Endothelial Dll4 overexpression reduces vascular response and inhibits tumor growth and metastasization in vivo.

BACKGROUND: The inhibition of Delta-like 4 (Dll4)/Notch signaling has been shown to result in excessive, nonfunctional vessel proliferation and significant tumor growth suppression. However, safety concerns emerged with the identification of side effects resulting from chronic Dll4/Notch blockade. Alternatively, we explored the endothelial Dll4 overexpression using different mouse tumor models. METHODS: We used a transgenic mouse model of endothelial-specific Dll4 overexpression, previously produced. Growth kinetics and vascular histopathology of several types of solid tumors was evaluated, namely Lewis Lung Carcinoma xenografts, chemically-induced skin papillomas and RIP1-Tag2 insulinomas. RESULTS: We found that increased Dll4/Notch signaling reduces tumor growth by reducing vascular endothelial growth factor (VEGF)-induced endothelial proliferation, tumor vessel density and overall tumor blood supply. In addition, Dll4 overexpression consistently improved tumor vascular maturation and functionality, as indicated by increased vessel calibers, enhanced mural cell recruitment and increased network perfusion. Importantly, the tumor vessel normalization is not more effective than restricted vessel proliferation, but was found to prevent metastasis formation and allow for increased delivery to the tumor of concomitant chemotherapy, improving its efficacy. CONCLUSIONS: By reducing endothelial sensitivity to VEGF, these results imply that Dll4/Notch stimulation in tumor microenvironment could be beneficial to solid cancer patient treatment by reducing primary tumor size, improving tumor drug delivery and reducing metastization. Endothelial specific Dll4 overexpression thus appears as a promising anti-angiogenic modality that might improve cancer control.

Endothelial Jagged1 antagonizes Dll4 regulation of endothelial branching and promotes vascular maturation downstream of Dll4/Notch1.

OBJECTIVE: Notch signaling controls cardiovascular development and has been associated with several pathological conditions. Among its ligands, Jagged1 and Dll4 were shown to have opposing effects in developmental angiogenesis, but the underlying mechanism and the role of Jagged1/Notch signaling in adult angiogenesis remain incompletely understood. The current study addresses the importance of endothelial Jagged1-mediated Notch signaling in the context of adult physiological angiogenesis and the interactions of Jagged1 and Dll4 on angiogenic response and vascular maturation processes. APPROACH AND RESULTS: The role of endothelial Jagged1 in wound healing kinetics and angiogenesis was investigated with endothelial-specific Jag1 gain-of-function and loss-of-function mouse mutants (eJag1OE and eJag1cKO). To study the interactions between the 2 Notch ligands, genetic mouse models were combined with pharmacological inhibition of Dll4 or Jagged1, respectively. Jagged1 overexpression in endothelial cells increased vessel density, maturation, and perfusion, thus accelerating wound healing. The opposite effect was seen in eJag1cKO animals. Interestingly, Dll4 blockade in these animals led to an increase in vascular density but induced a greater decrease in perivascular cell coverage. However, Jagged1 inhibition in Dll4 gain-of-function (eDll4OE) mutants, with reduced angiogenesis, further diminished angiogenic growth and hampered perivascular cell coverage. Our findings suggest that as Dll4 blocks endothelial activation through Notch1 signaling, it also induces Jagged1 expression. Jagged1 then blocks Dll4 signaling through Notch1, allowing endothelial activation by vascular endothelial growth factor and endothelial layer growth. Jagged1 also initiates maturation of the newly formed vessels, possibly by binding and activating endothelial Notch4. Importantly, mice administered with a Notch4 agonistic antibody mimicked the mural cell phenotype of eJag1OE mutants without affecting angiogenic growth, which is thought to be Notch1 dependent. CONCLUSIONS: Endothelial Jagged1 is likely to operate downstream of Dll4/Notch1 signaling to activate Notch4 and regulate vascular maturation. Thus, Jagged1 not only counteracts Dll4/Notch in the endothelium but also generates a balance between angiogenic growth and maturation processes in vivo.

Incomplete Dll4/Notch signaling inhibition promotes functional angiogenesis supporting the growth of skin papillomas.

BACKGROUND: In invasive malignancies, Dll4/Notch signaling inhibition enhances non-functional vessel proliferation and limits tumor growth by reducing its blood perfusion. METHODS: To assess the effects of targeted Dll4 allelic deletion in the incipient stages of tumor pathogenesis, we chemically induced skin papillomas in wild-type and Dll4 (+/-) littermates, and compared tumor growth, their histological features, vascularization and the expression of angiogenesis-related molecules. RESULTS: We observed that Dll4 down-regulation promotes productive angiogenesis, although with less mature vessels, in chemically-induced pre-cancerous skin papillomas stimulating their growth. The increase in endothelial activation was associated with an increase in the VEGFR2 to VEGFR1 ratio, which neutralized the tumor-suppressive effect of VEGFR-targeting sorafenib. Thus, in early papillomas, lower levels of Dll4 increase vascularization through raised VEGFR2 levels, enhancing sensitivity to endogenous levels of VEGF, promoting functional angiogenesis and tumor growth. CONCLUSION: Tumor promoting effect of low-dosage inhibition needs to be considered when implementing Dll4 targeting therapies.

Combination of Dll4/Notch and Ephrin-B2/EphB4 targeted therapy is highly effective in disrupting tumor angiogenesis.

