Delta-like Ligand 4 Expression Research Articles

Clinicopathologic significance of the delta-like ligand 4, vascular endothelial growth factor, and hypoxia-inducible factor-2α in gallbladder cancer.

Gallbladder cancer (GBC) is usually detected in advanced stages with a low 5-year survival rate. Delta-like ligand 4 (DLL4), vascular endothelial growth factor (VEGF), and hypoxia-inducible factor-2alpha (HIF2α) have been studied for their role in tumorigenesis and potential for therapeutic target, and multiple clinical trials of the agents targeting them are ongoing. We investigated the expression of these markers in surgically resected GBC and tried to reveal their association with the clinicopathologic features, mutual correlation of their expression, and prognosis of the GBC patients by their expression. We constructed the tissue microarray blocks of 99 surgically resected GBC specimens and performed immunohistochemistry of DLL4, VEGF, and HIF2α. We used the quantitative digital image analysis to evaluate DLL4 and VEGF expression, while the expression of HIF2α was scored manually. The expression of VEGF and HIF2α showed a significant trend with tumor differentiation (p= .028 and p= .006, respectively). We found that the high DLL4 and VEGF expression were significantly correlated with lymph node metastasis (p= .047, both). The expression of VEGF and HIF2α were significantly correlated (p < .001). The GBC patients with low HIF2α expression showed shorter recurrence-free survival than those with high HIF2α expression. This study suggested the possibility of the usage of DLL4 and VEGF to predict the lymph node metastasis and the possibility of VEGF and HIF2α to predict the expression level mutually. Further studies may be needed to validate our study results and eventually accelerate the introduction of the targeted therapy in GBC.

Development of anti DLL4 Nanobody fused to truncated form of Pseudomonas exotoxin: As a novel immunotoxin to inhibit of cell proliferation and neovascularization

Targeted tumor therapy is an attractive approach for cancer treatment. Delta-like ligand 4 (DLL4) is overexpressed in tumor vasculature and plays a pivotal role in tumor neovascular development and angiogenesis during tumor progression. Immunotoxins due to their superior cell-killing ability and the relative simplicity of their preparation, have great potential in the clinical treatment of cancer. The aim of this study was to develop a novel immunotoxin against DLL4 as a cell cytotoxic agent and angiogenesis maturation inhibitor. In present study, an immunotoxin, named DLL4Nb-PE, in which a Nanobody as targeting moiety fused to the Pseudomonas exotoxin A (PE) was constructed, expressed and assessed by SDS-PAGE, western blotting, ELISA and flowcytometry. The functional assessment was carried out via MTT, apoptosis and chicken chorioallantoic membrane (CAM) assays. It was demonstrated DLL4Nb-PE specifically binds to DLL4 and recognizes DLL4-expressing MKN cells. The cytotoxicity assays showed that this molecule could induce apoptosis and kill DLL4 positive MKN cells. In addition, it inhibited neovascularization in the chicken chorioallantoic membrane. Our findings indicate designed anti-DLL4 immunotoxin has valuable potential for application to the treatment of tumors with high DLL4 expression.

Abstract P02-03: Phase Ia/Ib dose-escalation study of ABL001 (CTX-009, bispecific antibody targeting DLL4 and VEGF-A) as a single agent in patients with advanced solid tumors

