Dongwang Zhu

miR-27a-3p suppresses tumor metastasis and VM by down-regulating VE-cadherin expression and inhibiting EMT: an essential role for Twist-1 in HCC

Twist-1 and miRNAs have been reported to be associated with tumor metastasis and angiogenesis. However, the relationship between Twist-1 and miRNAs and the function of miRNAs remain largely undefined. We aimed to reveal the Twist-1-related miRNA expression profile and to determine whether Twist-1 functions in tumor metastasis and vasculogenic mimicry (VM) by regulating miRNA expression in hepatocellular carcinoma (HCC). Results showed that the expression of miR-27a-3p was consistently down-regulated in HCC cell lines and tissue samples displaying high expression of Twist-1. Both loss- and gain-of-function assays revealed suppressive effects of miR-27a-3p. Low miR-27a-3p expression was significantly associated with early metastasis in HCC. Subsequent investigations revealed that miR-27a-3p mediated the inhibition of epithelial–mesenchymal transition (EMT). Additional experiments showed that VE-cadherin is a direct target of miR-27a-3p and further demonstrated the critical role of miR-27a-3p in suppressing tumor metastasis and VM. Conclusions: Twist-1 up-regulation in HepG2 cells resulted in the differential expression of 18 miRNAs. Among them, miR-27a-3p deregulation contributed to VM and metastasis. The miR-27a-3p-mediated down-regulation of VE-cadherin and inhibition of EMT may be essential for Twist-1 to induce tumor metastasis and VM. Our findings highlight the importance of miR-27a-3p and suggest a promising new strategy for anti-HCC therapy.

Twist1-related miR-26b-5p suppresses epithelial-mesenchymal transition, migration and invasion by targeting SMAD1 in hepatocellular carcinoma

Twist1 is well known to induce epithelial-mesenchymal transition (EMT) and promote tumor metastasis. MicroRNAs (miRNAs) are involved in the EMT process and are associated with metastasis in hepatocellular carcinoma (HCC). In the present study, microRNA-26b-5p (miR-26b-5p) expression was consistently and significantly downregulated in HepG2-Twist1 HCC cell lines compared with HepG2-vector cell lines using microarrays (the HepG2-Twist1 cell line can stably express Twist1). miR-26b- 5p downregulation was directly mediated by Twist1 through binding to the promoter region of miR-26b-5p in HepG2-Twist1 cells by ChIP-seq technology. Both gain- and loss-of-function studies showed that miR-26b-5p dramatically suppressed EMT and the invasion ability of HCC cells in vitro. Using mouse models, tumors derived from miR- 26b-5p-overexpressed HCC cells exhibited a significant reduction in tumorigenicity compared with the control group. Subsequent investigation revealed that miR-26b-5p directly inhibited SMAD family member 1 (SMAD1) expression. miR-26b-5p repressed BMP4/Smad1 signaling following SMAD1 inhibition. Overexpression of SMAD1 reversed the function of miR-26b-5p. In human HCC tissues and mouse xenograft tumors, miR-26b-5p levels were inversely correlated with SMAD1 expression as well as metastasis. Conclusion: miR-26b-5p suppresses Twist1-induced EMT, invasion, and metastasis of HCC cells by targeting SMAD1 and BMP4/Smad1 signaling. This suggests a promising application for miR-26b-5p in anti-HCC therapy.

Notch4+ cancer stem-like cells promote the metastatic and invasive ability of melanoma