BACKGROUND: Dll4/Notch and Ephrin-B2/EphB4 pathways play critical roles in tumor vessel development and maturation. This study evaluates the efficacy of the inhibition of both signaling pathways, alone and in combination, in reducing the growth of an autochthonous mouse tumor and assesses potential adverse effects. METHODS: We used the transgenic RIP1-Tag2 tumor model to study the effects of 1) inhibition of Dll4/Notch by either Dll4 allelic deletion or use of a soluble extracellular Dll4 (sDll4), 2) inhibition of Ephrin-B2/EphB4 signaling by a soluble extracellular EphB4 fused to albumin (sEphB4-Alb), and 3) inhibition of both pathways by sEphB4-Alb combined with either Dll4 allelic deletion or sDll4. To investigate adverse effects, we used inducible endothelial-specific Dll4 knock-out mice, treated with sEphB4-Alb, and carried out histopathological analysis. RESULTS: Dll4 allele deletion or soluble Dll4 treatment resulted in increased tumor vessel density, reduced mural cell recruitment and vessel perfusion which resulted in reduced tumor size. The soluble EphB4 instead reduced vessel density and vessel perfusion, leading to reduction of tumor size. Greater efficacy was observed when sEphB4-Alb was combined with either Dll4 allele deletion or sDll4 in regards to tumor size, vessel perfusion and mural cell recruitment. Induced endothelial specific Dll4 loss-of-function caused hepatic vascular alterations, which were prevented by concomitant sEphB4-Alb treatment. CONCLUSION: Combination targeting of Dll4/Notch and Ephrin-B2/EphB4 has potential for clinical investigation, providing cumulative efficacy and increased safety over Dll4/Notch inhibition alone.

Metastasis is impaired by endothelial-specific Dll4 loss-of-function through inhibition of epithelial-to-mesenchymal transition and reduction of cancer stem cells and circulating tumor cells.

Systemic inhibition of Dll4 has been shown to thoroughly reduce cancer metastasis. The exact cause of this effect and whether it is endothelial mediated remains to be clarified. Therefore, we proposed to analyze the impact of endothelial Dll4 loss-of-function on metastasis induction on three early steps of the metastatic process, regulation of epithelial-to-mesenchymal transition (EMT), cancer stem cell (CSC) frequency and circulating tumor cell (CTC) number. For this, Lewis Lung Carcinoma (LLC) cells were used to model mouse tumor metastasis in vivo, by subcutaneous transplantation into endothelial-specific Dll4 loss-of-function mice. We observed that endothelial-specific Dll4 loss-of-function is responsible for the tumor vascular regression that leads to the reduction of tumor burden. It induces an increase in tumoral blood vessel density, but the neovessels are poorly perfused, with increased leakage and reduced perivascular maturation. Unexpectedly, although hypoxia was increased in the tumor, the number and burden of macro-metastasis was significantly reduced. This is likely to be a consequence of the observed reduction in both EMT and CSC numbers caused by the endothelial-specific Dll4 loss-of-function. This multifactorial context may explain the concomitantly observed reduction of the circulating tumor cell count. Furthermore, our results suggest that endothelial Dll4/Notch-function mediates tumor hypoxia-driven increase of EMT. Therefore, it appears that endothelial Dll4 may constitute a promising target to prevent metastasis.

In vitro and in vivo biological characterization of the anti-proliferative potential of a cyclic trinuclear organotin(iv) complex.

Identification of novel molecules that can selectively inhibit the growth of tumor cells, avoid causing side effects to patients and/or intrinsic or acquired resistance, usually associated with common chemotherapeutic agents, is of utmost importance. Organometallic compounds have gained importance in oncologic chemotherapy, such as organotin(iv) complexes. In this study, we assessed the anti-tumor activity of the cyclic trinuclear organotin(iv) complex with an aromatic oximehydroxamic acid group [nBu2Sn(L)]3(H2L = N,2-dihydroxy-5-[N-hydroxyethanimidoyl]benzamide) - MG85 - and provided further characterization of its biological targets. We have previously shown the high anti-proliferative activity of this complex against human colorectal and hepatocellular carcinoma cell lines and lower cytotoxicity in neonatal non-tumor fibroblasts. MG85 induces tumor cell apoptosis and down-regulation of proteins related to tubulin dynamics (TCTP and COF1). Further characterization included the: (i) evaluation of interference in the cell cycle progression, including the expression of critical genes; (ii) affinity to DNA and the corresponding mode of binding; (iii) genotoxic potential in cells with deficient DNA repair pathways; and (iv) in vivo tumor reduction efficiency using mouse colorectal carcinoma xenografts.

Low-dosage inhibition of Dll4 signaling promotes wound healing by inducing functional neo-angiogenesis.

Recent findings regarding Dll4 function in physiological and pathological conditions indicate that this Notch ligand may constitute an important therapeutic target. Dll4 appears to be a major anti-angiogenic agent, occupying a central role in various angiogenic pathways. The first trials of anti-Dll4 therapy in mice demonstrated a paradoxical effect, as it reduced tumor perfusion and growth despite leading to an increase in vascular density. This is seen as the result of insufficient maturation of the newly formed vasculature causing a circulatory defect and increased tumor hypoxia. As Dll4 function is known to be closely dependent on expression levels, we envisioned that the therapeutic anti-Dll4 dosage could be modulated to result in the increase of adequately functional blood vessels. This would be useful in conditions where vascular function is a limiting factor for recovery, like wound healing and tissue hypoxia, especially in diabetic patients. Our experimental results in mice confirmed this possibility, revealing that low dosage inhibition of Dll4/Notch signaling causes improved vascular function and accelerated wound healing.