Target For Anti-angiogenic Therapy Research Articles

The role and molecular mechanism of GDF5 in regulating cell migration and angiogenesis via the Hippo-YAP signaling pathway in colorectal cancer.

292 Background: Distant metastasis is a leading cause of poor prognosis and mortality in colorectal cancer (CRC) patients, with angiogenesis playing a crucial role in tumor progression. The TGF-β superfamily has been shown to significantly influence tumor angiogenesis, yet the biological function and molecular mechanism of Growth Differentiation Factor 5 (GDF5), a member of this superfamily, remain unexplored in CRC. Given that the efficacy of current anti-angiogenic therapies is limited to a subset of patients, identifying new therapeutic targets involved in tumor angiogenesis is of great clinical importance. GDF5, with its potential role in angiogenesis regulation, presents a promising candidate to bridge this therapeutic gap. Methods: In this study, we analyzed GDF5 expression in CRC tissue samples using both the TCGA dataset and samples from our center, revealing significantly lower GDF5 expression in tumors compared to adjacent normal tissues. Moreover, GDF5 downregulation correlated with lymph node metastasis and disease recurrence. Functional assays in vitro demonstrated that GDF5 overexpression reduced tumor cell migration and invasion, while its knockdown produced the opposite effect. Co-culture assays showed that GDF5 knockdown significantly enhanced endothelial cell migration and angiogenesis, whereas overexpression diminished these effects. Additionally, F-actin cytoskeletal staining indicated that GDF5 overexpression led to cytoskeletal remodeling in endothelial cells. Mechanistically, we found that GDF5 overexpression in tumor cells downregulated the epithelial-mesenchymal transition (EMT) marker Slug and upregulated E-cadherin, while downregulating N-cadherin. In endothelial cells, GDF5 inhibited the Hippo-YAP signaling pathway, increasing YAP phosphorylation and reducing nuclear YAP levels. Conversely, GDF5 knockdown suppressed YAP phosphorylation and promoted its nuclear translocation. Results: Our findings demonstrate that GDF5 functions as a tumor suppressor in CRC by regulating cell migration and angiogenesis. This study reveals that GDF5 exerts its anti-cancer effects by modulating both EMT in tumor cells and cytoskeletal changes in endothelial cells, leading to the inhibition of the Hippo-YAP signaling pathway and reduced angiogenic potential. These results identify GDF5 as a promising new target for anti-angiogenic therapy in CRC, offering potential avenues for improving treatment strategies in patients resistant to current therapies. Conclusions: GDF5 acts as a tumor suppressor in colorectal cancer by inhibiting cell migration and angiogenesis through modulation of the Hippo-YAP signaling pathway, making it a promising therapeutic target for anti-angiogenic treatment in patients resistant to current therapies.

Immuno-PET Imaging of CD93 Expression with 64Cu-Radiolabeled NOTA-mCD93 ([64Cu]Cu-NOTA-mCD93) and Insulin-Like Growth Factor Binding Protein 7 ([64Cu]Cu-NOTA-IGFBP7).

CD93 is overexpressed in multiple solid tumor types, serving as a novel target for antiangiogenic therapy. The goal of this study was to develop a 64Cu-based positron emission tomography (PET) tracer for noninvasive imaging of CD93 expression. Antimouse-CD93 mAb (mCD93) and the CD93 ligand IGFBP7 were conjugated to a bifunctional chelator, p-isothiocyanatobenzyl-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-NOTA) and labeled with 64Cu. To evaluate the pharmacokinetic properties and tumor-targeting efficacy of [64Cu]Cu-NOTA-mCD93 and [64Cu]Cu-NOTA-IGFBP7, PET imaging and biodistribution were performed on both 4T1 murine breast tumor-bearing mice and MDA-MB-231 human breast tumor-bearing mice. The tumor model HT1080-FAP, which does not overexpress CD93, was used as a negative control. Fluorescent immunostaining was conducted on different tissues to correlate radiotracer uptake with CD93 expression. 64Cu-labeling was achieved with high yield and specific activity. Serial PET imaging revealed that the in vivo performance of [64Cu]Cu-NOTA-IGFBP7 was superior to that of [64Cu]Cu-NOTA-mCD93, and that the tracer [64Cu]Cu-NOTA-IGFBP7 exhibited elevated tumor uptake values and excellent tumor retention in MDA-MB-231 mice, rather than in 4T1 murine mice. The MDA-MB-231 tumor uptake of [64Cu]Cu-NOTA-IGFBP7 was 2.85 ± 0.15, 3.69 ± 0.60, 6.91 ± 0.88, and 6.35 ± 0.55%ID/g at 1, 4, 24, and 48 h p.i., respectively, which were significantly higher than that in the CD93-negative HT1080-FAP tumor (0.73 ± 0.15, 0.97 ± 0.31, 1.00 ± 0.07, and 1.02 ± 0.11%ID/g, respectively). The significant difference between positive and negative tumors indicated [64Cu]Cu-NOTA-IGFBP7 was specifically binding to CD93. Biodistribution data as measured by gamma counting were consistent with the PET analysis. Ex vivo histology further confirmed the high CD93 expression on MDA-MB-231 tumor tissues. Herein, we prepared two novel radiotracers, [64Cu]Cu-NOTA-mCD93 and [64Cu]Cu-NOTA-IGFBP7, for the first immune-PET imaging of CD93 expression. Our results suggest that [64Cu]Cu-NOTA-IGFBP7 is a more potential radiotracer for visualizing angiogenesis due to its sensitive, persistent, and CD93-specific characteristics.

