RhoE is frequently down-regulated in hepatocellular carcinoma (HCC) and suppresses HCC invasion through antagonizing the Rho/Rho-kinase/myosin phosphatase target pathway.

UNLABELLED: Deregulation of Rho guanosine triphosphatase (GTPase) pathways plays an important role in tumorigenesis and metastasis of hepatocellular carcinoma (HCC). RhoE/Rnd3 belongs to an atypical subfamily of the RhoGTPase, the Rnd family, as it lacks the intrinsic GTPase activity and remains always in its active GTP-bound form. In this study we investigated the role of RhoE in HCC. We examined the expression of RhoE in primary HCC samples from patients predominantly infected with the hepatitis B virus (HBV) and found that the RhoE messenger RNA (mRNA) level was frequently down-regulated (83.1%, 59/71) in HCCs. Low expression of RhoE in the tumors was significantly associated with shorter disease-free survival (P = 0.020) of the patients. Knockdown of RhoE by short-hairpin RNA using a lentiviral approach led to increased cell motility and invasiveness in SMMC7721 and BEL7402 HCC cells. Moreover, in vivo an orthotopic liver injection model in nude mice further demonstrated that knockdown of RhoE enhanced local invasion of HCC cells in the livers, with more invasive tumor front and increased incidence of venous invasion. Mechanistically, stable knockdown of RhoE in HCC cells significantly enhanced the phosphorylation of myosin phosphatase, promoted assembly of stress fibers, and increased the formation of plasma membrane blebbings, all these changes and activities being associated with activation of the Rho/Rho-kinase (ROCK) pathway. CONCLUSION: RhoE was frequently down-regulated in predominantly HBV-associated HCCs and this down-regulation was associated with a more aggressive HCC phenotype. RhoE regulated the cytoskeleton remodeling and suppressed HCC motility and invasiveness by way of inhibiting the Rho/ROCK axis.

Caveolin-1 overexpression is associated with hepatocellular carcinoma tumourigenesis and metastasis.

Caveolin-1 (Cav1) has been implicated in diverse human cancers, yet its role in hepatocellular carcinoma (HCC) tumourigenesis and metastasis remains elusive. In the current study, we aim to provide a comprehensive understanding regarding the functional role of Cav1 in HCC tumourigenesis and metastasis. Cav1 expression was examined in a panel of human HCC cell lines using western blotting analysis and quantitative RT-PCR and human tissues by immunohistochemistry. Cav1 was not detected in normal liver cell line and all non-tumourous liver tissues but exclusively expressed in HCC cell lines and tissues. Dramatic expression of Cav1 was found in metastatic HCC cell lines and tumours, indicating a progressive increase of Cav1 expression along disease progression. Cav1 overexpression was significantly correlated with venous invasion (p = 0.036). To investigate the functions of Cav1 in HCC, Cav1 overexpressing and knockdown stable clones were established in HCC cells and their tumourigenicity and metastatic potential were examined. Overexpression of Cav1 promoted HCC cell growth, motility, and invasiveness, as well as tumourigenicity in vivo. Conversely, knockdown of Cav1 in metastatic HCC cells inhibited the motility and invasiveness and markedly suppressed the tumour growth and metastatic potential in vivo. Collectively, our findings have shown the exclusive expression of Cav1 in HCC cell lines and clinical samples and revealed an up-regulation of Cav1 along HCC progression. The definitive role of Cav1 in promoting HCC tumourigenesis was demonstrated, and we have shown for the first time in a mouse model that Cav1 promotes HCC metastasis.

Significance of serum DKK1 as a diagnostic biomarker in hepatocellular carcinoma.

