Aberrant DNA methylation is a well-known feature of tumours and has been associated with metastatic melanoma. However, since melanoma cells are highly heterogeneous, it has been challenging to use affected genes to predict tumour aggressiveness, metastatic evolution, and patients’ outcomes. We hypothesized that common aggressive hypermethylation signatures should emerge early in tumorigenesis and should be shared in aggressive cells, independent of the physiological context under which this trait arises. We compared paired melanoma cell lines with the following properties: (i) each pair comprises one aggressive counterpart and its parental cell line and (ii) the aggressive cell lines were each obtained from different host and their environment (human, rat, and mouse), though starting from the same parent cell line. Next, we developed a multi-step genomic pipeline that combines the DNA methylome profile with a chromosome cluster-oriented analysis. A total of 229 differentially hypermethylated genes was commonly found in the aggressive cell lines. Genome localization analysis revealed hypermethylation peaks and clusters, identifying eight hypermethylated gene promoters for validation in tissues from melanoma patients. Five Cytosine-phosphate-Guanine (CpGs) identified in primary melanoma tissues were transformed into a DNA methylation score that can predict survival (log-rank test, p=0.0008). This strategy is potentially universally applicable to other diseases involving DNA methylation alterations.

• cancer biology
• chromosomes
• gene expression
• human

B-cell lymphoma-2 (Bcl-2)-like protein-10 (Bcl2L10) is the less studied member of Bcl-2 family proteins, with the controversial role in different cancer histotypes. Very recently, Bcl2L10 expression in melanoma tumor specimens and its role in melanoma response to therapy have been demonstrated. Here, the involvement of Bcl2L10 on the in vitro and in vivo properties associated with melanoma aggressive features has been investigated.

Endogenous Bcl2L10 protein expression was detected by western blotting analysis in a panel of patient-derived and commercially available human melanoma cells. In vitro assays to evaluate clonogenicity, cell proliferation, cell migration, cell invasion, and in vitro capillary-like structure formation [vasculogenic mimicry (VM)] have been performed by using human melanoma cells stably overexpressing Bcl2L10 or transiently transfected for loss/gain function of Bcl2L10, grown under two- or three-dimensional (3D) conditions Xenograft melanoma model was employed to evaluate in vivo tumor growth and angiogenesis.

Results demonstrated that Bcl2L10 acts as an inducer of in vitro cell migration, invasion, and VM, while in vitro cell proliferation, in vivo tumor growth, as well as colony formation properties were not affected. Dissecting different signaling pathways, it was found that Bcl2L10 positively affects the phosphorylation of extracellular-signal-regulated kinase (ERK) and the expression of markers of cell invasion, such as urokinase plasminogen activator receptor (uPAR) and matrix metalloproteinases (MMPs). Of note, Bcl2L10-dependent in vitro migration, invasion, and VM are linked to uPAR. Bcl2L10 also negatively regulates the intracellular calcium level. Finally, reduced invasion capability in 3D spheroid invasion assay of melanoma cells transiently overexpressing Bcl2L10 was observed after treatment with inhibitors of MMPs and uPAR.

Overall, data reported in this paper provide evidence supporting a positive role of Bcl2L10 in melanoma aggressive features.

• Bcl2l10
• ovarian cancer
• RNA sequencing
• SDHD
• IDH1

BCL2L10 is the sixth and less studied protein from the group of Bcl-2 anti-apoptotic proteins. These proteins are important therapeutic targets since they convey resistance to anticancer regimens. We describe here for the first time the role of BCL2L10 in melanoma. We found that BCL2L10 is abundantly and frequently expressed both in melanoma cell lines and tumor samples. This increased expression is due to the activity of the transcription factor STAT3 that positively regulate BCL2L10 transcription. We describe that Bcl2l10 is a pro-survival factor in melanoma, being able to protect cells from the cytotoxic effect of different drugs, including cisplatin, dacarbazine, and ABT-737. BCL2L10 also inhibited the cell death upon combination treatments of PLX-4032, a BRAF inhibitor, with ABT-737 or cisplatin. In summary, we determined that BCL2L10 is expressed in melanoma and contributes to cell survival. Hence, targeting BCL2L10 may enhance the clinical efficacy of other therapies for malignant melanoma.

