ANGPT2

Gene Summary

Gene:ANGPT2; angiopoietin 2
Aliases: ANG2, AGPT2
Location:8p23.1
Summary:The protein encoded by this gene is an antagonist of angiopoietin 1 (ANGPT1) and endothelial TEK tyrosine kinase (TIE-2, TEK). The encoded protein disrupts the vascular remodeling ability of ANGPT1 and may induce endothelial cell apoptosis. Three transcript variants encoding three different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
Databases:VEGA, OMIM, HGNC, Ensembl, GeneCard, Gene
Protein:angiopoietin-2
Source:NCBIAccessed: 15 March, 2017

Ontology:

What does this gene/protein do?
Show (29)

Cancer Overview

Research Indicators

Publications Per Year (1992-2017)
Graph generated 15 March 2017 using data from PubMed using criteria.

Literature Analysis

Mouse over the terms for more detail; many indicate links which you can click for dedicated pages about the topic.

  • Gene Expression
  • Case-Control Studies
  • Messenger RNA
  • Stomach Cancer
  • Endothelial Cells
  • Membrane Glycoproteins
  • Transcription
  • Chromosome 8
  • Angiopoietin-2
  • Proto-Oncogene Proteins
  • Disease Progression
  • Angiopoietins
  • Neuroblastoma
  • Cancer RNA
  • Receptor Protein-Tyrosine Kinases
  • Receptor, TIE-2
  • Immunohistochemistry
  • Up-Regulation
  • Angiogenesis
  • RNA Interference
  • Thrombocythemia, Essential
  • Cell Proliferation
  • Down-Regulation
  • Adenocarcinoma
  • Lymphokines
  • Hepatocellular Carcinoma
  • Liver Cancer
  • Trans-Activators
  • Young Adult
  • Biomarkers, Tumor
  • Angiogenesis Inhibitors
  • Angiopoietin-1
  • Cancer Gene Expression Regulation
  • Breast Cancer
  • Proteins
  • Gene Expression Profiling
  • VEGFA
  • Endothelial Growth Factors
  • Intercellular Signaling Peptides and Proteins
  • Oligonucleotide Array Sequence Analysis
  • RTPCR
Tag cloud generated 15 March, 2017 using data from PubMed, MeSH and CancerIndex

Specific Cancers (4)

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).

Latest Publications: ANGPT2 (cancer-related)

Karakurt N, Aksu T, Koksal Y, et al.
Angiopoietins in the bone marrow microenvironment of acute lymphoblastic leukemia.
Hematology. 2016; 21(6):325-31 [PubMed] Related Publications
OBJECTIVE: Angiogenesis have implications in leukemia biology. Angiopoietin 1 (Ang 1) is an angiogenic cytokine which is essential in survival and proliferation of endothelial cells. Angiopoietin 2 (Ang 2) promotes dissociation of pericytes and increases vascular permeability and stromal derived factor 1 alpha (SDF 1α) which is a key player in stem cell traffic in the bone marrow (BM), has stimulating effects on angiogenesis as well. Here, we investigated the role of the leukemic BM microenvironment and specifically, the role of SDF 1α-CXCR4 and Ang 1/Ang 2-Tie 2 axes.
METHODS: Here, Ang 1, Ang 2, and SDF 1α levels were measured in the BM plasma and in supernatants of mesenchymal stem/stromal cells (MSCs) of patients with ALL and compared with those of healthy controls.
RESULTS: The results showed that at diagnosis, BM plasma levels of Ang 1 and SDF 1α were significantly low and Ang 2 was high when compared to control values. Remission induction was associated with an increase in Ang 1/Ang 2 ratio and SDF levels in BM plasma.
DISCUSSION: The results suggest that BM microenvironment and leukemic cell-stroma interaction influences the secretion of Ang 1, 2 and SDF 1α, thus, may affect both angiogenesis, homing and mobilization of leukemic blasts.

Seifert M, Garbe M, Friedrich B, et al.
Comparative transcriptomics reveals similarities and differences between astrocytoma grades.
BMC Cancer. 2015; 15:952 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Astrocytomas are the most common primary brain tumors distinguished into four histological grades. Molecular analyses of individual astrocytoma grades have revealed detailed insights into genetic, transcriptomic and epigenetic alterations. This provides an excellent basis to identify similarities and differences between astrocytoma grades.
METHODS: We utilized public omics data of all four astrocytoma grades focusing on pilocytic astrocytomas (PA I), diffuse astrocytomas (AS II), anaplastic astrocytomas (AS III) and glioblastomas (GBM IV) to identify similarities and differences using well-established bioinformatics and systems biology approaches. We further validated the expression and localization of Ang2 involved in angiogenesis using immunohistochemistry.
RESULTS: Our analyses show similarities and differences between astrocytoma grades at the level of individual genes, signaling pathways and regulatory networks. We identified many differentially expressed genes that were either exclusively observed in a specific astrocytoma grade or commonly affected in specific subsets of astrocytoma grades in comparison to normal brain. Further, the number of differentially expressed genes generally increased with the astrocytoma grade with one major exception. The cytokine receptor pathway showed nearly the same number of differentially expressed genes in PA I and GBM IV and was further characterized by a significant overlap of commonly altered genes and an exclusive enrichment of overexpressed cancer genes in GBM IV. Additional analyses revealed a strong exclusive overexpression of CX3CL1 (fractalkine) and its receptor CX3CR1 in PA I possibly contributing to the absence of invasive growth. We further found that PA I was significantly associated with the mesenchymal subtype typically observed for very aggressive GBM IV. Expression of endothelial and mesenchymal markers (ANGPT2, CHI3L1) indicated a stronger contribution of the micro-environment to the manifestation of the mesenchymal subtype than the tumor biology itself. We further inferred a transcriptional regulatory network associated with specific expression differences distinguishing PA I from AS II, AS III and GBM IV. Major central transcriptional regulators were involved in brain development, cell cycle control, proliferation, apoptosis, chromatin remodeling or DNA methylation. Many of these regulators showed directly underlying DNA methylation changes in PA I or gene copy number mutations in AS II, AS III and GBM IV.
CONCLUSIONS: This computational study characterizes similarities and differences between all four astrocytoma grades confirming known and revealing novel insights into astrocytoma biology. Our findings represent a valuable resource for future computational and experimental studies.

Gatalica Z, Vranic S, Ghazalpour A, et al.
Multiplatform molecular profiling identifies potentially targetable biomarkers in malignant phyllodes tumors of the breast.
Oncotarget. 2016; 7(2):1707-16 [PubMed] Free Access to Full Article Related Publications
Malignant phyllodes tumor is a rare breast malignancy with sarcomatous overgrowth and with limited effective treatment options for recurrent and metastatic cases. Recent clinical trials indicated a potential for anti-angiogenic, anti-EGFR and immunotherapeutic approaches for patients with sarcomas, which led us to investigate these and other targetable pathways in malignant phyllodes tumor of the breast. Thirty-six malignant phyllodes tumors (including 8 metastatic tumors with two cases having matched primary and metastatic tumors) were profiled using gene sequencing, gene copy number analysis, whole genome expression, and protein expression. Whole genome expression analysis demonstrated consistent over-expression of genes involved in angiogenesis including VEGFA, Angiopoietin-2, VCAM1, PDGFRA, and PTTG1. EGFR protein overexpression was observed in 26/27 (96%) of cases with amplification of the EGFR gene in 8/24 (33%) cases. Two EGFR mutations were identified including EGFRvIII and a presumed pathogenic V774M mutation, respectively. The most common pathogenic mutations included TP53 (50%) and PIK3CA (15%). Cases with matched primary and metastatic tumors harbored identical mutations in both sites (PIK3CA/KRAS and RB1 gene mutations, respectively). Tumor expression of PD-L1 immunoregulatory protein was observed in 3/22 (14%) of cases. Overexpression of molecular biomarkers of increased angiogenesis, EGFR and immune checkpoints provides novel targeted therapy options in malignant phyllodes tumors of the breast.

