Research IndicatorsGraph generated 11 March 2017 using data from PubMed using criteria.
Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic. Tag cloud generated 11 March, 2017 using data from PubMed, MeSH and CancerIndex
Specific Cancers (6)
Data table showing topics related to specific cancers and associated disorders. Scope includes mutations and abnormal protein expression.
Note: list is not exhaustive. Number of papers are based on searches of PubMed (click on topic title for arbitrary criteria used).
OMIM, Johns Hopkin University
Referenced article focusing on the relationship between phenotype and genotype.
International Cancer Genome Consortium.
Summary of gene and mutations by cancer type from ICGC
Cancer Genome Anatomy Project, NCI
COSMIC, Sanger Institute
Somatic mutation information and related details
GEO Profiles, NCBI
Search the gene expression profiles from curated DataSets in the Gene Expression Omnibus (GEO) repository.
Latest Publications: DLL4 (cancer-related)
Pierscianek D, Wolf S, Keyvani K, et al.Study of angiogenic signaling pathways in hemangioblastoma.
Neuropathology. 2017; 37(1):3-11 [PubMed
] Related Publications
Hemangioblastoma (HB) is mainly located in the brain and the spinal cord. The tumor is composed of two major components, namely neoplastic stromal cells and abundant microvessels. Thus, hyper-vascularization is the hallmark of this tumor. Despite the identification of germline and/or epigenetic mutations of Von Hippel Lindau (VHL) gene as an important pathogenic mechanism of HB, little is known about the molecular signaling involved in this highly vascularized tumor. The present study investigated the key players of multiple angiogenic signaling pathways including VEGF/VEGFR2, EphB4/EphrinB2, SDF1α/CXCR4 and Notch/Dll4 pathways in surgical specimens of 22 HB. The expression of key angiogenic factors was detected by RT(2) -PCR and Western blot. Immunofluorescent staining revealed the cellular localization of these proteins. We demonstrated a massive upregulation of mRNA levels of VEGF and VEGFR2, CXCR4 and SDF1α, EphB4 and EphrinB2, as well as the main components of Dll4-Notch signaling in HB. An increase in the protein expression of VEGF, CXCR4 and the core-components of Dll4-Notch signaling was associated with an activation of Akt and Erk1/2 and accompanied by an elevated expression of PCNA. Immuofluorescent staining revealed the expression of VEGF and CXCR4 in endothelial cells as well as in tumor cells. Dll4 protein was predominantly found in tumor cells, whereas EphB4 immunoreactivity was exclusively detected in endothelial cells. We conclude that multiple key angiogenic pathways were activated in HB, which may synergistically contribute to the abundant vascularization in this tumor. Identification of these aberrant pathways provides potential targets for a possible future application of anti-angiogenic therapy for this tumor, particularly when a total surgical resection becomes difficult due to the localization or multiplicity of the tumor.
Yang S, Liu Y, Xia B, et al.DLL4 as a predictor of pelvic lymph node metastasis and a novel prognostic biomarker in patients with early-stage cervical cancer.
Tumour Biol. 2016; 37(4):5063-74 [PubMed
] Related Publications
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.
Zhang JF, Chen Y, Qiu XX, et al.The vascular delta-like ligand-4 (DLL4)-Notch4 signaling correlates with angiogenesis in primary glioblastoma: an immunohistochemical study.
Tumour Biol. 2016; 37(3):3797-805 [PubMed
] Related Publications
Delta-like ligand-4 (DLL4)-Notch signaling is known to play a pivotal role in the regulation of tumor angiogenesis. We had previously found that DLL4 was overexpressed, while Notch1 receptor, which binds to DLL4 during angiogenesis, was absent in the majority of human primary glioblastomas. Thus, DLL4-Notch signaling pathway in the regulation of tumor angiogenesis in primary glioblastoma remains unknown. Tumor tissues from 70 patients with primary glioblastoma were analyzed by immunohistochemistry for expression of components of DLL4-Notch signaling, vascular endothelial growth factor (VEGF), and microvessel density (MVD). Immunohistochemistry results showed that the positive staining of DLL4 and Notch4 was primarily distributed in tumor vascular endothelial cells but rarely detected in tumor cells. However, VEGF, hairy/enhancer of split-1 (HES1; a target gene of Notch signaling), and Notch1-3 expression was seen in both tumor vascular endothelial cells and tumor cells. Univariate analysis showed that the expression levels of VEGF and DLL4, HES1, and Notch4 in tumor endothelial cells were significantly associated with MVD in primary glioblastoma (P < 0.001). Binary logistic regression analysis showed that high expression levels of DLL4, HES1, and Notch4 in tumor endothelial cells were associated with a decrease of MVD in primary glioblastoma, while MVD increased with elevated VEGF expression in contrast. In addition, DLL4, Notch4, and HES1 expression were positively correlated in tumor vascular endothelial cells (P < 0.05). We conclude that the vascular DLL4-Notch4 signaling and VEGF signaling complementing each other plays an important role in the progression of tumor angiogenesis in primary glioblastoma. Graphical abstract A, positive staining of DLL4 in human kidney; B, positive staining of VEGF in human breast cancer; C, positive staining of CD34 in human lung cancer; D, positive staining of HES1 in human breast cancer; E-H, positive staining of Notch1-4: E-F in human lung cancer; G-H in human kidney.
BACKGROUND: The development of targeted therapies has undoubtedly broadened therapeutic options for patients with colorectal cancer (CRC). The use of bevacizumab to reduce angiogenesis has been associated with improved clinical outcomes. However, an urgent need for prognostic/predictive biomarkers for anti-angiogenic therapies still exists.
METHODS: Clinical data of 105 CRC patients treated with bevacizumab in conjunction with chemotherapy were analyzed. The expression of vascular endothelial growth factor (VEGF) receptors, NOTCH1 receptor and its ligand DLL4 were determined by immunohistochemistry. Tumor samples were arranged on a tissue microarray. The association between protein expression and clinicopathological characteristics and outcomes was determined.
