Gebo Wen

Identification and characterization of novel spliced variants of PRMT2 in breast carcinoma

Protein N‐arginine methyltransferases (PRMTs) participate in a number of cellular processes, including cell growth, nuclear/cytoplasmic protein shuttling, differentiation, RNA splicing and post‐transcriptional regulation. PRMT2 (also known as HRMT1L1) is clearly involved in lung function, the inflammatory response, apoptosis promotion, Wnt signaling and leptin signaling regulation through different mechanisms. In this study, we report the molecular and cell biological characterization of three novel PRMT2 splice variants isolated from breast cancer cells and referred to as PRMT2α, PRMT2β and PRMT2γ. Compared with the wild‐type PRMT2, these variants lack different motifs and therefore generate distinct C‐terminal domains. Confocal microscopy scanning revealed a distinct intracellular localization of PRMT2 variants, suggesting that the alternatively spliced C‐terminus of PRMT2 can directly influence its subcellular localization. Our findings reveal that these variants are capable of binding to estrogen receptor alpha (ERα) both in vitro and in vivo, and the N‐terminal regions of these variants contribute to ERα–PRMT2 interactions. Furthermore, these variants were proved to be able to enhance ERα‐mediated transactivation activity. Luciferase reporter assays showed that PRMT2s could modulate promoter activities of the ERα‐targeted genes of Snail and E‐cadherin. In addition, PRMT2 silencing could enhance 17β‐estradiol‐induced proliferation by regulating E2F1 expression and E2F1‐responsive genes in ERα‐positive breast cancer cells. Real‐time PCR and immunohistochemistry showed that overall PRMT2 expression was upregulated in breast cancer tissues and significantly associated with ERα positivity status both in breast cancer cell lines and breast cancer tissues. We speculate that PRMT2 and its splice variants may directly modulate ERα signaling and play a role in the progression of breast cancer.

Transforming growth factor-β signaling in tumor initiation, progression and therapy in breast cancer: an update

Transforming growth factor-β (TGF-β) is a ubiquitous cytokine playing an essential role in cell proliferation, differentiation, apoptosis, adhesion and invasion, as well as in cellular microenvironment. In malignant diseases, TGF-β signaling features a growth inhibitory effect at an early stage but aggressive oncogenic activity at the advanced malignant state. Here, we update the current understanding of TGF-β signaling in cancer development and progression with a focus on breast cancer. We also review the current approaches of TGF-β signaling-targeted therapeutics for human malignancies.

Adiponectin protects endothelial cells from the damages induced by the intermittent high level of glucose

Globular adiponectin (gAd) has anti-atherogenic effects on the vascular wall. Intermittent hyperglycemia induces endothelial cells (ECs) injury but the physiological factors that may protect against ECs damage are largely unknown. In the present study, we investigated the effect of gAd on ECs dysfunction induced by intermittent high glucose. The gAd significantly attenuated intermittent high glucose-induced apoptosis and oxidative stress in human umbilical vein endothelial cells. This was achieved by decreasing caspase-3 and 3-nitrotyrosine protein expression, increasing nitric oxide (NO) secretion and phosphorylation of Akt, AMPK, and endothelial nitric oxide synthase protein expression. Pretreatment with a phosphatidylinositol 3’ kinase (PI3K) inhibitor, LY294002, partly reversed adiponectin’s anti-apoptotic effect. Taken together, our results indicate that gAd acts as a critical physiological factor which protects against fluctuating high glucose-induced endothelial damage. It may act via attenuating apoptosis and increasing synthesis of NO through both the PI3K/AKT and AMPK signaling pathway to reduce oxidative stress and cell apoptosis.

