Yanlei Li

Diffusion-weighted imaging in predicting and monitoring the response of uterine cervical cancer to combined chemoradiation.

AIM To investigate the ability of diffusion-weighted imaging (DWI) to predict and monitor the response of uterine cervical cancer to combined chemoradiation using apparent diffusion coefficients (ADCs). MATERIALS AND METHODS Seventeen women (mean age 48.5 years) with uterine cervical cancer received conventional magnetic resonance imaging (MRI) and DWI prior to chemoradiation and after 1 and 2 months of therapy. A subgroup of eight also had MRI and DWI repeated after 15 days of therapy. Treatment response was determined according to changes in tumour size after 2 months of therapy and was classified as complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). Pretreatment ADCs were compared between the different disease response groups, and dynamic changes of ADCs in each group were observed. Pearsons correlation test was calculated between those ADC parameters and tumour response. RESULTS Pretreatment ADCs for CR were significantly lower than those of PR (p=0.005). Negative correlation was found between pretreatment ADCs and percentage size reduction after 2 months of chemoradiation (p=0.016). The percentage ADC change after 1 month correlated positively with percentage size reduction after 2 months of therapy (p=0.021). ADCs after 15 days of therapy increased significantly compared with pretreatment ones (p=0.001); however, the longest tumour diameter showed no statistically significant change (p=0.078). CONCLUSION ADCs may have the potential to be used to predict and monitor the response of uterine cervical cancer to therapy.

ZEB2 promotes vasculogenic mimicry by TGF-β1 induced epithelial-to-mesenchymal transition in hepatocellular carcinoma

AIMS Zinc finger E-box binding homeobox 2 (ZEB2), an epithelial-mesenchymal transition (EMT) regulator, has been involved in invasion and metastasis of human tumor. Although EMT may be involved in vasculogenic mimicry (VM) formation, no reports describing the relation between ZEB2 and VM are available. We hypothesize that ZEB2 may promote VM formation in hepatocellular carcinoma (HCC). METHODS AND RESULTS Paraffin-embedded tumor tissue samples from 92 patients were immunostained with anti-ZEB2 antibody. We found that the ZEB2 nuclear expression was significantly associated with VM formation and metastasis. Patients with VM and ZEB2 nuclear expression had a shorter survival period than those without expression. In vitro, ZEB2 overexpression significantly enhanced cell motility, invasiveness, and VM formation of HepG2 cells. ZEB2 upregulation also increased VE-cadherin, Flt-1, and Flk-1 expression and activated MMPs. ZEB2 knockdown inhibited cell motility, invasiveness, and VM formation in Bel7402 cells. ZEB2 knockdown also decreased VE-cadherin, Flt-1, and Flk-1 expression and MMP activity. In addition, EMT in HepG2 cells was induced by TGF-β1 treatment, and the kinetics of expression of EMT markers and regulators were assessed by Western blot analysis. The expression of ZEB2 increased significantly, and VM formation was promoted. CONCLUSION ZEB2 can promote VM formation through the EMT pathway. Our findings may represent a novel therapeutic target in HCC.

OCT4 Expression and Vasculogenic Mimicry Formation Positively Correlate with Poor Prognosis in Human Breast Cancer

To evaluate the prognostic value of OCT4 expression and vasculogenic mimicry (VM) in human breast cancer, we examined OCT4 expression and VM formation using immunohistochemistry and CD31/PAS (periodic acid-schiff) double staining on 90 breast cancer specimens. All patients were followed up for five–149 months following surgery. Survival curves were generated using Kaplan-Meier method. Multivariate analysis was performed using Cox regression model to assess the prognostic values. Results showed positive correlation between OCT4 expression and VM formation (p < 0.05). Both OCT4 expression and VM were also positively correlated with lymph node metastasis, higher histological grade, and Nottingham prognostic index (p < 0.05). Patients with OCT4 expression or VM formation exhibited poorer overall survival (OS) and disease-free survival (DFS) than OCT4-negative or VM-negative patients (p < 0.05). OCT4-positive/VM-positive patients also had the worst OS and DFS (p < 0.05). In multivariate survival analysis, VM, Nottingham prognostic index (NPI), and Her2 were independent prognostic factors related to OS and OCT4-positive/VM-positive patients, whereas NPI and Her2 were independent predictors of DFS. These results suggest that a combined OCT4 expression/VM could improve the prognostic judgment for breast cancer patients.

Regulation of proliferation, angiogenesis and apoptosis in hepatocellular carcinoma by miR-26b-5p.

MicroRNAs (miRNAs) play vital roles in cell proliferation, differentiation and apoptosis in hepatocellular carcinoma (HCC). miR-26b has been confirmed as an important regulator in carcinogenesis and other pathological processes. miR-26b-5p is one member of the mature miR-26 family, and its functional role in proliferation, angiogenesis and apoptosis in HCC remains unknown. Here, we demonstrate that miR-26b-5p expression was significantly decreased in HCC tissues and HCC cell lines compared with normal liver tissues and liver cells by quantitative real-time polymerase chain reaction (qRT-PCR). The relationships between miR-26b-5p and the clinical characteristics of HCC patients were further analysed, and miR-26b-5p was positively correlated with the differentiation of HCC cells. Computational searches were further used to identify the downstream targets and signalling pathways of miR-26b-5p in HCC cells. Cell viability, proliferation and tube formation abilities were assessed by scrape, 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and three-dimensional culture assays to confirm that miR-26b-5p inhibited HCC cell growth and impaired the tube formation ability of the HCC cells. Both in vitro and in vivo studies showed that miR-26b-5p could suppress vascular mimicry (VM) and angiogenesis by down-regulating the expression of VE-cadherin, Snail and MMP2 and could inhibit the apoptosis of HCC cells. Using mouse models, we revealed that tumours derived from miR-26b-5p-expressing HCC cells displayed a significant decrease in microvessel density compared with those derived from control cells. Therefore, our data provide further insight into the role of miR-26b-5p as a negative regulator of proliferation, angiogenesis, and apoptosis in HCC.