RESULTS: Bevacizumab was administered as a first-line of treatment in 70.5 % of our cases. The median progression-free survival (PFS) was 10.2 months. The median overall survival (OS) of the total cohort was 24.4 months. Bevacizumab, as the first-line of treatment, and the presence of liver metastasis were independently associated with objective response rate. Membrane VEGFR1 and VEGFR3 expressions were associated with the presence of lung metastasis; interestingly, VEGFR3 was associated with less liver metastasis. NOTCH1 expression was associated with lymph node metastasis. There was a trend toward association between improved PFS and lower NOTCH1 expression (p = 0.06). Improved OS was significantly associated with lower NOTCH1 expression (p = 0.01). In a multivariate analysis, ECOG (Eastern Cooperative Oncology Group) performance status, liver metastasis, histological grade, and NOTCH1 expression were independently associated with OS.
CONCLUSION: Our findings illustrated the expression profile of angiogenesis-related proteins and their association with clinicopathological characteristics and outcomes. NOTCH1 expression is a detrimental prognostic factor in metastatic CRC patients treated with chemotherapy plus bevacizumab.
Zhang YZ, Qin F, Han ZG, et al.Prognostic significance of DLL4 expression in papillary thyroid cancer.
Eur Rev Med Pharmacol Sci. 2015; 19(15):2901-5 [PubMed
] Related Publications
OBJECTIVE: Delta-like ligand 4 (DLL4)-Notch signaling has an important role in tumor neovascular development and angiogenesis during tumor growth. However, the clinical significance of DLL4 expression in papillary thyroid cancer remains unclear to date.
PATIENTS AND METHODS: 207 papillary thyroid cancer patients were in the present study. DLL4 expression in papillary thyroid cancer was analyzed and evaluated immunohistochemically. The correlation between DLL4 and clinicopathological factors was also evaluated.
RESULTS: DLL4 expression was showed in the cytoplasm of papillary thyroid cancer cells by immunohistochemical staining. DLL4 positivity in papillary thyroid cancer was found in 112 (54%) of the 207 papillary thyroid cancer patients. Papillary thyroid cancer DLL4 expression was significantly correlated with thyroid tumour invasion and metastasis.
CONCLUSIONS: Overexpression of DLL4 is associated with thyroid tumour invasion and metastasis and it may be an effective target of anti-DLL4 treatment in papillary thyroid cancer.
BACKGROUND: Tumor dormancy has been described as a state of hibernation. Dormancy can be switched to proliferation by different pathways, which may play a critical role in tumor recurrence. In this study, we investigated genetic variations within genes involved in tumor dormancy and their association with recurrence and outcome in patients with colorectal liver metastases (CLM) who underwent neoadjuvant bevacizumab-based chemotherapy.
PATIENTS AND METHODS: Genomic DNA was extracted from resected CLM (FFPE) from 149 patients. Single-nucleotide polymorphisms (SNPs) in 14 genes associated with dormancy were analyzed by direct Sanger DNA sequencing and evaluated for response, recurrence-free survival (RFS), overall survival (OS) and recurrence patterns.
RESULTS: NME1 rs34214448 C>A was significantly associated with RFS in univariable analysis (P = 0.039) and with intrahepatic recurrence (P = 0.014). NOTCH3 rs1044009 T>C and CD44 rs8193 C>T showed a significant difference in 3-year OS rates (P = 0.004 and P = 0.042, respectively). With respect to radiological response, CD44 rs8193 C>T variant genotypes were associated with a significantly higher response rate (P = 0.033). Recursive partitioning analyses revealed that Dll4 rs12441495 C>G, NME1 rs34214448 C>A and NOTCH3 rs1044009 T>C were the dominant SNPs predicting histological response, RFS and OS, respectively.
CONCLUSION: Our data suggest that gene variations within genes involved in tumor dormancy pathways are associated with response and outcome in patients with resected CLM. These data may lead to new and more effective treatment strategies targeting tumor dormancy.
Villa E, Critelli R, Lei B, et al.Neoangiogenesis-related genes are hallmarks of fast-growing hepatocellular carcinomas and worst survival. Results from a prospective study.
Gut. 2016; 65(5):861-9 [PubMed
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OBJECTIVE: The biological heterogeneity of hepatocellular carcinoma (HCC) makes prognosis difficult. We translate the results of a genome-wide high-throughput analysis into a tool that accurately predicts at presentation tumour growth and survival of patients with HCC.
DESIGN: Ultrasound surveillance identified HCC in 78 (training set) and 54 (validation set) consecutive patients with cirrhosis. Patients underwent two CT scans 6 weeks apart (no treatment in-between) to determine tumour volumes (V0 and V1) and calculate HCC doubling time. Baseline-paired HCC and surrounding tissue biopsies for microarray study (Agilent Whole Human Genome Oligo Microarrays) were also obtained. Predictors of survival were assessed by multivariate Cox model.
RESULTS: Calculated tumour doubling times ranged from 30 to 621 days (mean, 107±91 days; median, 83 days) and were divided into quartiles: ≤53 days (n=19), 54-82 days (n=20), 83-110 days (n=20) and ≥111 days (n=19). Median survival according to doubling time was significantly lower for the first quartile versus the others (11 vs 41 months, 42, and 47 months, respectively) (p<0.0001). A five-gene transcriptomic hepatic signature including angiopoietin-2 (ANGPT2), delta-like ligand 4 (DLL4), neuropilin (NRP)/tolloid (TLL)-like 2 (NETO2), endothelial cell-specific molecule-1 (ESM1), and nuclear receptor subfamily 4, group A, member 1 (NR4A1) was found to accurately identify rapidly growing HCCs of the first quartile (ROC AUC: 0.961; 95% CI 0.919 to 1.000; p<0.0001) and to be an independent factor for mortality (HR: 3.987; 95% CI 1.941 to 8.193, p<0.0001).
CONCLUSIONS: The hepatic five-gene signature was able to predict HCC growth in individual patient and the consequent risk of death. This implies a role of this molecular tool in the future therapeutic management of patients with HCC.
TRIAL REGISTRATION NUMBER: ClinicalTrials.gov Identifier: NCT01657695.
