Down‐regulating Myoferlin inhibits the vasculogenic mimicry of melanoma via decreasing MMP‐2 and inducing mesenchymal‐to‐epithelial transition

Vasculogenic mimicry (VM) constitutes a novel approach for tumour blood supply and contributes to tumour metastasis and poor prognosis in patients with melanoma. Myoferlin (MYOF), a type II membrane protein involved in membrane regeneration and repair, is elevated in several malignant tumours, especially in advanced melanomas. This study aims to investigate the role and mechanism of MYOF in the regulation of VM. VM structures were found in 14 of 52 tested melanoma samples, and high MYOF expression correlated with VM structures. According to Kaplan–Meier survival curves, VM channels and elevated MYOF expression both correlated with poor prognosis in melanoma patients. Down‐regulation of MYOF by siRNA severely impaired the capability of A375 cells to form VM structures in vitro. Further studies demonstrated MYOF knockdown inhibited cell migration and invasion, which is required for VM formation, via decreasing MMP‐2 expression as evidenced by Western blotting, RT‐RCP and ELISA results. SB‐3CT, a specific inhibitor of MMP‐2, showed similar inhibiting effects with siMYOF, further supporting that MYOF down‐regulation inhibits MMP‐2 expression to affect VM formation. Moreover, MYOF knockdown suppress VM formation by A375 cells by inducing mesenchymal‐to‐epithelial transition (MET). After down‐regulating MYOF, focal adhesions were enlarged and A375 cells developed into a clear epithelial morphology. Such cells acquired the expression of E‐cadherin at adherens junctions along with a loss of mesenchymal markers, such as Vimentin and Twist1. In conclusion, MYOF plays an important role in VM and knockdown of MYOF suppresses VM formation via decreasing MMP‐2 and inducing MET in A375 melanoma cells.     

Rictor regulates the vasculogenic mimicry of melanoma via the AKT‐MMP‐2/9 pathway

Vasculogenic mimicry (VM)‐positive melanomas are usually associated with poor prognosis. Rictor, the key component of the rapamycin‐insensitive complex of mTOR (mTORC2), is up‐regulated in several cancers, especially in melanomas with poor prognosis. The aim of this study was to investigate the role of Rictor in the regulation of VM and the mechanism underlying this possible regulation. VM channels were found in 35 of 81 tested melanoma samples and high Rictor expression correlated with VM structures. Moreover, Kaplan–Meier survival curves indicated that VM structures and high Rictor expression correlated with shorter survival in patients with melanoma. In vitro, Rictor knockdown by short hairpin RNA (shRNA) significantly inhibited the ability of A375 and MUM‐2B melanoma cells to form VM structures, as evidenced by most tubes remaining open. Cell cycle analysis revealed that Rictor knockdown blocked cell growth and resulted in the accumulation of cells in G2/M phase, and cell migration and invasion were greatly affected after Rictor down‐regulation. Western blotting assays indicated that down‐regulating Rictor significantly inhibited the phosphorylation of AKT at Ser⁴⁷³ and Thr³⁰⁸, which subsequently inhibited the expression and activity of downstream MMP‐2/9, as confirmed by real‐time PCR and gelatin Zymography. MK‐2206, a small‐molecule inhibitor of AKT, similarly inhibited the activity of AKT and secretion of MMP‐2/9, further supporting that Rictor down‐regulation inhibits the phosphorylation of AKT and activity of downstream MMP‐2/9 to affect VM formation. In conclusion, Rictor plays an important role in melanoma VM via the Rictor—AKT—MMP‐2/9 signalling pathway.     

