PFKFB4 Drives the Oncogenicity in TP53-Mutated Hepatocellular Carcinoma in a Phosphatase-Dependent Manner.

BACKGROUND & AIMS: Metabolic reprogramming is recognized as a cancer hallmark intimately linked to tumor hypoxia, which supports rapid tumor growth and mitigates the consequential oxidative stress. Phosphofructokinase-fructose bisphosphatase (PFKFB) is a family of bidirectional glycolytic enzymes possessing both kinase and phosphatase functions and has emerged as important oncogene in multiple types of cancer. However, its clinical relevance, functional significance, and underlying mechanistic insights in hepatocellular carcinoma (HCC), the primary malignancy that develops in the most important metabolic organ, has never been addressed. METHODS: PFKFB4 expression was examined by RNA sequencing in The Cancer Genome Atlas and our in-house HCC cohort. The up-regulation of PFKFB4 expression was confirmed further by quantitative polymerase chain reaction in an expanded hepatitis B virus-associated HCC cohort followed by clinicopathologic correlation analysis. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated PFKFB4 knockout cells were generated for functional characterization in vivo, targeted metabolomic profiling, as well as RNA sequencing analysis to comprehensively examine the impact of PFKFB4 loss in HCC. RESULTS: PFKFB4 expression was up-regulated significantly in HCC and correlated positively with TP53 and TSC2 loss-of-function mutations. In silico transcriptome-based analysis further revealed PFKFB4 functions as a critical hypoxia-inducible gene. Clinically, PFKFB4 up-regulation was associated with more aggressive tumor behavior. Functionally, CRISPR/Cas9-mediated PFKFB4 knockout significantly impaired in vivo HCC development. Targeted metabolomic profiling revealed that PFKFB4 functions as a phosphatase in HCC and its ablation caused an accumulation of metabolites in downstream glycolysis and the pentose phosphate pathway. In addition, PFKFB4 loss induced hypoxia-responsive genes in glycolysis and reactive oxygen species detoxification. Conversely, ectopic PFKFB4 expression conferred sorafenib resistance. CONCLUSIONS: PFKFB4 up-regulation supports HCC development and shows therapeutic implications.

Midline 1 interacting protein 1 promotes cancer metastasis through FOS-like 1-mediated matrix metalloproteinase 9 signaling in HCC.

BACKGROUND AND AIMS: Understanding the mechanisms of HCC progression and metastasis is crucial to improve early diagnosis and treatment. This study aimed to identify key molecular targets involved in HCC metastasis. APPROACH AND RESULTS: Using whole-transcriptome sequencing of patients' HCCs, we identified and validated midline 1 interacting protein 1 (MID1IP1) as one of the most significantly upregulated genes in metastatic HCCs, suggesting its potential role in HCC metastasis. Clinicopathological correlation demonstrated that MID1IP1 upregulation significantly correlated with more aggressive tumor phenotypes and poorer patient overall survival rates. Functionally, overexpression of MID1IP1 significantly promoted the migratory and invasive abilities and enhanced the sphere-forming ability and expression of cancer stemness-related genes of HCC cells, whereas its stable knockdown abrogated these effects. Perturbation of MID1IP1 led to significant tumor shrinkage and reduced pulmonary metastases in an orthotopic liver injection mouse model and reduced pulmonary metastases in a tail-vein injection model in vivo . Mechanistically, SP1 transcriptional factor was found to be an upstream driver of MID1IP1 transcription. Furthermore, transcriptomic sequencing on MID1IP1-overexpressing HCC cells identified FOS-like 1 (FRA1) as a critical downstream mediator of MID1IP1. MID1IP1 upregulated FRA1 to subsequently promote its transcriptional activity and extracellular matrix degradation activity of matrix metalloproteinase MMP9, while knockdown of FRA1 effectively abolished the MID1IP1-induced migratory and invasive abilities. CONCLUSIONS: Our study identified MID1IP1 as a regulator in promoting FRA1-mediated-MMP9 signaling and demonstrated its role in HCC metastasis. Targeting MID1IP1-mediated FRA1 pathway may serve as a potential therapeutic strategy against HCC progression.

Investigation of Functional Synergism of CENPF and FOXM1 Identifies POLD1 as Downstream Target in Hepatocellular Carcinoma.

