Mechanisms Of Hedgehog Signaling Pathway To Acquire EGFR-TKIs Resistance In EGFR-mutated Non-small Cell Lung Cancer Patients With Brain Metastasis

Lung cancer is the most deadly malignancy in the world.Non-small cell lung cancer(NSCLC)accounts for about 85%of all lung cancer cases.Platinum-based two-drug combination chemotherapy is the main first-line standard treatment for advanced NSCLC,and the progression free survival PFS is only about 5 months.The incidence of brain metastasis(BM)is about 10%in non-small cell lung cancer,while the incidence of brain metastasis is about 25%-38%in the whole development process.Once brain metastasis occurs,the traditional treatment of NSCLC brain metastasis is brain radiotherapy(CRT),including whole brain radiotherapy(WBRT),stereotactic radiosurgery(SRS),and so on.The median survival time of patients is only 1-3 months.EGFR mutation is a good prognostic marker of NSCLC.However,NSCLC patients with EGFR mutation have a higher risk of brain metastasis.The emergence of molecular targeted drugs has brought great hope for the treatment of lung cancer.For patients with epidermal growth factor receptor(EGFR)receptor sensitive mutation,EGFR tyrosine kinase inhibitor(EGFR-TKI)has an effective rate of 70%,and has become the first-line drug of EGFR-TKI for patients with advanced egfr-sensitive mutant NSCLC,which is also superior to the optimal supportive treatment for second-line or third-line treatment.However,drug resistance is inevitable in almost all patients,and the primary or secondary drug resistance of EGFR-TKI greatly restricts its clinical application.Currently,mechanisms related to EGFR-TKI resistance have been reported,including T790M secondary mutation,MET amplification,overexpression of hepatocyte growth factor HGF and KRAs mutation.Hedgehog(Hh)signaling pathway is one of the important signaling pathways in embryonic period,regulating cell proliferation,differentiation,transformation of epithelial mesenchymal(epithelial-to-mesencmal transition,EMT)and stem cells maintain,in order to make the normal development of tissues and organs.Abnormal activation of Hh signaling pathways in adult tissues is associated with the formation,self-renewal,and drug resistance of a variety of tumors,including lung cancer.There is growing evidence that EMT in malignant tumors can lead to increased invasion and migration of tumor cells and resistance to traditional therapies.Hh pathway plays an extremely important role in the occurrence,development and treatment of various tumors.At the same time,the intervention of different targets of these signaling pathways in the treatment of tumors also shows good results and application prospects.Several Hh signaling pathway inhibitors have entered clinical trials and achieved good results Daurismo,a Hh signaling pathway inhibitor,has been approved for the treatment of newly diagnosed acute myelogenous leukemia over 75 years old who can not accept high-intensity chemotherapy.Several Hh signaling pathway inhibitors have entered the clinical trial stage and achieved good clinical efficacy in the treatment of basal cell carcinoma of the skin and medulloblastoma.Therefore,we speculated that abnormal activation of Hh signaling pathway may be involved in the process of drug resistance inNSCLC EGFR-TKI treatment,and reduce the therapeutic effect of EGFR-TKI.The combination of Hh signaling pathway inhibitors and EGFR-TKI may effectively improve the response of patients with primary or secondary drug resistance to EGFR-TKI.This research mainly divided into two parts,the first part mainly discusses in different time whole brain radiotherapy combined therapy with EGFR-TKIs-with EGFR mutations of NSCLC with brain metastasis in patients with clinical curative effect and survival,The second partdividedinto three chapters is mainly to investigate the role and mechanism of Hedgehog signaling pathway in the resistance of NSCLC to EGFR-TKIs with brain metastasis and to construct human non-small cell lung cancer cell line PC9(EGFR mutant)with EGFR sensitive mutation and secondary drug-resistant NSCLC with brain metastasis lung cancer tissue.In vitro cell model of PC9-resistor