angiogenin and Breast-Neoplasms

Antiangiogenesis is a promising approach to cancer therapy but is limited by the lack of tumor-homing capability of the current antiangiogenic agents. Angiogenin, a protein overexpressed and secreted by tumors to trigger angiogenesis for their growth, has never been explored as an antiangiogenic target in cancer therapy. Here it is shown that filamentous fd phage, as a biomolecular biocompatible nanofiber, can be engineered to become capable of first homing to orthotopic breast tumors and then capturing angiogenin to prevent tumor angiogenesis, resulting in targeted cancer therapy without side effects. The phage is genetically engineered to display many copies of an identified angiogenin-binding peptide on its side wall and multiple copies of a breast-tumor-homing peptide at its tip. Since the tumor-homing peptide can be discovered and customized virtually toward any specific cancer by phage display, the angiogenin-binding phages are thus universal "plug-and-play" tumor-homing cancer therapeutics.

Topics: Bacteriophage M13; Breast Neoplasms; Cell Line, Tumor; Genetic Engineering; Humans; Molecular Targeted Therapy; Neovascularization, Pathologic; Peptide Library; Ribonuclease, Pancreatic

Angiogenin (ANG) is a secreted ribonuclease (RNase) with cell-type- and context-specific roles in growth, survival, and regeneration. Although these functions require receptor-mediated endocytosis and appropriate subcellular localization, the identity of the cell surface receptor remains undefined. Here, we show that plexin-B2 (PLXNB2) is the functional receptor for ANG in endothelial, cancer, neuronal, and normal hematopoietic and leukemic stem and progenitor cells. Mechanistically, PLXNB2 mediates intracellular RNA processing that contribute to cell growth, survival, and regenerative capabilities of ANG. Antibodies generated against the ANG-binding site on PLXNB2 restricts ANG activity in vitro and in vivo, resulting in inhibition of established xenograft tumors, ANG-induced neurogenesis and neuroprotection, levels of pro-self-renewal transcripts in hematopoietic and patient-derived leukemic stem and progenitor cells, and reduced progression of leukemia in vivo. PLXNB2 is therefore required for the physiological and pathological functions of ANG and has significant therapeutic potential in solid and hematopoietic cancers and neurodegenerative diseases.

Topics: Animals; Breast Neoplasms; Cell Proliferation; Female; Glioblastoma; Hematopoietic Stem Cells; Heterografts; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Nerve Tissue Proteins; Neurogenesis; Ribonuclease, Pancreatic

Angiogenesis is the process of new blood vessel formation, regulated by a number of pro- and antiangiogenic factors and usually begins in response to hypoxia. Exogenous administration of melatonin has shown numerous anti-tumor effects and appears to inhibit tumor angiogenesis. However, many factors involved in the anti-angiogenic effect of melatonin are still under investigation. Here, we evaluate the effects of melatonin on cell viability and expression of angiogenic factors in MCF-7 and MDA-MB-231 breast cancer cells under hypoxic conditions. Cell viability was investigated by MTT and gene and protein expression of the hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF-A) were verified by qPCR and immunocytochemistry after melatonin treatment (1 mM) under hypoxic conditions. Additionally, a protein array with 20 different cytokines/factors was performed on tumor cell lysates. The results showed that 1 mM of melatonin reduced the viability of MCF-7 and MDA-MB-231 cells (p < .05). This treatment also decreased both gene and protein expression of HIF-1α and VEGF-A under hypoxic conditions (p < .05). Among the proteins evaluated by protein array, melatonin treatment during hypoxia reduced VEGF-C, VEGFR receptors (VEGFR2 and VEGFR3), matrix metalloproteinase 9 (MMP9) and Angiogenin in MCF-7 cells. In MDA-MB-231 cells, a significant decrease was observed in VEGFR2, epidermal growth factor receptor (EGFR) and Angiogenin (p < .05). Taken together, these results showed that melatonin acts in the regulation of angiogenic factors in breast tumor cells and suggests an anti-angiogenic activity, particularly under hypoxic conditions.

