Role Of STAT3 In Wilms Tumor Cancer Stem Cell Progression And Apoptosis

Objective: Through investigate Immunohistochemistry expression level of p-AKT, GSK-3β,Snail in Wilms tumor and their correlation with tumor progression and metastasis,and study the effect of STAT3 suppression in the Wilms tumor cell line proliferation and apoptosis, which might provide a useful theory basis and therapy target for treatment of Wilms’ tumor.Methods: Our research includes three parts. In the first part Immunohistochemistry was used to investigate the correlation between the expression of p-AKT, GSK-3β, Snail in Wilms tumor and tumor clinicopathological factors. In the second part, MACS was used to isolate CD133+ cell population,;cell sphere forming assay, colony-formation assay, wound healing assay,RT-PCR, and nude mice xenograft model were used to prove that CD133+ cell population have properties similar to cancer stem cells. In the third part, STAT3 inhibitor Stattic was used to prevented the phosphorylation of STAT3, furthermore, MTT assay was used to explore the inhibitory proliferation effect of Stattic at different drug concentrations(0 μM,0.625 μM,1.25μM,2.5 μM,5 μM), the Annexin-V-FITC/PI double-staining assay was used to explore the apoptosis rate of Stattic, nude mice xenograft model injected with Stattic was used to probe the inhibitory effect of Stattic to nude mice tumors. Westernblot was used to prove the the effect of Stattic to STAT3 Downstream related proteins Bcl-2 and Bcl-xl.Results:1. Immunohistochemistry of p-AKT, GSK-3β and Snail showed that AKT expression was associated with Wilms tumor Stage and metastasis(p=0.029,p=0.005), Snail expression was associated with Wilms tumor Stage, metastasis, and relapse(p=0.007, p=0.001, p=0.042), however,GSK-3β was not significantly associated with any clinicopathological factors(p > 0.05); the prevalence of p-AKT expression was positively associated with the prevalence of Snail expression(p=0.039), but there was no significant correlation between p-AKT and GSK-3β, GSK-3β and Snail immunoreactivity in WT specimens(p>0.05). p-AKT and Snail positive Wilms tumor patients had significantly shorter disease-free survival(DFS) and overall survival(OS) compared with p-AKT and Snail negative tumors(p<0.05), while GSK-3β was not significantly associatedwith either DFS or OS(p>0.05).2. MACS sorting assay showed that CD133+ cell population accounts for 7.38% in G401 cell line before sorting, and after sorting, CD133+ cell population accounts for 99.97%.3. Cell sphere forming assay showed that CD133+ cell population has stronger cell sphere forming ability than CD133- cell population and parental cells(p<0.05).4. Colony-formation result showed that CD133+ cell population formed more colonies than CD133- cell population and parental cells(p<0.05).5. Wound healing assay showed that CD133+ cell population has stronger migration ability than CD133- cell population(p<0.05).6. RT-PCR assay revealed that CD133+ cell population has higher expression of SOX,NANOG, OCT4 than CD133- cell population(p<0.05).7. Nude mice xenograft model reavealed that nude mice injected CD133+ cell population formed larger tumor volume than CD133- cell population(p<0.05).8. MTT assay showed that the inhibition of CD133+ cell proliferation was increased with the extension of Stattic concentration increased, at the concentration of 0.625 μM and 1.25 μM Stattic group, the inhibitory rate was 25% and 46% at 24 h, respectively.9. Annexin V-FITC/PI assay showed that apoptosis of CD133+ cell population was significantly(p<0.05) inhibited by Stattic in a dose-dependent manner; as Stattic concentration increased, the apoptosis rate increased along.10. Nude mice xenograft model injected with Stattic showed that Stattic significantly inhibited the growth of CD133+ cell forming tumors.After 4 weeks injection of Stattic,CD133+/Stattic group formed smaller tumor volume than CD133+ control group(p < 0.05).Tumor HE staining results showed that CD133+/Stattic group appear obvious apoptosis phenomemon, such as chromatin condensation, karyopyknosis, and cellular structure disappearance, et al. While CD133+ control group showed nuclei hyperchromatic,karyokinesisis increasing, indicating cells are still proliferated actively.11. Westernblot assay showed that p-STAT3 expression decreased significantly after Stattic treatment, as well as Bcl-2 and Bcl-xl expression.Conclusion:1. AKT expression was associated with Wilms tumor Stage and metastasis,Snail expression was associated with Wilms tumor Stage, metastasis, and relapse, however. p-AKT and Snail positive Wilms tumor patients had significantly shorter disease-free survival(DFS) and overall survival(OS).2. CD133+ cells has cancer stem cell character in Wilms tumor G401 cell line, indicatingCD133 might be a cancer stem cell biomarker in Wilms tumor.3. By targeting STAT3 signaling pathway, we can effectively inhibit CD133+ cells proliferation, accelerate its apoptosis.