treatment subgroup and physiological saline treatment subgroup(P> 0.05).In H446/CDDP-shSHP2 group,the tumor volume was significantly smaller after cisplatin treatment than physiological saline treatment(P <0.05).This confirms that high expression of SHP2 can lead to the decreased sensitivity of small cell lung cancer to cisplatin,while partial knockout of SHP2 can increase SCLC’s sensitivity to cisplatin.4.Clinic analysis on the correlation between SHP2 and CA916798 expression and chemotherapy resistance(1)13 out of 20 patients with high expression of SHP2 have high expression of CA916798,and 3 out of 12 patients with low expression of SHP2 expression have high expression of CA916798(P <0.05).In clinical patients,the SHP2 expression has positive correlation with CA916798 expression in tumor tissue.(2)Survival analysis(OS)indicates that patients with high expression of SHP2 in tumor tissues have shorter survival period than patients with low expression in tumor tissues(P = 0.045).Compared with patients with low expression of CA916798 in tumor tissues,patients with high expression of CA916798 have shorter average survival period(P = 0.002).This indicates that high expression of SHP2 and CA916798 is related to the patient’s poor response to chemotherapy.Conclusion:Our present study indicates that SHP2 can promote SCLC resistance,which is mainly achieved via PI3K/AKT/CA916798 pathway.In clinical treatment,the expression of SHP2 and CA916798 in tumor tissues can be used as a predictor of chemotherapy effect.Our research clarifies the role of SHP2/ PI3K/AKT/CA916798 pathway in affecting the chemoresistance in SCLC,and provides potential intervention targets for developing strategies to increase sensitivity in clinical SCLC chemotherapy.