The vasculogenic mimicry, CD146+ and CD105+ microvessel density in the prognosis of endometrioid endometrial adenocarcinoma: a single-centre immunohistochemical study

The microvessel compartment is crucial in the tumour microenvironment of endometrioid adenocarcinoma (EA). This study investigated the role of vasculogenic mimicry (VM), CD146, and CD105 microvessel density in the clinical prognosis of EA. A total of 188 EA cases were analyzed, with VM channels and microvessels detected using PAS/CD31, CD146, and CD105 staining. Mann-Whitney and Fisher exact tests were used to compare the study groups according to the evaluated criteria. ROC analysis included determination of the confidence interval (CI) and area under the ROC curve. The Mantel-Cox test was used to analyze progression-free survival. Multivariate Cox proportional hazard analysis was performed using stepwise regression. Results showed that VM channels and CD146 and CD105 microvessels were significantly higher (p < 0.0001) in cases with unfavourable prognosis. Univariate survival analysis highlighted the significant role of these factors in progression-free survival, while multivariate Cox analysis identified VM and CD146+ vessels as predictive factors. This study demonstrates, for the first time, that VM, CD146, and CD105-positive vessels are involved in EA prognosis, suggesting their potential as independent prognostic indicators and targets for antiangiogenic therapy. However, these findings require further validation through large-scale studies.

The suppression of OTUD7B by miR-491-5p enhances the ubiquitination of VEGFA to suppress vascular mimicry in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the main type of lung cancer with high morbidity and mortality. Vascular mimicry (VM), a distinct microcirculation model in tumors that differs from classical angiogenesis, is strongly associated with poor clinical outcomes in cancer patients. miR-491-5p has been reported to prevent NSCLC progression, including proliferation, metastasis, and angiogenesis. However, the effect and mechanism of miR-491-5p on VM have not been studied in NSCLC. The expression of miR-491-5p was detected by quantitative reverse transcription PCR (qPCR) and fluorescence in situ hybridization (FISH). Cell counting kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) staining assays were used to examine cell growth. Tube formation assay was used to assess VM in NSCLC cells. Immunohistochemistry (IHC) and western blot were performed to detect protein expression. Immunoprecipitation was used to confirm the interaction between OTU deubiquitinase 7B (OTUD7B) and vascular endothelial growth factor A (VEGFA), and the level of ubiquitinated VEGFA. A nude mouse tumorigenesis model was used to evaluate the carcinogenic capacity of NSCLC cells in vivo. Luciferase reporter assay was used to identify the potential target of miR-491-5p. MiR-491-5p was found downregulated in NSCLC tissues, and miR-491-5p deficiency was strongly associated with angiogenesis. miR-491-5p mimics suppressed cell viability, migration, and VM. Conversely, an inhibitor of miR-491-5p had the opposite effect. OTUD7B, a deubiquitinase, was identified as a downstream target of miR-491-5p. A luciferase reporter assay indicated that miR-491-5p directly binds to the 3'UTR of OTUD7B. Moreover, mimics of miR-491-5p caused a significant reduction in the OTUD7B protein in NSCLC cells, and an inhibitor of miR-491-5p stabilized the OTUD7B protein. In addition, overexpression of OTUD7B promoted cell proliferation, migration, and VM, similar to the effects of an inhibitor of miR-491-5p. Further exploration revealed that OTUD7B interacts with VEGFA and that the miR-491-5p-OTUD7B axis modulates the ubiquitination of VEGFA. The rescue experiment indicated that OTUD7B compromised the inhibitory effects of miR-491-5p on the cellular function of NSCLC cells. Overall, our study first proved that miR-491-5p impedes VM by suppressing OUTD7B and promoting the ubiquitination of VEGFA. The miR-491-5p/OTUD7B axis may be a novel target for antiangiogenic therapy in NSCLC.