Abstract VEGF blockade is a validated therapeutic approach and anti-VEGF antibodies are established components of several standard regimens for various malignancies. However, the overall response rate to single agent VEGF blockers is extremely low and many patients develop resistance to regimens that include VEGF blockers. Hence, additional angiogenesis-targeted therapies are necessary to overcome the resistance to VEGF blockade and improve response rates. Delta-like ligand 4 (DLL4), a Notch pathway ligand, plays a key role in the overgrowth of dysfunctional microvasculature, and DLL4 is upregulated by VEGF. ABL001 (CTX-009) is a bispecific antibody targeting both DLL4 and VEGF-A with significant anti-tumor activity in preclinical studies. A phase 1, multicenter, open-label and first-in-human study was conducted to evaluate the safety and tolerability of ABL001 (CTX-009) in patients with advanced solid tumors. The dose escalation phase had 9 dose cohorts from 0.3 to 17.5 mg/kg (IV every 2 weeks), using a traditional 3+3 design. The study was expanded at dose levels of 7.5, 10, 12.5 and 15 mg/kg. Archived tumor tissue samples were collected to assess DLL4 expression levels using an immunohistochemistry (IHC) method. Key selection criteria included: age ≥19 years, metastatic or unresectable advanced solid tumors, ECOG ≤2, and adequate hematologic, hepatic and renal function. A total of 45 patients (31 patients in the dose escalation phase and 14 patients in the dose expansion phase) were enrolled in the study. The patients were heavily pretreated and received a median of 4 lines of prior therapies. 86.7% of the patients were ECOG 1. Gastric cancer (n=19) and colorectal cancer (n=18) were the most common tumors. No dose limiting toxicities (DLTs) were observed. The most common Treatment-Emergent Adverse Events (TEAEs) reported in ≥10% patients were hypertension (37.8%), headache (22.2%), anemia (13.3%), asthenia (13.3%) and fatigue (11.1%). Grade 3 or greater TEAEs reported in ≥5% patients were hypertension (15.6%) and anemia (6.7%). The most common Treatment Related Adverse Events (TRAEs) reported in ≥10% patients were hypertension (37.8%) and headache (15.6%). Of the 39 evaluable patients, there were 4 partial responses (PRs) and 3 of those responses were confirmed by RECIST. Responses began to appear at the 10 mg/kg dose level. The 3 confirmed partial responses included 2 colorectal cancer patients at 10 mg/kg and 1 gastric cancer patient at 12.5 mg/kg. All 3 patients with PRs exhibited high DLL4 expression by IHC on archived tumor tissue samples. The unconfirmed PR was in a patient with gastric cancer dosed at 15 mg/kg who was also DLL4 IHC positive. The recommended Phase 2 doses (RP2Ds) were determined to be 10 mg/kg and 12.5 mg/kg and the Overall Response Rate (ORR) at the RP2D was 18.8% (3/16) and the Disease Control Rate (DCR) was 62.5% (11/16). ABL001 (CTX-009) monotherapy is well tolerated and shows promising antitumor activity. Clinical trial information: NCT03292783 Citation Format: Jeeyun Lee, SeungTae Kim, JungYong Hong, YoungSuk Park, JoonOh Park, WonKi Kang, Keun-Wook Lee, Jin Won Kim, Ji-Won Kim, Se Hyun Kim, Eunsin Ha, Sangmi Lee, JongRan Kim, Weon-Kyoo You. Phase Ia/Ib dose-escalation study of ABL001 (CTX-009, bispecific antibody targeting DLL4 and VEGF-A) as a single agent in patients with advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P02-03.

The role of Notch ligand, Delta-like ligand 4 (DLL4), in cancer angiogenesis—implications for therapy

Summary Background It is generally accepted that angiogenesis is a complex and tightly regulated process characterized by the growth of blood vessels from existing vasculature. Activation of the Notch signalling pathway affects multiple aspects of vascular development. One of the components of the Notch signalling pathway, Delta-like ligand 4 (DLL4), has recently appeared as a critical regulator of tumour angiogenesis and thus as a promising therapeutic target. Methods This review article includes available data from peer-reviewed publications associated with the role of DLL4 in cancer angiogenesis. Searches were performed in PubMed, EMBASE, Google Scholar and Web of Science using the terms “tumour angiogenesis”, “DLL4”, “Notch signalling” and “anti-cancer therapy”. Results The survival curves of cancer patients revealed that the patients with high DLL4 expression levels had significantly shorter survival times than the patients with low DLL4 expression. Moreover, a positive correlation was also identified between DLL4 and VEGF receptorsʼ expression levels. It seems that inhibition of DLL4 may exert potent growth inhibitory effects on some tumours resistant to anti-VEGF therapies. A great number of blocking agents of DLL4/Notch signalling including anti-DLL4 antibodies, DNA vaccination, Notch antibodies and gamma-secretase inhibitors have been studied in preclinical tumour models. Conclusion DLL4 seems to be a promising target in anti-cancer therapy. Nevertheless, the careful evaluation of adverse effects on normal physiological processes in relation to therapeutic doses of anti-DLL4 drugs will be significant for advancement of DLL4 blocking agents in clinical oncology.