Topics: Angiogenesis Inhibitors; Antioxidants; Breast Neoplasms; Cell Hypoxia; Cell Survival; Cytokines; ErbB Receptors; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Matrix Metalloproteinase 9; MCF-7 Cells; Melatonin; Neovascularization, Pathologic; Ribonuclease, Pancreatic; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor C; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factor Receptor-3

Sex hormones and their receptors play critical roles in the development and progression of the breast and prostate cancers. Here we report that a novel type of transfer RNA (tRNA)-derived small RNA, termed Sex HOrmone-dependent TRNA-derived RNAs (SHOT-RNAs), are specifically and abundantly expressed in estrogen receptor (ER)-positive breast cancer and androgen receptor (AR)-positive prostate cancer cell lines. SHOT-RNAs are not abundantly present in ER(-) breast cancer, AR(-) prostate cancer, or other examined cancer cell lines from other tissues. ER-dependent accumulation of SHOT-RNAs is not limited to a cell culture system, but it also occurs in luminal-type breast cancer patient tissues. SHOT-RNAs are produced from aminoacylated mature tRNAs by angiogenin-mediated anticodon cleavage, which is promoted by sex hormones and their receptors. Resultant 5'- and 3'-SHOT-RNAs, corresponding to 5'- and 3'-tRNA halves, bear a cyclic phosphate (cP) and an amino acid at the 3'-end, respectively. By devising a "cP-RNA-seq" method that is able to exclusively amplify and sequence cP-containing RNAs, we identified the complete repertoire of 5'-SHOT-RNAs. Furthermore, 5'-SHOT-RNA, but not 3'-SHOT-RNA, has significant functional involvement in cell proliferation. These results have unveiled a novel tRNA-engaged pathway in tumorigenesis of hormone-dependent cancers and implicate SHOT-RNAs as potential candidates for biomarkers and therapeutic targets.

Topics: Amino Acids; Animals; Base Sequence; Bombyx; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Epithelial Cells; Female; Gene Knockdown Techniques; Gonadal Steroid Hormones; Humans; Hydroxylation; Male; Models, Biological; Molecular Sequence Data; Phosphates; Prostatic Neoplasms; Real-Time Polymerase Chain Reaction; Receptors, Androgen; Receptors, Estrogen; Ribonuclease, Pancreatic; RNA, Transfer; Sequence Analysis, RNA

Therapeutic strategies for targeting angiogenesis have been proven as successful treatments for divergent cancers. We previously discovered an anti-angiogenic miR-542-3p, which directly targeted the key angiogenesis-promoting protein Angiopoietin-2 to inhibit tumor angiogenesis in breast cancer models. In this study, to further investigate the mechanism of miR-542-3p induced angiogenic inhibition, we screened for tumor cell derived factors which were responsible for miR-542-3p alteration in endothelial cells. We found that tumor cell-derived angiogenin downregulated miR-542-3p in endothelial cells. Overexpression of angiogenin in tumor cells facilitated angiogenic activation in both in vitro and in vivo models via inhibition of miR-542-3p. Furthermore, our results showed that angiogenin could suppress CEBPB and POU2F1, which were transcription factors for miR-542-3p, suggesting a novel tumor cell-endothelial cell signal pathway. In addition, the level of angiogenin in primary breast carcinomas correlated with clinical progression. Serum levels of angiogenin were associated with metastatic development of breast cancer patients. Together, these findings reveal a novel regulatory pathway whereby tumor-derived angiogenin directly activates angiogenesis through inhibition of miR-542-3p, suggesting that angiogenin may represent a promising target for anti-angiogenic therapy and a potential marker for monitoring disease progression.

Topics: Animals; Breast Neoplasms; CCAAT-Enhancer-Binding Protein-beta; Cell Line, Tumor; Culture Media, Conditioned; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; HEK293 Cells; Human Umbilical Vein Endothelial Cells; Humans; Mice, Inbred BALB C; Mice, Nude; MicroRNAs; Neovascularization, Physiologic; Octamer Transcription Factor-1; Paracrine Communication; Ribonuclease, Pancreatic; Signal Transduction; Time Factors; Transcription, Genetic; Transfection; Tumor Burden

