ZIC2-dependent OCT4 activation drives self-renewal of human liver cancer stem cells.
ABSTRACT: Liver cancer stem cells (CSCs) have been identified and shown to have self-renewal and differentiation properties; however, the biology of these hepatic CSCs remains largely unknown. Here, we analyzed transcriptome gene expression profiles of liver CSCs and non-CSCs from hepatocellular carcinoma (HCC) cells lines and found that the transcription factor (TF) ZIC2 is highly expressed in liver CSCs. ZIC2 was required for the self-renewal maintenance of liver CSCs, as ZIC2 depletion reduced sphere formation and xenograft tumor growth in mice. We determined that ZIC2 acts upstream of the TF OCT4 and that ZIC2 recruits the nuclear remodeling factor (NURF) complex to the OCT4 promoter, thereby initiating OCT4 activation. In HCC patients, expression levels of the NURF complex were consistent with clinical severity and prognosis. Moreover, ZIC2 and OCT4 levels positively correlated to the clinicopathological stages of HCC patients. Altogether, our results indicate that levels of ZIC2, OCT4, and the NURF complex can be detected and used for diagnosis and prognosis prediction of HCC patients. Moreover, these factors may be potential therapeutic targets for eradicating liver CSCs.
Project description:Background: Accumulating evidence has revealed the importance of cancer stem cells (CSCs) in self-renewal and chemoresistance. Previous studies reported high expression of ZIC2 was closely associated with tumorigenesis and CSC traits. However, the role of ZIC2 as a crucial factor for regulating CSC properties in lung adenocarcinoma (LAC) remains elusive. Methods: RT-PCR and WB assay were employed to assess ZIC2 expression in 20 LAC tumor tissues and the matched non-cancerous tissues. The role of ZIC2 in LAC CSC were analyzed by evaluation of CSC-related markers expression and spheroid formation in vitro. Cisplatin and paclitaxel resistance capacities were evaluated by CCK8 assay, colony formation assay, and flow cytometry analysis. Subcutaneous NOD/SCID mice models were generated to assess in vivo CSC features. Results: High expression of ZIC2 was found in LAC tumor tissues and indicated a poor overall survival in LAC patients. ZIC2 upregulated an array of CSCs-related genes, including EpCAM, OCT4, SOX2, NANOG, C-Myc and Bmi-1. Knockdown of ZIC2 inhibited sphere-forming capacity and decreased cisplatin and paclitaxel resistance. However, overexpression of ZIC2 achieved opposite effects. Mechanically, ZIC2 acts upstream of OCT4 to promote its expression, resulting in enhancement of CSC traits in LAC. Conclusion: Our results demonstrated that ZIC2 was crucial for promoting CSC traits in LAC cells, and served as a potential biomarker for predicting prognosis. The ZIC2-OCT4 network will facilitate the evaluation of the potential therapeutic efficacy of chemotherapy and predict patient sensitivity to treatment.
Project description:Hepatocellular carcinoma (HCC) is the most prevalent liver cancer, characterized by a high rate of recurrence and heterogeneity. Liver cancer stem cells (CSCs) may well contribute to both of these pathological properties, but the mechanism underlying their self-renewal maintenance is poorly understood. Here, we identified a long noncoding RNA (lncRNA) termed HAND2-AS1 that is highly expressed in liver CSCs. Human HAND2-AS1 and its mouse ortholog lncHand2 display a high level of conservation. HAND2-AS1 is required for the self-renewal maintenance of liver CSCs to initiate HCC development. Mechanistically, HAND2-AS1 recruits the INO80 chromatin-remodeling complex to the promoter of BMPR1A, thereby inducing its expression and leading to the activation of BMP signaling. Importantly, interfering with expression of HAND2-AS1 by antisense oligonucleotides (ASOs) and BMPR1A by siRNAs has synergistic anti-tumorigenic effects on humanized HCC models. Moreover, knockout of lncHand2 or Bmpr1a in mouse hepatocytes impairs BMP signaling and suppresses the initiation of liver cancer. Our findings reveal that HAND2-AS1 promotes the self-renewal of liver CSCs and drives liver oncogenesis, offering a potential new target for HCC therapy.
