Cytokeratin 18-mediated disorganization of intermediate filaments is induced by degradation of plectin in human liver cells

Rho GDP dissociation inhibitor 2 (RhoGDI2) is a regulator of the Rho family GTPases. Recent work from our laboratory suggests that RhoGDI2 expression potentially enhances resistance to cisplatin as well as promotes tumor growth and malignant progression in gastric cancer. In this study, we demonstrate that phospholipase C-gamma (PLCγ) is required for RhoGDI2-mediated cisplatin resistance and cancer cell invasion in gastric cancer. The levels of phosphorylated PLCγ are markedly enhanced in RhoGDI2-overexpressing SNU-484 cells and, by contrast, repressed in RhoGDI2-depleted MKN-28 cells. Depletion of PLCγ expression or inhibition of its activity not only significantly increases cisplatin-induced apoptosis but also suppresses the invasive ability of RhoGDI2-overexpressing SNU-484 cells. Taken together, our results suggest that PLCγ plays a key role in RhoGDI2-mediated cisplatin resistance and cell invasion in gastric cancer cells.     

microRNA-377-3p downregulates the oncosuppressor T-cadherin in colorectal adenocarcinoma cells

Colorectal cancer (CRC) is the second leading cause of death of malignant tumors worldwide. Recent studies point to a role for the adiponectin-receptor axis in colorectal carcinogenesis, and in particular to the oncosuppressive properties of the T-cadherin receptor. In addition, the loss of T-cadherin expression in tumor tissues has been linked to cancer progression and attributed to aberrant methylation of its promoter. Recognizing the pivotal role of microRNAs in CRC, this study explores their possible contribution to the downregulation of T-cadherin. A systematic bioinformatics analysis, restricted by microRNA expression data in the colon or in cultured colorectal cell lines, predicted twelve top-ranking target miRNA sites within the 3ʹ UTR of T-cadherin. Experimental validation analyses based on luciferase reporter constructs and miRNA mimic or miRNA inhibitor transfections toward colorectal adenocarcinoma cell lines indicated that miR-377-3p was able to directly bind to the T-cadherin sequence, and thus downregulating its expression. Given the oncogenic activity of miR-377 and the oncosuppressive activity of T-cadherin in CRC, the regulatory circuit highlighted in this study may add new insights into molecular mechanisms driving colorectal carcinogenesis, and perspectively it could be exploited to identify novel biomarkers and therapeutic targets.     

Vascular endothelial growth factor-C promotes the growth and invasion of gallbladder cancer via an autocrine mechanism

Vascular endothelial growth factor-C (VEGF-C) has a well-defined action on neoplastic lymphangiogenesis and angiogenesis through VEGF receptor-3 (VEGFR-3) and VEGFR-2, respectively, which are generally expressed in endothelial cells. The function of the VEGF-C/receptors pathway in tumor cell types is largely unknown. In this study, we examined the expression and role of VEGF-C/receptors in gallbladder cancer (GBC) cells. We examined the expression of VEGF-C in 50 surgical specimens from gallbladder cancer and three human gallbladder cancer cell lines. Both siRNA and neutralizing antibody to deplete the expression of VEGF-C were used to characterize the biological effect of VEGF-C in GBC NOZ cells. Furthermore, we examined the expression of its receptors, VEGFR-3 and VEGFR-2, in three human GBC cell lines. Our results are as follows: The expression of VEGF-C in the invasive marginal portion was significantly higher than the expression in the central portions. All the three GBC cell lines expressed VEGF-C. Treatment of NOZ cells with VEGF-C siRNA or a neutralizing antibody suppressed cell proliferation and invasion. Moreover, all the three GBC cell lines expressed VEGFR3, but only the NOZ cells expressed VEGFR-2 mRNA. Treatment of NOZ cells with a VEGFR-3 neutralizing antibody suppressed cell invasion, but treatment of NOZ cells with a VEGFR-2 neutralizing antibody suppressed cell proliferation and invasion. In conclusion, GBC cells express both VEGF-C and its receptors. VEGF-C may have a role in the progressive growth and invasion of human GBC through an autocrine mechanism.     

miR-200b regulates cell migration via Zeb family during mouse palate development

Palate development requires coordinating proper cellular and molecular events in palatogenesis, including the epithelial–mesenchymal transition (EMT), apoptosis, cell proliferation, and cell migration. Zeb1 and Zeb2 regulate epithelial cadherin (E-cadherin) and EMT during organogenesis. While microRNA 200b (miR-200b) is known to be a negative regulator of Zeb1 and Zeb2 in cancer progression, its regulatory effects on Zeb1 and Zeb2 in palatogenesis have not yet been clarified. The aim of this study is to investigate the relationship between the regulators of palatal development, specifically, miR-200b and the Zeb family. Expression of both Zeb1 and Zeb2 was detected in the mesenchyme of the mouse palate, while miR-200b was expressed in the medial edge epithelium. After contact with the palatal shelves, miR-200b was expressed in the palatal epithelial lining and epithelial island around the fusion region but not in the palatal mesenchyme. The function of miR-200b was examined by overexpression via a lentiviral vector in the palatal shelves. Ectopic expression of miR-200b resulted in suppression of the Zeb family, upregulation of E-cadherin, and changes in cell migration and palatal fusion. These results suggest that miR-200b plays crucial roles in cell migration and palatal fusion by regulating Zeb1 and Zeb2 as a noncoding RNA during palate development.     

