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.

Sox9 confers stemness properties in hepatocellular carcinoma through Frizzled-7 mediated Wnt/ß-catenin signaling.

Sox9, an SRY-related HMG box transcription factor, is a progenitor/precursor cell marker of the liver expressed during embryogenesis and following liver injury. In this study, we investigated the role of Sox9 and its molecular mechanism with reference to stemness properties in hepatocellular carcinoma (HCC). Here, we observed upregulation of Sox9 in human HCC tissues compared with the non-tumorous liver counterparts (p < 0.001). Upregulation of Sox9 transcript level was associated with poorer tumor cell differentiation (p = 0.003), venous invasion (p = 0.026), advanced tumor stage (p = 0.044) and shorter overall survival (p = 0.042). Transcript levels of Sox9 and CD24 were positively correlated. Silencing of Sox9 in HCC cells inhibited in vitro cell proliferation and tumorsphere formation, sensitized HCC cells to chemotherapeutic agents, and suppressed in vivo tumorigenicity. In addition, knockdown of Sox9 suppressed HCC cell migration, invasion, and in vivo lung metastasis. Further studies showed that Sox9 endowed stemness features through activation of Wnt/ß-catenin signaling, which was confirmed by the partial rescue effect on tumorigenicity and self-renewal upon transfection of active ß-catenin in Sox9 knockdown cells. By ChIP and luciferase promoter assays, Frizzled-7 was identified to be the direct transcriptional target of Sox9. In conclusion, Sox9 confers stemness properties of HCC through Frizzled-7 mediated Wnt/ß-catenin pathway.

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).

TCGA whole-transcriptome sequencing data reveals significantly dysregulated genes and signaling pathways in hepatocellular carcinoma.

This study systematically evaluates the TCGA whole-transcriptome sequencing data of hepatocellular carcinoma (HCC) by comparing the global gene expression profiles between tumors and their corresponding nontumorous liver tissue. Based on the differential gene expression analysis, we identified a number of novel dysregulated genes, in addition to those previously reported. Top-listing upregulated (CENPF and FOXM1) and downregulated (CLEC4G, CRHBP, and CLEC1B) genes were successfully validated using qPCR on our cohort of 65 pairs of human HCCs. Further examination for the mechanistic overview by subjecting significantly upregulated and downregulated genes to gene set enrichment analysis showed that different cellular pathways were involved. This study provides useful information on the transcriptomic landscape and molecular mechanism of hepatocarcinogenesis for development of new biomarkers and further in-depth characterization.

PIM1 regulates glycolysis and promotes tumor progression in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is characteristically one of the most rapidly proliferating tumors which outgrows functional blood supply and results in regional oxygen deprivation. Overexpression of PIM1, a serine/threonine kinase, has been identified recently in human cancers. Knowledge on PIM1 in HCC is however, scarce. By immunohistochemical analysis on 56 human primary HCC samples, we observed overexpression of PIM1 in 39% of the cases. In two independent cohorts of paired primary and extra-hepatic metastatic HCC tissues, PIM1 expression was higher (p=0.002) in the extra-hepatic metastatic HCC tissues as compared with the corresponding primary HCCs. PIM1 was markedly up-regulated in multiple HCC cell lines in hypoxic condition (1% O2) versus normoxia (20% O2). Silencing of PIM1 suppressed HCC cell invasion in vitro as compared to non-target control, and decreased HCC cell proliferation in vitro and tumor growth and metastatic potential in vivo. Knockdown of PIM1 significantly reduced glucose uptake by HCC cells and was associated with decreased levels of p-AKT and key molecules in the glycolytic pathway. Taken together, PIM1 is up-regulated by hypoxia in HCC and promotes tumor growth and metastasis through facilitating cancer cell glycolysis. Targeting PIM1 may have potential role in the management of HCC.

Lysyl oxidase-like 2 is critical to tumor microenvironment and metastatic niche formation in hepatocellular carcinoma.

UNLABELLED: Poor prognosis of cancers, including hepatocellular carcinoma (HCC), is mainly associated with metastasis; however, the underlying mechanisms remain poorly understood. This article investigates the role of lysyl oxidase-like 2 (LOXL-2) in the biology of HCC metastasis. First, we showed that HCC metastasis relies on a collagen-modifying enzyme, LOXL2, which was significantly overexpressed in tumorous tissues and sera of HCC patients, indicating that LOXL2 may be a good diagnostic marker for HCC patients. Second, we delineated a complex, interlinked signaling network that involves multiple regulators, including hypoxia, transforming growth factor beta (TGF-ß), and microRNAs (miRNAs), converging to control the expression of LOXL2. We found not only that LOXL2 was regulated by hypoxia/hypoxia-inducible factor 1 alpha (HIF-1α), but also that TGF-ß activated LOXL2 transcription through mothers against decapentaplegic homolog 4 (Smad4), whereas two frequently underexpressed miRNA families, miR-26 and miR-29, cooperatively suppressed LOXL2 transcription through interacting with the 3' untranslated region of LOXL2. Third, we demonstrated the imperative roles of LOXL2 in modifying the extracellular matrix components in the tumor microenvironment and metastatic niche of HCC. LOXL2 promoted intrahepatic metastasis by increasing tissue stiffness, thereby enhancing the cytoskeletal reorganization of HCC cells. Furthermore, LOXL2 facilitated extrahepatic metastasis by enhancing recruitment of bone-marrow-derived cells to the metastatic site. CONCLUSION: These findings integrate the clinical relevance, molecular regulation, and functional implications of LOXL2 in HCC metastasis.

The impact of hypoxia in hepatocellular carcinoma metastasis.

Hypoxia is a common phenomenon in hepatocellular carcinoma (HCC). Hypoxia stabilizes transcription factor, hypoxia-inducible factor (HIF), to activate gene transcription. Expression of HIF is closely associated with metastasis and poor prognosis in HCC. HIF mediates expression of genes that are involved in every step of HCC metastasis including epithelial-mesenchymal transition, invasion of the extracellular matrix, intravasation, extravasation, and secondary growth of the metastases. Because HIF is the central regulator of HCC metastasis, HIF inhibitors are attractive tools when used alone or as combined treatment to curb HCC metastasis. This review will summarize the current findings on the impact of hypoxia/HIF in HCC, with a particular focus on cancer metastasis.

Regulation of hepatocarcinogenesis by microRNAs.

Liver cancer (hepatocellular carcinoma, HCC) is a major malignancy worldwide. Etiologically, hepatocarcinogenesis is closely associated with HBV and HCV infections; however, its underlying molecular mechanism is not completely understood. MicroRNAs are a class of small non-coding RNAs that negatively regulate gene expression by interacting with the 3'UTR of protein-coding mRNA. MicroRNAs are implicated in nearly all major biological and cellular events, and recent findings further link microRNA deregulation to human carcinogenesis. In this review, we will focus on the aberrant expression of miRNAs in liver cancer and the pathological implications and molecular functions of some well-characterized oncogenic and tumor suppressive miRNAs. Finally, the clinical prospect of miRNAs as a novel diagnostic and therapeutic intervention will be discussed.