CircRNA_103765 acts as a proinflammatory factor via sponging miR-30 family in Crohn\u2019s disease

Increasing evidence suggests that circular RNAs (circRNAs) play critical roles in various pathophysiological activities. However, the role of circRNAs in inflammatory bowel disease (IBD) remains unclear. Here we report the potential roles of hsa_circRNA_103765 in regulating cell apoptosis induced by TNF-α in Crohn’s disease (CD). We identify that CircRNA_103765 expression was significantly upregulated in peripheral blood mononuclear cells (PBMCs) of patients with active IBD. A positive correlation with TNF-α significantly enhanced circRNA_103765 expression in CD, which was significantly reversed by anti-TNF-α mAb (infliximab) treatment. In vitro experiments showed that TNF-α could induce the expression of circRNA_103765, which was cell apoptosis dependent, while silencing of circRNA_103765 could protect human intestinal epithelial cells (IECs) from TNF-α-induced apoptosis. In addition, circRNA_103765 acted as a molecular sponge to adsorb the miR-30 family and impair the negative regulation of Delta-like ligand 4 (DLL4). Collectively, CircRNA_103765 is a novel important regulator of the pathogenesis of IBD via sponging miR-30 family-mediated DLL4 expression changes. Blockade of circRNA_103765 could serve as a novel approach for the treatment of IBD patients.

&lt;i&gt;Mycobacterium tuberculosis&lt;/i&gt;&amp;nbsp;Impedes CD40-Dependent Notch Signaling to Restrict Th &lt;sub&gt;17&lt;/sub&gt; Polarization During Infection

Early Th17 responses are necessary to provide protection against Mycobacterium tuberculosis (Mtb). However, we previously demonstrated that Mtb restricts Th17 polarization and that engaging the CD40 costimulatory pathway on dendritic cells (DCs) enhances Th17 responses and augments Mtb control. Defining the molecular mechanisms that limit Th17 polarization during Mtb infection will facilitate the design of TB vaccination strategies that induce protective Th17 immunity. Here, we identify the Notch ligand, Delta-like ligand 4 (DLL4), as necessary for Th17 polarization and demonstrate that Mtb limits DLL4 signaling on DCs to prevent optimal Th17 responses. While Mtb infection induced only low levels of Dll4 mRNA in DCs, exogenously engaging CD40 on Mtb-infected DCs enhanced Dll4 expression and led to high frequencies of DLL4+ and DLL4+ Jagged1+ DCs. Antibody blockade of DLL4 on DCs reduced Th17 polarization but did not affect Th1 polarization. DLL4 was also required for inducing multifunctional CXCR3+ CCR6+ CD4 T cells co-expressing IL-17, IL-22 and IFN-γ in the lungs of mice, and DLL4-dependent Th17 responses inversely correlated with Mtb lung burdens. Additionally, we show that the Mtb Hip1 protease attenuates DLL4 expression on lung DCs by impeding CD40 signaling. Overall, our results demonstrate that Mtb impedes CD40-dependent Notch ligand signaling to restrict Th17 responses and identify the CD40-DLL4 pathways as targets for developing new Th17-inducing vaccines and adjuvants for TB.

Synergistic antitumor activity of a DLL4/VEGF bispecific therapeutic antibody in combination with irinotecan in gastric cancer

Notch signaling has been identified as a critical pathway in gastric cancer (GC) progression and metastasis, and inhibition of Delta-like ligand 4 (DLL4), a Notch ligand, is suggested as a potent therapeutic approach for GC. Expression of both DLL4 and vascular endothelial growth factor receptor 2 (VEGFR2) was similar in the malignant tissues of GC patients. We focused on vascular endothelial growth factor (VEGF), a known angiogenesis regulator and activator of DLL4. Here, we used ABL001, a DLL4/VEGF bispecific therapeutic antibody, and investigated its therapeutic effect in GC. Treatment with human DLL4 therapeutic antibody (anti-hDLL4) or ABL001 slightly reduced GC cell growth in monolayer culture; however, they significantly inhibited cell growth in 3D-culture, suggesting a reduction in the cancer stem cell population. Treatment with anti-hDLL4 or ABL001 also decreased GC cell migration and invasion. Moreover, the combined treatment of irinotecan with anti-hDLL4 or ABL001 showed synergistic antitumor activity. Both combination treatments further reduced cell growth in 3D-culture as well as cell invasion. Interestingly, the combination treatment of ABL001 with irinotecan synergistically reduced the GC burden in both xenograft and orthotopic mouse models. Collectively, DLL4 inhibition significantly decreased cell motility and stem-like phenotype and the combination treatment of DLL4/VEGF bispecific therapeutic antibody with irinotecan synergistically reduced the GC burden in mouse models. Our data suggest that ABL001 potentially represents a potent agent in GC therapy. Further biochemical and pre-clinical studies are needed for its application in the clinic.

Anti-Cancer Effect of Melatonin via Downregulation of Delta-like Ligand 4 in Estrogen-Responsive Breast Cancer Cells.