Sphere formation in conditioned serum‐free culture medium supplemented with epidermal growth factor and basic fibroblast growth factor (tumorospheres) is considered useful for the enrichment of cancer stem‐like cells, also known as tumor‐initiating cells. We used a gene expression microarray to investigate the gene expression profile of melanoma cancer stem‐like cells (MCSLCs). The results showed that MCSLCs highly expressed the following Notch signaling pathway molecules: Notch3 (NM_008716), Notch4 (NM_010929), Dtx4 (NM_172442), and JAG2 (NM_010588). Immunofluorescence staining showed tumorosphere cells highly expressed Notch4. Notch4high B16F10 cells were isolated by FACS, and Western blotting showed that high Notch4 expression is related to the expression of epithelial–mesenchymal transition (EMT)‐associated proteins. Reduced invasive and migratory properties concomitant with the downregulation of the EMT markers Twist1, vimentin, and VE‐cadherin and the overexpression of E‐cadherin was observed in human melanoma A375 and MUM‐2B cells. In these cells, Notch4 was also downregulated, both by Notch4 gene knockdown and by application of the γ‐secretase inhibitor, DAPT. Mechanistically, the re‐overexpression of Twist1 by the transfection of cells with a Twist1 expression plasmid led to an increase in VE‐cadherin expression and a decrease in E‐cadherin expression. Immunohistochemical analysis of 120 human melanoma tissues revealed a significant correlation between the high expression of Notch4 and the metastasis of melanoma. Taken together, our findings indicate that Notch4+ MCSLCs trigger EMT and promote the metastasis of melanoma cells.

C-myc overexpression drives melanoma metastasis by promoting vasculogenic mimicry via c-myc/snail/Bax signaling

Abstractc-Myc is a well-characterized proto-oncogene that induces cellular transformation and modulates programmed cell death. While recent studies have demonstrated high expression of c-Myc protein in advanced and metastatic melanoma, the clinical and biological implications remain to be fully elucidated. In this study, we investigated the effect of c-Myc overexpression in melanoma tumorigenesis. Clinicopathological analysis demonstrated that c-Myc expression positively correlated with the formation of vasculogenic mimicry (VM) and linearly patterned programmed cell necrosis (LPPCN). Clinically, high c-Myc expression was significantly associated with distant metastasis and poor prognosis, while biologically, c-Myc overexpression led to significant increases in cell motility, invasiveness and metastasis. Moreover, c-Myc induced the formation of VM and promoted the expression of epithelial-mesenchymal transition (EMT)-associated protein Snail both in vivo and in vitro. High expression of c-Myc increased Bax expression in hypoxic conditions and induced cell apoptosis. Taken together, we conclude that c-Myc overexpression promotes the formation of VM by EMT and LPPCN in melanoma. Our improved understanding of the clinical and biological effects of c-Myc overexpression in melanoma highlights the incomplete understanding of this oncogene, and indicates that c-Myc is a potential therapeutic target of this disease.Key messageHigh c-Myc expression is associated with tumor metastasis and poor prognosis in human melanoma.c-Myc upregulates Snail expression to promote EMT via the TGF-β/Snail/Ecadherin signal pathway.c-Myc leads to cell death by upregulating Bax expression causing a lower Bcl2/Bax ratio under severe hypoxic conditions.c-Myc promotes vasculogenic mimicry and linearly patterned programmed cell necrosis.

Subpopulations of uPAR+ contribute to vasculogenic mimicry and metastasis in large cell lung cancer

The urokinase plasminogen activator receptor (uPAR) is closely associated with poor prognosis in various aggressive cancers including large-cell lung cancer (LCLC). Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks involving the blood supply in early tumor formation. We demonstrate the statistically positive correlation of uPAR expression with VM formation, metastasis, and poor prognosis of LCLC patients. uPAR(+) cells sorted from the LCLC H460 cell line show higher invasion, migration capacity, and tube structure formation capability on Matrigel compared with uPAR(-) cells. uPAR(+) tumor cells highly expressed vimentin and VE-cadherin; the epithelial marker E-cadherin was low expressed. Higher EMT-regulated protein twist and snail expressions were also observed in these cells. uPAR(+) cells injected subcutaneously into nude mice markedly increased tumor growth, induced VM formation and liver metastasis; by contrast, uPAR(-) cells did not. The data suggest that uPAR expression may predict VM formation, tumor metastasis and poorer prognosis of LCLC patients. The uPAR gene may be used as a novel therapeutic target for inhibiting angiogenesis and metastasis in LCLC.

