Formation Of Tumor Vasculature Research Articles

EGFR Mutation-Harboring Lung Cancer Cells Produce CLEC11A with Endothelial Trophic and Tumor-Promoting Activities.

Simple SummaryTumor angiogenesis is an important step in the progression of solid tumors. Understanding the mechanisms involved in tumor vasculature formation is critical for improving anti-angiogenic strategies. In this study, we reported that EGFR mutation-containing lung cancer cells produced CLEC11A with endothelial trophic and tumor-promoting activities. CLEC11A could be a novel factor involved in tumor angiogenesis.The formation of new blood vessels in solid tumors is regulated by various endothelial trophic factors. We identified that CLEC11A, an extracellular C-type lectin, was over-expressed in lung cancer cell lines harboring mutated EGFR. CLEC11A expression was also frequently elevated in lung adenocarcinoma (LAC) tissues with EGFR mutation. CLEC11A-expressing H1299 cells formed larger tumors in nude mice than did the control cells. The CLEC11A-expressing tumors contained more CD31-positive cells, suggesting that they had a higher angiogenic activity. CLEC11A per se did not induce blood vessel formation, but enhanced angiogenesis triggered by VEGF-A or basic FGF in vivo. Additionally, the expression of small hairpin RNA against CLEC11A (shCLEC11A) in HCC827 LAC cells suppressed their tumorigenic ability. Purified CLEC11A exhibited a chemotactic ability, which is dependent on its integrin-binding RGD and LDT motifs, toward endothelial cells. This chemotactic activity was not affected by the presence of a VEGFR inhibitor. Conditioned medium produced by HCC827-shCLEC11A cells had diminished chemotactic ability toward endothelial cells. CLEC11A treatments increased the levels of active integrin β1 that were not associated with activation of focal adhesion kinases in endothelial cells. Our results indicated that CLEC11A was a factor of angiogenic potential and was involved in lung cancer tumorigenesis.

Gene Delivery of RNA Therapeutics for the Treatment of Brain Tumors

The proliferation of glioblastoma multiforme (GBM) is often coupled to the dysregulation, amplification and overexpression of tyrosine kinase receptors (TKRs) including epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) among others. TKRs are cell‐surface receptors that bind to their cognate ligand, peptide hormones known as growth factors. Binding of the growth factor ligand stimulates TKRs, activating a signal‐transduction cascade that leads to cell growth, migration and the formation of tumor vasculature. A look at TKRs reveals a general architecture of the receptor. They are comprised of three distinct domains: an extracellular, transmembrane and an intracellular domain. As these receptors are often upregulated in GBM, an RNA Therapy to modulate the expression, inhibit the translation of critical TKR domains or reduce its activation would reduce the functionality of these oncogenic transcripts and proteins in GBM. Our research focuses on the development of AAV gene therapy vectors that encode novel RNA therapeutics to reduce the expression of oncogenic transcripts as well as inhibit their activation in GBM, the most common malignant primary brain tumor in adults. Individuals diagnosed with GBM have a short life expectancy of 12–15 months. The standard care and treatment as well as the developmental pipeline of new therapeutic strategies are often limited by the blood‐brain barrier (BBB), restricting systemically administered therapies from reaching the brain (Hicks et al., 2015). Thus, a novel strategy that circumvents this barrier is necessary to effectively reach the CNS tumor microenvironment. Direct AAV delivery of genes that encode RNA therapeutics at the time of tumor resection would bypass the BBB to deliver a persistent and constant dosage from the inside‐out. With the advancement of nanopore sequencing technology, we are able to read longer transcripts (>200kb). With this technology, we are able to examine the architecture of nascent pre‐mRNA transcripts, detect lower abundant alternatively spliced and polyadenylated isoforms, as well take advantage of RNA structurome analysis to reveal RNA elements within TKRs which are more susceptible to RNA anti‐sense directed therapies. To this end, we have developed a strategy to identify novel targets within oncogenic transcripts and incorporate these therapies directed against these targets into our AAV directed RNA gene therapy platforms. In addition, we use RNA mimics to take advantage of the RNA interference pathway to modulate and reduce expression of oncogenic transcripts. This research has the potential to identify novel RNA elements and alternatively spliced and polyadenylated isoforms of TKRs in health and disease. The ultimate goal of our research project is to build on current strategies to identify even better RNA elements of the EGFR and VEGFR2 transcripts which are structurally available for both microRNA degradation and anti‐sense directed alternative splicing therapy.

Adhesion molecules and the extracellular matrix as drug targets for glioma.

