GNG2 acts as a tumor suppressor in breast cancer through stimulating MRAS signaling

G-protein gamma subunit 2 (GNG2) is involved in several cell signaling pathways, and is essential for cell proliferation and angiogenesis. However, the role of GNG2 in tumorigenesis and development remains unclear. In this study, 1321 differentially expressed genes (DEGs) in breast cancer (BC) tissues were screened using the GEO and TCGA databases. KEGG enrichment analysis showed that most of the enriched genes were part of the PI3K-Akt signaling pathway. We identified GNG2 from the first five DEGs, its expression was markedly reduced in all BC subtype tissues. Cox regression analysis showed that GNG2 was independently associated with overall survival in patients with luminal A and triple-negative breast cancers (TNBC). GNG2 over-expression could significantly block the cell cycle, inhibit proliferation, and promote apoptosis in BC cells in vitro. In animal studies, GNG2 over-expression inhibited the growth of BC cells. Further, we found that GNG2 significantly inhibited the activity of ERK and Akt in an MRAS-dependent manner. Importantly, GNG2 and muscle RAS oncogene homolog (MRAS) were co-localized in the cell membrane, and the fluorescence resonance energy transfer (FRET) experiment revealed that they had direct interaction. In conclusion, the interaction between GNG2 and MRAS likely inhibits Akt and ERK activity, promoting apoptosis and suppressing proliferation in BC cells. Increasing GNG2 expression or disrupting the GNG2–MRAS interaction in vivo could therefore be a potential therapeutic strategy to treat BC.Subject terms: Breast cancer, Breast cancer     

Cx31.1 acts as a tumour suppressor in non-small cell lung cancer (NSCLC) cell lines through inhibition of cell proliferation and metastasis

Reduced connexin expression and loss of gap junction function is a characteristic of many cancers, including lung cancer. However, there are little reports about the relation between Cx31.1 and lung cancer. This study was conducted to investigate the effect of Cx31.1 on non-small cell lung cancer (NSCLC). We found that the Cx31.1 was down-regulated in NSCLC cell lines, and the expression levels were reversely related with their metastatic potential. We ectopically expressed Cx31.1 in H1299 NSCLC cell line to examine the influence of Cx31.1 overexpression. The results showed that overexpression of Cx31.1 in H1299 cells reduced cell proliferation, induced a delay in the G(1) phase, inhibited anchorage-independent growth and suppressed cell migration and invasion. The cell cycle delay and cell migration and invasion suppressive effects of Cx31.1 were partially reversed by siRNA targeting mRNA of Cx31.1. Moreover, xenografts of Cx31.1 overexpressing H1299 cells showed reduced tumourigenicity. These results suggested that Cx31.1 has tumour-suppressive properties. Further investigation indicated that cyclin D3 may be responsible for Cx31.1-induced G(1) phase delay. Importantly, Cx31.1 increased the expression of epithelial markers, such as cytokeratin 18, and decreased expression of mesenchymal markers, such as vimentin, indicating a Cx31.1-mediated partial shift from a mesenchymal towards an epithelial phenotype. We concluded that Cx31.1 inhibit the malignant properties of NSCLC cell lines, the mechanisms under this may include regulation of EMT.     

Folic acid enforces DNA methylation-mediated transcriptional silencing of PTEN,APC and RARbeta2 tumour suppressor genes in breast cancer

Folate, one of the most studied dietary compounds, has recently become the main topic of debates on food fortification. Although low folate levels may be associated with increased risk of cancer development, simultaneously several reports indicate a detrimental effects mediated by high folate concentrations. Using the methylation sensitive restriction analysis (MSRA) and real-time RT-PCR we tested the effect of folic acid on DNA promoter methylation and expression of PTEN, APC and RARbeta2 tumour suppressor genes in MCF-7 and MDA-MB-231 breast cancer cell lines with different invasive capacity. The tested genes encode proteins involved in regulation of oncogenic intracellular signaling pathways. The results show that the increasing concentrations of folic acid lead to a dose-dependent down-regulation of tumour suppressor genes which may be linked to the increased DNA methylation detected within their promoter regions. The effects were more remarkable in non-invasive MCF-7 cells where we also observed 30% up-regulation of DNMT1 expression at the highest folate concentration used. Our findings show that caution need to be used when introducing folic acid supplementation since it may lead to cancer progression.     

ZNRF3 acts as a tumour suppressor by the Wnt signalling pathway in human gastric adenocarcinoma

E3 ubiquitin ligases regulate a variety of biological processes through the ubiquitin–proteasome system, together with ubiquitin activating enzyme E1 and ubiquitin-conjugating enzyme E2. Previous studies have demonstrated that zinc and ring finger 3 (ZNRF3), which belongs to the E3 ubiquitin ligases family is involved in the Wnt signalling pathway, which plays an important role in causing cancer. However, the expression and function of ZNRF3 in human gastric adenocarcinoma still remains unclear. Immunohistochemical and western blot analysis showed a significant down-regulation of ZNRF3 protein in gastric adenocarcinoma tissues compared with adjacent normal gastric tissues. In addition, there was a correlation between the down-regulation of ZNRF3 and poor tissue differentiation in gastric adenocarcinoma. To investigate the potential function of ZNRF3 in cell proliferation and apoptosis, a gastric cell line SGC7901 was employed. The over-expression of wild-type ZNRF3, which was accomplished by the transient transfection of recombinant pEGFP-ZNRF3 (or empty plasmids as control) into the cell line SGC7901, was confirmed by western blot analysis. Flow-cytometry-based and Cell Counting Kit-8 assays showed that over-expression of wt ZNRF3 induced apoptosis and suppressed proliferation. ZNRF3-overexpressing gastric cells displayed partly attenuated protein levels of beta-catenin and TCF-4 compared with those transfected with the empty plasmid. Our study demonstrates a novel gastric adenocarcinoma suppressor and reveals that ZNRF3 inhibits gastric cancer cell growth and promotes the cell apoptosis by affecting the Wnt/beta-catenin/TCF signalling pathway.     