Background: Lines of evidence implicate CENPF and FOXM1 may have novel co-operative roles in driving hepatocellular carcinoma (HCC). Objective: We investigated the clinicopathological correlation, functional characterization, molecular mechanism and translational significance of CENPF and FOXM1. Methods: We carried out integrative studies investigating functional synergism of CENPF and FOXM1 in HCC and its metastasis. Human HCC samples, HCC cell lines and mouse model were used in the studies. Stable knockdown, q-PCR, Western blotting, whole-transcriptomic sequencing (RNA-seq), as well as cell and mouse assays were performed. Results: Upon clinicopathological correlation, we found that co-overexpression of CENPF and FOXM1 in human HCCs was associated with more aggressive tumor behavior including presence of venous invasion, tumor microsatellite formation, and absence of tumor encapsulation. Moreover, co-silencing FOXM1 and CENPF using shRNA approach in HCC cell lines resulted in significantly reduced cell proliferation. Furthermore, our RNA-seq and differential gene expression analysis delineated that CENPF and FOXM1 co-regulated a specific set of target genes in various metabolic processes and oncogenic signaling pathways. Among them, POLD1, which encodes the catalytic subunit of DNA polymerase δ, was ranked as the top downstream target co-regulated by CENPF and FOXM1. POLD1 expression was positively correlated with that of FOXM1 and CENPF in HCCs. In addition, POLD1 expression was significantly upregulated in HCC tumors. Functionally, in vivo orthotopic injection model showed that stable knockdown of POLD1 in HCC cells suppressed tumor incidence and tumorigenicity and had a trend of diminished lung metastasis. Conclusion: Taken together, our data suggest that CENPF and FOXM1 could synergistically support hepatocarcinogenesis via the regulation of POLD1. CENPF and FOXM1 may represent new vulnerabilities to novel drug-based therapy in HCC.

Ephrin-A3/EphA2 axis regulates cellular metabolic plasticity to enhance cancer stemness in hypoxic hepatocellular carcinoma.

BACKGROUND & AIMS: The highly proliferative nature of hepatocellular carcinoma (HCC) frequently results in a hypoxic intratumoural microenvironment, which creates a therapeutic challenge owing to a lack of mechanistic understanding of the phenomenon. We aimed to identify critical drivers of HCC development and progression in the hypoxic microenvironment. METHODS: We performed integrative analysis of multiple transcriptomic and genomic profiles specific for HCC and hypoxia and identified the Ephrin-A3/Eph receptor A2 (EphA2) axis as a clinically relevant and hypoxia-inducible signalling axis in HCC. The functional significance and mechanistic consequences of the Ephrin-A3/EphA2 axis were examined in EFNA3- and EPHA2- knockdown/overexpressing HCC cells. The potential downstream pathways were investigated by transcriptome sequencing, quantitative reverse-transcription PCR, western blotting analysis and metabolomics. RESULTS: EFNA3 was frequently upregulated in HCC and its overexpression was associated with more aggressive tumour behaviours. HIF-1α directly and positively regulated EFNA3 expression under hypoxia. EFNA3 functionally contributed to self-renewal, proliferation and migration in HCC cells. EphA2 was identified as a key functional downstream mediator of EFNA3. Functional characterisation of the Ephrin-A3/EphA2 forward-signalling axis demonstrated a promotion of self-renewal ability and tumour initiation. Mechanistically, the Ephrin-A3/EphA2 axis promoted the maturation of SREBP1 and expression of its transcriptional target, ACLY, was significantly associated with the expression of EFNA3 and hypoxia markers in clinical cohorts. The metabolic signature of EPHA2 and ACLY stable knockdown HCC cells demonstrated significant overlap in fatty acid, cholesterol and tricarboxylic acid cycle metabolite profiles. ACLY was confirmed to mediate the self-renewal function of the Ephrin-A3/EphA2 axis. CONCLUSIONS: Our findings revealed the novel role of the Ephrin-A3/EphA2 axis as a hypoxia-sensitive modulator of HCC cell metabolism and a key contributor to HCC initiation and progression. LAY SUMMARY: Hepatocellular carcinoma (HCC) is a fast-growing tumour; hence, areas of the tumour often have insufficient vasculature and become hypoxic. The presence of hypoxia within tumours has been shown to negatively impact on the survival of patients with tumours, including HCC. Herein, we identified the Ephrin-A3/EphA2 axis as a key functional driver of tumour initiation and progression in response to hypoxia. Additionally, we showed that SREBP1-ACLY-mediated metabolic rewiring was an important downstream effector that induced cancer stemness in response to Ephrin-A3/EphA2 forward-signalling.