Project description:BACKGROUND:Liver cancer stem cells (CSCs) are critical determinants of HCC relapse and therapeutic resistance, but the mechanisms underlying the maintenance of CSCs are poorly understood. We aimed to explore the role of tumor repressor Zinc-fingers and homeoboxes 2 (ZHX2) in liver CSCs. METHODS:CD133+ or EPCAM+ stem-like liver cancer cells were sorted from tumor tissues of HCC patients and HCC cell lines by flow cytometry. In addition, sorafenib-resistant cells, tumor-sphere forming cells and side population (SP) cells were respectively cultured and isolated as hepatic CSCs. The tumor-initiating and chemoresistance properties of ZHX2-overexpressing and ZHX2-knockdown cells were analyzed in vivo and in vitro. Microarray, luciferase reporter assay, chromatin immunoprecipitation (ChIP) and ChIP-on-chip analyses were performed to explore ZHX2 target genes. The expression of ZHX2 and its target gene were determined by quantitative RT-PCR, western blot, immunofluorescence and immunohistochemical staining in hepatoma cells and tumor and adjacent tissues from HCC patients. RESULTS:ZHX2 expression was significantly reduced in liver CSCs from different origins. ZHX2 deficiency led to enhanced liver tumor progression and expansion of CSC populations in vitro and in vivo. Re-expression of ZHX2 restricted capabilities of hepatic CSCs in supporting tumor initiation, self-renewal and sorafenib-resistance. Mechanically, ZHX2 suppressed liver CSCs via inhibiting KDM2A-mediated demethylation of histone H3 lysine 36 (H3K36) at the promoter regions of stemness-associated transcription factors, such as NANOG, SOX4 and OCT4. Moreover, patients with lower expression of ZHX2 and higher expression of KDM2A in tumor tissues showed significantly poorer survival. CONCLUSION:ZHX2 counteracts stem cell traits through transcriptionally repressing KDM2A in HCC. Our data will aid in a better understanding of molecular mechanisms underlying HCC relapse and drug resistance.
Project description:Liver cancer stem cells (CSCs) harbour self-renewal and differentiation properties, accounting for chemotherapy resistance and recurrence. However, the molecular mechanisms to sustain liver CSCs remain largely unknown. In this study, based on analysis of several hepatocellular carcinoma (HCC) transcriptome datasets and our experimental data, we find that C8orf4 is weakly expressed in HCC tumours and liver CSCs. C8orf4 attenuates the self-renewal capacity of liver CSCs and tumour propagation. We show that NOTCH2 is activated in liver CSCs. C8orf4 is located in the cytoplasm of HCC tumour cells and associates with the NOTCH2 intracellular domain, which impedes the nuclear translocation of N2ICD. C8orf4 deletion causes the nuclear translocation of N2ICD that triggers the NOTCH2 signalling, which sustains the stemness of liver CSCs. Finally, NOTCH2 activation levels are consistent with clinical severity and prognosis of HCC patients. Altogether, C8orf4 negatively regulates the self-renewal of liver CSCs via suppression of NOTCH2 signalling.
Project description:Liver cancer stem cells (CSCs) may contribute to the high rate of recurrence and heterogeneity of hepatocellular carcinoma (HCC). However, the biology of hepatic CSCs remains largely undefined. Through analysis of transcriptome microarray data, we identify a long noncoding RNA (lncRNA) called lncBRM, which is highly expressed in liver CSCs and HCC tumours. LncBRM is required for the self-renewal maintenance of liver CSCs and tumour initiation. In liver CSCs, lncBRM associates with BRM to initiate the BRG1/BRM switch and the BRG1-embedded BAF complex triggers activation of YAP1 signalling. Moreover, expression levels of lncBRM together with YAP1 signalling targets are positively correlated with tumour severity of HCC patients. Therefore, lncBRM and YAP1 signalling may serve as biomarkers for diagnosis and potential drug targets for HCC.
Project description:Zinc finger protein X-linked (ZFX) is frequently upregulated in multiple human malignancies and also plays a critical role in the maintenance of self-renewal in embryonic stem cells. However, the role of ZFX in liver cancer stem cells (CSCs) remains obscure. We observed that the elevated expression of both ZFX and epithelial cell adhesion molecule (EpCAM) was associated with aggressive clinicopathological features and indicated poor prognosis in patients with hepatocellular carcinoma (HCC). ZFX was commonly enriched in liver EpCAM+ CSCs. Knockdown of ZFX decreased the proportion of EpCAM+ CSCs in HCC cells and suppressed their expression of stemness-related genes, self-renewal capacity, chemoresistance, metastatic potential, and tumorigenicity. Conversely, upregulation of ZFX in CSCs rescued these inhibitory effects and enhanced stem-like properties. Mechanistically, depletion of ZFX reduced nuclear translocation and transactivation of ?-catenin, thereby inhibiting the self-renewal capacity of EpCAM+ CSCs. Moreover, knockdown of ?-catenin attenuated the self-renewal of EpCAM+ HCC cells stably expressing ZFX, further indicating that ?-catenin is required for ZFX-mediated expansion and maintenance of EpCAM+ CSCs. Taken together, our findings indicate that ZFX activates and maintains EpCAM+ liver CSCs by promoting nuclear translocation and transactivation of ?-catenin. Furthermore, combination of ZFX and EpCAM may serve as a significant indicator for prognosis of patients with HCC.
