CA10 is associated with HBV-related hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is the main threat for the patients infected with hepatitis B virus (HBV), but the oncogenic mechanism of HBV-related HCC is still controversial. Previously, we have found that several HBV surface gene (HBS) non-sense mutations are oncogenic. Among these mutations, sW182* was found to have the most potent oncogenicity. In this study, we found that Carbonic Anhydrase X (CA10) level was specifically increased in sW182* mutant-expressing cells. CA10 overexpression was also associated with HBS nonsense mutation in HBV-related HCC tumor tissues. Transformation and tumorigenesis assays revealed that CA10 had significant oncogenic activity. In addition, CA10 overexpression resulted in dysregulation of apoptosis-related proteins, including Mcl-1, Bcl-2, Bcl-xL and Bad. While searching for the regulatory mechanism of CA10, miR-27b was found to downregulate CA10 expression by regulating its mRNA degradation and its expression was decreased in sW182* mutant cells. Moreover, CA10 overexpression was associated with down-regulation of miR-27b in human HBV-related HCC tumor tissues with sW182* mutation. Therefore, induction of the expression of CA10 through repression of miR-27b by sW182* might be one mechanism involved in HBS mutation-related hepatocarcinogenesis.

TARBP2-mediated destabilization of Nanog overcomes sorafenib resistance in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a lethal human malignancy and a leading cause of cancer-related death worldwide. Patients with HCC are often diagnosed at an advanced stage, and the prognosis is usually poor. The multikinase inhibitor sorafenib is the first-line treatment for patients with advanced HCC. However, cases of primary or acquired resistance to sorafenib have gradually increased, leading to a predicament in HCC therapy. Thus, it is critical to investigate the mechanism underlying sorafenib resistance. Transactivation response element RNA-binding protein 2 (TARBP2) is a multifaceted miRNA biogenesis factor that regulates cancer stem cell (CSC) properties. The tumorigenicity and drug resistance of cancer cells are often enhanced due to the acquisition of CSC features. However, the role of TARBP2 in sorafenib resistance in HCC remains unknown. Our results demonstrate that TARBP2 is significantly downregulated in sorafenib-resistant HCC cells. The TARBP2 protein was destabilized through autophagic-lysosomal proteolysis, thereby stabilizing the expression of the CSC marker protein Nanog, which facilitates sorafenib resistance in HCC cells. In summary, here we reveal a novel miRNA-independent role of TARBP2 in regulating sorafenib resistance in HCC cells.

Metabolic risk factors are associated with non-hepatitis B non-hepatitis C hepatocellular carcinoma in Taiwan, an endemic area of chronic hepatitis B.

Metabolic risk factors, such as obesity, fatty liver, high lipidemia, and diabetes mellitus are associated with increased risk for nonviral hepatocellular carcinoma (HCC); however, few nonviral HCC studies have stratified patients according to underlying etiologies. From 2005 to 2011, 3,843 patients with HCC were recruited into the Taiwan Liver Cancer Network. Of these patients, 411 (10.69%) who were negative for hepatitis B virus (HBV), surface antigen, HBV DNA, and anti-hepatitis C virus (HCV) antibody were classified as non-HBV non-HCV (NBNC)-HCC. Detailed clinical analyses of these patients were compared with age- and sex-matched patients with HBV-HCC or HCV-HCC for the associated metabolic risk factors. For this comparison, 420 patients with HBV-HCC and 420 patients with HCV-HCC were selected from the 3,843 patients with HCC. Multivariate analyses showed fatty liver (by echography), high triglyceride levels (>160 mg/dL), and diabetes mellitus history to be significantly associated only with NBNC-HCC and not with the matched patients with HBV- or HCV-HCC. When the patients with HCC were further divided into four groups based on history of alcoholism and cirrhotic status, the group without alcoholism and without cirrhosis exhibited the strongest association with the metabolic risk factors. Based on trend analyses, patients with NBNC-HCC with or without alcoholism were significantly different from the matched patients with HBV- or HCV-HCC, except for patients with alcoholism and cirrhosis, in having more than two of the above three risk factors. Conclusion: Metabolic risk factors are significantly associated with nonviral HCC, especially for patients without alcoholism in Taiwan. Because the prevalence of viral HCC is decreasing due to the success of universal vaccination and antiviral therapy, strategies for cancer prevention, prediction, and surveillance for HCC will require modification. (Hepatology Communications 2018;2:747-759).