Hyperactivation of the Wnt/ß-catenin signaling pathway has been implicated in cancers, including hepatocellular carcinoma (HCC). Dickkopf-1 (DKK1) is a secretory antagonist of the Wnt/ß-catenin signaling pathway and has been found to be upregulated in many cancer types. The functional role of DKK1 in cancer progression has been revealed in several studies but there is controversy with regard to its antitumor function; its oncogenic role seems to be context-dependent. Nevertheless, DKK1 has been proposed to be a potential new biomarker in several types of cancers. The paper by Shen et al. revealed the diagnostic accuracy of DKK1 as a serum biomarker for HCC in a large-scale, multicenter study. Their study demonstrated that serum DKK1 was high in both sensitivity and specificity in diagnosing HCC, especially early-stage HCC and α-fetoprotein-negative HCCs. The authors also demonstrated that combination of serum α-fetoprotein with serum DKK1 could further improve the diagnostic accuracy. Overall, their findings revealed the importance and significance of DKK1 in HCC diagnosis.

Novel MAGT1 Mutation Found in the First Chinese XMEN in Hong Kong.

The availability of next-generation sequencing (NGS) helps to resolve many of the diagnostic odysseys. Common variable immunodeficiency disease (CVID) is an entity encompassing a heterogenous group of conditions with hypogammaglobulinemia, and it is a diagnosis of exclusion. In recent years, with the advances of molecular diagnostics, more and more patients have been reclassified with more defined entities after their genetic causes were found. Here, we reported a young man, who was managed as CVID since childhood, presenting with recurrent infection, hypogammaglobulinemia, and immune thrombocytopenia (ITP). Finally, more than a decade after initial presentation, gene panel testing revealed a novel mutation in the MAGT1 gene. Collectively, the genetic findings and clinical presentations confirm the diagnosis of X-linked immunodeficiency with magnesium defect and Epstein-Barr virus infection and neoplasia (XMEN). MAGT1 is an evolutionarily conserved, magnesium-specific transporter expressed in all mammalian cells that plays an essential role in magnesium homeostasis. MAGT1 also acts as an accessory protein for STT3B, as catalytic subunits of the oligosaccharyltransferase protein complex, which carries out glycan chain transfer to proteins in the endoplasmic reticulum during N-glycosylation. Glycans play an essential role in the stability, maturation, and localization in glycoproteins that are important in our immune cells' function. Mutation of the gene resulted in a rare X-linked recessive condition XMEN. The disease has complete penetrance but variable expressivity. It is mainly associated with immunodeficiency, immunodysregulation, and predisposition to EBV-associated lymphoproliferation. Extraimmune manifestations have also been reported in some patient cohorts, including hepatic and neurological abnormalities. Overall, the presentation varies among patients and overlaps with other clinical entities, in which diagnosis is challenging. Before the era of NGS, traditional workup hinges heavily on phenotype studies, followed by single-gene sequencing. The diagnostic yield is low, and a significant delay in diagnosis is common. This case illustrated the importance of early consideration of molecular studies in complex immunological cases without obvious secondary causes as an integral part of patient management.

Deregulation of microRNA expression occurs early and accumulates in early stages of HBV-associated multistep hepatocarcinogenesis.

BACKGROUND & AIMS: Deregulation of microRNAs (miRNAs) plays an important role in human carcinogenesis. However, miRNA deregulation in the pre-malignant lesions and expression changes during multistep hepatocarcinogenesis remain elusive. METHODS: In this study, we investigated the expression changes of seven cancer-related miRNAs during the early stages of HBV related hepatocarcinogenesis. miRNA was extracted from formalin fixed paraffin embedded (FFPE) dysplastic nodules (DN), small HCCs, and their corresponding non-tumorous livers. Expression changes of miRNAs were examined by real-time RT-qPCR. RESULTS: We found that down-regulation of miR-145 and miR-199b and up-regulation of miR-224 were frequently observed in pre-malignant DNs and these changes persisted throughout HCC development. Restoration of miR-145 in both HepG2 and Hep3B HCC cells significantly inhibited cell proliferation and reduced cell migration and cell invasion. Furthermore, these inhibitory functions of miR-145 could be substantially reduced by an anti-miR-145 inhibitor. CONCLUSIONS: Our results showed that miRNA deregulation was an early event and accumulated throughout the various steps of HBV-associated hepatocarcinogenesis. Our findings also suggest that miR-145 is a candidate tumor suppressive miRNA and may play an important role in HCC development.