Frezzetti D, Gallo M, Roma C, et al.
Vascular Endothelial Growth Factor A Regulates the Secretion of Different Angiogenic Factors in Lung Cancer Cells.
J Cell Physiol. 2016; 231(7):1514-21 [PubMed] Related Publications
Vascular endothelial growth factor A (VEGFA) is one of the main mediators of angiogenesis in non-small cell lung cancer (NSCLC). Recently, it has been described an autocrine feed-forward loop in NSCLC cells in which tumor-derived VEGFA promoted the secretion of VEGFA itself, amplifying the proangiogenic signal. In order to investigate the role of VEGFA in lung cancer progression, we assessed the effects of recombinant VEGFA on proliferation, migration, and secretion of other angiogenic factors in A549, H1975, and HCC827 NSCLC cell lines. We found that VEGFA did not affect NSCLC cell proliferation and migration. On the other hand, we demonstrated that VEGFA not only produced a strong and persistent increase of VEGFA itself but also significantly induced the secretion of a variety of angiogenic factors, including follistatin (FST), hepatocyte growth factor (HGF), angiopoietin-2 (ANGPT2), granulocyte-colony stimulating factor (G-CSF), interleukin (IL)-8, leptin (LEP), platelet/endothelial cell adhesion molecule 1 (PECAM-1), and platelet-derived growth factor bb (PDGF-BB). PI3K/AKT, RAS/ERK, and STAT3 signalling pathways were found to mediate the effects of VEGFA in NSCLC cell lines. We also observed that VEGFA regulation mainly occurred at post-transcriptional level and that NSCLC cells expressed different isoforms of VEGFA. Collectively, our data suggested that VEGFA contributes to lung cancer progression by inducing a network of angiogenic factors, which might offer potential for therapeutic intervention.

Nie DM, Wu QL, Zhu XX, et al.
Angiogenic factors are associated with development of acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation.
J Huazhong Univ Sci Technolog Med Sci. 2015; 35(5):694-9 [PubMed] Related Publications
Acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, the mechanisms of aGVHD are not well understood. We aim to investigate the roles of the three angiogenic factors: angiopoietin-1 (Ang-1), Ang-2 and vascular endothelial growth factor (VEGF) in the development of aGVHD. Twenty-one patients who underwent allo-HSCT were included in our study. The dynamic changes of Ang-1, Ang-2 and VEGF were monitored in patients before and after allo-HSCT. In vitro, endothelial cells (ECs) were treated with TNF-β in the presence or absence of Ang-1, and then the Ang-2 level in the cell culture medium and the tubule formation by ECs were evaluated. After allo-HSCT, Ang-1, Ang-2 and VEGF all exhibited significant variation, suggesting these factors might be involved in the endothelial damage in transplantation. Patients with aGVHD had lower Ang-1 level at day 7 but higher Ang-2 level at day 21 than those without aGVHD, implying that Ang-1 may play a protective role in early phase yet Ang-2 is a promotion factor to aGVHD. In vitro, TNF-β promoted the release of Ang-2 by ECs and impaired tubule formation of ECs, which were both weakened by Ang-1, suggesting that Ang-1 may play a protective role in aGVHD by influencing the secretion of Ang-2, consistent with our in vivo tests. It is concluded that monitoring changes of these factors following allo-HSCT might help to identify patients at a high risk for aGVHD.

He T, Qi F, Jia L, et al.
Tumor cell-secreted angiogenin induces angiogenic activity of endothelial cells by suppressing miR-542-3p.
Cancer Lett. 2015; 368(1):115-25 [PubMed] Related Publications
Therapeutic strategies for targeting angiogenesis have been proven as successful treatments for divergent cancers. We previously discovered an anti-angiogenic miR-542-3p, which directly targeted the key angiogenesis-promoting protein Angiopoietin-2 to inhibit tumor angiogenesis in breast cancer models. In this study, to further investigate the mechanism of miR-542-3p induced angiogenic inhibition, we screened for tumor cell derived factors which were responsible for miR-542-3p alteration in endothelial cells. We found that tumor cell-derived angiogenin downregulated miR-542-3p in endothelial cells. Overexpression of angiogenin in tumor cells facilitated angiogenic activation in both in vitro and in vivo models via inhibition of miR-542-3p. Furthermore, our results showed that angiogenin could suppress CEBPB and POU2F1, which were transcription factors for miR-542-3p, suggesting a novel tumor cell-endothelial cell signal pathway. In addition, the level of angiogenin in primary breast carcinomas correlated with clinical progression. Serum levels of angiogenin were associated with metastatic development of breast cancer patients. Together, these findings reveal a novel regulatory pathway whereby tumor-derived angiogenin directly activates angiogenesis through inhibition of miR-542-3p, suggesting that angiogenin may represent a promising target for anti-angiogenic therapy and a potential marker for monitoring disease progression.