and in vivo tumor model of PC9-res xenograft in nude mice with secondary EGFR-TKIs as study subjects,the expression difference of Hh signaling pathway in the sensitive and secondary EGFR-TKIs resistance NSCLC BM was systematically detected,and the effects of up-regulation and low-knocking Hh signaling pathway on secondary EGFR-TKIs resistance were verified.The effect of combination therapy with Hh inhibitors and EGFR-TKIs on the secondary resistance to EGFR-TKIs in NSCLC BM was observed to provide a new direction and strategy for the clinical treatment of secondary drug-resistance to EGFR-TKIs in NSCLC BM with EGFR mutation.Methods:1.Concurrent vs.sequential whole brain radiotherapy and TKI in EGFR-mutated NSCLC patients with brain metastasisTo examine the outcomes of concurrent vs.sequential whole-brain radiotherapy(WBRT)and epidermal growth factor receptor-tyrosine kinase inhibitor(EGFR-TKI)in non-small cell lung cancer(NSCLC)patients with EGFR mutation.Retrospectively 105 patients in NSCLCwith brain metastasis,and EGFR mutation(Affiliated Hospital of Guangdong Medical University,01/2011 to 12/2014)were grouped as:EGFR-TKIs alone(n=39,group A),EGFR-TKIs+concurrent radiotherapy(n=34,group B),and radiotherapy followed by EGFR-TKIs(n=32,group C).2.Mechanisms of Hedgehog signaling pathway to acquire EGFR-TKIs resistance in EGFR-mutated non-small cell lung cancer patients with brain metastasis2.1 The expression and clinical significance of Hedgehog pathway-related protein in EGFR-TKI resistant and sensitive NSCLC associated with brain metastasis in lung cancer.2.1.1 The expression of Hedgehog pathway-related proteins in NSCLC tumors with EGFR-TKIs sensitivity and resistance was statistically analyzed according to The Cancer Genome Atlas(The Cancer Genome Atlas,TCGA),and the relationship between the expression of Hedgehog pathway-related proteins and prognosis and survival time of patients was analyzed.2.1.231 case NSCLC patients with brain metastasis(20 of whom were EGFR-TKIs sensitive and 11 were EGFR-TKIs resistant)was performed by q-PCR,to detect Hedgehog pathway related proteins.31 cases of NSCLC patients with brain metastasistissues were collected,from January 2016 to February 2018 were selected for this study.Among them,20 cases were sensitive to EGFR-TKIs and 11 cases were resistant to,using the immunohistochemistry and Western-blotmethods to detecte the expression.2.2 Effects of SHH and shRNA-GLI1 on biological characteristics of non-small cell lung cancer in vitroThe effects of Hedgehog signaling pathway on the activation and inactivation of EGFR-TKIs-resistant PC9-res cell and PC9 cell were detected by using the constructed PC9-res cell and PC9 cell as experimental objects.2.2.1 The effect of Hedgehog on the expression of GLI1 in human non-small cell lung cancer cell PC9 were tested by immunofluorescence.2.2.2 The effects of different EGFR-TKIs resistance on GLI1 and EMT were detected by western blot and immunofluorescence.2.2.3 The effect of exogenous SHH on GLI1 nucleus and expression in PC9 were detected by Immunofluorescence DAPI.2.2.4 The effects of Hedgehog signaling pathway activation on GLI1 and EMT-related proteins in PC9-sensitive non-small cell lung cancer cells were detected by qPCR,Western-blot and immunofluorescence.2.2.5 The effect of different Hedgehog signaling pathway activation on migration of PC9-sensitive cell were detected by Transwell assay;2.2.6 The effects of different hedgehog signaling pathway activation on apoptosis of PC9 sensitive cel 1 were detected by Immunofluorescence and Annexin V/PI.2.2.7 Non-small cell lung cancer cell line PC9-res was infected with shRNA Lentivirus Expression vector system.,The effect of inactivation of Hedgehog signaling pathway on PC9-res cell were detected.2.2.8The effect of inactivation of Hedgehog signaling pathway on GLI1 nucleus and expression after PC9-res strain by immunofluorescence DAPI.2.2.9 The effects of inactivation of Hedgehog signaling pathway on GLI1 and EMT in PC9-res cells were detected.by Western-blot.2.2.10 was used to detect the effects of inactivation of different Hedgehog signaling pathways on migration and invasion of PC9-res cells