miR‐515‐5p controls cancer cell migration through MARK4 regulation

Here, we show that miR‐515‐5p inhibits cancer cell migration and metastasis. RNA‐seq analyses of both oestrogen receptor receptor‐positive and receptor‐negative breast cancer cells overexpressing miR‐515‐5p reveal down‐regulation of NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNAs. We demonstrate that miR‐515‐5p inhibits MARK4 directly 3′ UTR interaction and that MARK4 knock‐down mimics the effect of miR‐515‐5p on breast and lung cancer cell migration. MARK4 overexpression rescues the inhibitory effects of miR‐515‐5p, suggesting miR‐515‐5p mediates this process through MARK4 down‐regulation. Furthermore, miR‐515‐5p expression is reduced in metastases compared to primary tumours derived from both in vivo xenografts and samples from patients with breast cancer. Conversely, miR‐515‐5p overexpression prevents tumour cell dissemination in a mouse metastatic model. Moreover, high miR‐515‐5p and low MARK4 expression correlate with increased breast and lung cancer patients' survival, respectively. Taken together, these data demonstrate the importance of miR‐515‐5p/MARK4 regulation in cell migration and metastasis across two common cancers.     

Matrix Metalloproteinase-13 (MMP-13) Directly and Indirectly Promotes Tumor Angiogenesis

Matrix metalloproteinases (MMPs) are extracellular zinc-dependent endopeptidases involved in the degradation and remodeling of extracellular matrix in physiological and pathological processes. MMPs also have a role in cell proliferation, migration, differentiation, angiogenesis, and apoptosis. We previously identified cancer invasion-related factors by comparing the gene expression profiles between parent and the highly invasive clone of cancer cells. Matrix metalloproteinase-13 (MMP-13) was identified as a common up-regulated gene by cancer invasion-related factors. Although MMP-13 slightly promoted tumor invasion, we found that MMP-13 was involved in tumor angiogenesis. Conditioned medium from MMP-13-overexpressing cells promoted capillary formation of immortalized human umbilical vein endothelial cells. Furthermore, treatment with recombinant MMP-13 protein enhanced capillary tube formation both in vitro and in vivo. MMP-13-promoted capillary tube formation was mediated by activation of focal adhesion kinase and ERK. Interestingly, MMP-13 promoted the secretion of VEGF-A from fibroblasts and endothelial cells. By immunohistochemical analysis, we found a possible correlation between MMP-13 expression and the number of blood vessels in human cancer cases. In summary, these findings suggest that MMP-13 may directly and indirectly promote tumor angiogenesis.     

lncRNA NR2F1‐AS1 promotes breast cancer angiogenesis through activating IGF‐1/IGF‐1R/ERK pathway

Long non‐coding RNAs (lncRNAs) take various effects in cancer mostly through sponging with microRNAs (miRNAs). lncRNA NR2F1‐AS1 is found to promote tumour progression in hepatocellular carcinoma, endometrial cancer and thyroid cancer. However, the role of lncRNA NR2F1‐AS1 in breast cancer angiogenesis remains unknown. In this study, we found lncRNA NR2F1‐AS1 was positively related with CD31 and CD34 in breast cancer through Pearson's correlation analysis, while lncRNA NR2F1‐AS1 transfection promoted human umbilical vascular endothelial cell (HUVEC) tube formation. In breast cancer cells, lncRNA NR2F1‐AS1 enhanced the HUVEC proliferation, tube formation and migration ability through tumour‐conditioned medium (TCM). In zebrafish model, lncRNA NR2F1‐AS1 increased the breast cancer cell‐related neo‐vasculature and subsequently promoted the breast cancer cell metastasis. In mouse model, lncRNA NR2F1‐AS1 promoted the tumour vessel formation, increased the micro vessel density (MVD) and then induced the growth of primary tumour. Mechanically, lncRNA NR2F1‐AS1 increased insulin‐like growth factor‐1 (IGF‐1) expression through sponging miRNA‐338‐3p in breast cancer cells and then activated the receptor of IGF‐1 (IGF‐1R) and extracellular signal‐regulated kinase (ERK) pathway in HUVECs. These results indicated that lncRNA NR2F1‐AS1 could promote breast cancer angiogenesis through IGF‐1/IGF‐1R/ERK pathway.     