CD93 maintains endothelial barrier function and limits metastatic dissemination.

Compromised vascular integrity facilitates extravasation of cancer cells and promotes metastatic dissemination. CD93 has emerged as a target for antiangiogenic therapy, but its importance for vascular integrity in metastatic cancers has not been evaluated. Here, we demonstrate that CD93 participates in maintaining the endothelial barrier and reducing metastatic dissemination. Primary melanoma growth was hampered in CD93-/- mice, but metastatic dissemination was increased and associated with disruption of adherens and tight junctions in tumor endothelial cells and elevated expression of matrix metalloprotease 9 at the metastatic site. CD93 directly interacted with vascular endothelial growth factor receptor 2 (VEGFR2) and its absence led to VEGF-induced hyperphosphorylation of VEGFR2 in endothelial cells. Antagonistic anti-VEGFR2 antibody therapy rescued endothelial barrier function and reduced the metastatic burden in CD93-/- mice to wild-type levels. These findings reveal a key role of CD93 in maintaining vascular integrity, which has implications for pathological angiogenesis and endothelial barrier function in metastatic cancer.

Chrysin alleviates DNA damage to improve disturbed immune homeostasis and pro-angiogenic environment in laser-induced choroidal neovascularization

Choroidal neovascularization (CNV) is a devastating pathology of numerous ocular diseases, such as wet age-related macular degeneration (wAMD), which causes irreversible vision loss. Although anti-vascular endothelial growth factor (VEGF) therapy has been widely used, poor response or no response still exists in some cases, suggesting that there are other components involved in the angiogenic process. Therefore, the underlying mechanism needs to be clarified and new target of anti-angiogenic therapy is urgently needed. It has been demonstrated that damaged retinal pigment epithelium (RPE) cells can activate inflammasome, driving a degenerative tissue environment and an enhanced pro-angiogenic response, which implies that RPE dysfunction may be a hallmark of the pathogenesis. Previously, we have shown that DNA damage can induce RPE dysfunction, triggering senescence-associated secretory phenotype (SASP) and local inflammation. In this study, we identify that chrysin can reduce DNA damage, especially telomere erosion in vitro, thus compromise the dysfunction of RPE and the decreased expression of SASP factor. Importantly, we find that DNA damage of RPE cells is remarkable in laser-induced CNV lesion, resulting in inflammatory response, which can be ameliorated by chrysin, mainly through IL-17 signaling pathway and its downstream signal transducer and activator of transcription 3 (STAT3) activities. In summary, our results indicate the interplay between DNA damage, perturbed RPE homeostasis, inflammatory response and angiogenesis in laser-induced CNV, and more importantly, chrysin may be an effective therapeutic supplement for CNV.

Golgi Protein 73 Promotes Angiogenesis in Hepatocellular Carcinoma.

Golgi protein 73 (GP73), a resident protein of the Golgi apparatus, is notably elevated in hepatocellular carcinoma (HCC). While its critical role in remodeling the tumor microenvironment (TME) is recognized, the intricate mechanisms are not fully understood. This study reveals that GP73 in HCC cells interacts with prolyl hydroxylase-2 (PHD-2) in a competitive manner, thereby impeding the hydroxylation of hypoxia-induced factor-1α (HIF-1α). The effect above promotes the production and secretion of vascular endothelial growth factor A (VEGFA). Moreover, exosomal GP73 derived from HCC cells can be internalized by human umbilical vein endothelial cells (HUVECs) and competitively interact with HECTD1, an E3 ubiquitin ligase targeting growth factor receptor-bound protein 2 (GRB2). This interaction stabilizes GRB2, thereby activating the Ras-mitogen-activated protein kinase (MAPK) signaling pathway. Consequently, escalated levels of GP73 intensify VEGF production in HCC cells and potentiate mitogenic signaling in vascular endothelial cells, fostering angiogenesis in the TME. Our findings propose that GP73 might serve as a novel target for anti-angiogenic therapy in HCC.

Bulk RNA-Seq Combined with Single-Cell Transcriptome Sequencing Reveals the Possible Mechanisms by Which HDGFL3 Involves in Prostate Cancer Growth and Metastasis.