were detected by Transwell assay.2.2.11 The effects of inactivation of different Hedgehog signaling pathways on apoptosis of PC9-res cells were detected by immunofluorescence and Annexin V/PI.2.3 Effects of SHH,shRNA-GLI1 and Hedgehog inhibitors on biological characteristics of non-small cell lung cancer in vivoEstablishment of PC9 cell transplantation model in nude mice and PC9-res cell transplantation model in nude mice to observe the effects of SHH,shRNA-GLI1 and Hedgehog inhibitors on the growth of PC9 cell transplantation in nude mice with subcutaneous non-small cell lung cancer.According to the different treatment methods of transplanted cell,The nude mice were divided into control(Ctr)group and SHH group.According to the different expression vectors of vectors and shRNA lentiviral expression vectors that were constructed to infect PC9-res transplanted lentiviral expression vectors,Nude mice were divided into vector group,shGLI-1 group and shGLI-2 group.After 2 weeks of inoculation,the tumor size was measured regularly until the tumor grew to 5 mm.After the tumor grew to 8 mm,the drug gefitinib was administered orally every day.The dosage was 20 mg/kg,5 days a week for continuous administration.2.3.1 The effect of the growth of transplanted tumors?the quality of gross specimens and survival of transplanted tumors in nude mice.2.3.2 The effect of SHH treatment on gefitinib-induced apoptosis of PC9 cells were detectedby Tunel staining.2.3.3 The effects of different Lentivirus Expression vector cellon the growth,mass and survival of transplanted tumors in nude mice.2.3.4 The effect of different Lentivirus Expression vector cell on gefitinib-induced PC9-res apoptosis by Tunel staining.The nude mouse model of PC9-res cell transplantation was established,According to the different treatment methods,nude mice were divided into gefitinib group,GDC-0449 group and gefitinib combined with GDC-0449 group.The dosage of gefitinib and GDC-0449 were 20 mg/kg and 32 mg/kg respectively,and excipients were used as control group.After 2 weeks of inoculation,the tumor size was measured regularly when the tumor grew to 3-5 mm.When the tumor grew to about 8 mm,the drug was given orally every day for 5 days a week for continuous observation.2.3.5 Observed the growth volume?survival and survival time of the transplanted tumors in nude miceeffects by different drugs.Results:1.Concurrent vs.sequential whole brain radiotherapy and TKI in EGFR-mutated NSCLC patients with brain metastasisThe ORRs of group A,group B and group C are 66.7%,85.3%and 75%respectively by observing the therapeutic effect of EGFR mutation in patients with brain metastasis from non-small cell lung cancer based on the EGFR-TKIs,and there are significant differences between the two groups(P<0.05).The intracranial DCR of group A,group B and group C are 79.5%,94.1%and 87.5%respectively.The DCR of group B was significantly higher than that of group A(P<0.05),but there is no significant difference with group C(P>0.05),and there's no significant difference of DCR between group A and group C(P>0.05).The median OS of group A,group B and group C are 16.9 months(95%CI:13.852-20.264),24.5 months(95%CI:19.967-28.984)and 21.3 months(95%CI:17.785-24.861)respectively.The difference among the three groups is significant(P<0.05),and group B is significantly higher than that of group A and C(P<0.05)The median intracranial iPFS of group A,group B and group C are 6.8 months(95%CI:5.143-7.857),12.4 months(95%CI:10.774-13.226)and 9.1 months(95%CI:6.937-11.063)respectively.The difference among the three groups is significant(P<0.05).The difference among the three groups is significant(P<0.05),and group B is significantly higher than that of group A and C(P<0.05).The median extracranial ePFS in group A,B and C is 7.8 months(95%CI:7.392-8.208),9.4 months(95%CI:7.867-10.533)and 8.3 months(95%CI:6.755-9.245)respectively,with no significant difference(P>0.05).Most of the adverse reactions in the three groups were grade 1 or grade 2,with no significant difference(P>0.05),which were tolerable.2.Mechanisms of hedgehog signal transfer in EGFR-TKI resistance through of non-small cell lung cancerwith