Ombretta Salvucci

The role of CXCR4 receptor expression in breast cancer: a large tissue microarray study.

SummaryThe chemokine receptor CXCR4 is an important factor in the migration, invasiveness, metastasis and proliferation of breast cancer cells. We have retrospectively analyzed the levels of expression of CXCR4 in a large cohort of breast cancers and pre-invasive breast samples linked to clinical data. A total of 1808 invasive breast carcinomas and 214 pre-invasive breast samples could be analyzed in correlation with basic clinico-pathological data such as hormone receptor status, HER2 status and tumor grade. The majority of breast cancers expressed either nuclear or cytoplasmic staining or both. CXCR4 cytoplasmic expression was associated with parameters of tumor aggressivity (tumor grade and lymph node status) and had prognostic value (age-adjusted hazard ratio=1.73; Confidence Interval: 1.07–2.77) with respect to disease-specific survival. CXCR4 positivity in the cytoplasm but not the nucleus was associated with HER2 expression and amplification as well as with hormone receptor negativity (both ER and PR). The percentage of nuclear staining increased from normal breast tissue (20%) to ductal carcinoma-in-situ DCIS (43%) to invasive cancer (67%) while CXCR4 was expressed in the cytoplasm of 67% of (DCIS) cases (double that in normal breast samples), suggesting an important role in breast tumor progression. The CXCR4 receptor is expressed in many breast cancers, justifying its development as a therapeutic target in breast cancer patients. Its cytoplasmic expression is associated with breast tumor progression, suggesting potential value as a diagnostic marker.

CXCR4 peptide antagonist inhibits primary breast tumor growth, metastasis and enhances the efficacy of anti-VEGF treatment or docetaxel in a transgenic mouse model

CXCR4 is a chemokine receptor implicated in the homing of cancer cells to target metastatic organs, which overexpress its ligand, stromal cell‐derived factor (SDF)‐1. To determine the efficacy of targeting CXCR4 on primary tumor growth and metastasis, we used a peptide inhibitor of CXCR4, CTCE‐9908, that was administered in a clinically relevant approach using a transgenic breast cancer mouse model. We first performed a dosing experiment of CTCE‐9908 in the PyMT mouse model, testing 25, 50 and 100 mg/kg versus the scrambled peptide in groups of 8–16 mice. We then combined CTCE‐9908 with docetaxel or DC101 (an anti‐VEGFR2 monoclonal antibody). We found that increasing doses of CTCE‐9908 alone slowed the rate of tumor growth, with a 45% inhibition of primary tumor growth at 3.5 weeks of treatment with 50 mg/kg of CTCE‐9908 (p = 0.005). Expression levels of VEGF were also found to be reduced by 42% with CTCE‐9908 (p = 0.01). In combination with docetaxel, CTCE‐9908 administration decreased tumor volume by 38% (p = 0.02), an effect that was greater than that observed with docetaxel alone. In combination with DC101, CTCE‐9908 also demonstrated an enhanced effect compared to DC101 alone, with a 37% decrease in primary tumor volume (p = 0.01) and a 75% reduction in distant metastasis (p = 0.009). In combination with docetaxel or an anti‐angiogenic agent, the anti‐tumor and anti‐metastatic effects of CTCE‐9908 were markedly enhanced, suggesting potentially new effective combinatorial therapeutic strategies in the treatment of breast cancer, which include targeting the SDF‐1/CXCR4 ligand/receptor pair.

EphrinB reverse signaling contributes to endothelial and mural cell assembly into vascular structures

EphrinB transmembrane ligands and their cognate EphB receptor tyrosine kinases regulate vascular development through bidirectional cell-to-cell signaling, but little is known about the role of EphrinB during postnatal vascular remodeling. We report that EphrinB is a critical mediator of postnatal pericyte-to-endothelial cell assembly into vascular structures. This function is dependent upon extracellular matrix-supported cell-to-cell contact, engagement of EphrinB by EphB receptors expressed on another cell, and Src-dependent phosphorylation of the intracytoplasmic domain of EphrinB. Phosphorylated EphrinB marks angiogenic blood vessels in the developing and hypoxic retina, the wounded skin, and tumor tissue, and is detected at contact points between endothelial cells and pericytes. Furthermore, inhibition ofEphrinB activity prevents proper assembly of pericytes and endothelial cells into vascular structures. These results reveal a role for EphrinB signaling in orchestrating pericyte/endothelial cell assembly, and suggest that therapeutic targeting of EphrinB may prove useful for disrupting angiogenesis when it contributes to disease.

Essential Roles of EphB Receptors and EphrinB Ligands in Endothelial Cell Function and Angiogenesis

Eph receptor tyrosine kinases and their Ephrin ligands represent an important signaling system with widespread roles in cell physiology and disease. Receptors and ligands in this family are anchored to the cell surface; thus Eph/Ephrin interactions mainly occur at sites of cell-to-cell contact. EphB4 and EphrinB2 are the Eph/Ephrin molecules that play essential roles in vascular development and postnatal angiogenesis. Analysis of expression patterns and function has linked EphB4/EphrinB2 to endothelial cell growth, survival, migration, assembly, and angiogenesis. Signaling from these molecules is complex, with the potential for being bidirectional, emanating both from the Eph receptors (forward signaling) and from the Ephrin ligands (reverse signaling). In this review, we describe recent advances on the roles of EphB/EphrinB protein family in endothelial cell function and outline potential approaches to inhibit pathological angiogenesis based on this understanding.

