Mechanism Of Hedgehog Signaling Pathway In EGFR-TKI Sensitive And Resistance NSCLC Cells

Lung cancer is still a leading cause of cancer death in malignant worldwide. Non-small cell lung cancer (NSCLC) accounts for about85%of all lung cancer cases. Platinum-based doublet chemotherapy is the standard first line treatment for advanced NSCLC, but the median progression free survival (PFS) is only about5months. Toxicity of chemotherapy can seriously affect the quality of life. The advent of target therapy shed the great light on the lung cancer treatment. For patients with epidermal growth factor receptor (EGFR) sensitive mutation, the efficiency of gefitinib up to71.2%, has now become the first-line treatment in advanced NSCLC that harbored EGFR sensitive mutations. The second or third line treatment with EGFR TKI is better than best supportive care. Several mechanisms including T790M secondary mutation, MET amplification, hepatocyte growth factor (HGF) overexpression and KRAS mutation have been reported.Hedgehog (Hh) signaling pathway is one of the important pathways during embryogenesis. It regulate proliferation, differentiation, epithelial-to-mesenchymal transition (EMT) and stem cell maintenance, make organ developed properly. Aberrant activation of Hh signaling in adult tissues leads to tumorigenesis and treatment resistance. Emerging evidences indicated that EMT in malignant results in migration, invasion and resistance to conventional therapy. Cancer stem-like cells (CSCs) is another reason for resistance. Recent study indicated that EMT process in cancers generated a lot of CSCs. Hh signaling pathway closely regulate EMT and CSCs through its downstream direct targets Snail and ABCG2respectively in tumors. Many Hh inhibitors has carried out phase I/II clinical trial, and acquired good response in basal cell carcinoma (BCC). Hh and EGFR signaling regulated proliferation of brain stem cells synergistically in embryo stage. Taken together, we supposed that stimulation of Hh signaling pathway may bypass or attenuate the therapeutic efficacy of EGFR-TKIs in NSCLC. The combination of Hh inhibitor and EGFR-TKI may effectively increase the response of EGFR-TKI in patients with EGFR-TKI primary and secondary resistance.Our research used the NSCLC cells with EGFR sensitive mutation, secondary resistance mutation or primary mutation as the cell model to systematically demonstrate the activity difference of Hh signaling pathway between EGFR-TKI sensitive and resistant NSCLC cells. Effect of Hh signaling on the sensitivity of EGFR-TKI and efficacy of the combination of Hh inhibitor and EGFR-TKI were evaluated.Chapter one Expression difference of Hh signaling pathway between cancer and adjacent normal lung tissues in NSCLCMethodExpression of the molecules of Hh signaling pathway including PTCH1, PTCH2, SMO, GLI1, GLI2and GLI3were detected in20NSCLC tumor tissues and matched adjacent non-neoplastic lung tissues by real time PCR.Statistical analyses were performed by SPSS13.0soft wear. Measurement data presented by mean±standard deviation. At first data were test by normal distribution and homoscedasticity test. The data of all molecule expression meet normal distribution and homoscedasticity, therefore, the difference between groups tested by independent t test, difference among multiple groups tested by one-way ANOVA, pairwise comparisons used with LSD method. Correlation of two groups tested by Pearson correlation if data meet normal distribution, and tested by Spearman correlation if data did not meet normal distribution. Survival difference between different groups tested by Kaplan-Meier survival analysis and log-rank test.P<0.05was considered significant difference.Result1. Clinicopathological characteristic of patients:The study is a preliminary study with a small sample. A total of20NSCLC tumor tissues and matched adjacent non-neoplastic lung tissues were included. All of the patients are adenocarcinoma.10cases are early stage (stage I-II), and10cases are advanced stage (stage Ⅲ-Ⅳ).6cases are EGFR mutation,2cases are KRAS mutation,5cases are wild type, and the gene status of another7cases is unknown.2. Expression of Hh signaling pathway molecules in cancer tissues was lower than in adjacent normal lung tissues. Totally, there was no significant difference of SMO expression between cancer and normal tissues (t=-0.689, P=0.499). Expression of PTCH1(t=-7.764, P<0.001), PTCH2(t=-2.806, P=0.011), GLI1(t=-3.725, P=0.001), GLI2(t=-4.648, P<0.001) and GLI3(t=-8.124, P<0.001) were statistically different between cancer and normal tissues. All of these five genes were higher expressed in normal tissues.3. Hh signaling pathway was aberrant activated only in a small subset groups of lung adenocarcinoma. GLI1is a marker of Hh signaling activation. The results indicated that GLI1was aberrantly upregulated