Based on publicly available transcriptome and single-cell sequencing data, the current study aimed to explore the molecular mechanisms underlying the involvement of hepatocellular carcinoma-derived growth factor-like 3 (HDGFL3) in prostate cancer (PCA) growth and metastasis. The Gene Expression Omnibus database was used to download the single cell transcriptome of PCA (GSE193337). Single-cell RNA sequencing (scRNA-seq) data were examined to identify which genes are essential for endothelial cell function. The Cancer Genome Atlas Prostate Adenocarcinoma database provided the RNA sequencing data, and univariate COX regression analysis was introduced to identify the genes that were associated with the prognosis of patients with PCA. Human PCA cell lines PC-3 and DU145 were used in in vitro cellular studies to test the effect of silencing HDGFL3. The results were validated using Transwell® assay, scratch assay, and cell counting kit-8 assay. To support the role of HDGFL3 in PCA, an in vivo animal model of PCA transplantation tumor in nude mice was established. Quantitative reverse transcription polymerase chain reaction was introduced to measure HDGFL3 messenger ribonucleic acid (mRNA) expression levels in tumor tissues from nude mice, and Hematoxylin and Eosin staining was used to identify lung metastasis. Immunohistochemical staining was employed to identify the expression levels of HDGFL3 and hematopoietic progenitor cell antigen CD34+. It was discovered through analysis of the scRNA-seq dataset that HDGFL3, a gene specific to endothelial cells, is linked to a poor prognosis in men with PCA. In addition, HDGFL3 and the expression of genes linked to angiogenesis have a substantial association. Studies on cells in vitro revealed that silencing HDGFL3 prevented PC-3 and DU145 cells from proliferation, migrating and invading. Silencing HDGFL3 decreased the weight of prostate tumors, the number of lung metastases, and the area occupied by CD34+ vascular endothelial cells, according to in vivo investigations. This study identified HDGFL3 as a key gene in endothelial cells that may stimulate tumor angiogenesis to increase the growth and spread of PCA. These results imply that HDGFL3 may represent a possible target for antiangiogenic therapy in the management of PCA.

A review article: The mysterious pericytes

Blood vessels are composed of two types of interacting cells. Endothelial cells form the inner lining of the vessel wall, and perivascular cells referred to as pericytes, vascular smooth muscle cells or mural cells which envelop the surface of the vascular tube. They are also called Rouget cells after their discoverer, Charles Rouget. Electron-microscope analyses first revealed the morphological character of pericytes. In general, pericytes possess a cell body with a prominent nucleus and a small content of cytoplasm with several long processes embracing the abluminal endothelium wall. They are embedded within the basement membrane of microvessels, which is formed by pericytes and endothelial cells. Pericytes play an integral role in the maintenance of the blood–brain barrier as well as several other homeostatic and hemostatic functions of the brain. These cells are also a key component of the neurovascular unit, which includes endothelial cells, astrocytes, and neurons. Pericytes provide a variety of functions such as capillary blood flow regulation, clearance and phagocytosis of cellular debris, angiogenesis formation of new blood vessels and regulating blood–brain barrier permeability. Recently, pericytes have gained new attention as functional and critical involvement to tumor angiogenesis and progression. Therefore as potential new targets for antiangiogenic therapies. Pericytes are complex. Their ontogeny is not completely understood, and they perform various functions throughout the body. This review article describes the current knowledge about the nature of pericytes and their functions during blood vessel growth, vessel maintenance, and pathological angiogenesis.

Аngiogenesis in Uveal Melanoma

Uveal melanoma (UM) is less than 0.5 % in the spectrum of human tumors, and less than 5 % among all types of melanoma, therefore, it is considered to be rare. At the same time, UM is recognized as the most common intraocular malignant neoplasm. Its share among all intraocular tumors is 60 %. Radical local treatments are considered effective, but the frequency of distant metastases is unacceptably high, and the life expectancy of patients with metastatic stage of the disease is short and on average is 4–5 months. Survival rates have remained stable for the past 40 years, reflecting the lack of current effective system strategies. The tumor metastasizes in a haematogenic way, so it is not surprising that angiogenesis is constantly in the focus of scientific developments. The importance of studying angiogenesis in UM is due to the ability to predict based on the quantitative indicators of vessels inside the tumor and to search for potential targets of antiangiogenic therapy in the future. The authors used two methods of studying angiogenesis in UM: morphological with quantitative vascular counting and immunohystochemical method (IHC) with markers of endothelial cells CD34 and CD31, VIII factor, VEGF molecules, bFGF, thrombospondin and others. IHC-staining of vessels in UM allowed to visualize vessels that were not visible due to intense pigmentation of tumors or compression of vessels by tumor cells. Comparison of data obtained by the two methods demonstrated the advantages of IHS analysis over classical morphological methods. It was found that UM, as a malignant solid tumor, differs high averages of vessels per unit area. The highest rates are recorded in epitheloid melanoma, which is associated with a higher rate of growth, and more frequent metastasis, compared to similar rates in revere cell melanoma. The number of vessels per unit area in the viewing area in UM decreases with age, which explained the development of metastases in more distant after enucleation time in elderly patients. Differences in vascular density in tumors of different localization were revealed and described: they were the maximum in pre-equatorial tumors, and minimal — in iris tumors.