Mesenchymal stem cells promote lymphangiogenic properties of lymphatic endothelial cells

Lymphatic metastasis is one of the main prognostic factors concerning long‐term survival of cancer patients. In this regard, the molecular mechanisms of lymphangiogenesis are still rarely explored. Also, the interactions between stem cells and lymphatic endothelial cells (LEC) in humans have not been well examined. Therefore, the main objective of this study was to assess the interactions between mesenchymal stem cells (MSC) and LEC using in vitro angiogenesis assays. Juvenile LEC were stimulated with VEGF‐C, bFGF, MSC‐conditioned medium (MSC‐CM) or by co‐culture with MSC. LEC proliferation was assessed using a MTT assay. Migration of the cells was determined with a wound healing assay and a transmigration assay. To measure the formation of lymphatic sprouts, LEC spheroids were embedded in collagen or fibrin gels. The LEC's capacity to form capillary‐like structures was assessed by a tube formation assay on Matrigel^®. The proliferation, migration and tube formation of LEC could be significantly enhanced by MSC‐CM and by co‐culture with MSC. The effect of stimulation with MSC‐CM was stronger compared to stimulation with the growth factors VEGF‐C and bFGF in proliferation and transmigration assays. Sprouting was stimulated by VEGF‐C, bFGF and by MSC‐CM. With this study, we demonstrate the potent stimulating effect of the MSC secretome on proliferation, migration and tube formation of LEC. This indicates an important role of MSC in lymphangiogenesis in pathological as well as physiological processes.     

Effects on in vitro and in vivo angiogenesis induced by small peptides carrying adhesion sequences

It is well known that tumor growth is strictly dependent on neo‐vessel formation inside the tumor mass and that cell adhesion is required to allow EC proliferation and migration inside the tumor. In this work, we have evaluated the in vitro and in vivo effects on angiogenesis of some peptides, originally designed to promote cell adhesion on biomaterials, containing RGD motif mediating cell adhesion via integrin receptors [RGD, GRGDSPK, and (GRGDSP)₄K] or the heparin‐binding sequence of human vitronectin that interacts with HSPGs [HVP(351–359)]. Cell adhesion, proliferation, migration, and capillary‐like tube formation in Matrigel were determined on HUVECs, whereas the effects on in vivo angiogenesis were evaluated using the CAM assay. (GRGDSP)₄K linear sequence inhibited cell adhesion, decreased cell proliferation, migration and morphogenesis in Matrigel, and induced anti‐angiogenic responses on CAM at higher degree than that determined after incubation with RGD or GRGDSPK. Moreover, it counteracted both in vitro and in vivo the pro‐angiogenic effects induced by the Fibroblast growth factor (FGF‐2). On the other hand, HVP was not able to affect cell adhesion and appeared less effective than (GRGDSP)₄K. Our data indicate that the activity of RGD‐containing peptides is related to their adhesive properties, and their effects are modulated by the number of cell adhesion motifs and the aminoacidic residues next to these sequences. The anti‐angiogenic properties of (GRGDSP)₄K seem to depend on its interaction with integrins, whereas the effects of HVP may be partially due to an impairment of HSPGs/FGF‐2. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Syndecan-2 is expressed in the microvasculature of gliomas and regulates angiogenic processes in microvascular endothelial cells

