endothelin-1 and Breast-Neoplasms

It has been long recognized that skeleton represents one of the most favored metastatic sites for common cancers like breast and prostate. During the last decade the molecular mechanisms that are responsible for the development of bone metastasis have been gradually illuminated. It appears that the bone microenvironment has a pivotal role in this process. Metastatic tumor cells interact with bone triggering a cascade of molecular events that produce osteolytic and/or osteoblastic phenomena. In this review, we summarize and discuss the most significant factors and signaling pathways implicated in bone colonization. Moreover, based on the recent literature and data, we foresee the need for designing novel agents that will efficiently disrupt these interactions among cancer cells and bone microenvironment, bringing hope for more effective treatments.

Topics: Animals; beta Catenin; Biphenyl Compounds; Bone Neoplasms; Bone Remodeling; Breast Neoplasms; Cathepsin K; Chemokine CXCL12; Diphosphonates; Endothelin-1; Female; Humans; Hypoxia; Male; Neoplastic Stem Cells; Osteoblasts; Osteoclasts; Osteolysis; Parathyroid Hormone-Related Protein; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Receptors, CXCR4; Signal Transduction; Urokinase-Type Plasminogen Activator; Wnt Proteins

Bone metastases are complications of multiple myeloma and solid tumors, including breast and prostate carcinomas. Several reports have demonstrated that the preference to metastasize to bone by tumor cells is not a casual but an addressed event, which relies on specific interactions among tumor cells, bone marrow microenvironment and bone cells. One of the features that gives tumor cells more chances to survive and proliferate into the bone tissue is osteomimicry, that is the ability to acquire a bone cell phenotype, especially osteoblast-like. As clearly demonstrated, prostate and breast cancer cells try to resemble osteoblasts by expressing bone matrix proteins, the specific marker alkaline phosphatase, and molecules regulating the osteoblast/osteoclast cross-talk. Based on this evidence it is crucial to dissect in more detail the molecular mechanisms underlying the osteomimetic properties of cancer cells and identify new therapeutic targets eventually leading to a better and prolonged life expectation for patients with bone.

Topics: Alkaline Phosphatase; beta 2-Microglobulin; Bone Matrix; Bone Morphogenetic Proteins; Bone Neoplasms; Breast Neoplasms; Core Binding Factor Alpha 1 Subunit; Endothelin-1; Female; Humans; Male; Models, Biological; Osteoblasts; Osteolysis; Phenotype; Prostatic Neoplasms; Signal Transduction; Urokinase-Type Plasminogen Activator; Wnt Proteins

The endothelin system comprises the three peptide hormones endothelin (ET)-1, -2, -3, their G protein-coupled receptors, endothelin-A-receptor (ET(A)R) and endothelin-B-receptor (ET(B)R), and the enzymes of endothelin biosynthesis and degradation. In the past two decades, an impressive amount of data has been accumulated investigating the role of the endothelin system in a variety of malignancies. In many cancers, ET-1/ET(A)R interaction induces proliferation, angiogenesis, antiapoptosis and resistance to chemotherapy. Data indicate a pivotal role of the endothelin system in tumorigenesis, local progression and metastasis. Subsequently, novel drugs have been designed inhibiting ET-1 biosynthesis or ET(A)R interaction. A wide range of preclinical data is available on the role of ET(A)R antagonists in gynecological, urological and breast cancers providing evidence for their antiangiogenic, proapoptotic and growth inhibitory effects. Of particular interest is the anti-invasive and antimetastatic efficacy of ET(A)R antagonists and synergism when co-administered with established cancer therapies. Data indicate a future role of ET(A)R antagonists in oncologic therapies.

Topics: Animals; Antineoplastic Agents; Aspartic Acid Endopeptidases; Breast Neoplasms; Endometrial Neoplasms; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Female; Humans; Kidney Neoplasms; Male; Metalloendopeptidases; Ovarian Neoplasms; Prostatic Neoplasms; Receptors, Endothelin; Urinary Bladder Neoplasms; Urogenital Neoplasms; Uterine Cervical Neoplasms

The endothelin axis (ET axis), comprising the three peptides endothelin (ET)-1, -2, -3 and their receptors ET(A)R and ET(B)R, is expressed in various cells and tissues. The biologically active ET-1 is formed by endothelin-converting enzyme (ECE) from inactive big-ET-1. ET-1 has emerged as an important peptide in a host of biological functions, including development, cellular proliferation, apoptosis and angiogenesis, thereby playing an important physiological and pathophysiological role. As these effects are mediated by ET(A)R, activation of ET(B)R prevents apoptosis, inhibits ECE expression and mediates the clearance of ET-1. Emerging data indicate that the ET axis is involved in tumourigenesis and tumour progression of various cancers. Expression of the ET axis has been demonstrated in a wide range of human tumours. Since most data have been reported for female malignancies, this review will focus on the role of the ET axis in cancers of the ovary, the cervix and the breast. In ovarian cancer, activation of ET(A)R by ET-1 is a key mechanism in the cellular signalling network promoting cancer growth and progression. Similar effects have been shown for cervical and endometrial cancer. In breast cancer, ET-1 via ET(A)R promotes proliferation and invasion, mediates bone metastases and predicts unfavourable response to chemotherapy. The outstanding role of ET-1 and ET(A)R in carcinogenesis and tumour progression has led to an extensive search for interfering agents, resulting in the development of selective ET(A)R antagonists on the one hand and inhibitors of the endothelin-converting enzyme (ECE) on the other. Targeting the ET axis via ET(A)R or ECE blockade seems to be a promising approach in the treatment of female malignancies.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Aspartic Acid Endopeptidases; Breast Neoplasms; Endothelin A Receptor Antagonists; Endothelin B Receptor Antagonists; Endothelin-1; Endothelin-2; Endothelin-3; Endothelin-Converting Enzymes; Enzyme Inhibitors; Female; Humans; Metalloendopeptidases; Molecular Sequence Data; Neoplasms; Ovarian Neoplasms; Receptor, Endothelin A; Receptor, Endothelin B; Uterine Cervical Neoplasms

Skeletal-related complications occur commonly in many solid tumors including breast, prostate and lung cancer as well as multiple myeloma. In addition, malignancies and their associated treatment may result in bone loss or osteoporosis. This review will focus solely on recent data associated with metastatic bone disease with a focus on breast cancer, prostate cancer and multiple myeloma. Bone loss or osteoporosis associated with cancer will be covered in a separate article in this issue.. Recent progress in understanding the pathophysiology of bone metastases has pointed to several novel pathways: transforming growth factor beta; receptor activator of nuclear factor beta ligand and osteoprotegerin; and Wnt signaling pathways and associated factors such as dickkopf-1 and endothelin-1.. The identification of new pathways is important in metastatic bone disease from cancer and has allowed for the development of novel therapeutics aimed at preventing the devastating complications of bone metastases. Bisphosphonates remain the predominant therapy in use for the treatment and prevention of skeletal-related adverse effects from cancer.

Topics: Bone Density Conservation Agents; Bone Neoplasms; Breast Neoplasms; Diphosphonates; Endothelin-1; Female; Humans; Intercellular Signaling Peptides and Proteins; Male; Multiple Myeloma; Osteoprotegerin; Prostatic Neoplasms; RANK Ligand; Receptor Activator of Nuclear Factor-kappa B; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Topics: Animals; Breast Neoplasms; Endothelin-1; Humans; Linoleic Acid; Melatonin; Neoplasms; Telomerase

Certain solid tumors metastasize to bone and cause an osteoblastic response. The mechanisms by which tumor cells stimulate this new bone formation are not completely understood.. The authors identified three breast cancer lines that cause osteoblastic metastases in female nude mice and provided evidence that tumor-produced endothelin-1 (ET-1) mediates the osteoblastic response.. Tumor conditioned media, as well as exogenous ET-1, stimulated osteoblast proliferation and new bone formation in cultures of mouse calvariae. These effects were blocked by antagonists of the endothelin A (ET(A)), but not ET(B), receptors. Mice inoculated with the ZR-75-1 breast cancer line and treated with a selective ET(A) receptor antagonist (ABT-627) had significantly fewer osteoblastic bone metastases and less tumor burden compared with untreated mice. In contrast, there was no effect of ABT-627 on osteolytic bone metastases caused by ET-1-negative breast cancer, MDA-MB-231. ABT-627 had no effect on growth in vitro or at the orthotopic site of ZR-75-1 or MDA-MB-231 cells.. Collectively, the data suggested that tumor-produced ET-1 mediates osteoblastic bone metastases by stimulating osteoblast proliferation and new bone formation. ET(A) receptor blockade may be useful for prevention and the treatment of osteoblastic bone metastases due to breast or prostate cancer.

Topics: Animals; Bone Neoplasms; Breast Neoplasms; Disease Models, Animal; Endothelin Receptor Antagonists; Endothelin-1; Female; Mice; Mice, Nude; Neoplasms, Experimental; Osteoblasts; Receptor, Endothelin A

EndoTAG-1 (ET), a novel formulation of cationic liposomes carrying embedded paclitaxel (Taxol), shows antitumoral activity, targeting tumor endothelial cells in solid tumors. Patients with advanced metastatic cancer were evaluated investigating effects on pharmacokinetics and tumor vasculature using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and contrast-enhanced ultrasound (CEUS).. The pharmacokinetic (PK) profile of ET (22 mg/m(2) i.v.) was evaluated after single and repeated doses. DCE-MRI and CEUS explored hepatic metastases before, during and after the 4-week treatment cycle. Angiogenic biomarkers were assessed. Tumor response was evaluated by modified RECIST.. The PK profile demonstrated slight accumulation of paclitaxel after repeated doses. DCE-MRI parameters K(trans) and/or iAUC(60) showed a trend to decrease. Changes of blood flow-dependent parameters of DCE-MRI and CEUS were well correlated. Angiogenic biomarkers revealed no clear trend. ET was generally well tolerated; common toxic effects were fatigue and hypersensitivity reactions. Nine (9 of 18) patients had stable disease after the first treatment cycle. Four patients without disease progression continued treatment.. This study including multiple pretreated patients with different metastatic cancer revealed individually distinctive hemodynamic alterations by DCE-MRI. The PK profiles of ET were similar as observed previously.