The Notch signaling pathway has a key role in angiogenesis and Delta - Like Ligand 4 (DLL4) is one of the main ligands of Notch involved in cell proliferation in sprouting vessels. In this study, we aimed to evaluate the expression of DLL4 in primary breast tumors and to examine the effect of melatonin on DLL4 expression in vitro. Eighty-five breast tumor and paired adjacent non-tumor tissue samples were collected. Apoptosis assay was performed on breast cancer cells to evaluate melatonin effects. Western blot and quantitative RT-PCR were used to measure DLL4 expression. Then, we investigated the effect of melatonin on the expression of DLL4 in four breast cancer cell lines at RNA and protein levels. We also performed a probabilistic neural network analysis to study genes closely associated with DLL4 expression. Our results showed a significantly higher expression of DLL4 in tumor tissues compared to non-tumor tissues (P = 0.027). Melatonin treatment substantially attenuated DLL4 expression in BT474 and MCF-7 cells, but not in SK-BR-3 and MDA-MB-231 cells. Also, melatonin induced apoptosis in all four cell lines. Network analysis revealed a set of 15 genes that had close association and interaction with DLL4. DLL4 was overexpressed in breast cancer tissues as compared to the non-tumor tissues. It can be concluded that melatonin treatment attenuated DLL4 expression only in estrogen- responsive breast cancer cells and is able to induce apoptosis in breast cancer cells.

Pattern of expression of Delta-like ligand 4 (DLL-4) in patients with pancreatic ductal and ampullary adenocarcinomas and its clinicopathological prognostic implications

There is an increasing interest for Notch signalling pathway and particularly Delta-like ligand 4 (DLL4), a Notch ligand as potential therapeutic target to improve outcome for patients with pancreatic ductal adenocarcinoma (PDAC). : characterize the expression of DLL4 in PDAC and ampullary adenocarcinoma (AA), evaluate their correlation with clinicopathologic features and patients’ survival. In a retrospective study, using immunohistochemistry, we assessed the expression of DLL4 in 62 cases composed of 39 cases of PDAC and 23 cases of AA undergone Whipple and received adjuvant chemotherapy and radiotherapy. We assessed the expression level of DLL4 both in tumor cells and stromal vascular endothelial cells. The relationships of DLL4 expression with clinico-pathologic parameters and clinical outcome were evaluated. There was no statistically significant relation between clinico-pathological parameters and DLL4 score expression in tumor cells of PDAC cases. However, there was statistically significant relation between DLL4 score expression in tumor cells of AA cases and tumor stage (p= 0.041). Also, there was no statistical significance regarding DLL4 expression in stromal cells in PDAC and AA cases and clinico-pathological parameters. Regarding survival functions for pancreatic &amp; ampullary tumor cases; the median overall survival (OS) was 10 and 22 months for pancreatic (95% CI: 1-45) and ampullary tumors (95% CI: 1-69) respectively. OS for pancreatic and ampullary tumors was higher in cases with low DLL4 expression versus cases with high expressions with no statistically significance (P=0.48 &amp; 0.09 respectively). The median Progression free survival (PFS) was 7 and 17.5 months for pancreatic (95% CI: 0-43) and ampullary tumors (95% CI: 0-96) respectively. PFS was higher in cases with low DLL4 expression rather than cases with high expressions with no statistically significant differences (P=0.52 and 0.19 respectively). High DLL4 expression in cancer cells was associated with worse OS and DFS than low DLL4 expression.

Inhibition of Delta-like Ligand 4 enhances the radiosensitivity and inhibits migration in cervical cancer via the reversion of epithelial\u2013mesenchymal transition