Akt phosphorylation of deleted in liver cancer 1 abrogates its suppression of liver cancer tumorigenesis and metastasis.

BACKGROUND & AIMS: Deleted in liver cancer 1 (DLC1), which encodes a Rho GTPase activating protein, is a bona fide tumor suppressor in hepatocellular carcinoma. Underexpression of DLC1 in cancer has been attributed to genomic deletion and epigenetic silencing. However, the regulatory mechanism of the tumor suppressive activity of DLC1 remains elusive. In this study, we elucidated a novel post-translational modification by which the activity of DLC1 is functionally regulated. METHODS: Molecular and biochemical approaches were employed to study Akt phosphorylation of DLC1. In vitro and in vivo functional assays were performed to elucidate the functional significance of Akt phosphorylation of DLC1. RESULTS: Phosphorylation of ectopically expressed and endogenous DLC1 was enhanced upon insulin induction or with Akt expression in liver cancer cell lines. Conversely, addition of a phosphatidylinositol 3-kinase/Akt pathway inhibitor or silencing of Akt attenuated the phosphorylation level of DLC1. Site-directed mutagenesis was employed to replace the serine residue of the consensus Akt substrate motifs of DLC1 with alanine. S567 of DLC1 was identified as the only target of Akt phosphorylation. S567 is well conserved in all DLC family members. DLC2 was phosphorylated by Akt at the corresponding residue. Functional assays demonstrated that the S567D phosphomimetic DLC1 mutant lost its inhibitory activities in tumorigenesis and metastasis of oncogenically transformed hepatoblasts in a mouse model. CONCLUSIONS: This study has revealed a novel post-translational modification that functionally deregulates the biologic activities of DLC1. Phosphorylation of DLC1 and DLC2 by Akt at the conserved residue points to a common regulatory mechanism of the DLC tumor suppressor family.

Clinicopathological and prognostic significance of serum and tissue Dickkopf-1 levels in human hepatocellular carcinoma.

BACKGROUND: Although Dickkopf-1 (DKK1) is known to be a negative regulator of the Wnt/ß-catenin pathway, it has been recently found to be upregulated in cancers. AIMS: We investigated the clinical and prognostic significance of both serum and transcript DKK1 and its functional roles in human hepatocellular carcinoma (HCC). METHODS: We evaluated the expression level of DKK1 in both tissue and serum samples from patients with HCC using GeneChip microarray and real-time-quantitative PCR and sandwich ELISA system respectively. The clinicopathological and prognostic significance of serum and tissue DKK1 levels was examined. Functional characterization of DKK1 with regard to cell migration, invasion and tumour growth was performed. RESULTS: Both DKK1 transcript and serum protein were upregulated in a stepwise manner in human HCCs. Its transcript levels were associated with more aggressive tumour behaviour, in terms of venous invasion (P = 0.003), advanced tumour stage (P = 0.003). DKK1 transcript correlated with shorter overall (P = 0.006) and disease-free survival (P = 0.012), and higher serum DKK1 levels correlated with shorter disease-free survival (P = 0.046). Knockdown of DKK1 significantly reduced both migratory and invasive abilities of HCC cells, whereas overexpression of DKK1 enhanced the tumour formation efficiency and tumour growth in vivo. CONCLUSIONS: Serum and tissue DKK1 levels increased in a stepwise manner in multistep hepatocarcinogenesis and had prognostic significance. DKK1 plays a functional role in cell migration, invasion and tumour growth.

Rho-kinase 2 is frequently overexpressed in hepatocellular carcinoma and involved in tumor invasion.

