Long Noncoding RNA URB1-Antisense RNA 1 (AS1) Suppresses Sorafenib-Induced Ferroptosis in Hepatocellular Carcinoma by Driving Ferritin Phase Separation.

Sorafenib, a first-line molecular-target drug for advanced hepatocellular carcinoma (HCC), has been shown to be a potent ferroptosis inducer in HCC. However, we found that there was a lower level of ferroptosis in sorafenib-resistant HCC samples than in sorafenib-sensitive HCC samples, suggesting that sorafenib resistance in HCC may be a result of ferroptosis suppression. Recent reports have shown that long noncoding RNAs (lncRNAs) are involved in programmed cell death (PCD), including apoptosis and ferroptosis. This study aimed to investigate the roles and underlying molecular mechanisms of lncRNAs in sorafenib-induced ferroptosis in HCC cells. Using lncRNA sequencing, we identified a ferroptosis-related lncRNA, URB1-antisense RNA 1 (AS1), which was highly expressed in sorafenib-resistant HCC samples and predicted poor survival in HCC. Furthermore, URB1-AS1 mitigates sorafenib-induced ferroptosis by inducing ferritin phase separation and reducing the cellular free iron content. Hypoxia inducible factor (HIF)-1α was identified as a key factor promoting URB1-AS1 expression in sorafenib-resistant HCC cells. Notably, we found that specifically inhibiting the expression of URB1-AS1 with N-acetylgalactosamine (GalNAc)-small interfering (si)URB1-AS1 successfully enhanced the sensitivity of HCC cells to sorafenib in an in vivo tumor model. Our study uncovered a critical role for URB1-AS1 in the repression of ferroptosis, suggesting URB1-AS1 targeting may represent a potential approach to overcome sorafenib resistance in HCC.

SERPINA12 promotes the tumorigenic capacity of HCC stem cells through hyperactivation of AKT/ß-catenin signaling.

BACKGROUND AND AIMS: HCC is an aggressive disease with poor clinical outcome. Understanding the mechanisms that drive cancer stemness, which we now know is the root cause of therapy failure and tumor recurrence, is fundamental for designing improved therapeutic strategies. This study aims to identify molecular players specific to CD133 + HCC to better design drugs that can precisely interfere with cancer stem cells but not normal stem cell function. APPROACH AND RESULTS: Transcriptome profiling comparison of epithelial-specific "normal" CD133 + cells isolated from fetal and regenerating liver against "HCC" CD133 + cells isolated from proto-oncogene-driven and inflammation-associated HCC revealed preferential overexpression of SERPINA12 in HCC but not fetal and regenerating liver CD133 + cells. SERPINA12 upregulation in HCC is tightly associated with aggressive clinical and stemness features, including survival, tumor stage, cirrhosis, and stemness signatures. Enrichment of SERPINA12 in HCC is mediated by promoter binding of the well-recognized ß-catenin effector TCF7L2 to drive SERPINA12 transcriptional activity. Functional characterization identified a unique and novel role of endogenous SERPINA12 in promoting self-renewal, therapy resistance, and metastatic abilities. Mechanistically, SERPINA12 functioned through binding to GRP78, resulting in a hyperactivated AKT/GSK3ß/ß-catenin signaling cascade, forming a positive feed-forward loop. Intravenous administration of rAAV8-shSERPINA12 sensitized HCC cells to sorafenib and impeded the cancer stem cell subset in an immunocompetent HCC mouse model. CONCLUSIONS: Collectively, our findings revealed that SERPINA12 is preferentially overexpressed in epithelial HCC CD133 + cells and is a key contributor to HCC initiation and progression by driving an AKT/ß-catenin feed-forward loop.

Glucose deprivation-induced aberrant FUT1-mediated fucosylation drives cancer stemness in hepatocellular carcinoma.

