Guanling Huang

The Long Noncoding RNA lncTCF7 Promotes Self-Renewal of Human Liver Cancer Stem Cells through Activation of Wnt Signaling

Hepatocellular carcinoma (HCC) is the most prevalent subtype of liver cancer, and it is 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 mechanisms underlying their self-renewal and maintenance are poorly understood. Here, using transcriptome microarray analysis, we identified a long noncoding RNA (lncRNA) termed lncTCF7 that is highly expressed in HCC tumors and liver CSCs. LncTCF7 is required for liver CSC self-renewal and tumor propagation. Mechanistically, lncTCF7 recruits the SWI/SNF complex to the promoter of TCF7 to regulate its expression, leading to activation of Wnt signaling. Our data suggest that lncTCF7-mediated Wnt signaling primes liver CSC self-renewal and tumor propagation. In sum, therefore, we have identified an lncRNA-based Wnt signaling regulatory circuit that promotes tumorigenic activity in liver cancer stem cells, highlighting the role that lncRNAs can play in tumor growth and propagation.

WASH inhibits autophagy through suppression of Beclin 1 ubiquitination

Autophagy degrades cytoplasmic proteins and organelles to recycle cellular components that are required for cell survival and tissue homeostasis. However, it is not clear how autophagy is regulated in mammalian cells. WASH (Wiskott–Aldrich syndrome protein (WASP) and SCAR homologue) plays an essential role in endosomal sorting through facilitating tubule fission via Arp2/3 activation. Here, we demonstrate a novel function of WASH in modulation of autophagy. We show that WASH deficiency causes early embryonic lethality and extensive autophagy of mouse embryos. WASH inhibits vacuolar protein sorting (Vps)34 kinase activity and autophagy induction. We identified that WASH is a new interactor of Beclin 1. Beclin 1 is ubiquitinated at lysine 437 through lysine 63 linkage in cells undergoing autophagy. Ambra1 is an E3 ligase for lysine 63‐linked ubiquitination of Beclin 1 that is required for starvation‐induced autophagy. The lysine 437 ubiquitination of Beclin 1 enhances the association with Vps34 to promote Vps34 activity. WASH can suppress Beclin 1 ubiquitination to inactivate Vps34 activity leading to suppression of autophagy.

Transient Activation of Autophagy via Sox2-Mediated Suppression of mTOR Is an Important Early Step in Reprogramming to Pluripotency

Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components. Here we show that autophagy is required for reprogramming of somatic cells to form induced pluripotent stem cells (iPSCs). Our data indicate that mammalian target of rapamycin (mTOR) is downregulated by Sox2 at an early stage of iPSC generation and that this transient downregulation of mTOR is required for reprogramming to take place. In the absence of Sox2, mTOR remains at a high level and inhibits autophagy. Mechanistically, Sox2 binds to a repressive region on the mTOR promoter and recruits the NuRD complex to mediate transcriptional repression. We also detected enhanced autophagy at the four- to eight-cell stage of embryonic development, and a similar Sox2 and mTOR-mediated regulatory pathway seems to operate in this context as well. Thus, our findings reveal Sox2-dependent temporal regulation of autophagy as a key step in cellular reprogramming processes.

LncBRM initiates YAP1 signalling activation to drive self-renewal of liver cancer stem cells

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.

lnc-β-Catm elicits EZH2-dependent β-catenin stabilization and sustains liver CSC self-renewal

Liver cancer stem cells (CSCs) may contribute to the high rate of recurrence and heterogeneity of hepatocellular carcinoma (HCC); however, the molecular mechanisms underlying their self-renewal and differentiation remain largely unknown. Through analysis of transcriptome microarray data, we identified a long noncoding RNA (lncRNA) called lnc-β-Catm, which is highly expressed in human HCC tumors and liver CSCs. We found that lnc-β-Catm is required for self-renewal of liver CSCs and tumor propagation in mice. lnc-β-Catm associates with β-catenin and the methyltransferase EZH2, thereby promoting β-catenin methylation. Methylation suppresses the ubiquitination of β-catenin and promotes its stability, thus leading to activation of Wnt–β-catenin signaling. Accordingly, the expression of lnc-β-Catm, EZH2 and Wnt–β-catenin targets is positively correlated with cancer severity and prognosis of people with HCC.

