Angiogenesis In Solid Tumors Research Articles

Correlations of vascular architecture and angiogenesis with pituitary adenoma histotype.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor in solid tumors. However, its role in angiogenesis in pituitary adenoma is controversial. Angiogenesis in solid tumors including pituitary adenoma is commonly evaluated by microvascular density (MVD). Here, we evaluated MVD and the role of VEGF in vascular architecture in 51 pituitary adenomas (24 nonfunctioning, 13 prolactin-secreting, 10 growth hormone-secreting, 3 adrenocorticotropic hormone-secreting, and 1 thyroid-stimulating hormone-secreting). Paraffin sections were stained with CD34 and VEGF. MVD and vascular architecture parameters (vessel area, diameter, perimeter, and roundness) were evaluated in CD34-stained sections. Immunohistochemistry showed 27/51 tumors (53%) were VEGF-positive. There were no significant differences in MVD, any vascular parameter, or adenoma volume between VEGF-positive and VEGF-negative tumors. VEGF mRNA expression was significantly higher in VEGF-positive tumors. There were no significant correlations between VEGF mRNA expression and MVD or vascular parameters. However, vessel diameter and perimeter were significantly larger in prolactin-secreting than nonfunctioning and growth hormone-secreting macroadenomas. The difference in vessel diameter was observed among both VEGF-positive and all adenomas (micro- and macroadenoma). Thus, VEGF may have limited roles in the development of vascular architecture and tumor angiogenesis in pituitary adenomas, but the differences in vessel architecture by histotype (i.e., larger vessel diameter and perimeter in prolactin-secreting adenomas) suggest the hormonal regulation of vessel architecture rather than angiogenesis

Emerging treatments in lung cancer - targeting the RLIP76 molecular transporter.

Multidrug resistance in lung cancer cells is a significant obstacle in the treatment of lung cancer. Resistance to chemotherapeutic agents is often the result of efflux of the drugs from cancer cells, mediated by adenosine triphosphate (ATP)-dependent drug transport across the plasma membrane. Thus, identifying molecular targets in the cancer cell transport machinery could be a key factor in successful combinatorial therapy, along with chemotherapeutic drugs. The transport protein Ral-interacting protein of 76 kDa (RLIP76), also known as Ral-binding protein 1 (RalBP1), is a highly promising target for lung cancer treatment. RLIP76 is an ATP-dependent non-ATP-binding cassette (ABC) transporter, responsible for the major transport function in many cells, including many cancer cell lines, causing efflux of glutathione-electrophile conjugates of both endogenous metabolites and environmental toxins. RLIP76 is expressed in most human tissues, and is overexpressed in non-small-cell lung cancer cell lines and in many tumor types. The blockade of RLIP76 by various approaches has been shown to increase the sensitivity to radiation and chemotherapeutic drugs, and leads to apoptosis in cells. In xenograft tumor models in mice, RLIP76 blockade or depletion results in complete and sustained regression across many cancer cell types, including lung cancer cells. In addition to its transport function, RLIP76 has many other cellular and physiological functions based on its domain structure, which includes a unique Ral-binding domain and a Rho GTPase activating protein (RhoGAP)-catalytic domain as well as docking sites for multiple signaling proteins. As a Ral effector, RhoGAP, and adapter protein, RLIP76 has been shown to play important roles in endocytosis, mitochondrial fission, cell spreading and migration, actin dynamics during gastrulation, and Ras-induced tumorigenesis. Additionally, RLIP76 is also important for stromal cell function in tumors, as it was recently shown to be required for efficient endothelial cell function and angiogenesis in solid tumors. However, RLIP76 knockout mice are viable, and blockade effects appear to be selective for implanted tumors in mice, suggesting the possibility that RLIP76-targeting drugs may be successful in clinical trials. In this review, we outline the many cellular and physiological functions of RLIP76 in normal and cancer cells, and discuss the potential for RLIP76-based therapeutics in lung cancer treatment.