The formation of tumor vasculature and cell invasion along white matter tracts have pivotal roles in the development and progression of glioma. A better understanding of the mechanisms of angiogenesis and invasion in glioma will aid the development of novel therapeutic strategies. The processes of angiogenesis and invasion cause the production of an array of adhesion molecules and extracellular matrix (ECM) components. This review focuses on the role of adhesion molecules and the ECM in malignant glioma. The results of clinical trials using drugs targeted against adhesion molecules and the ECM for glioma are also discussed.

MTOR Inhibitors Block Kaposi Sarcoma Growth by Inhibiting Essential Autocrine Growth Factors and Tumor Angiogenesis

Kaposi sarcoma originates from endothelial cells and it is one of the most overt angiogenic tumors. In Sub-Saharan Africa, where HIV and the Kaposi sarcoma-associated herpesvirus (KSHV) are endemic, Kaposi sarcoma is the most common cancer overall, but model systems for disease study are insufficient. Here, we report the development of a novel mouse model of Kaposi sarcoma, where KSHV is retained stably and tumors are elicited rapidly. Tumor growth was sensitive to specific allosteric inhibitors (rapamycin, CCI-779, and RAD001) of the pivotal cell growth regulator mTOR. Inhibition of tumor growth was durable up to 130 days and reversible. mTOR blockade reduced VEGF secretion and formation of tumor vasculature. Together, the results show that mTOR inhibitors exert a direct anti-Kaposi sarcoma effect by inhibiting angiogenesis and paracrine effectors, suggesting their application as a new treatment modality for Kaposi sarcoma and other cancers of endothelial origin.

Mechanisms of Tumor Development and Anti-angiogenic Therapy in Glioblastoma Multiforme

Despite advances in surgical and medical therapy, glioblastoma multiforme (GBM) remains a fatal disease. There has been no significant increase in survival for patients with this disease over the last 20 years. Tumor vasculature formation and glioma cell invasion along the white matter tracts both play a pivotal role in glioma development. Angiogenesis and invasion are the major factors believed to be responsible for treatment resistance in tumors, and a better understanding of the glioma invasion and angiogenesis mechanisms will lead to the development of potential new treatments. In this review, we focus on the molecular characteristics of angiogenesis and invasion in human malignant glioma. We discuss bevacizumab and cilengitide, which are used to inhibit angiogenesis in GBM.

Broad Expression Analysis of Human ANTXR1/TEM8 Transcripts Reveals Differential Expression and Novel Splizce Variants

Tumor endothelial marker 8 (TEM8; ANTXR1) is one of two anthrax toxin receptors; the other is capillary morphogenesis gene 2 protein (CMG2; ANTXR2). TEM8 shows enhanced expression in certain tumor endothelia, and is thought to be a player in tumor vasculature formation. However, a comprehensive expression profile of individual TEM8 variants in normal or cancerous tissues is lacking. In this work we carried out an extensive analysis of all splice variants of human TEM8 in 12 digestive tissues, and 8 each fetal and adult tissues, 6 of them cognate pairs. Using variant-specific primers, we first ascertained the status of full-length transcripts by nested PCR. We then carried out quantitative analysis of each transcript by real-time PCR. Three splice variants of TEM8 were reported before, two single-pass integral membrane forms (V1 and V2) and one secreted (V3). Our analysis has revealed two new variants, one encoding a membrane-bound form of the receptor and the other secreted, which we have designated V4 and V5, respectively. All tissues had V1, V2, V3, and V4, but only prostate had V5. Real-time PCR revealed that all variants are present at different levels in various tissues. V3 appeared the most abundant of all. To ascertain its functionality for anthrax toxin, we expressed the newly identified form V4 in a receptor-negative host cell, and included V1 and V2 for comparison. Cytotoxicity, toxin binding, and internalization assays showed V4 to be as efficient a receptor as V1 and V2.

Abstract LB-308: Combination of CTx-0294945 a highly selective inhibitor of focal adhesion kinase with bevacizumab in pre-clinical models of breast cancer