HIF-1α promoted vasculogenic mimicry formation in hepatocellular carcinoma through LOXL2 up-regulation in hypoxic tumor microenvironment

BackgroundThe incidence and mortality rates of hepatocellular carcinoma (HCC) have steadily increased in recent years. A hypoxic microenvironment is one of the most important characteristics of solid tumors which has been shown to promote tumor metastasis, epithelial-mesenchymal transition and angiogenesis. Epithelial-mesenchymal transition and vasculogenic mimicry have been regarded as crucial contributing factors to cancer progression. HIF-1α functions as a master transcriptional regulator in the adaptive response to hypoxia. Lysyl oxidases like 2 (LOXL2) is a member of the lysyl oxidase family, which main function is to catalyze the covalent cross-linkages of collagen and elastin in the extracellular matrix. Recent work has demonstrated that HIF-1α promotes the expression of LOXL2, which is believed to amplify tumor aggressiveness. LOXL2 has shown to promote metastasis and is correlated with poor prognosis in hepatocellular carcinoma. The purpose of our study is to explore the role of HIF-1α in progression and metastasis of hepatocellular carcinoma by promoting the expression of LOXL2 as well as the potential regulatory mechanism.MethodsHIF-1α, LOXL2 expression and CD31/periodic acid-Schiff double staining in HCC patient samples were examined by immunohistochemical staining. shRNA plasmids against HIF-1α was used to determine whether LOXL2 been increased by HIF-1α. We monitored a series of rescue assays to demonstrate our hypothesis that LOXL2 is required and sufficient for HIF-1α induced EMT and VM formation, which mediates cellular transformation and takes effect in cellular invasion. Then we performed GeneChip® Human Transcriptome Array (HTA) 2.0 in HepG2 cells, HepG2 cells overexpressed LOXL2 and HepG2 cells treated with CoCl2.ResultsIn clinical HCC tissues, it confirmed a positive relationship between HIF-1α and LOXL2 protein. Importantly, HIF-1α and LOXL2 high expression and the presence of vasculogenic mimicry were correlated to poor prognosis. HIF-1α was found to induce EMT, HCC cell migration, invasion and VM formation by regulating LOXL2. The results of microarray assays were analyzed.ConclusionHIF-1α plays an important role in the development of HCC by promoting HCC metastasis, EMT and VM through up-regulating LOXL2. This study highlights the potential therapeutic value of targeting LOXL2 for suppression of HCC metastasis and progression.

HMGA2 promotes vasculogenic mimicry and tumor aggressiveness by upregulating Twist1 in gastric carcinoma

High mobility group protein A2 (HMGA2) is a transcription factor that plays an important role in the invasion and metastasis of gastric carcinoma (GC). The term vasculogenic mimicry (VM) refers to the unique ability of aggressive tumour cells to mimic the pattern of embryonic vasculogenic networks. However, the relationship between HMGA2 and VM formation remains unclear. In the present study, we examined concomitant HMGA2 expression and VM in 228 human GC samples and 4 GC cell lines. Our data indicate that HMGA2 is not only significantly associated with VM formation but also influences the prognosis of patients with gastric carcinoma. Overexpression of HMGA2 significantly increased cell motility, invasiveness, and VM formation both in vitro and in vivo. A luciferase reporter assay, Co-IP and ChIP demonstrated that HMGA2 induced the expression of Twist1 and VE-cadherin by binding to the Twist1 promoter. Moreover, we observed a decrease in VE-cadherin following Twist1 knockdown in cells overexpressing HMGA2. This study indicates that HMGA2 promotes VM in GC via Twist1-VE-cadherin signalling and influences the prognosis of patients with GC.

HnRNPM and CD44s expression affects tumor aggressiveness and predicts poor prognosis in breast cancer with axillary lymph node metastases

