adrenomedullin and Breast-Neoplasms

Breast cancer is the most frequently diagnosed cancer and is the leading cause of cancer-associated mortality in women worldwide. Intermedin (IMD, also known as Adrenomedullin 2, ADM2) is an endogenous peptide that belongs to the calcitonin gene-related peptide family and has been reported to play important roles in several types of cancers, including breast cancer. In this study, we sought to investigate how IMD affects the behavior of breast cancer cells, the underlying mechanism of these effects, and whether blockade of IMD has a therapeutic effect against breast cancer.. Transcriptome sequencing (RNA-Seq), cell biological experiments, Western blotting, immunoprecipitation, and animal tumor models were used.. IMD expression was significantly increased in breast cancer samples, and the IMD level was positively correlated with lymph node metastasis and Ki67 expression. Cell biological experiments showed that IMD promoted the anchorage-independent growth, migration, and invasive ability of breast cancer cells. Inhibiting IMD activity with an anti-IMD monoclonal antibody blocked these tumor-promoting effects. In addition, blockade of IMD reduced in situ tumor growth and significantly decreased lung metastasis of 4T1 breast cancer in vivo. IMD induced Src kinase phosphorylation, which triggered the transcription of c-Myc, a major oncoprotein controlling the expression of genes that encode ribosomal components. Our data suggest that IMD is involved in breast cancer cell invasion and metastasis, potentially through increasing ribosome biogenesis and protein translation via the Src/c-Myc signaling pathway.. These results suggest that IMD may be a novel target for the treatment of breast cancer.

Topics: Adrenomedullin; Animals; Breast Neoplasms; Female; Humans; Neuropeptides; Peptide Hormones; Protein Biosynthesis; Proto-Oncogene Proteins c-myc; Ribosomes; Signal Transduction

Cancer cells cooperate with cells that compose their environment to promote tumor growth and invasion. Among them, adipocytes provide lipids used as a source of energy by cancer cells and adipokines that contribute to tumor expansion. Mechanisms supporting the dynamic interactions between cancer cells and stromal adipocytes, however, remain unclear.. We set-up a co-culture model with breast cancer cells grown in 3D as mammospheres and human adipocytes to accurately recapitulate intrinsic features of tumors, such as hypoxia and cancer cell-adipocytes interactions.. Herein, we observed that the lipid droplets' size was reduced in adipocytes adjacent to the mammospheres, mimicking adipocyte morphology on histological sections. We showed that the uncoupling protein UCP1 was expressed in adipocytes close to tumor cells on breast cancer histological sections as well as in adipocytes in contact with the mammospheres. Mammospheres produced adrenomedullin (ADM), a multifactorial hypoxia-inducible peptide while ADM receptors were detected in adipocytes. Stimulation of adipocytes with ADM promoted UCP1 expression and increased HSL phosphorylation, which activated lipolysis. Invalidation of ADM in breast cancer cells dramatically reduced UCP1 expression in adipocytes.. Breast tumor cells secreted ADM that modified cancer-associated adipocytes through paracrine signaling, leading to metabolic changes and delipidation. Hence, ADM appears to be crucial in controlling the interactions between cancer cells and adipocytes and represents an excellent target to hinder them.

Topics: Adipocytes; Adrenomedullin; Breast; Breast Neoplasms; Cell Hypoxia; Coculture Techniques; Female; Humans; Lipid Droplets; Lipolysis; MCF-7 Cells; Paracrine Communication; Spheroids, Cellular; Tumor Microenvironment; Uncoupling Protein 1

