The Study On The Expression Of HIF-1α And HIF-2α In Small Cell Lung Cancer Stem Cells And Tissues

Objective1. To study the expression and significance of HIFs in small cell lung cancer stem cells.2. To study the expression of HIFs in small cell lung cancer tissues, and analyze their clinicopathologic significance.Methods1. With the defined serum-free culture, we enriched the third passage tumor spheres cells from H446as the tumor stem cells.2. Real-time PCR was performed to determine the mRNA expression level of HIF-1α and HIF-2a in tumor spheres cells and parental cells from H446.3. We further determine the mRNA expression level of OCT-4, the target gene of HIF-2a, and other stem cell factor SOX2、c-Myc and NONOG.4. Immunofluorescence staining was performed to determine the protein expression level of HIF-1α、HIF-2a and OCT-4in tumor spheres cells and parental cells.5. Immunohistochemistry was used to detect the expression of HIF-1α and HIF-2α in small cell lung cancer tissues, and analyze their clinicopathologic significance.6. Spearman correlation analysis was performed to analyze the correlation of HIFs with the small cell lung cancer stem cell markers uPAR.7. Using Kaplan-Meier method to perform univariate analysis. The variables that were found to be significant in univariate analysis were put into a Cox proportional hazards model for multivariate analysis.Results1. The expression of HIF-la and HIF-2a in SCLC stem cells(1) Previous work from our laboratory showed that the third passage tumor spheres cells from H446were enriched with stem cells, here, we take the third passage tumor spheres as the tumor stem cells. Real-time PCR results showed the mRNA expression level of HIF-2a was up-regulated in tumor spheres cells compared to parental cells (P＜0.05); however, the mRNA expression level of HIF-la was down-regulated in tumor spheres cells (P <0.05), indicating the mRNA expression level of HIF-la and HIF-2a showied a complementary relationship.(2) We further detect the expression of stem cell factor that may be regulated by HIF-2α. The results showed that OCT-4mRNA was significantly upregulated in SCLC stem cells (P<0.05), but the mRNA expression level of SOX2, c-Myc and NONOG was not statistically significant in tumor spheres cells and parental cells.(3) Immunofluorescence staining showed HIF-2a was positive expressed in tumor spheres cells, but the staining was weak or even loss in parental cells. In contrast, HIF-la was very weak or even missing in tumor spheres cells, but it was positive expressed in parental cells.2. The expression and clinical significance of HIF-la and HIF-2a in SCLC tissues.(1) Expression of HIF-1α was detected in22/45(48.9%) of SCLC by immunohistochemistry, it was negative in normal tissue adjacent cnacer; HIF-la was mainly expressed in nuclear of cancer cells, HIF-la-positive cells were diffuse distribution in the nests of the tumor; HIF-2a were expressed in11/45(24.4%) of SCLC patients, it was negative in normal tissue adjacent cnacer; HIF-2a staining was mainly present in the nuclear of cancer cells, HIF-2a-positive cells were frequently distributed around necrotic regions. In tumor stromal cells, a subset of cells morphologically identified macrophages were frequently positive for HIF-2a.(2) Spearman correlation analysis of HIF-la/HIF-2a and the small cell lung cancer stem cell markers uPAR showed that there was no correlation between HIF-1α and uPAR expression (P=0.183). Interestingly, we found a significantly positive correlation between HIF-2a and uPAR expression (P=0.001). Serial sections staining for HIF-2a and uPAR showed uPAR and HIF-2a co-localized around necrotic regions to some degree, but they do not overlap exclusively.(3) The expression of HIF-2α was closely related to tumor size and distant metastasis:HIF-2a expression increased significantly with the development of tumor size (P<0.05), and the distant metastasis rate was higer in HIF-2a-posive group than HIF-2α-negative group (P＜0.05). In contrast, the expression of HIF-1α has no relationship with age, sexy, tumor size, lymph metastasis, pleural invasion and distant metastasis (P＞0.05).(4) Univariate survival analysis revealed that HIF-la, HIF-2α, age and distant metastasis were correlated with poor overall survival. However, in multivariate analysis, only HIF-2α expression and distant metastasis were the independent prognostic indicators (P=0.004,0.018, respectively).Conclusions1. HIF-1α and HIF-2α were positive expressed in tumor stem cells from H446, but the expression levels of HIF-2α were up-regulated, the expression levels of HIF-la were down-regulated instead, suggesting they showed a complementary relationship.2. HIF-1α and HIF-2α were positive expressed in SCLC tissues, but the positive rate of HIF-2α was lower than HIF-1α, they were negative expressed in normal tissue adjacent cnacer.3. The expression of HIF-2α may be related to SCLC stem cells.4. The expression of HIF-2α had close relationship with tumor size and distant metastasis, it can be used as the independent prognostic indicators of small cell lung cancer.In summary, HIF-2α was up-regulated in SCLC stem cells. The expression of HIF-2a may represent a more aggressive phenotype in SCLC.