Expression Of Hedgehog Signaling Molecules And Their Clinical Significance In Non-small Cell Lung Cancer

BackgroundLung cancer is one of the most common tumors in our country. Recent years, the morbidity of lung cancer is on the rise. This trend threats the human health seriously. Although the treatment of lung cancer has made a great progress in recent years, the problems including resistance, recurrence and metastasis which arise after treatment remain a huge obstacle to patients'long-term survival. The mechanisms of the occurrence and development of lung cancer still have many mysteries. With the answer revealed continually, it will provide more targets new for individualized therapy in the different lung cancer subtypes.Numerous studies show that many very important pathways in embryonic development, such as Wnt, Norch, TGF and hedgehog signaling pathways, also play the key roles in tumor development. Many studied for the function of hedgehog signaling pathway in tumors have shown that this important pathway for regulation of embryonic development is abberantly activated in a variety of human tumors, including skin basal cell carcinoma, pancreatic cancer, prostate cancer, stomach cancer, ovarian cancer and small cell lung cancer.Mammalian Hedgehog signaling pathway is mainly composed by three ligands: Sonic hedgehog (SHH), Desert hedgehog (DHH), Indian hedgehog (IHH), two transmembrane receptors:Petched (PTCH), Smothen (SMO), three transcription factors:GLI1, GLI2, GLI3, and downstream target genes. Currently, the mechanism of action of the Hedgehog signaling pathway in non-small cell lung cancer (NSCLC) is still unclear. Although there are several studies for the role of SHH-GLI1/GLI2 pathway in NSCLC, the expression pattern of HH/GLI3 pathway and its clinical significance in NSCLC is remain unknown.ObjectiveTo explore the expression pattern of HH/GLI3 signaling pathway proteins, SHH, DHH, IHH, SMO and GLI3, in the non-small cell lung cancer tissues, and its potential clinical significance. Systematically investigate the correlation of expression of three ligands, SHH, DHH, IHH, with downstream transcription factor, GLI3 protein. Our research may provide the theoretical basis for further illuminating the molecular mechanism of lung cancer development and finding the new target for lung cancer's diagnosis and treatment.MethodFour hundred eighteen paraffin-embedded non-small cell lung cancer (NSCLC) samples were obtained from patients who had been diagnosed with NSCLC between February 1994 and December 1997 at the GuangDong lung cancer research institute. The tissue microarray (TMA) was constructed after slides evaluation by the experienced pathologist. All of the patients had never received chemotherapy, radiotherapy and any other anti-tumour treatment. All of patients have complete clinicopathologic data. The protein expression level of SHH, IHH, DHH, SMO and GLI3 in lung cancer tissue were detected by immunohistochemistry staining. According to the positive percentage and intensity of staining, the degree of immunostaining of TMAs were scored. The staining intensity was scored as follows: 0 (no staining),1 (weak staining, light yellow),2 (moderate staining, yellowish brown), and 3 (strong staining, brown). The percentage of positively stained cells was scored as follows:0 (0-5%); 1 (5%-25%); 2 (26%-50%); 3 (51%-100%), both scores were multiplied; An optimal cutoff value was identified:<6 as low expression;≥6 as high expressionAll statistical analyses were performed using the SPSS 13.0 statistical software package. The Chi-square test, Fisher's exact test and Mann-Whitney U test were used to assess the relation between the proteins expression and clinicopathologic parameters. Overall survival curves between subgroups divided according to the proteins expression levels were drawn by using the Kaplan-Meier method, and significant differences among subgroups were compared by using the log-rank test. Multivariate analyses were performed using Cox proportional hazards model to identify independent prognostic factors. Correlations between GLI3 expression and upstream ligands, SHH, DHH, IHH, were analysed by using the partial correlation test. Two related sample Wilcoxon non-paramentric test were used to compare the protein expression level between 16 cases of paraffin-embeded tissue sections of NSCLC and paired adjacent non-neoplastic tissues.