Twist1 Influences the Expression of Leading Members of the IL-17 Signaling Pathway in HER2-Positive Breast Cancer Cells.

Breast cancer (BC) is a heterogeneous disease composed of multiple subtypes with different molecular characteristics and clinical outcomes. The metastatic process in BC depends on the transcription factors (TFs) related to epithelial-mesenchymal transition (EMT), including the master regulator Twist1. However, its role beyond EMT in BC subtypes remains unclear. Our study aimed to investigate the role of Twist1, beyond EMT, in the molecular subtypes of BC. In patients, we observed the overexpression of TWIST1 in the HER2+ group. The silencing of TWIST1 in HER2+ BC cells resulted in the upregulation of 138 genes and the downregulation of 174 genes compared to control cells in a microarray assay. In silico analysis revealed correlations between Twist1 and important biological processes such as the Th17-mediated immune response, suggesting that Twist1 could be relevant for IL-17 signaling in HER2+ BC. IL-17 signaling was then examined, and it was shown that TWIST1 knockdown caused the downregulation of leading members of IL-17 signaling pathway. Taken together, our findings suggest that Twist1 plays a role on IL-17 signaling in HER2+ BC.

Extracellular vesicles derived from human Wharton's jelly mesenchymal stem cells protect hippocampal neurons from oxidative stress and synapse damage induced by amyloid-ß oligomers.

BACKGROUND: Mesenchymal stem cells (MSCs) have been explored as promising tools for treatment of several neurological and neurodegenerative diseases. MSCs release abundant extracellular vesicles (EVs) containing a variety of biomolecules, including mRNAs, miRNAs, and proteins. We hypothesized that EVs derived from human Wharton's jelly would act as mediators of the communication between hMSCs and neurons and could protect hippocampal neurons from damage induced by Alzheimer's disease-linked amyloid beta oligomers (AßOs). METHODS: We isolated and characterized EVs released by human Wharton's jelly mesenchymal stem cells (hMSC-EVs). The neuroprotective action of hMSC-EVs was investigated in primary hippocampal cultures exposed to AßOs. RESULTS: hMSC-EVs were internalized by hippocampal cells in culture, and this was enhanced in the presence of AßOs in the medium. hMSC-EVs protected hippocampal neurons from oxidative stress and synapse damage induced by AßOs. Neuroprotection by hMSC-EVs was mediated by catalase and was abolished in the presence of the catalase inhibitor, aminotriazole. CONCLUSIONS: hMSC-EVs protected hippocampal neurons from damage induced by AßOs, and this was related to the transfer of enzymatically active catalase contained in EVs. Results suggest that hMSC-EVs should be further explored as a cell-free therapeutic approach to prevent neuronal damage in Alzheimer's disease.

Hypoxia regulates the expression of tissue factor pathway signaling elements in a rat glioma model.

Hypoxia and necrosis are fundamental features of glioma, and their emergence is critical for the rapid biological progression of this fatal tumor. The presence of vaso-occlusive thrombus is higher in grade IV tumors [glioblastoma multiforme (GBM)] compared with lower grade tumors, suggesting that the procoagulant properties of the tumor contribute to its aggressive behavior, as well as the establishment of tumor hypoxia and necrosis. Tissue factor (TF), the primary cellular initiator of coagulation, is overexpressed in GBMs and likely favors a thrombotic microenvironment. Phosphatase and tensin homolog (PTEN) loss and hypoxia are two common alterations observed in glioma that may be responsible for TF upregulation. In the present study, ST1 and P7 rat glioma lines, with different levels of aggressiveness, were comparatively analyzed with the aim of identifying differences in procoagulant mechanisms. The results indicated that P7 cells display potent procoagulant activity compared with ST1 cells. Flow cytometric analysis showed less pronounced levels of TF in ST1 cells compared with P7 cells. Notably, P7 cells supported factor X (FX) activation via factor VIIa, whereas no significant FXa generation was observed in ST1 cells. Furthermore, the exposure of phosphatidylserine on the surface of P7 and ST1 cells was investigated. The results supported the assembly of prothrombinase complexes, accounting for the production of thrombin. Furthermore, reverse transcription-quantitative polymerase chain reaction showed that CoCl2 (known to induce a hypoxic-like stress) led to an upregulation of TF levels in P7 and ST1 cells. Therefore, increased TF expression in P7 cells was accompanied by increased TF procoagulant activity. In addition, hypoxia increased the shedding of procoagulant TF-bearing microvesicles in both cell lines. Finally, hypoxic stress induced by treatment with CoCl2 upregulated the expression of the PAR1 receptor in both P7 and ST1 cells. In addition to PAR1, P7, but not ST1 cells, expressed higher levels of PAR2 under hypoxic stress. Thus, modulating these molecular interactions may provide additional insights for the development of more efficient therapeutic strategies against aggressive glioma.