Topics: Adult; Aged; Angiogenesis Inhibitors; Angiotensin II; Area Under Curve; Breast Neoplasms; Colorectal Neoplasms; Contrast Media; Endothelin-1; Female; Humans; Interleukins; Liposomes; Liver Neoplasms; Magnetic Resonance Imaging; Male; Middle Aged; Paclitaxel; Pancreatic Neoplasms; Ultrasonography; Vascular Endothelial Growth Factor A

Endothelin-1 (ET-1) and its receptors (ETAR and ETBR), referred to as the endothelin (ET) axis, are overexpressed in breast carcinomas and appear to influence tumour growth and progression. The objective of this study was to determine the effect of expression of the ET axis in breast carcinomas on response to cytotoxic chemotherapy. The study included 44 patients with locally advanced breast cancer participating in a prospective phase III study evaluating high-dose neoadjuvant chemotherapy of epirubicin and cyclophosphamide. Expression of ET-1, ETAR and ETBR was determined by semiquantitative immunohistochemical analysis of breast cancer tissue from prechemotherapy tru-cut biopsies. Immunohistochemical staining was positive for ET-1 in 61.5%, for ETAR in 35% and for ETBR in 35.9% of breast carcinomas. Pathological response to chemotherapy was significantly decreased in ETAR-positive patients (P=0.002). In total, 50% of ETAR-positive patients as compared to 7.7% of ETAR-negative patients attained pathologically 'no change'. Logistic regression confirmed ETAR as an independent predictive marker for pathological response (P=0.009). These data indicate that increased expression of ETAR in breast carcinomas is associated with resistance to chemotherapy. Determination of ETAR status may serve as a predictive marker for identifying patients less likely to be responsive to conventional chemotherapy.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Carcinoma; Chemotherapy, Adjuvant; Cyclophosphamide; Drug Resistance, Neoplasm; Endothelin-1; Epirubicin; Female; Gene Expression Profiling; Humans; Immunohistochemistry; Middle Aged; Neoadjuvant Therapy; Predictive Value of Tests; Receptor, Endothelin A; Receptor, Endothelin B; Sensitivity and Specificity; Treatment Outcome

Tamoxifen protects against myocardial infarction through mechanisms that are poorly understood. We studied the effects of tamoxifen and another antiestrogen, toremifene, on the production of vasoconstrictive endothelin-1 and of vasodilatory nitric oxide in 44 postmenopausal patients with breast cancer. These started treatment, in randomized order, with either tamoxifen (20 mg/day; n = 25) or toremifene (40 mg/day; n = 19). Plasma samples collected before treatment and after 6 and 12 months of both regimens were assayed for endothelin-1 with a specific radioimmunoassay and for nitrite/nitrate with a method based on the Griess reaction. The antiestrogen group as a whole showed a fall in endothelin-1 at 6 months (5.9 +/- 3.3%; p = 0.06) (mean +/- SE) and at 12 months (7.1 +/- 5.5%; p = 0.03). This fall was solely due to toremifene, the use of which was associated with falls in endothelin-1 at 6 months (12.9 +/- 4.7%; p = 0.01) and 12 months (9.2 +/- 6.2%; p = 0.06). The antiestrogen regimen failed to affect plasma nitric oxide significantly but nevertheless the ratio between nitric oxide and endothelin-1 rose by 31.6 +/- 13.3% at 6 months and by 35.6 +/- 15.3% at 12 months in the antiestrogen users, an effect similar in the tamoxifen and toremifene groups. We conclude that antiestrogens may protect against myocardial infarction by preventing the release of endothelin-1 and by shifting the balance between nitric oxide and endothelin-1 to the dominance of the former. Our data predict that toremifene and tamoxifen at the doses studied here will provide similar cardiovascular protection.

Topics: Aged; Aged, 80 and over; Breast Neoplasms; Endothelin-1; Estrogen Antagonists; Female; Humans; Middle Aged; Myocardial Infarction; Nitrates; Postmenopause; Tamoxifen; Toremifene

Endothelin-1 (ET-1) may contribute to vasoconstriction and flap blood flow during microvascular surgery. Calcium antagonists have suppressed ET-1 release in some studies. The effect of 5 mg of felodipine, a vasodilating calcium antagonist, administered the evening and morning before a microvascular TRAM (transverse rectus abdominis musculocutaneous) flap breast reconstruction, on perioperative plasma ET-1 concentrations, peripheral temperature gradient (Tgrad), rectal temperature (Trect), flap skin blood flow with transcutaneous oxygen tension (ptcO2), heart rate (HR) and mean arterial pressure (MAP) was evaluated in this clinical, randomised, double blind, prospective study. Felodipine did not cause any statistically significant changes in ET-1 levels, Tgrad, Trect, Ptc O2, flap survival or pre- or intraoperative MAP. HR was significantly higher in the felodipine group before induction and at 10 min postoperatively. We conclude that in patients undergoing a microvascular TRAM flap breast reconstruction, preoperatively administered felodipine has no effect on perioperative ET-1 levels, temperature changes, or flap skin blood flow.

Topics: Adult; Breast Neoplasms; Calcium Channel Blockers; Double-Blind Method; Endothelin-1; Felodipine; Female; Humans; Mammaplasty; Microcirculation; Middle Aged; Oxygen; Prospective Studies; Rectus Abdominis; Surgical Flaps; Vasoconstriction

The survival rates of women with breast cancer have improved significantly over the last four decades due to advances in breast cancer early diagnosis and therapy. However, breast cancer survivors have an increased risk of cardiovascular complications following chemotherapy. While this increased risk of later occurring structural cardiac remodeling and/or dysfunction has largely been attributed to the cardiotoxic effects of breast cancer therapies, the effect of the breast tumor itself on the heart prior to cancer treatment has been largely overlooked. Thus, the objectives of this study were to assess the cardiac phenotype in breast cancer patients prior to cancer chemotherapy and to determine the effects of human breast cancer cells on cardiomyocytes.. We investigated left ventricular (LV) function and structure using cardiac magnetic resonance imaging in women with breast cancer prior to systemic therapy and a control cohort of women with comparable baseline factors. In addition, we explored how breast cancer cells communicate with the cardiomyocytes using cultured human cardiac and breast cancer cells.. Our results indicate that even prior to full cancer treatment, breast cancer patients already exhibit relative LV hypertrophy (LVH). We further demonstrate that breast cancer cells likely contribute to cardiomyocyte hypertrophy through the secretion of soluble factors and that at least one of these factors is endothelin-1.. Overall, the findings of this study suggest that breast cancer cells play a greater role in inducing structural cardiac remodeling than previously appreciated and that tumor-derived endothelin-1 may play a pivotal role in this process.

Topics: Breast Neoplasms; Case-Control Studies; Cell Communication; Cell Line, Tumor; Cells, Cultured; Culture Media, Conditioned; Endothelin-1; Female; Humans; Hypertrophy; Hypertrophy, Left Ventricular; Magnetic Resonance Imaging; Middle Aged; Myocytes, Cardiac; Paracrine Communication; Retrospective Studies; Tumor Cells, Cultured; Ventricular Remodeling

GDF-15 is an established cardiovascular risk marker but is equally implicated in tumour biology. Elevated levels of GDF-15 have indeed been observed in distinct tumour entities. This study aimed to explore the relation of GDF-15 to other cardiac biomarkers and the general association of GDF-15 on prognosis in an unselected cohort of treatment-naïve cancer patients.. We prospectively enrolled 555 consecutive patients at time of diagnosis of malignant disease prior receiving anticancer therapy. Plasma GDF-15 concentrations were determined alongside other cardiac and routine laboratory markers. All-cause mortality was defined as primary endpoint.. GDF-15 levels were 338 ng/L (IQR:205-534) for the total cohort, and values were comparable for different tumour entities except breast cancer. Metastatic disease was characterized by higher plasma GDF-15 [435 ng/L (IQR:279-614) vs 266 ng/L (IQR:175-427), P < .001]. GDF-15 correlated positively with inflammatory status reflected by CRP, SAA and IL-6 [r = .31, P < .001, r = .23, P < .001 and r = .14, P = .002] and cardiac biomarkers as NT-proBNP, hsTnT, MR-proADM and CT-proET-1 [r = .46; r = .46; r = .59 and r = .50; P < .001 for all]. GDF-15 was significantly associated with all-cause mortality after multivariate adjustment [adj.HR for ln(GDF-15) 1.78, 95%CI:1.47-2.16, P < .001]. There was a significant interaction between solid and haematological malignancies with loss of association of GDF-15 with outcome in myelodysplastic and myeloproliferative disease.. Elevated plasma GDF-15 is associated with progressing disease severity and poor prognosis in solid tumours of treatment-naïve cancer patients. GDF-15 increase is accompanied by worsening systemic inflammation and a subclinical functional impairment of different organs including the heart. GDF-15 represents a promising target for our pathophysiologic understanding in cardio-oncology linking conditions of both cardiac and neoplastic disease.

Topics: Adrenomedullin; Aged; Breast Neoplasms; C-Reactive Protein; Cause of Death; Endothelin-1; Female; Gastrointestinal Neoplasms; Glycopeptides; Growth Differentiation Factor 15; Humans; Interleukin-6; Lung Neoplasms; Male; Middle Aged; Mortality; Myelodysplastic Syndromes; Myeloproliferative Disorders; Natriuretic Peptide, Brain; Neoplasm Metastasis; Neoplasms; Peptide Fragments; Prognosis; Proportional Hazards Models; Prospective Studies; Protein Precursors; Serum Amyloid A Protein; Troponin T

We report a 46-yr-old woman with a history of breast cancer who presented with diffuse myalgias, bone pain, and osteosclerosis. She was found to have recurrent breast cancer producing endothelin-1.. Acquired osteosclerosis can be caused by various disorders. Endothelin -1 is believed to contribute to osteosclerosis caused by breast cancer.. Although the bone marrow biopsy did not reveal breast cancer, she developed skin lesions consistent with metastatic breast cancer. She ultimately died from progressive disease. At autopsy immunohistochemistry for endothelin-1 was performed on a section from the L5 vertebral body.. The section from the L5 vertebral body showed small foci of cells consistent with metastatic carcinoma and a prominent sclerotic response. Immunohistochemistry for endothelin-1 was strongly positive.. Recurrent breast cancer may present with diffuse osteosclerosis. Endothelin-1 may be a paracrine factor responsible for increased bone formation and osteosclerosis.

Topics: Adenocarcinoma; Bone Neoplasms; Breast Neoplasms; Endothelin-1; Fatal Outcome; Female; Humans; Middle Aged; Osteosclerosis; Radiography; Skin Neoplasms

We examined the influence of microenvironment stimuli on molecular events relevant to the biological functions of 1833-bone metastatic clone and the parental MDA-MB231 cells. (i) In both the cell lines, hepatocyte growth factor (HGF) and the osteoblasts' biological products down regulated nuclear Ets-1-protein level in concomitance with endogenous miR-125b accumulation. In contrast, under hypoxia nuclear Ets-1 was unchanged, notwithstanding the miR-125b increase. (ii) Also, the 1833-cell invasiveness and the expression of Endothelin-1, the target gene of Ets-1/HIF-1, showed opposite patterns under HGF and hypoxia. We clarified the molecular mechanism(s) reproducing the high miR-125b levels with the mimic in 1833 cells. Under hypoxia, the miR-125b mimic maintained a basal level and functional Ets-1 protein, as testified by the elevated cell invasiveness. However, under HGF ectopic miR-125b downregulated Ets-1 protein and cell motility, likely involving an Ets-1-dominant negative form sensible to serum conditions; Ets-1-activity inhibition by HGF implicated HIF-1α accumulation, which drugged Ets-1 in the complex bound to the Endothelin-1 promoter. Altogether, 1833-cell exposure to HGF would decrease Endothelin-1 transactivation and protein expression, with the possible impairment of Endothelin-1-dependent induction of E-cadherin, and the reversion towards an invasive phenotype: this was favoured by Ets-1 overexpression, which inhibited HIF-1α expression and HIF-1 activity. (iii) In MDA-MB231 cells, HGF strongly and rapidly decreased Ets-1, hampering invasiveness and reducing Ets-1-binding to Endothelin-1 promoter; HIF-1α did not form a complex with Ets-1 and Endothelin-1-luciferase activity was unchanged. Overall, depending on the microenvironment conditions and endogenous miR-125b levels, bone-metastatic cells might switch from Ets-1-dependent motility towards colonization/growth, regulated by the balance between Ets-1 and HIF-1.