Rapidly growing tumors often experience hypoxia and nutrient (e.g., glucose) deficiency because of poor vascularization. Tumor cells respond to the cytotoxic effects of such stresses by inducing molecular adaptations that promote clonal selection of a more malignant tumor-initiating cell phenotype, especially in the innermost tumor regions. Here, we report a regulatory mechanism involving fucosylation by which glucose restriction promotes cancer stemness to drive drug resistance and tumor recurrence. Using hepatocellular carcinoma (HCC) as a model, we showed that restricted glucose availability enhanced the PERK/eIF2α/ATF4 signaling axis to drive fucosyltransferase 1 (FUT1) transcription via direct binding of ATF4 to the FUT1 promoter. FUT1 overexpression is a poor prognostic indicator for HCC. FUT1 inhibition could mitigate tumor initiation, self-renewal, and drug resistance. Mechanistically, we demonstrated that CD147, ICAM-1, EGFR, and EPHA2 are glycoprotein targets of FUT1, in which such fucosylation would consequently converge on deregulated AKT/mTOR/4EBP1 signaling to drive cancer stemness. Treatment with an α-(1,2)-fucosylation inhibitor sensitized HCC tumors to sorafenib, a first-line molecularly targeted drug used for advanced HCC patients, and reduced the tumor-initiating subset. FUT1 overexpression and/or CD147, ICAM-1, EGFR, and EPHA2 fucosylation may be good prognostic markers and therapeutic targets for cancer patients.

Chemotherapy-Enriched THBS2-Deficient Cancer Stem Cells Drive Hepatocarcinogenesis through Matrix Softness Induced Histone H3 Modifications.

The physical microenvironment is a critical mediator of tumor behavior. However, detailed biological and mechanistic insight is lacking. The present study reveals the role of chemotherapy-enriched CD133+ liver cancer stem cells (CSCs) with THBS2 deficiency. This subpopulation of cells contributes to a more aggressive cancer and functional stemness phenotype in hepatocellular carcinoma (HCC) by remodeling the extracellular matrix (ECM) through the regulation of matrix metalloproteinase (MMP) activity, collagen degradation, and matrix stiffness. The local soft spots created by these liver CSCs can enhance stemness and drug resistance and provide a route of escape to facilitate HCC metastasis. Interestingly, a positive feed-forward loop is identified where a local soft spot microenvironment in the HCC tumor is enriched with CD133 expressing cells that secrete markedly less ECM-modifying THBS2 upon histone H3 modification at its promoter region, allowing the maintenance of a localized soft spot matrix. Clinically, THBS2 deficiency is also correlated with low HCC survival, where high levels of CSCs with low THBS2 expression in HCC are associated with decreased collagen fiber deposits and an invasive tumor front. The findings have implications for the treatment of cancer stemness and for the prevention of tumor outgrowth through disseminated tumor cells.

PRMT6 deficiency induces autophagy in hostile microenvironments of hepatocellular carcinoma tumors by regulating BAG5-associated HSC70 stability.

Autophagy is a critical survival factor for cancer cells, whereby it maintains cellular homeostasis by degrading damaged organelles and unwanted proteins and supports cellular biosynthesis in response to stress. Cancer cells, including hepatocellular carcinoma (HCC), are often situated in a hypoxic, nutrient-deprived and stressful microenvironment where tumor cells are yet still able to adapt and survive. However, the mechanism underlying this adaptation and survival is not well-defined. We report deficiency of the post-translational modification enzyme protein arginine N-methyltransferase 6 (PRMT6) in HCC to promote the induction of autophagy under oxygen/nutrient-derived and sorafenib drug-induced stress conditions. Enhanced autophagic flux in HCC cells negatively correlated with PRMT6 expression, with the catalytic domain of PRMT6 critically important in mediating these autophagic activities. Mechanistically, PRMT6 physically interacts and methylates BAG5 to enhance the degradation of its interacting partner HSC70, a well-known autophagy player. The therapeutic potential of targeting BAG5 using genetic approach to reverse tumorigenicity and sorafenib resistance mediated by PRMT6 deficiency in HCC is also demonstrated in an in vivo model. The clinical implications of these findings are highlighted by the inverse correlative expressions of PRMT6 and HSC70 in HCC tissues. Collectively, deficiency of PRMT6 induces autophagy to promote tumorigenicity and cell survival in hostile microenvironments of HCC tumors by regulating BAG5-associated HSC70 stability through post-translational methylation of BAG5. Targeting BAG5 may therefore be an attractive strategy in HCC treatment by suppressing autophagy and inducing HCC cell sensitivity to sorafenib for treatment.