only in1case (5%)(GLI1expression level was2-fold high in cancer tissues than in adjacent normal tissues). This case was an early stage lung cancer harbor EGFR mutation. In addition,4samples showed high SMO expression (SMO expression level was2-fold high in cancer tissues than in adjacent normal tissues). Among these4samples,2cases was early stage, one of them had EGFR mutation;2cases was advance stage, both were wile type.1of early stage case with unknown gene status had the co-upregulation of SMO and GLI2,1of advanced stage with wild type gene status had the co-upregulation of SMO and PTCH2.4. The correlation between Hh signaling pathway gene expression in tumor tissues and clinicopathological parameters(1) Statistical analysis showed that Hh signaling pathway gene expression in tumor tissues was not associated with gender, age, smoking and gene status. GLI2expression was higher in early stage than in advanced stage (t=2.579, P=0.019). SMO expression was positively correlated with tumor volum (r=0.560, P=0.010).(2) Patients were divided into high-expression and low-expression groups sorted by the median of Hh signaling pathway gene expression in tumor tussues, and Kaplan-Meier survival analysis was performed. The results indicated that high GLI1high-expression group and GLI3high-expression group had the better overall survival than low expression groups. The median overall survival in GLI1high expression group was not reached, the median overall survival in GLI1low expression group was306days (Log-rank test,χ2=3.957,P=0.047,95%CI:130.9-481.1). The median overall survival in GLI3high expression group was also not reached, the median overall survival in GLI3low expression group was306days (Log-rank test,χ2=4.003, P=0.045,95%CI:0-794.1). However, the majority of patients in high expression of these two genes groups were early clinical stage (7/10), clinical stage may be the most important reason influencing survival.ConclusionTotally, expression of Hh signaling pathway molecules was lower in cancer tissues than in normal tissues. Hh signaling pathway may be aberrantly activated only in a small subset of lung adenocarcinoma. Hh signaling pathway may be worked in the transition of normal to malignant, and early stage of malignant. Chapter two Activity differences between EGFR-TKIs sensitive and resistance NSCLC cellsMethodThe activity of Hh signaling was detected in EGFR-TKIs sensitive, resistant cells and tumors by immunohistochemistry and western blot.Statistical analyses were performed by SPSS13.0soft wear. Measurement data presented by mean±standard deviation. At first, data were tested by normal distribution and homoscedasticity test. Western blot gray value between different groups was analyzed by one-way ANOVA, if the data was normal distribution and homoscedasticity. Intragroup difference was tested by LSD test. It was analyzed by welch test if the data was not homoscedasticity, and the intragroup difference was tested by Dunnett’s T3test. difference of Multiple group ranked data was tested by Kruskal-Wallis H test. Correlation of ranked data was analyzed by spearman rank correlation test. P<0.05was considered significant difference.Result1. Hh signaling pathway was aberrantly activated in EGFR-TKIs resistant cells.As previously mentioned, GLI1is the marker of activation of Hh signaling pathway. Firstly, GLI1expression was tested in EGFR-TKIs sensitive and resistant cells by immunohistochemistry. The results indicated that GLI1was negatively expressed in EGFR-TKIs sensitive cells PC9, while was positively expressed in EGFR-TKIs resistant cells H1975and A549. These results indicated that Hh signaling pathway was activated in EGFR-TKIs resistant cells, while was silenced in EGFR-TKIs sensitive cells.The results were confirmed by western blot that GLI1was upregulated in EGFR-TKIs resistant cells. Further demonstrated that activity of Hh signaling pathway was obviously difference between EGFR-TKIs sensitive and resistant cells, Hh signaling pathway was aberrantly activated in EGFR-TKIs resistant cells. 2. Hyper-activation of Hh signaling pathway accompanied by EMT phenotype and ABCG2upregualtion.Previous studies indicated that Hh signaling regulated EMT by upregulation of Snail and downregulation of E-cadherin. In addition, stem cell biomarker ABCG2was also the directed target gene of Hh signaling pathway. In order to further clarify the differences between EGFR-TKI sensitive and resistant cells, the target genes of Hh signaling pathway were detected by western blot in EGFR-TKI sensitive and resistant cells. The results showed that compared with EGFR-TKIs resistant cells, the expression of Snail was significantly downregulated in EGFR-TKIs sensitive cells PC9. Conversely, E-cadherin was obviously expressed in EGFR-TKIs sensitive cells PC9, and was almost absent in EGFR-TKIs resistant