Plasma Stromal Cell–Derived Factor-1: Host Derived Marker Predictive of Distant Metastasis in Breast Cancer

Purpose: Homing of breast cancer cells to metastatic sites may be regulated by the production of stromal cell–derived factor (SDF)-1 by specific target organs, which attracts CXCR4-expressing breast cancer cells. We investigated the value of SDF-1 as a predictive blood marker of distant metastasis in breast cancer, together with a common polymorphism of SDF-1, SDF-1-3′A. Experimental Design: Plasma samples were collected prospectively for 270 consecutive primary breast cancer patients with a median follow-up of 3.3 years. Plasma SDF-1 levels were measured using an ELISA, and the polymorphism was identified via PCR-RFLP analysis. Results: Plasma SDF-1 levels were divided into two groups, low and high, based on the median SDF-1 value of 2,661 pg/mL. Patients with low SDF-1 showed an increased risk of developing distant metastasis (relative risk, 1.94; P = 0.02) and poorer breast cancer–specific survival [adjusted hazard ratio (AHR), 3.92; P = 0.007]. Patients with both low plasma SDF-1 levels and the SDF-1-3′A polymorphism showed a poorer breast cancer–specific survival (AHR, 3.98; P = 0.001) and distant disease-free survival (AHR, 2.88; P = 0.003). In a separate cohort of 22 breast cancer patients, we found no significant difference in SDF-1 levels before and posttumor resection. Conclusion: We found that low plasma SDF-1 is an independent host-derived predictive marker of distant metastasis in breast cancer. The prognostic value of the combination of a low plasma SDF-1 level and the SDF-1-3′A polymorphism identifies a cohort of patients with an intrinsic susceptibility for poorer survival.

Kaposi Sarcoma Herpesvirus Promotes Endothelial-to-Mesenchymal Transition through Notch-Dependent Signaling

Endothelial-to-mesenchymal transition (EndMT) is now widely considered a pivotal contributor to cancer progression. In this study, we show that the Kaposis sarcoma (KS)-associated herpesvirus (KSHV) is a sufficient cause of EndMT, potentially helping to explain the aggressiveness of KS that occurs commonly in AIDS patients. Upon KSHV infection, primary dermal microvascular endothelial cells lost expression of endothelial markers and acquired expression of mesenchymal markers, displaying new invasive and migratory properties along with increased survival. KSHV activated Notch-induced transcription factors Slug and ZEB1, and canonical Notch signaling was required for KSHV-induced EndMT. In contrast, KSHV did not utilize the TGFβ signaling pathway, which has also been linked to EndMT. Within KS lesions, KSHV-infected spindle cells displayed features compatible with KSHV-induced EndMT including a complex phenotype of endothelial and mesenchymal properties, Notch activity, and nuclear ZEB1 expression. Our results show that KSHV engages the EndMT program to increase the invasiveness and survival of infected endothelial cells, traits that likely contribute to viral persistence and malignant progression. One important implication of our findings is that therapeutic approaches to disrupt the Notch pathway may offer novel approaches for KS treatment.

The Influence of Tumor-Host Interactions in the Stromal Cell-Derived Factor-1/CXCR4 Ligand/Receptor Axis in Determining Metastatic Risk in Breast Cancer

The chemokine stromal cell-derived factor-1 (SDF-1) may function to attract CXCR4-expressing cancer cells to metastatic organs. We have previously demonstrated that low plasma SDF-1, a host-derived marker, increases distant metastatic risk in breast cancer. We therefore hypothesized that tumors overexpressing the SDF-1 receptor CXCR4 have an enhanced ability to metastasize in patients with low plasma SDF-1 levels. In this study, we determined the prognostic significance of activated CXCR4, or phosphorylated CXCR4 (p-CXCR4), and CXCR7, another receptor for SDF-1. Immunohistochemistry was performed on a tissue microarray built using 237 samples from the same cohort of patients for which we measured plasma SDF-1 levels. We found that the prognostic value of p-CXCR4 expression (hazard ratio or HR, 3.95; P = 0.004) was superior to total CXCR4 expression (HR, 3.20; P = 0.03). The rate of breast cancer-specific mortality was much higher in patients with both high p-CXCR4 expression and low plasma SDF-1 levels (HR, 5.96; P < 0.001) than either low plasma SDF-1 (HR, 3.59; P = 0.01) or high p-CXCR4 expression (HR, 3.83; P = 0.005) alone. The added prognostic value of low plasma SDF-1 was only effective in patients with high p-CXCR4 expression, and as such, provides clinical validation for modulation of the metastatic potential of tumor cells by an inherent host-derived metastatic risk factor.