CRAF Methylation by PRMT6 Regulates Aerobic Glycolysis-Driven Hepatocarcinogenesis via ERK-Dependent PKM2 Nuclear Relocalization and Activation.

BACKGROUND AND AIMS: Most tumor cells use aerobic glycolysis (the Warburg effect) to support anabolic growth and promote tumorigenicity and drug resistance. Intriguingly, the molecular mechanisms underlying this phenomenon are not well understood. In this work, using gain-of-function and loss-of-function in vitro studies in patient-derived organoid and cell cultures as well as in vivo positron emission tomography-magnetic resonance imaging animal models, we showed that protein arginine N-methyltransferase 6 (PRMT6) regulates aerobic glycolysis in human hepatocellular carcinoma (HCC) through nuclear relocalization of pyruvate kinase M2 isoform (PKM2), a key regulator of the Warburg effect. APPROACH AND RESULTS: We found PRMT6 to methylate CRAF at arginine 100, interfering with its RAS/RAF binding potential, and therefore altering extracellular signal-regulated kinase (ERK)-mediated PKM2 translocation into the nucleus. This altered PRMT6-ERK-PKM2 signaling axis was further confirmed in both a HCC mouse model with endogenous knockout of PRMT6 as well as in HCC clinical samples. We also identified PRMT6 as a target of hypoxia through the transcriptional repressor element 1-silencing transcription factor, linking PRMT6 with hypoxia in driving glycolytic events. Finally, we showed as a proof of concept the therapeutic potential of using 2-deoxyglucose, a glycolysis inhibitor, to reverse tumorigenicity and sorafenib resistance mediated by PRMT6 deficiency in HCC. CONCLUSIONS: Our findings indicate that the PRMT6-ERK-PKM2 regulatory axis is an important determinant of the Warburg effect in tumor cells, and provide a mechanistic link among tumorigenicity, sorafenib resistance, and glucose metabolism.

Deficiency in Embryonic Stem Cell Marker Reduced Expression 1 Activates Mitogen-Activated Protein Kinase Kinase 6-Dependent p38 Mitogen-Activated Protein Kinase Signaling to Drive Hepatocarcinogenesis.

BACKGROUND AND AIMS: Embryonic stem-cell-related transcription factors are central to the establishment and maintenance of stemness and pluripotency, and their altered expression plays key roles in tumors, including hepatocellular carcinoma (HCC), a malignancy with no effective treatment. Here, we report on the embryonic stem cell marker, reduced expression 1 (REX1; also known as zinc finger protein 42), to be selectively down-regulated in HCC tumors. APPROACH AND RESULTS: Deficiency of REX1 in HCC was attributed to a combination of hypermethylation at its promoter as well as histone modification by methylation and acetylation. Clinically, hypermethylation of REX1 was closely associated with neoplastic transition and advanced tumor stage in humans. Functionally, silencing of REX1 potentiated the tumor-initiating and metastasis potential of HCC cell lines and xenografted tumors. Lentivirus-mediated Rex1 ablation in liver of male immunocompetent mice with HCC, induced by hydrodynamic tail vein injection of proto-oncogenes, enhanced HCC development. Transcriptome profiling studies revealed REX1 deficiency in HCC cells to be enriched with genes implicated in focal adhesion and mitogen-activated protein kinase (MAPK) signaling. From this lead, we subsequently found REX1 to bind to the promoter region of mitogen-activated protein kinase kinase 6 (MKK6), thereby obstructing its transcription, resulting in altered p38 MAPK signaling. CONCLUSIONS: Our work describes a critical repressive function of REX1 in maintenance of HCC cells by regulating MKK6 binding and p38 MAPK signaling. REX1 deficiency induced enhancement of p38 MAPK signaling, leading to F-actin reorganization and activation of nuclear factor erythroid 2-related factor 2-mediated oxidative stress response, which collectively contributed to enhanced stemness and metastatic capabilities of HCC cells.

PRMT6 Regulates RAS/RAF Binding and MEK/ERK-Mediated Cancer Stemness Activities in Hepatocellular Carcinoma through CRAF Methylation.

