The Function And Mechanism Of KIAA1199 Regulating Sorafenib Resistance In Hepatocellular Carcinoma

Background and Objective:In general,a late diagnosis,high risk of recurrence and metastasis,and tolerant to conventional chemotherapeutic agents are considered to the principle reasons for the high mortality rates of Hepatocellular carcinoma（HCC）.Sorafenib is the first and only targeted therapy for advanced HCC.However,the emergence of acquired sorafenib tolerance has prevented patients from benefiting from this drug.Up to now,the molecular mechanism of sorafenib tolerance is still poorly characterized.Cancer stem cells（CSCs）,a subset of cells within the tumor with the potential for self-renewal,differentiation and tumorigenicity,are thought to be the major cause of cancer therapy failure.Therefore,further investigated the relationship between sorafenib-resistant cells and CSCs,deeply analyzing the molecular mechanisms regulating sorafenib resistance and self-renewal of HCC CSCs are still required.Identifying the biomarkers involved sorafenib tolerance and CSCs self-renewal,and clarifying the regulation mechanisms are of great significance for targeting HCC drug resistance.To better identify the key gene involved in sorafenib tolerance and uncover potential targets for HCC therapy,the whole human genome microarray analysis was used to screen the differentially expressed genes in sorafenib-resistant HCC models and the corresponding counterparts.Our data showed that expression of KIAA1199 is significantly elevated in our established sorafenib-resistant HCC cells models in vitro and xenografts models in vivo.Although KIAA1199 is associated with aggressive tumor phenotype and poor survival in multiple tumor tissues,little is known about its functional role in sorafenib resistance and CSCs self-renewal.Based on our previous studies and literature review,we speculated that KIAA1199 was involved in the regulation of sorafenib resistance and the maintenance of CSCs self-renewal in HCC.Methods:1.Establishment of HCC sorafenib-resistant cell models in vitro and in vivo by long term exposed to sorafenib;2.The whole human genome microarray analysis was used to screen the differentially expressed genes in sorafenib-resistant models and the corresponding counterparts;3.Expression and location of protein in tissue and cells was detected by western blotting and immunofluorescence staining;4.m RNA expression in tissues and cells was detected by RT-PCR;5.The migration and invasion abilities of sorafenib-resistant HCC cells and the corresponding counterparts in vitro were performed by transwells assay;6.The liver metastasis foci in spleen-liver metastasis model was detected by HE staining;7.The effect of KIAA1199 on HCC CSCs self-renewal in vitro was determined by cloning and sphere formation efficiency assays;8.The effect of KIAA1199 on HCC CSCs tumor-initiating capability was determined by NOD/SCID mice tumor formation in vivo.Results:Section 1 Establishment and characterization of sorafenib-resistant HCC cell and xenograft modelsAt first,we established sorafenib-resistant HCC cell and xenograft models.Compared with the corresponding counterparts,our data showed that sorafenib-resistant HCC cells possess slow growth characteristic,and the STR analyses revealed that the two sorafenib-resistant cell lines matched perfectly with the original parental HCC cells.Furthermore,we also found that sorafenib-resistant HCC cells and xenograft models both were more resistant to sorafenib treatment.More importantly,the data also revealed that sorafenib-resistant HCC models possess higher self-renewal ability,and upregulation of several known HCC CSCs markers.Altogether,our data demonstrated the successful establishment of sorafenib-resistant HCC models suitable for further investigation of sorafenib resistance.Section 2 KIAA1199 is highly expressed in sorafenib-resistant HCC cell and xenograft modelsThe whole human genome microarray analysis was used to screen the differentially expressed genes in sorafenib-resistant HCC model and the corresponding counterparts.For the first time,we revealed that KIAA1199 was the most significantly elevated target in sorafenib-resistant HCC cells and xenograft models.Subsequently,KIAA1199 expression was verified by western blotting and RT-PCR.In addition,we also found KIAA1199 expression was obviously higher in sorafenib-resistant HCC patients than sorafenib-sensitive HCC patients（P<0.05）.Section 3 Expression of KIAA1199 in clinical HCC samples and its clinical significanceTo further unravel the role of KIAA1199 in HCC,we investigated the KIAA1199 expression in primary HCC tumor tissues and the corresponding adjacent noncancerous tissues.The data showed that KIAA1199 protein expression was obviously higher in HCC tumor tissues than in matched tumor adjacent noncancerous tissues（9/12,75%）.Furthermore,our data also indicated that KIAA1199 m RNA expression was 2.41-fold higher on average in primary HCC tumor tissues than in matched adjacent tissues（n?