Dual role of WNT5A in promoting endothelial differentiation of glioma stem cells and angiogenesis of glioma derived endothelial cells.

Glioma is a devastating cancer with a rich vascular network. No anti-angiogenic treatment is available for prolonging the overall survival of glioma patients. Recent studies have demonstrated that the endothelial differentiation of glioma stem cells (GSCs) into glioma-derived endothelial cells (GDECs) may be a novel target for anti-angiogenic therapy in glioma; however, the underlying mechanisms of this process remain unknown. Here, we report that wingless-related integration site (WNT) family member 5A (WNT5A) plays significant roles in GSC endothelial differentiation and GDECs angiogenesis. WNT5A is preferentially secreted by GDECs, and inhibition of WNT5A suppresses angiogenesis and tumorigenesis in GDECs. Silencing of WNT5A in GDECs also disrupts the impact of GDECs on stimulating GSC endothelial differentiation. Frizzled-4 is a receptor that mediates the effect of WNT5A on GSC endothelial differentiation and angiogenesis of GDECs via GSK3β/β-catenin/epithelial-mesenchymal transition signalling. The shWNT5A@cRGD-DDD liposomes, targeting WNT5A, exert anti-angiogenic effects in vivo. In this study, we identified that WNT5A has a dual functional role in modulating the endothelial differentiation of GSCs and angiogenesis of GDECs, indicating that WNT5A is a potential target for anti-angiogenesis-based therapeutics in glioma.

HOX Genes Family and Cancer: A Novel Role for Homeobox B9 in the Resistance to Anti-Angiogenic Therapies.

Simple SummaryThe inhibition of angiogenesis, relying on the use of drugs targeting the VEGF signaling pathway, has become one of the main strategies for cancer treatment. However, the intrinsic and acquired resistance to this type of therapy limit its efficacy. Thus, the identification of novel therapeutic targets is urgently needed. The resistance to anti-angiogenic treatment often occurs through the activation of alternative VEGF independent signaling pathways and recruitment of bone marrow-derived pro-angiogenic cells in the tumor microenvironment. HOX genes are key regulators of embryonic development, also involved in angiogenesis and in cancer progression. HOXB9 upregulation occurs in many types of cancer and it has been identified as a critical transcription factor involved in tumour resistance to anti-angiogenic drugs. Indeed, HOXB9 modulates the expression of alternative pro-angiogenic secreted factors in the tumour microenvironment leading tumor escape from the anti-angiogenic treatments. Hence, HOXB9 could serves as a novel therapeutic target to overcome the resistance to anti-angiogenic therapies.Angiogenesis is one of the hallmarks of cancer, and the inhibition of pro-angiogenic factors and or their receptors has become a primary strategy for cancer therapy. However, despite promising results in preclinical studies, the majority of patients either do not respond to these treatments or, after an initial period of response, they develop resistance to anti-angiogenic agents. Thus, the identification of a novel therapeutic target is urgently needed. Multiple mechanisms of resistance to anti-angiogenic therapy have been identified, including the upregulation of alternative angiogenic pathways and the recruitment of pro-angiogenic myeloid cells in the tumor microenvironment. Homeobox containing (HOX) genes are master regulators of embryonic development playing a pivotal role during both embryonic vasculogenesis and pathological angiogenesis in adults. The importance of HOX genes during cancer progression has been reported in many studies. In this review we will give a brief description of the HOX genes and their involvement in angiogenesis and cancer, with particular emphasis on HOXB9 as a possible novel target for anti-angiogenic therapy. HOXB9 upregulation has been reported in many types of cancers and it has been identified as a critical transcription factor involved in resistance to anti-angiogenic drugs.