Arginine methylation is a post-translational modification that plays pivotal roles in signal transduction and gene transcription during cell fate determination. We found protein methyltransferase 6 (PRMT6) to be frequently downregulated in hepatocellular carcinoma (HCC) and its expression to negatively correlate with aggressive cancer features in HCC patients. Silencing of PRMT6 promoted the tumor-initiating, metastasis, and therapy resistance potential of HCC cell lines and patient-derived organoids. Consistently, loss of PRMT6 expression aggravated liver tumorigenesis in a chemical-induced HCC PRMT6 knockout (PRMT6-/-) mouse model. Integrated transcriptome and protein-protein interaction studies revealed an enrichment of genes implicated in RAS signaling and showed that PRMT6 interacted with CRAF on arginine 100, which decreased its RAS binding potential and altered its downstream MEK/ERK signaling. Our work describes a critical repressive function for PRMT6 in maintenance of HCC cells by regulating RAS binding and MEK/ERK signaling via methylation of CRAF on arginine 100.

FSTL1 Promotes Metastasis and Chemoresistance in Esophageal Squamous Cell Carcinoma through NFκB-BMP Signaling Cross-talk.

Esophageal squamous cell carcinoma (ESCC) has a generally poor prognosis, and molecular markers to improve early detection and predict outcomes are greatly needed. Here, we report that the BMP-binding follistatin-like protein FSTL1 is overexpressed in ESCCs, where it correlates with poor overall survival. Genetic amplification of FSTL1 or chromosome 3q, where it is located, occurred frequently in ESCC, where FSTL1 copy number correlated positively with higher FSTL1 protein expression. Elevating FSTL1 levels by various means was sufficient to drive ESCC cell proliferation, clonogenicity, migration, invasion, self-renewal, and cisplatin resistance in vitro and tumorigenicity and distant metastasis in vivo Conversely, FSTL1 attenuation by shRNA or neutralizing antibody elicited the opposite effects in ESCC cells. mRNA profiling analyses suggested that FSTL1 drives ESCC oncogenesis and metastasis through various pathways, with deregulation of NFκB and BMP signaling figuring prominently. Cross-talk between the NFκB and BMP pathways was evidenced by functional rescue experiments using inhibitors of NFκB and TLR4. Our results establish the significance of FSTL1 in driving oncogenesis and metastasis in ESCC by coordinating NFκB and BMP pathway control, with implications for its potential use as a diagnostic or prognostic biomarker and as a candidate therapeutic target in this disease setting. Cancer Res; 77(21); 5886-99. ©2017 AACR.

TP53INP1 Downregulation Activates a p73-Dependent DUSP10/ERK Signaling Pathway to Promote Metastasis of Hepatocellular Carcinoma.

Identifying critical factors involved in the metastatic progression of hepatocellular carcinoma (HCC) may offer important therapeutic opportunities. Here, we report that the proapoptotic stress response factor TP53INP1 is often selectively downregulated in advanced stage IV and metastatic human HCC tumors. Mechanistic investigations revealed that TP53INP1 downregulation in early-stage HCC cells promoted metastasis via DUSP10 phosphatase-mediated activation of the ERK pathway. The DUSP10 promoter included putative binding sites for p73 directly implicated in modulation by TP53INP1. Overall, our findings show how TP53INP1 plays a critical role in limiting the progression of early-stage HCC, with implications for developing new therapeutic strategies to attack metastatic HCC. Cancer Res; 77(17); 4602-12. ©2017 AACR.

Octamer 4/microRNA-1246 signaling axis drives Wnt/ß-catenin activation in liver cancer stem cells.

Wnt/ß-catenin signaling is activated in CD133 liver cancer stem cells (CSCs), a subset of cells known to be a root of tumor recurrence and therapy resistance in hepatocellular carcinoma (HCC). However, the regulatory mechanism of this pathway in CSCs remains unclear. Here, we show that human microRNA (miRNA), miR-1246, promotes cancer stemness, including self-renewal, drug resistance, tumorigencity, and metastasis, by activation of the Wnt/ß-catenin pathway through suppressing the expression of AXIN2 and glycogen synthase kinase 3ß (GSK3ß), two key members of the ß-catenin destruction complex. Clinically, high endogenous and circulating miR-1246 was identified in HCC clinical samples and correlated with a worse prognosis. Further functional analysis identified octamer 4 (Oct4) to be the direct upstream regulator of miR-1246, which cooperatively drive ß-catenin activation in liver CSCs. CONCLUSION: These findings uncover the noncanonical regulation of Wnt/ß-catenin in liver CSCs by the Oct4/miR-1246 signaling axis, and also provide a novel diagnostic marker as well as therapeutic intervention for HCC. (Hepatology 2016;64:2062-2076).