Cellular heterogeneity and plasticity in liver cancer.

Hepatocarcinogenesis involves complex genetic and cellular dysregulations which drive the formation of hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, with extensive heterogeneity. In contrast to the broad spectrum of molecularly driven therapies available for defined patient groups in certain cancer types, unfortunately the treatment options for HCC are highly limited. The lack of representative molecular and cellular signatures in the heterogeneous HCC tumors that can effectively guide the choice of the most appropriate treatment among the patients unavoidably limits the treatment outcome. Advancement and wide availability of the next-generation sequencing technologies have empowered us to examine and capture not only the detailed genetic alterations of the HCC cells but also the precise composition of different cell types within the tumor microenvironment and their interactions with the HCC cells at an unprecedented level. The information generated has provided new insight and better defined the inter-patient intertumoral heterogeneity, intra-patient intratumoral heterogeneity as well as the plasticity of HCC cells. These collectively provide a robust scientific basis in guiding the development and use of targeted therapy and immunotherapy. To complement, liquid biopsy coupled with high-sensitivity sequencing could potentially be adopted as a more practical and safer approach to detect and reflect the tumor heterogeneity in HCC patients in guiding the choice of treatment and monitoring disease progression.

Dysregulation of RalA signaling through dual regulatory mechanisms exerts its oncogenic functions in hepatocellular carcinoma.

BACKGROUND AND AIMS: Ras-like (Ral) small guanosine triphosphatases (GTPases), RalA and RalB, are proto-oncogenes directly downstream of Ras and cycle between the active guanosine triphosphate-bound and inactive guanosine diphosphate-bound forms. RalGTPase-activating protein (RalGAP) complex exerts a negative regulation. Currently, the role of Ral up-regulation in cancers remains unclear. We aimed to examine the clinical significance, functional implications, and underlying mechanisms of RalA signaling in HCC. APPROACH AND RESULTS: Our in-house and The Cancer Genome Atlas RNA sequencing data and quantitative PCR data revealed significant up-regulation of RalA in patients' HCCs. Up-regulation of RalA was associated with more aggressive tumor behavior and poorer prognosis. Consistently, knockdown of RalA in HCC cells attenuated cell proliferation and migration in vitro and tumorigenicity and metastasis in vivo. We found that RalA up-regulation was driven by copy number gain and uncovered that SP1 and ETS proto-oncogene 2 transcription factor cotranscriptionally drove RalA expression. On the other hand, RalGAPA2 knockdown increased the RalA activity and promoted intrahepatic and extrahepatic metastasis in vivo. Consistently, we observed significant RalGAPA2 down-regulation in patients' HCCs. Intriguingly, HCC tumors showing simultaneous down-regulation of RalGAPA2 and up-regulation of RalA displayed a significant association with more aggressive tumor behavior in terms of more frequent venous invasion, more advanced tumor stage, and poorer overall survival. Of note, Ral inhibition by a Ral-specific inhibitor RBC8 suppressed the oncogenic functions in a dose-dependent manner and sensitized HCC cells to sorafenib treatment, with an underlying enhanced inhibition of mammalian target of rapamycin signaling. CONCLUSIONS: Our results provide biological insight that dysregulation of RalA signaling through dual regulatory mechanisms supports its oncogenic functions in HCC. Targeting RalA may serve as a potential alternative therapeutic approach alone or in combination with currently available therapy.

Antioxidant supplements promote tumor formation and growth and confer drug resistance in hepatocellular carcinoma by reducing intracellular ROS and induction of TMBIM1.