The formation of the vascular network requires a tightly controlled balance of pro-angiogenic and stabilizing signals. Perturbation of this balance can result in dysregulated blood vessel morphogenesis and drive pathologies including cancer. Here, we have identified a novel gene, ARHGAP18, as an endogenous negative regulator of angiogenesis, limiting pro-angiogenic signaling and promoting vascular stability. Loss of ARHGAP18 promotes EC hypersprouting during zebrafish and murine retinal vessel development and enhances tumor vascularization and growth. Endogenous ARHGAP18 acts specifically on RhoC and relocalizes to the angiogenic and destabilized EC junctions in a ROCK dependent manner, where it is important in reaffirming stable EC junctions and suppressing tip cell behavior, at least partially through regulation of tip cell genes, Dll4, Flk-1 and Flt-4. These findings highlight ARHGAP18 as a specific RhoGAP to fine tune vascular morphogenesis, limiting tip cell formation and promoting junctional integrity to stabilize the angiogenic architecture.
Minuzzo S, Agnusdei V, Pusceddu I, et al.DLL4 regulates NOTCH signaling and growth of T acute lymphoblastic leukemia cells in NOD/SCID mice.
Carcinogenesis. 2015; 36(1):115-21 [PubMed
] Related Publications
Activation of the NOTCH pathway occurs commonly in T acute lymphoblastic leukemia (T-ALL) mainly due to mutations in NOTCH1 or alterations in FBW7 and is involved in the regulation of cell proliferation and survival. Since mutations hit different domains of the receptor, they are predicted to heterogeneously perturb ligand-induced NOTCH1 activity. Moreover, T-ALL cells also co-express NOTCH3 receptors which could be triggered by different ligands. In this study, we aimed to investigate the role of DLL4 in the regulation of NOTCH signaling in T-ALL cells in the context of different types of NOTCH1 mutation or wild-type NOTCH receptor, as well as the effects of DLL4 neutralization on T-ALL engraftment in mice. We found that NOTCH signaling can be stimulated in T-ALL cells in vitro by either human or murine DLL4 with heterogeneous effects, according to NOTCH1/FBW7 mutation status, and that these effects can be blocked by antibodies neutralizing DLL4, NOTCH1 or NOTCH2/3. In vivo, DLL4 is expressed in the spleen and the bone marrow (BM) of NOD/SCID mice bearing T-ALL xenografts as well as the BM of T-ALL patients. Importantly, DLL4 blockade impaired growth of T-ALL cells in NOD/SCID mice and increased leukemia cell apoptosis. These results show that DLL4 is an important component of the tumor microenvironment which contributes to the early steps of T-ALL cell growth.
The Delta-like ligand 4 (Dll4) and Notch signaling pathway plays a key role in embryonic vascular development and tumor growth. In this study, we measured the expression of Dll4 in clear cell renal cell carcinoma (ccRCC) and explored the correlation between Dll4 and ccRCC. We used sh-Dll4 treatment in a nude mouse model to observe the effect that inhibition of the Dll4/Notch pathway had on angiogenesis and vasculogenesis. We found up-regulation of Dll4 to be closely correlated with distant metastasis and worse overall survival. Cox regression analysis showed that Dll4 might be a prognostic marker of ccRCC. Blockade of Dll4/Notch signaling inhibited tumor growth in the mouse model via anti-angiogenesis and anti-vasculogenesis effects. We concluded that Dll4 might be a novel therapeutic target for the treatment of ccRCC.
Wang H, Huang X, Zhang J, et al.The expression of VEGF and Dll4/Notch pathway molecules in ovarian cancer.
Clin Chim Acta. 2014; 436:243-8 [PubMed
] Related Publications
BACKGROUND: VEGF and Dll4/Notch pathways play important roles in tumor angiogenesis. The purpose of this study is to investigate the expression of these two pathway molecules in ovarian cancer and their possible relationships in carcinogenesis.
METHODS: Twenty-eight specimens of human ovarian carcinoma, 18 of benign ovarian and 20 of healthy ovarian tissues were subjected to immunohistochemical analysis for VEGF, VEGFR1, and VEGFR2, Dll4, Notch1, and Notch3 expression. Microvessel density (MVD) was evaluated by counting the number of CD34-stained microvessels in each pathologic specimen.
RESULTS: The expression of VEGF, VEGFR1, Dll4, Notch1, or Notch3 in ovarian tumor tissues was higher than that in normal ovary tissues as well as that in benign ovarian tumor tissues (P<0.05). In the tumor tissues, Dll4 was positively correlated with VEGFR1 expression and Notch1 was positively associated with VEGFR2 and MVD. Moreover, VEGFR2 expression was positively associated with ascites and distant metastasis (R=0.401, P=0.034).
CONCLUSIONS: Dll4 represents a potential biomarker and therapeutic target for ovarian angiogenesis. VEGFR2 is significantly related to ovarian metastasis and invasion. Therefore testing the key molecules of these two pathways expression may have some diagnostic and prognostic value for ovarian cancer.
Fatty acid-binding protein 4 (FABP4) is an adipogenic protein and is implicated in atherosclerosis, insulin resistance, and cancer. In endothelial cells, FABP4 is induced by VEGFA, and inhibition of FABP4 blocks most of the VEGFA effects. We investigated the DLL4-NOTCH-dependent regulation of FABP4 in human umbilical vein endothelial cells by gene/protein expression and interaction analyses following inhibitor treatment and RNA interference. We found that FABP4 is directly induced by NOTCH. Stimulation of NOTCH signaling with human recombinant DLL4 led to FABP4 induction, independently of VEGFA. FABP4 induction by VEGFA was reduced by blockade of DLL4 binding to NOTCH or inhibition of NOTCH signal transduction. Chromatin immunoprecipitation of the NOTCH intracellular domain showed increased binding to two specific regions in the FABP4 promoter. The induction of FABP4 gene expression was dependent on the transcription factor FOXO1, which was essential for basal expression of FABP4, and FABP4 up-regulation following stimulation of the VEGFA and/or the NOTCH pathway. Thus, we show that the DLL4-NOTCH pathway mediates endothelial FABP4 expression. This indicates that induction of the angiogenesis-restricting DLL4-NOTCH can have pro-angiogenic effects via this pathway. It also provides a link between DLL4-NOTCH and FOXO1-mediated regulation of endothelial gene transcription, and it shows that DLL4-NOTCH is a nodal point in the integration of pro-angiogenic and metabolic signaling in endothelial cells. This may be crucial for angiogenesis in the tumor environment.