BackgroundConcurrent chemoradiotherapy is the common first-line treatment for patients with advanced cervical cancer. However, radioresistance remains a major clinical challenge, which results in recurrence and poor survival. Many studies have shown the potential of Delta-like Ligand 4 (DLL4) as a novel prognostic biomarker and therapeutic target in many solid tumors. Previously, we have found that high DLL4 expression in tumor cells may predict the pelvic lymph node metastasis and poor prognosis in patients with cervical cancer. In our present study, we further studied the effects of DLL4 on the biological behavior and radiosensitivity of cervical cancer cells.MethodsThe expression of DLL4 and epithelial–mesenchymal transition (EMT) phenotype markers in cervical cancer cell lines or tissues were detected using Western blotting, and the expression of DLL4 mRNA in cervical cancer cell lines or tissues was detected using Quantitative real-time PCR. The effect of DLL4 on cell proliferation, migration, and radiosensitivity was evaluated using the CCK8 assay, flow cytometry, Transwell assays for cell invasion and migration, and Immunofluorescence staining in vitro.ResultsThe expression of DLL4 in radiotherapy-resistant SiHa cells was significantly higher than that in radiotherapy-sensitive Me-180 cells. Furthermore, downregulation of DLL4 enhanced the radiosensitivity of SiHa and Caski cells via the inhibition of cell proliferation, promotion of radiation-induced apoptosis, and inhibition of the DNA damage repair. Moreover, downregulation of DLL4 inhibited the EMT and reduced the proliferation, invasion, and migration ability in SiHa and Caski cells. Consistent with the DLL4 expression in the cell lines, the expression of DLL4 in the tissues of the radioresistant group was also higher than that of the radiosensitive group.ConclusionsDownregulation of DLL4 inhibited the progression and increased the radiosensitivity in cervical cancer cells by reversing EMT. These results indicated the promising prospect of DLL4 against the radioresistance and metastasis of cervical cancer and its potential as a predictive biomarker for radiosensitivity and prognosis in patients with cervical cancer patients receiving concurrent chemoradiotherapy (cCRT).

ABT-165 plus FOLFIRI versus bevacizumab plus FOLFIRI in patients with metastatic colorectal cancer (mCRC) previously treated with fluoropyrimidine/oxaliplatin and bevacizumab.

TPS720 Background: Dual variable domain immunoglobulin ABT-165 targets human vascular endothelial growth factor (VEGF) and delta-like ligand 4 (DLL4). Combined VEGF and DLL4 blockade increased inhibition of subcutaneous xenograft growth of human colon cancer-derived cell lines versus blockade of either axis alone. In vivo, ABT-165 plus chemotherapy (CT) induced tumor regression with improved efficacy, vs anti-VEGF monoclonal antibody plus CT. In a phase 1 study, tolerable recommended phase 2 dose was identified for ABT-165 plus FOLFIRI and showed promising efficacy. This phase 2 trial in progress assesses efficacy/safety of ABT-165 plus FOLFIRI vs bevacizumab (bev) plus FOLFIRI in patients with second-line mCRC. Methods: This is an open-label, multicenter, phase 2 randomized (1:1) trial (NCT03368859) in patients (≥ 18 years; Eastern Cooperative performance status: 0–1) with histologically/cytologically confirmed mCRC who progressed after fluoropyrimidine/oxaliplatin and bev. ABT-165 (2.5 mg/kg) plus FOLFIRI (irinotecan: 180 mg/m2; leucovorin: 400 mg/m2; fluorouracil bolus: 400 mg/m2, infusion: 2400 mg/m2) or bev (5 mg/kg) plus FOLFIRI are given intravenously on day 1 of each 14-day cycle, until disease progression/intolerable toxicity. Primary endpoint is progression-free survival (PFS). Secondary endpoints include overall survival (OS), objective response rate (ORR), and safety. Exploratory endpoints include biomarkers predictive for efficacy/safety, correlation of DLL4 levels with PFS, OS, and ORR, pharmacodynamic effects, and efficacy/safety-exposure relationships in ABT-165 arm. Hazard ratios of PFS and OS comparing the 2 groups are estimated using Cox proportional hazard model. Kaplan-Meier methodology is used to estimate PFS and OS curves, median PFS and OS, and their 90% confidence intervals. Safety is assessed by ABT-165 exposure, adverse events (AEs), serious AEs, all deaths, and changes in laboratory data and vital signs. Archival tissue is collected and evaluated for DLL4 expression and angiogenesis signature. Approximately 100 patients are planned to be enrolled, with recruitment initiated January 2018. Clinical trial information: NCT03368859.

Gamma-Secretase Inhibitor, DAPT, Prevents the Development of Retinopathy of Prematurity in a Rat Model by Regulating the Delta-Like Ligand 4/Notch Homolog-1 (DLL4/Notch-1) Pathway.