Topics: Bone Neoplasms; Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; DNA; Endothelin-1; Female; Hepatocyte Growth Factor; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; MicroRNAs; Models, Biological; Neoplasm Invasiveness; Proto-Oncogene Protein c-ets-1; Transcriptional Activation; Tumor Microenvironment

The expression of the transcription factor

Topics: Animals; Breast Neoplasms; Chromatin; Culture Media, Conditioned; Endothelin-1; Epigenesis, Genetic; Epithelial Cells; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; HEK293 Cells; Humans; Neoplasm Metastasis; Neovascularization, Pathologic; Promoter Regions, Genetic; RNA, Messenger; SOXC Transcription Factors; Survival Analysis; Trans-Activators; Transcription, Genetic; Xenograft Model Antitumor Assays; Zebrafish

Biomarkers for bevacizumab efficacy in metastatic breast cancer (MBC) are of urgent need. The genetic variability of genes involved in angiogenesis could explain the interpatient variability of drug effects. For this biomarker study DNA was extracted from tumor blocks or blood samples of patients with human epidermal growth factor receptor 2 (HER2)-negative MBC treated with bevacizumab in combination with chemotherapy (bevacizumab cohort, 163 patients) or chemotherapy only (control cohort, 105 patients). We assessed the correlation of 10 single-nucleotide polymorphisms (SNPs) in genes modulating angiogenesis (vascular endothelial growth factor-A (VEGF-A), VEGF receptor 1 (VEGFR-1), serine threonine kinase 39 (STK39)) or hypertension (endothelin-1 and uromodulin) with outcome and toxicity. In the bevacizumab cohort, the SNP rs5370-TT in endothelin-1 (EDN1) showed a significantly shorter median overall survival (OS, 6.3 vs 21.3 months; hazard ratio (HR) 2.89, 95% confidence interval (CI) 1.34-6.26; log-rank P=0.0069) and a trend toward worse median progression-free survival (3.5 vs 7.9 months; HR 2.05, 95% CI 0.96-4.39; log-rank P=0.065) compared with the alternate genotypes combined. Similarly, patients harboring the VEGF-936 (rs3025039) TT alleles showed a significantly shorter median OS than patients with VEGF-936 CC or CT (14.9 vs 21.3 months; HR 2.37, 95% CI 1.09-5.13; P=0.0286). In multivariate analysis including important clinical parameters like disease-free survival (DFS), adjuvant chemotherapy, ECOG (Eastern Cooperative Oncology Group) performance score, histologic subtype, grade, hormone receptor status, visceral metastases and treatment line, only the association of rs5370 (EDN1) with OS was still statistically significant (P=0.012). In the control cohort, no association of the EDN1 genotype with outcome was seen, suggesting a predictive value for bevacizumab. In conclusion, the SNP rs5370 in endothelin-1 could help identifying patients who unlikely gain any benefit from bevacizumab.

Topics: Adult; Aged; Aged, 80 and over; Angiogenesis Inhibitors; Antibodies, Monoclonal, Humanized; Antineoplastic Agents, Immunological; Bevacizumab; Biomarkers, Tumor; Breast Neoplasms; Disease-Free Survival; Endothelin-1; Female; Humans; Middle Aged; Neovascularization, Pathologic; Polymorphism, Single Nucleotide; Receptor, ErbB-2; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1

The pathogenesis of bone metastasis is unclear, and much focus in metastatic biology and therapy relays on epigenetic alterations. Since DNA-methyltransferase blockade with 5-aza-2'-deoxycytidine (dAza) counteracts tumour growth, here we utilized dAza to clarify whether molecular events undergoing epigenetic control were critical for bone metastatization. In particular, we investigated the patterns of secreted-protein acidic and rich in cysteine (SPARC) and of Endothelin 1, affected by DNA methyltransferases in tumours, with the hypothesis that in bone metastasis a coordinate function of SPARC and Endothelin 1, if any occurs, was orchestrated by DNA methylation. To this purpose, we prepared a xenograft model with the clone 1833, derived from human-MDA-MB231 cells, and dAza administration slowed-down metastasis outgrowth. This seemed consequent to the reductions of SPARC and Endothelin 1 at invasive front and in the bone marrow, mostly due to loss of Twist. In the metastasis bulk Snail, partly reduced by dAza, might sustain Endothelin 1-SPARC cooperativity. Both SPARC and Endothelin 1 underwent post-translational control by miRNAs, a molecular mechanism that might explain the in vivo data. Ectopic miR29a reduced SPARC expression also under long-term dAza exposure, while Endothelin 1 down-regulation occurred in the presence of endogenous-miR98 expression. Notably, dAza effects differed depending on in vivo and in vitro conditions. In 1833 cells exposed to 30-days dAza, SPARC-protein level was practically unaffected, while Endothelin 1 induction depended on the 3'-UTR functionality. The blockade of methyltransferases leading to SPARC reduction in vivo, might represent a promising strategy to hamper early steps of the metastatic process affecting the osteogenic niche.

Topics: Animals; Azacitidine; Bone Neoplasms; Breast Neoplasms; DNA Methylation; DNA, Neoplasm; Endothelin-1; Female; Humans; Mice; Mice, Nude; MicroRNAs; Neoplasm Metastasis; Neoplasm Proteins; Osteonectin; RNA, Neoplasm; Tumor Microenvironment

In order to become established in the skeleton, metastatic cells disseminating from the breast carcinoma need to acquire organ-specific traits. There are no effective predictors for who will develop bone metastasis to guide long-term predictive therapy. Our purpose was to individuate events critical for bone colonization to make a molecular classification of breast carcinoma useful for bone-metastasis outcome. In dysplasia adjacent to carcinoma and in pair-matched specimens of bone metastasis we examined SPARC expression and localization as well as Endothelin 1/ETAR signals by immunohistochemistry, and the evaluation of plasma levels of SPARC by ELISA was also performed. In patients with breast carcinoma metastasizing to bone, SPARC and Endothelin 1/ETAR axis were highly expressed from dysplasia until bone metastasis, but the SPARC plasma level was as low as that of normal women, in contrast to patients that never develop bone metastasis, suggesting that circulating SPARC was counter adhesive. Altogether, the early identification of SPARC/Endothelin 1/ETAR in dysplastic lesions would be important to devise therapies preventing metastasis engraftment, since often carcinoma cells spread to distant organs at the time or even before patients present with cancer.

Topics: Adult; Biomarkers, Tumor; Bone Neoplasms; Breast Neoplasms; Carcinoma; Case-Control Studies; Endothelin-1; Female; Humans; Middle Aged; Osteonectin; Receptor, Endothelin A

The present study was undertaken to clarify the function(s) of Endothelin-1 and its receptors ETAR and ETBR in osteolytic-bone metastasis from breast cancer, and their regulation by hepatocyte and transforming growth factors (HGF, TGF-β) and hypoxia. The aim was to evaluate the adaptability of bone metastasis to microenvironmental stimuli through Endothelin-1-mediated epithelial-mesenchymal transition (EMT), or the reverse process MET, and through osteomimicry possible key features for bone colonization. We compared low (MCF-7) and high (MDA-MB231) invasive-breast carcinoma cells, and 1833-bone metastatic clone, with human pair-matched primary breast-carcinomas and bone metastases. Parental MDA-MB231 and the derived 1833-clone responded oppositely to the stimuli. In 1833 cells, TGF-β and hypoxia increased Endothelin-1 release, altogether reducing invasiveness important for engraftment, while Endothelin-1 enhanced MDA-MB231 cell invasiveness. The Endothelin-1-autocrine loop contributed to the cooperation of intracellular-signaling pathways and extracellular stimuli triggering MET in 1833 cells, and EMT in MDA-MB231 cells. Only in 1833 cells, HGF negatively influenced transactivation and release of Endothelin-1, suggesting a temporal sequence of these stimuli with an initial role of HGF-triggered Wnt/β-catenin pathway in metastatization. Then, Endothelin-1/ETAR conferred MET and osteomimetic phenotypes, with Runt-related transcription factor 2 activation and metalloproteinase 9 expression, contributing to colonization and osteolysis. Findings with human pair-matched primary ductal carcinomas and bone metastases gave a translational significance to the molecular study. Endothelin-1, ETAR and ETBR correlated with the acquisition of malignant potential, because of high expression already in the in situ carcinoma. These molecular markers might be used as predictive index of aggressive behavior and invasive/metastatic phenotype.

Topics: Bone Neoplasms; Breast Neoplasms; Endothelin-1; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Neoplasm Invasiveness; Receptor, Endothelin A; Receptor, Endothelin B; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment; Wnt Signaling Pathway

Elevated macrophage infiltration in tumor tissues is associated with breast cancer metastasis. Cancer cell migration/invasion toward angiogenic microvasculature is a key step in metastatic spread. We therefore studied how macrophages stimulated breast cancer cell interactions with endothelial cells. Macrophages produced cytokines, such as interleukin-8 and tumor necrosis factor-α, to stimulate endothelin (ET) and ET receptor (ETR) expression in breast cancer cells and human umbilical vascular endothelial cells (HUVECs). ET-1 was induced to a greater extent from HUVECs than from breast cancer cells, resulting in a density difference that facilitated cancer cell chemotaxis toward HUVECs. Macrophages also stimulated breast cancer cell adhesion to HUVECs and transendothelial migration, which were repressed by ET-1 antibody or ETR inhibitors. The ET axis induced integrins, such as αV and β1, and their counterligands, such as intercellular adhesion molecule-2 and P-selectin, in breast cancer cells and HUVECs, and antibodies against these integrins efficiently suppressed macrophage-stimulated breast cancer cell interactions with HUVECs. ET-1 induced Ets-like kinase-1 (Elk-1), signal transducer and activator of transcription-3 (STAT-3), and nuclear factor-κB (NF-κB) phosphorylation in breast cancer cells. The use of inhibitors to prevent their phosphorylation or ectopic overexpression of dominant-negative IκBα perturbed ET-1-induced integrin αV and integrin β1 expression. The physical associations of these three transcriptional factors with the gene promoters of the two integrins were furthermore evidenced by a chromatin immunoprecipitation assay. Finally, our mouse orthotopic tumor model revealed an ET axis-mediated lung metastasis of macrophage-stimulated breast cancer cells, suggesting that the ET axis was involved in macrophage-enhanced breast cancer cell endothelial interactions.

Topics: Animals; Antigens, CD; Breast Neoplasms; Cell Adhesion Molecules; Cell Line, Tumor; Cell Movement; Endothelin-1; ets-Domain Protein Elk-1; Female; Gene Expression Regulation, Neoplastic; Heterografts; Human Umbilical Vein Endothelial Cells; Humans; Integrin alphaV; Integrin beta1; Macrophages; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Transplantation; P-Selectin

Human mesenchymal stem cells (hMSCs) are currently investigated for a variety of therapeutic applications. However, MSCs isolated from primary tissue cannot meet clinical grade needs and should be expanded in vitro for several passages. Although hMSCs show low possibility for undergoing oncogenic transformation, they do, similar to other somatic cells, undergo cellular senescence and their therapeutic potential is diminished when cultured in vitro. However, the role of senescent MSCs in tumor progression remains largely elusive. In the current study, by establishing senescent human umbilical cord mesenchymal stem cells (s-UCMSCs) through the replicative senescence model and genotoxic stress induced premature senescence model, we show that s-UCMSCs significantly stimulate proliferation and migration of breast cancer cells in vitro and tumor progression in a co-transplant xenograft mouse model compared with 'young' counterparts (defined as MSCs at passage 5, in contrast to senescent MSCs at passage 45). In addition, we identified IL-6, a known pleiotropic cytokine, as a principal mediator for the tumor-promoting activity of s-UCMSCs by induction of STAT3 phosphorylation. Depletion of IL-6 from s-UCMSCs conditioned medium partially abrogated the stimulatory effect of s-UCMSCs on the proliferation and migration of breast tumor cells.