Neuropilin-2 promotes tumourigenicity and metastasis in oesophageal squamous cell carcinoma through ERK-MAPK-ETV4-MMP-E-cadherin deregulation.

Oesophageal squamous cell carcinoma (ESCC) is the most common histological subtype of oesophageal cancer. The disease is particularly prevalent in southern China. The incidence of the disease is on the rise and its overall survival rate remains dismal. Identification and characterization of better molecular markers for early detection and therapeutic targeting are urgently needed. Here, we report levels of transmembrane and soluble neuropilin-2 (NRP2) to be significantly up-regulated in ESCC, and to correlate positively with advanced tumour stage, lymph node metastasis, less favourable R category and worse overall patient survival. NRP2 up-regulation in ESCC was in part a result of gene amplification at chromosome 2q. NRP2 overexpression promoted clonogenicity, angiogenesis and metastasis in ESCC in vitro, while NRP2 silencing by lentiviral knockdown or neutralizing antibody resulted in a contrary effect. This observation was extended in vivo in animal models of subcutaneous tumourigenicity and tail vein metastasis. Mechanistically, overexpression of NRP2 induced expression of ERK MAP kinase and the transcription factor ETV4, leading to enhanced MMP-2 and MMP-9 activity and, as a consequence, suppression of E-cadherin. In summary, NRP2 promotes tumourigenesis and metastasis in ESCC through deregulation of ERK-MAPK-ETV4-MMP-E-cadherin signalling. NRP2 represents a potential diagnostic or prognostic biomarker and therapeutic target for ESCC. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

C-terminal truncated hepatitis B virus X protein promotes hepatocellular carcinogenesis through induction of cancer and stem cell-like properties.

Tumor relapse after chemotherapy typifies hepatocellular carcinoma (HCC) and is believed to be attributable to residual cancer stem cells (CSCs) that survive initial treatment. Chronic infection with hepatitis B virus (HBV) has long been linked to the development of HCC. Upon infection, random HBV genome integration can lead to truncation of hepatitis B virus X (HBx) protein at the C-terminus. The resulting C-terminal-truncated HBx (HBx-ΔC) was previously shown to confer enhanced invasiveness and diminished apoptotic response in HCC cells. Here, we found HBx-ΔC to promote the appearance of a CD133 liver CSC subset and confer cancer and stem cell-like features in HCC. HBx-ΔC was exclusively detected in HCC cell lines that were raised from patients presented with a HBV background with concomitant CD133 expression. Stable overexpression of the naturally occurring HBx-ΔC mutants, HBx-Δ14 or HBx-Δ35, in HCC cells Huh7 and immortalized normal liver cells MIHA resulted in a significant increase in the cells ability to self-renew, resist chemotherapy and targeted therapy, migrate and induce angiogenesis. MIHA cells with the mutants stably overexpressed also resulted in the induction of CD133, mediated through STAT3 activation. RNA sequencing profiling of MIHA cells with or without HBx-ΔC mutants stably overexpressed identified altered FXR activation. This, together with rescue experiments using a selective FXR inhibitor suggested that C-terminal truncated HBx can mediate cancer stemness via FXR activation. Collectively, we find C-terminal truncated HBx mutants to confer cancer and stem cell-like features in vitro and to play an important role in driving tumor relapse in HCC.

ANXA3/JNK Signaling Promotes Self-Renewal and Tumor Growth, and Its Blockade Provides a Therapeutic Target for Hepatocellular Carcinoma.