UNLABELLED: Deregulation of Rho family small guanosine triphosphatases has been implicated in human carcinogenesis. Rho-kinases are downstream effectors of Rho guanosine triphosphatases in the regulation of cytoskeletal reorganization and cell motility. However, their functions in human cancers remain elusive. In this study, we aimed to investigate the role of Rho-kinases in hepatocellular carcinoma (HCC) tumor progression and invasion. We first examined the expression of the two Rho-kinases (ROCK1 and ROCK2) in human HCC, and found that ROCK2 was frequently overexpressed in primary HCCs (22/41 [53.66%]). Clinico-pathological analysis revealed that overexpression of ROCK2 was significantly associated with the presence of tumor microsatellite formation (P = 0.005), suggesting that deregulation of ROCK2 may contribute to the intrahepatic metastasis of HCC. Consistently, we demonstrated that stable overexpression of ROCK2 significantly enhanced cell motility and invasiveness in HCC cells. Conversely, stable knockdown of ROCK2 by short hairpin RNA approach remarkably reduced HCC cell migration and invasion. Moreover, orthotopic liver xenograft models provided further support that stable knockdown of ROCK2 suppressed HCC invasion in vivo. Stable knockdown of ROCK2 in HCC cells significantly inhibited Golgi reorientation, myosin phosphatase phosphorylation, and formations of stress fibers, filopodia, and lamellipodia; these molecular and cellular events are crucial for cell motility and cancer invasion. CONCLUSION: Our results indicate that ROCK2 was overexpressed in human HCCs, and this overexpression was associated with a more aggressive biological behavior. Our findings also demonstrate that ROCK2 played a significant role in regulating cytoskeletal events and contributed to the invasion of HCC.

Rapamycin and CCI-779 inhibit the mammalian target of rapamycin signalling in hepatocellular carcinoma.

BACKGROUND: The mammalian target of rapamycin (mTOR), which phosphorylates p70S6K and 4EBP1 and activates the protein translation process, is upregulated in cancers and its activation may be involved in cancer development. AIMS: In this study, we investigated the tumour-suppressive effects of rapamycin and its new analogue CCI-779 on hepatocellular carcinoma (HCC). METHODS: Rapamycin and its new analogue CCI-779 were applied to treat HCC cells. Cell proliferation, cell cycle profile and tumorigenicity were analysed. RESULTS: In human HCCs, we observed frequent (67%, 37/55) overexpression of mTOR transcripts using real-time reverse transcriptase-polymerase chain reaction. Upon drug treatment, PLC/PRF/5 showed the greatest reduction in cell proliferation using the colony formation assay, as compared with HepG2, Hep3B and HLE. Rapamycin was a more potent antiproliferative agent than CCI-779 in HCC cell lines. Proliferation assays by cell counting showed that the IC(50) value of rapamycin was lower than that of CCI-779 in PLC/PRF/5 cells. Furthermore, flow cytometric analysis showed that both drugs could arrest HCC cells in the G(1) phase but did not induce apoptosis of these cells, suggesting that these mTOR inhibitors are cytostatic rather than cytotoxic. Upon rapamycin and CCI-779 treatment, the phosphorylation level of mTOR and p70S6K in HCC cell lines was significantly reduced, indicating that both drugs can suppress mTOR activity in HCC cells. In addition, both drugs significantly inhibited the growth of xenografts of PLC/PRF/5 cells in nude mice. CONCLUSIONS: Our findings indicate that rapamycin and its clinical analogue CCI-779 possess tumour-suppressive functions towards HCC cells.

HAI-2 is epigenetically downregulated in human hepatocellular carcinoma, and its Kunitz domain type 1 is critical for anti-invasive functions.

Pharmacological demethylation-based gene expression profile analysis is a useful tool to identify epigenetically silenced tumour suppressor genes. HGF activator inhibitor 2 (HAI-2), a serine protease inhibitor, has been identified as one of the candidate tumour suppressor genes in human hepatocellular carcinoma (HCC) with this technique. In this study, we aimed to characterise the epigenetic status and tumour suppressive function of HAI-2 in HCC. We validated that HAI-2 expression was either absent or low in most of the HCC cell lines tested, and 5-Aza-2'-deoxycytidine treatment significantly restored its expression in 9 (75%) of these 12 cell lines. HAI-2 was found to be frequently underexpressed in human HCCs (p < 0.001). With bisulphite DNA sequencing and methylation-specific PCR, we found that the promoter of the HAI-2 gene was frequently hypermethylated in both HCC cell lines and human HCCs. Ectopic expression of HAI-2 significantly inhibited cell migration and invasiveness of HCC cells in vitro and suppressed tumourigenicity in vivo. In addition, we also provided the first evidence that HAI-2 mediated its tumour suppressor function via the Kunitz domain 1 (KD-1), as KD-1 but not KD-2 inactivating mutant abolished its anti-tumour invasiveness in vitro. Our findings suggest that HAI-2 is a candidate tumour suppressor gene that is frequently hypermethylated and underexpressed in human HCCs, and the KD-1 domain of HAI-2 is the key region responsible for its anti-invasive function.