Angiogenesis is the formation of new blood vessels from the existing vasculature and is necessary for tumor growth. Syndecan-2 (S2) is highly expressed in the microvasculature of mouse gliomas. When S2 expression was down-regulated in mouse brain microvascular endothelial cells (MvEC), this inhibited cell motility and reduced the formation of capillary tube-like structures in vitro. Pro-angiogenic growth factors and enzymes up-regulated during glioma tumorigenesis stimulated shedding of the S2 ectodomain from endothelial cells in vitro. The effect of shed S2 on angiogenic processes was investigated by incorporating recombinant S2 ectodomain (S2ED) into in vitro angiogenesis assays. S2ED promoted membrane protrusion, migration, capillary tube formation, and cell-cell interactions. We therefore propose that S2 is necessary for angiogenesis of MvEC, proangiogenic factors expressed during glioma progression regulate S2 shedding, and shed S2 ectodomain may increase endothelial cell angiogenic processes.     

CoREST1 Promotes Tumor Formation and Tumor Stroma Interactions in a Mouse Model of Breast Cancer

Regulators of chromatin structure and gene expression contribute to tumor formation and progression. The co-repressor CoREST1 regulates the localization and activity of associated histone modifying enzymes including lysine specific demethylase 1 (LSD1) and histone deacetylase 1 (HDAC1). Although several CoREST1 associated proteins have been reported to enhance breast cancer progression, the role of CoREST1 in breast cancer is currently unclear. Here we report that knockdown of CoREST1 in the basal-type breast cancer cell line, MDA-MB-231, led to significantly reduced incidence and diminished size of tumors compared to controls in mouse xenograft studies. Notably, CoREST1-depleted cells gave rise to tumors with a marked decrease in angiogenesis. CoREST1 knockdown led to a decrease in secreted angiogenic and inflammatory factors, and mRNA analysis suggests that CoREST1 promotes expression of genes related to angiogenesis and inflammation including VEGF-A and CCL2. CoREST1 knockdown decreased the ability of MDA-MB-231 conditioned media to promote endothelial cell tube formation and migration. Further, tumors derived from CoREST1-depleted cells had reduced macrophage infiltration and the secretome of CoREST1 knockdown cells was deficient in promoting macrophage migration and macrophage-mediated angiogenesis. Taken together, these findings reveal that the epigenetic regulator CoREST1 promotes tumorigenesis in a breast cancer model at least in part through regulation of gene expression patterns in tumor cells that have profound non-cell autonomous effects on endothelial and inflammatory cells in the tumor microenvironment.     

Identification of IGPR-1 as a novel adhesion molecule involved in angiogenesis

Angiogenesis-the growth of new blood vessels from preexisting vessels-is an important physiological process and is considered to play a key role in tumor growth and metastasis. We identified the immunoglobulin-containing and proline-rich receptor-1 (IGPR-1, also called TMIGD2) gene as a novel cell adhesion receptor that is expressed in various human organs and tissues, mainly in cells with epithelium and endothelium origins. IGPR-1 regulates cellular morphology, homophilic cell aggregation, and cell-cell interaction. IGPR-1 activity also modulates actin stress fiber formation and focal adhesion and reduces cell migration. Silencing of expression of IGPR-1 by small interfering RNA (siRNA) and by ectopic overexpression in endothelial cells showed that IGPR-1 regulates capillary tube formation in vitro, and B16F melanoma cells engineered to express IGPR-1 displayed extensive angiogenesis in the mouse Matrigel angiogenesis model. Moreover, IGPR-1, through its proline-rich cytoplasmic domain, associates with multiple Src homology 3 (SH3)-containing signaling proteins, including SH3 protein interacting with Nck (SPIN90/WISH), bullous pemphigoid antigen-1, and calcium channel β2. Silencing of expression of SPIN90/WISH by siRNA in endothelial cells showed that SPIN90/WISH is required for capillary tube formation. These features of IGPR-1 suggest that IGPR-1 is a novel receptor that plays an important role in cell-cell interaction, cell migration, and angiogenesis.     