brain metastasis EGFR-mutation2.1 Expression and clinical significance of Hedgehog pathway-related protein in EGFR-TKI resistant and sensitive NSCLC associated with brain metastasis in lung cancer2.1.1 The expression of Hedgehog pathway-related proteins and its relationship with prognosis and survival time of patients in EGFR-TKIs sensitive and resistant NSCLC tumor tissues are analyzed by TCGA database.The results show that the expression of Hedgehog pathway-related proteins is significantly increased in P-resistance tumor tissues,such as GLI1,SMO,BCL2 and SNAIL,and the difference between groups is statistically significant(P<0.05).The progression free survival and overall survival time of NSCLC patients with high GLI1 expression is shorter than those with low GLI1 expression,and the difference between groups is statistically significant(P<0.05).Therefore,these results suggest that Hedgehog pathway-related proteins are highly expressed in EGFR-TKIs resistant NSCLC and negatively correlated with patient prognosis.2.1.2 GLI1,SMO,BCL2,PTCH1,HHIP,SNAIL and CYCLIND were detected by qPCR.Compared with P-sensitive tumor tissue,Hedgehog pathway-related proteins such as GLI1,SMO,BCL2,PTCH1,HHIP,SNAIL and CYCLIND in P-resistance tumors are significantly increased,which suggest us that Hedgehog pathway might be involved in regulating the drug resistance of NSCLC to EGFR-TKIs.The results of,immunohistochemistry and Western-blot are further verified.Compared with P-sensitive tumor tissue,the expression of Hedgehog pathway-related protein in P-resistance tumors are significantly up-regulated,and the difference between groups is statistically significant(P<0.05).2.2Effects of SHH,shRNA-GLI1 and Hedgehog inhibitors on biological characteristics of non-small cell lung cancer2.2Effects of SHH and shRNA-GLI1 on biological characteristics of non-small cell lung cancer in vitro2.2.1 The results of immunofluorescence show that compared with PC9,the GLI1 in R-1 and R-2 cells of PC9-res is mainly expressed in the nucleus,and nuclear aggregation increased.Besides,the expression of GLI1 increased and the difference is significant and the difference between groups has statistical significance(P<0.05).2.2.2 Western blot and immunofluorescence are used to detect the effects of different EGFR-TKIs resistance on GLI1 and EMT.Western blot results show that compared with PC9,the GLI1 expression in R-1 and R-2 cells of PC9-res is significantly higher than that in PC9 cells(P<0.05).Compared with PC9,the expression of E-cadherin in PC9-res decreased significantly,and the difference between groups is statistically significant(P<0.05);N-cadherin and vimentin expression increased significantly,and the difference between groups is statistically significant(P<0.05).2.2.3 The results of immunofluorescence DAPI staining showed that compared with blank control,the expression of GLI1 in PC9 cells treated with SHH is mainly concentrated in the nucleus,the nuclear aggregation increased,the expression of GLI1 increased significantly,and the difference between groups is statistically significant(P<0.05).2.2.4 The effects of Hedgehog signaling pathway activation on GLI1 and EMT-related proteins in PC9-sensitive cell lines of non-small cell lung cancer are observed by qPCR,Western-blot and immunofluorescence,and the results of qPCR,Western-blot and immunofluorescence are analyzed:Compared with the blank control,the expression of E-cadherin and ZO-1 in PC9 cells of SHH treatment group decreased significantly,and the difference between groups is statistically significant(P<0.05);N-cadherin and vimentin expression increased significantly,and the difference between groups is statistically significant(P<0.05)..2.2.5 Transwell assay is used to detect the effects of different Hedgehog signaling pathway activation on migration of PC9-sensitive cell lines from non-small cell lung cancer.Transwell assay shows that compared with blank control,the expression of PC9 cells in SHH treatment group is significantly increased,and the difference between groups is statistically significant(P<0.05)..2.2.6 Immunofluorescence and Annexin V#PI methods are used to observe the effect of