Inhibition of Cancer Cell Migration and Invasion through Suppressing the Wnt1-mediating Signal Pathway by G-quadruplex Structure Stabilizers

WNT1 encodes a multifunctional signaling glycoprotein that is highly expressed in several malignant tumors. Patients with Wnt1-positive cancer are usually related to advanced metastasis. Here, we found that a stretch of G-rich sequences located at the WNT1 promoter region is capable of forming G-quadruplex structures. The addition of G-quadruplex structure stabilizers, BMVC and BMVC4, raises the melting temperature of the oligonucleotide formed by the WNT1 promoter G-rich sequences. Significantly, the expression of WNT1 was repressed by BMVC or BMVC4 in a G-quadruplex-dependent manner, suggesting that they can be used to modulate WNT1 expression. The role of G-quadruplex stabilizers on Wnt1-mediated cancer migration and invasion was further analyzed. The protein levels of β-catenin, a mediator of the Wnt-mediated signaling pathway, and the downstream targets MMP7 and survivin were down-regulated upon BMVC or BMVC4 treatments. Moreover, the migration and invasion activities of cancer cells were inhibited by BMVC and BMVC4, and the inhibitory effects can be reversed by WNT1-overexpression. Thus the Wnt1 expression and its downstream signaling pathways can be regulated through the G-quadruplex sequences located at its promoter region. These findings provide a novel approach for future drug development to inhibit migration and invasion of cancer cells.     

Twist1 accelerates tumour vasculogenic mimicry by inhibiting Claudin15 expression in triple‐negative breast cancer

The up‐regulation of EMT regulator Twist1 has been implicated in vasculogenic mimicry (VM) formation in human triple‐negative breast cancer (TNBC). Twist1 targets the Claudin15 promoter in hepatocellular carcinoma cells. Claudin family members are related with TNBC. However, the relationship between Claudin15 and VM formation is not clear. In this study, we first found that Claudin15 expression was frequently down‐regulated in human TNBC, and Claudin15 down‐regulation was significantly associated with VM and Twist1 nuclear expression. Claudin15 down‐regulation correlated with shorter survival compared with high levels. Claudin15 silence significantly enhanced cell motility, invasiveness and VM formation in the non‐TNBC MCF‐7 cells. Conversely, an up‐regulation of Claudin15 remarkably reduced TNBC MDA‐MB‐231 cell migration, invasion and VM formation. We also showed that down‐regulation of Claudin15 was Twist1‐dependent, and Twist1 repressed Claudin15 promoter activity. Furthermore, GeneChip analyses of mammary glands of Claudin15‐deficient mice indicated that Claudin18 and Jun might be downstream factors of Twist1‐Claudin15. Our results suggest that Twist1 induced VM through Claudin15 suppression in TNBC, and Twist1 inhibition of Claudin15 might involve Claudin18 and Jun expression.     

Novel function of transcription factor Nrf2 as an inhibitor of RON tyrosine kinase receptor-mediated cancer cell invasion

Recepteur d' origine nantais (RON), a tyrosine kinase receptor, is aberrantly expressed in human tumors and promotes cancer cell invasion. RON receptor activation is also associated with resistance to tamoxifen treatment in breast cancer cells. Nrf2 is a positive regulator of cytoprotective genes. Using chromatin immunoprecipitation (ChIP) and site-directed mutagenesis studies of the RON promoter, we identified Nrf2 as a negative regulator of RON gene expression. High Nrf2 and low RON expression was observed in normal mammary tissue whereas high RON and low or undetectable expression of Nrf2 was observed in breast tumors. The Nrf2 inducer sulforaphane (SFN) as well as ectopic Nrf2 expression or knock-down of the Nrf2 negative regulator keap1, which stabilizes Nrf2, inhibited RON expression and invasion of carcinoma cells. Consequently, our studies identified a novel functional role for Nrf2 as a "repressor" and RON kinase as a molecular target of SFN, which mediates the anti-tumor effects of SFN. These results are not limited to breast cancer cells since the Nrf2 inducer SFN stabilized Nrf2 and inhibited RON expression in carcinoma cells from various tumor types.     