Tabernero J, Lenz HJ, Siena S, et al.
Analysis of circulating DNA and protein biomarkers to predict the clinical activity of regorafenib and assess prognosis in patients with metastatic colorectal cancer: a retrospective, exploratory analysis of the CORRECT trial.
Lancet Oncol. 2015; 16(8):937-48 [PubMed] Related Publications
BACKGROUND: Tumour mutational status is an important determinant of the response of metastatic colorectal cancer to targeted treatments. However, the genotype of the tissue obtained at the time of diagnosis might not accurately represent tumour genotype after multiple lines of treatment. This retrospective exploratory analysis investigated the clinical activity of regorafenib in biomarker subgroups of the CORRECT study population defined by tumour mutational status or plasma protein levels.
METHODS: We used BEAMing technology to identify KRAS, PIK3CA, and BRAF mutations in DNA obtained from the plasma of 503 patients with metastatic colorectal cancer who enrolled in the CORRECT trial. We quantified total human genomic DNA isolated from plasma samples for 503 patients using a modified version of human long interspersed nuclear element-1 (LINE-1) quantitive real-time PCR. We also measured the concentration of 15 proteins of interest-angiopoietin 2, interleukin 6, interleukin 8, placental growth factor, soluble TIE-1, soluble VEGFR1, VEGF-A, VEGF-C, VEGF-D, VEGF-A isoform 121, bone morphogenetic protein 7, macrophage colony-stimulating factor, stromal cell-derived factor-1, tissue inhibitor of metalloproteinase 2, and von Willebrand factor-in plasma samples from 611 patients. We did correlative analyses of overall survival and progression-free survival in patient subgroups based on mutational status, circulating DNA concentration, and protein concentrations. The CORRECT trial was registered with ClinicalTrials.gov, number NCT01103323.
FINDINGS: Tumour-associated mutations were readily detected with BEAMing of plasma DNA, with KRAS mutations identified in 349 (69%) of 503 patients, PIK3CA mutations in 84 (17%) of 503 patients, and BRAF mutations in 17 (3%) of 502 patients. We did not do correlative analysis based on BRAF genotype because of the low mutational frequency detected for this gene. Some of the most prevalent individual hot-spot mutations we identified included: KRAS (KRAS G12D, 116 [28%] of 413 mutations; G12V, 72 [17%]; and G13D, 67 [16%]) and PIK3CA (PIK3CA E542K, 27 [30%] of 89 mutations; E545K, 37 [42%]; and H1047R, 12 [14%]). 41 (48%) of 86 patients who had received anti-EGFR therapy and whose archival tumour tissue DNA was KRAS wild-type in BEAMing analysis were identified as having KRAS mutations in BEAMing analysis of fresh plasma DNA. Correlative analyses suggest a clinical benefit favouring regorafenib across patient subgroups defined by KRAS and PIK3CA mutational status (progression-free survival with regorafenib vs placebo: hazard ratio [HR] 0·52, 95% CI 0·35-0·76 for KRAS wild-type; HR 0·51, 95% CI 0·40-0·65 for KRAS mutant [KRAS wild type vs mutant, pinteraction=0·74]; HR 0·50, 95% CI 0·40-0·63 for PIK3CA wild-type; HR 0·54, 95% CI 0·32-0·89 for PIK3CA mutant [PIK3CA wild-type vs mutant, pinteraction=0·85]) or circulating DNA concentration (progression-free survival with regorafenib vs placebo: HR 0·53, 95% CI 0·40-0·71, for low circulating DNA concentrations; HR 0·52, 95% CI 0·40-0·70, for high circulating DNA concentrations; low vs high circulating DNA, pinteraction=0·601). With the exception of von Willebrand factor, assessed with the median cutoff method, plasma protein concentrations were also not associated with regorafenib activity in terms of progression-free survival. In univariable analyses, the only plasma protein that was associated with overall survival was TIE-1, high concentrations of which were associated with longer overall survival compared with low TIE-1 concentrations. This association was not significant in multivariable analyses.
INTERPRETATION: BEAMing of circulating DNA could be a viable approach for non-invasive analysis of tumour genotype in real time and for the identification of potentially clinically relevant mutations that are not detected in archival tissue. Additionally, the results show that regorafenib seems to be consistently associated with a clinical benefit in a range of patient subgroups based on mutational status and protein biomarker concentrations.
FUNDING: Bayer HealthCare Pharmaceuticals.

Bessho H, Wong B, Huang D, et al.
Effect of Ang-2-VEGF-A Bispecific Antibody in Renal Cell Carcinoma.
Cancer Invest. 2015; 33(8):378-86 [PubMed] Related Publications
The blockade of VEGF pathway has been clinically validated as an initial treatment for renal cell carcinoma (RCC). Angiopoietin-2 (Ang-2) has been indicated as a key regulator for angiogenesis escape. The effect of a novel bispecific antibody (A2V CrossMab) against both Ang-2 and VEGF was investigated in comparison with either factor. A2V CrossMab significantly reduced tumor volume, vessel density, and interstitial fluid pressure compared to either monotherapy of anti-VEGF or anti-Ang-2. Host-derived angiogenesis-related genes have been significantly down-regulated in A2V CrossMab group. These data demonstrate that A2V CrossMab has additive anti-tumor effect for the treatment of RCC.

Jung S, Sielker S, Purcz N, et al.
Analysis of angiogenic markers in oral squamous cell carcinoma-gene and protein expression.
Head Face Med. 2015; 11:19 [PubMed] Free Access to Full Article Related Publications
PURPOSE: Therapeutic strategies attacking oral squamous cell carcinoma have not essentially succeeded to improve long-term prognosis and overall survival over the last decades. Therefore, in this study, we aimed to illuminate the molecular regulation of angiogenesis in this tumour entity in order to demask novel markers of prognosis or therapeutic approach.
MATERIALS AND METHODS: A panel of significant transcriptional alterations in angiogenic genes of 83 cancer samples was established by comparison to 30 samples of healthy oral mucosa with microarray technique. Immunohistochemistry (IHC) was performed to trace the signalling cascade from gene to protein level.
RESULTS: A distinctive expression profile of VEGFA, EFNB2, PECAM1/CD31, ANGPT1 and ANGPT2 was revealed: VEGFA, EFNB2, and ANGPT2 were found overexpressed in 84 % to 95 % of tumour samples. In contrast, the expression of CD31 and ANGPT1 was downregulated in 80 % to 95 % of tumour samples. IHC confirmed results of the microarray analysis. Tumours with lymphatic spread showed higher gene expression rates of VEGFA, EFNB2 and ANGPT2 in moderately differentiated tumours and of VEGFA and EFNB2 in small tumours, respectively. The ANGPT1/ ANGPT2 transcription ratio was found decreased in larger tumours and especially in tumours without lymphatic spread.
CONCLUSIONS: A characteristic expression profile of angiogenic markers was established. The specific overexpression of EFNB2 in small tumours with lymphatic spread and the typical decrease of the ANGPT1/ ANGPT2 ratio in larger tumours give weight to EFNB2 and angiopoietins as prognostic factors and potential therapeutic targets.

Teichert M, Stumpf C, Booken N, et al.
Aggressive primary cutaneous B-cell lymphomas show increased Angiopoietin-2-induced angiogenesis.
Exp Dermatol. 2015; 24(6):424-9 [PubMed] Related Publications
Primary cutaneous large B-cell lymphomas, leg type (PCLBCL/LT) are primary cutaneous B-cell lymphoma (PCBCL) with an intermediate prognosis. Therefore, antracycline-based polychemotherapy combined with rituximab has been recommended as first-line treatment. Yet, despite this regimen, the 5-year survival rate remains 50-66% only. Angiogenesis, the formation of a vascular network, is essential for the pathogenesis of nodal lymphomas. So far, no study has analysed angiogenesis and its key factors in PCLBCL/LT. The present study was aimed at characterizing angiogenesis in PCLBCL/LT to identify the angiogenic molecules as potential therapeutic targets. The intra-tumoral microvessel density (MVD) was assessed by immunohistochemical studies of CD20 and CD31. The MVD was higher in PCLBCL/LT compared with indolent PCBCL. Analyses of open-source microarray data showed correlation between the angiogenic molecule angiopoietin-2 (Ang-2) and pan-endothelial cell markers. ELISA studies determined a shift between Ang-2 and Ang-1 towards Ang-2 in the peripheral blood of PCLBCL/LT patients. Immunofluorescence costainings against the Ang receptor Tie2/angiogenic integrins/CD34 revealed that the vasculature in both aggressive and indolent PCBCL tumors harbours an endothelial cell subpopulation with reduced expression of Tie2. In contrast, the alternative Ang-2 binding partners, angiogenic integrins, are strongly expressed in PCBCL. In line with these findings, downstream targets of Ang-2-integrin signalling, that is phosphorylation of focal adhesion kinase at Tyr397, and sprouting angiogenesis are enhanced in PCLBCL/LT. Our data present Ang-2 as a promising therapeutic target and anti-angiogenic therapy as a new line in treatment of PCLBCL/LT as a hitherto intractable disease.