different activation states of Hedgehog signaling pathway on apoptosis of PC9 sensitive cell lines of non-small cell lung cancer.The results show that compared with the blank control,the apoptosis of PC9 cells in SHH treatment group is significantly reduced,and the difference between groups is statistically significant(P<0.05).2.2.7 The shRNA lentivirus expression vector system is successfully constructed to infect PC9-res cell line of non-small cell lung cancer.2.2.8 The results of immunofluorescence DAPI staining show that compared with blank control,the nuclear aggregation and expression of GLI1 in shRNA knockdown PC9-res group are significantly lower than those in blank control(P<0.05).2.2.9 Western-blot results show that compared with the blank control,the expression of GLI1 in PC9-res knocked down by shRNA is significantly lower,and the difference between groups was statistically significant(P<0.05)..The expression of E-cadherin and ZO-1 increased significantly,and the difference between groups is statistically significant(P<0.05).The expression of N-cadherin and vimentin decreased significantly,and the difference between groups is statistically significant(P<0.05).2.2.10 Transwell assay shows that compared with blank control,the expression of shRNA in PC9-res knockdown group is significantly lower than that in blank control(P<0.05).2.2.11 The results of immunofluorescence and Annexin V/PI assay show that compared with the blank control,the apoptosis of PC9-res group is significantly increased in shRNA knockdown group,and the difference between groups is statistically significant(P<0.05).2.3Effects of SHH,shRNA-GLI1 and Hedgehog inhibitors on biological features of non-small cell lung cancer2.3.1 Observe the effect of SHH treatment on the growth,mass and survival of PC-9 cell transplanted tumors in nude mice.The transplanted tumors appeared in the axillary inoculation area of nude mice about 2 weeks on average.All the animals survived and formed tumors in vivo.No inflammation such as swelling and ulceration occurred at the inoculation site.From the 3rd week,the volume of transplanted tumors in SHH group is significantly higher than that in control group(P<0.05=),with significant difference(P<0.05).,the significant effect of promoting the tumor growth is observed.In SHH group,the nude mice had poor mental status,sluggish,poor appetite,faster growth rate and larger volume of transplanted tumors.There is no obvious boundary between the tumors and the surrounding tissues,and the boundary is not clear.There's no complete envelope around the tumors.In the control group,the nude mice had good mental spirit,active,good appetite,and the size of transplanted tumors is small.Besides,there are clear and complete envelopes around the tumors,no obvious infiltration and adhesion,and the tumors can be dissected completely.The weight of transplanted tumors of nude mice in SHH group is significantly higher than that in control group(P<0.05).,which has statistical significance.2.3.2 The results of Tunel staining show that compared with control group,the apoptosis of transplanted tumors of nude mice in SHH group is significantly decreased,and the difference between groups is statistically significant(P<0.05).2.3.3 Observe the effect of shGLI transfection on the growth,mass and survival of PC9-res cell transplanted tumors of nude mice.The transplanted tumors appeared in the axillary inoculation area of nude mice about 2 weeks on average.All the inoculated animals survived and formed tumors in vivo.No inflammation such as swelling or ulceration occurred at the inoculation site.From the 3rd week,the volume of transplanted tumors of nude mice in shGLI-1 and shGLI-2 groups is significantly lower than that in vector group(P<0.05).The difference is significant,with statistical significance((P<0.05),and significant inhibition of tumor growth is observed.The nude mice in shGLI-1 and shGLI-2 groups had good mental status,active,good appetite,small size of transplanted tumors,obvious and complete envelope formation around the tumors,clear boundaries,no obvious infiltration and adhesion,which can dissect the tumors completely.However,in the vector