Angiogenin (ANG), a 14-kDa pro-angiogenic secreted protein, has been shown to play a role in cell migration and tumor invasion, which involve proteolytic cleavage of plasminogen to generate plasmin. However, the mechanism by which ANG regulates plasmin formation and cell migration was not known. Our studies here detected elevated levels of secreted and cell surface-bound ANG in highly invasive metastatic breast cancer cells. ANG was also detected at very high levels in the tumor cells in infiltrating ductal carcinomas. By immunofluorescence and immunoprecipitation analysis, ANG was detected at the leading edges of the cell surfaces where it colocalized and interacted with members of the plasminogen activation system (PAS) such as annexin A2 (A2), calpactin (S100-A10) and urokinase plasminogen activator receptor (uPAR). Analysis of lipid raft (LR) and non-lipid raft (NLR) regions of the cell membranes showed the predominance of ANG, A2 and S100-A10 in the LR regions. In contrast, uPAR was detected predominantly in the NLR fractions, suggesting that ANG interacts with uPAR at the junctions of LR and NLR regions. ANG knockdown in T47D and MDA-MB-231 breast cancer cell lines did not affect the cellular expression of A2, S100-A10 and uPAR but decreased cell migration and plasmin formation. Neutralization of ANG with monoclonal antibodies similarly decreased the migration of MDA-MB-231 cells. In the presence of ANG, uPAR was observed to interact with uPA, which is necessary for plasmin formation. Conversely, in the absence of ANG, uPAR did not interact with uPA and FAK and Src kinases were observed to be dephosphorylated. Exogenous addition of recombinant ANG to ANG knocked down MDA-MB-231 cells restored FAK phosphorylation, uPAR interactions with uPA, plasmin formation as well as migration of these cells. Taken together, our results identified a novel role for ANG as a member of the uPAR interactome that facilitates the interaction of uPAR with uPA, leading to plasmin formation and cell migration necessary for tumor invasion and metastasis of breast cancer cells.

Topics: Breast; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Female; Humans; Neoplasm Invasiveness; Plasminogen; Protein Interaction Maps; Receptors, Urokinase Plasminogen Activator; Ribonuclease, Pancreatic; Urokinase-Type Plasminogen Activator

Nuclear translocation of angiogenin (ANG) is essential for the proliferation of its target cells. ANG promotes rRNA synthesis, while whether it regulates mRNA transcription remains unknown. Using the chromatin immunoprecipitation method, we have identified 12 ANG-binding sequences. One of these sequences lies in the estrogen receptor-related receptor gamma (ERRγ) gene which we designated as ANG-Binding Sequence within ERRγ (ABSE). ABSE exhibited ANG-dependent repressor activity in the luciferase reporter system. Down-regulation of ANG increased ERRγ expression, and active gene marker level at the ABSE region. The expression levels of ERRγ targets genes, p21(WAF/CIP) and p27(KIP1), and the occupation of ERRγ on their promoter regions were increased in ANG-deficient cells accordingly. Furthermore, knockdown of ERRγ promoted the proliferation rate in ANG-deficient breast cancer cells. Finally, immunohistochemistry staining showed negative correlation between ANG and ERRγ in breast cancer tissue. Altogether, our study provides evidence that nuclear ANG directly binds to the ABSE of ERRγ gene and inhibits ERRγ transcription to promote breast cancer cell proliferation.

Topics: Base Sequence; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromatin Immunoprecipitation; Down-Regulation; Female; Gene Expression Regulation, Neoplastic; Histones; Humans; Immunohistochemistry; Male; Protein Binding; Protein Processing, Post-Translational; Receptors, Estrogen; Ribonuclease, Pancreatic; RNA Polymerase II; Transcription, Genetic