BackgroundRetinopathy of prematurity (ROP), or retrolental fibroplasia, affects premature infants who have undergone intensive care with oxygen therapy. This study aimed to investigate the inhibitory effect of the gamma-secretase inhibitor, DAPT, on neovascularization and its mechanism in a rat model of ROP.Material/MethodsSixty neonatal Sprague-Dawley (SD) rats included the control group (n=20), the model group (n=20), and the DAPT-treated group (n=20). The rat model of ROP was established using repeat cycles of oxygen inhalation. Enzyme-linked immunosorbent assay (ELISA) measured serum levels of vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR-1), and VEGFR-2. Histology of the retinal tissue included immunohistochemistry for the expression of Notch homolog-1 (Notch-1) and delta-like ligand 4 (DLL4). Retinal mRNA levels of DLL4, Notch-1, VEGF, VEGFR-1, and VEGFR-2 were evaluated with quantitative real-time polymerase chain reaction (qRT-PCR).ResultsThe rat model of ROP showed increased serum levels of VEGF, VEGFR-1, and VEGFR-2 compared with the control group, which were decreased in the DAPT group. Histology of the retinal tissue in the model group showed degeneration of the retinal ganglion cells, and immunohistochemistry showed increased expression of Notch-1 and DLL4 compared with the control group and DAPT group. Retinal tissue in the model group had increased mRNA levels of DLL4, Notch-1, VEGF, VEGFR-1, and VEGFR-2 compared with the control group, and the DAPT group.ConclusionsIn a rat model, treatment with DAPT reduced the retinal changes associated with ROP with a mechanism that involved VEGF and its receptors through the DLL4/Notch-1 pathway.

High delta-like ligand 4 expression correlates with a poor clinical outcome in gastric cancer.

Background: Emerging evidence suggests that delta-like ligand 4 (DLL4) and other members of the Notch pathway may offer new targets for development of anti-angiogenesis drugs for the treatment of several tumor types. However, the role of DLL4 in gastric cancer (GC) remains unclear. In this study, we investigated the impact of DLL4 overexpression on recurrence and survival in gastric cancer (GC) patients.Methods: DLL4 expression levels were evaluated by immunohistochemistry in tissue samples from 336 GC patients. Samples were classified into high and low DLL4 expression according to a cut-off of 50% positively stained cells. The correlation between DLL4 expression and clinicopathological parameters, disease-free survival (DFS), and overall survival (OS) were statistically analyzed.Results: High DLL4 expression was observed in 67 (19.9%) of the 336 GC patients. After a median follow-up duration of 54.97 months [95% confidence interval (CI), 52.40-57.55 months), patients at stage II-IV with high DLL4 expression showed significantly poorer DFS compared with those at the same stage but with low DLL4 expression [not reached (NR) for both cohorts, hazard ratio (HR) 0.73 (95% CI, 0.38-1.40); p = 0.007]. Likewise, GC patients with high DLL4 expression had a significantly shorter OS following curative surgery compared to those with low DLL4 expression [NR for both groups, HR 0.56 (95% CI, 0.32-0.96; p = 0.002]. High DLL4 expression had a greater influence on DFS in stage IIIb/IV patients than in patients at early stages [34.87 vs. 10.1 months; HR, 0.44 (95% CI, 0.19-0.96); p = 0.004]. Moreover, stage IIIb/IV patients with high DLL4 expression had a significantly shorter OS after surgery than those with low DLL4 expression [58.87 vs. 16.93 months, HR 0.39 (95% CI, 0.16-0.99), p = 0.001).Conclusion: High DLL4 expression was observed in 19.9% of GC patients and was significantly associated with poor survival following curative surgery. Given its prevalence in the GC cohort with a poor prognosis, DLL4 is a potential therapeutic target.

Delta-like ligand 4 in hepatocellular carcinoma intrinsically promotes tumour growth and suppresses hepatitis B virus replication.

AIMTo investigate the role of Delta-like ligand 4 (DLL4) on tumour growth in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) in vivo.METHODSWe suppressed DLL4 expression in an HBV expressing HCC cell line, HepG2.2.15 and analysed the growth ability of cells as subcutaneous tumours in nude mice. The expression of tumour angiogenesis regulators, VEGF-A and VEGF-R2 in tumour xenografts were examined by western blotting. The tumour proliferation and neovasculature were examined by immunohistochemistry. The viral replication and viral protein expression were measured by quantitative PCR and western blotting, respectively.RESULTSEighteen days after implantation, tumour volume in mice implanted with shDLL4 HepG2.2.15 was significantly smaller than in mice implanted with control HepG2.2.15 (P < 0.0001). The levels of angiogenesis regulators, VEGF-A and VEGF-R2 were significantly decreased in implanted tumours with suppressed DLL4 compared with the control group (P < 0.001 and P < 0.05, respectively). Furthermore, the suppression of DLL4 expression in tumour cells reduced cell proliferation and the formation of new blood vessels in tumours. Unexpectedly, increased viral replication was observed after suppression of DLL4 in the tumours.CONCLUSIONThis study demonstrates that DLL4 is important in regulating the tumour growth of HBV-associated HCC as well as the neovascularization and suppression of HBV replication.