group,the nude mice had poor spirit,sluggish,poor appetite,the size of the transplanted tumors is larger,and there's no obvious boundary between the tissue and the surrounding area.The boundary is not clear,and there's no complete envelope around the tumors.The weight of transplanted tumors of nude mice in shGLI-1 group and shGLI-2 injection group is significantly lower than that in vector group(P<0.05).2.3.4 Tunel staining detection shows that compared with vector group,the apoptosis of transplanted tumors of nude mice in shGLI-1 group and shGLI-2 injection group increased significantly,and the difference between groups is statistically significant(P<0.05).2.3.5 Observe the effects of EGFR-TKIs combined with Hh inhibitors on the growth volume,survival and survival time of PC9-res transplanted tumors in nude mice.The average axillary inoculation area of nude mice showed transplanted tumors about 2 weeks later.After 3 weeks,the diameter was about 0.3-0.5 cm.All the inoculated animals survived and formed tumors in vivo.There was no inflammation reaction such as swelling and ulceration at the inoculation site.The nude mice in gefitinib combined with GDC-0449 group had good spirit and activity,good appetite,and the nude mice in other three groups had poor spirit,performance,and spirit.The volume of transplanted tumors of nude mice in gefitinib combined with GDC-0449 group is significantly lower than that in the other three groups,and the difference is significant,which has statistical significance(P<0.05=,but there's no difference among the other three groups,and only significant tumor inhibition is observed in the combination group.The survival time of transplanted tumors of nude mice in gefitinib combined with GDC-0449 group is significantly higher than that in the other three groups(P<0.05),but there's no difference among the other three groups,only the prolongation of survival time has been observed in the combined group.Conclusion:Compared with synchronous application of whole brain radiotherapy,EGFR-TKIs alone or sequential application of EGFR-TKIs after whole brain radiotherapy can effectively improve the short-term and long-term efficacy of NSCLC patients with EGFR mutation,without increasing other adverse reactions,but the emergence of EGFR-TKIs resistance is inevitable.There are significant differences in the expression of Hh signaling pathway between EGFR-TKI sensitive and secondary drug-resistant NSCLC brain metastasis lung cancer tissues.The expression of Hh signaling pathway is inactivated in sensitive cells and abnormally up-regulated in secondary drug-resistant cells.The abnormal activation of Hh signaling pathway may be one of the mechanisms of resistance to EGFR-TKIs in NSCLC with EGFR mutation.Hedgehog pathway-related proteins are highly expressed in EGFR-TKIs resistant NSCLC and negatively correlated with prognosis of patients.Hh signaling pathway is abnormally up-regulated in EGFR-TKI resistant cells.Meanwhile,EMT manifestations such as inhibiting the expression of E-cadherin and promoting the expression of N-cadherin and vimentin appear.Hh signaling pathway was activated and PC9 was resistant to gefitinib by SHH treatment.After the constructed shRNA Lentivirus Expression vector System infects PC9-res,Hh signaling pathway inactivates,reverses the EMT phenotype of PC9-res cells,inhibits the invasion and metastasis of PC9-res cells,which promotes apoptosis induced by EGFR-TKI.The transplantation model of lung cancer PC9 cells and PC9-res cells in nude mice are successfully constructed.Hh signaling pathway was activated by SHH treatment promotes gefitinib resistance,promotes the growth of transplanted tumors in nude mice,and inhibits the apoptosis of tumor cells.Hh signaling pathway was Inactivated can reversal of gefitinib resistance.significantly inhibit the growth of xenografts in nude mice and promote apoptosis of tumor cells by down-regulation of shRNA-GLI1.The combination of EGFR-TKI and Hh signaling pathway inhibitors can reverse the sensitivity of PC9-res cells to EGFR-TKI,inhibit the size of tumors and prolong the survival time of transplanted tumors of EGFR-TKI resistant cells in nude mice.