ZEB1‐mediated vasculogenic mimicry formation associates with epithelial–mesenchymal transition and cancer stem cell phenotypes in prostate cancer

The zinc finger E‐box‐binding homeobox 1 (ZEB1) induced the epithelial–mesenchymal transition (EMT) and altered ZEB1 expression could lead to aggressive and cancer stem cell (CSC) phenotypes in various cancers. Tissue specimens from 96 prostate cancer patients were collected for immunohistochemistry and CD34/periodic acid–Schiff double staining. Prostate cancer cells were subjected to ZEB1 knockdown or overexpression and assessment of the effects on vasculogenic mimicry formation in vitro and in vivo. The underlying molecular events of ZEB1‐induced vasculogenic mimicry formation in prostate cancer were then explored. The data showed that the presence of VM and high ZEB1 expression was associated with higher Gleason score, TNM stage, and lymph node and distant metastases as well as with the expression of vimentin and CD133 in prostate cancer tissues. Furthermore, ZEB1 was required for VM formation and altered expression of EMT‐related and CSC‐associated proteins in prostate cancer cells in vitro and in vivo. ZEB1 also facilitated tumour cell migration, invasion and clonogenicity. In addition, the effects of ZEB1 in prostate cancer cells were mediated by Src signalling; that is PP2, a specific inhibitor of the Src signalling, dose dependently reduced the p‐Src⁵²⁷ level but not p‐Src⁴¹⁶ level, while ZEB1 knockdown also down‐regulated the level of p‐Src⁵²⁷ in PC3 and DU‐145 cells. PP2 treatment also significantly reduced the expression of VE‐cadherin, vimentin and CD133 in these prostate cancer cells. Src signalling mediated the effects of ZEB1 on VM formation and gene expression.     

Britannin stabilizes T cell activity and inhibits proliferation and angiogenesis by targeting PD-L1 via abrogation of the crosstalk between Myc and HIF-1α in cancer

BackgroundProgrammed cell death-ligand 1 (PD-L1) is overexpressed in tumor cells, which causes tumor cells to escape T cell killing, and promotes tumor cell survival, cell proliferation, migration, invasion, and angiogenesis. Britannin is a natural product with anticancer pharmacological effects.PurposeIn this work, we studied the anticancer potential of britannin and explored whether britannin mediated its effect by inhibiting the expression of PD-L1 in tumor cells.MethodsIn vitro, the mechanisms underlying the inhibition of PD-L1 expression by britannin were investigated by MTT assay, homology modeling and molecular docking, RT-PCR, western blotting, co-immunoprecipitation, and immunofluorescence. The changes in tumor killing activity, cell proliferation, cell cycle, migration, invasion, and angiogenesis were analyzed by T cell killing assays, EdU labeling, colony formation, flow cytometry, wound healing, matrigel transwell invasion, and tube formation, respectively. In vivo, the antitumor activity of britannin was evaluated in the HCT116 cell xenograft model.ResultsBritannin reduced the expression of PD-L1 in tumor cells by inhibiting the synthesis of the PD-L1 protein but did not affect the degradation of the PD-L1 protein. Britannin also inhibited HIF-1α expression through the mTOR/P70S6K/4EBP1 pathway and Myc activation through the Ras/RAF/MEK/ERK pathway. Mechanistically, britannin inhibited the expression of PD-L1 by blocking the interaction between HIF-1α and Myc. In addition, britannin could enhance the activity of cytotoxic T lymphocytes and inhibit tumor cell proliferation and angiogenesis by inhibiting PD-L1. Finally, in vivo observations were confirmed by demonstrating the antitumor activity of britannin in a murine xenograft model.ConclusionBritannin inhibits the expression of PD-L1 by blocking the interaction between HIF-1α and Myc. Moreover, britannin stabilizes T cell activity and inhibits proliferation and angiogenesis by inhibiting PD-L1 in cancer. The current work highlights the anti-tumor effect of britannin, providing insights into the development of cancer therapeutics via PD-L1 inhibition.     