The Hepatitis Viral Status in Patients With Hepatocellular Carcinoma: a Study of 3843 Patients From Taiwan Liver Cancer Network.

Hepatocellular carcinoma (HCC) is the leading cancer death in Taiwan. Chronic viral hepatitis infections have long been considered as the most important risk factors for HCC in Taiwan. The previously published reports were either carried out by individual investigators with small patient numbers or by large endemic studies with limited viral marker data. Through collaboration with 5 medical centers across Taiwan, Taiwan liver cancer network (TLCN) was established in 2005. All participating centers followed a standard protocol to recruit liver cancer patients along with their biosamples and clinical data. In addition, detailed viral marker analysis for hepatitis B virus (HBV) and hepatitis C virus (HCV) were also performed. This study included 3843 HCC patients with available blood samples in TLCN (recruited from November 2005 to April 2011). There were 2153 (56.02%) patients associated with HBV (HBV group); 969 (25.21%) with HCV (HCV group); 310 (8.07%) with both HBV and HCV (HBV+HCV group); and 411 (10.69%) were negative for both HBV and HCV (non-B non-C group). Two hundred two of the 2463 HBV patients (8.20%) were HBsAg(-), but HBV DNA (+). The age, gender, cirrhosis, viral titers, and viral genotypes were all significantly different between the above 4 groups of patients. The median age of the HBV group was the youngest, and the cirrhotic rate was lowest in the non-B non-C group (only 25%). This is the largest detailed viral hepatitis marker study for HCC patients in the English literatures. Our study provided novel data on the interaction of HBV and HCV in the HCC patients and also confirmed that the HCC database of TLCN is highly representative for Taiwan and an important resource for HCC research.

CARMA3 Represses Metastasis Suppressor NME2 to Promote Lung Cancer Stemness and Metastasis.

RATIONALE: CARD-recruited membrane-associated protein 3 (CARMA3) is a novel scaffold protein that regulates nuclear factor (NF)-κB activation; however, the underlying mechanism of CARMA3 in lung cancer stemness and metastasis remains largely unknown. OBJECTIVES: To investigate the molecular mechanisms underlying the involvement of CARMA3 in non-small cell lung cancer progression. METHODS: The expression levels of CARMA3 and NME2 in a cohort of patients with lung cancer (n = 91) were examined by immunohistochemistry staining and assessed by Kaplan-Meier survival analysis. The effects of CARMA3, microRNA-182 (miR-182), and NME2 on cancer stemness and metastasis were measured in vitro and in vivo. Chromatin immunoprecipitation and luciferase reporter assays were performed to determine the mechanisms of NF-κB-driven miR-182 expression and NME2 regulation. MEASUREMENTS AND MAIN RESULTS: We observed that CARMA3 inversely correlated with NME2 expression in patients with lung cancer (Pearson correlation coefficient: R = -0.24; P = 0.022). NME2 levels were significantly decreased in tumor tissues compared with adjacent normal lung tissues (P < 0.001), and patients with lung cancer with higher levels of NME2 had longer survival outcomes (overall survival, P < 0.01; disease-free survival, P < 0.01). Mechanistically, CARMA3 promoted cell motility by reducing the level of NME2 through the NF-κB/miR-182 pathway and by increasing cancer stem cell properties and metastasis in lung cancer. CONCLUSIONS: We identified a novel mechanism of CARMA3 in lung cancer stemness and metastasis through the negative regulation of NME2 by NF-κB-dependent induction of miR-182. Our findings provide an attractive strategy for targeting the CARMA3/NF-κB/miR-182 pathway as a potential treatment for lung cancer.