BACKGROUND: Controversy over the benefits of antioxidants supplements in cancers persists for long. Using hepatocellular carcinoma (HCC) as a model, we investigated the effects of exogenous antioxidants N-acetylcysteine (NAC) and glutathione (GSH) on tumor formation and growth. METHODS: Multiple mouse models, including diethylnitrosamine (DEN)-induced and Trp53KO/C-MycOE-induced HCC models, mouse hepatoma cell and human HCC cell xenograft models with subcutaneous or orthotopic injection were used. In vitro assays including ROS assay, colony formation, sphere formation, proliferation, migration and invasion, apoptosis, cell cycle assays were conducted. Western blot was performed for protein expression and RNA-sequencing to identify potential gene targets. RESULTS: In these multiple different mouse and cell line models, we observed that NAC and GSH promoted HCC tumor formation and growth, accompanied with significant reduction of intracellular reactive oxygen species (ROS) levels. Moreover, NAC and GSH promoted cancer stemness, and abrogated the tumor-suppressive effects of Sorafenib both in vitro and in vivo. Exogenous supplementation of NAC or GSH reduced the expression of NRF2 and GCLC, suggesting the NRF2/GCLC-related antioxidant production pathway might be desensitized. Using transcriptomic analysis to identify potential gene targets, we found that TMBIM1 was significantly upregulated upon NAC and GSH treatment. Both TCGA and in-house RNA-sequence databases showed that TMBIM1 was overexpressed in HCC tumors. Stable knockdown of TMBIM1 increased the intracellular ROS; it also abolished the promoting effects of the antioxidants in HCC cells. On the other hand, BSO and SSA, inhibitors targeting NAC and GSH metabolism respectively, partially abrogated the pro-oncogenic effects induced by NAC and GSH in vitro and in vivo. CONCLUSIONS: Our data implicate that exogenous antioxidants NAC and GSH, by reducing the intracellular ROS levels and inducing TMBIM expression, promoted HCC formation and tumor growth, and counteracted the therapeutic effect of Sorafenib. Our study provides scientific insight regarding the use of exogenous antioxidant supplements in cancers.

Single-cell RNA sequencing shows the immunosuppressive landscape and tumor heterogeneity of HBV-associated hepatocellular carcinoma.

Interaction between tumor cells and immune cells in the tumor microenvironment is important in cancer development. Immune cells interact with the tumor cells to shape this process. Here, we use single-cell RNA sequencing analysis to delineate the immune landscape and tumor heterogeneity in a cohort of patients with HBV-associated human hepatocellular carcinoma (HCC). We found that tumor-associated macrophages suppress tumor T cell infiltration and TIGIT-NECTIN2 interaction regulates the immunosuppressive environment. The cell state transition of immune cells towards a more immunosuppressive and exhaustive status exemplifies the overall cancer-promoting immunocellular landscape. Furthermore, the heterogeneity of global molecular profiles reveals co-existence of intra-tumoral and inter-tumoral heterogeneity, but is more apparent in the latter. This analysis of the immunosuppressive landscape and intercellular interactions provides mechanistic information for the design of efficacious immune-oncology treatments in hepatocellular carcinoma.

RSK2-inactivating mutations potentiate MAPK signaling and support cholesterol metabolism in hepatocellular carcinoma.

BACKGROUND & AIMS: Mutational profiling of patient tumors has suggested that hepatocellular carcinoma (HCC) development is mainly driven by loss-of-function mutations in tumor suppressor genes. p90 ribosomal S6 kinase 2 (RSK2) functions as a direct downstream kinase of ERK1/2 and elevated RSK2 expression has been reported to support oncogenic functions in some cancers. We investigated if RSK2 was also dysregulated by inactivating mutations in cancers including HCC. METHODS: We performed exome sequencing and targeted DNA sequencing on HBV-associated HCCs to examine recurrent RSK2 mutations. The functional significance and mechanistic consequences of RSK2 mutations were examined in natural RSK2-null HCC cells, and RSK2-knockout HCC cells. The potential downstream pathways underlying RSK2 mutations were investigated by RNA sequencing, qRT-PCR and mass spectrometry. RESULTS: We detected recurrent somatic RSK2 mutations at a rate of 6.3% in our HCC cohorts and revealed that, among many cancer types, HCC was the cancer most commonly harboring RSK2 mutations. The RSK2 mutations were inactivating and associated with a more aggressive tumor phenotype. We found that, functionally, restoring RSK2 expression in natural RSK2-null HBV-positive Hep3B cells suppressed proliferation and migration in vitro and tumorigenicity in vivo. Mechanistically, RSK2-inactivating mutations attenuated a SOS1/2-dependent negative feedback loop, leading to the activation of MAPK signaling. Of note, this RSK2 mutation-mediated MAPK upregulation rendered HCC cells more sensitive to sorafenib, a first-line multi-kinase inhibitor for advanced HCC. Furthermore, such activation of MAPK signaling enhanced cholesterol biosynthesis-related gene expression in HCC cells. CONCLUSIONS: Our findings reveal the mechanistic and functional significance of RSK2-inactivating mutations in HCC. These inactivating mutations may serve as an alternative route to activate MAPK signaling and cholesterol metabolism in HCC. LAY SUMMARY: In this study, we identified and functionally characterized RSK2-inactivating mutations in human hepatocellular carcinoma and demonstrated their association with aggressive tumor behavior. Mutations in RSK2 drive signaling pathways with known oncogenic potential, leading to enhanced cholesterol biosynthesis and potentially sensitizing tumors to sorafenib treatment.