Project description:Liver cancer stem cells contribute to tumorigenesis, progression, recurrence and drug resistance of hepatocellular carcinoma (HCC). However, the underlying mechanism for the propagation of liverCSCs is not fully understood yet. Here we show that miR-219 is upregulated in liver CSCs. Knockdown of miR-219 attenuates the self-renewal and tumorigenicity of liver CSCs. Conversely, miR-219 overexpressing enhances the self-renewal and tumorigenicity of liver CSCs.Mechanistically,miR-219 downregulates E-cadherin via itsmRNA 3'UTR in liver CSCs. The correlation between miR-219 and E-cadherin is validated in human HCC tissues. Furthermore, the miR-219 expression determines the responses of hepatoma cells to sorafenib treatment. Our findings indicate that miR-219 plays a critical role in liver CSCs expansion and sorafenib response, rendering miR-219 as an optimal target for the prevention and intervention of HCC.Abbreviations: HCC: Hepatocellular carcinoma; CSCs: cancer stem cells; DMEM: Dulbecco's modified Eagle's medium; FBS: fetal bovine serum; OS: overall survival.
Project description:Background:MicroRNAs (miRNAs) play a critical role in the regulation of cancer stem cells (CSCs). However, the role of miRNAs in liver CSCs has not been fully elucidated. Methods:Real-time PCR was used to detect the expression of miR-miR-28-5p in liver cancer stem cells (CSCs). The impact of miR-28-5p on liver CSC expansion was investigated both in vivo and in vitro. The correlation between miR-28-5p expression and sorafenib benefits in HCC was further evaluated in patient-derived xenografts (PDXs). Results:Our data showed that miR-28-5p was downregulated in sorted EpCAM- and CD24-positive liver CSCs. Biofunctional investigations revealed that knockdown miR-28-5p promoted liver CSC self-renewal and tumorigenesis. Consistently, miR-28-5p overexpression inhibited liver CSC's self-renewal and tumorigenesis. Mechanistically, we found that insulin-like growth factor-1 (IGF-1) was a direct target of miR-28-5p in liver CSCs, and the effects of miR-28-5p on liver CSC's self-renewal and tumorigenesis were dependent on IGF-1. The correlation between miR-28-5p and IGF-1 was confirmed in human HCC tissues. Furthermore, the miR-28-5p knockdown HCC cells were more sensitive to sorafenib treatment. Analysis of patient-derived xenografts (PDXs) further demonstrated that the miR-28-5p may predict sorafenib benefits in HCC patients. Conclusion:Our findings revealed the crucial role of the miR-28-5p in liver CSC expansion and sorafenib response, rendering miR-28-5p an optimal therapeutic target for HCC.
Project description:Hepatocellular carcinoma (HCC) is the third leading cause of cancer death. This high mortality has been commonly attributed to the presence of residual cancer stem cells (CSCs). Meanwhile, MEK1 signaling is regarded as a key molecular in HCC maintenance and development. However, nobody has figured out the particular mechanisms that how MEK1 signaling regulates liver CSCs self-renewal. In this study, we show that inhibition or depletion of MEK1 can significantly decrease liver CSCs self-renewal and tumor growth both in vitro and vivo conditions. Furthermore, we demonstrate that MEK1 signaling promotes liver CSCs self-renewal and tumorigenicity by maintaining SIRT1 level. Mechanistically, MEK1 signaling keeps SIRT1 protein stabilization through activating SIRT1 ubiquitination, which inhibits proteasomal degradation. Clinical analysis shows that patients co-expression of MEK1 and SIRT1 are associated with poor survival. Our finding indicates that MEK1-SIRT1 can act as a novel diagnostic biomarker and inhibition of MEK1 may be a viable therapeutic option for targeting liver CSCs treatment.
Project description:Developmental cell fate specification is a unidirectional process that can be reverted in response to injury or experimental reprogramming. Whether differentiation and de-differentiation trajectories intersect mechanistically is unclear. Here, we performed comparative screening in lineage-related mouse naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), and identified the constitutively expressed zinc finger transcription factor (TF) Zfp281 as a bidirectional regulator of cell state interconversion. We showed that subtle chromatin binding changes in differentiated cells translate into activation of the histone H3 lysine 9 (H3K9) methyltransferase Ehmt1 and stabilization of the zinc finger TF Zic2 at enhancers and promoters. Genetic gain-of-function and loss-of-function experiments confirmed a critical role of Ehmt1 and Zic2 downstream of Zfp281 both in driving exit from the ESC state and in restricting reprogramming of EpiSCs. Our study reveals that cell type-invariant chromatin association of Zfp281 provides an interaction platform for remodeling the cis-regulatory network underlying cellular plasticity.