Vascular endothelial growth factor (VEGF) is an important signaling protein and a predominant mediator of angiogenesis in tumor growth and metastasis. Therefore, antagonism of the VEGF pathway results in inhibition of abnormal angiogenesis, then suppression of tumor growth and metastasis. VEGF-Trap, a high-affinity soluble decoy receptor, is currently in phase II clinical trails, and has demonstrated more efficacy in different types of solid tumors by intravenous injection every two weeks. In our study, we used recombinant AAV2 as a delivery vehicle to achieve long-lasting expression of VEGF Trap protein in a mouse model for the first time. We report that AAV2-VEGF-Trap can be safely administered and sustained expression in vivo via a single intravenously administration, simultaneously suppressing primary tumor growth and preventing the pulmonary metastases of 4T1 tumors. Decreased microvessel density and increased tumor cell apoptosis were observed in the treatment group. AAV2-VEGF-Trap can obviously decrease not only the concentration of VEGF in sera, but also the concentration of other angiogenic factors, such as aFGF, bFGF, angiopoietin-1 and others. These studies suggest that AAV-mediated long-term expression of VEGF-Trap is a useful and safe tool to block tumor progression and inhibit spontaneous pulmonary metastases.

Topics: Animals; Apoptosis; Breast Neoplasms; Dependovirus; Disease Models, Animal; Female; Gene Transfer Techniques; Genetic Therapy; Injections, Intravenous; Lung Neoplasms; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; Receptors, Vascular Endothelial Growth Factor; Recombinant Fusion Proteins; Ribonuclease, Pancreatic; Thymidine Phosphorylase; Vascular Endothelial Growth Factor A

Tumoural angiogenesis is essential for the growth and spread of breast cancer cells. Therefore the aim of this study was to assess the diagnostic performance of angiogenesis markers in tumours and there reflecting levels in serum of breast cancer patients. Angiogenin, Ang2, fibroblast growth factor basic, intercellular adhesion molecule (ICAM)-1, keratinocyte growth factor (KGF), platelet-derived growth factor-BB, and VEGF-A were measured using a FASTQuant angiogenic growth factor multiplex protein assay. We observed that breast cancer tumours exhibited high levels of PDGF-BB, bFGF and VEGF, and extremely high levels of TIMP-1 and Ang-2, whereas in serum we found significantly higher levels of Ang-2, PDGF-BB, bFGF, ICAM-1 and VEGF in patients with breast cancer compared to the benign breast diseases patients. Moreover, some of these angiogenesis markers evaluated in tumour and serum of breast cancer patients exhibited association with standard clinical parameters, ER status as well as MVD of tumours. Angiogenesis markers play important roles in tumour growth, invasion and metastasis. Our results suggest that analysis of angiogenesis markers in tumour and serum of breast cancer patients using multiplex protein assay can improve diagnosis and prognosis in this diseases.

Topics: Adult; Aged; Aged, 80 and over; Angiopoietin-2; Becaplermin; Biomarkers, Tumor; Breast Neoplasms; Female; Fibroblast Growth Factor 7; Fibroblast Growth Factors; Humans; Intercellular Adhesion Molecule-1; Middle Aged; Neovascularization, Pathologic; Prognosis; Proto-Oncogene Proteins c-sis; Ribonuclease, Pancreatic; Vascular Endothelial Growth Factor A

Angiogenin, a 14.2-kDa polypeptide member of the RNase A superfamily, has potent angiogenic effects. Nuclear accumulation of angiogenin is essential for its angiogenic activity. Increased angiogenin expression has been associated with the transition of normal breast tissue into invasive breast carcinoma. In this article, we investigated whether estradiol (E(2)) affected angiogenin in breast tissue.. We used microdialysis for sampling of extracellular angiogenin in vivo. In vitro cultures of whole normal breast tissue, breast cancer cells, and endothelial cells were used.. We show that extracellular angiogenin correlated significantly with E(2) in normal human breast tissue in vivo and that exposure of normal breast tissue biopsies to E(2) stimulated angiogenin secretion. In breast cancer patients, the in vivo angiogenin levels were significantly higher in tumors compared with the adjacent normal breast tissue. In estrogen receptor-positive breast cancer cells, E(2) increased and tamoxifen decreased angiogenin secretion. Moreover, E(2)-induced angiogenin derived from cancer cells significantly increased endothelial cell proliferation. Tamoxifen reversed this increase as well as inhibited nuclear translocation of angiogenin. In vivo, in experimental breast cancer, tamoxifen decreased angiogenin levels and decreased angiogenesis. Additionally, treating tumor-bearing mice with an antiangiogenin antibody resulted in tumor stasis, suggesting a role for angiogenin in estrogen-dependent breast cancer growth.. Our results suggest previously unknown mechanisms by which estrogen and antiestrogen regulate angiogenesis in normal human breast tissue and breast cancer. This may be important for estrogen-driven breast cancer progression and a molecular target for therapeutic interventions.