Lee SH, Lee S, Yang H, et al.Notch pathway targets proangiogenic regulator Sox17 to restrict angiogenesis.
Circ Res. 2014; 115(2):215-26 [PubMed
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RATIONALE: The Notch pathway stabilizes sprouting angiogenesis by favoring stalk cells over tip cells at the vascular front. Because tip and stalk cells have different properties in morphology and function, their transcriptional regulation remains to be distinguished. Transcription factor Sox17 is specifically expressed in endothelial cells, but its expression and role at the vascular front remain largely unknown.
OBJECTIVE: To specify the role of Sox17 and its relationship with the Notch pathway in sprouting angiogenesis.
METHODS AND RESULTS: Endothelial-specific Sox17 deletion reduces sprouting angiogenesis in mouse embryonic and postnatal vascular development, whereas Sox17 overexpression increases it. Sox17 promotes endothelial migration by destabilizing endothelial junctions and rearranging cytoskeletal structure and upregulates expression of several genes preferentially expressed in tip cells. Interestingly, Sox17 expression is suppressed in stalk cells in which Notch signaling is relatively high. Notch activation by overexpressing Notch intracellular domain reduces Sox17 expression both in primary endothelial cells and in retinal angiogenesis, whereas Notch inhibition by delta-like ligand 4 (Dll4) blockade increases it. The Notch pathway regulates Sox17 expression mainly at the post-transcriptional level. Furthermore, endothelial Sox17 ablation rescues vascular network from excessive tip cell formation and hyperbranching under Notch inhibition in developmental and tumor angiogenesis.
CONCLUSIONS: Our findings demonstrate that the Notch pathway restricts sprouting angiogenesis by reducing the expression of proangiogenic regulator Sox17.
Deng J, Liu X, Rong L, et al.IFNγ-responsiveness of endothelial cells leads to efficient angiostasis in tumours involving down-regulation of Dll4.
J Pathol. 2014; 233(2):170-82 [PubMed
] Related Publications
Although IFNγ is regarded as a key cytokine in angiostatic response, our poor understanding of its effective cellular target drastically limits its clinical trials against angiogenesis-related disorders. Here, we investigated the effect of IFNγ on endothelial cells (ECs) and possible molecular mechanisms in angiostasis. By employing Tie2(IFNγR) mice, in which IFNγR expression was reconstituted under the control of Tie2 promoter in IFNγR-deficient mice, we found that the response of ECs to IFNγ was highly effective in inhibiting blood supply and retarding tumour growth. Interestingly, the expression of IFNγR on Tie2(-) cells did not inhibit, but promoted tumour growth in control wild-type mice. Mechanism studies showed that IFNγ reacting on ECs down-regulated the delta-like ligand 4 (Dll4)/Notch signalling pathway. Accordingly, overexpression of Dll4 in human ECs diminished the effect of IFNγ on ECs. This study demonstrates that the action of IFNγ on ECs, but not other cells, is highly effective for tumour angiostasis, which involves down-regulating Dll4. It provides insights for EC-targeted angiostatic therapy in treating angiogenesis-associated disorders in the clinic.
Pastò A, Serafin V, Pilotto G, et al.NOTCH3 signaling regulates MUSASHI-1 expression in metastatic colorectal cancer cells.
Cancer Res. 2014; 74(7):2106-18 [PubMed
] Related Publications
MUSASHI-1 (MSI-1) is a well-established stem cell marker in both normal and malignant colon cells and it acts by positively regulating the NOTCH pathway through inactivation of NUMB, a NOTCH signaling repressor. To date, the mechanisms of regulation of MSI-1 levels remain largely unknown. Here, we investigated the regulation of MSI-1 by NOTCH signaling in colorectal cancer cell lines and in primary cultures of colorectal cancer metastases. Stimulation by the NOTCH ligand DLL4 was associated with an increase of MSI-1 mRNA and protein levels, and this phenomenon was prevented by the addition of an antibody neutralizing NOTCH2/3 but not NOTCH1. Moreover, forced expression of activated NOTCH3 increased MSI-1 levels, whereas silencing of NOTCH3 by short hairpin RNA reduced MSI-1 levels in both colorectal cancer cells and CRC tumor xenografts. Consistent with these findings, enforced NOTCH3 expression or stimulation by DLL4 increased levels of activated NOTCH1 in colorectal cell lines. Finally, treatment of colorectal cancer cells with anti-NOTCH2/3 antibody increased NUMB protein while significantly reducing formation of tumor cell spheroids. This novel feed-forward circuit involving DLL4, NOTCH3, MSI-1, NUMB, and NOTCH1 may be relevant for regulation of NOTCH signaling in physiologic processes as well as in tumor development. With regard to therapeutic implications, NOTCH3-specific drugs could represent a valuable strategy to limit NOTCH signaling in the context of colorectal cancers overexpressing this receptor.
BACKGROUND: RE1-silencing transcription factor (REST), a neuronal repressor gene, regulates neuronal stem cell differentiation. Ewing sarcoma may originate from neural crest cells. In the current study, the authors investigated whether REST plays a role in the growth of this tumor.
METHODS: REST expression was determined by Western blot analysis and reverse transcription-polymerase chain reaction in 3 human Ewing sarcoma cell lines and 7 patient tumor samples. The role of REST in tumor growth and tumor vascular morphology was determined using a Ewing sarcoma xenograft model. Immunofluorescence staining, Hypoxyprobe, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assays were performed to investigate the impact of REST on pericyte marker expression, hypoxia, and apoptosis in vivo.