Dishevelled-3 phosphorylation is governed by HIPK2/PP1Cα/ITCH axis and the non-phosphorylated form promotes cancer stemness via LGR5 in hepatocellular carcinoma.

Dishevelled-3 (Dvl3) is regarded as a binding hub with many different interacting partners. However, its regulation and mechanism on cancer stemness remain to be explored. In this study, we showed that Dvl3 was significantly overexpressed in human hepatocellular carcinomas (HCCs) and promoted cancer stemness both in vitro and in vivo. We found that the non-phosphorylated (NP)-Dvl3 was more stable than the phosphorylated form, more active in activating ß-catenin transcriptional activity, and more potent in enhancing self-renewal ability in HCC cells. Mechanistically, we confirmed that the homeodomain-interacting protein kinase-2 (HIPK2) and E3 ubiquitin ligase ITCH were able to physically bind to Dvl3 protein. Knockdown of HIPK2 and the protein phosphatase regulatory unit C-alpha (PP1Cα) resulted in sustained Dvl3 phosphorylation and hence decrease in the NP form of Dvl3. On the other hand, knockdown of E3 ubiquitin ligase ITCH reduced the phosphorylation-induced degradation and stabilized the phosphorylated Dvl3 protein. Furthermore, the NP-Dvl3 enhanced the LGR5 promoter activity to upregulate LGR5 expression, which was associated with increased cancer stemness in HCC. Our findings established that HIPK2/PP1Cα/ITCH axis sustains the de-phosphorylation of Dvl3. This post-translational modification of Dvl3 in turn maintains LGR5 expression and enhances the cancer stemness properties in HCC.

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.

Upregulation of the Wnt co-receptor LRP6 promotes hepatocarcinogenesis and enhances cell invasion.

BACKGROUND: Activation of the Wnt/ß-catenin signaling pathway plays a crucial role in hepatocellular carcinoma (HCC). Low-density lipoprotein (LDL) receptor-related protein-6 (LRP6) is one of the co-receptors of the Wnt/ß-catenin pathway and forms a signaling complex with Wnt ligand and Frizzled receptor to activate downstream signaling. However, the role of LRP6 in hepatocarcinogenesis is unclear. In this study, we examined its expression and roles in human HCC. METHODOLOGY/PRINCIPAL FINDINGS: Using real-time quantitative RT-PCR, we found that LRP6 was frequently (45%) overexpressed in human HCCs (P = 0.003). In vitro studies showed that ectopic expression of LRP6 increased the protein level of ß-catenin. Moreover, overexpression of the full-length and constitutively active LRP6, respectively, activated the WNT/ß-catenin signaling pathway, as shown by the TCF/ß-catenin reporter assay. With regard to the effects of LRP6 overexpression in HCC cells, stable overexpression of the constitutively active LRP6 in BEL-7402 HCC cells enhanced cell proliferation, cell migration, and invasion in vitro as well as tumorigenicity in nude mice. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that overexpression of LRP6 contributes to the hyperactivation of the Wnt/ß-catenin signaling pathway in human HCCs and suggest it may play a role in hepatocarcinogenesis.

Deleted in liver cancer 2 (DLC2) was dispensable for development and its deficiency did not aggravate hepatocarcinogenesis.