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.

USP44+ Cancer Stem Cell Subclones Contribute to Breast Cancer Aggressiveness by Promoting Vasculogenic Mimicry

Vasculogenic mimicry (VM), a newly defined pattern of tumor blood supply, describes the functional plasticity of aggressive cancer cells that form vascular networks. In our previous study, breast cancer stem cells (CSC) were shown to potentially participate in VM formation. In this study, breast CSCs presented centrosome amplification (CA) phenotype and ubiquitin-specific protease 44 (USP44) upregulation. USP44 expression contributed to the establishment of bipolar spindles in breast CSCs with supernumerary centrosomes by localizing at pole-associated centrosomes. The bipolar spindle patterns of breast CSCs with CA, including planar-like and apico-basal–like, functioned differently during the VM process of CSCs. Moreover, the ability of transendothelial migration in VM-forming cells was increased. In vivo experiment results showed that CSC xenografts presented linearly patterned programmed cell necrosis, which provided a spatial foundation for VM formation as well as angiogenesis. Breast CSCs further showed increased levels of IL6 and IL8. However, USP44 silencing induced spindle multipolarity, abated VM, reduced transendothelial migration, and consequently decreased IL6 and IL8 levels in breast CSCs. Finally, USP44+ CSC subclones (ALDH1+/USP44+/IL6+/IL8+) were identified in breast cancer specimens through consecutive sections scanning. The subclones were related not only to CA, but also to VM. Statistical analysis suggested that USP44+ CSC subclones could be used as an independent prognostic biomarker of poor clinical outcomes in patients with breast cancer. Collectively, the identification of USP44+ CSC subclones may contribute to the prediction of VM formation and aggressive behavior. This study provides novel insights into the therapy for advanced breast cancer. Mol Cancer Ther; 14(9); 2121–31. ©2015 AACR.

Hypoxia promotes vasculogenic mimicry formation by the Twist1-Bmi1 connection in hepatocellular carcinoma

Aggressive tumor cells can mimic embryonic vasculogenic networks and form vasculogenic mimicry (VM). Preliminary studies demonstrated that hypoxia can promote VM formation; however, the underlying mechanism remains unclear. The present study aimed to investigate the role of the Twist1‑Bmi1 connection in hypoxia‑induced VM formation and the underlying mechanism. In the in vitro experiments, western blot analysis demonstrated that hypoxia upregulated the expression of Twist1, Bmi1, epithelial‑mesenchymal transition (EMT) markers, stem cell markers and VM‑associated markers. The 3D culture assay showed that hypoxia promoted VM formation in hepatocellular carcinoma (HCC) cell lines. Using transfection and in vitro cell experiments, the Twist1‑Bmi1 connection was confirmed to have an important role in inducing EMT, cell stemness and VM formation. In the in vivo experiments, the murine hypoxia models were established via incomplete femoral artery ligation and the mechanism by which hypoxia promoted Twist1 and Bmi1 expression and led to VM formation was demonstrated by immunohistochemistry staining and endomucin/periodic acid Schiff double‑staining. In conclusion, hypoxia upregulate the expression of Twist1 and Bmi1, and these two proteins have an important role in inducing EMT and cancer cell stemness, which contributed to VM formation.

MRI findings in Duane's ocular retraction syndrome

AIM To investigate the innervation pattern of extra-ocular muscles in patients with clinically diagnosed Duanes ocular retraction syndrome (DRS) using magnetic resonance imaging (MRI). MATERIALS AND METHODS The study population consisted of 11 patients. Six patients had type I DRS (eight eyes), four patients had type II DRS (five eyes) and one patient had inverse DRS. Images were acquired using a Siemens 3 T MRI system. The type of DRS, corresponding innervation findings, and condition of the affected muscles were evaluated by two experienced neuroradiologists in consensus. RESULTS All patients with clinically diagnosed DRS type I showed absence of the abducens nerve (CN6), hypoplasia of the superior oblique muscle (SOM), and aberrant innervation of lateral rectus muscle (LRM) by an extra branch of oculomotor nerve (CN3). All patients with type II DRS show dual-innervation of the LRM (by CN6 and an aberrant CN3 branch) and hypoplasia of SOM. The single patient with inverse DRS showed hypoplasia of CN3, the medial rectus muscle (MRM), the inferior rectus muscle (IRM), and the inferior oblique muscle (IOM). CONCLUSION Each type of DRS has characteristic MRI appearances. Therefore, MRI is a useful diagnostic tool for the confirmation and classification of suspected cases of DRS.

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.