T-cell Immunoglobulin and ITIM Domain (TIGIT) Receptor/Poliovirus Receptor (PVR) Ligand Engagement Suppresses Interferon-γ Production of Natural Killer Cells via β-Arrestin 2-mediated Negative Signaling

Background: T-cell immunoglobulin and ITIM domain (TIGIT) was recently defined as an inhibitory receptor that is expressed on NK cells and T cells. Results: TIGIT/poliovirus receptor (PVR) ligation signaling mediates suppression of IFN-γ production through NF-κB pathway via β-arrestin 2-mediated negative signaling. Conclusion: TIGIT/PVR signaling suppresses IFN-γ production of NK cells. Significance: TIGIT/PVR signaling acts as a potent negative mediator to down-regulate NK cell response for immune homeostasis. Natural killer (NK) cell activation is well orchestrated by a wide array of NK cell receptor repertoire. T-cell immunoglobulin and ITIM domain (TIGIT) receptor was recently defined as an inhibitory receptor that is expressed on NK cells and T cells. TIGIT receptor/poliovirus receptor (PVR) ligand engagement signaling inhibits cytotoxicity mediated by NK and CD8+ T cells. However, it is unclear how TIGIT/PVR signaling regulates cytokine secretion in NK cells. Here we show that TIGIT/PVR engagement suppresses interferon-γ (IFN-γ) production of NK cells. TIGIT transgenic NK cells generate less IFN-γ undergoing TIGIT/PVR ligation. Moreover, TIGIT knock-out NK cells produce much more IFN-γ. TIGIT/PVR ligation signaling mediates suppression of IFN-γ production via the NF-κB pathway. We identified a novel adaptor β-arrestin 2 that associates with phosphorylated TIGIT for further recruitment of SHIP1 (SH2-containing inositol phosphatase 1) through the ITT-like motif. Importantly, SHIP1, but not other phosphatases, impairs the TNF receptor-associated factor 6 (TRAF6) autoubiquitination to abolish NF-κB activation, leading to suppression of IFN-γ production in NK cells.

Long noncoding RNA lncKdm2b is required for ILC3 maintenance by initiation of Zfp292 expression

Innate lymphoid cells (ILCs) communicate with other hematopoietic and nonhematopoietic cells to regulate immunity, inflammation and tissue homeostasis. How ILC lineages develop and are maintained remains largely unknown. In this study we observed that a divergent long noncoding RNA (lncRNA), lncKdm2b, was expressed at high levels in intestinal group 3 ILCs (ILC3s). LncKdm2b deficiency in the hematopoietic system led to reductions in the number and effector functions of ILC3s. LncKdm2b expression sustained the maintenance of ILC3s by promoting their proliferation through activation of the transcription factor Zfp292. Mechanistically, lncKdm2b recruited the chromatin organizer Satb1 and the nuclear remodeling factor (NURF) complex onto the Zfp292 promoter to initiate its transcription. Deletion of Zfp292 or Bptf also abrogated the maintenance of ILC3s, leading to susceptibility to bacterial infection. Therefore, our findings reveal that lncRNAs may represent an additional layer of regulation of ILC development and function.

Sox2 functions as a sequence-specific DNA sensor in neutrophils to initiate innate immunity against microbial infection

Neutrophils express Toll-like receptors (TLRs) for the recognition of conserved bacterial elements to initiate antimicrobial responses. However, whether other cytosolic DNA sensors are expressed by neutrophils remains elusive. Here we found constitutive expression of the transcription factor Sox2 in the cytoplasm of mouse and human neutrophils. Neutrophil-specific Sox2 deficiency exacerbated bacterial infection. Sox2 directly recognized microbial DNA through its high-mobility-group (HMG) domain. Upon challenge with bacterial DNA, Sox2 dimerization was needed to activate a complex of the kinase TAK1 and its binding partner TAB2, which led to activation of the transcription factors NF-κB and AP-1 in neutrophils. Deficiency in TAK1 or TAB2 impaired Sox2-mediated antibacterial immunity. Overall, we reveal a previously unrecognized role for Sox2 as a cytosolic sequence-specific DNA sensor in neutrophils, which might provide potential therapeutic strategies for the treatment of infectious diseases.

ZIC2-dependent OCT4 activation drives self-renewal of human liver cancer stem cells

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.

C8orf4 negatively regulates self-renewal of liver cancer stem cells via suppression of NOTCH2 signalling

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.