The role of the metastasis suppressor gene KAI1 in melanoma angiogenesis

The tetraspan protein KAI1 (CD82) has been previously shown to have important roles in cell migration, invasion, and melanoma prognosis. In this study, we investigated the role of KAI1 regarding melanoma angiogenesis. KAI1 overexpression strongly suppressed the growth of the human umbilical vein endothelial cells and their tubular structure formation in vitro. Also, KAI1 was able to inhibit both interleukin‐6 (IL‐6) and VEGF at mRNA and protein levels. Using nude mice in the in vivo study, we showed that KAI1, through the regulation of ING4, inhibited blood vessel formation in matrigel plugs along with the downregulation of IL‐6 and VEGF, and the recruitment of CD31‐positive cells. Finally, we found that KAI1 was able to suppress the activity of a serine/threonine kinase Akt by suppressing Akt phosphorylation (Ser473). Taken together, our results suggested that KAI1 was able to suppress melanoma angiogenesis by downregulating IL‐6 and VEGF expression, and the restoration of KAI1 functionality offered a new approach in human melanoma treatment.     

Diallyl trisulfide inhibits migration,invasion and angiogenesis of human colon cancer HT‐29 cells and umbilical vein endothelial cells,and suppresses murine xenograft tumour growth

Angiogenesis inhibitors are beneficial for the prevention and treatment of angiogenesis‐dependent diseases including cancer. We examined the cytotoxic, anti‐metastatic, anti‐cancer and anti‐angiogenic effects of diallyl trisulfide (DATS). In HT29 cells, DATS inhibited migration and invasion through the inhibition of focal adhesion kinase (FAK), extracellular signal‐regulated kinase, c‐Jun N‐terminal kinase and p38 which was associated with inhibition of matrix metalloproteinases‐2, ‐7 and ‐9 and VEGF. In human umbilical vein endothelial cells (HUVEC), DATS inhibited the migration and angiogenesis through FAK, Src and Ras. DATS also inhibited the secretion of VEGF. The capillary‐like tube structure formation and migration by HUVEC was inhibited by DATS. The chicken egg chorioallantoic membrane (CAM) assay indicated that DATS treatment inhibited ex‐vivo angiogenesis. We investigated the anti‐tumour effects of DATS against human colon cancer xenografts in BALB/c^nu/nu mice and its anti‐angiogenic activity in vivo. In this in‐vivo study, DATS also inhibited the tumour growth, tumour weight and angiogenesis (decreased the levels of haemoglobin) in HT29 cells. In conclusion, the present results suggest that the inhibition of angiogenesis may be an important mechanism in colon cancer chemotherapy by DATS.     

n-Butyl Benzyl Phthalate Promotes Breast Cancer Progression by Inducing Expression of Lymphoid Enhancer Factor 1

Environmental hormones play important roles in regulating the expression of genes involved in cell proliferation, drug resistance, and breast cancer risk; however, their precise role in human breast cancer cells during cancer progression remains unclear. To elucidate the effect of the most widely used industrial phthalate, n-butyl benzyl phthalate (BBP), on cancer progression, we evaluated the results of BBP treatment using a whole human genome cDNA microarray and MetaCore software and selected candidate genes whose expression was changed by more than ten-fold by BBP compared with controls to analyze the signaling pathways in human breast cancer initiating cells (R2d). A total of 473 genes were upregulated, and 468 were downregulated. Most of these genes are involved in proliferation, epithelial-mesenchymal transition, and angiogenesis signaling. BBP induced the viability, invasion and migration, and tube formation in vitro, and Matrigel plug angiogenesis in vivo of R2d and MCF-7. Furthermore, the viability and invasion and migration of these cell lines following BBP treatment was reduced by transfection with a small interfering RNA targeting the mRNA for lymphoid enhancer-binding factor 1; notably, the altered expression of this gene consistently differentiated tumors expressing genes involved in proliferation, epithelial-mesenchymal transition, and angiogenesis. These findings contribute to our understanding of the molecular impact of the environmental hormone BBP and suggest possible strategies for preventing and treating human breast cancer.     