RESULTS: High levels of REST were expressed in all 3 human Ewing sarcoma cell lines and in 6 of the 7 patient tumor samples. Overexpression of EWS-FLI-1 in human mesenchymal stem cells and human neural progenitor cells was found to increase REST expression. Inhibition of EWS-FLI-1 using small interfering RNA decreased REST expression in human Ewing sarcoma cells. Inhibition of REST did not affect EWS-FLI-1, but significantly suppressed tumor growth in vivo, reduced the tumor vessel pericyte markers α- smooth muscle actin (SMA) and desmin, increased hypoxia and apoptosis in tumor tissues, and decreased the expression of delta-like ligand 4 (DLL4) and Hes1.
CONCLUSIONS: Inhibition of REST suppressed tumor growth, inhibited pericyte marker expression, and increased tumor hypoxia and apoptosis. Because tumor vessel function has been linked to tumor growth and metastases, REST may be a new therapeutic target in patients with Ewing sarcoma.
Clatot F, Gouérant S, Mareschal S, et al.The gene expression profile of inflammatory, hypoxic and metabolic genes predicts the metastatic spread of human head and neck squamous cell carcinoma.
Oral Oncol. 2014; 50(3):200-7 [PubMed
] Related Publications
OBJECTIVES: To assess the prognostic value of the expression profile of the main genes implicated in hypoxia, glucose and lactate metabolism, inflammation, angiogenesis and extracellular matrix interactions for the metastatic spread of head and neck squamous cell carcinoma.
PATIENTS AND METHODS: Using a high-throughput qRT-PCR, we performed an unsupervised clustering analysis based on the expression of 42 genes for 61 patients. Usual prognostic factors and clustering analysis results were related to metastasis free survival.
RESULTS: With a median follow-up of 48months, 19 patients died from a metastatic evolution of their head and neck squamous cell carcinoma and one from a local recurrence. The unsupervised clustering analysis distinguished two groups of genes that were related to metastatic evolution. A capsular rupture (p=0.005) and the "cluster CXCL12 low" (p=0.002) were found to be independent prognostic factors for metastasis free survival. Using a Linear Predictive Score methodology, we established a 9-gene model (VHL, PTGER4, HK1, SLC16A4, DLL4, CXCL12, CXCR4, PTGER3 and CA9) that was capable of classifying the samples into the 2 clusters with 90% accuracy.
CONCLUSION: In this cohort, our clustering analysis underlined the independent prognostic value of the expression of a panel of genes involved in hypoxia and tumor environment. It allowed us to define a 9-gene model which can be applied routinely to classify newly diagnosed head and neck squamous cell carcinoma. If confirmed by an independent prospective study, this approach may help future clinical management of these aggressive tumors.
Mezquita B, Mezquita J, Barrot C, et al.A truncated-Flt1 isoform of breast cancer cells is upregulated by Notch and downregulated by retinoic acid.
J Cell Biochem. 2014; 115(1):52-61 [PubMed
] Related Publications
We have previously reported that the major isoform of Flt1/VEGFR-1 expressed in MDA-MB-231 breast cancer cells was a truncated intracellular isoform transcribed from intron 21 (i21 Flt1). This isoform upregulated the active form of Src and increased breast cancer cell invasiveness. Since expression of the transmembrane and soluble Flt1 isoforms of HUVEC is activated by Notch signaling, we wondered whether the expression of the intracellular isoform i21 Flt1 was also dependent on Notch activation. We report here that the expression of i21 Flt1 in HUVEC and MDA-MB-231 cells is downregulated by the γ-secretase inhibitor DAPT. In addition, treatment of MDA-MB-231 cells with siRNA specific for Notch-1 and Notch-3 downregulates the expression of i21 Flt1. In agreement with these findings, HUVEC and MDA-MB-231 breast cancer cells, cultured on dishes coated with recombinant human Dll4 extracellular domain, express higher levels of i21 Flt1. In cancer cells, Flt1 is a target of the micro RNA family miR-200. In MDA-MB-231 breast cancer cells, the truncated intracellular isoform i21 Flt1 is also negatively regulated by miR-200c. Retinoic acid interferes i21 Flt1 expression by downregulating Notch-3 and upregulating miR-200 expression. Treatment of MDA-MB-231 breast cancer cells with both a γ-secretase inhibitor and retinoic acid suppresses the expression of i21 Flt1, providing a new mechanism to explain the effectiveness of this therapeutic approach.
AIM: To investigate the potential roles of Delta-like ligand 4 (DLL4) on the biological behavior of gastric cancer cells and its molecular mechanisms.
METHODS: A recombinant eukaryotic expression vector containing human DLL4 gene was constructed and transfected into the human gastric cancer cell line SGC7901. Clones with up-regulated DLL4 were selected and amplified. The effect of DLL4 up-regulation on gastric cancer cell growth was assessed using cell growth assay. The migration and invasion were assessed using a transwell migration assay and matrigel invasion assay. Matrix metalloproteinases were detected using the zymogram technique. Cells were implanted subcutaneously into male BALB/c nu/nu mice. Tumor volumes were then calculated and compared. DLL4 staining in the implanted tumor was performed using immunohistochemistry technique.
RESULTS: Growth curves over a six-day time course showed significantly promoted cell proliferation of SGC7901 cells with up-regulated DLL4. DLL4 up-regulation in SGC7901 cells promoted the migration (205.4 ± 15.2 vs 22.3 ± 12.1, P < 0.05) and invasion (68.8 ± 5.3 vs 18.2 ± 6.0, P < 0.05) in vitro and tumorigenicity in vivo (2640.5 ± 923.6 mm(3) vs 1115.1 ± 223.8 mm(3), P < 0.05). Furthermore, significantly increased mRNA level and increased secretion of matrix metalloproteinase-2 (MMP-2) proenzyme were observed in SGC7901 cells with up-regulated DLL4. However, increased MMP-9 mRNA level but decreased extracellular MMP-9 proenzyme level was observed.
CONCLUSION: Our observations indicated a mechanism by which activation of DLL4-mediated Notch signaling promotes the expression and secretion of MMP-2 proenzyme and influences the progress of gastric cancer.