Topics: Animals; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cells, Cultured; Cellular Senescence; Culture Media, Conditioned; Endothelin-1; Female; Fetal Blood; Humans; Hydrogen Peroxide; Interleukin-6; MCF-7 Cells; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Neovascularization, Pathologic; Oxidants; Phosphorylation; STAT3 Transcription Factor; Xenograft Model Antitumor Assays

Here, we report a complex regulation of endothelin-1 (ET-1) axis driven by epigenetic reactions in 1833-bone metastatic cells, emphasizing the importance in skeletal metastasis from breast carcinoma. Inhibitors of histone deacetylases, trichostatin A (TSA), and of DNA methylases, 5'-Azacytidine (Aza), caused, respectively, reduction and increase in 1833 cell invasiveness, without affecting the basal migration of parental MDA-MB231 cells. Of note, in the two cell lines exposed to Aza the blockade of the ET-1 receptor ETAR with BQ-123 oppositely changed invasive properties. Even if in MDA-MB231 cells the ET-1 axis was scarcely influenced by epigenetic reactions, ETAR remarkably decreased after Aza. In contrast, in 1833 cells Aza exposure enhanced ET-1 coupled to ETAR wild type, being also ETAR truncated form increased, and invasiveness was stimulated. Under demethylation, the increase in ET-1 steady state protein level in 1833 clone seemed regulated at transcriptional level principally via Ets1 transcription factor. In fact, actinomycin D almost completely prevented ET-1 mRNA induction due to Aza. Only in 1833 cells, TSA exposure inactivated ET-1 axis, with reduction of the expression of ET-1 and ETAR mutated form, in agreement with Matrigel invasion decrease. This treatment favoured the ET-1 repressional control, taking place at the level of mRNA stability due to the 3'-untranslated region in the ET-1 gene, and also decreased transcription via NF-kB. Environmental conditions that alter the balance between epigenetic reactions might, therefore, affect metastasis migratory mode influencing ET-1 axis.

Topics: Azacitidine; Bone Neoplasms; Breast Neoplasms; Carcinoma; Cell Line, Tumor; Cell Movement; Collagen; Dactinomycin; DNA Methylation; Drug Combinations; Endothelin A Receptor Antagonists; Endothelin-1; Enzyme Inhibitors; Epigenesis, Genetic; Female; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Laminin; Mutation; Neoplasm Invasiveness; Peptides, Cyclic; Proteoglycans; Proto-Oncogene Protein c-ets-1; Receptor, Endothelin A; RNA Stability; RNA, Messenger; Transcription, Genetic

Deregulation of the endothelin system, comprised of endothelin-1 (ET-1), its isoforms (ET-2 and ET-3) and their receptors (ET(A)R and ET(B)R), is under investigation in various types of human cancer. ET-1 has been suggested to participate in breast cancer development and progression, while Big ET-1, its biological precursor, has also been found elevated in breast cancer patients. In the present study, we investigated plasma ET-1 and Big ET-1 levels in patients with suspicious mammographic lesions, in order to assess their potential application as diagnostic biomarkers in the early estimation of breast disease. The study consisted of 94 patients (Group A to 30 patients with invasive ductal carcinoma: Group B, 30 with ductal carcinoma in situ; and group C, 34 with papilloma or ductal hyperplasia), who underwent an image-guided vacuum-assisted breast biopsy, and 30 healthy controls (group D). ET-1 and Big ET-1 plasma levels were measured with enzyme-linked immunosorbent assay. ET-1 levels did not exhibit significant differences between patients and healthy controls (Group A to 0.92 fmol/mL; Group B: 0.90 fmol/mL; Group C: 0.66 fmol/mL; and Group D: 0.86 fmol/mL). In contrast, Big ET-1 levels were significantly higher in patients with invasive or in situ carcinoma compared to healthy controls (Group A: 0.69 fmol/mL; Group B, 0.62 fmol/mL; and group D: 0.39 fmol/mL; p < 0.001 and p < 0.01). Plasma Big ET-1 may provide a useful tool for the early detection of invasive or noninvasive ductal breast cancer. The utilization of such a diagnostic tool would greatly assist in the modern management of breast cancer.

Topics: Adult; Aged; Biomarkers, Tumor; Breast Neoplasms; Carcinoma, Ductal, Breast; Carcinoma, Intraductal, Noninfiltrating; Early Detection of Cancer; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Female; Humans; Middle Aged; Prognosis; Protein Precursors; Sensitivity and Specificity

Triple-negative breast cancer (TNBC) is characterized by the lack of expression of estrogen receptor-α (ER-α), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER-2). However, pathways responsible for downregulation of therapeutic receptors, as well as subsequent aggressiveness, remain unknown. In this study, we discovered that lactoferrin (Lf) efficiently downregulates levels of ER-α, PR, and HER-2 in a proteasome-dependent manner in breast cancer cells, and it accounts for the loss of responsiveness to ER- or HER-2-targeted therapies. Furthermore, we found that lactoferrin increases migration and invasiveness of both non-TNBC and TNBC cell lines. We discovered that lactoferrin directly stimulates the transcription of endothelin-1 (ET-1), a secreted proinvasive polypeptide that acts through a specific receptor, ET(A)R, leading to secretion of the bioactive ET-1 peptide. Interestingly, a therapeutic ET-1 receptor-antagonist blocked lactoferrin-dependent motility and invasiveness of breast cancer cells. The physiologic significance of this newly discovered Lf-ET-1 axis in the manifestation of TNBC phenotypes is revealed by elevated plasma and tissue lactoferrin and ET-1 levels in patients with TNBC compared with those in ER(+) cases. These findings describe the first physiologically relevant polypeptide as a functional determinant in downregulating all three therapeutic receptors in breast cancer, which uses another secreted ET-1 system to confer invasiveness. Results presented in this article provide proof-of-principle evidence in support of the therapeutic effectiveness of ET-1 receptor antagonist to completely block the lactoferrin-induced motility and invasiveness of the TNBC as well as non-TNBC cells, and thus, open a remarkable opportunity to treat TNBC by targeting the Lf-ET-1 axis using an approved developmental drug.

Topics: Breast Neoplasms; Caco-2 Cells; Cell Line, Tumor; Cell Movement; Down-Regulation; Endothelin A Receptor Antagonists; Endothelin-1; Estrogen Receptor alpha; Female; Humans; Lactoferrin; Neoplasm Invasiveness; Phenotype; Proteasome Endopeptidase Complex; Receptor, Endothelin A; Receptor, ErbB-2; Receptors, Progesterone; RNA Processing, Post-Transcriptional

Endothelin-1 (ET-1) and endothelin A receptor (ETAR) contribute to the development and progression of breast carcinomas by modulating cell proliferation, angiogenesis, and anti-apoptosis. We investigated antitumoral effects of the specific ETAR antagonist ZD4054 in breast cancer cells and xenografts, and assessed antitumoral efficacy of the combinations of ZD4054 with aromatase inhibitors and fulvestrant. Gene expression changes were assessed by quantitative real-time PCR. Cell proliferation was measured using alamarBlue; migration and invasion assays were performed using modified Boyden chambers. Evaluating the antitumoral efficacy of ZD4054 in vivo, different breast cancer models were employed using nude mice xenografts. ZD4054 reduced ET-1 and ETAR expression in MCF-7, MDA-MB-231, and MDA-MB-468 breast cancer cells in a concentration-dependent manner. ZD4054 inhibited invasion by up to 37.1% (P = 0.022). Combinations of ZD4054 with either anastrozole or letrozole produced significant reductions in migration of aromatase-overexpressing MCF-7aro cells (P < 0.05). Combination of ZD4054 with fulvestrant reduced MCF-7 cell migration and invasion by 36.0% (P = 0.027) and 56.7% (P < 0.001), respectively, with effects significantly exceeding those seen with either compound alone. Regarding tumor volume reduction in vivo, ZD4054 (10 mg/kg) was equipotent to fulvestrant (200 mg/kg) and exhibited additive effects with anastrozole (0.5 mg/kg). These data are the first indicating that selective ETAR antagonism by ZD4054 displays antitumoral activity on breast cancer cells in vitro and in vivo. Our data strongly support a rationale for the clinical use of ZD4054 in combination with endocrine therapies.

Topics: Anastrozole; Animals; Antineoplastic Combined Chemotherapy Protocols; Aromatase Inhibitors; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Dose-Response Relationship, Drug; Endothelin A Receptor Antagonists; Endothelin-1; Estradiol; Estrogen Antagonists; Female; Fulvestrant; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Nude; Neoplasm Invasiveness; Nitriles; Pyrrolidines; Receptor, Endothelin A; Tamoxifen; Time Factors; Triazoles; Tumor Burden; Xenograft Model Antitumor Assays

Human epidermal growth factor receptor 2 (HER2) is frequently overexpressed in human breast cancers. It is known to drive tumor growth and progression and represents a prominent target in breast cancer therapy. The endothelin (ET) system, in particular ET-1 and its receptor ET(A)R, is of major relevance for breast cancer growth and invasion. Having previously demonstrated coexpression of ET(A)R and HER2 in breast tumors, this study was designed to investigate molecular interactions of HER2 (including the epidermal growth factor receptor EGFR as its major coreceptor) and ET signaling, and the potential benefit of a combined anti-HER2/ET(A)R treatment in human breast cancer cells. Dual HER2-ET(A)R targeting utilizing trastuzumab (monoclonal anti-HER2 antibody) and the ET(A)R antagonist atrasentan was superior to each agent alone in inhibiting basal and EGF-induced proliferation and invasion of HER2-overexpressing BT-474 and SK-BR-3 cells. EGF-induced invasion was partially inhibited by atrasentan alone, suggesting the involvement of ET(A)R in EGF receptor mediated invasion of breast cancer cells. Moreover, secretion of the pro-invasive ET-1 was shown to be induced by EGF via EGFR and HER2, including MAPK-dependent signaling. In turn, an ET-1/ET(A)R-dependent regulation of EGFR protein expression and phosphorylation (at Tyr845) was observed, which may contribute to the additional anti-proliferative and anti-invasive effects of atrasentan on trastuzumab treated cells; reconfirming, atrasentan failed to enhance inhibitory effects of EGFR-targeted agents. This study suggests complex interactions between HER2/EGFR and ET pathways in breast cancer and supports the hypothesis that dual HER2-ET(A)R targeting may represent a highly effective approach in breast cancer treatment.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Atrasentan; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Down-Regulation; Drug Synergism; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Female; Genes, erbB-2; Humans; Neoplasm Metastasis; Phosphorylation; Pyrrolidines; Receptor, Endothelin A; Trastuzumab

The DLX gene family encodes for homeobox transcription factors involved in the control of morphogenesis and tissue homeostasis. Their expression can be regulated by Endothelin1 (ET1), a peptide associated with breast cancer invasive phenotype. Deregulation of DLX gene expression was found in human solid tumors and hematologic malignancies. In particular, DLX4 overexpression represents a possible prognostic marker in ovarian cancer. We have investigated the role of DLX genes in human breast cancer progression.. MDA-MB-231 human breast carcinoma cells were grown in vitro or injected in nude mice, either subcutaneously, to mimic primary tumor growth, or intravenously, to mimic metastatic spreading. Expression of DLX2, DLX5 and DLX6 was assessed in cultured cells, either treated or not with ET1, tumors and metastases by RT-PCR. In situ hybridization was used to confirm DLX gene expression in primary tumors and in lung and bone metastases. The expression of DLX2 and DLX5 was evaluated in 408 primary human breast cancers examining the GSE1456 and GSE3494 microarray datasets. Kaplan-Meier estimates for disease-free survival were calculated for the patients grouped on the basis of DLX2/DLX5 expression.. Before injection, or after subcutaneous growth, MDA-MB-231 cells expressed DLX2 but neither DLX5 nor DLX6. Instead, in bone and lung metastases resulting from intravenous injection we detected expression of DLX5/6 but not of DLX2, suggesting that DLX5/6 are activated during metastasis formation, and that their expression is alternative to that of DLX2. The in vitro treatment of MDA-MB-231 cells with ET1, resulted in switch from DLX2 to DLX5 expression. By data mining in microarray datasets we found that expression of DLX2 occurred in 21.6% of patients, and was significantly correlated with prolonged disease-free survival and reduced incidence of relapse. Instead, DLX5 was expressed in a small subset of cases, 2.2% of total, displaying reduced disease-free survival and high incidence of relapse which was, however, non-significantly different from the other groups due to the small size of the DLX+ cohort. In all cases, we found mutually exclusive expression of DLX2 and DLX5.. Our studies indicate that DLX genes are involved in human breast cancer progression, and that DLX2 and DLX5 genes might serve as prognostic markers.