cells H1975and A549. ABCG2was over expressed in EGFR-TKI resistant cells. The results indicated that Hh signaling pathway was aberrantly activated along with EMT and ABCG2upregulation.3. Activity of Hh signaling pathway in EGFR-TKI sensitive and resistant NSCLC tissues.In order to further understand the difference between EGFR-TKI sensitive and resistant NSCLC,12NSCLC tumor tissues matched with gene status of these three cell lines were employed, expression of GLI1, ABCG2, E-cadherin and Snail were test by immunohistochemistry. Kruskal-Wallis H test results showed that the difference of GLI1expression between three groups was not statistical significance (χ2=4.460, P=0.108) because of small sample size. However, the mean rank of GLI1expression in EGFR-TKI sensitive mutation group was5.25, in EGFR-TKI secondary resistance group was4.88, in primary EGFR-TKI resistance group was9.38. There was the trend that GLI1was higher expressed in primary resistant group which contained KRAS mutation. The tissues which contained KRAS mutation may have the higher activity of Hh signaling pathway way.The mean rank of ABCG2expression in EGFR-TKI sensitive mutation group was5.13, in EGFR-TKI secondary resistance group was6.00, in primary EGFR-TKI resistance group was8.38, there was no statistical significance(χ2=2.155, P=0.340).The mean rank of E-cadherin expression in EGFR-TKI sensitive mutation group was8.00, in EGFR-TKI secondary resistance group was5.75, in primary EGFR-TKI resistance group was5.75, there was no statistical significance (χx2=1.179, P=0.555). The mean rank of Snail expression in EGFR-TKI sensitive mutation group was5.38, in EGFR-TKI secondary resistance group was6.75, in primary EGFR-TKI resistance group was7.38, there was no statistical significance (χ2=0.838, P=0.658).The results of correlation analysis showed that E-cadherin expression was significantly negative correlated with Snail expression (r=-0.582, P=0.047). Additionally, GLI1expression was negatively correlated with E-cadherin expression (r=-0.408, P=0.188), positively correlated with Snail (r=0.327, P=0.300) and ABCG2(r=0.386, P=0.215) expression. Because of small sample size, the correlation was no statistical significance. However, the trend was coincidence with cell experiment.Conclusion1Activity of Hh signaling pathway was significantly different between EGFR-TKI sensitive and resistant cells. It was silenced in sensitive cells, while aberrantly activated in resistant cells.2Aberrant activation of Hh signaling pathway accompanied by EMT phenotype and ABCG2upregulation in EGFR-TKIs resistant cells.3Hh signaling pathway was aberrantly activated in a subset of NSCLC. Hh signaling pathway may play a more important role in EGFR-TKIs primary resistant NSCLC with KRAS mutations. Chapter three Up-regulation of Hh signaling pathway lead to EGFR-TKIs resistance in NSCLC cellsMethodActivity of Hh signaling pathway was detected after EGFR-TKI sensitive cells PC9was treating with extrinsic SHH for oh,24,48h, and cell proliferation was evaluated with MTT.Statistical analyses were performed by SPSS13.0soft wear. Measurement data presented by mean±standard deviation. At first data were test by normal distribution and homoscedasticity test. All of data meet normal distribution and homoscedasticity, therefore, cell proliferation difference between different groups tested by factorial analysis, pairwise comparisons used with LSD method. P<0.05was considered significant difference.Result1. Activity of Hh signaling pathway in EGFR-TKIs sensitive cells PC9was upregulated by extrinsic SHH exposure.Hh signaling pathway was silenced in PC9cells. After extrinsic SHH treatment24h and48h, GLI1was obviously up-regulated in EGFR-TKIs sensitive cells PC9detected by immunohistochemistry and western blot.2. Up-regulation of Hh signaling pathway induced EMT phenotype and up-regulated ABCG2.E-cadherin, Snail and ABCG2were detected by western blot after SHH treatment for Oh,24h and48h, in PC9cells. The results showed that E-cadherin was down-regulated after SHH treatment24h and48h, while Snail expression was up-regulated gradually. ABCG2expression was also up-regulated after SHH treatment for24h and48h. The results indicated that aberrant activation of Hh signaling pathway contributed to EMT phenotype and up-regulated ABCG2expression. 