SOCS3 Methylation Predicts a Poor Prognosis in HBV Infection-Related Hepatocellular Carcinoma

Suppressor of cytokine signaling 3 (SOCS3) plays crucial roles in JAK/STAT signaling pathway inhibition in hepatocellular carcinoma (HCC). However, the methylation status of SOCS3 in HBV infection-related HCC and the relationship between SOCS3 methylation and the clinical outcome remain unknown. Here, we reported that in HCC tumor tissues, two regions of the CpG island (CGI) in the SOCS3 promoter were subjected to methylation analysis and only the region close to the translational start site of SOCS3 was hypermethylated. In HCC tumor tissues, SOCS3 showed an increased methylation frequency and intensity compared with that in the adjacent non-tumor tissues. Moreover, SOCS3 expression was significantly down-regulated in HCC cell lines and tumor tissues, and this was inversely correlated with methylation. Kaplan–Meier curve analysis revealed that in patients with an hepatitis B virus (HBV) infection background, SOCS3 hypermethylation was significantly correlated with a poor clinical outcome of HCC patients. Our findings indicated that SOCS3 hypermethylation has already happened in non-tumor tissues and increased in both frequency and intensity in tumor tissues. This suggests that the methylation of SOCS3 could predict a poor prognosis in HBV infection-related HCC patients.     

SMG1 Acts as a Novel Potential Tumor Suppressor with Epigenetic Inactivation in Acute Myeloid Leukemia

Suppressor with morphogenetic effect on genitalia family member (SMG1) belongs to a family of phosphoinositide 3-kinase-related kinases and is the main kinase involved in nonsense-mediated mRNA decay. Recently, SMG1 was suggested as a novel potential tumor suppressor gene, particularly in hypoxic tumors. To investigate the function of SMG1 in acute myeloid leukemia (AML), we performed methylation-specific polymerase chain reaction and found that SMG1 was hypermethylated in the promoter region. SMG1 hypermethylation was found in 66% (33/50) of AML samples compared with none (0/14) of the normal controls. SMG1 mRNA was down-regulated in AML patients with hypermethylation status whereas it was readily expressed in patients without methylation. Moreover, treatment of AML cells with demethylating agent 5-aza-2''-deoxycytidine (decitabine) inhibited AML cell growth and induced apoptosis by reversing SMG1 methylation status and restoring SMG1 expression. On the other hand, knockdown of SMG1 by RNA interference inhibited apoptosis. We also found that mTOR expression level was negatively correlated to SMG1 expression in AML patients which indicated that SMG1 and mTOR maybe act antagonistically to regulate AML cell growth. In conclusion, our results indicate that SMG1 acts as a potential tumor suppressor with epigenetic regulation in AML.     

Frequent Epigenetic Suppression of Tumor Suppressor Gene Glutathione Peroxidase 3 by Promoter Hypermethylation and Its Clinical Implication in Clear Cell Renal Cell Carcinoma

The goal of this study is to identify novel tumor suppressor genes silenced by promoter methylation in clear cell renal cell carcinoma (ccRCC) and discover new epigenetic biomarkers for early cancer detection. Reactive oxygen species (ROS) is a major cause of DNA damage that correlates with cancer initiation and progression. Glutathione peroxidase 3 (GPX3), the only known extracellular glycosylated enzyme of GPXs, is a major scavenger of ROS. GPX3 has been identified as a tumor suppressor in many cancers. However, the role of GPX3 in ccRCC remains unclear. This study aimed to investigate its epigenetic alteration in ccRCC and possible clinicopathological association. In our study, GPX3 methylation and down-regulation were detected in 5 out of 6 ccRCC cell lines and the GPX3 mRNA and protein expression level in ccRCC tumors was significantly lower than in adjacent non-malignant renal tissues (p < 0.0001). Treatment with 5-Aza-2''-deoxycytidine restored GPX3 expression in ccRCC cells. Aberrant methylation was further detected in 77.1% (162/210) of RCC primary tumors, but only 14.6% (7/48) in adjacent non-malignant renal tissues. GPX3 methylation status was significantly associated with higher tumor nuclear grade (p = 0.014). Thus, our results showing frequent GPX3 inactivation by promoter hypermethylation in ccRCC may reveal the failure in the cellular antioxidant system in ccRCC and may be associated with renal tumorigenesis. GPX3 tumor specific methylation may serve as a biomarker for early detection and prognosis prediction of ccRCC.     