DLC2 (deleted in liver cancer 2), a Rho GTPase-activating protein, was previously shown to be underexpressed in human hepatocellular carcinoma and has tumor suppressor functions in cell culture models. We generated DLC2-deficient mice to investigate the tumor suppressor role of DLC2 in hepatocarcinogenesis and the function of DLC2 in vivo. In this study, we found that, unlike homologous DLC1, which is essential for embryonic development, DLC2 was dispensable for embryonic development and DLC2-deficient mice could survive to adulthood. We also did not observe a higher incidence of liver tumor formation or diethylnitrosamine (DEN)-induced hepatocarcinogenesis in DLC2-deficient mice. However, we observed that DLC2-deficient mice were smaller and had less adipose tissue than the wild type mice. These phenotypes were not due to reduction of cell size or defect in adipogenesis, as observed in the 190B RhoGAP-deficient mouse model. Together, these results suggest that deficiency in DLC2 alone does not enhance hepatocarcinogenesis.

Tissue factor pathway inhibitor-2 as a frequently silenced tumor suppressor gene in hepatocellular carcinoma.

UNLABELLED: In HCC, inactivation of tumor suppressor genes plays a significant role in carcinogenesis. Apart from deletions and mutations, growing evidence has indicated that epigenetic alterations including aberrant promoter methylation and histone deacetylation are also implicated in inactivation of tumor suppressor genes. The goal of this study was to identify epigenetically silenced candidate tumor suppressor genes in human HCC by comparing the changes in oligonucleotide microarray gene expression profiles in HCC cell lines upon pharmacological treatment with the demethylating agent 5-Aza-2'-deoxycytidine (5-Aza-dC). By analyzing the gene expression profiles, we selected tissue factor pathway inhibitor-2 (TFPI-2), a Kunitz-type serine protease inhibitor, for validation and further characterization. Our results showed that TFPI-2 was frequently silenced in human HCC and HCC cell lines. TFPI-2 was significantly underexpressed in approximately 90% of primary HCCs when compared with their corresponding nontumorous livers. TFPI-2 promoter methylation was detected in 80% of HCC cell lines and 47% of human HCCs and was accompanied by reduced TFPI-2 messenger RNA expression. In addition, TFPI-2 expression in HCC cell lines can be robustly restored by combined treatment with 5-Aza-dC and histone deacetylase inhibitor trichostatin A. These findings indicate that TFPI-2 is frequently silenced in human HCC via epigenetic alterations, including promoter methylation and histone deacetylation. Moreover, ectopic overexpression of TFPI-2 significantly suppressed the proliferation and invasiveness of HCC cells. CONCLUSION: Our findings suggest that TFPI-2 is a candidate tumor suppressor gene in human HCC.

Tensin2 variant 3 is associated with aggressive tumor behavior in human hepatocellular carcinoma.

Tensins are a new family of proteins that act as an important link among extracellular matrix, actin cytoskeleton, and signal transduction and have been implicated in human cancers. Tensin2 was initially identified in a search for new tensin family members that share extensive sequence homology with tensin1. Tensin2 was highly expressed in liver tissues. A recent study reported that one of the splicing variants of tensin2, variant 3, promotes cell migration. In the present study, we aimed to elucidate the role of variant 3 in hepatocarcinogenesis by assessing the expression of variant 3 mRNA in hepatocellular carcinoma (HCC) tissue and ectopically expressing variant 3 in HCC cell lines. Analysis of variant 3 expression in human HCC tissue revealed it was overexpressed in 46% (23/50) of tumor tissues as compared with the corresponding nontumorous livers. High expression of variant 3 was significantly associated with venous invasion (P = .037), tumor microsatellite formation (P = .022), and tumor nonencapsulation (P = .049). Our ectopic expression study showed that variant 3 significantly promoted the cell growth and motility of HCC cells. The clonal transfectants of variant 3 were more closely packed and resulted in a higher saturation density than in the control vector transfectants. Variant 3 expression also enhanced the proliferation rate in culture and in vivo tumorigenicity in nude mice. In conclusion, we reveal a novel role for variant 3 in the progression of HCC and suggest the feasibility of elevated variant 3 expression as a tumor progression marker for HCC.