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

Tumor- or cancer-associated fibroblasts (TAFs or CAFs) are active players in tumorigenesis and exhibit distinct angiogenic and tumorigenic properties. Adrenomedullin (AM), a multifunctional peptide plays an important role in angiogenesis and tumor growth through its receptors calcitonin receptor-like receptor/receptor activity modifying protein-2 and -3 (CLR/RAMP2 and CLR/RAMP3). We show that AM and AM receptors mRNAs are highly expressed in CAFs prepared from invasive breast carcinoma when compared to normal fibroblasts. Immunostaining demonstrates the presence of immunoreactive AM and AM receptors in the CAFs (n = 9). The proliferation of CAFs is decreased by anti-AM antibody (αAM) and anti-AM receptors antibody (αAMR) treatment, suggesting that AM may function as a potent autocrine/paracrine growth factor. Systemic administration of αAMR reduced neovascularization of in vivo Matrigel plugs containing CAFs as demonstrated by reduced numbers of the vessel structures, suggesting that AM is one of the CAFs-derived factors responsible for endothelial cell-like and pericytes recruitment to built a neovascularization. We show that MCF-7 admixed with CAFs generated tumors of greater volume significantly different from the MCF-7 xenografts in nude mice due in part to the induced angiogenesis. αAMR and AM22-52 therapies significantly suppressed the growth of CAFs/MCF-7 tumors. Histological examination of tumors treated with AM22-52 and aAMR showed evidence of disruption of tumor vasculature with depletion of vascular endothelial cells, induced apoptosis and decrease of tumor cell proliferation. Our findings highlight the importance of CAFs-derived AM pathway in growth of breast carcinoma and in neovascularization by supplying and amplifying signals that are essential for pathologic angiogenesis.

Topics: Adrenomedullin; Animals; Antibodies, Neutralizing; Blotting, Western; Breast Neoplasms; Cancer-Associated Fibroblasts; Cell Line; Cell Proliferation; Cells, Cultured; Chemokine CXCL12; Female; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Mice, Inbred C57BL; Mice, Nude; Neovascularization, Pathologic; Receptors, Adrenomedullin; Reverse Transcriptase Polymerase Chain Reaction; Transplantation, Heterologous; Tumor Burden

Adrenomedullin (AM) is secreted by breast cancer cells and increased by hypoxia. It is a multifunctional peptide that stimulates angiogenesis and proliferation. The peptide is also a potent paracrine stimulator of osteoblasts and bone formation, suggesting a role in skeletal metastases-a major site of treatment-refractory tumor growth in patients with advanced disease.. The role of adrenomedullin in bone metastases was tested by stable overexpression in MDA-MB-231 breast cancer cells, which cause osteolytic bone metastases in a standard animal model. Cells with fivefold increased expression of AM were characterized in vitro, inoculated into immunodeficient mice and compared for their ability to form bone metastases versus control subclones. Bone destruction was monitored by X-ray, and tumor burden and osteoclast numbers were determined by quantitative histomorphometry. The effects of AM overexpression on tumor growth and angiogenesis in the mammary fat pad were determined. The effects of AM peptide on osteoclast-like multinucleated cell formation were tested in vitro. A small-molecule AM antagonist was tested for its effects on AM-stimulated ex vivo bone cell cultures and co-cultures with tumor cells, where responses of tumor and bone were distinguished by species-specific real-time PCR.. Overexpression of AM mRNA did not alter cell proliferation in vitro, expression of tumor-secreted factors or cell cycle progression. AM-overexpressing cells caused osteolytic bone metastases to develop more rapidly, which was accompanied by decreased survival. In the mammary fat pad, tumors grew more rapidly with unchanged blood vessel formation. Tumor growth in the bone was also more rapid, and osteoclasts were increased. AM peptide potently stimulated bone cultures ex vivo; responses that were blocked by small-molecule adrenomedullin antagonists in the absence of cellular toxicity. Antagonist treatment dramatically suppressed tumor growth in bone and decreased markers of osteoclast activity.. The results identify AM as a target for therapeutic intervention against bone metastases. Adrenomedullin potentiates osteolytic responses in bone to metastatic breast cancer cells. Small-molecule antagonists can effectively block bone-mediated responses to tumor-secreted adrenomedullin, and such agents warrant development for testing in vivo.

Topics: Adenocarcinoma; Adrenomedullin; Animals; Bone and Bones; Bone Neoplasms; Breast Neoplasms; Cell Line, Tumor; Disease Progression; Female; Humans; Mice; Mice, Nude; Neoplasm Transplantation; RNA, Messenger

Topics: Adrenomedullin; Animals; Breast Neoplasms; Humans; Male; Mice; Neovascularization, Pathologic; Peptides; Severe Combined Immunodeficiency; Splenic Neoplasms