Topics: Adult; Aged; Aged, 80 and over; Animals; Breast Neoplasms; Cell Nucleus; Cell Proliferation; Disease Models, Animal; Estradiol; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Microdialysis; Middle Aged; Neoplasm Transplantation; Neovascularization, Pathologic; Ribonuclease, Pancreatic; Tamoxifen; Tumor Cells, Cultured; Xenograft Model Antitumor Assays; Young Adult

The study which we performed was to determine serum concentrations of angiogenic factors including VEGF, angiogenin and TGF-beta 1 in early stage breast cancer patients. These parameters were measured by ELISA in sera of 90 patients with breast cancer and 75 healthy controls. The mean serum VEGF concentration in patients compared to controls was not significantly different (0.33ng/mL vs 0.43ng/mL, respectively; p=0.156). Likewise, the insignificant change in mean values in patients vs controls was also observed for serum TGF-beta 1 (0.19ng/mL vs 0.19ng/mL, respectively; p=0.215) and serum angiogenin (243.24ng/mL vs 244.5ng/mL, respectively; p=0.976). Statistically significant correlation was found only between the tests, such as VEGF and angiogenin in patients who were included in the study (p<0.001). In conclusion, we couldn't find any diagnostic value between the early stage breast cancer and the three angiogenic parameters.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Female; Humans; Middle Aged; Ribonuclease, Pancreatic; ROC Curve; Sensitivity and Specificity; Transforming Growth Factor beta1; Vascular Endothelial Growth Factor A

Angiogenin is a potent angiogenic factor that is known to play an important role in tumor angiogenesis. In this paper, we investigate the cellular internalization of angiogenin conjugated with its highly specific aptamer. By using fluorophore-labeled aptamer and confocal laser scanning microscopy, we have developed a novel and simple method by which to visualize the real-time process of angiogenin internalization. Specifically, when aptamer-angiogenin conjugates were added into cell cultures, conjugates could be selectively bound to HUVE cells (human umbilical vein endothelial cells) and MCF-7 cells (human breast cancer cells). Nuclear staining and Z-axis scanning studies demonstrated that the aptamer-angiogenin conjugates were internalized to intracellular organelles, and dynamic confocal imaging studies indicated that the conjugates were quickly internalized. These results provide the first evidence that a fluorophore-labeled aptamer can be used as a fluorescent probe to visualize the spatiotemporal process of protein internalization in real time.

Topics: Animals; Aptamers, Nucleotide; Breast Neoplasms; Cell Line, Tumor; Chlorocebus aethiops; COS Cells; Endothelial Cells; Fluorescent Dyes; Humans; Microscopy, Confocal; Microscopy, Fluorescence; Ribonuclease, Pancreatic; SELEX Aptamer Technique

Angiogenin, a 14.2 kD polypeptide that was originally noted for its angiogenic activity, is now increasingly recognized to have a multiplicity of biological roles in both physiological and pathological conditions. In breast cancer, there are conflicting studies questioning the role of angiogenin. Here, the pattern of expression of angiogenin during the transition from normal breast tissue to ductal carcinoma in situ and invasive carcinoma is reported together with the correlates between the level of angiogenin in 239 invasive carcinomas and standard clinicopathological parameters, hypoxia-inducible factor (HIF)-1 alpha and the HIF-1 alpha target gene DEC-1. This study shows that angiogenin expression is up-regulated in the cytoplasmic and nuclear compartments in in situ carcinoma and invasive carcinoma compared with normal breast tissue and that angiogenin expression in invasive carcinomas is significantly positively associated with high tumour grade (p = 0.03), positive oestrogen receptor (ER) status (p = 0.01), HIF-1 alpha (p = 0.001) and DEC 1 (p = 0.001), but not with patient age (p = 0.8), tumour size (p = 0.25), lymph node status (p = 0.69), epidermal growth factor receptor (p = 0.56) or microvessel density (p = 0.32). No difference in relapse-free (p = 0.26) or overall (p = 0.63) survival was observed in patients stratified by angiogenin expression. This study suggests that angiogenin may be important in breast cancer progression and that, through its relationship with ER, it may be a target for tamoxifen.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Cell Hypoxia; Cell Nucleus; Cytoplasm; Disease Progression; DNA-Binding Proteins; Female; Humans; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Lymphatic Metastasis; Middle Aged; Neoplasm Proteins; Neovascularization, Pathologic; Nuclear Proteins; Receptors, Estrogen; Ribonuclease, Pancreatic; Survival Analysis; Transcription Factors; Up-Regulation