Kopparapu PK, Miranda C, Fogelstrand L, et al.
MCPH1 maintains long-term epigenetic silencing of ANGPT2 in chronic lymphocytic leukemia.
FEBS J. 2015; 282(10):1939-52 [PubMed] Related Publications
The microcephalin gene (MCPH1) [also known as inhibitor of human telomerase reverse transcriptase (hTERT) expression] is a tumor suppressor gene that is functionally involved in the DNA damage response. Angiopoietin 2 (ANGPT2) is a crucial factor regulating tumor angiopoiesis. Deregulation of angiogenesis is one of the hallmarks of many cancers, including chronic lymphocytic leukemia (CLL). In CLL, ANGPT2 is a well-studied potential prognostic marker. As MCPH1 overlaps with the ANGPT2 transcription unit on the same chromosome but in the opposite orientation, we wanted to study the functional role of MCPH1 in regulation of ANGPT2 in CLL. The mRNA expression levels of MCPH1 and ANGPT2, including the MCPH1 target gene hTERT, showed significant differences between two prognostic groups, i.e. IGHV-mutated and IGHV-unmutated (P = 0.007 for MCPH1, P = 0.0002 for ANGPT2, and P = 0.00001 for hTERT), in which the expression level of MCPH1 was inversely correlated with the expression levels of hTERT and ANGPT2. Downregulation of MCPH1 resulted in upregulation of ANGPT2, accompanied by loss of its promoter methylation. Using chromatin immunoprecipitation and coimmunoprecipitation assays, we found that MCPH1 binds to the ANGPT2 promoter and recruits DNA methyltransferases, thereby silencing ANGPT2. Thus, our data suggest a novel function for MCPH1 in regulating and maintaining ANGPT2 silencing in CLL through regulation of promoter DNA methylation.

Stremitzer S, Zhang W, Yang D, et al.
Genetic variations in angiopoietin and pericyte pathways and clinical outcome in patients with resected colorectal liver metastases.
Cancer. 2015; 121(11):1898-905 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Genes involved in the angiopoietin and pericyte pathways may become escape mechanisms under antivascular endothelial growth factor (anti-VEGF) therapy. The authors investigated whether variations within genes in these pathways are associated with clinical outcome in patients with colorectal liver metastases who undergo liver resection and receive perioperative, bevacizumab-based chemotherapy.
METHODS: Single nucleotide polymorphisms (SNPs) in 9 genes (angiopoietin-1 [ANGPT1]; ANGPT2; TEK tyrosine kinase, endothelial [TEK]; platelet-derived growth factor β [PDGFB]; β-type platelet-derived growth factor receptor [PDGFRB]; insulin-like growth factor 1 [IGF1]; transforming growth factor β1 [TGFB1]; RalA binding protein 1 [RALBP1]; and regulator of G-protein signaling 5 [RGS5]) were analyzed in samples of genomic DNA from 149 patients and were evaluated for associations with clinical outcome.
RESULTS: RALBP1 reference SNP 329007 (rs329007) A>G resulted in a significant difference in recurrence-free survival (A/A genotype, 14.0 months; A/G or G/G genotype, 9.2 months; hazard ratio [HR], 1.60; P = .024). PDGFB rs1800818 A>G was associated with 3-year overall survival rates (A/A genotype, 78%; A/G genotype, 69%; [HR 1.37]; G/G genotype, 53%; [HR 2.12]; P = .048). In multivariate analysis, RALBP1 rs329007 A>G remained significant (HR, 1.99; P = .002). PDGFB rs1800818 A>G and RALBP1 rs329007 A>G were correlated with radiologic response (A/A or A/G genotype, 86%; G/G genotype, 71% [P = .042]; A/A genotype, 78%; A/G or G/G genotype, 94% [P = .018], respectively). RALBP1 rs329007 A>G demonstrated significantly different rates of histologic response (A/A genotype: major histologic response, 35%; partial histologic response, 34%; no histologic response, 30%; A/G or G/G genotype: 46%, 13%, and 41%, respectively; P = .029). Recursive partitioning analysis revealed that ANGPT2 rs2442599 T>C and RALBP1 rs329007 A>G were the main SNPs that predicted histologic response and recurrence-free survival, whereas PDGFB rs1800818 A>G was the leading SNP that predicted overall survival. ANGPT2 rs2916702 C>T and rs2442631 G>A were significantly associated with the probability of achieving a cure.
CONCLUSIONS: The current data suggest that variations in genes involved in the angiopoietin and pericyte pathways may be predictive and/or prognostic biomarkers in patients with resected colorectal liver metastases who receive bevacizumab-based chemotherapy.

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] Related Publications
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.

Lieb W, Chen MH, Larson MG, et al.
Genome-wide association study for endothelial growth factors.
Circ Cardiovasc Genet. 2015; 8(2):389-97 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Endothelial growth factors including angiopoietin-2 (Ang-2), its soluble receptor Tie-2 (sTie-2), and hepatocyte growth factor play important roles in angiogenesis, vascular remodeling, local tumor growth, and metastatic potential of various cancers. Circulating levels of these biomarkers have a heritable component (between 13% and 56%), but the underlying genetic variation influencing these biomarker levels is largely unknown.
METHODS AND RESULTS: We performed a genome-wide association study for circulating Ang-2, sTie-2, and hepatocyte growth factor in 3571 Framingham Heart Study participants and assessed replication of the top hits for Ang-2 and sTie-2 in 3184 participants of the Study of Health in Pomerania. In multivariable-adjusted models, sTie-2 and hepatocyte growth factor concentrations were associated with single-nucleotide polymorphisms in the genes encoding the respective biomarkers (top P=2.40×10(-65) [rs2273720] and 3.64×10(-19) [rs5745687], respectively). Likewise, rs2442517 in the MCPH1 gene (in which the Ang-2 gene is embedded) was associated with Ang-2 levels (P=5.05×10(-8) in Framingham Heart Study and 8.39×10(-5) in Study of Health in Pomerania). Furthermore, single-nucleotide polymorphisms in the AB0 gene were associated with sTie-2 (top single-nucleotide polymorphism rs8176693 with P=1.84×10(-33) in Framingham Heart Study; P=2.53×10(-30) in Study of Health in Pomerania) and Ang-2 (rs8176746 with P=2.07×10(-8) in Framingham Heart Study; P=0.001 in Study of Health in Pomerania) levels on a genome-wide significant level. The top genetic loci were explained between 1.7% (Ang-2) and 11.2% (sTie-2) of the interindividual variation in biomarker levels.
CONCLUSIONS: Genetic variation contributes to the interindividual variation in growth factor levels and explains a modest proportion of circulating hepatocyte growth factor, Ang-2, and Tie-2. This may potentially contribute to the familial susceptibility to cancer, a premise that warrants further studies.