Upregulation of HYAL1 expression in breast cancer promoted tumor cell proliferation, migration, invasion and angiogenesis

Hyaluronic acid (HA) is a component of the Extra-cellular matrix (ECM), it is closely correlated with tumor cell growth, proliferation, metastasis and angiogenesis, etc. Hyaluronidase (HAase) is a HA-degrading endoglycosidase, levels of HAase are elevated in many cancers. Hyaluronidase-1 (HYAL1) is the major tumor-derived HAase. We previously demonstrated that HYAL1 were overexpression in human breast cancer. Breast cancer cells with higher HAase expression, exhibited significantly higher invasion ability through matrigel than those cells with lower HAase expression, and knockdown of HYAL1 expression in breast cancer cells resulted in decreased cell growth, adhesion, invasion and angiogenesis. Here, to further elucidate the function of HYAL1 in breast cancer, we investigated the consequences of forcing HYAL1 expression in breast cancer cells by transfection of expression plasmid. Compared with control, HYAL1 up-regulated cells showed increased the HAase activity, and reduced the expression of HA in vitro. Meantime, upregulation of HYAL1 promoted the cell growth, migration, invasion and angiogenesis in vitro. Moreover, in nude mice model, forcing HYAL1 expression induced breast cancer cell xenograft tumor growth and angiogenesis. Interestingly, the HA expression was upregulated by forcing HYAL1 expression in vivo. These findings suggested that HYAL1-HA system is correlated with the malignant behavior of breast cancer.     

LOXL2 promotes vasculogenic mimicry and tumour aggressiveness in hepatocellular carcinoma

Lysyl oxidase‐like 2 (LOXL2) has shown to promote metastasis and poor prognosis in hepatocellular carcinoma (HCC). Also, we have previously reported that vasculogenic mimicry (VM) is associated with invasion, metastasis and poor survival in HCC patients. In the present study, we investigated molecular function of LOXL2 in HCC and VM. We used the immunohistochemical and CD31/periodic acid‐Schiff double staining to detect the relationship between LOXL2 and VM formation. We performed the gain and loss of function studies and analysed the migratory, invasion and tube formation in HCC cell lines. We analysed the function of LOXL2 in VM formation and HCC metastasis both in vitro and in vivo. We have showed that LOXL2 was overexpression in HCC and was positively correlated with tumour grade, metastasis, VM formation and poor survival in 201 HCC patients. Secondly, our studies have showed that LOXL2 overexpression in HCC cells significantly promoted migration, invasion and tube formation. Finally, we found that LOXL2 may increase SNAIL expression, thereby enabling VM. Our study indicated that LOXL2 may promote VM formation and tumour metastasis by collaborating with SNAIL in HCC. What's more, the overexpression of LOXL2 indicated a poor prognosis in HCC patients.     

CircHIPK3 sponges miR‐558 to suppress heparanase expression in bladder cancer cells

Increasing evidences suggest that circular RNAs (circRNAs) exert crucial functions in regulating gene expression. In this study, we perform RNA‐seq and identify 6,154 distinct circRNAs from human bladder cancer and normal bladder tissues. We find that hundreds of circRNAs are significantly dysregulated in human bladder cancer tissues. We further show that circHIPK3, also named bladder cancer‐related circular RNA‐2 (BCRC‐2), is significantly down‐regulated in bladder cancer tissues and cell lines, and negatively correlates with bladder cancer grade, invasion as well as lymph node metastasis, respectively. Over‐expression of circHIPK3 effectively inhibits migration, invasion, and angiogenesis of bladder cancer cells in vitro and suppresses bladder cancer growth and metastasis in vivo. Mechanistic studies reveal that circHIPK3 contains two critical binding sites for the microRNA miR‐558 and can abundantly sponge miR‐558 to suppress the expression of heparanase (HPSE). Taken together, our findings provide evidence that circRNAs act as “microRNA sponges”, and suggest a new therapeutic target for the treatment of bladder cancer.