Topics: Animals; Biomarkers, Tumor; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Disease-Free Survival; Endothelin-1; Female; Gene Expression Profiling; Homeodomain Proteins; Humans; In Situ Hybridization; Kaplan-Meier Estimate; Lung Neoplasms; Mice; Mice, Nude; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Prognosis; Reverse Transcriptase Polymerase Chain Reaction; Time Factors; Transcription Factors

Endothelin (EDN) signalling plays a crucial role in cell differentiation, proliferation and migration processes. There is compelling evidence that altered EDN signalling is involved in carcinogenesis by modulating cell survival and promoting invasiveness. To date, most reports have focused on the oncogenic potential of EDN1 and EDN2, both of which are overexpressed in various tumour entities. Here, we aimed at a first comprehensive analysis on EDN3 expression and its implication in human breast cancer.. EDN3 mRNA expression was assessed by Northern blotting in normal human tissues (n = 9) as well as in matched pairs of normal and tumourous tissues from breast specimens (n = 50). EDN3 mRNA expression in breast cancer was further validated by real-time polymerase chain reaction (PCR) (n = 77). A tissue microarray was used to study EDN3 protein expression in breast carcinoma (n = 150) and normal breast epithelium (n = 44). EDN3 promoter methylation was analysed by methylation-specific PCR in breast cell lines (n = 6) before and after demethylating treatment, normal breast tissues (n = 17) and primary breast carcinomas (n = 128). EDN3 expression and methylation data were statistically correlated with clinical patient characteristics and patient outcome.. Loss of EDN3 mRNA expression in breast cancer, as initially detected by array-based expression profiling, could be confirmed by Northern blot analysis (> 2-fold loss in 96%) and real-time PCR (> 2-fold loss in 78%). Attenuated EDN3 expression in breast carcinoma was also evident at the protein level (45%) in association with adverse patient outcome in univariate (P = 0.022) and multivariate (hazard ratio 2.0; P = 0.025) analyses. Hypermethylation of the EDN3 promoter could be identified as the predominant mechanism leading to gene silencing. Reversion of the epigenetic lock by 5-aza-2'-deoxycytidine and trichostatin A resulted in EDN3 mRNA re-expression in vitro. Furthermore, EDN3 promoter hypermethylation was detected in 70% of primary breast carcinomas with significant association to loss of EDN3 mRNA expression (P = 0.005), whilst normal matched breast tissues revealed no EDN3 promoter methylation.. EDN3 is a frequent target of epigenetic inactivation in human breast cancer, potentially contributing to imbalanced EDN signalling commonly found in this disease. The clinical implication supports the view that EDN3, in contrast to EDN1 and EDN2, may act as natural tumour suppressor in the human mammary gland.

Topics: Breast Neoplasms; Cell Differentiation; Cell Movement; Cell Proliferation; Cell Survival; Endothelin-1; Endothelin-2; Endothelin-3; Epigenesis, Genetic; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Invasiveness; Oligonucleotide Array Sequence Analysis; Signal Transduction

Endothelin-1[ET-1] acts as a growth factor in various malignancies. Big endothelin-1 (big ET-1) is the precursor of ET-1. The aim of this study was to determine the importance of serum big ET-1 levels as a novel marker of disease in female patients with breast cancer of various clinical stages. The study consisted of 75 female patients with breast cancer who were diagnosed and treated at the Gazi University Department of Medical Oncology and 20 controls. The patients were classified as follows: group A [n=26], patients with newly diagnosed primary breast cancer but without metastasis; group B [n=33], patients with metastatic breast cancer who had undergone treatment for their diseases and in whom metastasis was detected during follow-up; group C [n=16], off-therapy patients whose cancer had been in remission for at least 5 years; and group D [n=20] healthy controls. Serum big ET-1 level were measured with an enzyme immunoassay kit. The median serum big ET-1 levels of the 75 patients with breast cancer [10.96+/-1.36 ng/ml] were statistically significantly higher than those of controls [8.97+/-1.55 ng/ml]. The median serum big ET-1 levels of the patients with primary breast cancer patients [group A] were statistically significantly different from those in the controls, the off-therapy patients and the patients with metastatic disease [11.56+/-0.78 ng/ml, 8.97+/-1.55 ng/ml, 9.76+/-1.52 ng/ml, and 10.83+/-1.18 ng/ml respectively, P=.001]. There was no statistically significant difference in the serum big ET-1 levels of patients in group A in terms of tumor stage, hormone receptor status or lymph node status. Serum big ET-1 levels were statistically significantly higher in patients with metastatic disease than in controls or off-therapy patients (P=.001). The serum big ET-1 levels of off-therapy patients whose disease was in remission were not statistically significantly different from those in controls (P>.05). Serum big ET-1 levels seemed to represent the activation of ET-1 in female patients with breast cancer. Serum big ET- 1 levels can be an indicator of the breast cancer. Further studies are needed to demonstrate the prognostic importance of serum big ET-1 in patients with breast cancer.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Endothelin-1; Female; Humans; Middle Aged; Neoplasm Metastasis; Neoplasm Staging

Tumour hypoxia, being widespread in solid tumours, is related to an increased risk of invasion and metastasis as well as to resistance to chemotherapy and radiotherapy. Hypoxia-inducible factor-1alpha (HIF-1alpha) has emerged as the key regulator of the cellular response to hypoxia. In primary breast cancers, HIF-1alpha is overexpressed, and high levels of HIF-1alpha predict for early relapse and increased metastasis. The endothelin (ET) axis, comprising the peptides ET-1, -2, -3 and their receptors A (ETAR) and B (ETBR), is another regulatory system of major relevance in human breast cancer. However, little is known about the interaction of HIF-1alpha and the ET axis in breast carcinomas. Therefore, we analysed expression of HIF-1alpha and the ET axis in 600 breast cancer tissue samples by immunohistochemistry, observing a significant correlation between expression of HIF-1alpha and ET-1 (P<0.001). In vitro, hypoxia was found to double ET-1 secretion of MCF-7 breast cancer cells (203.5% of controls; P<0.001), thereby promoting an invasive phenotype. Of note, real-time PCR analysis revealed that the increase of ET-1 was not due to enhanced transcription of the ET-1 gene. In invasion assays, breast cancer cell invasiveness was strongly increased by hypoxia (150.0% of controls; P=0.007). Most important, this increase was completely inhibited by the selective ETAR antagonist atrasentan. In conclusion, we provide evidence for a relevant interaction between hypoxia and the ET axis in breast cancer cells. Our data suggest that tumour hypoxia induces breast carcinoma invasiveness by releasing intracellularly stored ET-1. However, induction of invasiveness may be inhibited by selective ETAR antagonism, thus emphasising the promising status of the ET axis as a therapeutic target in breast cancer.

Topics: Antineoplastic Agents; Aspartic Acid Endopeptidases; Atrasentan; Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Cobalt; Endothelin A Receptor Antagonists; Endothelin-1; Endothelin-Converting Enzymes; Female; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Metalloendopeptidases; Neoplasm Invasiveness; Neoplasm Staging; Pyrrolidines; Receptor, Endothelin A; Time Factors; Up-Regulation

Breast cancer is the prevalent cancer worldwide. Excessive exposure to endogenous estrogen across a woman's lifespan contributes to and may be a causal factor in breast cancer. Tamoxifen is a mixed estrogen agonist and antagonist, which is used in treatment and prevention of breast cancer as an estrogen antagonist. Many patients experience resistance to tamoxifen for which many mechanisms have been suggested. Endothelin-1 acts as a mitogen for human breast fibroblasts and it affects tumor cell proliferation, invasion, angiogenesis, neovascularization, mitogenesis, and apoptosis inhibition. Previous studies have shown that estradiol is effective in inhibiting endothelin synthesis in breast tissue and cardiovascular system. Tamoxifen as an estrogen receptor (ER) agonist in cardiovascular system has a cardioprotective effect and decreases endothelin level as a vasoconstrictor in cardiovascular system. But in breast tissue tamoxifen acts as an ER antagonist. According to the role of endothelin in breast cancer and inhibitory effect of estrogen on endothelin, we hypothesized that tamoxifen causes increasing in endothelin level or endothelin receptors probably by inhibitory effect on ER in breast tissue, leading to tamoxifen resistance. Therefore a combination of tamoxifen with endothelin antagonist seems to be a reasonable therapeutic strategy.

Topics: Antineoplastic Agents, Hormonal; Breast Neoplasms; Drug Resistance, Neoplasm; Endothelin-1; Female; Humans; Tamoxifen

Endothelin-1 expression is increased in infiltrating duct carcinoma and is associated with larger tumour size, higher histological grade and lymphovascular permeation. This has not been evaluated in phyllodes tumours, which are uncommon fibroepithelial lesions with potential for local recurrences or distant metastasis. While the grading of phyllodes tumours depends on a combination of histological parameters, prediction of their behaviour remains difficult.. A large series of 461 phyllodes tumours (291 benign, 115 borderline malignant and 55 frankly malignant) were evaluated for endothelin-1 expression in both the epithelial cells and stromal cells by immunohistochemistry; results were correlated with the tumour grade.. For benign phyllodes tumours, the epithelial staining of endothelin was negative, weak, moderate and strong in 6%, 26%, 15% and 53% of cases respectively; results were 4%, 18%, 19% and 59% respectively for borderline and 6%, 18%, 6% and 70% respectively for frankly malignant tumours. For the stromal staining, the negative, weak, moderate and strong staining was 32%, 19%, 18% and 31% respectively for benign phyllodes, 24%, 13%, 10% and 53% respectively for borderline and 8%, 16%, 17% and 59% respectively for frankly malignant tumours. There was correlation between epithelial and stromal staining, and the stromal staining correlated with histological features of stromal cellularity, stromal cell nuclear pleomorphism, margin status and stromal overgrowth.. These observations suggest a close relationship between the epithelial and stromal elements in phyllodes tumours; endothelin may play a significant role in the malignant progression of phyllodes tumours.