BACKGROUND: Delta-like ligand 4 (DLL4)-Notch signaling plays a key role in tumor neovascular development and angiogenesis during tumor growth. The clinical significance of DLL4 expression in gastric cancer has not been clarified.
METHODS: Gastric cancer cell lines and 180 gastric cancer patients were enrolled. DLL4 expression in gastric cancer cells and stroma was identified and evaluated immunohistochemically. The association between DLL4 and clinicopathological factors was also assessed.
RESULTS: DLL4 expression was identified in the cellular membrane and cytoplasm of gastric cancer cells by immunoblotting and immunohistochemical staining. DLL4 positivity in cancer cells and stroma was found in 88 (48%) and 41 (22%) of the 180 gastric cancer patients respectively. Both cancer and stromal DLL4 expression significantly correlated with more advanced tumor depth, nodal involvement, and lymphatic and venous invasion. A strongly positive association between cancerous and stromal DLL4 expression was identified (p < 0.01). Both cancerous and stromal DLL4 expression were prognostic markers in gastric cancer as determined by univariate analysis.
CONCLUSIONS: Cancerous and stromal DLL4 expression was found in 48% and 22% in gastric cancer, and significantly affected postoperative clinical outcomes. Cancerous and stromal DLL4 expression may be an effective target of anti-DLL4 treatment in gastric cancer.
Iyer AK, Singh A, Ganta S, Amiji MMRole of integrated cancer nanomedicine in overcoming drug resistance.
Adv Drug Deliv Rev. 2013; 65(13-14):1784-802 [PubMed
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Cancer remains a major killer of mankind. Failure of conventional chemotherapy has resulted in recurrence and development of virulent multi drug resistant (MDR) phenotypes adding to the complexity and diversity of this deadly disease. Apart from displaying classical physiological abnormalities and aberrant blood flow behavior, MDR cancers exhibit several distinctive features such as higher apoptotic threshold, aerobic glycolysis, regions of hypoxia, and elevated activity of drug-efflux transporters. MDR transporters play a pivotal role in protecting the cancer stem cells (CSCs) from chemotherapy. It is speculated that CSCs are instrumental in reviving tumors after the chemo and radiotherapy. In this regard, multifunctional nanoparticles that can integrate various key components such as drugs, genes, imaging agents and targeting ligands using unique delivery platforms would be more efficient in treating MDR cancers. This review presents some of the important principles involved in development of MDR and novel methods of treating cancers using multifunctional-targeted nanoparticles. Illustrative examples of nanoparticles engineered for drug/gene combination delivery and stimuli responsive nanoparticle systems for cancer therapy are also discussed.
Bazan-Peregrino M, Sainson RC, Carlisle RC, et al.Combining virotherapy and angiotherapy for the treatment of breast cancer.
Cancer Gene Ther. 2013; 20(8):461-8 [PubMed
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A breast cancer-selective oncolytic adenovirus was engineered to express antagonists of vascular endothelial growth factor (VEGF) and Notch signaling to combine direct anticancer activity with disruption of tumor-associated angiogenesis. Replication of the parental virus, AdEHE2F, is stimulated by estrogen receptor (ER), E2F1 and hypoxia, and it mediates selective lysis of breast cancer cells in vitro and in vivo. Here, we encoded soluble Flt-1 (sFlt1) and soluble Dll4 (sDll4) under control of the E3 promoter. sFlt1 (the extra-cellular domain of VEGF receptor 1) binds VEGF-A and inhibits stimulation of VEGFR2, decreasing angiogenic stimulus. Conversely, sDll4 (the extracellular domain of Delta-like 4) antagonizes Notch signaling to prevent endothelial maturation. We hypothesized that these agents might show additive or synergistic activity. In vitro, sFlt1 inhibited endothelial cell proliferation and sprouting, whereas sDll4 increased the number of vascular branchpoints. In ER-positive ZR75.1 tumors in vivo AdEHE2F showed the potent direct virotherapy with no augmentation owing to sFlt1 or sDll4; however, in ER-negative MDA-231 tumors efficacy was enhanced by encoding sFlt1 or sDll4, with survival time extending to double that of controls. There was also a dramatic decrease in the total number of tumour blood vessels, as well as the number of perfused vessels, suggesting that improved efficacy reflects combined anti-tumour and anti-vascular effects.
BACKGROUND: Glioblastoma multiforme (GBM) is a highly aggressive brain tumor characterized by massive neovascularization, necrosis, and intense resistance to therapy. Deregulated Notch signaling has been implicated in the formation and progression of different malignancies. The present study attempted to investigate the activation status of Dll4-Notch signaling in primary human GBM and its association with vascular and clinical parameters in patients.
METHODS: Major components of Dll4-Notch signaling were examined by real-time reverse-transcription polymerase chain reaction (PCR), Western blotting, and immunohistochemistry in GBM (n = 26) and control (n = 11) brain tissue. The vascular pattern (VP) and microvascular density (MVD) were analyzed after laminin immunostaining. O6-Methylguanine-methyltransferase (MGMT) promoter methylation in GBM samples was detected by methylation-specific PCR.
RESULTS: The mRNA levels of Dll4, Jagged1, Notch1, Notch4, Hey1, Hey2, Hes1, and VEGF were 3.12-, 3.58-, 3.37-, 5.77-, 4.89-, 3.13-, 6.62-, and 32.57-fold elevated, respectively, in GBM samples, compared with the controls. Western blotting revealed a 4-, 3.7-, and 45.6-fold upregulation of Dll4, Notch1, and Hey1, respectively, accompanied by a downregulation of PTEN expression and an increase in the expression of p-Akt and VEGF. Immunostaining located the immunoreactivity of Dll4 and Notch1 in endothelial cells, microglia/macrophages, tumor cells, and astrocytes. Furthermore, the upregulation of Dll4-Notch signaling components was correlated to a low MVD and was potentially related to a classic VP, tumor edema, and MGMT promoter methylation.
CONCLUSIONS: The upregulation of Dll4-Notch signaling components was found in a subset of GBM samples and was associated with some angiogenic and clinical parameters. These findings highlight this signaling pathway as a potential therapeutic target for patients with GBM who show an activation of Dll4-Notch signaling.