Hepatitis B Virus-Telomerase Reverse Transcriptase Promoter Integration Harnesses Host ELF4, Resulting in Telomerase Reverse Transcriptase Gene Transcription in Hepatocellular Carcinoma.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) integrations are common in hepatocellular carcinoma (HCC). In particular, alterations of the telomerase reverse transcriptase (TERT) gene by HBV integrations are frequent; however, the molecular mechanism and functional consequence underlying TERT HBV integration are unclear. APPROACH AND RESULTS: We adopted a targeted sequencing strategy to survey HBV integrations in human HBV-associated HCCs (n = 95). HBV integration at the TERT promoter was frequent (35.8%, n = 34/95) in HCC tumors and was associated with increased TERT mRNA expression and more aggressive tumor behavior. To investigate the functional importance of various integrated HBV components, we employed different luciferase reporter constructs and found that HBV enhancer I (EnhI) was the key viral component leading to TERT activation on integration at the TERT promoter. In addition, the orientation of the HBV integration at the TERT promoter further modulated the degree of TERT transcription activation in HCC cell lines and patients' HCCs. Furthermore, we performed array-based small interfering RNA library functional screening to interrogate the potential major transcription factors that physically interacted with HBV and investigated the cis-activation of host TERT gene transcription on viral integration. We identified a molecular mechanism of TERT activation through the E74 like ETS transcription factor 4 (ELF4), which normally could drive HBV gene transcription. ELF4 bound to the chimeric HBV EnhI at the TERT promoter, resulting in telomerase activation. Stable knockdown of ELF4 significantly reduced the TERT expression and sphere-forming ability in HCC cells. CONCLUSIONS: Our results reveal a cis-activating mechanism harnessing host ELF4 and HBV integrated at the TERT promoter and uncover how TERT HBV-integrated HCCs may achieve TERT activation in hepatocarcinogenesis.

Cancer stemness in hepatocellular carcinoma: mechanisms and translational potential.

Cancer stemness, referring to the stem-cell-like phenotype of cancer cells, has been recognised to play important roles in different aspects of hepatocarcinogenesis. A number of well-established cell-surface markers already exist for liver cancer stem cells, with potential new markers of liver cancer stem cells being identified. Both genetic and epigenetic factors that affect various signalling pathways are known to contribute to cancer stemness. In addition, the tumour microenvironment-both physical and cellular-is known to play an important role in regulating cancer stemness, and the potential interaction between cancer stem cells and their microenvironment has provided insight into the regulation of the tumour-initiating ability as well as the cellular plasticity of liver CSCs. Potential specific therapeutic targeting of liver cancer stemness is also discussed. With increased knowledge, effective druggable targets might be identified, with the aim of improving treatment outcome by reducing chemoresistance.

Single-cell transcriptomics reveals the landscape of intra-tumoral heterogeneity and stemness-related subpopulations in liver cancer.