miR‐655 suppresses epithelial‐to‐mesenchymal transition by targeting Prrx1 in triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) is a highly aggressive breast cancer subtype that lacks effective targeted therapies. The epithelial‐to‐mesenchymal transition (EMT) is a key contributor in the metastatic process. In this study, we found that miR‐655 was down‐regulated in TNBC, and its expression levels were associated with molecular‐based classification and lymph node metastasis in breast cancer. These findings led us to hypothesize that miR‐655 overexpression may inhibit EMT and its associated traits of TNBC. Ectopic expression of miR‐655 not only induced the up‐regulation of cytokeratin and decreased vimentin expression but also suppressed migration and invasion of mesenchymal‐like cancer cells accompanied by a morphological shift towards the epithelial phenotype. In addition, we found that miR‐655 was negatively correlated with Prrx1 in cell lines and clinical samples. Overexpression of miR‐655 significantly suppressed Prrx1, as demonstrated by Prrx1 3′‐untranslated region luciferase report assay. Our study demonstrated that miR‐655 inhibits the acquisition of the EMT phenotype in TNBC by down‐regulating Prrx1, thereby inhibiting cell migration and invasion during cancer progression.     

Dickkopf‐1‐promoted vasculogenic mimicry in non‐small cell lung cancer is associated with EMT and development of a cancer stem‐like cell phenotype

To characterize the contributions of Dickkopf‐1 (DKK1) towards the induction of vasculogenic mimicry (VM) in non‐small cell lung cancer (NSCLC), we evaluated cohorts of primary tumours, performed in vitro functional studies and generated xenograft mouse models. Vasculogenic mimicry was observed in 28 of 205 NSCLC tumours, while DKK1 was detected in 133 cases. Notably, DKK1 was positively associated with VM. Statistical analysis showed that VM and DKK1 were both related to aggressive clinical course and thus were indicators of a poor prognosis. Moreover, expression of epithelial‐mesenchymal transition (EMT)‐related proteins (vimentin, Slug, and Twist), cancer stem‐like cell (CSC)‐related proteins (nestin and CD44), VM‐related proteins (MMP2, MMP9, and vascular endothelial‐cadherin), and β‐catenin‐nu were all elevated in VM‐positive and DKK1‐positive tumours, whereas the epithelial marker (E‐cadherin) was reduced in the VM‐positive and DKK1‐positive groups. Non‐small cell lung cancer cell lines with overexpressed or silenced DKK1 highlighted its role in the restoration of mesenchymal phenotypes and development of CSC characteristics. Moreover, DKK1 significantly promotes NSCLC tumour cells to migrate, invade and proliferate. In vivo animal studies demonstrated that DKK1 enhances the growth of transplanted human tumours cells, as well as increased VM formation, mesenthymal phenotypes and CSC properties. Our results suggest that DKK1 can promote VM formation via induction of the expression of EMT and CSC‐related proteins. As such, we feel that DKK1 may represent a novel target of NSCLC therapy.     

Promotion of hepatocellular carcinoma metastasis through matrix metalloproteinase activation by epithelial‐mesenchymal transition regulator Twist1

E‐cadherin loss is a key biological mechanism in tumour invasion. As a main regulator of epithelial‐mesenchymal transition (EMT) mechanism‐mediated invasion and metastasis, Twist1 plays an important role through its regulation of E‐cadherin expression. However, whether or not Twist2 has the same function in tumour metastasis remains unclear. The purpose of this study is to investigate the expressions and different roles of Twist1 and Twist2 in human hepatocellular carcinoma (HCC). The expressions of Twist1 and Twist2 in HCC tissue were evaluated by immunohistochemical staining. The role of Twist1 and Twist2 in invasiveness was also evaluated in vitro by using HCC cell lines. Twist1 nuclear overexpression is found to be correlated with HCC metastasis, and its expression is negatively correlated with E‐cadherin expression in human tissue. Twist2, a Twist1 homology protein, only expresses in the cytoplasm and shows no significant correlation with HCC metastasis. By ectopic transfection of Twist1 and Twist2 into the HCC cells, HepG2 and PLC, Twist1 is able to down‐regulate E‐cadherin expression and promote matrix metalloproteinase (MMP) activation, specifically in MMP2 and MMP9. In functional assays, Twist1 is found to promote invasion in HepG2 and PLC cells, but the invasion ability of the groups is not affected Twist2. Our findings indicate that Twist1 induces HCC invasion via increased activity in MMPs, leading to poor clinical prognoses. The results of this study also demonstrate a novel cogitation in Twist2, which has no effect on HCC invasion and metastasis. Twist1 may contribute to HCC invasion and metastasis and may be used as a novel therapeutic target for the inhibition of HCC metastasis.     