Alamo P, Gallardo A, Di Nicolantonio F, et al.
Higher metastatic efficiency of KRas G12V than KRas G13D in a colorectal cancer model.
FASEB J. 2015; 29(2):464-76 [PubMed] Related Publications
Although all KRas (protein that in humans is encoded by the KRas gene) point mutants are considered to have a similar prognostic capacity, their transformation and tumorigenic capacities vary widely. We compared the metastatic efficiency of KRas G12V (Kirsten rat sarcoma viral oncogene homolog with valine mutation at codon 12) and KRas G13D (Kirsten rat sarcoma viral oncogene homolog with aspartic mutation at codon 13) oncogenes in an orthotopic colorectal cancer (CRC) model. Following subcutaneous preconditioning, recombinant clones of the SW48 CRC cell line [Kras wild-type (Kras WT)] expressing the KRas G12V or KRas G13D allele were microinjected in the mouse cecum. The percentage of animals developing lymph node metastasis was higher in KRas G12V than in KRas G13D mice. Microscopic, macroscopic, and visible lymphatic foci were 1.5- to 3.0-fold larger in KRas G12V than in KRas G13D mice (P < 0.05). In the lung, only microfoci were developed in both groups. KRas G12V primary tumors had lower apoptosis (7.0 ± 1.2 vs. 7.4 ± 1.0 per field, P = 0.02), higher tumor budding at the invasion front (1.2 ± 0.2 vs. 0.6 ± 0.1, P = 0.04), and a higher percentage of C-X-C chemokine receptor type 4 (CXCR4)-overexpressing intravasated tumor emboli (49.8 ± 9.4% vs. 12.8 ± 4.4%, P < 0.001) than KRas G13D tumors. KRas G12V primary tumors showed Akt activation, and β5 integrin, vascular endothelial growth factor A (VEGFA), and Serpine-1 overexpression, whereas KRas G13D tumors showed integrin β1 and angiopoietin 2 (Angpt2) overexpression. The increased cell survival, invasion, intravasation, and specific molecular regulation observed in KRas G12V tumors is consistent with the higher aggressiveness observed in patients with CRC expressing this oncogene.

Motzer RJ, Hutson TE, Hudes GR, et al.
Investigation of novel circulating proteins, germ line single-nucleotide polymorphisms, and molecular tumor markers as potential efficacy biomarkers of first-line sunitinib therapy for advanced renal cell carcinoma.
Cancer Chemother Pharmacol. 2014; 74(4):739-50 [PubMed] Free Access to Full Article Related Publications
PURPOSE: Sunitinib is a first-line advanced renal cell carcinoma (RCC) standard of care. In a randomized phase II trial comparing sunitinib treatment schedules, separate exploratory biomarker analyses investigated the correlations of efficacy with selected serum, germ line single-nucleotide polymorphism (SNP), or tumor markers.
METHODS: Advanced RCC patients received first-line sunitinib 50 mg/day on the approved 4-week-on-2-week-off schedule (n = 146) or 37.5 mg/day continuous dosing (n = 146). The following correlation analyses were performed: (1) response evaluation criteria in solid tumors-defined tumor response with serum soluble protein levels via two distinct multiplex (n < 1,000) platforms; (2) response and time-to-event outcomes with germ line SNPs in vascular endothelial growth factor (VEGF)-A and VEGF receptor (VEGFR)3 genes; and (3) response and time-to-event outcomes with tumor immunohistochemistry status for hypoxia-inducible factor 1-alpha (HIF-1α) and carbonic anhydrase-IX or tumor Von Hippel-Lindau (VHL) gene inactivation status.
RESULTS: Lower baseline angiopoietin-2 (Ang-2) and higher baseline matrix metalloproteinase-2 (MMP-2) levels were identified by both platforms as statistically significantly associated with tumor response. There were no significant correlations between VEGF-A or VEGFR3 SNPs and outcomes. Progression-free survival was longer for HIF-1α percent of tumor expression groups 0-2 (HIF-1α low) versus 3-4 (HIF-1α high; p = 0.034). There were no significant correlations between outcomes and each VHL inactivation mechanism [mutation (86% of VHL-inactive patients), methylation (14%), and large deletion (7%)] or mechanisms combined.
CONCLUSIONS: Serum Ang-2 and MMP-2 and tumor HIF-1α were identified as relevant baseline biomarkers of sunitinib activity in advanced RCC, warranting further research into their prognostic versus predictive value.

McCorkle JR, Leonard MK, Kraner SD, et al.
The metastasis suppressor NME1 regulates expression of genes linked to metastasis and patient outcome in melanoma and breast carcinoma.
Cancer Genomics Proteomics. 2014 Jul-Aug; 11(4):175-94 [PubMed] Free Access to Full Article Related Publications
NME1 is a well-documented metastasis suppressor gene, with suppressor activity demonstrated across a wide spectrum of human cancers including melanoma and carcinomas of the breast, stomach and thyroid. A primary aim of the current study was to identify profiles of genes whose expression is regulated by NME1 in cell lines of melanoma and thyroid carcinoma origin. Impact of NME1 was determined by forcing its expression transiently in cell lines using a novel Ad5-based adenoviral vector (Ad5-NME1), followed 48 h later by analysis of RNA expression profiles using the U133A microarray chip. Robust NME1 expression was achieved following infection with the Ad5-NME1 adenovirus in the human metastasis-derived cell lines WM1158 (melanoma) and WRO82 (follicular thyroid carcinoma), resulting in wide-ranging effects on gene expression in both settings. A substantial proportion of the NME1-regulated genes identified in the analyses were of clear potential relevance to metastasis, such as matrix metalloproteinase-1 (MMP1), angiopoietin-2 (ANGPT2), SERPINB9 and colony stimulating factor receptor-2B (CSFR2B). Nine genes were identified (false discovery rate <0.1) that were regulated by NME1 in both the WM1158 and WRO82 cell lines, each possessing one or more such metastasis-relevant activities as stress fiber formation and focal adhesion (PPM1E, ZYX, PFN1), chemotaxis (CCR1) epithelial-mesenchymal signaling (WNT6), differentiation and morphogenesis (TBX4, ZFP36L2), and G protein modulation (GPR52 and PFN1). In addition, a number of the NME1-regulated genes were shown to be of prognostic value for distant disease-free survival and overall survival in melanoma and breast cancer. The combined expression of three NME1-regulated genes CSFR2B, MSF4A1 and SERPINB9 provided a strongly synergistic correlation with distant disease-free survival in the basal subtype of breast cancer (p<3.5e(-5), hazard ratio=0.33). Our study demonstrates that analysis of NME1-dependent gene expression is a powerful approach for identifying potential modulators of metastatic potential in multiple cancer types, which in turn may represent useful therapeutic targets. The study also highlights NME1-dependent genes as potential prognostic/diagnostic indices, which are profoundly lacking at present in melanoma.