DEC1, also known as SHARP-2 or Stra13, plays important roles in embryonic development, proliferation, apoptosis and cell differentiation in the mouse. DEC1 was recently identified as hypoxically induced in cDNA microarray studies of the human renal carcinoma cell line RCC4, to be regulated through hypoxia-inducible factor (HIF)-1alpha and via HIF-1alpha, able to block adipocyte differentiation. Nevertheless, its distribution and role in hypoxia and differentiation in human breast cancer are unknown. We therefore examined the pattern and level of expression of DEC1 using immunohistochemistry in whole tissue sections in normal, in situ and invasive breast carcinomas, and correlated the level of expression of DEC1 and clinicopathological factors and hypoxic tumour markers in 253 invasive carcinomas on tissue microarrays. We observed an increase in DEC1 expression during progression from normal to in situ and invasive carcinoma. Expression was not restricted to the tumour cell element but was also observed in endothelial, fibroblasts and inflammatory cells. There was a significant positive correlation between DEC1 and tumour grade (P=0.01), HIF-1alpha (P=0.04) and the hypoxically regulated gene angiogenin (P<0.0001), but no significant associations were observed with patient age (P=0.15), lymph node status (P=0.8), tumour size (P=0.3), oestrogen receptor (P=0.45), epidermal growth factor receptor (P=0.27) or Chalkley vessel count (P=0.45). There was no difference in relapse-free (P=0.84) or overall (P=0.78) survival. These findings suggest that DEC1 plays an important role in the progression to invasive breast cancer and that it may provide a mechanism by which hypoxia blocks tumour differentiation, and may contribute to a more aggressive phenotype. Reversing this phenotype may alter the biological behaviour of individual tumours.

Topics: Adult; Basic Helix-Loop-Helix Transcription Factors; Biomarkers, Tumor; Breast; Breast Neoplasms; Carcinoma in Situ; Cell Transformation, Neoplastic; Disease Progression; Female; Homeodomain Proteins; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; Middle Aged; Prognosis; Ribonuclease, Pancreatic; Transcription Factors; Tumor Suppressor Proteins

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis and vascular permeability. We hypothesized that malignant pleural effusions may contain high levels of VEGF protein as well as other cytokines implicated in these processes. Pleural effusions cytologically proven to be malignant were collected from 39 patients with various types of cancer, and VEGF, interleukin-8, and angiogenin levels in the effusions were determined by immunoassay. Negative controls were nonmalignant ascites and serum samples from healthy individuals. VEGF levels were significantly higher than those of control samples in pleural effusions secondary to breast, mesothelioma, and non-small cell lung cancer and when all malignant pleural effusion samples were pooled. Neither interleukin-8 nor angiogenin levels were elevated in malignant pleural effusions relative to the control samples. Vascular permeability, which was measured by using the Miles assay in nude mice, was increased proportionately with VEGF levels in the malignant pleural effusions; this increase in permeability induced by injection of recombinant VEGF or the malignant effusions was reduced by pretreating the mice with a VEGF receptor antibody.

