tetrathiomolybdate and Breast-Neoplasms

Although anti- angiogenesis strategies have generated much enthusiasm for therapeutic applications, it is still unknown whether they would be feasible for prevention. The possibility of interfering very early in tumor progression by modulating the cancer angiogenic switch is appealing. In this chapter, we review progress with in vitro and in vivo models that show that anti-angiogenic interventions may be amenable to long- term chemopreventive measures. In particular, some approaches that are nearly ready for major applications are anti-oxidant nutraceuticals and copper deficiency. We use these strategies as paradigms of how to make progress in this difficult but important area of translational research.

Topics: Angiogenesis Inhibitors; Animals; Anticarcinogenic Agents; Antioxidants; Breast Neoplasms; Carotenoids; Cell Transformation, Neoplastic; Chelating Agents; Copper; Dietary Supplements; Female; Flavonoids; Head and Neck Neoplasms; Humans; Lung Neoplasms; Male; Molybdenum; Neoplasms; Neovascularization, Pathologic; NF-kappa B; Phenols; Polyphenols; Prostatic Neoplasms; Reactive Oxygen Species

Bone marrow-derived progenitor cells, including VEGFR2. Patients with stage II triple-negative breast cancer (TNBC), stage III and stage IV without any evidence of disease (NED), received oral tetrathiomolybdate to maintain ceruloplasmin (Cp) between 8 and 17 mg/dL for 2 years or until relapse. Endpoints were effect on EPCs and other biomarkers, safety, event-free (EFS), and overall survival (OS). For laboratory studies, MDA-LM2-luciferase cells were implanted into CB17-SCID mice and treated with tetrathiomolybdate or water. Tumor progression was quantified by bioluminescence imaging (BLI), copper depletion status by Cp oxidase levels, lysyl oxidase (LOX) activity by ELISA, and collagen deposition.. Seventy-five patients enrolled; 51 patients completed 2 years (1,396 cycles). Most common grade 3/4 toxicity was neutropenia (3.7%). Lower Cp levels correlated with reduced EPCs (P = 0.002) and LOXL-2 (P < 0.001). Two-year EFS for patients with stage II-III and stage IV NED was 91% and 67%, respectively. For patients with TNBC, EFS was 90% (adjuvant patients) and 69% (stage IV NED patients) at a median follow-up of 6.3 years, respectively. In preclinical models, tetrathiomolybdate decreased metastases to lungs (P = 0.04), LOX activity (P = 0.03), and collagen crosslinking (P = 0.012).. Tetrathiomolybdate is safe, well tolerated, and affects copper-dependent components of the tumor microenvironment. Biomarker-driven clinical trials in high risk for patients with recurrent breast cancer are warranted. Clin Cancer Res; 23(3); 666-76. ©2016 AACR.

Topics: Adenocarcinoma; Amino Acid Oxidoreductases; Animals; Breast Neoplasms; Cell Line, Tumor; Ceruloplasmin; Chelating Agents; Copper; Disease Progression; Disease-Free Survival; Endothelial Progenitor Cells; Female; Follow-Up Studies; Humans; Lung Neoplasms; Mice, SCID; Molybdenum; Neoplasm Proteins; Neovascularization, Pathologic; Neutropenia; Risk; Triple Negative Breast Neoplasms; Tumor Microenvironment; Xenograft Model Antitumor Assays

Bone marrow-derived endothelial progenitor cells (EPCs) are critical for metastatic progression. This study explores the effect of tetrathiomolybdate (TM), an anti-angiogenic copper chelator, on EPCs in patients at high risk for breast cancer recurrence.. This phase 2 study enrolled breast cancer patients with stage 3 and stage 4 without evidence of disease (NED), and stage 2 if triple-negative. TM 100 mg orally was administered to maintain ceruloplasmin <17 mg/dl for 2 years or until relapse. The primary end point was change in EPCs.. Forty patients (28 stage 2/3, 12 stage 4 NED) were enrolled. Seventy-five percent patients achieved the copper depletion target by 1 month. Ninety-one percent of triple-negative patients copper-depleted compared with 41% luminal subtypes. In copper-depleted patients only, there was a significant reduction in EPCs/ml by 27 (P = 0.04). Six patients relapsed while on study, of which only one patient had EPCs maintained below baseline. The 10-month relapse-free survival was 85.0% (95% CI 74.6%-96.8%). Only grade 3/4 toxicity was hematologic: neutropenia (3.1% of cycles), febrile neutropenia (0.2%), and anemia (0.2%).. TM is safe and appears to maintain EPCs below baseline in copper-depleted patients. TM may promote tumor dormancy and ultimately prevent relapse.