Paired box 5 is a frequently methylated lung cancer tumour suppressor gene interfering β‐catenin signalling and GADD45G expression

Recent studies suggest that paired box 5 (PAX5) is down‐regulated in multiple tumours through its promoter methylation. However, the role of PAX5 in non‐small cell lung cancer (NSCLC) pathogenesis remains unclear. The aim of this study is to examine PAX5 expression, its methylation status, biological functions and related molecular mechanism in NSCLC. We found that PAX5 was widely expressed in normal adult tissues but silenced or down‐regulated in 88% (7/8) of NSCLC cell lines. PAX5 expression level was significantly lower in NSCLC than that in adjacent non‐cancerous tissues (P = 0.0201). PAX5 down‐regulation was closely associated with its promoter hypermethylation status and PAX5 expression could be restored by demethylation treatment. Frequent PAX5 promoter methylation in primary tumours (70%) was correlated with lung tumour histological types (P = 0.006). Ectopic expression of PAX5 in silenced lung cancer cell lines (A549 and H1975) inhibited their colony formation and cell viability, arrested cell cycle at G2 phase and suppressed cell migration/invasion as well as tumorigenicity in nude mice. Restoration of PAX5 expression resulted in the down‐regulation of β‐catenin and up‐regulation of tissue inhibitors of metalloproteinase 2, GADD45G in lung tumour cells. In summary, PAX5 was found to be an epigenetically inactivated tumour suppressor that inhibits NSCLC cell proliferation and metastasis, through down‐regulating the β‐catenin pathway and up‐regulating GADD45G expression.     

MMP‐2 and MMP‐13 affect vasculogenic mimicry formation in large cell lung cancer

Matrix metalloproteinases (MMPs) have critical functions in tumour vasculogenic mimicry (VM). This study explored the mechanisms underlying MMP‐13 and MMP‐2 regulation of tumour VM formation in large cell lung cancer (LCLC). In our study, laminin5 (Ln‐5) fragments cleaved by MMP‐2 promoted tubular structure formation by the LCLC cell lines H460 and H661 in three‐dimensional (3D) cultures. Transient up‐regulation of MMP‐13 or treatment with recombinant MMP‐13 protein abrogated tubular structure formation of H460 cells in 3D culture. Treated cells with Ln‐5 fragments cleaved by MMP‐2 stimulated EGFR and F‐actin expression. Ln‐5 fragments cleaved by MMP‐13 decreased EGFR/F‐actin expression and disrupted VM formation. MMP‐13 expression was negatively correlated with VM, Ln‐5 and EGFR in LCLC tissues and xenograft. In vivo experiments revealed that VM was decreased when the number of endothelium‐dependent vessels (EDVs) increased during xenograft tumour growth, whereas MMP‐13 expression was progressively increased. In conclusion, MMP‐2 promoted and MMP‐13 disrupted VM formation in LCLC by cleaving Ln‐5 to influence EGFR signal activation. MMP‐13 may regulate VM and EDV formation.     