Leptin Protein Expression and Promoter Methylation in Ovarian Cancer: A Strong Prognostic Value with Theranostic Promises

Ovarian cancer (OC) is the deadliest among all gynecological cancers. Epidemiological studies showed that obesity might influence many cancers including OC. One of the key factors that may link obesity and OC is leptin (LEP), known as an adipokine with pleiotropic effects on body homeostasis. This study aims to investigate the expression pattern of LEP, assess the methylation profiles of LEP and their associations with clinicopathological features including survival outcomes of OC patients. The protein expression of LEP was evaluated in 208 samples using both tissue microarray and immunohistochemistry techniques. The methylation profiles of LEP were measured in 63 formalin-fixed, paraffin-embedded tumor tissues by quantitative polymerase chain reaction using a MethyLight assay. Our results showed a significant association of LEP protein overexpression with several clinicopathological variables, mainly tumor subtype, LVI, age of menarche, tumor size and stage (p < 0.04). Kaplan–Meier analysis (using low expression versus high expression as a discriminator) indicated that LEP protein overexpression is a powerful positive prognosticator of both OC recurrence (DFS) and disease-specific survival (DSS) in our OC cohort (log-rank p = 0.01 and p = 0.002, respectively). This implies that patients with high LEP expression profiles live longer with less recurrence rates. Methylation analysis results demonstrated a clear association between no/low LEP protein expression pattern (38%) and LEP promoter CpG island hypermethylation (43%). Results of this study suggest that LEP is a powerful prognosticator of OC recurrence and DSS. LEP expression in OC seems to be regulated by its promoter hypermethylation through gene partial/total silencing. Further multi-institutional studies using larger cohorts are required to demystify the intricate molecular functions of this leptin-driven effects in OC pathophysiology and to accurately assess its theranostic potential and validate its prognostic/predictive power in OC onset, progression towards more effective and personalized management of OC patients.     

ANK2 Hypermethylation in Canine Mammary Tumors and Human Breast Cancer

Canine mammary tumors (CMT) constitute the most common tumor types found in female dogs. Understanding this cancer through extensive research is important not only for clinical veterinary applications, but also in the scope of comparative oncology. The use of DNA methylation as a biomarker has been noted for numerous cancers in the form of both tissue and liquid biopsies, yet the study of methylation in CMT has been limited. By analyzing our canine methyl-binding domain sequencing (MBD-seq) data, we identified intron regions of canine ANK2 and EPAS1 as differentially methylated regions (DMGs) in CMT. Subsequently, we established quantitative methylation specific PCR (qMSP) of ANK2 and EPAS1 to validate the target hypermethylation in CMT tissue, as well as cell free DNA (cfDNA) from CMT plasma. Both ANK2 and EPAS1 were hypermethylated in CMT and highlighted as potential tissue biomarkers in CMT. ANK2 additionally showed significant hypermethylation in the plasma cfDNA of CMT, indicating that it could be a potential liquid biopsy biomarker as well. A similar trend towards hypermethylation was indicated in HBC at a specific CpG of the ANK2 target on the orthologous human region, which validates the comparative approach using aberrant methylation in CMT.     