PDZRN4 acts as a suppressor of cell proliferation in human liver cancer cell lines

Recently, some reports show that Ligand of Numb Protein‐X 1 (LNX1) could be a suppressor gene in gliomas, while our current research has firstly shown that PDZ domain containing ring finger 4 (PDZRN4), another member of LNX family, could also be a potential suppressor in hepatocellular carcinoma (HCC). PDZRN4, also named LNX4 (Ligand of Numb Protein‐X 4), is a member of the LNX family. We recently found that PDZRN4, but not LNX1, was down‐regulated in HCC samples, and the role of PDZRN4 in the progression of HCC had not been studied before. To address this question, firstly, we evaluated the expression of PDZRN4 in HCC samples and adjacent non‐cancerous tissues. Semi‐quantitative polymerase chain reaction showed that PDZRN4 was down‐regulated in 24/36 (66.7%) HCC samples separately. In addition, our research shows that PDZRN4 is silenced in all of the 12 HCC cell lines tested. Subsequently, cell‐based functional assay exhibited that ectopic expression of PDZRN4 inhibits the proliferation, plate colony formation and anchorage‐independent colony formation of HCC cells. Collectively, our results showed that PDZRN4 might be a potential tumour suppressor gene and had anti‐proliferative effect on HCC cell proliferation, which would be of great significance to the researches on HCC. Copyright © 2015 John Wiley & Sons, Ltd.     

TG-interacting factor 1 acts as a transcriptional repressor of sterol O-acyltransferase 2

Acat2 [gene name: sterol O-acyltransferase 2 (SOAT2)] esterifies cholesterol in enterocytes and hepatocytes. This study aims to identify repressor elements in the human SOAT2 promoter and evaluate their in vivo relevance. We identified TG-interacting factor 1 (Tgif1) to function as an important repressor of SOAT2. Tgif1 could also block the induction of the SOAT2 promoter activity by hepatocyte nuclear factor 1α and 4α. Women have ∼30% higher hepatic TGIF1 mRNA compared with men. Depletion of Tgif1 in mice increased the hepatic Soat2 expression and resulted in higher hepatic lipid accumulation and plasma cholesterol levels. Tgif1 is a new player in human cholesterol metabolism.     

2-Mercaptoethanol acts as a potentiating factor of interleukin-2-dependent lymphocyte proliferation

An interleukin-2 (IL-2) dependent cell line which could be grown continuously with crude concanavalin A-stimulated rat or mouse spleen cell culture supernatant could not survive for over 48 hr in the culture medium supplemented with partially purified or recombinant IL-2. The cell growth was restored by adding another factor obtained from the same crude culture supernatant. This potentiating activity which was physicochemically inseparable from serum albumin was also obtained from the culture medium containing 2-mercaptoethanol (2-ME) and incubated at 37 C for 24 hr without the spleen cells. By further experiments, it was demonstrated that 2-ME itself had the potentiating activity on the IL-2-dependent proliferation of this cell line and cysteine mediated the activity of 2-ME. The cells could not enter S-phase in the absence of 2-ME even in the presence of IL-2.     

Cutting edge: vasoactive intestinal peptide acts as a potent suppressor of inflammation in vivo by trans-deactivating chemokine receptors

Chemokines mediate trafficking of leukocytes to sites of inflammation and immune responses through activation of G protein-coupled receptors, which thereby provide appealing targets for novel anti-inflammatory agents. Vasoactive intestinal peptide (VIP) is an immunosuppressive neurotransmitter. We show that VIP inhibited the function of chemokine receptors on monocytes and CD4(+) T lymphocytes, with impaired chemotaxis and calcium flux in response to the cognate chemokine ligands CXCL12, CCL3, CCL4, and CCL5. This was mediated by VIP receptor type 1 and was not caused by chemokine receptor internalization. However, VIP caused dose-dependent phosphorylation of the chemokine receptor CCR5. This trans-deactivation process was studied in a murine model of delayed-type hypersensitivity: continuous infusion of VIP resulted in significant abrogation of monocyte and lymphocyte infiltration. Circulating mononuclear cells from VIP-infused mice were unable to respond to chemokines. VIP may provide a novel approach to treatment of inflammatory diseases through inhibition of chemokine-dependent leukocyte recruitment.     

Ethanol acts as a potent agent sensitizing colon cancer cells to the TRAIL-induced apoptosis

Identification of mechanisms of modulation of the TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis is important for its potential use in anticancer therapy. Ethanol can induce cell death in vitro and in vivo by different signalling pathways. Its effect in combination with death ligands is unknown. We investigated how ethanol modulates the effects of TRAIL in colon cancer cells. After combined TRAIL and ethanol treatment, a potentiation of caspase-8, -9, -3 activation, a proapoptotic Bid protein cleavage, a decrease of mitochondrial membrane potential, a complete poly(ADP)ribose polymerase cleavage, and disappearance of antiapoptotic Mcl-1 protein were demonstrated. Ethanol acts as a potent agent sensitizing colon cancer cells to TRAIL-induced apoptosis.