SRSF10 inhibits biogenesis of circ-ATXN1 to regulate glioma angiogenesis via miR-526b-3p/MMP2 pathway

BackgroundAngiogenesis plays an important role in the progress of glioma. RNA-binding proteins (RBPs) and circular RNAs (circRNAs), dysregulated in various tumors, have been verified to mediate diverse biological behaviors including angiogenesis.MethodsQuantitative real-time PCR (qRT-PCR) and western blot were performed to detect the expression of SRSF10, circ-ATXN1, miR-526b-3p, and MMP2/VEGFA. The potential function of SRSF10/circ-ATXN1/miR-526b-3p axis in glioma-associated endothelial cells (GECs) angiogenesis was further studied.ResultsSRSF10 and circ-ATXN1 were significantly upregulated in GECs compared with astrocyte-associated endothelial cells (AECs). Knockdown of SRSF10 or circ-ATXN1 significantly inhibited cell viability, migration and tube formation of GECs where knockdown of SRSF10 exerted its function by inhibiting the formation of circ-ATXN1. Moreover, the combined knockdown of SRSF10 and circ-ATXN1 significantly enhanced the inhibitory effects on cell viability, migration and tube formation of GECs, compared with knockdown of SRSF10 and circ-ATXN1, respectively. MiR-526b-3p was downregulated in GECs. Circ-ATXN1 functionally targeted miR-526b-3p in an RNA-induced silencing complex. Up-regulation of miR-526b-3p inhibited cell viability, migration and tube formation of GECs. Furthermore, miR-526b-3p affected the angiogenesis of GECs via negatively regulating the expression of MMP2/VEGFA.ConclusionSRSF10/circ-ATXN1/miR-526b-3p axis played a crucial role in regulating the angiogenesis of GECs. The above findings provided new targets for anti-angiogenic therapy in glioma.

Endothelial Small GTPase RhoA: A Potential Target for Anti‐angiogenic Therapy

ObjectiveTumor vessels display abnormal structure and function due to imbalanced angiogenesis. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the best known pro‐angiogenic factors in this process. The goal of antiangiogenic therapy is to inhibit tumor growth by oxygen and nutrient deprivation. Current antiangiogenic therapies target VEGF/VEGFR signaling pathways. However development of resistance due to upregulation of bFGF remains a serious disadvantage of current approaches, leading to efforts of simultaneous targeting of both VEGF and FGF pathways. A small GTPase RhoA has been reported to regulate VEGF‐induced angiogenesis. In the present study we investigate the role of endothelial RhoA in bFGF‐induced angiogenesis to identify whether RhoA can be considered a target for both VEGF‐ and FGF‐induced angiogenesis.MethodsWe performed RhoA pulldown assays to identify RhoA activation downstream of bFGF treatment. We used Boyden chamber cell migration assay, 2D tube formation assay using matrigel and 3D sprouting assay with collagen to assess angiogenic potential. Endothelial RhoA was knocked down by siRNAs or blocked by pharmacological inhibitors. Biochemical assays were performed to identify the molecular mechanism of bFGF‐induced RhoA activation. In vivo, endothelial‐specific inducible RhoA‐deficient mice with a fluorescent reporter were used in a newly modified matrigel plug angiogenesis model allowing identification of endothelial RhoA excision efficiency.ResultsbFGF treatment activates RhoA in human umbilical vein endothelial Cells (HUVEC). RhoA inhibition with C3 toxin or RhoA knockdown abrogated bFGF‐induced endothelial cell migration and tube formation in vitro. In vivo, endothelial RhoA deficiency blocked bFGF‐induced angiogenesis in matrigel plug assay. Among the bFGF receptors, FGFR1 is predominantly expressed in HUVECs and FGFR1 knockdown abrogated bFGF‐induced RhoA activation and angiogenesis. RhoA inhibition partially blocked bFGF‐induced JNK phosphorylation but did not affect ERK and p38 activation. Ongoing experiments are aiming to delineate the precise role of endothelial RhoA in the FGF downstream pathway.ConclusionCollectively, our data suggest that RhoA pathway participates in bFGF‐induced angiogenesis and can be a potential target for anti angiogenic therapy.

YKL-39 as a Potential New Target for Anti-Angiogenic Therapy in Cancer.

YKL-39 belongs to the evolutionarily conserved family of Glyco_18-containing proteins composed of chitinases and chitinase-like proteins. Chitinase-like proteins (CLPs) are secreted lectins that lack hydrolytic activity due to the amino acid substitutions in their catalytic domain and combine the functions of cytokines and growth factors. One of the major cellular sources that produce CLPs in various pathologies, including cancer, are macrophages. Monocytes recruited to the tumor site and programmed by tumor cells differentiate into tumor-associated macrophages (TAMs), which are the primary source of pro-angiogenic factors. Tumor angiogenesis is a crucial process for supplying rapidly growing tumors with essential nutrients and oxygen. We recently determined that YKL-39 is produced by tumor-associated macrophages in breast cancer. YKL-39 acts as a strong chemotactic factor for monocytes and stimulates angiogenesis. Chemotherapy is a common strategy to reduce tumor size and aggressiveness before surgical intervention, but chemoresistance, resulting in the relapse of tumors, is a common clinical problem that is critical for survival in cancer patients. Accumulating evidence indicates that TAMs are essential regulators of chemoresistance. We have recently found that elevated levels of YKL-39 expression are indicative of the efficiency of the metastatic process in patients who undergo neoadjuvant chemotherapy. We suggest YKL-39 as a new target for anti-angiogenic therapy that can be combined with neoadjuvant chemotherapy to reduce chemoresistance and inhibit metastasis in breast cancer patients.