Hollern DP, Honeysett J, Cardiff RD, Andrechek ER
The E2F transcription factors regulate tumor development and metastasis in a mouse model of metastatic breast cancer.
Mol Cell Biol. 2014; 34(17):3229-43 [PubMed] Free Access to Full Article Related Publications
While the E2F transcription factors (E2Fs) have a clearly defined role in cell cycle control, recent work has uncovered new functions. Using genomic signature methods, we predicted a role for the activator E2F transcription factors in the mouse mammary tumor virus (MMTV)-polyomavirus middle T oncoprotein (PyMT) mouse model of metastatic breast cancer. To genetically test the hypothesis that the E2Fs function to regulate tumor development and metastasis, we interbred MMTV-PyMT mice with E2F1, E2F2, or E2F3 knockout mice. With the ablation of individual E2Fs, we noted alterations of tumor latency, histology, and vasculature. Interestingly, we noted striking reductions in metastatic capacity and in the number of circulating tumor cells in both the E2F1 and E2F2 knockout backgrounds. Investigating E2F target genes that mediate metastasis, we found that E2F loss led to decreased levels of vascular endothelial growth factor (Vegfa), Bmp4, Cyr61, Nupr1, Plod 2, P4ha1, Adamts1, Lgals3, and Angpt2. These gene expression changes indicate that the E2Fs control the expression of genes critical to angiogenesis, the remodeling of the extracellular matrix, tumor cell survival, and tumor cell interactions with vascular endothelial cells that facilitate metastasis to the lungs. Taken together, these results reveal that the E2F transcription factors play key roles in mediating tumor development and metastasis in addition to their well-characterized roles in cell cycle control.

Yamamura T, Matsumoto N, Matsue Y, et al.
Sodium butyrate, a histone deacetylase inhibitor, regulates Lymphangiogenic factors in oral cancer cell line HSC-3.
Anticancer Res. 2014; 34(4):1701-8 [PubMed] Related Publications
AIM: Tumor angiogenesis is a focus of molecularly-targeted therapies. This study investigated the effect of sodium butyrate (SB), a histone deacetylase inhibitor, on the synthesis of antiangiogenic and lymphangiogenic factors in oral squamous cell carcinoma.
DESIGN: Gene alterations in HSC-3 cells were assessed using cDNA microarrays before and after treatment with SB. The mRNA and protein expression of lymphangiogenic factors were also assessed by quantitative PCR, western blotting and immunocytochemistry.
RESULTS: Microarray analysis revealed that treatment with SB led to altered expression of angiogenesis-related gene expression. The quantitative polymerase chain reaction showed that platelet-derived growth factor-B, angiopoietin-2, vascular endothelial growth factor (VEGF)-C, and VEGFD were down-regulated. Western blotting and immunocytochemistry confirmed reduced protein synthesis of VEGFC.
CONCLUSION: SB inhibits expression of lymphangiogenic factors in HSC-3 cells. Within the limitations of the present study, SB may have potential as an anti-metastatic pro-drug for oral cancer.

Coelho AL, Araújo A, Gomes M, et al.
Circulating Ang-2 mRNA expression levels: looking ahead to a new prognostic factor for NSCLC [corrected].
PLoS One. 2014; 9(2):e90009 [PubMed] Free Access to Full Article Related Publications
Non-small cell lung cancer (NSCLC) is the most common cancer and the leading cause of death from cancer worldwide. Antiangiogenic strategies directed towards tumor stroma are becoming gold standard in NSCLC treatment and researchers have been searching for biomarkers to identify patients for whom therapy with antiangiogenic inhibitors may be most beneficial and the importance of these as prognostic factors in NSCLC. The purpose of this study was to evaluate the prognostic value of circulating Ang-2 mRNA levels prior to treatment in NSCLC patients. The mRNA levels were determined by quantitative real-time PCR in the peripheral blood of 92 NSCLC patients. Our results demonstrate that patients with high circulating Ang-2 mRNA levels have diminished overall survival when compared to those with low mRNA levels (20.3 months vs 34.3 months, respectively; Log Rank Test, p = 0.016), when considering all NSCLC stages and this difference is even bigger when considering only patients with stage IV (15.9 months vs 31.3 months, respectively; Log Rank Test, p = 0.036). Moreover, circulating Ang-2 mRNA levels independently determine overall survival, and the concordance (c) index analysis showed that the definition of a nomogram that contains information regarding tumor stage, patients' smoking status and circulating Ang-2 mRNA levels present an increased capacity to predict overall survival in NSCLC patients (c-index 0.798). These results suggest that this nomogram could serve as a unique and practical tool to determine prognosis in NSCLC, not relying on the availability of adequate surgical or biopsy specimens of NSCLC. Attending to our results, the circulating Ang-2 mRNA levels should also be included in the design of preclinical studies and clinical trials involving antiangiogenic drugs targeting Ang-2, to guide adequate patient stratification and dose selection and increasing the likelihood of benefit to a level that is acceptable to patients and clinicians.

Arbiser JL
PHIPing out: a genetic basis for tumor ulceration.
J Invest Dermatol. 2014; 134(3):600-2 [PubMed] Free Access to Full Article Related Publications
Ulceration is a common negative prognostic marker of solid tumors including melanoma. The signaling basis of ulceration is being elucidated. PHIP has been found to be amplified in wild-type melanomas, resulting in Akt activation and aerobic glycolysis (Warburg effect), associated with ulceration. The ulceration phenotype likely represents the genotype of the reactive oxygen driven tumor, in which reactive oxygen drives angiopoietin-2 production, tumor growth, and invasion. This phenotype is amenable to pharmacologic intervention.

He T, Qi F, Jia L, et al.
MicroRNA-542-3p inhibits tumour angiogenesis by targeting angiopoietin-2.
J Pathol. 2014; 232(5):499-508 [PubMed] Related Publications
Angiopoietin-2 (Angpt2) plays a critical role in angiogenesis and tumour progression. Therapeutic targeting of Angpt2 has been implicated as a promising strategy for cancer treatment. Whereas miRNAs are emerging as important modulators of angiogenesis, regulation of Angpt2 by miRNAs has not been established. Here we firstly report that Ang2 is targeted by a microRNA, miRNA-542-3p, which inhibits tumour progression by impairing Ang2's pro-angiogenic activity. In cultured endothelial cells, miR-542-3p inhibited translation of Angpt2 mRNA by binding to its 3' UTR, and addition of miR-542-3p to cultured endothelial cells attenuated angiogenesis. Administration of miR-542-3p to tumour-bearing mice reduced tumour growth, angiogenesis and metastasis. Furthermore, the level of miR-542-3p in primary breast carcinomas correlated inversely with clinical progression in primary tumour samples from stage III and IV patients. Together, these findings uncover a novel regulatory pathway whereby an anti-angiogenic miR-542-3p directly targets the key angiogenesis-promoting protein Angpt2, suggesting that miR-542-3p may represent a promising target for anti-angiogenic therapy and a potential marker for monitoring disease progression.