The vascular endothelial growth factor as a predictive and prognostic factor in patients with advanced gastric cancer treated with docetaxel, cisplatin, and 5-florouracil (DCF) regimen.

e15191 Background: Vascular endothelial growth factor (VEGF) is a key element stimulating the angiogenesis in solid tumors. In the current study, we aimed to assess the role of plasma VEGF levels as a prognostic and predictive marker for advanced gastric cancer patients treated with a modified docetaxel, cisplatinum and 5-florouracil (DCF) regimen. Methods: Thirty consecutive patients treated with a modified DCF regimen were included in the study. The plasma VEGF levels of the patients before treatment and following two cycles of chemotherapy were assayed. Results: The modified DCF regimen was well tolerated and yielded a median overall survival (OS) time of 9,0 months and 2-year OS rate of 20%. Good performance status, lower pre-treatment VEGF levels,and decrease in VEGF levels following chemotherapy were found to be independent prognostic factors for OS. Conclusions: The results of the current study suggest that the corrected plasma levels of VEGF could be used as a prognostic factor for advanced gastric cancer patients and a decrease in VEGF levels following 2 cycles of chemotherapy as a predictive marker for patients receiving DCF chemotherapy regimen.

Abstract 1104: Malignant stroma regulates luminal breast cancer-cell growth versus basal breast cancer cell migration through different growth factor pathways.

Abstract The stromal microenvironment plays an important role in growth and aggressiveness of breast cancers. We have generated a reactive mouse mammary stromal cell line called BJ3Z that is tumorigenic in nude mice. In mixed-cell xenografts, BJ3Z cells enhance growth and angiogenesis of solid tumors established from MCF-7 human breast cancer cells. To minimize extensive dependence on in vivo studies, we have developed Matrigel-based 3D colony co-culture assays to analyze the role of the stromal microenvironment on epithelial-cell derived breast cancers in vitro. First, to define the properties of reactive BJ3Z cells, they were contrasted to normal mouse mammary gland fibroblasts (NMF), both grown in 3D. Expression profiling showed that Transforming Growth Factor-β1 (TGF-β1) and Platelet-Derived Growth Factor ligands A and B (PDGF-A & PDGF-B) are upregulated in BJ3Z cells. Next, we contrasted effects of BJ3Z and NMF cells on the three major breast cancer subtypes: Luminal MCF-7 cells; HER2 amplified BT-474 cells; Basal-like MDA-MB-231 and BT-20 cells. We find that malignant stroma has differential effects depending on the breast cancer subtype. 1. With regard to PDGF: Reactive stroma generated by BJ3Z cells in 3D, increases the growth of Luminal and HER2-amplified breast cancer cells as measured by BrdU incorporation. In contrast, BJ3Z cells suppress growth of Basal-like breast cancer cells. Similar results were obtained using BJ3Z-conditioned media. This suggests that the effects of BJ3Z cells on Luminal tumor-cell growth involve soluble factors. In that regard, use of Imatinib Mesylate (IM) a tyrosine kinase inhibitor against PDGF receptors completely blocks the rise in MCF-7 cell proliferation caused by BJ3Z-cell co-culture, or by BJ3Z cell-conditioned medium. In order to demonstrate that this is not due to a mouse-specific factor we showed that recombinant human PDGF-BB enhances 3D growth of MCF-7 cells. Parenthetically, BJ3Z-conditioned medium also enhances an in vitro angiogenesis model by a PDGF-dependent pathway that can be inhibited by IM. 2. With regard to TGF-β this pathway is not involved in Luminal tumor-cell growth. Rather in Basal-like cancer cells, BJ3Z or their conditioned medium use TGF-β pathway-dependent Smad 2/3 phosphorylation to enhance cell migration. This effect can be blocked by a TGF-βR1 inhibitor. We conclude that the stromal microenvironment uses the PDGF pathway to regulate Luminal tumor-cell growth and angiogenesis; the TGF-β pathway to regulate Basal tumor-cell aggressiveness. Citation Format: Mauricio P. Pinto, Britta M. Jacobsen, Kathryn B. Horwitz. Malignant stroma regulates luminal breast cancer-cell growth versus basal breast cancer cell migration through different growth factor pathways. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1104. doi:10.1158/1538-7445.AM2013-1104

Fibrin and Collagen Differentially but Synergistically Regulate Sprout Angiogenesis of Human Dermal Microvascular Endothelial Cells in 3-Dimensional Matrix

Angiogenesis is a highly regulated event involving complex, dynamic interactions between microvascular endothelial cells and extracellular matrix (ECM) proteins. Alteration of ECM composition and architecture is a hallmark feature of wound clot and tumor stroma. We previously reported that during angiogenesis, endothelial cell responses to growth factors are modulated by the compositional and mechanical properties of a surrounding three-dimensional (3D) extracellular matrix (ECM) that is dominated by either cross-linked fibrin or type I collagen. However, the role of 3D ECM in the regulation of angiogenesis associated with wound healing and tumor growth is not well defined. This study investigates the correlation of sprout angiogenesis and ECM microenvironment using in vivo and in vitro 3D angiogenesis models. It demonstrates that fibrin and type I collagen 3D matrices differentially but synergistically regulate sprout angiogenesis. Thus blocking both integrin alpha v beta 3 and integrin alpha 2 beta 1 might be a novel strategy to synergistically block sprout angiogenesis in solid tumors.

RALBP1/RLIP76 Depletion in Mice Suppresses Tumor Growth by Inhibiting Tumor Neovascularization

RalBP1/RLIP76 is a widely expressed multifunctional protein that binds the Ral and R-Ras small GTPases. In the mouse, RLIP76 is nonessential but its depletion or blockade promotes tumorigenesis and heightens the sensitivity of normal and tumor cells to radiation and cytotoxic drugs. However, its pathobiologic functions, which support tumorigenesis, are not well understood. Here, we show that RLIP76 is required for angiogenesis and for efficient neovascularization of primary solid tumors. Tumor growth from implanted melanoma or carcinoma cells was blunted in RLIP76(-/-) mice. An X-ray microcomputed tomography-based method to model tumor vascular structures revealed defects in both the extent and form of tumor angiogenesis in RLIP76(-/-) mice. Specifically, tumor vascular volumes were diminished and vessels were fewer in number, shorter, and narrower in RLIP76(-/-) mice than in wild-type mice. Moreover, we found that angiogenesis was blunted in mutant mice in the absence of tumor cells, with endothelial cells isolated from these animals exhibiting defects in migration, proliferation, and cord formation in vitro. Taken together, our results establish that RLIP76 is required for efficient endothelial cell function and angiogenesis in solid tumors.

Peripheral blood biomarkers of solid tumor angiogenesis in dogs: A polychromatic flow cytometry pilot study

A subset of peripheral blood hematopoietic stem and progenitor cells of bone marrow origin is elevated in humans with solid cancers before treatment and declines with therapy. This biomarker of angiogenesis is not specific to tumor type and has great potential in the objective assessment of treatment response in clinical trials. This pilot study was designed to develop a biomarker of neoangiogenesis in dogs for the diagnosis of cancer, the measurement of treatment response, and the provision of objective data in clinical trials. Polychromatic flow cytometry was used to quantify two subsets of circulating hematopoietic stem and progenitor cells in dogs with spontaneous solid tumors before (n=8) and after (n=3) treatment, and normal controls (n=6).Pro-angiogenic peripheral blood cells of bone marrow origin were detected in all eight cases and the six normal controls; however, there was no statistically significant difference between the two groups. Interestingly, an apparent decline in pro-angiogenic cells was observed after treatment. Bone marrow derived hematopoietic cells appear to contribute to tumor angiogenesis in dogs, as has been previously reported in humans. While the methodology for pro-angiogenic cell quantification in a small number of dogs in the current study did not result in a significant difference from normal controls, an optimized canine polychromatic flow cytometry protocol holds great promise in the development of a canine cancer model and for the objective measurements of treatment response in clinical trials.

Increased Angiogenesis-associated Poor Outcome in Acute Lymphoblastic Leukemia

Angiogenesis in solid tumors is important for tumor growth, invasion, and metastasis. However, angiogenesis plays also an important role in hematological malignancies. We have analyzed the expression of vascular endothelial growth factor (VEGF) in the leukemic blast cells and microvessel density (MVD) in the bone marrow biopsy samples of the patients with acute lymphoblastic leukemia (ALL). Bone marrow MVD of the patients with ALL was significantly higher compared with normal controls and complete remission (P<0.001), but slightly lower than in patients with relapsed ALL (P>0.05). The bone marrow blast VEGF expression was significantly higher in newly diagnosed ALL patients, with predominant strong VEGF expression as compared with complete remission patients (who had negative or weak VEGF expression) (P<0.05), whereas initial values were slightly lower than in relapsed patients. There was a strong positive correlation between VEGF expression and MVD at presentation of ALL. Stronger expression of VEGF on blast cells indicates shorter overall survival in ALL. Furthermore, initial values of MVD had positive correlation with overall survival and leukemia-free survival (P=0.024 and P=0.017, respectively). Our data suggest that increased angiogenesis (confirmed by immunohistochemical expression of VEGF in leukemic blasts), and MVD may play an important role in the pathophysiology of ALL with prognostic implications. Thus, targeting VEGF pathway may bring the new approach for ALL treatment-using antiangiogenic drugs and tyrosine kinase inhibitors in combination with standard chemotherapy regimens.

New Gene Therapy Strategies for Cancer Treatment: A Review of Recent Patents

Cancer is the second leading cause of death in the Western world. The limited successes of available treatments for cancer mean that new strategies need to be developed. The possibility of modifying the cancer cell with the introduction of genetic material opens the way to a new approach based on gene therapy. There are still many technical difficulties to be overcome, but recent advances in the molecular and cellular biology of gene transfer have made it likely that gene therapy will soon start to play an increasing role in clinical practice, particularly in the treatment of cancer. Gene therapy will probably be the therapeutic option in cases in which conventional treatments such as surgery, radiotherapy and chemotherapy have failed. The development of modified vectors, and an improved understanding of interactions between the vector and the human host, are generating inventions that are being protected by patents due to the considerable interest of industry for their possible commercialization. We review the latest strategies, patented and/or under clinical trial, in cancer gene therapy. These include patents that cover the use of modified vectors to increase the security and specificity, recombining adenovirus that leads to loss or gain of gene function, activation of the patient's own immune cells to eliminate cancer cells by expression of molecules that enhance immune responses, silencing genes related to the development of drug resistance in patients, inhibition of angiogenesis of solid tumors by targeting the tumor vasculature, and the development of enzymes that destroy viral or cancerous genetic material.

Interstitial fluid pressure as an alternate regulator of angiogenesis independent of hypoxia driven HIF‐1α in solid tumors

We previously showed that interstitial fluid pressure (IFP) may be an alternate regulator of angiogenesis in solid tumors. Given the accepted link between hypoxia-induced factor and angiogenesis this study investigated the effect of IFP on hypoxia-inducible factor (HIF-1α) and vascular endothelial growth factor (VEGF) in human osteosarcoma xenografts in SCID mice and in different hypoxic environments. Tumors were grown either at heterotopic (flank) or orthotopic (medullary canal of the proximal tibia) sites in the host animal. Microfluidic probes determined pH, O(2)-saturation, IFP, and peripheral blood flow perfusion continuously. We assessed tumor growth in the orthotopic site (n = 15) by softex radiographs weekly, 3D microCT, histological evaluation, and for molecular responses. An increased cytoplasmic immunohistostaining of cells for HIF-1α (p = 0.03) and VEGF-A (p = 0.004) on the outer periphery was noted compared to the tumor center, with VEGFR2 uniformly stained throughout. This paralleled a raised state of interstitial hypertension (p = 0.007) in the tumor center relative to the peripheral surface but was inconsistent with a state of hypoxia (p = 0.03) in the tumor center. In vitro culture of human osteosarcoma cell lines (HOS, U2OS) and a human osteoblast control at 0- and 20-mmHg of hydrostatic pressure revealed suppression of HIF-1α (p = 0.02) and VEGF-A (p = 0.02) gene expression when IFP was raised, while the effect on VEGFR1 was equivocal. This study proposes an alternative regulatory angiogenic pathway via the influence of IFP on cancer cell function. The identification of a mechanistic cellular link to the physical parameter becomes an important tool to evaluate cancer cell growth within solid tumors.

Targeting the Tumor Microenvironment: Focus on Angiogenesis

Tumorigenesis is a complex multistep process involving not only genetic and epigenetic changes in the tumor cell but also selective supportive conditions of the deregulated tumor microenvironment. One key compartment of the microenvironment is the vascular niche. The role of angiogenesis in solid tumors but also in hematologic malignancies is now well established. Research on angiogenesis in general, and vascular endothelial growth factor in particular, is a major focus in biomedicine and has led to the clinical approval of several antiangiogenic agents including thalidomide, bevacizumab, sorafenib, sunitinib, pazopanib, temesirolimus, and everolimus. Indeed, antiangiogenic agents have significantly changed treatment strategies in solid tumors (colorectal cancer, renal cell carcinoma, and breast cancer) and multiple myeloma. Here we illustrate important aspects in the interrelationship between tumor cells and the microenvironment leading to tumor progression, with focus on angiogenesis, and summarize derived targeted therapies.

Heme oxygenase-1 promotes angiogenesis in urothelial carcinoma of the urinary bladder

Angiogenesis is necessary for the growth, invasion, and metastasis of solid tumors. Previous studies have shown that heme oxygenase-1 (HO-1) plays an important role in angiogenesis in both normal and cancerous cells, such as vascular endothelial cells and pancreatic cancer cells, respectively. In this study, we analyzed the role of HO-1 and other angiogenic factors in urothelial carcinoma of the bladder. Specifically, we used real-time reverse transcription polymerase chain reaction (PCR) and Western blotting to investigate the upregulation of 7 angiogenic factors, namely, HO-1, vascular endothelial growth factor (VEGF), hypoxia-inducible factor (HIF)-1α, HIF-2α, cyclooxygenase-2 (COX-2), interleukin-8 (IL-8), and basic fibroblast growth factor (bFGF) under hypoxic conditions in the T24 urothelial carcinoma cell line. We also used enzyme-linked immunosorbent assay (ELISA) to measure the amount of VEGF secreted into the growth media. In addition, we administered an HO-1 inhibitor, zinc protoporphyrin IX, to mice with subcutaneous T24 tumors to assess the modulation of angiogenesis in solid tumors in vivo. We also performed immunohistochemical analyses of 23 primary bladder cancer specimens with high-grade tumors infiltrating into the stroma (pT1) for expression of HO-1, VEGF, HIF-1α, HIF-2α, COX-2, and CD31. Image analysis of CD31 staining was performed to estimate microvessel density (MVD), a measure of angiogenesis. Hypoxic conditions induced upregulation of HO-1, VEGF, HIF-1α, HIF-2α, and COX-2 in T24 cells and increased VEGF secretion, which could be suppressed by zinc protoporphyrin IX. In vivo, inhibition of HO-1 decreased tumor growth and MVD by suppressing angiogenic factors, particularly VEGF and HIF-1α. In clinical specimens of bladder cancer, high expression of HO-1 was correlated with high expression of HIF-1α (P=0.027) and high MVD (P=0.005), but not with VEGF expression (P=0.19). In conclusion, since overexpression of HO-1 promotes angiogenesis in urothelial carcinoma cells, HO-1 inhibitors could be used as novel therapeutics for urothelial carcinoma of the urinary bladder.

Examining the safety profile of angiogenesis inhibitors: implications for clinical practice

Dysregulation of angiogenesis in solid tumors enables tumor growth and metastasis. Receptors that mediate angiogenesis, and their ligands, have been identified; among these, vascular endothelial growth factor (VEGF) and its receptor tyrosine kinase play a central role. In non-small cell lung cancer (NSCLC), clinical benefit has been shown with bevacizumab, which blocks VEGF binding to its receptors. In addition, small-molecule tyrosine kinase inhibitors are currently in clinical practice or development in several indications. The potential clinical benefit of antiangiogenic agents is accompanied by the need to understand their safety profiles, seek suitable combination regimens and identify patients able to tolerate one treatment over another. This is particularly relevant in cancers for which patients often present with advanced disease and suffer co-morbidities, requiring an agent that is both efficacious and well tolerated over long-term continuous treatment. Using literature from peer-reviewed journals, this review considers different antiangiogenic agents and their safety profiles, focusing on the potential impact of these data on the treatment of patients with NSCLC.

Highlights of This Issue

Tumorigenesis is influenced by natural selection processes, thus molecular evolutionary methodologies could be useful for understanding cancer biology. Zhang and colleagues describe a phylogenetic analysis of 353 cancer cell lines based on the alignment of 3252 variant nucleotides from 494 genes from 353 cancer cell lines. Reconstructed phylogenetic trees clustered cell lines by shared DNA variant patterns rather than tissue, suggesting that certain diverse cancer types have similar oncogenic pathways. Positive selection analysis revealed specific DNA variants that might be potential driver mutations. Their study supports the broader application of evolutionary approaches in the analyses of the cancer genome.The clinical efficacy of lapatinib, a small molecule inhibitor of ErbB2 and EGFR tyrosine kinases, is limited by the development of therapeutic resistance. Xia and colleagues show that lapatinib unexpectedly activated RelA/NF-κB in ErbB2+ breast cancer cell lines and in ErbB2+ breast cancer patients. Inhibition of RelA phosphorylation led to increased apoptosis in lapatinib-treated ErbB2+ breast cancer cells. Furthermore, increased phospho-RelA expression in tumors treated with lapatinib monotherapy correlated with a poorer clinical outcome in ErbB2+ breast cancer patients. These findings identify RelA as a promising therapeutic target to enhance the clinical efficacy of lapatinib in ErbB2+ breast cancers.Approaches to improve standard chemotherapeutic regimens by defining combinations are still empiric. Jimeno and colleagues evaluated a rational way of identifying relevant targets involved in the vulnerability of pancreatic cancer to gemcitabine. Pancreatic xenograft samples with known gemcitabine sensitivity were dynamically interrogated by exposure to gemcitabine and analysis of a preselected set of druggable genes. This identified Plk1 as a target dictating the susceptibility of pancreatic cancer to gemcitabine. Mechanistic studies aimed at testing this hypothesis were conducted utilizing gene interference and pharmacologic Plk1 inhibition, followed by in vivo testing in a direct xenograft model of pancreatic cancer.Brivanib is an oral, dual selective inhibitor of VEGF and FGF signaling under clinical investigation. Bhide and colleagues evaluated the antitumor activity and antiangiogenic activities of brivanib and its prodrug brivanib alaninate in xenograft models and Matrigel plug assays in athymic mice. Brivanib and brivanib alaninate demonstrated potent antitumor and antiangiogenic activity over multiple dose levels in a broad range of tumor types. Further, brivanib alaninate inhibited angiogenesis driven by VEGF or bFGF alone, or when both growth factors were combined. This data supports the utility of targeting both VEGF- and FGF-induced angiogenesis in solid tumors.

Oxygen Tension in the Bone Marrow (BM) of Patients with Malignant and Indolent Monoclonal Gammopathy: Role of Hypoxia and Hypoxia-Inducible Factor (HIF)-1α in the Regulation of Gene Expression and Pro-Angiogenic Profiles of CD138+ Cells.

Abstract 422Multiple myeloma (MM) is characterized by the accumulation of malignant plasmacells into the bone marrow (BM) microenvironment that supports their growth and survival. Particularly, an increase of BM angiogenesis occurs in relationship with plasmacells infiltration playing a critical role in the progression on monoclonal gammopathy. Hypoxia is known to be associated to angiogenesis in solid tumors as well as the hypoxia-inducible factor (HIF)-1α is a critical trigger and regulator of the angiogenic switch. Actually, the oxygen levels in the BM of patients with monoclonal gammopathy as well the effects of hypoxia and HIF-1α on the gene expression and pro-angiogenic profile of CD138+ cells are not known.In this study, first, we investigated the level of BM oxygen saturation (sO2) and partial pressure (pO2) in a cohort of 44 patients with monoclonal gammopathy at the diagnosis including active MM (n°=25), smoldering MM (n° = 8) and MGUS (n°=11) showing that BM of MM patients was hypoxic (mean ± SD of pO2: 52.5 ± 8.69 mmHg; sO2 of 83.7 ± 11%) even if any significant difference in both pO2 and sO2 was not observed in comparison with smoldering MM patients or MGUS as well as in relationship with the stage of disease (p=0.7). Next, we evaluated how hypoxia exposition could modify the gene expression profile of CD138+cells isolated form MM patients by U133 Plus2.0 Arrays. By supervised analysis performed on 11 paired samples we found that hypoxia significantly modulated 714 genes in CD138+ cells isolated from MM patients. Interestingly, genes belonging either to oxidative stress and hypoxia signaling, including heme oxygenase 1 (HMOX1) and the heat shock proteins 90kDa alpha (HSP90AA1; HSP90AB1), or to protein ubiquitination pathway, such as XIAP, were significantly up-regulated by hypoxia. Among the pro-angiogenic genes, we found that both VEGFA and IL8 were induced by hypoxia. Following, given that HIF-1α accumulates only in hypoxic condition, we checked HIF-1α protein expression in the BM of MM patients by immunohistochemistry on bone biopsies immediately after fixation. A strong HIF-1α immunostaining was demonstrated in MM cells at nuclear level in all patients analyzed. Interestingly, the presence of HIF-1α protein was also observed in isolated CD138+ MM cells of about 28% of MM patients in normoxic condition.In order to investigate the potential role of HIF-1α on MM cell gene expression and angiogenic profile we performed HIF-1α silencing in MM cells by siRNA anti-HIF-1α and that exposed cells to normoxic or hypoxic conditions. HIF-1α suppression was associated to the modulation of pro-angiogenic molecules (VEGFA, VEGFB, IL-8 and PGF) and oxidative stress (SOD2, PTGS2, TXT) and glycogenolysis regulating (ENO2, ALDOC, PFKFB3, HK2, PDK1, PFKFB4) genes. Data obtained were than validated by real time PCR and at protein level by western blot and ELISA assay. Consistently we found that HIF-1α suppression significantly inhibited the pro-angiogenic properties, evaluated in an in vitro angiogenesis model as capillary junctions and tubules formation and tubule length.In conclusion, we demonstrate that the MM-BM environment is hypoxic and that HIF-1α protein expressed by MM cells. Consistently we show that hypoxia and HIF-1α significantly modulate the gene expression profiles of MM cells regulating the expression of the pro-angiogenic factors by MM cells and their pro-angiogenic properties. Disclosures:No relevant conflicts of interest to declare.

Numerical simulation of solid tumor angiogenesis with Endostatin treatment: a combined analysis of inhibiting effect of anti-angiogenic factor and micro mechanical environment of extracellular matrix

To investigate the influence of anti-angiogenesis drug Endostatin on solid tumor angiogenesis, a mathematical model of tumor angiogenesis was developed with combined influences of local extra-cellular matrix mechanical environment, and the inhibiting effects of Angiostatin and Endostatin. Simulation results show that Angiostatin and Endostatin can effectively inhibit the process of tumor angiogenesis, and decrease the number of blood vessels in the tumor. The present model could be used as a valid theoretical method in the investigation of anti-angiogenic therapy of tumors.

Secreted Frizzle-Related Protein 2 Stimulates Angiogenesis via a Calcineurin/NFAT Signaling Pathway

Secreted frizzle-related protein 2 (SFRP2), a modulator of Wnt signaling, has recently been found to be overexpressed in the vasculature of 85% of human breast tumors; however, its role in angiogenesis is unknown. We found that SFRP2 induced angiogenesis in the mouse Matrigel plug assay and the chick chorioallantoic membrane assay. SFRP2 inhibited hypoxia induced endothelial cell apoptosis, increased endothelial cell migration, and induced endothelial tube formation. The canonical Wnt pathway was not affected by SFRP2 in endothelial cells; however, a component of the noncanonical Wnt/Ca2+ pathway was affected by SFRP2 as shown by an increase in NFATc3 in the nuclear fraction of SFRP2-treated endothelial cells. Tacrolimus, a calcineurin inhibitor that inhibits dephosphorylation of NFAT, inhibited SFRP2-induced endothelial tube formation. Tacrolimus 3 mg/kg/d inhibited the growth of SVR angiosarcoma xenografts in mice by 46% (P = 0.04). In conclusion, SFRP2 is a novel stimulator of angiogenesis that stimulates angiogenesis via a calcineurin/NFAT pathway and may be a favorable target for the inhibition of angiogenesis in solid tumors.

Enterolactone but not genistein decreases estradiol-induced tumor growth, angiogenesis, and secreted VEGF in vivo in human breast cancer.

Abstract Abstract #5081 Phytoestrogens have been shown to be potential compounds in breast cancer prevention and treatment by poorly understood mechanisms. There are two major classes of phytoestrogens in the diet; lignans and isoflavones. One main phytoestrogen in Western diet is the mammalian lignan enterolactone (ENL) whereas soy is the major source of the isoflavone genistein (GEN) usually found in Eastern diets. In this paper we investigated the effects of ENL and GEN on estradiol induced breast cancer growth in nude mice. Ovariectomized mice treated with a continuous release of estradiol were used. MCF-7 tumors were established and mice were fed with basal diet (BD), or BD with added ENL, GEN, or a combination of ENL and GEN. Tumor growth was followed and microdialysis was used to sample extracellular VEGF and FGF-2 in vivo after 2-3 weeks of treatment. We show that ENL alone or in combination with GEN, counteracted estradiol-induced growth and angiogenesis of solid tumors. GEN alone did not affect tumor growth or angiogenesis compared with BD. The pro-angiogenic extracellular levels of VEGF in vivo were significantly decreased by ENL and ENL+GEN diets compared with tumors in the BD group whereas GEN diet alone did not affect the levels compared with BD. FGF-2 levels were similar in all groups. Our in vivo findings were confirmed in vitro. By adding ENL and/or GEN, estradiol-induced VEGF secretion in MCF-7 cells decreased significantly in the ENL and ENL+GEN groups whereas GEN alone had no effects compared with estradiol exposed cells. FGF-2 was unaffected by estrogen and phytoestrogen exposure compared with control cells without added hormones. Estradiol up-regulated VEGFR-2 in human umbilical vein endothelial cells and this up-regulation was inhibited by ENL suggesting a pro-angiogenic effect by estradiol by two different mechanisms, both of which were inhibited by the addition of ENL. Our results suggest that ENL has potent anti-estrogenic effects on ER+ breast cancer whereas GEN failed to inhibit estradiol-induced tumor growth and VEGF secretion. Various phytoestrogens may have very different effects on breast cancer. ENL may prove to be beneficial in breast cancer prevention strategies in the future while GEN may cause opposite effects. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 5081.

The hepatocyte growth factor/c-Met signaling pathway as a therapeutic target to inhibit angiogenesis

Angiogenesis in tumors is driven by multiple growth factors that activate receptor tyrosine kinases. An important driving force of angiogenesis in solid tumors is signaling through vascular endothelial growth factor (VEGF) and its receptors (VEGFRs). Angiogenesis inhibitors that target this signaling pathway are now in widespread use for the treatment of cancer. However, when used alone, inhibitors of VEGF/VEGFR signaling do not destroy all blood vessels in tumors and do not slow the growth of most human cancers. VEGF/VEGFR signaling inhibitors are, therefore, used in combination with chemotherapeutic agents or radiation therapy. Additional targets for inhibiting angiogenesis would be useful for more efficacious treatment of cancer. One promising target is the signaling pathway of hepatocyte growth factor (HGF) and its receptor (HGFR, also known as c-Met), which plays important roles in angiogenesis and tumor growth. Inhibitors of this signaling pathway have been shown to inhibit angiogenesis in multiple in vitro and in vivo models. The HGF/c-Met signaling pathway is now recognized as a promising target in cancer by inhibiting angiogenesis, tumor growth, invasion, and metastasis.

PEG-conjugated Hemoglobin Combination with Cisplatin Enforced the Antiangiogeic Effect in a Cervical Tumor Xenograft Model

Angiogenesis, an essential event involved in a tumor's progression and metastasis, is regulated by hypoxia. Hypoxia widely exists in solid tumors due to the abnormal vasculature of tumor tissue and insufficiency of tissue oxygenation. We speculate that hemoglobin-based oxygen carriers (HBOCs) can attenuate tissue hypoxia, thereby suppressing the angiogenesis in solid tumor in the context that HBOCs have the ability to increase tissue oxygenation. In the present study, PEG-conjugated hemoglobin solution (0.3 g/kg i.v. or 0.6 g/kg i.v.) was intravenously administrated to BALB/c nude mice bearing the cervical tumor twice a week with or without the treatment of cisplatin (5mg/kg i.p.) to investigate whether PEG-conjugated hemoglobin has a chemo-sensitization effect though anti-angiogenesis pathway. Tumor volume was measured every three days and tumor hypoxia was detected by immunohistochemistry for Hypoxyprobe-1. Anti-angiogenic effect was accessed by detection of mRNA and protein levels of vascular endothelial growth factor (VEGF), the most important angiogenic factor. Our results showed that high concentration of PEG-conjugated hemoglobin solution significantly impeded the growth of tumor when compared with the control group. Moreover, VEGF expression was declined when treated with PEG-conjugated hemoglobin, possibly through the HIF regulation system. Collectively, treatment of PEG-conjugated hemoglobin combination with cisplatin has an antiangiogenic effect, but the underlying mechanism should be further studied.