Huizhi Sun

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.

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.

Anti-angiogenic treatment promotes triple-negative breast cancer invasion via vasculogenic mimicry

ABSTRACT Agents that target angiogenesis have shown limited efficacy for human triple-negative breast cancer (TNBC) in clinical trials. Along with endothelium-dependent vessels, there is also vasculogenic mimicry (VM) in the microcirculation of malignant tumors. The role of VM is not completely understood regarding anti-angiogenic treatment. In this study, human TNBC MDA-MB-231 and Hs578T and non-TNBC MCF-7 and BT474 tumor-bearing mice were treated with sunitinib, an anti-angiogenic drug, using a clinically relevant schedule. The drug was administered for one week and then discontinued. Tumor growth and invasion were observed, and the microcirculation patterns were detected with PAS/endomucin staining. Moreover, hypoxia and VM-associated proteins were evaluated with Hypoxyprobe kits and immunohistochemistry, respectively. Sunitinib significantly inhibited tumor growth in the TNBC and non-TNBC tumors. However, MDA-MB-231 and Hs578T tumors regrew and were more aggressive when the treatment was stopped. The discontinuation had no significant effect on the behavior of the non-TNBC MCF-7 and BT474 tumors. The growth of endothelium-dependent vessels in the TNBC MDA-MB-231 and Hs578T tumors were blocked by sunitinib, during which the number of VM channels significantly increased and resulted in a rebound of endothelium-dependent vessels after sunitinib discontinuation. Moreover, the VM-associated proteins VE-cadherin and Twist1 upregulated in the sunitinib-treated MDA-MB-231 and Hs578T tumors. Furthermore, the clinical significance of this upregulation was validated in 174 human breast cancers. The results from human breast cancer specimens indicated that there were more VM-positive TNBC cases than those in non-TNBC cases. HIF-1α, MMP2, VE-cadherin, and Twist1 were also expressed in a higher level in human TNBC compared with non-TNBC. In aconclusion, sunitinib promoted TNBC invasion by VM. The VM status could be helpful to predict the efficacy of anti-angiogenic therapy in patients with TNBC.

Significance of Vasculogenic Mimicry Formation in Gastric Carcinoma

Background: The purpose of this study was to evaluate the clinical significance and prognostic roles of vasculogenic mimicry (VM) and elucidate their intrinsic association with molecular markers. Methods: 89 human gastric cancer cases with detailed follow-up and clinicopathologic data were collected. CD34/periodic acid-Schiff double staining was performed to validate the existence of VM. Immunohistochemistry was performed to explore the expression of different molecular factors. Results: VM was observed in 24 gastric cancer patients. They were prone to higher histological grade, hematogenous metastasis, distant recurrence, and chance of progression to stage III or IV (p < 0.05). The VM group had shorter overall and disease-free survival (p < 0.05). VM negativity was independently prognostic for prolonged overall or disease-free survival (p < 0.05). VM was positively associated with levels of matrix metalloproteinase-2, matrix metalloproteinase-9, vascular endothelial growth factor, and vascular endothelial growth factor receptor-1 (p < 0.05), but not with vascular endothelial growth factor receptor-2 (p > 0.05). Conclusion: VM should be regarded as a good marker to indicate pathobiological behaviors of gastric cancer. Using antibodies against matrix metalloproteinases, vascular endothelial growth factor, or vascular endothelial growth factor receptors could be strategies to counteract VM formation.

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.

Spheres derived from the human SN12C renal cell carcinoma cell line are enriched in tumor initiating cells

BackgroundRecently, tumor initiating cells (TICs), which possess self-renewal and other stem cell properties, are regarded as the cause of tumor initiation, recurrence and metastasis. The isolation and identification of TICs could help to develop novel therapeutic strategies.MethodsIn this study, we isolated spheroid cells from human renal cell carcinoma (RCC) cell line SN12C in stem cell-conditioned medium. The stemness characteristics of spheroid cells, including tumorigenicity, self-renewal, proliferation and aldehyde dehydrogenase (ALDH) activity were evaluated; the expression levels of stemness genes and related proteins were assessed. Furthermore, study examined the differentiation of TICs into endothelial cells and the relationship between TICs and EMT.ResultsOur data demonstrated that spheroid cells cultured in defined serum-free medium possessed TIC properties, such as high tumorigenic capacity, upregulation of TIC-related genes and proteins, persistent self-renewal and extensive proliferation. Furthermore, spheroid cells were more aggressive in growth, invasion, scratch recovery, clonogenic survival and high aldehyde dehydrogenase (ALDH) activity. Interestingly, a marked increase in tumor vascularity compared to adherent tumors in vivo, and spheroid cells can differentiate into functional endothelial-like cells in vitro suggesting a role of tumor initiating cells in tumor angiogenesis. The spheroid cells also demonstrated down-regulated E-cadherin and up-regulated Vimentin expression, which is the typical phenotype of EMT.ConclusionsThese results suggest that spheroid cells with tumor initiating cells-like characteristics contributed to tumor generation, progression, high tumorigenicity, pro-angiogenic capability and relationship with EMT. Further experiments using more refined selection criteria such as a combination of two or multiple markers would be useful to specifically identify and purify TICs.

TP53INP1 inhibits hypoxia‐induced vasculogenic mimicry formation via the ROS/snail signalling axis in breast cancer

Tumour protein p53‐inducible nuclear protein 1 (TP53INP1) is a tumour suppressor associated with malignant tumour metastasis. Vasculogenic mimicry (VM) is a new tumour vascular supply pattern that significantly influences tumour metastasis and contributes to a poor prognosis. However, the molecular mechanism of the relationship between TP53INP1 and breast cancer VM formation is unknown. Here, we explored the underlying mechanism by which TP53INP1 regulates VM formation in vitro and in vivo. High TP53INP1 expression was not only negatively correlated with a poor prognosis but also had a negative relationship with VE‐cadherin, HIF‐1α and Snail expression. TP53INP1 overexpression inhibited breast cancer invasion, migration, epithelial‐mesenchymal transition (EMT) and VM formation; conversely, TP53INP1 down‐regulation promoted these processes in vitro by functional experiments and Western blot analysis. We established a hypoxia model induced by CoCl2 and assessed the effects of TP53INP1 on hypoxia‐induced EMT and VM formation. In addition, we confirmed that a reactive oxygen species (ROS)‐mediated signalling pathway participated in TP53INP1‐mediated VM formation. Together, our results show that TP53INP1 inhibits hypoxia‐induced EMT and VM formation via the ROS/GSK‐3β/Snail pathway in breast cancer, which offers new insights into breast cancer clinical therapy.