Regulatory Mechanism Of Tcf7l1 In The Self-renewal Of Liver Cancer Stem Cells

Hepatocellular carcinoma(HCC)is one of the most common malignant tumors worldwide,while nearly half of the new cases and the cancer deaths worldwide are estimated to occur in China.In addition to the prevalent incidence,the 5-year relative survival rate of HCC is as low as 15%.Research on the initiation and development of HCC will provide theoretical basis for the early diagnosis,the effective targets,and the development of new therapeutic strategies.Cancer stem cells(CSCs)are a population of cells within tumors capable of cancer initiation,multi-directional differentiation,radiotherapy and chemotherapy resistance,immune tolerance as well as high invasion and metastasis,therefore been believed to be responsible for tumor initiation,development,relapse and metastasis.Understanding the maintenance mechanisms of CSCs could allow the development of effective therapeutics for cancers including HCC.However,eventhough several liver CSCs markers have been identified,including CD133,EpCAM,CD13,CD90,CD24 and Nanog,the complex mechanism of self-renewal of liver CSCs is elusive.The Wnt/β-catenin pathway plays major roles in a wide range of developmental processes and cancer progression.Tcf7l1,which is a key effector molecule of the Wnt/β-catenin signaling pathway,regulates the self-renewal of embryonic stem cells(ESCs)and adult stem cells as well as promotes tumor growth in several types of malignant tumors.Nevertheless,the expression and role of Tcf7l1 in HCC remains largely unknown.Our previous study found that miRNA-449 a regulates the self-renewal of liver CSCs through translational repression of Tcf7l1,however,the detailed mechanism and signaling pathway associated with Tcf7l1 in liver CSCs remain unelucidated.Therefore,in this study,we investigated the role of Tcf7l1 in HCC and the underlying mechanisms.The main results of this study are as follows: 1.Tcf7l1 expression is negatively correlated with clinicopathological parameters and positively correlated with patient outcomes.Immunohistochemistry for 90 cases of HCC specimens showed that the expression levels of Tcf7l1 in the carcinoma tissues were significantly less than the adjacent normal tissue.In addition,Tcf7l1 expression level was negatively correlated with pathological grade and tumor stage.Kaplan-Meier survival analysis showed that patients with low expression levels of Tcf7l1 in tumors have a significantly poorer prognosis than patients with high expression levels of Tcf7l1 in tumors.These data showed that Tcf7l1 is associated with HCC progression and may play an important role in HCC.2.Tcf7l1 acts as a repressor in controlling the self-renewal of liver CSCsBy comparing Tcf7l1 expression in liver CSCs and non-CSCs,we found that the expression level of Tcf7l1 was significantly lower in liver CSCs than in non-CSCs.Furthermore,the Tcf7l1 expression gradually increased during liver CSCs differentiation in vitro,which implies a negative correlation between Tcf7l1 expression and the stemness of liver CSCs.Functionally,Tcf7l1 overexpression resulted in significant downregulation of stemness genes and upregulation of the differentiation genes.Overexpression of Tcf7l1 also reduced the colony formation and sphere formation,increased the sensitivity to the therapeutic agent in vitro,implying that Tcf711 suppressed the stemness of liver CSCs.In vivo,overexpression of Tcf711 impaired liver CSC tumor initiation and growth,which is in accordance with in vitro assays to support that Tcf711 is associated with differentiation of liver CSCs.3.Tcf7l1 represses the self-renewal of liver CSCs through transcriptional inhibition of NanogBy analyzing Tcf7l1 and Nanog expression in HCC tissues,we found that the expression of Nanog and Tcf7l1 was negatively correlated.ChIP assays and luciferase reporter assay confirmed that Tcf7l1 regulates Nanog transcription.Knockdown of Tcf7l1 significantly increased the colony and sphere formation of liver non-CSCs in vitro.Importantly,these effects were completely abolished by simultaneously knocking down of Nanog,which demonstrated that Tcf7l1 plays an important role in regulating the stemness of liver CSCs through transcriptional inhibition of Nanog.To ask whether Tcf7l1 mediated stemness of liver CSCs through β-catenin-dependent pathway,we constructed a lentiviral vector expressing β-catenin binding-deficient Tcf7l1(△NTcf7l1).The results showed that both full-length Tcf7l1 and △NTcf7l1 overexpression significantly repressed the expression of Nanog.Functionally,△NTcf7l1 overexpression also significantly reduced the colony and sphere formation abilities.Collectively,our data indicated that Tcf7l1-suppressed self-renewal of liver CSCs independent of β-catenin.4.Tcf7l1 is negatively regulated by extrinsic IGF signaling in HCC and participates in IGF signaling mediated self-renewal of liver CSCs.EGF,bFGF,IGF2 and IL6,which have been reported to participate in modulating the self-renewal of liver CSCs,were analyzed to investigate the involvement of Tcf7l1 in extracellular factor-induced self-renewal.We found that Tcf7l1 expression was regulated by IGF signaling.Functionally,IGF signaling inhibition to reduce liver CSCs sphere formation was partially recovered after simultaneously knocking down of Tcf7l1,implying that Tcf7l1 is an important downstream of IGF signaling in regulating the self-renewal of liver CSCs.5.IGF signaling regulates Tcf7l1 phosphorylation and protein degradation through MEK/ERK pathwayBy analyzing Tcf7l1 mRNA and protein levels after IGF signaling stimulation,we found that IGF signaling regulates protein degradation of Tcf7l1.Mechanistically,IGF signaling stimulates the phosphorylation and ubiquitylation of Tcf7l1 through the MEK/ERK pathway.The results demonstrated for the first time that IGF MEK/ERK signaling regulates Tcf7l1 degradation.6.Regulation of Tcf7l1 by IGF signaling is independent of β-cateninTo elucidate whether Tcf7l1 degradation involves β-catenin in HCC,we knocked out β-catenin by CRISPR/Cas9 technology and then sorted and verified the clones.β-catenin knockout did not affect IGF signaling-mediated ubiquitination and expression of Tcf7l1,which suggested that IGF-mediated Tcf7l1 protein degradation is β-catenin-independent.In summary,we demonstrated that unlike the tumor-promoting effects reported in several other types of cancers,Tcf7l1 plays a suppressive role in tumor initiation and progression in HCC.Tcf7l1 is expressed at low levels in HCC,and its expression is negatively correlated with a poor prognosis.Tcf7l1 attenuates the self-renewal of liver CSCs through transcriptional repression of the Nanog gene.Importantly,we found that Tcf7l1 responded to environmental IGF2 and is regulated by MEK/ERK signaling.Mechanistically,IGF/MEK/ERK stimulates Tcf7l1 phosphorylation and ubiquitylation,thereby regulating its degradation.Moreover,IGF signaling that is involved in Tcf7l1-mediated self-renewal of liver CSCs is β-catenin-independent.In conclusion,our results provided new insights into how extracellular signals modulate the self-renewal of liver CSCs and highlighted the inhibitory role of Tcf7l1 in cancer.