Topics: Angiogenesis Inducing Agents; Animals; Breast Neoplasms; Capillary Permeability; Carcinoma, Non-Small-Cell Lung; Endothelial Growth Factors; Female; Humans; Interleukin-8; Lung Neoplasms; Lymphokines; Lymphoma; Male; Mesothelioma; Mice; Mice, Nude; Pleural Effusion, Malignant; Proteins; Receptor Protein-Tyrosine Kinases; Receptors, Growth Factor; Receptors, Vascular Endothelial Growth Factor; Recombinant Proteins; Ribonuclease, Pancreatic; Sarcoma; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: Angiogenesis Inducing Agents; Biomarkers, Tumor; Breast Neoplasms; Colorectal Neoplasms; Disease Progression; Female; Humans; Proteins; Ribonuclease, Pancreatic

Angiogenin (Ang), an inducer of neovascularization, is secreted by several types of human tumor cells and appears critical for their growth. The murine anti-Ang monoclonal antibody (mAb) 26-2F neutralizes the activities of Ang and dramatically prevents the establishment and metastatic dissemination of human tumor cell xenografts in athymic mice. However, for use clinically, the well-documented problem of the human anti-globulin antibody response known to occur with murine antibodies requires resolution. As a result, chimeric as well as totally humanized antibodies are currently being evaluated as therapeutic agents for the treatment of several pathological conditions, including malignancy. Therefore, we have constructed a chimeric mouse/human antibody based on the structure of mAb 26-2F. Complementary DNAs from the light and heavy chain variable regions of mAb 26-2F were cloned, sequenced, and genetically engineered by PCR for subcloning into expression vectors that contain human constant region sequences. Transfection of these vectors into nonproducing mouse myeloma cells resulted in the secretion of fully assembled tetrameric molecules. The chimeric antibody (cAb 26-2F) binds to Ang and inhibits its ribonucleolytic and angiogenic activities as potently as mAb 26-2F. Furthermore, the capacities of cAb 26-2F and its murine counterpart to suppress the formation of human breast cancer tumors in athymic mice are indistinguishable. Thus cAb 26-2F, with its retained neutralization capability and likely decreased immunogenicity, may be of use clinically for the treatment of human cancer and related disorders where pathological angiogenesis is a component.

Topics: Amino Acid Sequence; Angiogenesis Inducing Agents; Animals; Antibodies, Monoclonal; Base Sequence; Breast Neoplasms; Cloning, Molecular; Female; Humans; Immunoglobulin Heavy Chains; Immunoglobulin Light Chains; Immunoglobulin Variable Region; Mice; Mice, Nude; Molecular Sequence Data; Neovascularization, Pathologic; Plasmacytoma; Polymerase Chain Reaction; Proteins; Recombinant Fusion Proteins; Ribonuclease, Pancreatic; Transplantation, Heterologous; Tumor Cells, Cultured