Intercellular transfer of tissue factor via the uptake of tumor-derived microvesicles.

Coagulation proteins play a critical role in numerous aspects of tumor biology. Cancer cells express tissue factor (TF), the protein that initiates blood clotting, which frequently correlates with processes related to cell aggressiveness, including primary tumor growth, invasion, and metastasis. It has been demonstrated that TF gets incorporated into tumor-derived microvesicles (MVs), a process that has been correlated with cancer-associated thrombosis. Here, we describe the exchange of TF-bearing MVs between breast cancer cell lines with different aggressiveness potential. The highly invasive and metastatic MDA-MB-231 cells displayed higher surface levels of functional TF compared with the less aggressive MCF-7 cells. MVs derived from MDA-MB-231 cells were enriched in TF and accelerated plasma coagulation, but MCF-7 cell-derived MVs expressed very low levels of TF. Incubating MCF-7 cells with MDA-MB-231 MVs significantly increased the TF activity. This phenomenon was not observed upon pretreatment of MVs with anti-TF or annexin-V, which blocks phosphatidylserine sites on the surface of MVs. Our data indicated that TF-bearing MVs can be transferred between different populations of cancer cells and may therefore contribute to the propagation of a TF-related aggressive phenotype among heterogeneous subsets of cells in a tumor.

Glycoinositolphospholipids from Trypanosomatids subvert nitric oxide production in Rhodnius prolixus salivary glands.

BACKGROUND: Rhodnius prolixus is a blood-sucking bug vector of Trypanosoma cruzi and T. rangeli. T. cruzi is transmitted by vector feces deposited close to the wound produced by insect mouthparts, whereas T. rangeli invades salivary glands and is inoculated into the host skin. Bug saliva contains a set of nitric oxide-binding proteins, called nitrophorins, which deliver NO to host vessels and ensure vasodilation and blood feeding. NO is generated by nitric oxide synthases (NOS) present in the epithelium of bug salivary glands. Thus, T. rangeli is in close contact with NO while in the salivary glands. METHODOLOGY/PRINCIPAL FINDINGS: Here we show by immunohistochemical, biochemical and molecular techniques that inositolphosphate-containing glycolipids from trypanosomatids downregulate NO synthesis in the salivary glands of R. prolixus. Injecting insects with T. rangeli-derived glycoinositolphospholipids (Tr GIPL) or T. cruzi-derived glycoinositolphospholipids (Tc GIPL) specifically decreased NO production. Salivary gland treatment with Tc GIPL blocks NO production without greatly affecting NOS mRNA levels. NOS protein is virtually absent from either Tr GIPL- or Tc GIPL-treated salivary glands. Evaluation of NO synthesis by using a fluorescent NO probe showed that T. rangeli-infected or Tc GIPL-treated glands do not show extensive labeling. The same effect is readily obtained by treatment of salivary glands with the classical protein tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate (SO). This suggests that parasite GIPLs induce the inhibition of a salivary gland PTP. GIPLs specifically suppressed NO production and did not affect other anti-hemostatic properties of saliva, such as the anti-clotting and anti-platelet activities. CONCLUSIONS/SIGNIFICANCE: Taken together, these data suggest that trypanosomatids have overcome NO generation using their surface GIPLs. Therefore, these molecules ensure parasite survival and may ultimately enhance parasite transmission.

Inhibition of tissue factor by ixolaris reduces primary tumor growth and experimental metastasis in a murine model of melanoma.

Melanoma is a highly metastatic cancer and there is strong evidence that the clotting initiator protein, tissue factor (TF), contributes to its aggressive pattern. TF inhibitors may attenuate primary tumor growth and metastasis. In this study, we evaluated the effect of ixolaris, a TF inhibitor, on a murine model of melanoma B16F10 cells. Enzymatic assays performed with B16F10 and human U87-MG tumor cells as the TF source showed that ixolaris inhibits the generation of FX in either murine, human or hybrid FVIIa/TF complexes. The effect of ixolaris on the metastatic potential was further estimated by intravenous injection of B16F10 cells in C57BL/6 mice. Ixolaris (250 µg/kg) dramatically decreased the number of pulmonary tumor nodules (4 ± 1 compared to 47 ± 10 in the control group). Furthermore, a significant decrease in tumor weights was observed in primary tumor growth assays in animals treated with ixolaris (250 µg/kg) from days 3 to 18 after a subcutaneous inoculation of melanoma cells. Remarkably, immunohistochemical analyses showed that inhibition of melanoma growth by ixolaris is accompanied by a significant downregulation of both vascular endothelial growth factor (VEGF) expression and microvascular density in the tumor mass. Our data demonstrate that ixolaris targets B16F10 cell-derived TF, resulting in the reduction of both the primary tumor growth and the metastatic potential of melanoma, as well as the inhibition of tumor angiogenesis. Therefore TF may be a potential target for the treatment of this aggressive malignancy.