α=0.05 (two-sided) as the difference level, and P＜0.05 was considered indicative of statistical significance.Result1. SHH high expression is associated with well differentiation lung cancerSHH is expressed mainly in tumour cells, and subcellular location is at the cytoplasm. Among 343 evaluable tissues, expression of SHH is high in 87.2% (299/343) of cases, and is low in 12.8%(44/343) of cases. SHH expression level is not associated with age, gender, pathologic type, TNM stage, T stage, distance metastasis, lymph node metastasis, but the highe expression of SHH is significantly associated with well differentiation (nonparametric Mann-Whitney U test, P=0.044). Further cohort analysis in NSCLC pathologic subtypes show that SHH expression is not associated with any clinicopathologic parameters in adenocarcinoma, but highe expression is significantly associated with well differentication (nonparametric Mann-Whitney U test, P=0.005) in squamouns cell carcinoma.2. DHH expression is not associated with any clinicopathologic parameterDHH is expressed mainly in tumour cells of NSCLC tissues, and subcellular location is at the cytoplasm. Among 316 evaluable tissues, expression of DHH is high in 81.3%(257/316) of cases, is low in 18.7%(59/316) of cases. DHH expression level is not associated with any clinicopathologic parameters including age, gender, pathologic type, TNM stage, T stage, distance metastasis, lymph node metastasis and differentiation. Further cohort analysis in NSCLC pathologic subtype shows that DHH expression is still not associated with any clinicopathologic parameter in adenocarcinoma and squamous cell carcinoma.3. Stroma IHH higher expression is associated with higher stageIHH is expressed both in tumour epithelial cells and stroma cells of NSCLC tissues, and subcellular location is at cellular membrane and cytoplasm. Among 343 evaluable cases in tumor epithelial cells, expression of IHH is high in 51.9% (178/343) of cases, and is low in 48.1%(165/343) of cases. Among 326 evaluable cases in stroma cells, expression of IHH is high in 76.7%(250/326) of cases, and low in 23.3%(76/326) of cases. Expression level in epithelial cells is not associated with any clinicopathologic parameter. But the high expression of IHH in stroma cells is significantly associated with higher age (chi-squared test, P=0.046), TNM stage (chi-squared test, P=0.036) and T stage (chi-squared test, P=0.020). Further cohort analysis in NSCLC pathologic subtypes show that IHH expression at tumor epithelial cells is not associated with any clinicopathologic parameters in adenocarcinoma and squamous cell carcinoma; but high expression of IHH at stroma cells is significantly associated with higher age (chi-squared test, P=0.021) in adenocarcinoma; and with higher T stage (chi-squared test, P=0.011) in squamous cell carcinoma.4. High expression of SMO is associated with well differentiation squamous cell carcinomaSMO is expressed mainly in tumour cells of NSCLC tissues, and subcellular location is at cellular membrane and cytoplasm. Among 372 evaluable SMO expression tissues, expression of SMO is high in 87.2%(326/374) of cases, and is low in 12.8%(48/374) of cases. SMO expression level is not associated with age, gender, pathologic type, TNM stage, T stage, distance metastasis, lymph node metastasis, but the high expression of SMO is significantly associated with well differentiation (nonparametric Mann-Whitney U test, P=0.049). Further cohort analysis in NSCLC pathologic subtype shows that SMO expression is not associated with any clinicopathologic parameters in adenocarcinoma, but high expression is significantly associated with well defferentiation (nonparametric Mann-Whitney U test, P=0.034) in squamouns cell carcinoma.5. Higher expression of GLI3 is associated with lymph node metastasisGLI3 is expressed mainly in tumour cells of NSCLC tissues, and subcellular location is at the cytoplasm and cell nuclus. Among 365 evaluable GLI3 tissues, expression of GLI3 is high in 44.7%(163/365) of cases, and is low in 55.3% (202/365) of cases. GLI3 expression level is not associated with age, gender, pathologic type, TNM stage, T stage, distance metastases and differentiation, but the higher expression of IHH is significantly associated with lymph node metastases (chi-squared test, P=0.018). Further cohort analysis in NSCLC pathologic subtype shows that GLI3 expression is not associated with age, gender, TNM stage, T stage and distance metastases, but higher expression is significantly associated with lymph node metastases (chi-squared test, P=0.036) and differentiation (nonparametric Mann-Whitney test, P=0.021) in adenocarcinoma. GLI3 expression is not associated with any clinicopathologic parameters in squamous cell carcinoma.6. Expression of GLI3 is correlated with tumor epithelial IHH and SMOThe partial correlation analysis shows that GLI3 expression is positively correlated with expression of tumor epithelial IHH (r=0.216, P=0.017) and SMO (r=0.316, P=0.000), while expression of tumor epithelial