3. Up-regulation of Hh signaling by exposure to SHH lead to Gefitinib tolerance.Cell proliferation was tested by MTT after N-SHH exposure for24h followed by Gefitinib treatment. The results indicated that the difference of cell proliferation was significant between extrinsic SHH exposure and non-exposure groups (F=76.677, P <0.001). The difference of cell proliferation was also significant among different Gefitinib concentration (F=32.785, P<0.001). In low concentration group (Gefitinib20nM), cell proliferation in experimental group was higher than in control group with marginal statistical difference (t=-2.7, P=0.054). In median and high concentration groups (Gefitinib40uM and80uM respectively), cell proliferation in experimental group was significantly higher than in control group with significant statistical difference (t=-8.146, P=0.001and t=-6.855, P=0.002respectively). The results demonstrated that aberrant activation of Hh signaling pathway contributed to EGFR-TKIs resistance in NSCLC cells.ConclusionUp regulation of Hh signaling pathway activation lead to EMT induction and ABCG2up-regulation, contribute to EGFR-TKIs tolerance in EGFR-TKIs sensitive NSCLC cells.Chapter four combination of Hh signaling pathway inhibitor and EGFR-TKI synergistically inhibited cell proliferationMethodHh signaling pathway activity after SANT-1treatment was test by western blot in EGFR-TKIs resistant cells. Difference of cell proliferation in Gefitinib single agent, or SANT-1single agent, or combination groups were tested by cell clone formation assay and MTT. Statistical analyses were performed by SPSS13.0soft wear. Measurement data presented by mean±standard deviation. At first data were test by normal distribution and homoscedasticity test. All of data meet normal distribution and homoscedasticity, therefore, cell clone difference test by one-way ANOVA, cell proliferation difference between different groups tested by factorial analysis, pairwise comparisons used with LSD method. P<0.05was considered significant difference.Result1. Inhibition of Hh signaling pathway in EGFR-TKIs resistant cells can inverse EMT phenotype, down-regulate ABCG2expression and inhibit cell migrationTo further determine the molecular mechanisms of Hh signaling with EGFR-TKIs resistance in NSCLC cells, we examined the GLI1, Snail, E-cadherin and ABCG2expression in0,24and48hours after EGFR-TKI resistant cell lines HI975and A549were treated with SANT-1(40uM). The results indicated that after treatment with SANT-1for24hours, GLI1expression was down regulated and Snail expression was significantly weakened in both two cell lines, and after treatment with SANT-1for48hours, Snail expression was lost in H1975. Inversely, E-cadherin expression was significantly elevated after EGFR-TKI resistant cell lines were treated with SANT-1for48hours. ABCG2expression was invisible after SANT-1treated the cells for24hours. These findings demonstrated that Hh inhibition effectively inversed EMT and decreased the abundance of CSCs in EGFR-TKIs resistant NSCLC cells. Wound healing assay showed that migration ability in EGFR-TKIs resistant cells were obviously weakened after SANT-1treatment for48h.2. Combination of Gefitinib and SANT1synergistically inhibited tumorigenesis and proliferation in EGFR-TKIs resistant NSCLC cell linesTo further demonstrate the role of Hh signaling pathway in NSCLC cells, we blocked Hh signaling using SMO inhibitor SANT-1. Previous study reported that the IC50of SANT-1in most NSCLC cell lines were about40uM, and the IC50of Gefitinib in EGFR-TKI sensitive cells were about20nM. Therefore, the EGFR-TKI resistant cell lines H1975and A549were treated with40uM SANT-1single agent, or 20nM Gefitinib single agent, or the combination of SANT-1and Gefitinib. As shown in figure4A and B, SANT-1or Gefitinib single agent didn’t effectively inhibit the clonogenic growth, however, colonies almost were not formed in the group of combination of SANT-1and Gefitinib (P<0.001). These results indicated that SANT-1and Gefitinib may have the synergistic effect in EGFR-TKI resistant NSCLC cells.To further confirm this result, we treated the EGFR-TKI resistance NSCLC cell lines H1975and A549with increasing concentration of SANT-1single agent, or Gefitinib single agent, or the combinations of SANT-1and Gefitinib, and then cell proliferation were measured. The results showed that A549cells not only resisted to Gefitinib, but also resisted to SANT-1. This result was coincidence with previous reports that A549cells possessed hyperactivation of Hh signaling, but were Hh signaling inhibitor resistance. However, the combination of Gefitinib and SANT-1effectively inhibited proliferation of A549cells (P<0.001). Although compared with Gefitinib, SANT-1was more effective to H1975cells (P=0.002), H1975cells has the best response to the combination of Gefitinib and SANT1(P<0.001). Taken together, these results confirmed that the combination of Hh signaling inhibitor and EGFR-TKIs had dramatic synergistic effects in EGFR-TKI resistant NSCLC cells.ConclusionInhibition of Hh signaling pathway in NSCLC can inverse EMT phenotype, down-regulate ABCG2expression and inhibit cell migration. Combination of Hh signaling pathway inhibitor and EGFR-TKIs synergistically increased EGFR-TKIs sensitivity in NSCLCs, and effectively decreased the cell proliferation of EGFR-TKIs resistant cells.