Exosomes derived from 5-fluorouracil-resistant colon cancer cells are enriched in GDF15 and can promote angiogenesis.

Background: Angiogenesis is important for tumor proliferation and distant metastasis. However, the role of drug-resistant tumor cells in angiogenesis remains largely unknown. Current anti-angiogenic strategies also have limitations and it would be useful to develop novel targets and treatment strategies.Methods: Differential ultracentrifugation was used to isolate conditioned medium-derived exosomes from 5-flurouracil (5-FU)-sensitive or -resistant colon cancer cells. Exosome endocytosis into human umbilical vein endothelial cells was observed via immunofluorescence. Differentially expressed proteins in the exosomes were confirmed via qRT-PCR and Western blotting. The angiogenic capacity of endothelial cells was evaluated using cell function assays and a rat model of abdominal aortic neovascularization. The underlying mechanisms were verified using qRT-PCR and Western blotting assays. Immunohistochemistry was used to evaluate in vivo angiogenesis.Results: We observed that the conditioned medium and exosomes from 5-FU-resistant colon cancer cells could promote angiogenesis. Exosomal growth/differentiation factor 15 (GDF15) was a potent inducer of this angiogenesis in vitro by inhibiting the Smad signaling pathway, thus increasing periostin (POSTN) levels. Moreover, 5-FU-resistant colon cancer cells showed high microvascular density in vivo. TGF-β1, an activator of the Smad signaling pathway, could partly eliminate those effects.Conclusions: Our study reveals the molecular regulation of angiogenesis in 5-FU-resistant colon cancer and suggests that the GDF15-POSTN axis may be a novel target for anti-angiogenic therapies in colon cancer.

Evaluation of Changes in the Expression Pattern of EDIL3 in Different Grades of Endometrial Cancer.

Background: EDIL3 is an extracellular matrix protein that plays a key role in angiogenesis. Changes in the pattern of its expression also affect cellular processes and the tumor microenvironment. Elevated level of EDIL3 is considered an unfavorable prognostic marker of survival.Objective: The aim of this study was to evaluate the changes in EDIL3 expression in endometrial cancer at various degrees of its differentiation (G1-G3) and to discuss its potential role as a molecular diagnostic marker and therapeutic target.Methods: The study group consisted of 45 patients with endometrial cancer: G1, 17; G2, 15; G3, 13. The control group (C) included 15 patients without neoplastic changes. The expression of EDIL3 was assessed using immunohistochemistry. Statistical analysis was performed using the Statistica 12 PL software (p<0.05).Results: Analysis of EDIL3 expression showed that the average optical density of the reaction product in G1 reached 130% of the control, while the values in G2 and G3 were 153% and 158%, respectively. Regardless of the endometrial cancer grade, an increase in EDIL3 level was observed compared to the control.Conclusion: In our study, we demonstrated overexpression of EDIL3 protein in endometrial cancer. Differences in expression between degrees of tumor differentiation suggest the potential of using changes in EDIL3 level as a new complementary diagnostic marker and target for anti-angiogenic therapy.

Mammalian Eps15 homology domain 1 potentiates angiogenesis of non-small cell lung cancer by regulating \u03b22AR signaling