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

Adrenomedullin (ADM) is an angiogenic factor that has also been shown to be a mitogen and a hypoxia survival factor for tumour cells. These properties point to ADM as a potential promoter of human malignancies, but little data are available concerning the expression of ADM in human breast cancer. In the present work, we have examined ADM peptide expression in a series of malignant breast tumours by immunohistochemistry using a newly developed anti-ADM monoclonal antibody. In addition, ADM plasma concentrations in breast cancer patients and healthy controls were determined by radioimmunoassay. Of the examined breast cancer samples, 27/33 (82%) showed a moderate to strong staining intensity. ADM-peptide expression in breast tumours was significantly correlated with axillary lymph node metastasis (P=0.030). Analysis of ADM plasma concentrations showed no significant difference between the circulating ADM levels of breast cancer patients and healthy controls. However, a significant positive correlation was found between tumour size and plasma ADM levels (r=0.641, P=0.017). Moreover, ADM levels in breast cancer patients correlated with the presence of lymph node metastasis (P=0.002). In conclusion, we have shown for the first time that ADM peptide is widely expressed in breast cancer and that the degree of expression is associated with lymph node metastasis. ADM peptide in plasma of breast cancer patients reflects the size of the primary tumour, but is unlikely to be a useful tumour marker for the detection of breast cancer. Plasma ADM might represent an independent predictor of lymph node metastasis. The clinical implications of these findings remain to be evaluated.

Topics: Adrenomedullin; Adult; Aged; Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Carcinoma; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression Regulation; Humans; Immunohistochemistry; Lymphatic Metastasis; Middle Aged; Peptides; Prognosis; Vasodilator Agents

Adrenomedullin is a secreted peptide hormone with multiple activities. Several reports have indicated that adrenomedullin may be involved in tumor survival, but this has not been directly shown. Here we evaluate the in vitro and in vivo effects of adrenomedullin overexpression in human breast cancer cells.. The human breast cancer cell lines T47D and MCF7, both of which express low basal levels of adrenomedullin, were stably transfected with an expression construct that contained the coding region of the human adrenomedullin gene or with empty expression vector. Properties of the transfected cells were assessed by proliferation and apoptosis assays, in vitro and in vivo angiogenesis assays, cell migration experiments, and xenograft implants. The effect of synthetic adrenomedullin on human ovarian (ECV) cancer cell motility was also tested. Western blot analysis was used to compare expression levels of several genes whose products are associated with cell growth and regulation of apoptosis.. T47D and MCF7 cells transfected with the adrenomedullin construct both expressed high levels of adrenomedullin mRNA and protein. Compared with cells transfected with empty vector, cells that overexpressed adrenomedullin displayed a more pleiotropic morphology, an increased angiogenic potential both in vitro and in vivo, and less apoptosis after serum deprivation. T47D and MCF7 cells did not display measurable motility, but ECV ovarian cancer cells treated with synthetic adrenomedullin were more motile than saline-treated ECV cells. Adrenomedullin-overexpressing T47D cells had higher levels of proteins involved in oncogenic signal transduction pathways (such as Ras, Raf, PKC, and MAPKp49) and lower levels of pro-apoptotic proteins (such as Bax, Bid, and caspase 8) than T47D cells transfected with empty vector. In a preliminary in vivo experiment, three of 10 nude mice injected with adrenomedullin-overexpressing T47D cells developed xenograft tumors, whereas none of the 10 nude mice injected with cells carrying the empty plasmid developed tumors.. These results further support the role of adrenomedullin as a survival factor for tumors. Development of physiologically efficient inhibitors of adrenomedullin may prove useful in the clinical management of cancer.

Topics: Adrenomedullin; Aorta; Apoptosis; Biomarkers, Tumor; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Division; Cell Survival; DNA, Neoplasm; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Neovascularization, Pathologic; Peptides; Plasmids; RNA, Messenger; RNA, Neoplasm; Transfection; Tumor Cells, Cultured; Up-Regulation

Tamoxifen is the current therapy of choice for patients with oestrogen receptor positive breast cancer, and it is currently under evaluation as a prophylactic for women at high risk of developing the disease. However, tamoxifen is also known to induce proliferative changes in the endometrium increasing the risk of developing endometrial hyperplasia, polyps and carcinoma. Angiogenesis is an intimate part of this process. For this reason, we have examined the expression of several well characterized angiogenic factors, namely, acidic and basic fibroblast growth factor, thymidine phosphorylase, vascular endothelial growth factor and adrenomedullin in both normal and tamoxifen exposed pre- and postmenopausal endometrium. Vascular density and endothelial proliferation index were also quantified. We found increased expression of acidic and basic fibroblast growth factor and adrenomedullin after treatment with tamoxifen mainly in premenopausal tissue. Vascular density was significantly increased in pre- but not post-menopausal endometrium (P=0.0018) following tamoxifen treatment. These results support the notion that angiogenesis is integral to the response to tamoxifen exposure, and is a potential target with which to block these side effects of tamoxifen.