HBx‐dependent activation of twist mediates STAT3 control of epithelium‐mesenchymal transition of liver cells

This study investigated the molecular mechanisms of liver cells with HBx expression on epithelium–mesenchymal transition (EMT) change using Western blot analysis and Transwell assay to assess EMT‐related protein expression and cell mobility. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were used to test the Twist promoter containing different STAT3 binding loci. Electrophoretic mobility band‐shift assay (EMSA) was used to detect Twist activity. Results showed that HBx expression affected the EMT‐related protein expression and the cell mobility of liver cancer cells (MHCC97) and liver cells (HL‐7702) in vitro or in vivo. These proteins exhibited reversed expression to a certain extent after Twist inhibition. In addition, the wound‐healing capability and the mobility of HL‐7702/HBx cells were lower than those treated with control‐siRNA. The expressions of p‐STAT3 and Twist were positively correlated with HBx expression. The second STAT‐3 binding sequence in the Twist promoter region of the HL‐7702/HBx cells was the first locus. Twist activity in the HL‐7702/HBx2 cells was higher than that in HL‐7702 cells. Moreover, the activity decreased when the cells were treated with HBx‐siRNA to inhibit HBx expression, or with STAT3 inhibitor to reduce STAT3 activation. Therefore, Twist is essential for the regulation of the mobility of liver cells with HBx expression. HBx activates the Twist promoter by activating STAT3 and promotes EMT occurrence in liver cells. J. Cell. Biochem. 114: 1097–1104, 2013. © 2012 Wiley Periodicals, Inc.     

Epstein-Barr virus-encoded latent membrane protein 1 modulates cyclin D1 by c-Jun/Jun B heterodimers

In our recent studies, we found that LMP1 encoded by Epstein-Barr virus could accelerate the formation of active c-Jun/Jun B heterodimer. We studied the regulation of cyclinD1 by c-Jun/Jun B heterodimers by laser scanning confocal influorescence microscopy, Western blot, luciferase activity assay, super-EMSA and flow cytometry in the Tet-on-LMP1 HNE2 cell line, in which LMP1 expression was regulated by Tet-on system. c-Jun/Jun B heterodimers induced by LMP1 could up regulate cyclin D1 promoter activity and expression. Overexpression of cyclinD1 accelerated the progression of cell cycle.     

Twist2 regulates CD7 expression and galectin-1-induced apoptosis in mature T-cells

In the periphery, a galectin-1 receptor, CD7, plays crucial roles in galectin-1-mediated apoptosis of activated T-cells as well as progression of T-lymphoma. Previously, we demonstrated that NF-κB downregulated CD7 gene expression through the p38 MAPK pathway in developing immature thymocytes. However, its regulatory pathway is not well understood in functional mature T-cells. Here, we show that CD7 expression was downregulated by Twist2 in Jurkat cells, a human acute T-cell lymphoma cell line, and in EL4 cells, a mature murine T-cell lymphoma cell line. Furthermore, ectopic expression of Twist2 in Jurkat cells reduced galectin-1-induced apoptosis. While full-length Twist2 decreased CD7 promoter activity, a C-terminal deletion form of Twist2 reversed its inhibition, suggesting an important role of the C-terminus in CD7 regulation. In addition, CD7 expression was enhanced by histone deacetylase inhibitors such as trichostatin A and sodium butyrate, which indicates that Twist2 might be one of candidate factors involved in histone deacetylation. Based on these results, we conclude that upregulation of Twist2 increases the resistance to galectin-1-mediated-apoptosis, which may have significant implications for the progression of some T-cells into tumors such as Sezary cells.     

HER2/neu expression correlates with vasculogenic mimicry in invasive breast carcinoma

Vasculogenic mimicry (VM) refers to the condition in which tumour cells mimic endothelial cells to form extracellular matrix‐rich tubular channels. VM is more extensive in more aggressive tumours. The human epidermal growth factor receptor 2 (HER2) gene is amplified in 20–30% of human breast cancers and has been implicated in mediating aggressive tumour growth and metastasis. However, thus far, there have been no data on the role of HER2 in VM formation. Immunohistochemical and histochemical double‐staining methods were performed to display VM in breast cancer specimens. Transfection in MCF7 cells was performed and clones were selected by G418. The three‐dimensional Matrigel culture was used to evaluate VM formation in the breast cancer cell line. According to statistical analysis, VM was related to the presence of a positive nodal status and advanced clinical stage. The positive rate of VM increased with increased HER2 expression. In addition, cases with HER2 3+ expression showed significantly greater VM channel count than those in other cases. The exogenous HER2 overexpression in MCF‐7 cells induced vessel‐like VM structures on the Matrigel and increased the VM mediator vascular endothelial (VE) cadherin. Our data provide evidence for a clinically relevant association between HER2 and VM in human invasive breast cancer. HER2 overexpression possibly induces VM through the up‐regulation of VE cadherin. Understanding the key molecular events may provide therapeutic intervention strategies for HER2+ breast cancer.     