The anti-apoptotic proteins from the Bcl-2 family are important therapeutic targets since they convey resistance to anticancer regimens. Despite the suspected functional redundancy among the six proteins of this subfamily, both basic studies and therapeutic approaches have focused mainly on BCL2, Bcl-xL, and MCL1. The role of BCL2L10, another member of this group, has been poorly studied in cancer and never has been in melanoma. We describe here that BCL2L10 is abundantly and frequently expressed both in melanoma cell lines and tumor samples. We established that BCL2L10 expression is driven by STAT3-mediated transcription, and by using reporter assays, site-directed mutagenesis, and ChIP analysis, we identified the functional STAT3 responsive elements in the BCL2L10 promoter. BCL2L10 is a pro-survival factor in melanoma since its expression reduced the cytotoxic effects of cisplatin, dacarbazine, and ABT-737 (a BCL2, Bcl-xL, and Bcl-w inhibitor). Meanwhile, both genetic and pharmacological inhibition of BCL2L10 sensitized melanoma cells to cisplatin and ABT-737. Finally, BCL2L10 inhibited the cell death upon combination treatments of PLX-4032, a BRAF inhibitor, with ABT-737 or cisplatin. In summary, we determined that BCL2L10 is expressed in melanoma and contributes to cell survival. Hence, targeting BCL2L10 may enhance the clinical efficacy of other therapies for malignant melanoma.

• ABT-737
• BCL2L10
• Bcl-2 family
• ML258
• STAT3
• cytotoxicity
• melanoma
• survival

Aberrant methylation in DNA regulatory regions could downregulate tumor suppressor genes without changing the sequences. However, our knowledge of secreted protein acidic and rich in cysteine (SPARC) and its aberrant methylation in gastric cancer (GC) is still inadequate. In the present research, we performed fundamental research to clarify the precise function of methylation on SPARC and its significance in GC.

To investigate promoter methylation and the effects of the SPARC gene in GC cells and tissues and to evaluate its clinical significance.

Plasmids that overexpressed the SPARC gene were transfected into human GC BGC-823 cells; non-transfected cells were used as a control group (NC group). Quantitative real-time polymerase chain reaction and western blotting (WB) were then used to detect the expression of SPARC. Methylation-specific polymerase chain reaction was executed to analyze the gene promoter methylation status. Cell viability was measured by the cell counting kit-8 assay. The migration and invasion ability of cells were detected by scratch assays and transwell chamber assays, respectively. Cell cycle events and apoptosis were observed with a flow cytometer.

The expression of SPARC mRNA in GC tissues and cells was significantly lower and showed differing degrees of hypermethylation, respectively, than that in normal adjacent tissues and control cells. Treatment with 5-Aza-2’-deoxycytidine (5-Aza-Cdr) was able to restore the expression of SPARC and reverse promoter hypermethylation. Overexpression of the SPARC gene significantly inhibited proliferation, migration, and invasion of GC cells, while also causing cell cycle arrest and apoptosis; the NC group exhibited the opposite effects.

This study demonstrated that SPARC could function as a tumor suppressor and might be silenced by promoter hypermethylation. Furthermore, in GC cells, SPARC inhibited migration, invasion, and proliferation, caused cell cycle arrest at the G₀/G₁ phase, and promoted apoptosis.

• Gastric cancer
• Secreted protein acidic and rich in cysteine
• Hypermethylation
• Clinicopathological features
• Tumor suppressor gene

• Carcinoma/metabolism
• Cell Line, Tumor
• DNA Methylation
• Decitabine
• Female
• Humans
• Male
• Middle Aged
• Osteonectin/physiology
• Stomach Neoplasms/metabolism
• Transfection

• Locally advanced rectal cancer
• Neoadjuvant chemoradiotherapy
• Next-generation sequencing
• Pathologic complete response
• Whole-exome sequencing

Background: The promoter methylation of MLH1 gene and gastric cancer (GC)has been investigated previously. To get a more credible conclusion, we performed a systematic review and meta and bioinformatic analysis to clarify the role of MLH1 methylation in the prediction and prognosis of GC.

Methods: Eligible studies were targeted after searching the PubMed, Web of Science, Embase, BIOSIS, CNKI and Wanfang Data to collect the information of MLH1 methylation and GC. The link strength between the two was estimated by odds ratio with its 95% confidence interval. The Newcastle-Ottawa scale was used for quantity assessment. Subgroup and sensitivity analysis were conducted to explore sources of heterogeneity. The Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were employed for bioinformatics analysis on the correlation between MLH1 methylation and GC risk, clinicopathological behavior as well as prognosis.