Topics: Adrenomedullin; Adult; Aged; Aged, 80 and over; Antineoplastic Agents, Hormonal; Breast Neoplasms; Endometrium; Female; Fibroblast Growth Factor 1; Fibroblast Growth Factor 2; Gene Expression Regulation, Neoplastic; Humans; Middle Aged; Neovascularization, Pathologic; Peptides; Postmenopause; Premenopause; Tamoxifen

We investigated tamoxifen's effects on the expression of growth regulatory genes in the endometrium to identify the mechanism by which tamoxifen induces proliferation.. Using immunohistochemical techniques, we analyzed 39 endometrial specimens for expression of Ki-67, lactoferrin, transforming growth factor-alpha, tumor necrosis factor receptor-II, adrenomedullin, estrogen receptors, and progesterone receptors. Twenty specimens were obtained from postmenopausal breast cancer patients treated with tamoxifen (20 mg/day) for at least 6 months to include two endometrial adenocarcinoma specimens. Five secretory phase, three proliferative phase, and seven atrophic endometrial specimens were used as controls. In addition, four endometrial adenocarcinoma specimens were reviewed from patients not treated with tamoxifen. Intensity of immunostaining was quantified using digitized imaging techniques.. Overexpression of both estrogen receptors and progesterone receptors, and an elevated proliferative index were the most consistent effects observed in benign endometrial specimens from tamoxifen-treated patients compared with atrophic controls (P <. 003). This staining pattern was also evident in adenocarcinomas from patients who received tamoxifen. Benign endometrium from tamoxifen-treated patients also expressed transforming growth factor-alpha, tumor necrosis factor receptor-II, lactoferrin, and adrenomedullin at levels comparable with those found in proliferative endometrial specimens.. These data provide further documentation that the uterotropic effects of tamoxifen may be due, at least in part, to the induction of estrogen receptors and progesterone receptors, as well as other genes associated with the proliferative phase. Furthermore, analysis of estrogen receptors, progesterone receptors, and Ki-67 may be useful in identifying postmenopausal individuals on tamoxifen, who are at increased risk for developing endometrial cancer.

Topics: Adrenomedullin; Antineoplastic Agents, Hormonal; Breast Neoplasms; Endometrial Neoplasms; Endometrium; Female; Gene Expression Regulation, Neoplastic; Genes, Regulator; Humans; Hyperplasia; Immunohistochemistry; Ki-67 Antigen; Lactoferrin; Peptides; Receptors, Estrogen; Receptors, Progesterone; Receptors, Tumor Necrosis Factor; Tamoxifen; Transforming Growth Factor alpha

Human adrenomedullin (hADM), human calcitonin gene-related peptide (hCGRP), and salmon calcitonin (sCT)-activated adenylyl cyclase with EC50 values of 132, 764, and 0.5 nM, respectively, in human breast cancer cell line, T 47D. Treatment of T 47D cell membranes with near maximal concentrations of sCT, hADM and hCGRP had no additive effect on adenylyl cyclase activity. Salmon calcitonin (8-32)[sCT (8-32)], selective antagonist of calcitonin receptor, inhibited the activation of adenylyl cyclase by these three peptides. On the other hand, the putative ADM receptor antagonist, ADM (22-52), and CGRP receptor antagonist, CGRP (8-37), failed to inhibit ADM-, CGRP- or sCT-activated adenylyl cyclase. These results suggest that in T47D cells, both ADM and CGRP activated adenylyl cyclase through sCT receptors.

Topics: Adenylyl Cyclases; Adrenomedullin; Breast Neoplasms; Calcitonin; Calcitonin Gene-Related Peptide; Calcitonin Gene-Related Peptide Receptor Antagonists; Cell Membrane; Drug Interactions; Enzyme Activation; Female; Humans; Membrane Proteins; Peptide Fragments; Peptides; Receptors, Adrenomedullin; Receptors, Calcitonin; Receptors, Peptide; Tumor Cells, Cultured