Identification and expression analysis of a novel transcript of the human PRMT2 gene resulted from alternative polyadenylation in breast cancer

The arginine N-methyltransferase 2 protein (PRMT2, also known as HRMT1L1) is thought to act as a coactivator of ERα. The present results show the occurrence of a novel transcript by alternative polyadenylation in the human PRMT2 gene. We demonstrated that the newly identified intron-retaining PRMT2L2 transcript is functionally intact, efficiently translated into protein in vivo. PRMT2 and PRMT2L2 mRNA expression profiles overlap with the distribution of ERα, with the strongest abundance in estrogen target tissues. Transient co-transfection assays demonstrated that PRMT2L2 enhance ERα-mediated transactivation activity of ERE-Luc in a ligand-dependent manner. Confocal microscopy scanning revealed a distinct intra-cellular localization of their fusion proteins, suggesting that the C-terminal region absent in PRMT2L2 is critical for the localization. Statistical analysis further showed that both PRMT2 and PRMT2L2 mRNAexpressions were up-regulated in breast cancer tissues, and significantly associated with ERα positivity status. Thus, post-transcriptional processing mechanism as alternative polyadenylation and splicing may play a crucial role in regulating human PRMT2 gene expression.

Chemical Genetics of Acetyl-CoA Carboxylases

Chemical genetic studies on acetyl-CoA carboxylases (ACCs), rate-limiting enzymes in long chain fatty acid biosynthesis, have greatly advanced the understanding of their biochemistry and molecular biology and promoted the use of ACCs as targets for herbicides in agriculture and for development of drugs for diabetes, obesity and cancers. In mammals, ACCs have both biotin carboxylase (BC) and carboxyltransferase (CT) activity, catalyzing carboxylation of acetyl-CoA to malonyl-CoA. Several classes of small chemicals modulate ACC activity, including cellular metabolites, natural compounds, and chemically synthesized products. This article reviews chemical genetic studies of ACCs and the use of ACCs for targeted therapy of cancers.

TGF-β1 induces HMGA1 expression in human breast cancer cells: Implications of the involvement of HMGA1 in TGF-β signaling

Transforming growth factor-β1 (TGF-β1) signaling and high mobility group A (HMGA1) are known to play essential roles in the progression of breast cancer by inducing epithelial-mesenchymal transition. However, the correlation between TGF-β1 and HMGA1 in breast cancer cell is not yet well understood. In this study, we determined the effects of TGF-β1 on HMGA1 expression in breast cancer cells and examined the role of HMGA1 in breast cancer progression. Our results demonstrated that TGF-β1 induced the expression of HMGA1 in both MCF-7 and MDA-MB-231 breast cancer cells, as shown by RT-qPCR and immunofluorescence staining; however, the TGF-β1-induced expression of HMGA was blocked by treatment of the cells with phosphatidylinositol-3 kinase (PI3K) signaling inhibitors. Moreover, the HMGA1 promoter activity was found to be activated by TGF-β1 in the MCF-7 and MDA-MB-231 cells and we found that specificity protein 1 (Sp1) was involved in the TGF-β1-induced HMGA1 promoter activity, as shown by luciferase activity assay. Furthermore, the enforced expression of HMGA1 by transfection with a HMGA1 promoter enhanced cellular oncogenic properties, including proliferation, migration and invasion, and a tissue microarray revealed that breast tumors expressing human epidermal growth factor receptor 2 (HER2) showed higher expression levels of HMGA1 (P=0.007). In addition, higher HMGA1 expression levels were also observed in the ductal breast cancer cases compared with the lobular breast cancer cases (P=0.000). These findings establish the first link between HMGA1 and TGF-β1 in breast cancer, providing further evidence of the pivotal role of HMGA1 in breast cancer progression.

Zinc alpha2 glycoprotein alleviates palmitic acid-induced intracellular lipid accumulation in hepatocytes.

Zinc alpha2 glycoprotein (ZAG) plays an important role in stimulating fat mobilization and lipolysis in adipose tissue, but its role in hepatic lipid metabolism remains unclear. Palmitic acid (PA) was used to stimulate HepG2 cells with ZAG overexpression or ZAG knock down (shRNA). Overexpression of ZAG significantly inhibited lipogenesis, promoted lipolysis and fatty acid β-oxidation, and attenuated PA-induced intracellular fat accumulation. Moreover, ZAG overexpression dramatically stimulated adiponectin expression in HepG2 cells. In contrast, knockdown of ZAG notably inhibited fatty acid β-oxidation, increased lipogenesis and lipid accumulation. Collectively, these data suggest that ZAG has the potential to alleviate hepatosteatosis, making it a promising therapeutic target for fatty liver.