Lu R, Ji Z, Li X, et al.
miR-145 functions as tumor suppressor and targets two oncogenes, ANGPT2 and NEDD9, in renal cell carcinoma.
J Cancer Res Clin Oncol. 2014; 140(3):387-97 [PubMed] Related Publications
PURPOSE: Abnormal expression of miRNAs is closely related to a variety of human cancers. The purpose of this study is to identify new tumor suppressor miRNA and elucidate its physiological function and mechanism in renal cell carcinoma (RCC).
METHODS: The expression of miR-145 in 45 RCC and adjacent normal tissues was performed by quantitative RT-PCR. Cell proliferation, migration, invasion, apoptosis and cycle assays were carried out for functional analysis after miR-145 transfection. Two target genes of miR-145 were identified by luciferase reporter assay. The altered expression of 84 epithelial to mesenchymal transition (EMT)-related genes after miR-145 transfection was detected by RT(2) Profiler EMT PCR array.
RESULTS: The expression of miR-145 was downregulated in RCC compared to their normal adjacent tissues. Restoring miR-145 expression in RCC cell lines dramatically suppressed cell proliferation, migration and invasion, and induced cell apoptosis and G2-phase arrest. We further validated those miR-145 targets two oncogenes, ANGPT2 and NEDD9 in RCC. In addition, miR-145 was found to regulate numerous genes involved in the EMT.
CONCLUSIONS: These findings demonstrate that miR-145 functions as tumor suppressor in RCC, suggesting that miR-145 may be a potential therapeutic target for RCC.

Reyes-Botero G, Dehais C, Idbaih A, et al.
Contrast enhancement in 1p/19q-codeleted anaplastic oligodendrogliomas is associated with 9p loss, genomic instability, and angiogenic gene expression.
Neuro Oncol. 2014; 16(5):662-70 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: The aim of this study was to correlate MRI features and molecular characteristics in anaplastic oligodendrogliomas (AOs).
METHODS: The MRI characteristics of 50 AO patients enrolled in the French national network for high-grade oligodendroglial tumors were analyzed. The genomic profiles and IDH mutational statuses were assessed using high-resolution single-nucleotide polymorphism arrays and direct sequencing, respectively. The gene expression profiles of 25 1p/19q-codeleted AOs were studied on Affymetrix expression arrays.
RESULTS: Most of the cases were frontal lobe contrast-enhanced tumors (52%), but the radiological presentations of these cases were heterogeneous, ranging from low-grade glioma-like aspects (26%) to glioblastoma-like aspects (22%). The 1p/19q codeletion (n = 39) was associated with locations in the frontal lobe (P = .001), with heterogeneous intratumoral signal intensities (P = .003) and with no or nonmeasurable contrast enhancements (P = .01). The IDH wild-type AOs (n = 7) more frequently displayed ringlike contrast enhancements (P = .03) and were more frequently located outside of the frontal lobe (P = .01). However, no specific imaging pattern could be identified for the 1p/19q-codeleted AO or the IDH-mutated AO. Within the 1p/19q-codeleted AO, the contrast enhancement was associated with larger tumor volumes (P = .001), chromosome 9p loss and CDKN2A loss (P = .006), genomic instability (P = .03), and angiogenesis-related gene expression (P < .001), particularly for vascular endothelial growth factor A and angiopoietin 2.
CONCLUSION: In AOs, the 1p/19q codeletion and the IDH mutation are associated with preferential (but not with specific) imaging characteristics. Within 1p/19q-codeleted AO, imaging heterogeneity is related to additional molecular alterations, especially chromosome 9p loss, which is associated with contrast enhancement and larger tumor volume.

Kahlert C, Pecqueux M, Halama N, et al.
Tumour-site-dependent expression profile of angiogenic factors in tumour-associated stroma of primary colorectal cancer and metastases.
Br J Cancer. 2014; 110(2):441-9 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: Tumour-associated stroma has a critical role in tumour proliferation. Our aim was to determine a specific protein expression profile of stromal angiogenic cytokines and matrix metalloproteinases (MMPs) to identify potential biomarkers or new therapy targets.
METHODS: Frozen tissue of primary colorectal cancer (n=25), liver (n=25) and lung metastases (n=23) was laser-microdissected to obtain tumour epithelial cells and adjacent tumour-associated stroma. Protein expression of nine angiogenic cytokines and eight MMPs was analysed using a multiplex-based protein assay.
RESULTS: We found a differential expression of several MMPs and angiogenic cytokines in tumour cells compared with adjacent tumour stroma. Cluster analysis displayed a tumour-site-dependent stromal expression of MMPs and angiogenic cytokines. Univariate analysis identified stromal MMP-2 and MMP-3 in primary colorectal cancer, stromal MMP-1, -2, -3 and Angiopoietin-2 in lung metastases and stromal MMP-12 and VEGF in liver metastases as prognostic markers (P>0.05, respectively). Furthermore, stroma-derived Angiopoietin-2 proved to be an independent prognostic marker in colorectal lung metastases.
CONCLUSION: Expression of MMPs and angiogenic cytokines in tumour cells and adjacent tumour stroma is dependent on the tumour site. Stroma-derived MMPs and angiogenic cytokines may be useful prognostic biomarkers. These data can be helpful to identify new agents for a targeted therapy in patients with colorectal cancer.

Triana-Baltzer G, Pavlicek A, Goulart A, et al.
Predictive markers of efficacy for an angiopoietin-2 targeting therapeutic in xenograft models.
PLoS One. 2013; 8(11):e80132 [PubMed] Free Access to Full Article Related Publications
The clinical efficacy of anti-angiogenic therapies has been difficult to predict, and biomarkers that can predict responsiveness are sorely needed in this era of personalized medicine. CVX-060 is an angiopoietin-2 (Ang2) targeting therapeutic, consisting of two peptides that bind Ang2 with high affinity and specificity, covalently fused to a scaffold antibody. In order to optimize the use of this compound in the clinic the construction of a predictive model is described, based on the efficacy of CVX-060 in 13 cell line and 2 patient-derived xenograft models. Pretreatment size tumors from each of the models were profiled for the levels of 27 protein markers of angiogenesis, SNP haplotype in 5 angiogenesis genes, and somatic mutation status for 11 genes implicated in tumor growth and/or vascularization. CVX-060 efficacy was determined as tumor growth inhibition (TGI%) at termination of each study. A predictive statistical model was constructed based on the correlation of these efficacy data with the marker profiles, and the model was subsequently tested by prospective analysis in 11 additional models. The results reveal a range of CVX-060 efficacy in xenograft models of diverse tissue types (0-64% TGI, median = 27%) and define a subset of 3 proteins (Ang1, EGF, Emmprin), the levels of which may be predictive of TGI by Ang2 blockade. The direction of the associations is such that better efficacy correlates with high levels of target and low levels of compensatory/antagonizing molecules. This effort has revealed a set of candidate predictive markers for CVX-060 efficacy that will be further evaluated in ongoing clinical trials.