Down-Regulation of Filamin Ainteracting protein 1-like Is Associated with Promoter Methylation and an Invasive Phenotype in Breast,Colon, Lung and Pancreatic Cancers

Identifying key mediators of cancer cell invasion and metastasis is critical to the development of more effective cancer therapies. We previously identified Filamin A interacting protein 1-like (FILIP1L) as an important inhibitor of cell migration and invasion in ovarian cancer. FILIP1L expression was inversely correlated with the invasive potential of ovarian cancer cell lines and ovarian cancer specimens. We also demonstrated that DNA methylation in the FILIP1L promoter was a mechanism by which FILIP1L was down-regulated in ovarian cancer. In our present study, we tested this observation in other cancer histologies: breast, colon, lung and pancreatic cancers. Both mRNA and protein expression of FILIP1L were down-regulated in these cancer cells compared with their normal epithelial cells. As in ovarian cancer, DNA methylation is a mechanism by which FILIP1L is down-regulated in these cancer histologies. Methylation status of the FILIP1L promoter was inversely correlated with FILIP1L expression. Reduced methylation in the FILIP1L promoter following treatment with a DNA demethylating agent was associated with restoration of FILIP1L expression in these cancer cells. Further, FILIP1L expression was inversely correlated with the invasive potential of these cancer cells. Re-expression of FILIP1L in FILIP1L-low expressing, highly-invasive cancer cell lines resulted in inhibition of cell invasion. Correspondingly, knockdown of FILIP1L in FILIP1L-high expressing, low-invasive cancer cell lines resulted in increase of cell invasion. Overall, these findings suggest that down-regulation of FILIP1L associated with DNA methylation is related with the invasive phenotype in various cancers. Thus, modulation of FILIP1L expression has the potential to be a target for cancer therapy.     

Downregulated long noncoding RNA LINC00313 inhibits the epithelial–mesenchymal transition,invasion, and migration of thyroid cancer cells through inhibiting the methylation of ALX4

Thyroid cancer represents one of the prevalent endocrine cancer with relatively high incidence rate around the world, accompanied by unchanged fatality rate. We probe into the specific role of LINC00313 in mediation of cellular processes of thyroid cancer including proliferation, migration, and invasion through the methylation of aristaless-like homeobox 4 (ALX4). Thyroid cancer-related long noncoding RNAs (lncRNAs) and genes were analyzed by microarray-based analysis. The antitumor effect of LINC00313 was examined with the gain- and loss-of-function experiments. In addition, the binding of LINC00313 and the promoter region of ALX4, and the interaction of LINC00313 with methylation-related proteins were detected. Later, xenograft tumors in nude mice were induced expecting to dig out the modulatory function of LINC00313 in tumor growth of thyroid carcinoma. The microarray-based analysis manifested that LINC00313 was overexpressed, whereas ALX4 was downregulated in thyroid cancer, the results of which were also verified in thyroid cancer tissues. Besides, our results demonstrated that LINC00313 bound to the ALX4 promoter region, and LINC00313 recruited DNMT1 and DNMT3B proteins to promote the methylation of ALX4 promoter region, thus suppressing the ALX4 expression. Finally, the downregulation of LINC00313 and upregulation of ALX4 repressed the AKT/mTOR signaling axis, thus inhibiting proliferative, migratory, invasive abilities as well as epithelial-to-mesenchymal transition (EMT) of thyroid cancer cells. Collectively, downregulated LINC00313 suppresses cell proliferation, migration, as well as invasion of thyroid cancer by inhibiting the methylation of ALX4 and increasing its expression by inactivation of the AKT/mTOR signaling pathway.     

Expression of adhesion molecule T-cadherin is increased during neointima formation in experimental restenosis

Phenotypic modulation, migration and proliferation of vascular smooth muscle cells (SMCs) are major events in restenosis after percutaneous transluminal angioplasty. Surface cell adhesion molecules, essential to morphogenesis and maintenance of adult tissue architecture, are likely to be involved, but little is known about cell adhesion molecules expressed on SMCs. T-cadherin is a glycosyl phosphatidylinositol-anchored member of the cadherin superfamily of adhesion molecules. Although highly expressed in vascular and cardiac tissues, its function in these tissues is unknown. We previously reported increased expression of T-cadherin in intimal SMCs in atherosclerotic lesions and proposed a role for T-cadherin in phenotype control. Here we performed immunohistochemical analysis of spatial and temporal changes in vascular T-cadherin expression following balloon catheterisation of the rat carotid artery. T-cadherin expression in SMCs markedly increases in the media early (1-4 days) after injury, and later (day 7-28) in forming neointima, especially in its preluminal area. Staining for monocyte/macrophage antigen ED-1, proliferating cell nuclear antigen and smooth muscle alpha-actin revealed that spatial and temporal changes in T-cadherin level coincided with the peak in cell migration and proliferation activity during neointima formation. In colchicine-treated cultures of rat aortic SMCs T-cadherin expression is increased in dividing M-phase cells but decreased in non-dividing cells. Together the data support an association between T-cadherin expression and SMC phenotype.