Effect of human S100A13 gene silencing on FGF-1 transportation in human endothelial cells.

BACKGROUND/PURPOSE The S100 protein is part of a Ca(2+) binding protein superfamily that contains an EF hand domain, which is involved in the onset and progression of many human diseases, especially the proliferation and metastasis of tumors. S100A13, a new member of the S100 protein family, is a requisite component of the fibroblast growth factor-1 (FGF-1) protein release complex, and is involved in human tumorigenesis by interacting with FGF-1 and interleukin-1. In this study, experiments were designed to determine the direct role of S100A13 in FGF-1 protein release and transportation. METHODS We successfully constructed the lentiviral vectors containing shRNA targeting the human S100A13 gene. Human umbilical vein endothelial cells (HUVECs) were transfected with lentiviral RNAi vectors for S100A13. Then immunofluorescence staining, real-time quantitative polymerase chain reaction and Western blotting were used to detect the inhibition efficiency of the vectors and to monitor the release and transportation of FGF-1 protein. RESULTS Lentiviral RNAi vectors induced suppression efficiency of S100A13 gene by 90% in HUVECs. FGF-1 protein was found to be transported from the cytoplasm to the cell membrane, and then released from cells when HUVECs were deprived of serum. The release of FGF-1 protein was blocked by the downregulation of S100A13, but the transportation was not affected, suggesting that S100A13 is a key cargo protein for FGF-1 release. CONCLUSION S100A13 promotes the release of FGF-1 protein, but does not affect the transportation of FGF-1 protein in HUVECs.

Wnt/β-catenin signaling pathway and lipolysis enzymes participate in methylprednisolone induced fat differential distribution between subcutaneous and visceral adipose tissue.

Glucocorticoids (GCs) are well known to induce fat distribution, which is consistent with the central adiposity phenotype seen in Cushings syndrome. GCs have been proposed to be both adipogenic and lipolytic in action within adipose tissues. Different adipogenic and lipolytic effects between subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) are likely to play a role in GCs induced fat differential distribution. Wnt/β-catenin signaling pathway is one of the most important regulators in adipogenesis. Adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) are the major lipases contributing to lipolysis. In the present study, we measured fat depot masses and the expression of Wnt/β-catenin signaling pathway and lipolytic enzymes of female Sprague-Dawley rats treated with or without methylprednisolone. We assessed the roles of Wnt/β-catenin signaling pathway and lipolytic enzymes in fat differential distribution between SAT and VAT. Our data suggested that methylprednisolone could inhibit Wnt/β-catenin signaling pathway in SAT and VAT, increase the expression of ATGL and HSL in SAT, and decrease the expression of ATGL and HSL in VAT. The differential expression of lipolysis enzymes induced by methylprednisolone between SAT and VAT might play a crucial role in fat distribution. Those findings would offer novel insights into the mechanisms of GCs induced fat distribution.

GABARAPL1 acts as a potential marker and promotes tumor proliferation and metastasis in triple negative breast cancer

GABAA-receptor-associated protein like-1 (GABARAPL1) is involved in a variety of cancers. The purpose of this study was to investigate the expression, prognostic roles and functions of GABARAPL1 in triple negative breast cancer (TNBC). Quantitative real-time PCR (qRT-PCR) showed that GABARAPL1 was up regulated in both TNBC cell lines and clinical TNBC tissues. High GABARAPL1 expression level was associated with shorter overall survival (OS) and disease free survival (DFS). Furthermore, inhibition of GABARAPL1 suppressed cell proliferation, tumorigenesis, invasion and metastasis, and induced cell apoptosis. We found that metadherin (MTDH) was a downstream target of GABARAPL1. Inhibition of GABARAPL1 suppressed the mRNA and protein expression of MTDH, and overexpression of MTDH could reverse the effects of GABARAPL1 inhibition, which meant GABARAPL1 performed its function partly through MTDH. Our findings demonstrate that GABARAPL1 acts as a tumor promoter in TNBC partly through MTDH. Targeting at GABARAPL1 could be a potential therapeutic strategy for TNBC.