Epigenetic Determinants of CYP1A1 Induction by the Aryl Hydrocarbon Receptor Agonist 3,3',4,4',5-Pentachlorobiphenyl (PCB 126)

Many enzymes involved in xenobiotic metabolism, including cytochrome P450 (CYP) 1A1, are regulated by the aryl hydrocarbon receptor (AhR). 3,3'',4,4'',5-Pentachlorobiphenyl (PCB 126) is a potent ligand for AhR and can thus induce the expression of CYP1A1. Interestingly, we observed that human carcinoma cell lines derived from different types of epithelial cells displayed divergent degrees of CYP1A1 induction after exposure to PCB 126. Since epigenetic mechanisms are known to be involved in cell type-specific gene expression, we sought to assess the epigenetic determinants of CYP1A1 induction in these carcinoma cell lines. In contrast to HepG2 hepatocarcinoma cells, HeLa cervical carcinoma cells showed significantly lower levels of CYP1A1 mRNA expression following PCB 126 exposure. Our results show that the two cell lines maintained differences in the chromatin architecture along the CYP1A1 promoter region. Furthermore, treatment with the epigenetic modifiers, trichostatin A (TSA) and 5-aza-2''-deoxycytidine (5-Aza-dC), significantly increased the expression of CYP1A1 after PCB 126 treatment in HeLa cells. However, we did not observe apparent differences in methylation levels or specific location of CpG DNA methylation between the two cell lines in the analyzed CYP1A1 promoter region. Taken together, our findings suggest that the differences in CYP1A1 expression between HepG2 and HeLa cells are due to differences in the chromatin architecture of the CYP1A1 promoter and thus establish a role of epigenetic regulation in cell-specific CYP1A1 expression.     

Lentiviral-Mediated Short Hairpin RNA Knockdown of MTDH Inhibits Cell Growth and Induces Apoptosis by Regulating the PTEN/AKT Pathway in Hepatocellular Carcinoma

The activation of oncogenes and the loss of tumor suppressor genes are believed to play critical roles in the pathogenesis of human hepatocellular carcinoma (HCC). Metaherin (MTDH), also called astrocyte elevated gene-1 (AEG-1), is frequently amplified in a variety of cancers, but the roles of MTDH with regard to growth and apoptosis in HCC have not yet been studied. In the present study, we first analyzed the expression of MTDH in HCC samples. We found that MTDH protein levels are higher in most HCC cancerous tissues compared with their matched adjacent non-tumor tissues. Additionally, the MTDH mRNA was also higher in HCC tissues compared to their matched adjacent non-tumor tissues. Knockdown of the endogenous MTDH using small interfering RNA further showed that deficiency of MTDH suppressed cell growth and caused apoptosis in HCC cells. Knockdown MTDH promoted PTEN and p53 expression in HCC cells and inhibited AKT phosphorylation. Knockdown MTDH also inhibited tumor growth in vivo. All these results indicated that MTDH protein levels in most HCC tissues are higher than non-tumor tissues, and knockdown of MTDH inhibited growth and induced apoptosis in HCC cells through the activation of PTEN. Therefore, MTDH might be an effective targeted therapy gene for HCC.     

TPX2 Is a Prognostic Marker and Contributes to Growth and Metastasis of Human Hepatocellular Carcinoma

Targeting protein for Xenopus kinesin-like protein 2 (TPX2), a microtubule-associated protein, impacts spindle assembly in human cells. Several studies have demonstrated that TPX2 is overexpressed in different types of human cancers and promotes tumor growth and metastasis. In this study, we found that the expression level of TPX2 was obviously higher in hepatocellular carcinoma (HCC) tissues than in matched nontumor tissues. Elevated expressions of TPX2 mRNA were observed in all HCC cell lines (HepG2, Hep3B, SMMC-7721, Bel-7402 and Huh7) as compared with that in a non-transformed hepatic cell line (LO2). Clinical analysis indicated that the positive expression of TPX2 was significantly correlated with venous infiltration, high Edmondson-Steiner grading and advanced TNM tumor stage in HCC. Furthermore, TPX2 was a novel prognostic marker for predicting 5-year overall survival (OS) and disease-free survival (DFS) of HCC patients. In vitro studies found that TPX2 knockdown significantly inhibited cell proliferation and viability in both Hep3B and HepG2 cells. Moreover, TPX2 knockdown obviously slowed down tumor growth in a nude mouse xenograft model. Otherwise, TPX2 knockdown prominently suppressed HCC cell invasion and migration. In conclusion, these results indicate that TPX2 may serve as a prognostic marker and promotes tumorigenesis and metastasis of HCC.     