E1A-mediated inhibition of HSPA5 suppresses cell migration and invasion in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is defined by reduced expression of the estrogen receptor, progesterone receptor, and HER2. TNBC is an especially aggressive group of breast cancers with poor prognosis. There are currently no validated molecular targets to effectively treat this disease. Thus, it is necessary to identify effective molecular targets and therapeutic strategies for TNBC patients. METHODS: The expression of HSPA5 in patients with breast cancer was examined by immunohistochemistry. The association of HSPA5 expression with tumor grade and metastatic events in TNBC patients was analyzed using the Oncomine database. The knockdown and overexpression of HSPA5 protein were performed to investigate the effects on E1A-suppressed cell migration/invasion of TNBC using in vitro transwell assays and tumor growth/experimental metastasis studies in animal models. RESULTS: The expression of HSPA5 was positively correlated with high-grade tumors, metastatic events, and poor overall survival in breast cancer patients with TNBC. E1A-inhibited HSPA5 expression suppressed cell migration/invasive ability of TNBC cell lines. Moreover, E1A significantly abolished lung metastases from breast cancer cells by inhibiting HSPA5 expression in a xenograft tumor model. CONCLUSIONS: The overexpression of HSPA5 is critical for high-risk metastasis of breast cancer and TNBC. The results of our study suggest that HSPA5 may be a crucial mediator of E1A-suppressed metastatic ability of breast cancer cells. Thus, E1A may be a potential target for diagnosis and individualized treatment in clinical practice.

De-acetylation and degradation of HSPA5 is critical for E1A metastasis suppression in breast cancer cells.

Elevated expression of heat shock protein 5 (HSPA5) promotes drug resistance and metastasis and is a marker of poor prognosis in breast cancer patients. Adenovirus type 5 E1A gene therapy has demonstrated antitumor efficacy but the mechanisms of metastasis-inhibition are unclear. Here, we report that E1A interacts with p300 histone acetyltransferase (HAT) and blocks p300-mediated HSPA5 acetylation at K353, which in turn promotes HSPA5 ubiquitination by GP78 (E3 ubiquitin ligase) and subsequent proteasome-mediated degradation. Our findings point out the Ying-Yang regulation of two different post-translational modifications (ubiquitination and acetylation) of HSPA5 in tumor metastasis.

Vascular endothelial growth factor-C upregulates cortactin and promotes metastasis of esophageal squamous cell carcinoma.

BACKGROUND: Vascular endothelial growth factor-C (VEGF-C) plays an important role during cancer progression and metastasis through activation of VEGF receptors. However, the role of VEGF-C in esophageal squamous cell carcinoma (ESCC) remains unclear. METHODS: The expression of VEGF-C in advanced stages of esophageal cancer was examined by immunohistochemistry and its expression was correlated with the protein level of cortactin (CTTN) by Western blot. Knockdown and overexpression of the CTTN protein were respectively performed to investigate the effects on VEGF-C-enhanced ESCC migration/invasion by in vitro transwell assay, cell tracing assay, and tumor growth/experimental metastasis in animal models. RESULTS: The expression of VEGF-C was positively correlated with tumor status and poor clinical prognosis in patient with esophageal cancer. VEGF-C-upregulated CTTN expression contributed the migration/invasive abilities of ESCC cell lines through Src-mediated downregulation of miR-326. Moreover, knockdown of CTTN expression significantly abolished VEGF-C-induced tumor growth and experimental lung metastasis in vivo. CONCLUSIONS: Upregulation of CTTN is critical for VEGF-C-mediated tumor growth and metastasis of ESCC. These finding suggest that VEGF-C upregulated CTTN expression through Src-mediated downregulation of miR-326. CTTN may be a crucial mediator of VEGF-C-involved ESCC metastasis, which provides a potential target for diagnosis and individualized treatment in clinical practice.

miR326 maturation is crucial for VEGF-C-driven cortactin expression and esophageal cancer progression.

Esophageal cancer is an aggressive human malignancy with increasing incidence in the developed world. VEGF-C makes crucial contributions to esophageal cancer progression that are not well understood. Here, we report the discovery of regulatory relationship in esophageal cancers between the expression of VEGF-C and cortactin (CTTN), a regulator of the cortical actin cytoskeleton. Upregulation of CTTN expression by VEGF-C enhanced the invasive properties of esophageal squamous cell carcinoma in vitro and tumor metastasis in vivo. Mechanistic investigations showed that VEGF-C increased CTTN expression by downregulating Dicer-mediated maturation of miR326, thereby relieving the suppressive effect of miR326 on CTTN expression. Clinically, expression of Dicer and miR326 correlated with poor prognosis in patients with esophageal cancer. Our findings offer insights into how VEGF-C enhances the robust invasive and metastatic properties of esophageal cancer, which has potential implications for the development of new biomarkers or therapies in this setting.