Frequent tumor relapse in hepatocellular carcinoma (HCC) has been commonly attributed to the presence of residual cancer stem cells (CSCs) after conventional treatments. We have previously identified and characterized CD133 to mark a specific CSC subset in HCC. In the present study, we found endogenous and secretory annexin A3 (ANXA3) to play pivotal roles in promoting cancer and stem cell-like features in CD133+ liver CSCs through a dysregulated JNK pathway. Blockade of ANXA3 with an anti-ANXA3 monoclonal antibody in vitro as well as in human HCC xenograft models resulted in a significant reduction in tumor growth and self-renewal. Clinically, ANXA3 expression in HCC patient sera closely associated with aggressive clinical features. Our results suggest that ANXA3 can serve as a novel diagnostic biomarker and that the inhibition of ANXA3 may be a viable therapeutic option for the treatment of CD133+ liver-CSC-driven HCC.

Regulatory role of miR-142-3p on the functional hepatic cancer stem cell marker CD133.

Tumor relapse after therapy typifies hepatocellular carcinoma (HCC) and is believed to be attributable to residual cancer stem cells (CSCs) that survive treatment. We have previously identified a CSC population derived from HCC that is characterized by CD133. Despite our growing knowledge of the importance of this subset of cells in driving HCC, the regulatory mechanism of CD133 is not known. Epigenetic changes are believed to be essential in the control of cancer and stem cells. Here, we report the epigenetic regulation of CD133 by miR-142-3p. The interaction between CD133 and miR-142-3p was identified by in silico prediction and substantiated by luciferase reporter analysis. Expression of CD133 was found to be inversely correlated with miR-142-3p in HCC clinical samples as well as in cell lines. Importantly, lower miR-142-3p expression in HCC was significantly associated with worst survival. Functional studies with miR-142-3p stably transduced in HCC cells demonstrated a diminished ability to self-renew, initiate tumor growth, invade, migrate, induce angiogenesis and resist chemotherapy. Rescue experiments whereby CD133 and miR-142-3p is simultaneously overexpressed compensated the deregulated ability of the cells to confer these features. Thus, miR-142-3p directly targets CD133 to regulate its ability to confer cancer and stem cell-like features in HCC.

Glial cells isolated from dorsal root ganglia express prostaglandin E(2) (EP(4)) and prostacyclin (IP) receptors.

Isolated cells from adult rat dorsal root ganglia (DRG) are frequently used as a model system to study responses of primary sensory neurons to nociceptor sensitizing agents such as prostaglandin E(2) and prostacyclin, which are presumed to act only on the neurons in typical mixed cell cultures. In the present study, we evaluated the expression of prostaglandin E(2) (EP(4)) and prostacyclin (IP) receptors in cultures of mixed DRG cells and in purified DRG glia. We show here that EP(4) and IP receptor agonists stimulated adenylyl cyclase activity in both mixed DRG cells and in purified DRG glia, and that these responses were specifically inhibited by EP(4) and IP receptor antagonists, respectively. The presence of EP(4) and IP receptors in DRG glia was further confirmed by the expression of EP(4) and IP receptor immunoreactivity and mRNA. With the increasing awareness of neuron-glial interactions within intact DRG and the use of isolated DRG cells in the study of mechanisms underlying nociception, it will be essential to consider the role played by EP(4) and IP receptor-expressing glial cells when evaluating prostanoid-induced sensitization of DRG neurons.

The role of glial cells in influencing neurite extension by dorsal root ganglion cells.

When pretreated with pertussis toxin (PTX), the neurites of adult rat dorsal root ganglion (DRG) cells in mixed cell cultures retract over a period of 2 h following the initial stimulus of removal from the cell culture incubator for brief periods of observation. The purpose of this investigation was to determine whether this PTX-dependent response was specific to any one of the three subpopulations of DRG neurons. However, no neurite retraction response was observed in neuron-enriched populations of cells, or in cultures enriched in isolectin B4 (IB4)-positive neurons or in IB4-negative neurons. But, the addition of non-neuronal cells, and/or medium conditioned by non-neuronal cells, was sufficient to restore the PTX-dependent neurite retraction response, but only in large diameter IB4-negative neurons. In conclusion, we have identified a regulatory response, mediated by Gi/o-proteins, which prevents retraction of neurites in large diameter IB4-negative cells of adult rat DRG. The non-neuronal cells of adult rat DRG constitutively release factor/s that can stimulate neurite retraction of a subset of isolated DRG neurons, but this property of non-neuronal cells is only observed when the Gi/o-proteins of large diameter IB4-negative cells are inhibited.