You C, Sandalcioglu IE, Dammann P, et al.Loss of CCM3 impairs DLL4-Notch signalling: implication in endothelial angiogenesis and in inherited cerebral cavernous malformations.
J Cell Mol Med. 2013; 17(3):407-18 [PubMed
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CCM3, a product of the cerebral cavernous malformation 3 or programmed cell death 10 gene (CCM3/PDCD10), is broadly expressed throughout development in both vertebrates and invertebrates. Increasing evidence indicates a crucial role of CCM3 in vascular development and in regulation of angiogenesis and apoptosis. Furthermore, loss of CCM3 causes inherited (familial) cerebral cavernous malformation (CCM), a common brain vascular anomaly involving aberrant angiogenesis. This study focused on signalling pathways underlying the angiogenic functions of CCM3. Silencing CCM3 by siRNA stimulated endothelial proliferation, migration and sprouting accompanied by significant downregulation of the core components of Notch signalling including DLL4, Notch4, HEY2 and HES1 and by activation of VEGF and Erk pathways. Treatment with recombinant DLL4 (rhDLL4) restored DLL4 expression and reversed CCM3-silence-mediated impairment of Notch signalling and reduced the ratio of VEGF-R2 to VEGF-R1 expression. Importantly, restoration of DLL4-Notch signalling entirely rescued the hyper-angiogenic phenotype induced by CCM3 silence. A concomitant loss of CCM3 and the core components of DLL4-Notch signalling were also demonstrated in CCM3-deficient endothelial cells derived from human CCM lesions (CCMEC) and in a CCM3 germline mutation carrier. This study defined DLL4 as a key downstream target of CCM3 in endothelial cells. CCM3/DLL4-Notch pathway serves as an important signalling for endothelial angiogenesis and is potentially implicated in the pathomechanism of human CCMs.
Yu S, Sun J, Zhang J, et al.Aberrant expression and association of VEGF and Dll4/Notch pathway molecules under hypoxia in patients with lung cancer.
Histol Histopathol. 2013; 28(2):277-84 [PubMed
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Tumor angiogenesis plays important roles in the pathogenesis and prognosis of lung cancer. Both vascular endothelial growth factor (VEGF) and Dll4/Notch pathways are critical for angiogenesis, whereas their relationship under hypoxia in lung cancer remains unknown. Thus, in the present study, we evaluated the expression of VEGF and Dll4/Notch signaling molecules, and assessed their association with the microvessel density (CD31) and hypoxia (HIF1a) in lung cancer and normal lung tissues using immunohistochemical and Real-time RT-PCR techniques. Then, we investigated the biological function of Dll4 by transfecting Dll4 into HUVECs. In lung cancer tissues, Notch pathway molecules (HES1) and VEGF pathway molecules (VEGFR1 and VEGFR2) were significantly up-regulated, while the ratio of VEGFR1/VEGFR2 was decreased. CD31 and HIF1a were also found to be elevated in lung cancer. VEGFR1 was negatively correlated with Notch1 while positively correlated with Dll4. CD31 was positively correlated with HIF1a but negatively correlated with VEGFR1. Moreover, HIF1a was nearly positively correlated with HES1 in lung cancer tissues. After transfection, Dll4, Notch1 and VEGFR1 were up-regulated while VEGF and VEGFR2 were down-regulated in Dll4-transfected HUVECs compared with controls. Also, our findings suggest that the expression of VEGF and VEGFR2 increased gradually with the disease progression of lung cancer. In summary, VEGF and Notch signaling pathway molecules were overexpressed in lung cancer, which positively correlates with hypoxia (HIF1a) and angiogenesis (CD31). There might be a negative feedback loop between VEGF and Dll4/Notch signaling pathway in lung tumor angiogenesis.
Zhang J, Ye J, Ma D, et al.Cross-talk between leukemic and endothelial cells promotes angiogenesis by VEGF activation of the Notch/Dll4 pathway.
Carcinogenesis. 2013; 34(3):667-77 [PubMed
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Angiogenesis is suggested to be important for leukemogenesis and chemosensitivity in acute myeloid leukemia (AML). The vascular endothelial growth factor (VEGF) and Notch/Dll4 pathways have been identified as critical in the regulation of embryonic vascular development and tumor angiogenesis. However, the potential role of the Notch/Dll4 pathway in leukemia-endothelium cross-talk and its functional link with VEGF remains obscure. This study assessed the expression of VEGF and Notch/Dll4 pathway molecules in primary AML and investigated their biological function in the coculture of endothelial cells with AML cells. The results demonstrated that bone marrow vascularity in the newly diagnosed AML patients was increased and correlated with high VEGF and Dll4 expression. Patients with untreated AML expressed higher levels of VEGFR2, Notch1, Dll4 and Hes1 than healthy controls. Moreover, the activation of the Notch/Dll4 pathway is associated with poor prognosis in AML. In addition, AML cells were shown to increase endothelial cell proliferation in Transwell coculture. This was associated with concomitant activation of the Notch/Dll4 pathway and upregulation of its downstream genes, such as matrix metalloproteinases, resulting in the enhancement of endothelial cell migration and tube formation. Our study also showed that upregulation of Dll4 expression in AML cells by cDNA transfection suppressed VEGF-induced endothelial cell proliferation and angiogenesis in direct contact coculture. These results elucidate a novel mechanism by which the interplay between AML and endothelial cells promotes angiogenesis through the Notch/Dll4 pathway. Modulation of this pathway may, therefore, hold promise as a novel antiangiogenic strategy for the treatment of AML.
BACKGROUND: Sirtuin 1 (SIRT1) acts as a key regulator of vascular endothelial homeostasis, angiogenesis, and endothelial dysfunction. However, the underlying mechanism for SIRT1-mediated lung carcinoma angiogenesis remains unknown. Herein, we report that the nicotinamide adenine dinucleotide 1 (NAD1)-dependent deacetylase SIRT1 can function as an intrinsic negative modulator of Delta-like ligand 4 (DLL4)/Notch signaling in Lewis lung carcinoma (LLC) xenograft-derived vascular endothelial cells (lung cancer-derived ECs).