Arsenic trioxide inhibits CXCR4-mediated metastasis by interfering miR-520h/PP2A/NF-κB signaling in cervical cancer.

BACKGROUND: Arsenic apparently affects numerous intracellular signal transduction pathways and causes many alterations leading to apoptosis and differentiation in malignant cells. We and others have demonstrated that arsenic inhibits the metastatic capacity of cancer cells. Here we present additional mechanistic studies to elucidate the potential of arsenic as a promising therapeutic inhibitor of metastasis. METHODS: The effects of arsenic trioxide (ATO) on human cervical cancer cell lines migration and invasion were observed by transwell assays. In experimental metastasis assays, cancer cells were injected into tail veins of severe combined immunodeficient mice for modeling metastasis. The mechanisms involved in ATO regulation of CXCR4 were analyzed by immunoblot, real-time polymerase chain reaction, and luciferase reporter assays. Immunohistochemistry was utilized to identify PP2A/C and CXCR4 protein expressions in human cervical cancer tissues. RESULTS: ATO inhibited CXCR4-mediated cervical cancer cell invasion in vitro and distant metastasis in vivo. We determined that ATO modulates the pivotal nuclear factor-kappa B (NF-κB)/CXCR4 signaling pathway that contributes to cancer metastasis. Substantiating our findings, we demonstrated that ATO activates PP2A/C activity by downregulating miR-520h, which results in IKK inactivation, IκB-dephosphorylation, NF-κB inactivation, and, subsequently, a reduction in CXCR4 expression. Furthermore, PP2A/C was reduced during cervical carcinogenesis, and the loss of PP2A/C expression was closely associated with the nodal status of cervical cancer patients. CONCLUSIONS: Our results indicate a functional link between ATO-mediated PP2A/C regulation, CXCR4 expression, and tumor-suppressing ability. This information will be critical in realizing the potential for synergy between ATO and other anti-cancer agents, thus providing enhanced benefit in cancer therapy.

Oxidative stress enhances Axl-mediated cell migration through an Akt1/Rac1-dependent mechanism.

Persistent oxidative stress is common in cancer cells because of abnormal generation of reactive oxygen species (ROS) and has been associated with malignant phenotypes, such as chemotherapy resistance and metastasis. Both overexpression of Axl and abnormal ROS elevation have been linked to cell transformation and increased cell migration. However, the relationship between Axl and ROS in malignant cell migration has not been previously evaluated. Using an in vitro human lung cancer model, we examined the redox state of lung adenocarcinoma cell lines of low metastatic (CL1-0) and high metastatic (CL1-5) potentials. Here we report that Axl activation elicits ROS accumulation through the oxidase-coupled small GTPase Rac1. We also observed that oxidative stress could activate Axl phosphorylation to synergistically enhance cell migration. Further, Axl signaling activated by H2O2 treatment results in enhancement of cell migration via a PI3K/Akt-dependent pathway. The kinase activity of Axl is required for the Axl-mediated cell migration and prolongs the half-life of phospho-Akt under oxidative stress. Finally, downregulation of Akt1, but not Akt2, by RNAi in Axl-overexpressing cells inhibits the amount of activated Rac1 and the ability to migrate induced by H2O2 treatment. Together, these results show that a novel Axl-signaling cascade induced by H2O2 treatment triggers cell migration through the PI3K/Akt1/Rac1 pathway. Elucidation of redox regulation in Axl-related malignant migration may provide new molecular insights into the mechanisms underlying tumor progression.

Angiopoietin-like protein 1 suppresses SLUG to inhibit cancer cell motility.

Angiopoietin-like protein 1 (ANGPTL1) is a potent regulator of angiogenesis. Growing evidence suggests that ANGPTL family proteins not only target endothelial cells but also affect tumor cell behavior. In a screen of 102 patients with lung cancer, we found that ANGPTL1 expression was inversely correlated with invasion, lymph node metastasis, and poor clinical outcomes. ANGPTL1 suppressed the migratory, invasive, and metastatic capabilities of lung and breast cancer cell lines in vitro and reduced metastasis in mice injected with cancer cell lines overexpressing ANGPTL1. Ectopic expression of ANGPTL1 suppressed the epithelial-to-mesenchymal transition (EMT) by reducing the expression of the zinc-finger protein SLUG. A microRNA screen revealed that ANGPTL1 suppressed SLUG by inducing expression of miR-630 in an integrin α(1)ß(1)/FAK/ERK/SP1 pathway-dependent manner. These results demonstrate that ANGPTL1 represses lung cancer cell motility by abrogating the expression of the EMT mediator SLUG.