Aurora kinase A stabilizes FOXM1 to enhance paclitaxel resistance in triple‐negative breast cancer

Triple‐negative breast cancer (TNBC) has a relatively poor outcome. Acquired chemoresistance is a major clinical challenge for TNBC patients. Previously, we reported that kinase‐dead Aurora kinase A (Aurora‐A) could effectively transactivate the FOXM1 promoter. Here, we demonstrate an additional pathway through which Aurora‐A stabilizes FOXM1 by attenuating its ubiquitin in TNBC. Specifically, Aurora‐A stabilizes FOXM1 in late M phase and early G1 phase of the cell cycle, which promotes proliferation of TNBC cells. Knock‐down of Aurora‐A significantly suppresses cell proliferation in TNBC cell lines and can be rescued by FOXM1 overexpression. We observe that paclitaxel‐resistant TNBC cells exhibit high expression of Aurora‐A and FOXM1. Overexpression of Aurora‐A offers TNBC cells an additional growth advantage and protection against paclitaxel. Moreover, Aurora‐A and FOXM1 could be simultaneously targeted by thiostrepton. Combination of thiostrepton and paclitaxel treatment reverses paclitaxel resistance and significantly inhibits cell proliferation. In conclusion, our study reveals additional mechanism through which Aurora‐A regulates FOXM1 and provides a new therapeutic strategy to treat paclitaxel‐resistant triple‐negative breast cancer.     

Expression analysis of epithelial cadherin and related proteins in IBH‐6 and IBH‐4 human breast cancer cell lines

Epithelial cadherin (E‐cadherin) is a 120 kDa cell–cell adhesion molecule involved in the establishment of epithelial adherens junctions. It is connected to the actin cytoskeleton by adaptor proteins such as β‐catenin. Loss of E‐cadherin expression/function has been related to tumor progression and metastasis. Several molecules associated with down‐regulation of E‐cadherin have been described, within them neural cadherin, Twist and dysadherin. Human breast cancer cell lines IBH‐6 and IBH‐4 were developed from ductal primary tumors and show characteristic features of malignant epithelial cells. In this study expression of E‐cadherin and related proteins in IBH‐6 and IBH‐4 cell lines was evaluated. In IBH‐6 and IBH‐4 cell extracts, only an 89 kDa E‐cadherin form (Ecad89) was detected, which is truncated at the C‐terminus and is present at low levels. Moreover, no accumulation of the 86 kDa E‐cadherin ectodomain and of the 38 kDa CTF1 fragment was observed. IBH‐6 and IBH‐4 cells showed an intracellular scattered E‐cadherin localization. β‐catenin accompanied E‐cadherin localization, and actin stress fibers were identified in both cell types. E‐cadherin mRNA levels were remarkably low in IBH‐6 and IBH‐4 cells. The E‐cadherin mRNA and genomic sequence encoding exons 14–16 could not be amplified in either cell line. Neither the mRNA nor the protein of neural cadherin and dysadherin were detected. Up‐regulation of Twist mRNA was found in both cell lines. In conclusion, IBH‐6 and IBH‐4 breast cancer cells show down‐regulation of E‐cadherin expression with aberrant protein localization, and up‐regulation of Twist; these features can be related to their invasive/metastatic characteristics. J. Cell. Physiol. 222: 596–605, 2010. © 2009 Wiley‐Liss, Inc.