BackgroundNon-small cell lung cancer (NSCLC) is a devastating disease with a heterogeneous prognosis, and the molecular mechanisms underlying tumor progression remain elusive. Mammalian Eps15 homology domain 1 (EHD1) plays a promotive role in tumor progression, but its role in cancer angiogenesis remains unknown. This study thus explored the role of EHD1 in angiogenesis in NSCLC.MethodsThe changes in angiogenesis were evaluated through human umbilical vein endothelial cell (HUVEC) proliferation, migration and tube formation assays. The impact of EHD1 on β2-adrenoceptor (β2AR) signaling was evaluated by Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR) analysis, and enzyme-linked immunosorbent assay (ELISA). The interaction between EHD1 and β2AR was confirmed by immunofluorescence (IF) and coimmunoprecipitation (Co-IP) experiments, and confocal microscopy immunofluorescence studies revealed that β2AR colocalized with the recycling endosome marker Rab11, which indicated β2AR endocytosis. Xenograft tumor models were used to investigate the role of EHD1 in NSCLC tumor growth.ResultsThe microarray analysis revealed that EHD1 was significantly correlated with tumor angiogenesis, and loss- and gain-of-function experiments demonstrated that EHD1 potentiates HUVEC proliferation, migration and tube formation. EHD1 knockdown inhibited β2AR signaling activity, and EHD1 upregulation promoted vascular endothelial growth factor A (VEGFA) and β2AR expression. Interestingly, EHD1 interacted with β2AR and played a novel and critical role in β2AR endocytic recycling to prevent receptor degradation. Aberrant VEGFA or β2AR expression significantly affected EHD1-mediated tumor angiogenesis. The proangiogenic role of EHD1 was confirmed in xenograft tumor models, and immunohistochemistry (IHC) analysis confirmed that EHD1 expression was positively correlated with VEGFA expression, microvessel density (MVD) and β2AR expression in patient specimens.ConclusionCollectively, the data obtained in this study suggest that EHD1 plays a critical role in NSCLC angiogenesis via β2AR signaling and highlight a potential target for antiangiogenic therapy.

Gain of affinity for VEGF165 binding within the VEGFR2/NRP1 cellular complex detected by an HTRF-based binding assay

Neuroplin 1 (NRP1), a transmembrane protein interacting with Vascular Endothelial Growth Factor VEGF-A165 (called here VEGF165) and the tyrosine kinase Receptor 2 (VEGFR2) promote angiogenesis and vascular homeostasis. In a pathophysiological context, several studies suggested that VEGFR2 and NRP1 mediate tumor development and progression. Given the involvement of the VEGF165 network in promoting tumor angiogenesis, NRP1, VEGFR2 and VEGF165 have been identified as targets for anti-angiogenic therapy. No binding assay exists to monitor specifically the binding of VEGF165 to the VEGFR2/NRP1 complex in intact cells. We established a binding assay based on the homogenous time-resolved fluorescence (HTRF®) technology. This unique binding assay enables to assess the interaction of VEGF165 with VEGFR2 or NRP1 within the VEGFR2/NRP1 complex. Ligand binding saturation experiments revealed that VEGF165 binds the VEGFR2/NRP1 complex at the cell surface with a ten to twenty-fold higher affinity compared to SNAP-VEGFR2 or SNAP-NRP1 receptors alone not engaged in the heteromeric complex. The assay allows characterizing the impact of NRP1 ligands on VEGF165 to the complex. It shows high specificity, reproducibility and robustness, making it compatible with high throughput screening (HTS) applications for identifying new VEGF165 antagonists selective for NRP1 or the VEGFR2/NRP1 complex.

308 EGFR Gene Amplification Predicts a Poor Response to Bevacizumab in Recurrent Glioblastoma

Abstract INTRODUCTION The highly vascular malignant brain tumor glioblastoma multiforme (GBM) appears to be an ideal target for anti-angiogenic therapy; however, clinical trials to date suggest the VEGF antibody bevacizumab affects only progression free survival without any benefit on overall survival. Here we analyze a group of patients with GBM who received bevacizumab treatment and are stratified according to tumor molecular and genomic profile (TCGA classification), with the goal of identifying molecular predictors of the response to bevacizumab. METHODS We performed a retrospective review of patients with a diagnosis of glioblastoma multiforme who were treated with bevacizumab in the recurrent setting at Memorial Sloan-Kettering Cancer Center, from 2006 to 2014. Treatment was discontinued by individual neuro-oncologists, based on clinical and radiographic criteria. Pre- and post- treatment imaging and genomic subtype were available on 83 patients; We analyzed clinical progression, and time to radiographic progression (rP) on MRI flair and T1 weighted images with contrast, quantified tumor volumes as well as the development of multifocal lesions. EGFR gene amplification and MGMT promotor methylation status were determined by FISH. RESULTS &gt;EGFR gene amplification was significantly associated with a shorter time to progression both in univariate (P = &lt; 0.001) and multivariate analysis (P = 0.011). Patients with classical tumors had a significantly shorter time to progression than mesenchymal and proneural patients (2.5 vs 6.32 and 5.2 months respectively, P = 0.001), although it lost its significance (P = 0.262) on multivariate analysis when correcting for EGFR status. Overall survival was significantly shorter in patients with EGFR- amplified GBM treated with bevacizumab. CONCLUSION EGFR gene amplification and classical subtype by TCGA analysis are associated with significantly shorter time to progression for patients with recurrent GBM when treated with bevacizumab. These findings can have a significant impact on decision making and should be further validated prospectively.