LDB2 regulates the expression of DLL4 through the formation of oligomeric complexes in endothelial cells.

Delta-like ligand 4 (DLL4) expression in endothelial cells is intimately associated with angiogenic sprouting and vascular remodeling, but the precise mechanism of transcriptional regulation of DLL4 remains incompletely understood. Here, we showed that LIM-domain binding protein 2 (LDB2) plays an important role in regulating basal DLL4 and VEGF-induced DLL4 expression. Knockdown of LDB2 using siRNA enhanced endothelial sprouting and tubular network formation in vitro. Injection of ldb2-morpholino resulted in defective development of intersegmental vessels in zebrafish. Reduction or overexpression of LDB2 in endothelial cells decreased or increased DLL4 expression. LDB2 regulated DLL4 promoter activity by binding to its promoter region and the same promoter region was occupied and regulated by the LMO2/TAL1/GATA2 complex. Interestingly, LDB2 also mediated VEGF-induced DLL4 expression in endothelial cells. The regulation of DLL4 by the LDB2 complex provides a novel mechanism of DLL4 transcriptional control that may be exploited to develop therapeutics for aberrant vascular remodeling.

The delta-like ligand 4-notch signaling inhibits tumor angiogenesis but promotes tumor growth in primary glioblastoma: An immunohistochemical study

Background: Delta-like ligand 4 (DLL4) is a key Notch ligand implicated in tumor angiogenesis. However, previous studies have shown that the DLL4-Notch signaling inhibits tumor angiogenesis but promotes tumor progression in primary glioblastoma. The underlying mechanism remains unknown. Methods: Tumor tissues from 70 patients with primary glioblastoma were analyzed by immunohistochemistry for the expression of DLL4, microvessel density (MVD), and Ki67 labeling index. The degree of tumor contrast enhancement (DTCE) on preoperative magnetic resonance imaging was also evaluated. The effect on prognosis was assessed based on Kaplan–Meier survival and Cox proportional hazard models. Results: Results showed that the DTCE was negatively correlated with DLL4 but positively correlated with MVD (r = −0.260, 0.593, P Conclusion: We conclude that the DLL4-Notch signaling improves tumor vascular function and contributes to the survival of malignant cells, resulting in less MVD but more tumor progression in primary glioblastoma.

MicroRNA-30 modulates metabolic inflammation by regulating Notch signaling in adipose tissue macrophages.

Abstract Macrophages are innate immune cells that play integral roles in maintenance of adipose tissue homeostasis. Visceral obesity stimulates pro-inflammatory macrophages to infiltrate into adipose tissue causing chronic low-grade inflammation that can lead to insulin resistance and cardiometabolic disorders. Notch signaling is elevated in obesity and serves as a form of communication between macrophages and adipocytes. Blockade of this pathway promotes adipose tissue browning while reducing inflammation and therefore, has therapeutic potential. MicroRNAs (miRNA, miR) are non-coding RNAs that bind the 3′UTR of target mRNAs to repress their translation and miRNA based therapies are presently being developed for clinical purposes. In the current study, we identified differentially expressed miRNAs in adipose tissue macrophages (ATMs) between lean and obese mice by microarray. Array analysis and PCR validation revealed miRNAs -30a-5p, -30c-5p, and -30e-5p were downregulated in obese ATMs suggesting the miR-30 family plays an important role in macrophage phenotype. Pathway analysis demonstrated that miR-30 targeted Notch signaling genes including Delta-like ligand-4 (DLL4), a previously identified therapeutic target for cardiometabolic disorders. It was noted that DLL4 expression was increased on obese ATMs and in vitro miRNA transfection studies further demonstrated that miR-30 modulates Notch signaling-induced inflammation. These data demonstrate for the first time that the miR-30 family may play a critical role in the regulation of DLL4-mediated Notch signaling in ATMs, thereby modulating metabolic inflammation. (Supported in part by NIH grants P01AT003961, R01AT006888, R01ES019313, R01MH094755 and P20GM103641).

DLL4 expression is a prognostic marker and may predict gemcitabine benefit in resected pancreatic cancer.