The role of the VEGF-C/VEGFRs axis in tumor progression and therapy.

Vascular endothelial growth factor C (VEGF-C) has been identified as a multifaceted factor participating in the regulation of tumor angiogenesis and lymphangiogenesis. VEGF-C is not only expressed in endothelial cells, but also in tumor cells. VEGF-C signaling is important for progression of various cancer types through both VEGF receptor-2 (VEGFR-2) and VEGF receptor-3 (VEGFR-3). Likewise, both receptors are expressed mainly on endothelial cells, but also expressed in tumor cells. The dimeric VEGF-C undergoes a series of proteolytic cleavage steps that increase the protein binding affinity to VEGFR-3; however, only complete processing, removing both the N- and C-terminal propeptides, yields mature VEGF-C that can bind to VEGFR-2. The processed VEGF-C can bind and activate VEGFR-3 homodimers and VEGFR-2/VEGFR-3 heterodimers to elicit biological responses. High levels of VEGF-C expression and VEGF-C/VEGFRs signaling correlate significantly with poorer prognosis in a variety of malignancies. Therefore, the development of new drugs that selectively target the VEGF-C/VEGFRs axis seems to be an effective means to potentiate anti-tumor therapies in the future.

Receptor tyrosine kinase AXL is induced by chemotherapy drugs and overexpression of AXL confers drug resistance in acute myeloid leukemia.

By using a novel profiling analysis of protein tyrosine kinases differentially expressed in the sensitive and refractory leukemia from the same patients we found that AXL was upregulated in drug-resistant leukemia. Furthermore, AXL could be induced by chemotherapy drugs in the acute myeloid leukemia U937 cells and this induction was dependent on the CCWGG methylation status of the AXL promoter. In U937 cells ectopically overexpressing AXL, addition of exogenous Gas6 induced AXL phosphorylation and activation of the Akt and ERK1/2 survival pathways. The Gas6-AXL activation pathway of drug resistance was associated with increased expression of Bcl-2 and Twist. These results show that upregulation of AXL by chemotherapy might induce drug resistance in acute myeloid leukemia in the presence of Gas6 stimulation.

Carbonic anhydrase III promotes transformation and invasion capability in hepatoma cells through FAK signaling pathway.

Carbonic anhydrase III (CAIII) is distinguished from the other members of the CA family by low carbon dioxide hydratase activity, resistance to the CA inhibitor acetazolamide, and a predominant expression in the liver of males. In this report the effects of CAIII expression on liver cancer cells invasiveness were explored. Overexpression of CAIII in the HCC cell line SK-Hep1 resulted in increased anchorage-independent growth and invasiveness. And siRNA-mediated silencing of CAIII expression decreased the invasive ability of SK-Hep1 cells. Furthermore, CAIII transfectants showed elevated focal adhesion kinase (FAK) and Src activity. Silencing of FAK expression in CAIII transfectants led to suppression of HCC cell invasion. More importantly, the CAIII transfectants acidified the culture medium at an accelerated speed than the control cells did. Taken together, these data suggest that the CAIII-promoted invasive ability of HCC cells may probably be mediated through, at least in part, the FAK signaling pathway via intracellular and/or extracellular acidification.

CTGF enhances the motility of breast cancer cells via an integrin-alphavbeta3-ERK1/2-dependent S100A4-upregulated pathway.