MicroRNA-130b Promotes Cell Aggressiveness by Inhibiting Peroxisome Proliferator-Activated Receptor Gamma in Human Hepatocellular Carcinoma

MircroRNA-130b (miR-130b) is proposed as a novel tumor-related miRNA and has been found to be significantly dysregulated in tumors. In this study, the expression level of miR-130b was found to be obviously higher in hepatocellular carcinoma (HCC) tissues than that in nontumor tissues. Further, miR-130b was expressed at significantly higher levels in aggressive and recurrent tumor tissues. Clinical analysis indicated that high-expression of miR-130b was prominently correlated with venous infiltration, high Edmondson-Steiner grading and advanced tumor-node-metastasis (TNM) tumor stage in HCC. Elevated miR-130b expression was observed in all HCC cell lines (HepG2, SMMC-7721, Huh7, Hep3B and MHCC97H) as compared with that in a nontransformed hepatic cell line (LO2). Furthermore, an inverse correlation between miR-130b and E-cadherin and a positive correlation between miR-130b and Vimentin were observed in HCC tissues. Down-regulation of miR-130b expression reduced invasion and migration in both Hep3B and MHCC97H cells. Peroxisome proliferator-activated receptor gamma (PPAR-γ) was inversely correlated with miR-130b expression in HCC tissues. In addition, down-regulation of miR-130b restored PPAR-γ expression and subsequently suppressed epithelial-mesenchymal transition (EMT) in HCC cells. We identified PPARγ as a direct target of miR-130b in HCC in vitro. Notably, PPAR-γ knockdown abolished down-regulation of miR-130b-inhibited EMT in MHCC97H cells. In conclusion, miR-130b may promote HCC cell migration and invasion by inhibiting PPAR-γ and subsequently inducing EMT.     

Overexpression of MicroRNA-30b Improves Adenovirus-Mediated p53 Cancer Gene Therapy for Laryngeal Carcinoma

MicroRNAs play important roles in laryngeal carcinoma and other cancers. However, the expression of microRNAs in paracancerous tissue has been studied less. Here, using laser capture microdissection (LCM), we detected the expression of microRNAs in paracancerous tissues. Among all down-regulated microRNAs in the center area of tumor tissues, only miR-30b expression was significantly reduced in paracancerous tissues compared to surgical margins. Therefore, to further investigate the effect of miR-30b on laryngeal carcinoma, we stably overexpressed miR-30b in laryngeal carcinoma cell line HEp-2 cells. It was found that although there was no significant difference in cell viability between miR-30b overexpressed cells and control HEp-2 cells, p53 expression was obviously enhanced in miR-30b overexpressed cells. Whether miR-30b could improve the anti-tumor effect of adenovirus-p53 (Ad-p53) in laryngeal carcinoma and other cancer cell lines was also evaluated. It was found that in miR-30b overexpressed HEp-2 cells, p53-mediated tumor cell apoptosis was obviously increased both in vitro and in vivo. MDM2-p53 interaction might be involved in miR-30b-mediated anti-tumor effect. Together, results suggested that miR-30b could modulate p53 pathway and enhance p53 gene therapy-induced apoptosis in laryngeal carcinoma, which could provide a novel microRNA target in tumor therapy.     