Hepatocellular carcinoma (HCC) is heterogeneous, rendering its current curative treatments ineffective. The emergence of single-cell genomics represents a powerful strategy in delineating the complex molecular landscapes of cancers. In this study, we demonstrated the feasibility and merit of using single-cell RNA sequencing to dissect the intra-tumoral heterogeneity and analyze the single-cell transcriptomic landscape to detect rare cell subpopulations of significance. Exploration of the inter-relationship among liver cancer stem cell markers showed two distinct major cell populations according to EPCAM expression, and the EPCAM+ cells had upregulated expression of multiple oncogenes. We also identified a CD24+/CD44+-enriched cell subpopulation within the EPCAM+ cells which had specific signature genes and might indicate a novel stemness-related cell subclone in HCC. Notably, knockdown of signature gene CTSE for CD24+/CD44+ cells significantly reduced self-renewal ability on HCC cells in vitro and the stemness-related role of CTSE was further confirmed by in vivo tumorigenicity assays in nude mice. In summary, single-cell genomics is a useful tool to delineate HCC intratumoral heterogeneity at better resolution. It can identify rare but important cell subpopulations, and may guide better precision medicine in the long run.

APOBEC3B promotes hepatocarcinogenesis and metastasis through novel deaminase-independent activity.

Cytidine deaminase APOBEC3B (A3B) is known to play important roles in creating de novo genomic C-to-T mutations in cancers and contribute to induction of genomic instability. Our study evaluated the roles of A3B in the progression and metastasis of human hepatocellular carcinoma (HCC). Using whole-transcriptome and whole-exome sequencing, and quantitative PCR, we found that A3B was overexpressed in human HCCs and A3B expression was significantly correlated with the proportion of genomic C-to-A and G-to-T mutations. Upon clinicopathological correlation, higher A3B expression was associated with more aggressive tumor behavior. Wild-type A3B (wt-A3B) overexpression in HCC cells promoted cell proliferation, and cell migratory and invasive abilities in vitro, and tumorigenicity and metastasis in vivo. On the other hand, knockdown of A3B suppressed cell proliferation, migratory, and invasive abilities of HCC cells with high endogenous A3B level. However, to our surprise, overexpression of A3B deaminase-dead double mutant (E68A/E255Q) led to similar results as wt-A3B in HCC. Furthermore, overexpression of wt-A3B and mutant A3B both enhanced cell cycle progression in HCC cells. Altogether, our data demonstrated a novel deaminase-independent role of A3B in contributing to HCC tumorigenesis and metastasis.

Asymmetric localization of DLC1 defines avian trunk neural crest polarity for directional delamination and migration.

Following epithelial-mesenchymal transition, acquisition of avian trunk neural crest cell (NCC) polarity is prerequisite for directional delamination and migration, which in turn is essential for peripheral nervous system development. However, how this cell polarization is established and regulated remains unknown. Here we demonstrate that, using the RHOA biosensor in vivo and in vitro, the initiation of NCC polarization is accompanied by highly activated RHOA in the cytoplasm at the cell rear and its fluctuating activity at the front edge. This differential RHOA activity determines polarized NC morphology and motility, and is regulated by the asymmetrically localized RhoGAP Deleted in liver cancer (DLC1) in the cytoplasm at the cell front. Importantly, the association of DLC1 with NEDD9 is crucial for its asymmetric localization and differential RHOA activity. Moreover, NC specifiers, SOX9 and SOX10, regulate NEDD9 and DLC1 expression, respectively. These results present a SOX9/SOX10-NEDD9/DLC1-RHOA regulatory axis to govern NCC migratory polarization.

SENP1 promotes hypoxia-induced cancer stemness by HIF-1α deSUMOylation and SENP1/HIF-1α positive feedback loop.

OBJECTIVE: We investigated the effect and mechanism of hypoxic microenvironment and hypoxia-inducible factors (HIFs) on hepatocellular carcinoma (HCC) cancer stemness. DESIGN: HCC cancer stemness was analysed by self-renewal ability, chemoresistance, expression of stemness-related genes and cancer stem cell (CSC) marker-positive cell population. Specific small ubiquitin-like modifier (SUMO) proteases 1 (SENP1) mRNA level was examined with quantitative PCR in human paired HCCs. Immunoprecipitation was used to examine the binding of proteins and chromatin immunoprecipitation assay to detect the binding of HIFs with hypoxia response element sequence. In vivo characterisation was performed in immunocompromised mice and stem cell frequency was analysed. RESULTS: We showed that hypoxia enhanced the stemness of HCC cells and hepatocarcinogenesis through enhancing HIF-1α deSUMOylation by SENP1 and increasing stabilisation and transcriptional activity of HIF-1α. Furthermore, we demonstrated that SENP1 is a direct target of HIF-1/2α and a previously unrecognised positive feedback loop exists between SENP1 and HIF-1α. CONCLUSIONS: Taken together, our findings suggest the significance of this positive feedback loop between HIF-1α and SENP1 in contributing to the increased cancer stemness in HCC and hepatocarcinogenesis under hypoxia. Drugs that specifically target SENP1 may offer a potential novel therapeutic approach for HCC.