IHH is correlated with expression of stroma IHH (r=0.187,P=0.038)in adenocarcinoma. GLI3 expression is positively correlated with expression of DHH (r=0.283, P=0.009), SMO (r=0.228, P=0.037) and tumor epithelial IHH (r=0.259, P=0.017), but tumor epithelial IHH expression is not correlated with stroma IHH expression (r=0.188, P=0.087) in squamous cell carcinoma.7. GLI3 expression in cancer tissues is significantly higher than in adjacent normal tissuesIn order to further explore the difference of expression of transcription factor GLI3 between cancer tissues and adjacent normal tissues, we tested the expression level of GLI3 in 16 pair lung cancer tissue and adjacent normal tissues, and using two related samples Wilcoxon test analyzed their primary scores. Rank mark is decided by "expression level of cancer tissue-expression level of adjacent normal tissue". There are 2 cases of negative ranks,11 cases of positive ranks and 3 cases of ties. The mean rank of negative ranks is 3.00, and the mean rank of positive ranks is 7.73. The domination of positive ranks demonstrated that GLI3 expression in cancer tissues is significantly higher than in adjacent normal tissues, the difference is statistic significance (paired nonparametric Wilcoxon test, P=0.005).8. The median overall survival of the GLI3 low expression group is significantly higher than the high expression groupThe Kaplan-Meier univariate survival analyses show that the expression level of SHH, DHH and SMO is not associated with overall survival (OS) of NSCLC patients. The further cohort analyses dose not show any association between their expression levels and overall survival of patients with adenocarcinoma or squamous cell carcinoma. But GLI3 expression is significantly associated with survival, the median OS of GLI3 low expression patients and GLI3 high expression patients are 37.2mon and 27.9mon respectively, demonstrating a significantly poor prognosis for GLI3 high expression(Log-rank test, P=0.021). Because GLI3 is one of the transcription factors of HH signaling pathway, we analyzed the association between nuclear expression level of GLI3 and OS alone. The result shows that the median OS of GLI3 low and high nuclear expression patients are 33.9mon and 28.4mon respectively, demonstrating a significantly poor prognosis for GLI3 high nuclear expression patients (Log-rank test, P=0.049). The further analyses in adenocarcinoma and squamous cell carcinoma, two of the most frequent pathologic subtypes of NSCLC, indicate that the effect of GLI3 overexpression to prognosis is mainly displaied in adenocarcinoma. In adenocarcinoma, the median OS of GLI3 low expression patients is 41.7mon, while the median OS of GLI3 high expression patients is only 25.6mon, demonstrating a significantly poor prognosis for high expression of GLI3 in adenocarcinma (Log-rank test, P=0.013). And the median OS of GLI3 low and high nuclear expression patients are 40.8mon and 26.2mon respectively, demonstrating a significantly poor prognosis for high nuclear expression of GLI3 in adenocarcinoma (Log-rank test, P=0.016). Furthermore, stroma expression of IHH in adenocarcinoma is also significantly associated with survival. The median OS of Stroma IHH low and high expression patients are 50.8mon and 27.9mon respectively, demonstrating a significantly poor prognosis for high expression of stroma IHH in adenocarcinoma (Log-rank test, P=0.021). There are not any association between HH signaling pathway moleculars and survival in squamous cell carcinoma.The further COX regression analysis brought into variates including sex, age, differentiation, distance metastases, lymph node metastases, T stage, expression level of IHH and GLI3. The result show that nuclear expression level of GLI3 is the independent and significant factors to predict a poor prognosis for lung adenocarcinoma (HR=1.640,95% CI=1.100-2.433, P=0.015).Conclusion1. The moleculars of HH signaling pathway, SHH, DHH, IHH, SMO and GLI3, is widely expressed in NSCLC tissues.2. GLI3 expression in cancer tissues is significantly higher than in adjacent normal tissues, and it is associated with lymph node metastasis. Therefore the overexpression of GLI3 may play a role of promoting lung cancer development.3. The patients of adenocarcinoma with high expression of GLI3 have a poor prognosis. Nuclear expression level of GLI3 is the independent and significant factors to predict a poor prognosis for lung adenocarcinoma.4. The HH signaling pathway may have different transduction mechanisms in lung squamous cell carcinoma and adenocarcinoma. In adenocarcinima, the pathway may be regulated by paracrine mechanism, while in squamous cell carcinoma, it may be regulated by autocrine.