MGMT-Methylation in Non-Neoplastic Diseases of the Central Nervous System

Quantifying O⁶-methylguanine-DNA methyltransferase (MGMT) promoter methylation plays an essential role in assessing the potential efficacy of alkylating agents in the chemotherapy of malignant gliomas. MGMT promoter methylation is considered to be a characteristic of subgroups of certain malignancies but has also been described in various peripheral inflammatory diseases. However, MGMT promoter methylation levels have not yet been investigated in non-neoplastic brain diseases. This study demonstrates for the first time that one can indeed detect slightly enhanced MGMT promoter methylation in individual cases of inflammatory demyelinating CNS diseases such as multiple sclerosis and progressive multifocal leucencephalopathy (PML), as well as in other demyelinating diseases such as central pontine and exptrapontine myelinolysis, and diseases with myelin damage such as Wallerian degeneration. In this context, we identified a reduction in the expression of the demethylase TET1 as a possible cause for the enhanced MGMT promoter methylation. Hence, we show for the first time that MGMT hypermethylation occurs in chronic diseases that are not strictly associated to distinct pathogens, oncogenic viruses or neoplasms but that lead to damage of the myelin sheath in various ways. While this gives new insights into epigenetic and pathophysiological processes involved in de- and remyelination, which might offer new therapeutic opportunities for demyelinating diseases in the future, it also reduces the specificity of MGMT hypermethylation as a tumor biomarker.     

Effect of Salt Stress on the Expression and Promoter Methylation of the Genes Encoding the Mitochondrial and Cytosolic Forms of Aconitase and Fumarase in Maize

The influence of salt stress on gene expression, promoter methylation, and enzymatic activity of the mitochondrial and cytosolic forms of aconitase and fumarase has been investigated in maize (Zea mays L.) seedlings. The incubation of maize seedlings in 150-mM NaCl solution resulted in a several-fold increase of the mitochondrial activities of aconitase and fumarase that peaked at 6 h of NaCl treatment, while the cytosolic activity of aconitase and fumarase decreased. This corresponded to the decrease in promoter methylation of the genes Aco1 and Fum1 encoding the mitochondrial forms of these enzymes and the increase in promoter methylation of the genes Aco2 and Fum2 encoding the cytosolic forms. The pattern of expression of the genes encoding the mitochondrial forms of aconitase and fumarase corresponded to the profile of the increase of the stress marker gene ZmCOI6.1. It is concluded that the mitochondrial and cytosolic forms of aconitase and fumarase are regulated via the epigenetic mechanism of promoter methylation of their genes in the opposite ways in response to salt stress. The role of the mitochondrial isoforms of aconitase and fumarase in the elevation of respiration under salt stress is discussed.     

The Tumor Suppressor Roles of miR-433 and miR-127 in Gastric Cancer

The discovery of microRNAs (miRNAs) provides a new and powerful tool for studying the mechanism, diagnosis and treatment of human cancers. Currently, the methylation epigenetic silencing of miRNAs with tumor suppressor features by CpG island hypermethylation is emerging as a common hallmark of different tumors. Here we showed that miR-433 and miR-127 were significantly down-regulated in gastric cancer (GC) tissues compared with the adjacent normal regions in 86 paired samples. Moreover, the lower level of miR-433 and miR-127 was associated with pM or pTNM stage in clinical gastric cancer patients. The restored expression of miR-433 and miR-127 in GC cells upon 5-Aza-CdR and TSA treatment suggested the loss of miR-433 and miR-127 was at least partly regulated by epigenetic modification in GC. Furthermore, the ectopic expression of miR-433 and miR-127 in gastric cancer cell lines HGC-27 inhibits cell proliferation, cell cycle progression, cell migration and invasion by directly interacting with the mRNA encoding oncogenic factors KRAS and MAPK4 respectively. Taken together, our results showed that miR-433 and miR-127 might act as tumor suppressors in GC, and it may provide novel diagnostic and therapeutic options for human GC clinical operation in the near future.