HnRNPM is an essential splicing factor and its expression is closely correlated with invasion and metastasis of tumor cells. The CD44 cell adhesion molecule is aberrantly expressed in many breast tumors and CD44 splice variants have been implicated in specific oncogenic signaling pathways. To investigate the clinical significance and biological function of hnRNPM, immunohistochemistry, quantitative, and semiquantitative polymerase chain reaction, lentiviral transfection system and transwell invasion assays were performed. We found that hnRNPM expression was significantly upregulated in breast cancer tissues compared with benign breast lesions. Although there was no significant correlation between hnRNPM and total CD44 protein or mRNA level, there was a negative correlation between hnRNPM and CD44v6. HnRNPM and CD44s expression showed positive correlation and in particular, they were dually expressed in breast cancer tissues. Interestingly, cancer stem cells marker, ALDH1+ phenotype was positively associated with overexpression of CD44s or hnRNPM and negatively related to CD44v6. Patients with high hnRNPM tended to have higher levels of CD44s, shorter overall survival (OS) and higher rates of lymph node metastases (LNM). Remarkably, Kaplan‐Meier and Cox regression analyses displayed that hnRNPM+ or CD44shigh was a poor prognostic factor for OS of patients with LNM. Upregulation of hnRNPM in MCF‐7 cells caused a significant increase in cell invasion, and this effect may occur through the regulation of CD44s expression. In conclusion, overexpression of hnRNPM promotes breast cancer aggressiveness by regulating the level of CD44s, indicates a poor prognosis for patients with LNM, and has potential as therapeutic targets.

TRA2A promoted paclitaxel resistance and tumor progression in triple-negative breast cancers via regulating alternative splicing

Treatment of triple-negative breast cancer (TNBC) has been challenging, and paclitaxel resistance is one of the major obstacles to the better prognosis. Deregulation of alternative splicing (AS) may contribute to tumor progression and chemotherapy resistance. Human AS factor TRA2 has two separate gene paralogs encoding TRA2A and TRA2B proteins. TRA2B is associated with cancer cell survival and therapeutic sensitivity. However, the individual role of TRA2A in cancer progression has not been reported. Here we report that TRA2A facilitates proliferation and survival and migration and invasion of TNBC cells. In addition, TRA2A promotes paclitaxel resistance of TNBC by specifically controlling cancer-related splicing, which is independent of other splicing factors. TRA2A overexpression could promote AS of CALU, RSRC2, and PALM during paclitaxel treatment of TNBC cells. The isoform shift of RSRC2 from RSRC2s to RSRC2l leads to a decreased RSRC2 protein expression, which could contribute to TNBC paclitaxel resistance. TRA2A can regulate RSRC2 AS by specifically binding upstream intronic sequence of exon4. Strikingly, TRA2A expression is increased dramatically in patients with TNBC, and has a close relationship with decreased RSRC2 expression; both are associated with poor survival of TNBC. Collectively, our findings suggest that paclitaxel targets the TRA2A–RSRC2 splicing pathway, and deregulated TRA2A and RSRC2 expression may confer paclitaxel resistance. In addition to providing a novel molecular mechanism of cancer-related splicing dysregulation, our study demonstrates that expression of TRA2A in conjunction with RSRC2 may provide valuable molecular biomarker evidence for TNBC clinical treatment decisions and patient outcome. Mol Cancer Ther; 16(7); 1377–88. ©2017 AACR.

Long noncoding RNA n339260 promotes vasculogenic mimicry and cancer stem cell development in hepatocellular carcinoma

Vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. Cancer stem cells (CSC) represent a subpopulation of tumor cells endowed with the capacity for self‐renewal and multilineage differentiation. Previous studies have indicated that CSC may participate in the formation of VM. With the advance of high‐resolution microarrays and massively parallel sequencing technology, long noncoding RNAs (lncRNAs) are suggested to play a critical role in tumorigenesis and, in particular, the development of human hepatocellular carcinoma (HCC). Currently, no definitive relationship between lncRNA and VM formation has been described. In the current study, we demonstrated that expression of the lncRNA, n339260, is associated with CSC phenotype in HCC, and n339260 level correlated with VM, metastasis, and shorter survival time in an animal model. Overexpression of n339260 in HepG2 cells was associated with a significant increase in CSC. Additionally, the appearance of VM and vascular endothelial (VE)‐cadherin, a molecular marker of VM, was also induced by n339260 overexpression. Using a short hairpin RNA approach, n339260 was silenced in tumor cells, and knockdown of n339260 was associated with reduced VM and CSC. The results of this study indicate that n339260 promotes VM, possibly by the development of CSC. The related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis.