Challenges facing antiangiogenesis therapy: The significant role of hypoxia-inducible factor and MET in development of resistance to anti-vascular endothelial growth factor-targeted therapies

It is now fully recognized that along with multiple physiological functions, angiogenesis is also involved in the fundamental process and pathobiology of several disorders including cancer. Recent studies have fully established the role of angiogenesis in cancer progression as well as invasion and metastasis. Consequently, many therapeutic agents such as monoclonal antibodies targeting angiogenesis pathway have been introduced in clinic with the hope for improving the outcomes of cancer therapy. Bevacizumab (Avastin®) was the first anti-vascular endothelial growth factor (VEGF) targeting monoclonal antibody developed with this purpose and soon received its accelerated US Food and Drug Administration (FDA) approval for treatment of patients with metastatic breast cancer in 2008. However, the failure to meet expecting results in different follow-up studies, forced FDA to remove bevacizumab approval for metastatic breast cancer. Investigations have now revealed that while suppressing VEGF pathway initially decreases tumor progression rate and vasculature density, activation of several interrelated pathways and signaling molecules following VEGF blockade compensate the insufficiency of VEGF and initially blocked angiogenesis, explaining in part the failure observed with bevacizumab single therapy. In present review, we introduce some of the main pathways and signaling molecules involved in angiogenesis and then propose how their interconnection may result in development of resistance to bevacizumab.     

Pro-angiogenic activity and vasculogenic mimicry in the tumor microenvironment by leptin in cancer

The acquired ability to induce the formation of a functional vasculature is a hallmark of cancer. Blood vessels in tumors are formed through various mechanisms, among the most important in cancer biology, angiogenesis, and vasculogenic mimicry have been described. Leptin is one of the main adipokines secreted by adipocytes in normal breast tissue and the tumor microenvironment. Here, we provide information on the relationship between leptin and the development of angiogenesis and vasculogenic mimicry in different types of cancer. Here, we report that leptin activates different pathways such as JAK-STAT3, MAPK/ERK, PKC, JNK, p38, and PI3K-Akt to induce the expression of various angiogenic factors and vasculogenic mimicry. In vivo models, leptin induces blood vessel formation through the PI3K-Akt-mTOR pathway. Interestingly, the relationship between leptin and vasculogenic mimicry was more significant in breast cancer. The information obtained suggests that leptin could be playing an essential role in tumor survival and metastasis through the induction of vascular mechanisms such as angiogenesis and vasculogenic mimicry; thus, leptin-induced pathways could be suggested as a promising therapeutic target.     

The Role of Respiratory Microbiota in Lung Cancer

Recently, the impact of microorganisms on tumor growth and metastasis has attracted great attention. The pathogenesis and progression of lung cancer are related to an increase in respiratory bacterial load as well as changes in the bacterial community because the microbiota affects tumors in many ways, including canceration, metastasis, angiogenesis, and treatment. The microbiota may increase tumor susceptibility by altering metabolism and immune responses, promoting inflammation, and increasing toxic effects. The microbiota can regulate tumor metastasis by altering multiple cell signaling pathways and participate in tumor angiogenesis through vascular endothelial growth factors (VEGF), endothelial cells (ECs), inflammatory factors and inflammatory cells. Tumor angiogenesis not only maintains tumor growth at the primary site but also promotes tumor metastasis and invasion. Therefore, angiogenesis is an important mediator of the interaction between microorganisms and tumors. The microbiota also plays a part in antitumor therapy. Alteration of the microbiota caused by antibiotics can regulate tumor growth and metastasis. Moreover, the microbiota also influences the efficacy and toxicity of tumor immunotherapy and chemotherapy. Finally, the effects of air pollution, a risk factor for lung cancer, on microorganisms and the possible role of respiratory microorganisms in the effects of air pollution on lung cancer are discussed.     

R-Ras Protein Inhibits Autophosphorylation of Vascular Endothelial Growth Factor Receptor 2 in Endothelial Cells and Suppresses Receptor Activation in Tumor Vasculature

Abnormal angiogenesis is associated with a broad range of medical conditions, including cancer. The formation of neovasculature with functionally defective blood vessels significantly impacts tumor progression, metastasis, and the efficacy of anticancer therapies. Vascular endothelial growth factor (VEGF) potently induces vascular permeability and vessel growth in the tumor microenvironment, and its inhibition normalizes tumor vasculature. In contrast, the signaling of the small GTPase R-Ras inhibits excessive angiogenic growth and promotes the maturation of regenerating blood vessels. R-Ras signaling counteracts VEGF-induced vessel sprouting, permeability, and invasive activities of endothelial cells. In this study, we investigated the effect of R-Ras on VEGF receptor 2 (VEGFR2) activation by VEGF, the key mechanism for angiogenic stimulation. We show that tyrosine phosphorylation of VEGFR2 is significantly elevated in the tumor vasculature and dermal microvessels of VEGF-injected skin in R-Ras knockout mice. In cultured endothelial cells, R-Ras suppressed the internalization of VEGFR2, which is required for full activation of the receptor by VEGF. Consequently, R-Ras strongly suppressed autophosphorylation of the receptor at all five major tyrosine phosphorylation sites. Conversely, silencing of R-Ras resulted in increased VEGFR2 phosphorylation. This effect of R-Ras on VEGFR2 was, at least in part, dependent on vascular endothelial cadherin. These findings identify a novel function of R-Ras to control the response of endothelial cells to VEGF and suggest an underlying mechanism by which R-Ras regulates angiogenesis.     

Antiangiogenic activity of the endocannabinoid anandamide: correlation to its tumor-suppressor efficacy

Endocannabinoids are now emerging as suppressors of key cell-signaling pathways involved in cancer cell growth, invasion, and metastasis. We have previously observed that the metabolically stable anandamide analog, 2-methyl-2'-F-anandamide (Met-F-AEA) can inhibit the growth of thyroid cancer in vivo. Our hypothesis was that the anti-tumor effect observed could be at least in part ascribed to inhibition of neo-angiogenesis. Therefore, the aim of this study was to assess the anti-angiogenic activity of Met-F-AEA, to investigate the molecular mechanisms underlying this effect and whether Met-F-AEA could antagonize tumor-induced endothelial cell sprouting. We show that Met-F-AEA inhibited bFGF-stimulated endothelial cell proliferation, in a dose-dependent manner, and also induced apoptosis, both effects reliant on cannabinoid CB1 receptor stimulation. Analyzing the signaling pathways implicated in angiogenesis, we observed that the bFGF-induced ERK phosphorylation was antagonized by Met-F-AEA, and we found that p38 MAPK was involved in Met-F-AEA-induced apoptosis. Moreover, Met-F-AEA was able to inhibit bi-dimensional capillary-like tube formation and activity of matrix metalloprotease MMP-2, a major matrix degrading enzyme. Importantly, we demonstrated that Met-F-AEA is also functional in vivo since it inhibited angiogenesis in the chick chorioallantoic neovascularization model. Finally, Met-F-AEA inhibited tumor-induced angiogenesis in a three-dimensional model of endothelial and thyroid tumor cell (KiMol) spheroids co-cultures in different 3-D polymeric matrices that resemble tumor microenvironment and architecture. Thus, our results suggest that anandamide could be involved in the control of cancer growth targeting both tumor cell proliferation and the angiogenic stimulation of the vasculature.     

The ephrins and Eph receptors in angiogenesis.

Eph receptors are a unique family of receptor tyrosine kinases that play critical roles in embryonic patterning, neuronal targeting, vascular development and adult neovascularization. Engagement of Eph receptors by ephrin ligands mediates critical steps of angiogenesis, including juxtacrine cell-cell contacts, cell adhesion to extracellular matrix, and cell migration. Recent evidence from in vitro angiogenesis assays and analysis of mice deficient for one or more members of the Eph family establishes the role of Eph signaling in sprouting angiogenesis and blood vessel remodeling during vascular development. Furthermore, elevated expression of Eph receptors and ephrin ligands is associated with tumors and associated tumor vasculature, suggesting that Eph receptors and their ephrin ligands also play critical roles in tumor angiogenesis and tumor growth. This review will focus on the relevance of Eph receptor signaling in embryonic and adult neovascularization, and possible contributions to tumor growth and metastasis.     

Hyaluronan promotes tumor lymphangiogenesis and intralymphantic tumor growth in xenografts

Hyaluronan （HA）, a high molecular weight glycosaminoglycan in the extracellular matrix, has been implicated in the promotion of malignant phenotypes, including tumor angiogenesis. However, little is known about the effect of HA on tumor-associated lymphangiogenesis. In this study, mouse hepatocellular carcinoma Hca-F cells combined with or without HA were injected subcutaneously into C3H/Hej mice, then angiogenesis and lymphangiogenesis of implanted tumors were examined by immunostaining for plateletendothelial cell adhesion molecule-1 and lymphatic vascular endothelial hyaluronan receptor-1 respectively. Interestingly, we found HA promotes tumor lymphangiogenesis and the occurrence of intratumoral lymphatic vessels, but has little effect on tumor angiogenesis. Moreover, HA also promotes intralymphatic tumor growth, although it is not sufficient to potentiate lymphatic metastasis. These results suggest that HA, which is elevated in most malignant tumor stroma, may also play a role in tumor progression by promoting lymphangiogenesis.     

Molecular mechanisms of lymphangiogenesis in development and cancer

The lymphatic system, also named the second vascular system, plays a critical role in tissue homeostasis and immunosurveillance. The past two decades of intensive research have led to the identification and detailed understanding of many molecular players and mechanisms regulating the formation of the lymphatic vasculature during embryonic development. Furthermore, clinical and experimental data clearly demonstrate that the formation of new lymphatic vessels by sprouting lymphangiogenesis from pre-existing lymphatic vessels, or by the de novo formation of lymphatic capillaries also occurs in various pathological conditions, such as cancer and organ transplant rejection, while lymphangiogenesis is non-functional in primary edema. In cancer, lymphatic vessels are one major gateway for invasive tumor cells to leave the primary tumor site and to establish distant organ metastasis. Therefore, the specific targeting of the lymphatic vasculature at the tumor site could be a promising approach to prevent metastasis formation.     

Plasmalemmal vacuolar H+-ATPases in angiogenesis, diabetes and cancer

Angiogenesis, i.e., new blood vessel formation, is required in normal and pathological states. A dysfunction in the microvascular endothelium occurs in diabetes, leading to decreased blood flow and limb amputation. In cancer, angiogenesis is increased to allow for growth, invasion, and metastasis of tumor cells. Better understanding of the molecular events that cause or are associated with either of these diseases is needed to develop therapies. The tumor and angiogenic cells micro-environment is acidic and not permissive for growth. We have shown that to survive this environment, highly metastatic and angiogenic cells employ vacuolar H⁺-ATPase at their plasma membranes (pmV-ATPases) to maintain an alkaline pH^cyt. However, in lowly metastatic and in microvascular endothelial cells from diabetic model, the density of pmV-ATPase and the cell invasiveness are decreased. Therefore, the overexpression of the pmV-ATPase is important for cell invasion, and essential for tumor progression, angiogenesis and metastasis. Both, cancer and diabetes are heterogenous diseases that involve many different proteins and signaling pathways. Changes in pH^cyt have been associated with the regulation of a myriad of proteins, signaling molecules and pathways affecting many if not all cellular functions. Since changes in pH^cyt are pleiotropic, we hypothesize that alteration in a single protein, pmV-ATPase, that can regulate pH^cyt may explain the dysfunction of many proteins and cellular pathways in diabetes and cancer. Our long term goal is to determine the molecular mechanisms by which pmV-ATPase expression regulates tumor angiogenesis and metastasis. Such knowledge would be useful to identify targets for cancer therapy.     

Rapid In Vitro Derivation of Endothelium Directly From Human Cancer Cells

The development of an independent blood supply by a tumor is essential for maintaining growth beyond a certain limited size and for providing a portal for metastatic dissemination. Host-derived endothelial cells (ECs) residing in and compromising the tumor vasculature originate via distinct processes known as sprouting angiogenesis and vasculogenesis. More recently ECs originating directly from the tumor cells themselves have been described although the basis for this phenomenon remains poorly understood. Here we describe in vitro conditions that allow lung and ovarian cancer cells to undergo a rapid and efficient transition into ECs that are indistinguishable from those obtained in vivo. A variety of methods were used to establish that the acquired phenotypes and behaviors of these tumor-derived ECs (TDECs) closely resemble those of authentic ECs. Xenografts arising from co-inoculated in vitro-derived TDECs and tumor cells were also more highly vascularized than control tumors; moreover, their blood vessels were on average larger and frequently contained admixtures of host-derived ECs and TDECs derived from the initial inoculum. These results demonstrate that cancer cells can be manipulated under well-defined in vitro conditions to initiate a tumor cell-to-EC transition that is largely cell-autonomous, highly efficient and closely mimics the in vivo process. These studies provide a suitable means by which to identify and perhaps modify the earliest steps in TDEC generation.     

PEDF inhibits lymphatic metastasis of nasopharyngeal carcinoma as a new lymphangiogenesis inhibitor

Nasopharyngeal carcinoma (NPC) is one of the most malignant tumors in southern China and Asia, and lymph node metastasis is an important cause for treatment failure. Lymphangiogenesis is a crucial step in lymphatic metastasis of NPC, while little is known about lymphangiogenesis in NPC. Similar to angiogenesis, lymphangitic neovascularization is a process of balance between pro-lymphangiogenesis and anti-lymphangiogenesis factors, but there are few studies on endogenous lymphangiogenesis inhibitors. Pigment epithelium-derived factor (PEDF) is a well-known effective endogenous angiogenesis inhibitor. However, the relationship between PEDF and lymphangiogenesis remains unknown. Our present study reveals that PEDF is lowly expressed in human NPC tissues with poor prognosis and is negatively correlated with lymphatic vessel density (LVD). Consistently, PEDF inhibits lymphangiogenesis and lymphatic metastasis of NPC in vivo experiments. Mechanistically, PEDF inhibits the proliferation, migration, and tube formation of lymphatic endothelial cells and promotes cell apoptosis. On the other hand, PEDF reduces the expression and secretion of vascular endothelial growth factor C (VEGF-C) of NPC cells through the nuclear factor-κB (NF-κB) signaling pathway. Our findings indicate that PEDF plays a vital role in lymphatic metastasis by targeting both lymphatic endothelial cells and NPC cells, and PEDF may represent a novel therapeutic target for NPC.Subject terms: Metastasis, Head and neck cancer     

Cancer stem cells and angiogenesis

Cancer stem cells (CSCs) or tumor initiating cells were identified and characterized as a unique subpopulation with stem cell features in many types of cancer. Current CSC studies provide novel insights regarding tumor initiation, progression, angiogenesis, resistance to therapy and interplay with the tumor micro-environment. A cancer stem cell niche has been proposed based on these findings. The niche provides the soil for CSC self-renewal and maintenance, stimulating essential signaling pathways in CSCs and leading to secretion of factors that promote angiogenesis and long term growth of CSCs. We present evidence which has emerged over the past 5 years indicating interaction of CSCs with angiogenesis in the proposed "vascular niche". Based on these findings, targeting the "cancer stem cell niche" by combining an individualized anti-CSC approach with treatment of their microenvironment may represent a novel therapeutic strategy against solid tumor systems.     

Remodeling of angiogenesis and lymphangiogenesis in cervical cancer development

The ability to stimulate angiogenesis/lymphangiogenesis is an intrinsic property of cancer cells, providing them necessary conditions for growth and metastasis. The “angiogenic switch” is one of the earliest consequences of malignant transformation; it involves altered expression of numerous genes and triggers a complex set of signaling pathways in endothelial cells. Processes of tumor microvascular network formation are closely associated with the stages of carcinogenesis (from appearance of benign lesions to invasive forms) and occur with numerous deviations from the norm. Analysis of expression of proangiogenic factors during sequential steps of cervical cancer development (intraepithelial neoplasia, cancer in situ, microinvasive, and invasive cancer) provides opportunity to reconstruct the regulatory mechanisms of angiogenesis/lymphangiogenesis with emphasis on the most important components. This review summarizes literature data on expression of key regulators of angiogenesis in cervical intraepithelial neoplasia and cervical cancer and analyses their possible involvement in molecular mechanisms of neoplastic transformation of epithelial cells, as well as invasion and tumor metastasis. Correlation between expression of proangiogenic molecular factors and various clinicopathological parameters is considered in the context of their possible use in molecular diagnostics and targeted therapy of cervical cancer. Special attention is paid to rather poorly studied regulators of lymphangiogenesis and “non-VEGF dependent,” or alternative, angiogenic pathways that constitute the prospect of future research in the field.     

Targeting vasculature in urologic tumors: mechanistic and therapeutic significance

Recent advances toward understanding the molecular mechanisms regulating cancer initiation and progression provide new insights into the therapeutic value of targeting tumor vascularity by interfering with angiogenic signaling pathways. The functional contribution of key angiogenic factors toward increased vascularity characterizing metastatic tumors and their therapeutic exploitation is considered in three major urologic malignancies, renal, bladder, and prostate cancer. With the realization that the success of the therapeutic efficacy of the various anti-angiogenic approaches for the treatment of urologic tumors has yet to be proven clinically, the challenge remains to select critical angiogenesis pathways that can be targeted for an individual tumor. Here we discuss the major mechanisms that support formation of vasculature in renal, bladder, and prostate tumors and the current results of targeting of specific molecules/regulators for therapeutic intervention against metastastic disease.     

Endothelial cells-targeted soluble human Delta-like 4 suppresses both physiological and pathological ocular angiogenesis

Due to its essential roles in angiogenesis, Notch pathway has emerged as an attractive target for the treatment of pathologic angiogenesis. Although both activation and blockage of Notch signal can impede angiogenesis, activation of Notch signal may be more promising because it was shown that long-term Notch signal blockage resulted in vessel neoplasm. However, an in vivo deliverable Notch ligand with highly efficient Notch-activating capacity has not been developed. Among all the Notch ligands, Delta-like4(Dll4) is specifically involved in angiogenesis. In this study, we generated a novel soluble Notch ligand h D4 R, which consists of the Delta-Serrate-Lag-2 fragment of human Dll4 and an arginine-glycine-aspartate(RGD) motif targeting endothelial cells(ECs). We demonstrated that h D4 R could bind to ECs through its RGD motif and effectively triggered Notch signaling in ECs. Further, we confirmed that h D4 R could suppress angiogenesis in vitro as manifested by network formation assay and sprouting assay. More importantly, h D4 R efficiently repressed neonatal retinal angiogenesis and laser-induced choroidal neovascularization(CNV) as well in vivo. In conclusion, we have developed an in vivo deliverable Notch ligand h D4 R, which suppresses angiogenesis both in vitro and in vivo, thus providing a new approach to tackle excessive angiogenesis relevant disease such as CNV.     

Preclinical Efficacy of Cystatin C to Target the Oncogenic Activity of Transforming Growth Factor β in Breast Cancer

We previously identified cystatin C (CystC) as a novel antagonist of transforming growth factor β (TGF-β) signaling in normal and malignant cells. However, whether the anti-TGF-β activities of CystC can be translated to preclinical animal models of breast cancer growth and metastasis remains unproven. Assessing the preclinical efficacy of CystC was accomplished using metastatic 4T1 breast cancer cells, whose oncogenic responses to TGF-β were inhibited both in vitro and in vivo. Indeed, we observed CystC to prevent TGF-β from stimulating the growth and pulmonary metastasis of 4T1 tumors in mice in part by reducing the extent of Smad2, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase 1/2 phosphorylation present in 4T1 tumors. We also found CystC to significantly antagonize angiogenesis in developing 4T1 tumors, suggesting a novel role for CystC in uncoupling TGF-β signaling in endothelial cells (ECs). Accordingly, CystC dramatically reduced murine and human EC responsiveness to TGF-β, including their ability to regulate the expression of 1) TGF-β signaling components, 2) inhibitor of differentiation (ID) family members, and 3) matrix metalloproteinases and their inhibitors (TIMPs) and to undergo cell invasion and angiogenic sprouting stimulated by TGF-β. Importantly, CystC prevented TGF-β from stimulating vessel development in Matrigel plugs implanted into genetically normal mice. Collectively, our findings provide the first preclinical evidence that CystC is efficacious in preventing breast cancer progression and angiogenesis stimulated by the oncogenic TGF-β signaling system and suggest that CystC-based chemotherapeutics possesses translational efficacy to one day treat and improve the clinical course of late-stage breast cancers.     

肿瘤浸润转移分子机制的研究进展

肿瘤浸润转移是多因素参与、多步骤完成的生物化学变化过程。人们已经逐渐认识到浸润转移不仅与肿瘤细胞有关,更是肿瘤细胞和肿瘤组织微环境复杂的相互作用的结果,其过程涉及多个分子作用机制和信号转导途径,包括细胞和细胞的黏附分子、细胞外基质降解、生长因子、趋化因子和淋巴血管生成因子等。本文综述了肿瘤浸润转移的分子机制。     

Tie2: a journey from normal angiogenesis to cancer and beyond

The tyrosine kinase receptor Tie2 was initially identified as a specific vascular growth factor that governed several properties of endothelial cells under both physiological and pathological conditions. It was subsequently found that angiopoietins, the natural ligands of Tie2, modulate Tie2-dependent signaling, which in turn regulates the survival and apoptosis of endothelial cells, controls vascular permeability, and regulates the capillary sprouting that occurs during normal angiogenesis such as through development and ovarian remodeling. Tie2 also seems to play a crucial role in several vascular abnormalities, such as familial venous malformations. Beyond its critical role in angiogenesis, Tie2 also appears to maintain the long-term population and quiescent status of hematopoietic stem cells in the bone marrow stem cell niche. In cancer, Tie2 was originally found to be overexpressed in tumoral vessels. More recently, our laboratory and others have found that Tie2 is also expressed outside the vascular compartment in several types of cancer, including leukemia and solid neoplasms such as gastric tumors, breast tumors, and gliomas. The role of Tie2 in these tumoral cells is currently being explored. In this regard, our group reported the importance of Tie2-expressing glioma cells in their adhesion to the tumoral microenvironment. Because cancer may be considered as a complex organ with several cellular lineages coexisting in the same tumor, the expression of Tie2 by different tumoral compartments makes this cellular receptor an attractive target for cancer therapy.