Abstract Background: Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that provides a critical hub for signalling from at least four different classes of cellular sensing mechanisms including growth factor receptors, GPCRs, integrins and mechanical stress forces. By temporal and spatial integration of signals from these sources, FAK plays a central role in cell migration, invasion and proliferation; processes vital for tumorigenesis. The significance of FAK to the function of signal transduction pathways provides a strong rationale for the combination of FAK inhibitors with other targeted agents to achieve improved efficacy against a range of cancers. Others have demonstrated the importance of FAK in angiogenesis and therefore combining a FAKi with anti-VEGF agents is attractive as it employs two complementary mechanisms of suppressing the formation of tumor vasculature. Here we present results from the co-administration of CTx-0294945, a highly selective FAKi, and bevacizumab (bev) in an orthotopic model of human breast cancer. Methods and Results: CTx-0294945 is an orally bioavailable small molecule ATP competitive inhibitor of focal adhesion kinase (FAK KD=0.21 nM). It exhibits high selectivity against a diverse panel of 125 kinases including the closely related Pyk2. CTx-0294945 inhibits autophosphorylation of 397Y-FAK in MDA-MB-231 cells with an IC50 = 7 nM and exhibits low general cellular toxicity (IC50 = 2.7 µM, MDA-MB-231 cells). CTx-0294945 is suitable for oral administration (%F=58 and t1/2=5.1 h at 20 mg/Kg in rat) and does not inhibit (IC50 >20 µM) any of the cytochrome p450 isoforms tested to date. To assess the co-administration of CTx-0294945 with bev, mice were injected orthotopically with MDA-MB-231 cells (106). After 14 days, when tumors were palpable, mice were randomized into 4 groups and dosing commenced. The groups were treated with CTx-0294945 (80 mg/kg QD, PO), bev (12.5 mg/Kg IP, x2/week), CTx-0294945 (80 mg/Kg QD, PO) and bev (12.5 mg/Kg IP, x2/week) or vehicle. Tumor growth was monitored and on day 28 animals in the vehicle and CTx-0294945 arms were culled when the size of the tumors reached ethical end point (1000 mm3). Tumor growth in the bev only and the CTx-0294945 + bev arms was significantly inhibited (75% and 88% TGI respectively). At this time the treatment regimes for both cohorts were stopped and tumor growth allowed to progress. After an additional 14 days the experiment was terminated when the bev treatment group reached ethical end point; however the average size of the tumors in the CTx-0294945 + bev cohort was still was only 562 mm3. Conclusions: Our data suggest the potential utility of combining a selective FAK inhibitor with bevacizumab to prevent tumour progression and enhance the durability of response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-308. doi:1538-7445.AM2012-LB-308

Abstract B46: Effective therapeutic response of MDA-MB-231 cell line to rapamycin.

Abstract The assessment of alternative therapeutics agents can develop more effective treatments in breast cancer, resulting in molecular-targeted drugs, developed from the knowledge of molecules involved in specific pathways that provide growth and progression of cancer. A large number of studies have suggested that immunosuppressive agents can act as powerful anti-cancer drugs. Rapamycin, a potent immunosupressor used routinely in transplant patients, also has antiproliferative, antifungal and antimicrobial activity. Recent studies show that rapamycin can alter the function of blood vessels by promoting thrombosis in tumor vessels and thus prevents the formation of tumor vasculature. In addition, experiments have focused on the antiangiogenic effects, antiproliferative and antimetastatic target protein inhibitors of rapamycin (mTOR), affecting the expression of genes involved in metabolism and protein synthesis. Thus, believing in the inhibitory effect of rapamycin in tumor progression in diferent tumor types, this study evaluated its antitumor activity in mammary tumoral cell line. Samples of breast cancer cell line, MDA-MB-231 (estrogen receptor negative) were cultured in medium DMEM with high glucose concentration (Cultilab), and incubated at 37° C in 5% CO2. All culture medium used was enriched with 20% BSA, 1% penicillin/streptomycin and 1% L-glutamine. It was established five groups, four of them received different concentrations of rapamycin (group I - 1nM, group II - 10nM, group III - 50nM and group IV - 100nM) and one group was classified as control group (control - group V). After the interval of 72 hours of application of rapamycin, cells were stained with Trypan Blue (cell vitality). The growth curve was performed by counting cells in a Neubauer chamber, in order to evaluate the effects of drug. After 72 hours, the group V contained 200 cells. In the treated groups I and II, there was a small decrease in cell number, reaching 180 and 150 cells, respectively. In group III, there was a reduction in cell number reached 118 cells and in group IV was observed 50% reduction in the number of cells compared to group V. These results prove that rapamycin is effective in controlling cell proliferation and causes a decrease in the number of cells. In addition, the action of rapamycin may be dose dependent, as increasing the dose applied in the same cell concentration there was a decrease in the amount of cells. This fact suggests that rapamycin can be a possible alternative to the establishment of therapeutic strategies for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B46.

Inhibition of ascites tumor growth in vivo by sTie‐2 is potentiated by a combinatorial therapy with sFLT‐1