Evidence for the involvement of SDF‐1 and CXCR4 in the disruption of endothelial cell‐branching morphogenesis and angiogenesis by TNF‐α and IFN‐γ

Vigorous inflammatory responses are associated with tissue damage, particularly when toxic levels of inflammatory cytokines are produced. Despite proangiogenic factors being present early at sites of inflammation, vascular repair occurs toward the end of the inflammatory response, suggesting modulation of the proangiogenic response. Endogenous inhibitors of angiogenesis induced during acute inflammation are poorly characterized. Here, we looked for endothelial cell‐derived modulators of angiogenesis that may account for delayed neovascularization during inflammation. Gene profiling of endothelial cells showed that the inflammatory cytokines tumor necrosis factor α (TNF‐α) and interferon‐γ (IFN‐γ) selectively promote expression of the antiangiogenic molecules, IFN‐inducible protein‐10, monokine induced by IFN‐γ, tryptophanyl‐tRNA synthetase, and tissue inhibitor of metalmetalloproteinase‐1, and inhibit expression of the proangiogenic molecules, platelet‐endothelial cell adhesion molecule‐1, vascular endothelial growth factor receptor‐2, stromal cell‐derived factor‐1 (SDF‐1), collagen type IV, endothelial cell growth factor‐1, and carcinoembryonic antigen‐related cell adhesion molecule‐1. Reduced endothelial cell expression of SDF‐1 protein by TNF‐α and IFN‐γ disrupts extracellular matrix‐dependent endothelial cell tube formation, an in vitro morphogenic process that recapitulates critical steps in angiogenesis. Replacement of SDF‐1 onto the endothelial cell surface reconstitutes this morphogenic process. In vivo, TNF‐α and IFN‐γ inhibit growth factor‐induced angiogenesis and SDF‐1 expression in endothelial cells. These results demonstrate that SDF‐1/CXC chemokine receptor‐4 constitutes a TNF‐α‐ and IFN‐γ‐regulated signaling system that plays a critical role in mediating angiogenesis inhibition by these inflammatory cytokines.

Semaphorin 6A regulates angiogenesis by modulating VEGF signaling

Formation of new vessels during development and in the mature mammal generally proceeds through angiogenesis. Although a variety of molecules and signaling pathways are known to underlie endothelial cell sprouting and remodeling during angiogenesis, many aspects of this complex process remain unexplained. Here we show that the transmembrane semaphorin6A (Sema6A) is expressed in endothelial cells, and regulates endothelial cell survival and growth by modulating the expression and signaling of VEGFR2, which is known to maintain endothelial cell viability by autocrine VEGFR signaling. The silencing of Sema6A in primary endothelial cells promotes cell death that is not rescued by exogenous VEGF-A or FGF2, attributable to the loss of prosurvival signaling from endogenous VEGF. Analyses of mouse tissues demonstrate that Sema6A is expressed in angiogenic and remodeling vessels. Mice with null mutations of Sema6A exhibit significant defects in hyaloid vessels complexity associated with increased endothelial cell death, and in retinal vessels development that is abnormally reduced. Adult Sema6A-null mice exhibit reduced tumor, matrigel, and choroidal angiogenesis compared with controls. Sema6A plays important roles in development of the nervous system. Here we show that it also regulates vascular development and adult angiogenesis.

MicroRNA126 contributes to granulocyte colony-stimulating factor-induced hematopoietic progenitor cell mobilization by reducing the expression of vascular cell adhesion molecule 1.

Background Mobilization of hematopoietic stem/progenitor cells from the bone marrow to the peripheral blood by granulocyte colony-stimulating factor is the primary means to acquire stem cell grafts for hematopoietic cell transplantation. Since hematopoietic stem/progenitor cells represent a minority of all blood cells mobilized by granulocyte colony-stimulating factor, the underlying mechanisms need to be understood in order to develop selective drugs. Design and Methods We analyzed phenotypic, biochemical and genetic changes in bone marrow cell populations from granulocyte colony-stimulating factor-mobilized and control mice, and linked such changes to effective mobilization of hematopoietic stem/progenitor cells. Results We show that granulocyte colony-stimulating factor indirectly reduces expression of surface vascular cell adhesion molecule 1 on bone marrow hematopoietic stem/progenitor cells, stromal cells and endothelial cells by promoting the accumulation of microRNA-126 (miR126)-containing microvescicles in the bone marrow extracellular compartment. We found that hematopoietic stem/progenitor cells, stromal cells and endothelial cells readily incorporate these miR126-loaded microvescicles, and that miR126 represses vascular cell adhesion molecule 1 expression on bone marrow hematopoietic stem/progenitor cells, stromal cells and endothelial cells. In line with this, miR126-null mice displayed a reduced mobilization response to granulocyte colony-stimulating factor. Conclusions Our results implicate miR126 in the regulation of hematopoietic stem/progenitor cell trafficking between the bone marrow and peripheral sites, clarify the role of vascular cell adhesion molecule 1 in granulocyte colony-stimulating factor-mediated mobilization, and have important implications for improved approaches to selective mobilization of hematopoietic stem/progenitor cells.