Connective tissue growth factor (CTGF) expression is elevated in advanced stages of breast cancer, but the regulatory role of CTGF in invasive breast cancer cell phenotypes is unclear. Presently, overexpression of CTGF in MCF-7 cells (MCF-7/CTGF cells) enhanced cellular migratory ability and spindle-like morphological alterations, as evidenced by actin polymerization and focal-adhesion-complex aggregation. Reducing the CTGF level in MDA-MB-231 (MDA231) cells by antisense CTGF cDNA (MDA231/AS cells) impaired cellular migration and promoted a change to an epithelial-like morphology. A neutralizing antibody against integrin alphavbeta3 significantly attenuated CTGF-mediated ERK1/2 activation and cellular migration, indicating that the integrin-alphavbeta3-ERK1/2 signaling pathway is crucial in mediating CTGF function. Moreover, the cDNA microarray analysis revealed CTGF-mediated regulation of the prometastatic gene S100A4. Transfection of MCF-7/CTGF cells with AS-S100A4 reversed the CTGF-induced cellular migratory ability, whereas overexpression of S100A4 in MDA231/AS cells restored their high migratory ability. Genetic and pharmacological manipulations suggested that the CTGF-mediated S100A4 upregulation was dependent on ERK1/2 activation, with expression levels of CTGF and S100A4 being closely correlated with human breast tumors. We conclude that CTGF plays a crucial role in migratory/invasive processes in human breast cancer by a mechanism involving activation of the integrin-alphavbeta3-ERK1/2-S100A4 pathway.

Sulfasalazine suppresses drug resistance and invasiveness of lung adenocarcinoma cells expressing AXL.

Metastasis and drug resistance are the major causes of mortality in patients with non-small cell lung cancer (NSCLC). Several receptor tyrosine kinases (RTKs), including AXL, are involved in the progression of NSCLC. The AXL/MER/SKY subfamily is involved in cell adhesion, motility, angiogenesis, and signal transduction and may play a significant role in the invasiveness of cancer cells. Notably, no specific inhibitors of AXL have been described. A series of CL1 sublines with progressive invasiveness established from a patient with NSCLC has been identified that positively correlates with AXL expression and resistance to chemotherapeutic drugs. The ectopic overexpression of AXL results in elevated cell invasiveness and drug resistance. Nuclear factor-kappaB (NF-kappaB) signaling activity is associated with AXL expression and may play an important role in the enhancement of invasiveness and doxorubicin resistance, as shown by using the NF-kappaB inhibitor, sulfasalazine, and IkappaB dominant-negative transfectants. In the current study, sulfasalazine exerted a synergistic anticancer effect with doxorubicin and suppressed cancer cell invasiveness in parallel in CL1 sublines and various AXL-expressing cancer cell lines. Phosphorylation of AXL and other RTKs (ErbB2 and epidermal growth factor receptor) was abolished by sulfasalazine within 15 min, suggesting that the inhibition of NF-kappaB and the kinase activity of RTKs are involved in the pharmacologic effects of sulfasalazine. Our study suggests that AXL is involved in NSCLC metastasis and drug resistance and may therefore provide a molecular basis for RTK-targeted therapy using sulfasalazine to enhance the efficacy of chemotherapy in NSCLC.

Identification and characterization of a novel gene Saf transcribed from the opposite strand of Fas.

Apoptosis is a morphologically distinct form of cell death involved in many physiological and pathological processes. The regulation of Fas/Apo-1 involved in membrane-mediated apoptosis has also been known to play crucial roles in many systems. More and more naturally occurring antisense RNAs are now known to regulate, at least in part, a growing number of eukaryotic genes. In this report, we describe the findings of a novel RNA transcribed from the opposite strand of the intron 1 of the human Fas gene. Using orientation-specific RT-PCR and northern blot analysis, we show that this transcript is 1.5 kb in length and was expressed in several human tissues and cell lines. This transcript was cloned by 5'- and 3'-RACE (rapid amplification of cDNA ends) and the transcription start site was determined by primer extension. This novel gene was named Saf. To assess the functions of Saf, Jurkat cells transfected with human Saf or control vector was prepared. The stable Saf-transfectant was highly resistant to Fas-mediated but not to TNF-alpha-mediated apoptosis. Although the overall mRNA expression level of Fas was not affected, expression of some novel forms of Fas transcripts was increased in Saf-transfectant, especially the inhibitory soluble forms. These findings collectively suggest that Saf might protect T lymphocytes from Fas-mediated apoptosis by blocking the binding of FasL or its agonistic Fas antibody. Saf might regulate the expression of Fas alternative splice forms through pre-mRNA processing.