=?45）.In addition,we further analyzed the correlation between KIAA1199 expression and the clinicopathological features of HCC.The data showed that enhanced KIAA1199 expression was positively correlated with metastatic probability（P=0.042）and TNM stage（P=0.002）.Section 4 The function and mechanism of KIAA1199 regulating sorafenib resistance in hepatocellular carcinomaThe endogenous KIAA1199 expression in HCC cells was positively correlated with migration ability.Furthermore,the in vitro and in vivo experiments revealed that up-regulation of KIAA1199promotes sorafenib-resistant HCC cell migration,invasion,and metastasis.At the meantime,the invasiveness and metastatic ability of sorafenib-resistant HCC cells were evidently impaired on KIAA1199 depletion.More importantly,migration potential was restored by rescuing KIAA1199expression via the exp-KIAA1199 lentivirus in KIAA1199-depleted sorafenib-resistant HCC cells.Moreover,the enhanced migratory and invasive potentials of sorafenib-resistant cells correlated with EMT phenotypic changes,as indicated by increased mesenchymal markers（Vimentin and Fibronectin）and decreased epithelial markers（E-cadherin,ZO-1 and Occludin）.In addition,we also found that exogenous EGF could significantly induce KIAA1199 expression after treated for 12 h,suggesting that KIAA1199 is an early response factor to EGF-induced EMT.Our data further revealed that overexpression of KIAA1199 promoted,whereas downregulation of KIAA1199expression impaired,EGFR phosphorylation,EGF-dependent STAT3 and ERK1/2 phosphorylation.Moreover,disrupted EGFR expression was successfully restored by rescuing KIAA1199 expression in sorafenib-resistant HCC cells,suggesting that KIAA1199-mediated EGF/EGFR cascade signaling activation regulates sorafenib-resistant HCC cell invasion and metastasis.Section 5 The function and mechanism of KIAA1199 regulating HCC CSCs self-renewal and tumor-initiatingKIAA1199 was significantly upregulated in Nanog^Pos cells,CD24^Pos cells,CD13^Pos cells and CD133^Pos cells compared with the corresponding negative cells,suggesting that overexpression of KIAA1199 is closely related to HCC CSCs.In both Nanog promoter-driven Nanog/GFP and CRISPR/Cas9/Nanog-GFP endogenous Nanog-labeled HCC CSCs models,knock-down of KIAA1199 expression could obviously reduce the cell proliferation rate of Nanog^Pos CSCs,and enhance their sensitivity to sorafenib treatment.In addition,our data also showed that knock-down of KIAA1199 expression could significantly impaired the colony-formation,sphere-formation capabilities of Nanog^Pos CSCs in vitro and the tumor initiating abilities in vivo.Furthermore,our data also demonstrated that upregulation of KIAA1199 promoted proliferation,self-renewal capabilities and the tumor-initiating abilities of Nanog^Negnon-CSCs.Next,we confirm KIAA1199 whether could increase the Nanog promoter activity.We chose different lengths of luciferase report lentivirus（named P-Nanog-500/1000/1500）,which carries Nanog promoter from TSS to-500/-1000/-1500 bp upstream,respectively.Our results showed that KIAA1199 could increase Nanog promoter activities,especially in-1000～-1500 bp regions.Furthermore,we found that overexpression of KIAA1199activate Wnt signaling pathway,subsequently lead to up-regulating the expression ofβ-catenin,maintaining the high activity of Nanog promoter and promoting its expression.Altogether,KIAA1199 participate in the regulation of self-renewal of HCC CSCs and promoting the progression of HCC.Conclusion:Our study suggests that KIAA1199 is a novel potential candidate drug-tolerant accelerant in HCC cell sorafenib resistance,tumor metastasis and self-renewal.This study enriches the understanding of important molecules involved in drug resistance and metastasis of HCC,and provides a theoretical basis for finding novel therapeutic targets specifically targeting HCC CSCs.