Background:There is an increasing interest for Notch signalling pathway and particularly Delta-like ligand 4 (DLL4) as potential therapeutic target to improve outcome for patients with pancreatic ductal adenocarcinoma (PDAC).Methods:Using immunohistochemistry (IHC) and tissue microarray (TMA), we assessed the expression patterns of DLL4, Notch1 and Notch3 in 151 patients from two independent cohorts of resected PDAC. We investigated the prognostic and the predictive significance of these proteins.Results:High IHC DLL4 expression in cancer cells was associated with worse overall survival (OS) and disease-free survival (DFS) than low DLL4 expression (median OS: 12.9 vs 30.4 months, P=0.004 and median DFS: 8.8 vs 17.4 months, P=0.02). High DLL4 expression remained a significant negative prognostic factor in multivariate analysis (HR for OS: 2.1, P=0.02 and HR for DFS: 2.0, P=0.02). Low DLL4 abundance was associated with a longer OS–only for patients who received an adjuvant gemcitabine-based chemotherapy (P<0.001) but not for patients who did not receive gemcitabine (P=0.72). Furthermore, the interaction test for adjuvant gemcitabine therapy was statistically significant (P<0.001). The validating cohort recapitulated the findings of the training cohort.Conclusions:Low DLL4 abundance in tumour cells may predict the benefit from adjuvant gemcitabine therapy after PDAC resection.

Altered ratios of pro- and anti-angiogenic VEGF-A variants and pericyte expression of DLL4 disrupt vascular maturation in infantile haemangioma.

Infantile haemangioma (IH), the most common neoplasm in infants, is a slowly resolving vascular tumour. Vascular endothelial growth factor A (VEGF‐A), which consists of both the pro‐ and anti‐angiogenic variants, contributes to the pathogenesis of IH. However, the roles of different VEGF‐A variants in IH progression and its spontaneous involution is unknown. Using patient‐derived cells and surgical specimens, we showed that the relative level of VEGF‐A165b was increased in the involuting phase of IH and the relative change in VEGF‐A isoforms may be dependent on endothelial differentiation of IH stem cells. VEGFR signalling regulated IH cell functions and VEGF‐A165b inhibited cell proliferation and the angiogenic potential of IH endothelial cells in vitro and in vivo. The inhibition of angiogenesis by VEGF‐A165b was associated with the extent of VEGF receptor 2 (VEGFR2) activation and degradation and Delta‐like ligand 4 (DLL4) expression. These results indicate that VEGF‐A variants can be regulated by cell differentiation and are involved in IH progression. We also demonstrated that DLL4 expression was not exclusive to the endothelium in IH but was also present in pericytes, where the expression of VEGFR2 is absent, suggesting that pericyte‐derived DLL4 may prevent sprouting during involution, independently of VEGFR2. Angiogenesis in IH therefore appears to be controlled by DLL4 within the endothelium in a VEGF‐A isoform‐dependent manner, and in perivascular cells in a VEGF‐independent manner. The contribution of VEGF‐A isoforms to disease progression also indicates that IH may be associated with altered splicing. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

DLL4 as a predictor of pelvic lymph node metastasis and a novel prognostic biomarker in patients with early-stage cervical cancer.

Delta-like ligand 4 (DLL4), one of the five Notch signaling ligands in mammals, has an important function in proliferation, invasion, metastasis, progression, and angiogenesis of malignancies. This study aimed to investigate DLL4 expression level in early-stage cervical carcinoma and to evaluate its clinical significance. We used fresh frozen and paraffin-embedded cervical cancer tissues to analyze DLL4 expression and its clinical significance. DLL4 expression at both mRNA and protein levels in cervical cancer tissues was significantly higher than that in normal cervical tissues. High DLL4 protein level was clearly correlated with high International Federation of Gynecology and Obstetrics (FIGO) stage (P = 0.044), lymphovascular space involvement (LVSI) (P = 0.015), pelvic lymph node metastasis (PLNM) (P = 0.001), and recurrence (P < 0.001). Univariate and multivariate logistic regression analyses demonstrated that DLL4 overexpression was strongly associated with lymph node metastasis (odds ratio, 2.790; 95% CI, 1.344-5.791; P = 0.006). Moreover, survival analysis revealed that DLL4 expression was an independent factor of unfavorable overall survival (hazard ratio, 2.130; 95% CI, 1.108-4.097; P = 0.023) and disease-free survival (hazard ratio, 1.965; 95% CI, 1.085-3.560; P = 0.026) in patients with cervical cancer. Overall, our data indicate that high DLL4 expression predicts pelvic lymph node metastasis and poor survival in cervical cancer. Therefore, DLL4 may be a potential clinical diagnostic marker for patients with early-stage cervical cancer.