Topics: Adolescent; Adult; Aged; Breast Neoplasms; Cell Nucleus; Endothelin-1; Epithelial Cells; Female; Humans; Middle Aged; Neoplasm Proteins; Phyllodes Tumor; Stromal Cells

Endothelin-1 (ET-1) and its receptors, ET(A)R and ET(B)R, are overexpressed in breast carcinomas. However, little is known about the relevance of endothelin-converting enzyme-1 (ECE-1) and ET-1 degrading neprilysin (NEP). In this study, expression of ECE-1 and NEP was determined in 600 breast cancer tissue samples by immunohistochemistry; staining results were correlated with clinicopathological parameters. For ECE-1 expression, we found a significant correlation with VEGF (P < 0.001) and ET(A)R expression (P = 0.048). While patients with ECE-1 overexpressing tumours had more frequent disease recurrence (P = 0.03), NEP overexpression correlated with improved disease-free survival (DFS) (P = 0.023) and less frequent metastasis (P = 0.046). Also, a decrease of NEP expression with malignant progression (G1-G3) was found. ECE-1 inhibition using the selective ECE-1 inhibitor RO 67-7447 in MCF-7 breast cancer cells led to a significantly decreased ET-1 expression and reduced cell invasiveness (54.3% of controls, P = 0.014). Our results indicate that overexpression of ECE-1 is associated with unfavourable outcome, whereas NEP positively influences survival. Thus, expression of ECE-1 and NEP may have prognostic relevance. Due to the anti-invasive effect of the selective ECE-1 inhibitor, targeting ECE-1 may represent an innovative option in future breast cancer therapy.

Topics: Aspartic Acid Endopeptidases; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Endothelin-1; Endothelin-Converting Enzymes; Female; Humans; Immunohistochemistry; Metalloendopeptidases; Neoplasm Invasiveness; Neprilysin; Prognosis; RNA, Messenger

The betacellulin precursor (pro-BTC) is a novel substrate for ADAM10-mediated ectodomain shedding. In this report, we investigated the ability of novel physiologically relevant stimuli, including G-protein coupled receptor (GPCR) agonists and reactive oxygen species (ROS), to stimulate pro-BTC shedding. We found that in breast adenocarcinoma MCF7 cells overexpressing pro-BTC, hydrogen peroxide (H2O2) was a powerful stimulator of ectodomain shedding. The stimulation of pro-BTC shedding by H2O2 was blocked by the broad-spectrum metalloprotease inhibitor TAPI-0 but was still functional in ADAM17 (TACE)-deficient stomach epithelial cells indicating the involvement of a distinct metalloprotease. H2O2-induced pro-BTC shedding was blocked by co-culturing cells in the anti-oxidant N-acetyl-L-cysteine but was unaffected by culture in calcium-deficient media. By contrast, calcium ionophore, which is a previously characterized activator of pro-BTC shedding, was sensitive to calcium depletion but was unaffected by co-culture with the anti-oxidant, identifying a clear distinction between these stimuli. We found that in vascular smooth muscle cells overexpressing pro-BTC, the GPCR agonist endothelin-1 (ET-1) was a strong inducer of ectodomain shedding. This was blocked by a metalloprotease inhibitor and by overexpression of catalytically inactive E385A ADAM10. However, overexpression of wild-type ADAM10 or ADAM17 led to an increase in ET-1-induced pro-BTC shedding providing evidence for an involvement of both enzymes in this process. This study identifies ROS and ET-1 as two novel inducers of pro-BTC shedding and lends support to the notion of activated shedding occurring under the control of physiologically relevant stimuli.

Topics: ADAM Proteins; ADAM10 Protein; ADAM17 Protein; Amyloid Precursor Protein Secretases; Animals; Betacellulin; Breast Neoplasms; Calcimycin; Cell Line, Tumor; Cells, Cultured; Dipeptides; Endothelin-1; Female; Humans; Hydrogen Peroxide; Hydroxamic Acids; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Metalloendopeptidases; Mice; Muscle, Smooth, Vascular; Protein Precursors; Protein Structure, Tertiary; Rabbits; Receptors, G-Protein-Coupled

Angiogenic factors including endothelin-1 (ET-1) play a key role in the progression of breast metastases to bone. We investigated the impact of ET-1 on the development of bone metastases in an immunocompetent murine skin-fold chamber model. Murine mammary carcinoma 4T1 was injected in a skin-fold chamber implanted on CB6 mice along with bone explants. Furthermore, mice were treated with or without a dual selective antagonist of both ET-1 receptors. The progression of the vascularization within the chamber was monitored over time by intravital microscopy (IVM). The tumor growth and the development of bone metastases were assessed by cytokeratin-19 gene expression and histological studies. Results indicate that this new model associated with IVM allows for the continuous monitoring of the change in vascularization associated with the development of bone metastases. Additionally, treatment with an antagonist of both ET-1 receptors was associated with the presence of significantly less vessels near the tumor mass compared to control mice. These changes were correlated with smaller tumor masses and reduced bone invasion (P < 0.05). Thus, in an immunocompetent murine model of breast carcinoma metastases to bone, our data support the hypothesis that vascularization plays a role in tumor development and progression and that ET-1 specifically modulates the angiogenesis associated with breast metastases to the bone.

Topics: Angiogenesis Inhibitors; Animals; Bone Neoplasms; Bosentan; Breast Neoplasms; Cell Division; Endothelin-1; Mammary Neoplasms, Animal; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Models, Biological; Neoplasm Metastasis; RNA, Messenger; Skin; Sulfonamides

Phyllodes tumors (PTs) of the breast are biphasic neoplasms composed of epithelium and a spindle-cell stroma. Currently, PTs are classified as benign, borderline, or malignant based on histopathologic features. However, histologic classification does not always predict outcome. Objective.-To determine the prognostic value of a variety of clinicopathologic features and immunoreactivities in PTs.. Sixteen benign, 8 borderline, and 6 malignant PTs with follow-up were examined for reactivity across a panel of immunohistochemical stains, including c-Kit, endothelin 1, p16, p21, p53, and Ki-67. Clinicopathologic features, including stromal cellularity, mitotic rate, and margin status, were also assessed. Tumor variables were compared among tumor subgroups and between tumors that did and did not recur.. Of the 30 PTs, 4 recurred (1 benign, 2 borderline, 1 malignant). One patient with a malignant tumor died of metastatic disease 34 months after initial diagnosis. The overall positive rate of c-Kit immunoreactivity was 13% in benign, 63% in borderline, and 67% in malignant PTs. Endothelin 1 epithelial cytoplasmic staining was seen in 100% of benign, 50% of borderline, and 17% of malignant PTs. Additionally, p16, p21, p53, and Ki-67 were differentially expressed among benign, borderline, and malignant tumors. Positive surgical resection margins was the only variable that significantly predicted recurrent disease (P = .02).. Stromal c-Kit positivity and epithelial endothelin 1 negativity are more often associated with malignant PTs; however, only positive margin status is significantly associated with tumor behavior.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers, Tumor; Breast Neoplasms; Disease Progression; Endothelin-1; Epithelium; Female; Humans; Immunohistochemistry; Ki-67 Antigen; Mastectomy; Mastectomy, Segmental; Middle Aged; Neoplasm Recurrence, Local; Phyllodes Tumor; Prognosis; Proto-Oncogene Proteins c-kit; Staining and Labeling; Tumor Suppressor Protein p53

Endothelin expression is increased in breast tumors and is associated with invasion and metastasis, whereas CCR7 expression by breast tumor cells may have a role in the organ specificity of breast cancer spread. In this article, we have analyzed whether endothelins influence breast tumor cell expression of the chemokine receptor CCR7. Stimulation of human breast tumor cell lines with endothelins increased cell surface expression of CCR7 via endothelin receptor A. The iron chelators desferrioxamine and cobalt chloride, which induce hypoxia-inducible factor (HIF)-mediated transcription, also increased CCR7 expression; transfection of a dominant-negative version of the HIF regulatory subunit, HIF-1alpha, into MCF-7 cells abolished CCR7 induction by endothelins, indicating that increased expression is due to HIF-1 stabilization. Endothelin stimulation promoted invasion toward the CCR7 ligands CCL19 and CCL21. Endothelin-mediated chemokine-independent invasion itself is dependent on CCR7 activity and could be abolished using a CCR7-neutralizing monoclonal antibody. In human breast carcinomas, mRNA expression of endothelins correlated with the level of CCR7 expression, both of which were associated with the presence of lymph node metastases. Expression of the CCR7 ligands CCL19 and CCL21 was also higher in breast cancer patients with lymph node involvement compared with those without, but expression of these chemokines did not correlate with endothelin expression. These data show that CCR7 may be regulated by the breast tumor microenvironment and further support the use of endothelin receptor antagonists in the treatment of invasive and metastatic breast cancer.

Topics: Breast Neoplasms; Cell Line, Tumor; Cell Movement; DNA Primers; Endothelin-1; Female; Gene Expression Regulation, Neoplastic; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Neoplasm Invasiveness; Polymerase Chain Reaction; Receptors, CCR7; Receptors, Chemokine; RNA, Messenger; RNA, Neoplasm

The objective of this study was to investigate expression of various growth factors associated with angiogenesis and lymphangiogenesis and of their receptors in ductal carcinomas in situ of the breast (DCIS). We studied protein expression of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF)-A, endothelin (ET)-1, and VEGF-C, and their receptors bFGF-R1, Flt-1, KDR, ET(A)R, ET(B)R, and Flt-4 immunohistochemically in 200 DCIS (pure DCIS: n=96; DCIS adjacent to an invasive component: n=104) using self-constructed tissue microarrays. Basic fibroblast growth factor-R1, VEGF-C, Flt-4, and ET(A)R were expressed in the tumour cells in the majority of cases, whereas bFGF and Flt-1 expression was rarely observed. VEGF-A, KDR, ET-1, and ET(B)R were variably expressed. The findings of VEGF-C and its receptor Flt-4 as lymphangiogenic factors being expressed in tumour cells of nearly all DCIS lesions and the observed expression of various angiogenic growth factors in most DCIS suggest that in situ carcinomas are capable of inducing angiogenesis and lymphangiogenesis. Moreover, we found a higher angiogenic activity in pure DCIS as compared to DCIS with concomitant invasive carcinoma. This association of angiogenic factors with pure DCIS was considerably more pronounced in the subgroup of non-high-grade DCIS (n=103) as compared with high-grade DCIS (n=94). Determination of these angiogenic markers may therefore facilitate discrimination between biologically different subgroups of DCIS and could help to identify a particularly angiogenic subset with a potentially higher probability of recurrence or of progression to invasiveness. For these DCIS, targeting angiogenesis may represent a feasible therapeutic approach for prevention of progression of DCIS to invasion.