Inhibition of tumor angiogenesis is a promising approach for cancer therapy and the Tie-2/angiopoietin pathway appears to play an important role. In the present study, we have developed strategies to explore the therapeutic potential of blocking the Tie-2/angiopoietin pathway by sTie-2. Ehrlich ascites tumor (EAT) cells were stably transfected to overexpress a truncated form of sTie-2. Transfectants were characterized for their in vitro growth behavior and transplanted into nude mice. Furthermore, recombinant sTie-2 produced by the baculovirus expression system was used to sequester angiopoietins in the murine ascites carcinoma model. The effect of sTie-2 treatment alone or in combination with sFLT-1 on the weight of the animal, ascites cell number and volume was studied. EAT cells stably transfected with a truncated form of sTie-2 showed no change in cell proliferation in vitro and colony forming in soft agar compared to control cells. However, sTie-2 transfected EAT cells transplanted into nude mice reduced tumor burden and demonstrated a reduction in ascites formation and peritoneal angiogenesis. Recombinant sTie-2 showed angiogenic activity in the tube formation and wound healing assay in vitro. sTie-2 treatment alone or in combination with sFLT-1 in an ascites tumor mouse model resulted in reduced peritoneal angiogenesis, with a concomitant decrease in tumor cell number, volume of ascites and the number of invasive tumor cells, as assayed by CD31 staining. The findings of the present study demonstrate an important role for the Tie-2/angiopoietin pathway in the formation of tumor vasculature and suggest that sTie-2 might yield useful anticancer therapy.

From Vessel Sprouting to Normalization

The accepted model of vessel branching distinguishes several endothelial cell fates. At the forefront of a vessel sprout, "tip cells" guide the sprouting vessel toward an angiogenic stimulus. Behind the tip, "stalk cells" proliferate to elongate the vessel branch and create a lumen. In mature vessels, endothelial cells acquire a streamlined shape to optimally conduct blood flow. For this purpose, endothelial cells switch to the "phalanx" cell fate, which is characterized by quiescent and nonproliferating cells aligned in a tight cobblestonelike layer. Vessel maturation also requires the recruitment of mural cells (ie, smooth muscle cells and pericytes). These cell fates are often altered in pathological conditions, most prominently during the formation of tumor vasculature. Given the essential role of hypoxia as the driving force for initiating angiogenesis, it is not surprising that the hypoxia-sensing machinery controls key steps in physiological and pathological angiogenesis.

Design, Structure−Activity Relationships and in Vivo Characterization of 4-Amino-3-benzimidazol-2-ylhydroquinolin-2-ones: A Novel Class of Receptor Tyrosine Kinase Inhibitors

The inhibition of key receptor tyrosine kinases (RTKs) that are implicated in tumor vasculature formation and maintenance, as well as tumor progression and metastasis, has been a major focus in oncology research over the last several years. Many potent small molecule inhibitors of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) kinases have been evaluated. More recently, compounds that act through the complex inhibition of multiple kinase targets have been reported and may exhibit improved clinical efficacy. We report herein a series of potent, orally efficacious 4-amino-3-benzimidazol-2-ylhydroquinolin-2-one analogues as inhibitors of VEGF, PDGF, and fibroblast growth factor (FGF) receptor tyrosine kinases. Compounds in this class, such as 5 (TKI258), are reversible ATP-competitive inhibitors of VEGFR-2, FGFR-1, and PDGFRbeta with IC(50) values <0.1 microM. On the basis of its favorable in vitro and in vivo properties, compound 5 was selected for clinical evaluation and is currently in phase I clinical trials.

Bone Marrow Subsets Differentiate into Endothelial Cells and Pericytes Contributing to Ewing's Tumor Vessels

Hematopoietic progenitor cells arising from bone marrow (BM) are known to contribute to the formation and expansion of tumor vasculature. However, whether different subsets of these cells have different roles in this process is unclear. To investigate the roles of BM-derived progenitor cell subpopulations in the formation of tumor vasculature in a Ewing's sarcoma model, we used a functional assay based on endothelial cell and pericyte differentiation in vivo. Fluorescence-activated cell sorting of human cord blood/BM or mouse BM from green fluorescent protein transgenic mice was used to isolate human CD34+/CD38(-), CD34+/CD45+, and CD34(-)/CD45+ cells and mouse Sca1+/Gr1+, Sca1(-)/Gr1+, VEGFR1+, and VEGFR2+ cells. Each of these progenitor subpopulations was separately injected intravenously into nude mice bearing Ewing's sarcoma tumors. Tumors were resected 1 week later and analyzed using immunohistochemistry and confocal microscopy for the presence of migrated progenitor cells expressing endothelial, pericyte, or inflammatory cell surface markers. We showed two distinct patterns of stem cell infiltration. Human CD34+/CD45+ and CD34+/CD38(-) and murine VEGFR2+ and Sca1+/Gr1+ cells migrated to Ewing's tumors, colocalized with the tumor vascular network, and differentiated into cells expressing either endothelial markers (mouse CD31 or human vascular endothelial cadherin) or the pericyte markers desmin and alpha-smooth muscle actin. By contrast, human CD34(-)/CD45+ and mouse Sca1(-)/Gr1+ cells migrated predominantly to sites outside of the tumor vasculature and differentiated into monocytes/macrophages expressing F4/80 or CD14. Our data indicate that only specific BM stem/progenitor subpopulations participate in Ewing's sarcoma tumor vasculogenesis.