To compare the prognostic impact of tumor angiogenesis factors (vascular endothelial growth factor [VEGF], angiogenin, and basic fibroblast growth factor [bFGF]), tumor proteolysis factors (urokinase-type plasminogen activator [uPA] and plasminogen activator inhibitor-1 [PAI-1]), and conventional tumor markers (stage, grade, and steroid receptors) in early breast cancer.. In the primary clinical study, tumor angiogenesis and other factors were detected in frozen biopsies from 305 primary breast tumors. VEGF expression was assessed by chemiluminescence immunosorbent assay (ICMA); angiogenin, bFGF, uPA, and PAI-1 by enzyme-linked immunosorbent assay (ELISA); and steroid receptors (estrogen receptor [ER] and progesterone receptor [PgR]) by enzyme immunoassay (EIA). In the validating clinical study, another set of 190 node-negative primary breast tumor samples were collected at a separate institution.. Univariate analysis of the primary study showed that VEGF levels were positively correlated with recurrence (P < .001). Angiogenin levels were positively correlated with disease relapse (P < .005) for the overall collective group, but not within the node-negative subset. No significant correlations were found between tumor bFGF levels and patient survival. In multivariate regression analysis, the only independent predictors of relapse-free survival (RFS) were VEGF, uPA, and lymph node status. In the validation set, the distribution of VEGF and uPA values were similar to those in the primary study; low expression of both VEGF and uPA identified patients with a < or = 20% likelihood of recurrence within 7 years.. Separate primary and validating clinical studies concur that tumor VEGF level is the most important prognostic parameter among several markers of tumor angiogenesis and proteolysis.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Endothelial Growth Factors; Enzyme-Linked Immunosorbent Assay; Female; Fibroblast Growth Factor 2; Humans; Immunosorbent Techniques; Luminescent Measurements; Lymph Nodes; Lymphokines; Middle Aged; Neovascularization, Pathologic; Plasminogen Activator Inhibitor 1; Proteins; Receptors, Estrogen; Receptors, Progesterone; Reproducibility of Results; Retrospective Studies; Ribonuclease, Pancreatic; Risk Factors; Urokinase-Type Plasminogen Activator; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Angiogenin is a protein originally isolated as an inducer of new blood vessel growth, and it has been reported to be an effective substrate for tumor cell adhesion. To understand the role of angiogenin in cancer progression, we evaluated the expression of angiogenin in 459 cases with primary breast carcinoma and in 40 benign breast specimens using an immunoassay. Higher angiogenin concentrations were observed in carcinomas in comparison with fibrocystic disease (mean, 17.3 versus 10.9 ng/mg; P = 0.008), but not with fibroadenomas. We selected 5 ng/mg cytosol protein of angiogenin as the normal cutoff for primary breast carcinoma. Eighty-eight percent of carcinomas expressed elevated angiogenin levels and 12% had low levels. We observed an association between elevated levels of angiogenin and low/ moderate histological grade (P = 0.001) and small tumor size (P = 0.026), but not with age, menopausal status, lymph node status, stage of disease, or hormonal receptor status. With a median follow-up of 31 months, breast cancer patients with elevated angiogenin levels had significantly longer disease-free survival (DFS) than patients with low angiogenin (log-rank, P = 0.003). This effect was equally observed in node-negative and node-positive cases. In a multivariate analysis of DFS, only angiogenin, tumor size, and histological grade showed statistical significance. A multivariate analysis of overall survival showed that angiogenin and tumor size were the only significant variables. Serum samples from the breast cancer patients at the time of surgery were available in 194 cases. We evaluated the levels of circulating angiogenin using the same immunoassay as in tumor tissue. Serum angiogenin levels were higher in cancer patients than in 40 healthy controls (mean, 401.2 versus 206.0 ng/ml; P < 0.0001). In breast cancer patients, we observed no correlation between the serum concentrations and the tissue levels of angiogenin (r = 0.115; P = 0.110). In addition, serum levels of angiogenin did not have a prognostic impact on the DFS of breast cancer patients (log-rank, P = 0.581). Our results indicate that elevated levels of tissue angiogenin, but not of circulating angiogenin, are a favorable prognostic factor in primary breast carcinoma, which is consistent with a role of angiogenin as a cancer cell substrate.

Topics: Adult; Aged; Aged, 80 and over; Breast Neoplasms; Female; Follow-Up Studies; Humans; Middle Aged; Neoplasm Staging; Prognosis; Protein Biosynthesis; Ribonuclease, Pancreatic; Survival Analysis

We investigated whether blood angiogenic factor (vascular endothelial growth factor, VEGF; angiogenin; basic fibroblast growth factor, bFGF; platelet-derived growth factor-AB, PDGF-AB) levels change during menstrual cycle of healthy premenopausal females or after menopause. We also measured the serum angiogenic factor levels in 34 operable breast cancer patients and compared them to those of healthy volunteer controls. No differences in the four angiogenic factor levels were found between the follicular and luteal phases of normal menstruation. However, angiogenin and bFGF levels were higher in pre-menopausal females than post-menopausal female and young male healthy volunteers. In cancer patients, the sero-positivity rate of the bFGF was 8.8% with menstrual-state-unmatched cut-off points, which increased to 36.4% with menstrual-state-matched cut-off points. This discrepancy was especially high in post-menopausal cancer patients. In conclusion, physiological elevation of the bFGF during normal menstruation can influence the precise interpretation of the pathological elevation of the bFGF in pre-menopausal breast cancer patients.

Topics: Adult; Aged; Angiogenesis Inducing Agents; Breast Neoplasms; Female; Fibroblast Growth Factor 2; Follicular Phase; Humans; Luteal Phase; Male; Menstrual Cycle; Middle Aged; Platelet-Derived Growth Factor; Postmenopause; Premenopause; Reference Values; Ribonuclease, Pancreatic; Vascular Endothelial Growth Factor A