Topics: Adolescent; Adult; Aged; Biomarkers; Breast Neoplasms; Carcinoma in Situ; Carcinoma, Ductal, Breast; Endothelin-1; Female; Fibroblast Growth Factor 2; Humans; Middle Aged; Protein Array Analysis; Receptor Protein-Tyrosine Kinases; Receptor, Fibroblast Growth Factor, Type 1; Receptors, Fibroblast Growth Factor; Receptors, Vascular Endothelial Growth Factor; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor C; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2

There is increased staining of endothelins (ET-1, -2, and -3) and receptors (ET-RA and -RB) in invasive breast tumors compared to nonneoplastic tissue, and ETs stimulate MCF-7 cell invasion in vitro. We analyzed ETstimulation of benign and transformed mammary epithelial cells, and whether expression of ETs is sufficient to induce invasiveness. In breast cancer patient serum, ET-1 was increased in those patients with lymph node metastases compared to those with no lymph node involvement; ETs, however, had no mitogenic effect on breast tumor cell lines in vitro. The benign mammary epithelial cell line, hTERT-HME1, and the poorly invasive breast tumor cell line MCF-7 secreted low levels of ET-1, while the invasive cell lines SKBR3 and MDAMB231 secreted high levels. Expression of the ETs and receptors by the cell lines broadly correlated with their in vitro invasiveness; overexpression of ETs in MCF-7 cells increased basal invasion. ET-mediated invasion involved both receptors and a calcium influx to induce a pertussis toxin-sensitive MAPK pathway. MMP-14 activity was induced via ET-RA in an autocrine manner. In contrast to transformed cells, ET stimulation or overexpression did not induce an invasive phenotype in benign cells. Benign cells do not respond to ETs, and ET expression is not sufficient to induce invasion; however, the level of ET production by tumor cells correlates with their invasiveness, and increasing expression of the ET axis promotes breast tumor cell invasion via both receptors, while MMP-14 is induced via ET-RA.

Topics: Breast Neoplasms; Calcium; Cell Line; Cell Line, Tumor; Endothelin-1; Endothelins; Enzyme Induction; Epithelial Cells; Female; Humans; Lymphatic Metastasis; Mammary Glands, Human; Matrix Metalloproteinases; Neoplasm Invasiveness; Phenotype; Receptor, Endothelin A; Receptor, Endothelin B; Signal Transduction

Endothelin-1 (ET-1) is overexpressed in breast carcinomas and influences via its receptors (ETAR and ETBR) transformation, differentiation and growth processes in the human breast, but little is known about the ET expression in breast cancer precursors. On this basis we evaluated the expression of ET-1, ETAR and ETBR in a series of breast carcinomas, ductal (DCIS) and lobular carcinoma in situ (LCIS) and normal breast tissue by immunohistochemical (IH) methods. IH staining of ET-1, ETAR and ETBR was performed in 88 invasive breast carcinomas, with adjacent carcinoma in situ and concomitant normal breast tissue. Moderate or strong cytoplasmic immunostaining was observed for ET-1 in 33.3%, for ETAR in 45.3% and for ETBR in 55.7% of invasive breast carcinomas. Comparative analysis of invasive cancer (CA), concomitant carcinoma in situ (CIS) and normal breast epithelium (NBE) revealed a stepwise increase of ET-1 and ETAR expression in the sequence NBE < CIS < CA. ETBR expression tended to be slightly higher in CIS than in CA (NBE versus CIS and NBE versus CA, for ETAR and ETBR, p<0.001, respectively; NBE versus CA for ET-1, p=0.035). Our data suggest that the expression of ET-1, ETAR and ETBR correlates with the acquisition of malignant potential and may be used as a prognostic indicator of aggressive behaviour and invasive potential of premalignant breast lesions.

Topics: Biomarkers, Tumor; Breast Neoplasms; Carcinoma; Carcinoma in Situ; Carcinoma, Intraductal, Noninfiltrating; Carcinoma, Lobular; Endothelin-1; Female; Humans; Immunochemistry; Receptor, Endothelin A; Receptor, Endothelin B; Retrospective Studies

We have studied the role of endothelins (ET-1, ET-2 and ET-3) and ET receptors (ET-RA and ET-RB) in the invasive capacity of breast tumor cells, which express ET-1 and ET-2 as well as ET-RA and ET-RB. Of five human breast tumor cell lines tested, all expressed mRNAs for ET-1, ET-2, and ET-RB. ET-RA mRNA was expressed by four of five tumor cell lines. Breast tumor cells migrated toward ET-1 and ET-2 but not toward ET-3. Chemotaxis involved signaling via both receptors, and a pertussis toxin-sensitive p42/p44 mitogen-activated protein kinase (MAPK)-mediated pathway that could be inhibited by MAPK kinase (MEK)1/2 antagonists. Chemotaxis toward ETs did not involve p38 or stress-activated protein kinase (SAPK)/Jun N-terminal kinase (JNK) and was not inhibited by hypoxia. Incubation of tumor cells with ET-2 also increased chemotaxis toward the chemokines CXCL12 and CCL21. As well as inducing chemotaxis of tumor cells, ET-1 and ET-2 increased tumor cell invasion through Matrigel. Furthermore, stimulation of macrophage/tumor cell cocultures with ETs led to increased matrix metalloproteinase (MMP)-2 and -9 production by macrophages and a marked increase in invasion of tumor cells. Antagonism of either ET-RA or ET-RB decreased the invasion seen in ET-stimulated cocultures, as did a broad-spectrum MMP inhibitor. Immunohistochemical staining of human breast tumor sections showed increased ET and ET receptor protein expression by tumor cells in invasive ductal carcinoma compared with normal breast tissue or ductal carcinoma in situ. Furthermore, tumor cell ET and receptor expression was stronger at the invasive margin of invasive ductal carcinomas, in the lymphovascular space, and in lymph node metastases. ET expression often colocalized with ET-RB expression in all neoplastic tissue indicating a possible autocrine action of ETs. We suggest that expression of ETs and their receptors by human breast tumors, particularly in conjunction with a high macrophage infiltrate, may have a role in the progression of breast cancer and the invasion of tumor cells.

Topics: Breast Neoplasms; Carcinoma in Situ; Carcinoma, Ductal; Cell Line, Tumor; Chemotaxis; Coculture Techniques; Endothelin-1; Endothelin-2; Humans; Isoenzymes; Macrophages; MAP Kinase Signaling System; Matrix Metalloproteinases; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Neoplasm Invasiveness; Receptor, Endothelin A; Receptor, Endothelin B; RNA, Messenger

Endothelin-1 (ET-1) and its receptors (ET(A)R and ET(B)R), referred to as the endothelin (ET) axis, are overexpressed in breast carcinomas, and influence tumorigenesis and tumor progression by various mechanisms, including angiogenesis. The objective of the study was to clarify if expression of the ET axis participates in angiogenesis of breast carcinoma. We analyzed expression of ET-1, ET(A)R, ET(B)R, and vascular endothelial growth factor (VEGF) immunohistochemically in 600 tissue array specimens from 200 paraffin-embedded breast carcinomas performing tissue microarray technology. Microvessel density (MVD) was determined by counting microvessels (identified by factor VIII) in each core specimen.. Moderate or strong immunostaining was observed for ET-1 in 25.4%, for ET(A)R in 43.7%, and for ET(B)R in 22.2% of breast carcinomas. Of all cases, 44.7% showed significant expression of VEGF. MVD varied between different tumor specimens (range, 0-80; median, 17). We observed a statistically significant correlation between MVD and ET expression status with higher MVD in ET-positive tumors. Moreover, expression of VEGF was found more frequently in tumors with overexpression of the ET axis (each P < 0.001). Staining of VEGF was correlated positively with MVD CONCLUSIONS: These results indicate that increased ET-1, ET(A)R, and ET(B)R expression is associated with increased VEGF expression and higher vascularity of breast carcinomas and, thus, could be involved in the regulation of angiogenesis in breast cancer. Our findings provide evidence that the expression pattern of the ET-axis and in particular of ET(A)R may have clinical relevance in future antiangiogenic targeted therapies for breast cancer.

Topics: Breast Neoplasms; Carcinoma; Cell Line, Tumor; Disease Progression; Endothelin-1; Factor VIII; Humans; Immunohistochemistry; Microcirculation; Neoplasm Metastasis; Neovascularization, Pathologic; Platelet Endothelial Cell Adhesion Molecule-1; Receptor, Endothelin A; Receptor, Endothelin B; Tissue Distribution; Vascular Endothelial Growth Factor A

The vasodilator hydralazine, used clinically in cardiovascular therapy, relaxes arterial smooth muscle by inhibiting accumulation of intracellular free Ca2+ via an unidentified primary target. Collagen prolyl hydroxylase is a known target of hydralazine. We therefore investigated whether inhibition of other members of this enzyme family, namely the hypoxia-inducible factor (HIF)-regulating O2-dependent prolyl hydroxylase domain (PHD) enzymes, could represent a novel mechanism of action. Hydralazine induced rapid and transient expression of HIF-1alpha and downstream targets of HIF (endothelin-1, adrenomedullin, haem oxygenase 1, and vascular endothelial growth factor [VEGF]) in endothelial and smooth muscle cells and induced endothelial cell-specific proliferation. Hydralazine dose-dependently inhibited PHD activity and induced nonhydroxylated HIF-1alpha, evidence for HIF stabilization specifically by inhibition of PHD enzyme activity. In vivo, hydralazine induced HIF-1alpha and VEGF protein in tissue extracts and elevated plasma VEGF levels. In sponge angiogenesis assays, hydralazine increased stromal cell infiltration and blood vessel density versus control animals. Thus, hydralazine activates the HIF pathway through inhibition of PHD activity and initiates a pro-angiogenic phenotype. This represents a novel mechanism of action for hydralazine and presents HIF as a potential target for treatment of ischemic disease.

Topics: Adrenomedullin; Angiogenesis Inducing Agents; Animals; Breast Neoplasms; Carcinoma; Carcinoma, Renal Cell; Cell Hypoxia; Cell Line, Tumor; Cells, Cultured; DNA-Binding Proteins; Dose-Response Relationship, Drug; Endothelial Cells; Endothelin-1; Enzyme Inhibitors; Gene Expression Regulation; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Humans; Hydralazine; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Implants, Experimental; Kidney Neoplasms; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Nude; Myocytes, Smooth Muscle; Neovascularization, Physiologic; Nuclear Proteins; Peptides; Procollagen-Proline Dioxygenase; Transcription Factors; Vascular Endothelial Growth Factor A; Vasodilator Agents

Endothelin-1 (ET-1) is overexpressed in breast carcinomas and stimulates tumor cell growth in an autocrine and paracrine fashion via its receptors, ET(A)R and ET(B)R. In this study, we evaluated the expression of ET-1 and ET receptors in breast carcinomas and determined its clinical and prognostic significance.. We analyzed expression of ET-1, ET(A)R, and ET(B)R in 176 breast carcinomas using a semiquantitative immunohistochemical approach. Statistical analysis of clinicopathological variables such as pT stage, pN stage, hormone receptor status, Her-2/neu amplification, histological grade, and long-term follow-up data were performed.. We observed a moderate to strong cytoplasmic staining for ET-1 in 69 (43.1%), for ET(A)R in 74 (46.5%), and for ET(B)R in 86 (53.4%) cases of primary breast cancer. A correlation was found between increased ET-1 expression and its receptors with several clinicopathological parameters that characterize aggressive types of breast cancer, with the exception of increased ET(A)R and ET(B)R expression with positive estrogen receptor status. Elevated expression of ET-1, ET(A)R, and ET(B)R was more common in breast carcinomas of patients with lower disease-free survival time and overall survival. In addition, a statistically significant correlation was observed between ET(A)R expression and reduced disease-free survival time (P = 0.041). Interestingly, the prognostic impact of ET(A)R expression was shown to be more pronounced in the subgroup of patients with a putative favorable prognosis according to classic prognostic factors.. Therefore, analysis of ET(A)R expression may improve the prediction of relapse and death and facilitate an individually based risk-directed adjuvant therapy in breast cancer patients.