Lyu T, Jia N, Wang J, et al.
Expression and epigenetic regulation of angiogenesis-related factors during dormancy and recurrent growth of ovarian carcinoma.
Epigenetics. 2013; 8(12):1330-46 [PubMed] Free Access to Full Article Related Publications
The initiation of angiogenesis can mark the transition from tumor dormancy to active growth and recurrence. Mechanisms that regulate recurrence in human cancers are poorly understood, in part because of the absence of relevant models. The induction of ARHI (DIRAS3) induces dormancy and autophagy in human ovarian cancer xenografts but produces autophagic cell death in culture. The addition of VEGF to cultures maintains the viability of dormant autophagic cancer cells, thereby permitting active growth when ARHI is downregulated, which mimics the "recurrence" of growth in xenografts. Two inducible ovarian cancer cell lines, SKOv3-ARHI and Hey-ARHI, were used. The expression level of angiogenesis factors was evaluated by real-time PCR, immunohistochemistry, immunocytochemistry and western blot; their epigenetic regulation was measured by bisulfite sequencing and chromatin immunoprecipitation. Six of the 15 angiogenesis factors were upregulated in dormant cancer cells (tissue inhibitor of metalloproteinases-3, TIMP3; thrombospondin-1, TSP1; angiopoietin-1; angiopoietin-2; angiopoietin-4; E-cadherin, CDH1). We found that TIMP3 and CDH1 expression was regulated epigenetically and was related inversely to the DNA methylation of their promoters in cell cultures and in xenografts. Increased H3K9 acetylation was associated with higher TIMP3 expression in dormant SKOv3-ARHI cells, while decreased H3K27me3 resulted in the upregulation of TIMP3 in dormant Hey-ARHI cells. Elevated CDH1 expression during dormancy was associated with an increase in both H3K4me3 and H3K9Ac in two cell lines. CpG demethylating agents and/or histone deacetylase inhibitors inhibited the re-growth of dormant cancer cells, which was associated with the re-expression of anti-angiogenic genes. The expression of the anti-angiogenic genes TIMP3 and CDH1 is elevated during dormancy and is reduced during the transition to active growth by changes in DNA methylation and histone modification.

Wang YH, Dong YY, Wang WM, et al.
Vascular endothelial cells facilitated HCC invasion and metastasis through the Akt and NF-κB pathways induced by paracrine cytokines.
J Exp Clin Cancer Res. 2013; 32(1):51 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: It is well documented that cancer cells secrete angiogenic factors to recruit and sustain tumor vascular networks. However, little is known about the effects of endothelial cells on the behavior of tumor cells. The study here was to determine the roles of endothelial cells in HCC cell growth, migration and invasion.
METHODS: A mixture of highly metastatic MHCC97H cells and HUVEC cells, as well as MHCC97H cells alone were subcutaneously injected into nude mice to observe the effects of HUVECs on HCC growth. The biological characteristics of MHCC97H cells respectively treated with conditioned medium (CM) derived from HUVECs and endothelial cell basal medium (EBM) in vitro, such as proliferation, migration and invasion, invasion/metastasis associated gene expression, were comparatively analyzed. Differential cytokines between CM and EBM were screened and identified using human cytokine array. Effects of the interested differential cytokine CCL2, IL-8 and CXCL16 and its related signaling pathways were further investigated in HCC cells.
RESULTS: Subcutaneous tumorigenicity of MHCC97H cells in nude mice was promoted by HUVECs and its invasion/metastasis associated genes were significantly upregulated. The in vitro, proliferation, migration and invasion of HCC cells treated with CM were all significantly enhanced as compared to those with EBM stimulation. Simultaneously, PI3K/Akt and ERK1/2 pathway in HCC cells were activated by CM. Total of 25 differential cytokines were identified between CM and EBM such as angiopoietin-2, CCL2 (MCP-1), uPA, endostatin, CXCL16, IL-8, pentraxin 3 etc. The selected differential cytokines CCL2, IL-8 and CXCL16 all modulated the expressions of HCC invasion/metastasis genes, especially MMP2 and MMP9. In exposure to CCL2 or CXCL16 alone, upregulation in AKT phosphorylation but no change in ERK phosphorylation were found in MHCC97H cells, moreover the contents of nuclear transcription factor NF-κB were increased as compared to the control. However, no effects on the activation of Akt and ERK pathway in MHCC97H were found in exposure to IL-8.
CONCLUSION: This study expands the contribution of endothelial cells to the progression of HCC. It unveils a new paradigm in which endothelial cells function as initiators of molecular crosstalks that enhance survival, migration and invasion of HCC cells.

Gaál EI, Tammela T, Anisimov A, et al.
Comparison of vascular growth factors in the murine brain reveals placenta growth factor as prime candidate for CNS revascularization.
Blood. 2013; 122(5):658-65 [PubMed] Related Publications
Vascular bypass procedures in the central nervous system (CNS) remain technically challenging, hindered by complications and often failing to prevent adverse outcome such as stroke. Thus, there is an unmet clinical need for a safe and effective CNS revascularization. Vascular endothelial growth factors (VEGFs) are promising candidates for revascularization; however, their effects appear to be tissue-specific and their potential in the CNS has not been fully explored. To test growth factors for angiogenesis in the CNS, we characterized the effects of endothelium-specific growth factors on the brain vasculature and parenchyma. Recombinant adeno-associated virus (AAV) vectors encoding the growth factors were injected transcranially to the frontoparietal cerebrum of mice. Angiogenesis, mural cell investment, leukocyte recruitment, vascular permeability, reactive gliosis and neuronal patterning were evaluated by 3-dimensional immunofluorescence, electron microscopy, optical projection tomography, and magnetic resonance imaging. Placenta growth factor (PlGF) stimulated robust angiogenesis and arteriogenesis without significant side effects, whereas VEGF and VEGF-C incited growth of aberrant vessels, severe edema, and inflammation. VEGF-B, angiopoietin-1, angiopoietin-2, and a VEGF/angiopoietin-1 chimera had minimal effects on the brain vessels or parenchyma. Of the growth factors tested, PlGF emerged as the most efficient and safe angiogenic factor, hence making it a candidate for therapeutic CNS revascularization.

Martinelli S, Kanduri M, Maffei R, et al.
ANGPT2 promoter methylation is strongly associated with gene expression and prognosis in chronic lymphocytic leukemia.
Epigenetics. 2013; 8(7):720-9 [PubMed] Free Access to Full Article Related Publications
Increasing evidence suggests a key role for angiopoietin-2 (ANGPT2) in influencing the aggressiveness of chronic lymphocytic leukemia (CLL). In the presence of vascular endothelial growth factor (VEGF), ANGPT2 causes vessel destabilization leading to neoangiogenesis. Accordingly, high expression levels of ANGPT2 and high degree of angiogenesis have consistently been associated with poor prognosis in CLL; however, the molecular mechanisms behind the variability in ANGPT2 expression are still to be discovered. Here, for the first time, we investigated the DNA methylation status of the ANGPT2 promoter in a large CLL cohort (n = 88) using pyrosequencing and correlated methylation data with ANGPT2 expression levels, prognostic factors and outcome. Importantly, methylation levels of the ANGPT2 gene correlated inversely with its mRNA expression levels (p<0.001). Moreover, low ANGPT2 methylation status was highly associated with adverse prognostic markers, shorter time to first treatment and overall survival. Finally, treatment with methyl inhibitors induced re-expression of ANGPT2 in two B-cell lymphoma cell lines, underscoring the importance of DNA methylation in regulating transcriptional silencing of this gene. In conclusion, we believe that the known variability in ANGPT2 expression among CLL patients could be explained by differential promoter DNA methylation and that low methylation levels of the ANGPT2 promoter have an adverse prognostic impact in CLL.

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