PRINCIPAL FINDINGS: SIRT1 negatively regulates Notch1 intracellular domain (N1IC) and Notch1 target genes HEY1 and HEY2 in response to Delta-like ligand 4 (DLL4) stimulation. Furthermore, SIRT1 deacetylated and repressed N1IC expression. Quantitative chromatin immunoprecipitation (qChIP) analysis and gene reporter assay demonstrated that SIRT1 bound to one highly conserved region, which was located at approximately -500 bp upstream of the transcriptional start site of Notch1,and repressed Notch1 transcription. Inhibition of endothelial cell growth and sprouting angiogenesis by DLL4/Notch signaling was enhanced in SIRT1-silenced lung cancer-derived EC and rescued by Notch inhibitor DAPT. In vivo, an increase in proangiogenic activity was observed in Matrigel plugs from endothelial-specific SIRT1 knock-in mice. SIRT1 also enhanced tumor neovascularization and tumor growth of LLC xenografts.
CONCLUSIONS: Our results show that SIRT1 facilitates endothelial cell branching and proliferation to increase vessel density and promote lung tumor growth through down-regulation of DLL4/Notch signaling and deacetylation of N1IC. Thus, targeting SIRT1 activity or/and gene expression may represent a novel mechanism in the treatment of lung cancer.
Kuramoto T, Goto H, Mitsuhashi A, et al.Dll4-Fc, an inhibitor of Dll4-notch signaling, suppresses liver metastasis of small cell lung cancer cells through the downregulation of the NF-κB activity.
Mol Cancer Ther. 2012; 11(12):2578-87 [PubMed
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Notch signaling regulates cell-fate decisions during development and postnatal life. Little is known, however, about the role of Delta-like-4 (Dll4)-Notch signaling between cancer cells, or how this signaling affects cancer metastasis. We, therefore, assessed the role of Dll4-Notch signaling in cancer metastasis. We generated a soluble Dll4 fused to the IgG1 constant region (Dll4-Fc) that acts as a blocker of Dll4-Notch signaling and introduced it into human small cell lung cancer (SCLC) cell lines expressing either high levels (SBC-3 and H1048) or low levels (SBC-5) of Dll4. The effects of Dll4-Fc on metastasis of SCLC were evaluated using a mouse model. Although Dll4-Fc had no effect on the liver metastasis of SBC-5, the number of liver metastasis inoculated with SBC-3 and H1048 cells expressing Dll4-Fc was significantly lower than that injected with control cells. To study the molecular mechanisms of the effects of Dll4-Fc on liver metastasis, a PCR array analysis was conducted. Because the expression of NF-κB target genes was affected by Dll4-Fc, we conducted an electrophoretic mobility shift assay and observed that NF-κB activities, both with and without stimulation by TNF-α, were downregulated in Dll4-Fc-overexpressing SBC-3 and H1048 cells compared with control cells. Moreover, Dll4-Fc attenuates, at least in part, the classical and alternative NF-κB activation pathway by reducing Notch1 signaling. These results suggest that Dll4-Notch signaling in cancer cells plays a critical role in liver metastasis of SCLC by regulating NF-κB signaling.
Yen WC, Fischer MM, Hynes M, et al.Anti-DLL4 has broad spectrum activity in pancreatic cancer dependent on targeting DLL4-Notch signaling in both tumor and vasculature cells.
Clin Cancer Res. 2012; 18(19):5374-86 [PubMed
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PURPOSE: We previously showed that targeting Delta-like ligand 4 (DLL4) in colon and breast tumors inhibited tumor growth and reduced tumor initiating cell frequency. In this report, we have extended these studies to pancreatic cancer and probed the mechanism of action in tumor and stromal cells involved in antitumor efficacy.
EXPERIMENTAL DESIGN: Patient-derived pancreatic xenograft tumor models were used to evaluate the antitumor effect of anti-DLL4. To investigate the mechanism of action, we compared the activity of targeting DLL4 in tumor cells with an anti-human DLL4 antibody (anti-hDLL4) and in the host stroma/vasculature with an anti-mouse DLL4 antibody (anti-mDLL4). The effect of these antibodies on cancer stem cell frequency was examined by in vivo limiting dilution assays.
RESULTS: The combination of anti-hDLL4 and anti-mDLL4 was efficacious in a broad spectrum of pancreatic tumor xenografts and showed additive antitumor activity together with gemcitabine. Treatment with either anti-hDLL4 or anti-mDLL4 delayed pancreatic tumor recurrence following termination of gemcitabine treatment, and the two together produced an additive effect. Anti-hDLL4 had a pronounced effect in reducing the tumorigenicity of pancreatic cancer cells based on serial transplantation and tumorsphere assays. In contrast, disruption of tumor angiogenesis with anti-mDLL4 alone or with anti-VEGF had minimal effects on tumorigenicity. Gene expression analyses indicated that anti-DLL4 treatment regulated genes that participate in Notch signaling, pancreatic differentiation, and epithelial-to-mesenchymal transition.
CONCLUSIONS: Our findings suggest a novel therapeutic approach for pancreatic cancer treatment through antagonism of DLL4/Notch signaling.
Duncan WC, Nio-Kobayashi JTargeting angiogenesis in the pathological ovary.
Reprod Fertil Dev. 2013; 25(2):362-71 [PubMed
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The ovary is a key tissue in the study of physiological neo-vascularisation in the adult and its study has highlighted important molecules involved in the regulation of angiogenesis in vivo. These include vascular endothelial growth factor, delta-like ligand 4, thrombospondin-1, prokineticin-1 and prostaglandin E2. Targeting these molecular pathways has therapeutic potential and their manipulation has an increasing preclinical and clinical role in the management of the pathological ovary. Targeting angiogenic pathways has utility in the promotion of ovarian angiogenesis to improve tissue and follicle survival and function as well as the prevention and management of ovarian hyperstimulation syndrome. There is a theoretical possibility that targeting angiogenesis may improve the function of the polycystic ovary and a real role for targeting angiogenesis in ovarian cancer.