Topics: Breast Neoplasms; Disease-Free Survival; Endothelin-1; Female; Humans; Immunohistochemistry; Lymphatic Metastasis; Multivariate Analysis; Neoplasm Metastasis; Neoplasm Staging; Receptor, Endothelin A; Receptor, Endothelin B; Reproducibility of Results; Survival Analysis; Time Factors

Endothelins (ET-1, ET-2 and ET-3) are 21-amino acid vasoactive peptides that bind to G-protein-linked transmembrane receptors, ET-RA and ET-RB. As well as modulating vasoconstriction, endothelins regulate growth in several cell types and may also affect differentiation, inflammation and angiogenesis. Both macrophages and endothelins are found in areas of hypoxia in solid tumors and ET-2 expression may be modulated by hypoxia in some tumors. As the peptide structure of mature endothelins is similar to that of CXC chemokines, we asked if endothelins contribute to control of macrophage distribution in tumors. We found that ET-2 is a chemoattractant for macrophages and THP-1 monocytic cells, but not for freshly isolated monocytes. The chemotactic response to ET-2 shows a typical bell-shaped response curve. Experiments with endothelin receptor antagonists showed that migration to ET-2 is mediated via the ET-RB receptor. Moreover, monocytes do not express ET-RB. Chemotaxis towards ET-2 is via the MAPK pathway: p44 and p42 are phosphorylated when THP-1 cells are stimulated with ET-2, and the MAPKK inhibitor PD98059 stops chemotaxis. As with 'classical' chemokines, migration toET-2 is also inhibited by hypoxia and by pertussis toxin. As well as its chemotactic properties, ET-2 leads to activation of macrophages. In human breast tumors that express ET-2, endothelins and ET-RB expressing macrophages often co-localized. While shorter than 'classical' chemokines, ET-2 shares a similar peptide sequence with chemokines and may signal via a similar receptor and MAPK-mediated pathway. Furthermore, ET-2 expression by tumors may modulate the behavior of macrophages such that activated cells accumulate in areas of hypoxia.

Topics: Bacterial Proteins; Breast Neoplasms; Carcinoma, Ductal, Breast; Cell Hypoxia; Cell Line; Chemokines, CXC; Chemotactic Factors; Chemotaxis; Endothelin Receptor Antagonists; Endothelin-1; Endothelin-2; Enzyme Inhibitors; Female; Flavonoids; Humans; Macrophage Activation; Macrophages; MAP Kinase Signaling System; Membrane Proteins; Monocytes; Neoplasm Proteins; Oligopeptides; Peptides, Cyclic; Phosphorylation; Piperidines; Protein Processing, Post-Translational; Receptor, Endothelin A; Receptor, Endothelin B; Receptors, Endothelin; Structure-Activity Relationship

Endothelins (ETs) are peptides expressed in many tumours which may stimulate angiogenesis and desmoplasia. Because ETs have not been extensively studied mammary neoplasia, we assessed ET protein and mRNA expression and receptor mRNA expression in normal and neoplastic breast tissues.. Tissues from five normal breasts, six fibroadenomas, seven ductal carcinomas in situ (DCIS) and 25 invasive carcinomas were stained with anti-ET-1 and anti-ET-3 antibodies and analysed using a grading system. ET-1, ET-3, ETA and ETB mRNA expression was assessed by quantitative RT-PCR from eight carcinomas and five normals. Weak staining for ET-1 and ET-3 was detected in all normals. Moderate to strong staining was seen in 72% and 64% of carcinomas for ET-1 and ET-3, respectively. Most fibroadenomas showed weak positivity for ET-1 (83%) and ET-3 (67%). ET-1 and ET-3 mRNA levels were upregulated in carcinomas compared with normal breast. No ETA mRNA was not detected in any tissue. ETB mRNA was detected in normal breast and was increased in carcinomas.. These results suggest that the ET system is altered in breast carcinomas and this may be of importance in the progression from in-situ to invasive carcinoma.

Topics: Blotting, Southern; Breast; Breast Neoplasms; Carcinoma in Situ; Carcinoma, Ductal, Breast; Endothelin-1; Endothelin-3; Female; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Receptor, Endothelin B; Receptors, Endothelin; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger

Ectopic hormone secretion in tumor cells is here described as an amplification of hormone production already present in normal, nonendocrine tumor-originated tissue. This idea is tested on the available data regarding endothelin-1 (ET-1) secreting tumors. The endothelins are ubiquitous regulatory peptides produced by various tissues. The precursor cells of many tumor types secrete endothelins. ET-1 protein expression was detected in situ in all tested prostate cancers as well as in normal prostate tissue. The majority of hepatocellular carcinomas produce ET-1, while ET-1 is secreted by the normal hepatic stellate cells. Human breast cancer cells produce immunoreactive ET-1. Similar data exist for pancreatic tissue, the thyroid and large bowel. We can conclude that tumor cells might sustain endothelin secretions already present in the normal tumor-originated tissue. The model that is presented of the pseudoectopic hormone secretion consists of relations between a few parameters. The proportion of hormone-secreting tumors (Th) among all tumors (T) of that organ depends on the amount of the hormone-secreting cells (Ch) among all cells (C) susceptible to malignant transformation. The corrective factor (k) was introduced in the expression Th/T=Ch/C*k, to represent specific conditions altering the malignant transformation probability for a certain normal hormone-secreting cell. In prostate, breast and colon, the kvalue is predicted to be approximately 1, suggesting that ET-1-secreting normal cells are not more prone to the malignant transformation than their neighbours. In liver and pancreas, the incidence of ET-1-secreting tumors outnumbers the proportions of normal ET-1-secreting cells (k values >1). In these organs, normal ET-1-secreting cells seem more likely to turn malignant in comparison to their neighbours, perhaps due to their function, position and exposition to oncogenic factors, or even due to their ET-1 secretion. There are similar data for thyroid and adrenal glands. No ET-1 secretion was reported in kidney neoplasms. Normal renal ET-1 secreting cells might be less prone to turn malignant than other renal cells. Unlse the normal lung tissue, small cell lung cancers often secrete adrenocorticotrophic hormone (ACTH). The pancreatic islet cells do not secrete gastrin, but their tumors often do. Constant k would exceed 1 in both cases. We speculate that these tumors might originate from a small subset of cells with the described feature. Tumor cells s

Topics: Animals; Breast Neoplasms; Endothelin-1; Female; Hormones; Humans; Kidney Neoplasms; Male; Models, Biological; Neoplasms

Breast cancer cells secrete endothelin-1 (ET-1), which may act as a paracrine mitogen in breast tumours. The paracrine factors and signal transduction pathways responsible for regulating ET-1 production in breast cancer are unknown. In this study we have examined the involvement of the protein kinase A (PKA) signalling pathway in the control of ET-1 secretion in the human breast cancer cell line MCF-7. Treatment of MCF-7 cells with various agents that activate protein kinase A (PKA) through increases in intracellular cAMP levels including forskolin, cholera toxin (ChT), the cAMP analogue 8-Br-cAMP, or the cAMP phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX) all markedly increased ET-1 release. Prostaglandin E2 (PGE2) while stimulating cAMP production, but not inositol lipid hydrolysis also significantly stimulated ET-1 release. Activation of PKC by 2-O-tetradecanoyl phorbol 13-acetate (TPA) also stimulated ET-1 secretion in MCF-7 cells. The PKA inhibitor H-89 attenuated the ET-1 response to PGE2, forskolin and ChT, but not that due to the PKC agonist TPA. The possibility that human breast fibroblasts (HBFs) are a target for ET-1 action with regard to PGE2 production was also investigated, and revealed that while HBFs were unresponsive to ET-1 alone, pretreatment with the cytokine IL-beta greatly potentiated PGE2 release in response to ET-1. In conclusion our results show that activation of either the PKA or PKC signalling pathways in human breast cancer cells increases ET-1 secretion. We also found that HBFs release PGE2 after treatment with ET-1 and that PGE2 itself stimulates ET-1 production in MCF-7 cells. The implication of this potential novel paracrine loop may be significant in view of the high levels of PGE2 and ET-1 found in malignant breast tissues.

Topics: Breast Neoplasms; Cyclic AMP-Dependent Protein Kinases; Dinoprostone; Endothelin-1; Enzyme Activation; Female; Fibroblasts; Humans; Signal Transduction; Tumor Cells, Cultured

Endothelin-1 (ET-1) levels are elevated in human breast tumours compared with normal and benign tissues, and in the presence of insulin-like growth factor 1 (IGF-I) ET-1 is a potent mitogen for human breast fibroblasts. In this study we have examined the ability of intact human breast cancer cell lines to process exogenously added big ET-1 (1-38) to the active mature ET-1 peptide by using a specific radioimmunometric assay. In both hormome-dependent (MCF-7, T47-D) and hormone-independent (MDA-MB-231) breast cancer cell lines the putative endothelin-converting enzyme (ECE) exhibited apparent Michaelis-Menten kinetics when converting added big ET-1 to ET-1. Both basal ET-1 production and exogenously added big ET-1 to ET-1 conversion were greatly reduced in all three cell lines in response to the metalloproteinase inhibitor phosphoramidon but were insensitive to other classes of protease inhibitors. Inhibition was also observed when cells were incubated in the presence of the divalent cation chelators 1,10-phenanthroline and EDTA. In MCF-7 cells the optimal pH for the ECE activity using a saponin cell permeabilisation procedure was found to residue within a narrow range of 6.2-7.26. Our results indicate that human breast cancer cells contain a neutral phosphoramidon-sensitive metalloproteinase which can process big ET-1 to ET-1. In the breast this conversion could contribute substantially to the local extracellular levels of this proposed paracrine breast fibroblast mitogen.

Topics: Aspartic Acid Endopeptidases; Breast; Breast Neoplasms; Chromatography, High Pressure Liquid; Culture Media; Edetic Acid; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Fibroblasts; Glycopeptides; Growth Substances; Humans; Hydrogen-Ion Concentration; Iron Chelating Agents; Kinetics; Metalloendopeptidases; Mitogens; Phenanthrolines; Protease Inhibitors; Protein Precursors; Sensitivity and Specificity; Tumor Cells, Cultured

This study performed immunohistochemical staining for human ET-1, utilizing avidinbiotin-peroxidase complex detection to examine 47 surgically resected primary breast cancers. Positive immunoreactivity was demonstrated in 19 of the 47 breast cancers (40.4%). There was no significant relationship between the expression of ET-1 and clinicopathological findings. A significant difference was found between ET-1 positive and negative groups in the incidence of recurrence and distant metastasis (P < 0.05). The 5-year overall survival rate was significantly poorer in patients with ET-1-positive cancer (84.2%), compared to 96.4% in patients with ET-1 negative cancer (P < 0.01). The 5-year disease-free survival rate was significantly poorer in patients with ET-1-positive cancer (73.7%) compared to 96.4% in patients with ET-1-negative cancer (P < 0.05). These results suggest that the expression of ET-1 could be used as a possible indicator for predicting the metastatic potential of breast cancer.

Topics: Adult; Aged; Breast Neoplasms; Culture Techniques; Endothelin-1; Female; Humans; Immunohistochemistry; Middle Aged; Prognosis; Reference Values; Sensitivity and Specificity; Survival Rate