Malignant transformation in melanocytes is associated with increased production of procoagulant microvesicles.

Shedding of microvesicles (MVs) by cancer cells is implicated in a variety of biological effects, including the establishment of cancer-associated hypercoagulable states. However, the mechanisms underlying malignant transformation and the acquisition of procoagulant properties by tumour-derived MVs are poorly understood. Here we investigated the procoagulant and prothrombotic properties of MVs produced by a melanocyte-derived cell line (melan-a) as compared to its tumourigenic melanoma counterpart Tm1. Tumour cells exhibit a two-fold higher rate of MVs production as compared to melan-a. Melanoma MVs display greater procoagulant activity and elevated levels of the clotting initiator protein tissue factor (TF). On the other hand, tumour- and melanocyte-derived MVs expose similar levels of the procoagulant lipid phosphatidylserine, displaying identical abilities to support thrombin generation by the prothrombinase complex. By using an arterial thrombosis model, we observed that melanoma- but not melanocyte-derived MVs strongly accelerate thrombus formation in a TF-dependent manner, and accumulate at the site of vascular injury. Analysis of plasma obtained from melanoma-bearing mice showed the presence of MVs with a similar procoagulant pattern as compared to Tm1 MVs produced in vitro. Remarkably, flow-cytometric analysis demonstrated that 60% of ex vivo MVs are TF-positive and carry the melanoma-associated antigen, demonstrating its tumour origin. Altogether our data suggest that malignant transformation in melanocytes increases the production of procoagulant MVs, which may contribute for a variety of coagulation-related protumoural responses.

Simultaneous tissue factor expression and phosphatidylserine exposure account for the highly procoagulant pattern of melanoma cell lines.

A correlation between cancer and hypercoagulability has been described for more than a century. Patients with cancer are at increased risk for thrombotic complications, and the clotting initiator protein, tissue factor (TF), is possibly involved in this process. In addition to TF, the presence of negatively charged phospholipids, particularly phosphatidylserine (PS), is necessary to support some of the blood-clotting reactions. There are few reports describing PS exposure by tumor cells. In this study, we characterized the procoagulant properties of the murine B16F10 and the human WM-266-4 melanoma cell lines. Flow cytometry analyses showed constitutive TF expression by both cell lines, in contrast to negative staining observed for the nontumorigenic melanocyte lineage, melan-A. In addition, tumor cells accelerate plasma clotting in a number-dependent manner. For WM-266-4, this ability was partially reversed by an anti-TF antibody but not by aprotinin, a nonspecific serine-protease inhibitor. Furthermore, flow-cytometric analyses showed the presence of PS at the outer leaflet of both cell lines. This phenomenon was determinant for the assembly of the intrinsic tenase (FIXa/FVIIIa) and prothrombinase (FXa/FVa) complexes, resulting in the activation of FX to FXa and prothrombin to thrombin, respectively. As a result, incubation of WM-266-4 with human plasma produces robust thrombin generation. In conclusion, simultaneous TF expression and PS exposure are responsible for the highly procoagulant pattern of the aggressive melanoma cell lines B16F10 and WM-266-4. Therefore, these cell lines might be regarded as useful models for studying the role of blood coagulation proteins in tumor biology.

Tumor-derived microvesicles modulate the establishment of metastatic melanoma in a phosphatidylserine-dependent manner.

Exposure of phosphatidylserine (PS) on cellular membranes and membrane-derived microvesicles stimulates a number of anti-inflammatory responses involved in malignant processes. Herein we show that B16F10 cells, a highly metastatic melanoma cell line, produce large quantities of PS-containing microvesicles in vitro. Tumor microvesicles increased TGF-beta(1) production by cultured macrophages and, in vivo, enhanced the metastatic potential of B16F10 cells in C57BL/6 mice, both effects being reversed by annexin V. Most strikingly, microvesicles induced melanoma metastasis in BALB/c mice, which are normally resistant to this tumor cell line. Altogether, this is the first demonstration that tumor-derived microvesicles favor the establishment of melanoma metastasis in a PS-dependent manner, possibly by down-regulating the host's inflammatory and/or anti-tumoral immune responses.