Molecular Pathogenesis of Hepatocellular Carcinoma.

The pathogenesis of hepatocellular carcinoma (HCC) is a multistep process involving the progressive accumulation of molecular alterations pinpointing different molecular and cellular events. The next-generation sequencing technology is facilitating the global and systematic evaluation of molecular landscapes in HCC. There is emerging evidence supporting the importance of cancer metabolism and tumor microenvironment in providing a favorable and supportive niche to expedite HCC development. Moreover, recent studies have identified distinct surface markers of cancer stem cell (CSC) in HCC, and they also put forward the profound involvement of altered signaling pathways and epigenetic modifications in CSCs, in addition to the concomitant drug resistance and metastasis. Taken together, multiple key genetic and non-genetic factors, as well as liver CSCs, result in the development and progression of HCC.

RhoE/ROCK2 regulates chemoresistance through NF-κB/IL-6/ STAT3 signaling in hepatocellular carcinoma.

Small Rho GTPase (Rho) and its immediate effector Rho kinase (ROCK) are reported to regulate cell survival, but the detailed molecular mechanism remains largely unknown. We had previously shown that Rho/ROCK signaling was highly activated in hepatocellular carcinoma (HCC). In this study, we further demonstrated that downregulation of RhoE, a RhoA antagonist, and upregulation of ROCK enhanced resistance to chemotherapy in HCC in both in vitro cell and in vivo murine xenograft models, whereas a ROCK inhibitor was able to profoundly sensitize HCC tumors to cisplatin treatment. Specifically, the ROCK2 isoform but not ROCK1 maintained the chemoresistance in HCC cells. Mechanistically, we demonstrated that activation of ROCK2 enhanced the phosphorylation of JAK2 and STAT3 through increased expression of IL-6 and the IL-6 receptor complex. We also identified IKKß as the direct downstream target of Rho/ROCK, and activation of ROCK2 significantly augmented NF-κB transcription activity and induced IL-6 expression. These data indicate that Rho/ROCK signaling activates a positive feedback loop of IKKß/NF-κB/IL-6/STAT3 which confers chemoresistance to HCC cells and is a potential molecular target for HCC therapy.

Down-regulation of TIMP2 by HIF-1α/miR-210/HIF-3α regulatory feedback circuit enhances cancer metastasis in hepatocellular carcinoma.

UNLABELLED: Cancer metastasis is a multistep process that involves a series of tumor-stromal interaction, including extracellular matrix (ECM) remodeling, which requires a concerted action of multiple proteolytic enzymes and their endogenous inhibitors. This study investigated the role of tissue inhibitor of metalloproteinases (TIMP) 2 in the context of hepatocellular carcinoma (HCC) metastasis. We found that TIMP2 was the most significantly down-regulated member among the TIMP family in human HCCs. Moreover, TIMP2 underexpression was frequent (41.8%; 23 of 55) in human HCCs and was significantly associated with liver invasion and poorer survival outcomes of HCC patients. Furthermore, stable silencing of TIMP2 in HCC cell lines enhanced cell invasive ability and ECM degradation associated with formation of invadopodia-like feature, suggesting that TIMP2 is a negative regulator of HCC metastasis. Using an orthotopic tumor xenograft model, we demonstrated that ectopic expression of TIMP2 open reading frame in the highly metastatic HCC cell line, MHCC-97L, significantly reduced HCC progression as well as pulmonary metastasis. Mechanistically, TIMP2 suppression, in a hypoxic environment, was induced through a regulatory feedback circuit consisting of hypoxia-inducible factor (HIF) 1 alpha, microRNA-210 (miR-210), and HIF-3α. CONCLUSION: TIMP2 is frequently down-regulated in human HCCs and its down-regulation is associated with aggressive tumor behavior and poorer patient outcome. Its suppression is under the regulation of a novel feedback circuit consisting of HIF-1α/miR-210/HIF-3α. TIMP2 is an important regulator of ECM degradation and HCC metastasis. (Hepatology 2016;64:473-487).