Results: 2365 GC and 1563 controls were included in the meta-analysis. The pooled OR of MLH1 methylation in GC was 4.895 (95% CI: 3.149-7.611, P<0.001), which considerably associated with increased GC risk. No significant difference was found in relation to Lauren classification, tumor invasion, lymph node/distant metastasis and tumor stage in GC. Analysis based on GEO and TCGA showed that high MLH1 methylation enhanced GC risk but might not related with GC clinicopathological features and prognosis.

Conclusion:MLH1 methylation is an alive biomarker for the prediction of GC and it might not affect GC behavior. Further study could be conducted to verify the impact of MLH1 methylation on GC prognosis.

• MLH1
• gastric cancer
• methylation
• prognosis
• risk

Background: Human mutL homolog 1 (hMLH1) is located on chromosome 3q21-23. As a classic tumor suppressor gene, many researchers have studied the association between hMLH1 promoter methylation and gastric cancer, but their conclusions were not always consistent. Therefore, we performed a meta-analysis to make a more integrated and precise estimate of the associations.

Method: PubMed, EMBASE, and Cochrane Library were retrieved without language restrictions. Data were analyzed by Review Manager 5.2 and Stata 12.0 software. Odds ratio (OR) with 95% confidence interval (95%CI) was used to assess the statistical associations.

Result: A total of 39 studies published before January 20, 2018 were included in this study. The results indicated that the frequency of hMLH1 promoter methylation in gastric cancers was substantially higher than that in non-cancer controls (OR = 7.94, 95%CI = 4.32–14.58, P < 0.001). Furthermore, hMLH1 promoter methylation had considerable associations with lymph node metastasis, microsatellite instability (MSI), and low expression of hMLH1 protein (OR = 1.53, 95%CI = 1.04–2.26, P = 0.03; OR = 15.33, 95%CI = 9.26–25.36, P < 0.001; OR = 37.86, 95%CI = 18.03–79.50, P < 0.001, respectively). No association was found between hMLH1 promoter methylation and Lauren classification or Helicobacter pylori (HP) infection status.

Conclusion: The present study provides evidence that promoter methylation of hMLH1 is a major causative event in the occurrence and development of human gastric cancer.

• MSI
• gastric cancer
• hMLH1
• meta-analysis
• methylation

• RUNX3 gene
• gastriccancer
• hypermethylation
• meta-analysis

• deregulation and overexpression/gene inactivation
• desoxyribonucleic acid methylation
• gastric cancer
• inflammation
• polymorphisms

• gastric cancer
• methylation
• tumor suppressor genes
• epigenetics

• Apoptosis
• Cell Adhesion
• Cell Cycle
• CpG Islands/genetics
• DNA Methylation
• DNA Repair
• Epigenesis, Genetic
• Genes, Tumor Suppressor
• Helicobacter Infections/genetics
• Helicobacter pylori
• Humans
• Neoplasm Invasiveness
• Promoter Regions, Genetic/genetics
• Stomach Neoplasms/genetics
• Stomach Neoplasms/metabolism
• Stomach Neoplasms/microbiology
• Stomach Neoplasms/pathology
• Tumor Suppressor Proteins/genetics
• Tumor Suppressor Proteins/metabolism

Runt-related transcription factor 3 (RUNX3) is a member of the runt-domain family of transcription factors and has been reported to be a candidate tumor suppressor in gastric cancer. However, the association between RUNX3 promoter methylation and gastric cancer remains unclear.

We systematically reviewed studies of RUNX3 promoter methylation and gastric cancer published in English or Chinese from January 2000 to January 2011, and quantified the association between RUNX3 promoter methylation and gastric cancer using meta-analysis methods.

A total of 1740 samples in 974 participants from seventeen studies were included in the meta-analysis. A significant association was observed between RUNX3 promoter methylation and gastric cancer, with an aggregated odds ratio (OR) of 5.63 (95%CI 3.15, 10.07). There was obvious heterogeneity among studies. Subgroup analyses (including by tissue origin, country and age), meta-regression were performed to determine the source of the heterogeneity. Meta-regression showed that the trend in ORs was inversely correlated with age. No publication bias was detected. The ORs for RUNX3 methylation in well-differentiated vs undifferentiated gastric cancers, and in intestinal-type vs diffuse-type carcinomas were 0.59 (95%CI: 0.30, 1.16) and 2.62 (95%CI: 1.33, 5.14), respectively. There were no significant differences in RUNX3 methylation in cancer tissues in relation to age, gender, TNM stage, invasion of tumors into blood vessel or lymphatic ducts, or tumor stage.

This meta-analysis identified a strong association between methylation of the RUNX3 promoter and gastric cancer, confirming the role of RUNX3 as a tumor suppressor gene.