Topics: Adult; Aged; Breast Neoplasms; Chelating Agents; Copper; Endothelial Cells; Female; Humans; Middle Aged; Molybdenum; Neoplasm Recurrence, Local; Risk Factors; Stem Cells

Cisplatin is an effective breast cancer drug but resistance often develops over prolonged chemotherapy. Therefore, we performed a candidate approach RNAi screen in combination with cisplatin treatment to identify molecular pathways conferring survival advantages. The screen identified ATP7A as a therapeutic target. ATP7A is a copper ATPase transporter responsible for intercellular movement and sequestering of cisplatin. Pharmaceutical replacement for ATP7A by ammonium tetrathiomolybdate (TM) enhanced cisplatin treatment in breast cancer cells. Allograft and xenograft models in athymic nude mice treated with cisplatin/TM exhibited retarded tumor growth, reduced accumulation of cancer stem cells and decreased cell proliferation as compared to mono-treatment with cisplatin or TM. Cisplatin/TM treatment of cisplatin-resistant tumors reduced ATP7A protein levels, attenuated cisplatin sequestering by ATP7A, increased nuclear availability of cisplatin, and subsequently enhanced DNA damage and apoptosis. Microarray analysis of gene ontology pathways that responded uniquely to cisplatin/TM double treatment depicted changes in cell cycle regulation, specifically in the G1/S transition. These findings offer the potential to combat platinum-resistant tumors and sensitize patients to conventional breast cancer treatment by identifying and targeting the resistant tumors' unique molecular adaptations.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cisplatin; Copper-Transporting ATPases; Drug Synergism; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; MCF-7 Cells; Mice, Nude; Molybdenum; Neoplastic Stem Cells; RNA Interference; Xenograft Model Antitumor Assays

Cu/Zn superoxide dismutases (SODs) like the extracellular SOD3 and cytoplasmic SOD1 regulate cell proliferation by generating hydrogen peroxide (H2O2). This pro-oxidant inactivates essential cysteine residues in protein tyrosine phosphatases (PTP) helping receptor tyrosine kinase activation by growth factor signaling, and further promoting downstream MEK/ERK linked cell proliferation. Disulfiram (DSF), currently in clinical cancer trials is activated by copper chelation, being potentially capable of diminishing the copper dependent activation of MEK1/2 and SOD1/SOD3 and promoting reactive oxygen species (ROS) toxicity. However, copper (Cu) overload may occur when co-administered with DSF, resulting in toxicity and mutagenicity against normal tissue, through generation of the hydroxyl radical (•OH) by the Fenton reaction.. To investigate: a) whether sub-toxic DSF efficacy can be increased without Cu overload against human melanoma cells with unequal BRAF(V600E) mutant status and Her2-overexpressing SKBR3 breast cancer cells, by increasing H2O2 from exogenous SOD; b) to compare the anti-tumor efficacy of DSF with that of another clinically used copper chelator, tetrathiomolybdate (TTM).. a) without copper supplementation, exogenous SOD potentiated sub-toxic DSF toxicity antagonized by sub-toxic TTM or by the anti-oxidant N-acetylcysteine; b) exogenous glucose oxidase, another H2O2 generator resembled exogenous SOD in potentiating sub-toxic DSF.. potentiation of sub-lethal DSF toxicity by extracellular H2O2 against the human tumor cell lines investigated, only requires basal Cu and increased ROS production, being unrelated to non-specific or TTM copper chelator sequestration.. These findings emphasize the relevance of extracellular H2O2 as a novel mechanism to improve disulfiram anticancer effects minimizing copper toxicity.

Topics: Acetylcysteine; Antineoplastic Agents; Blotting, Western; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Chelating Agents; Copper; Disulfiram; Free Radical Scavengers; Humans; Hydrogen Peroxide; MAP Kinase Signaling System; Melanoma; Molybdenum; Mutation, Missense; Proto-Oncogene Proteins B-raf; Reactive Oxygen Species; Receptor, ErbB-2; Superoxide Dismutase

Infiltration by macrophages (Mphi) indicates a poor prognosis in breast cancers, in particular by inducing angiogenesis. Our study aimed 1) to investigate the mechanism by which cooperation between Mphi and aggressive breast cancer cells (MDA-MB-231) induces angiogenesis; 2) to examine the effect of tetrathiomolybdate (TM) on this angiogenic activity.. Mphi coincubated with MDA-MB-231 were used as a model to mimic the inflammatory microenvironment. Angiogenesis induced by the culture media was tested in the chick chorioallantoic membrane (CAM). Mphi phenotype was evaluated by 1) expression of the M1 marker CD80, and secretion of interleukin 10 (IL-10), an M2 marker; 2) capacity to secrete Tumour Necrosis Factor alpha (TNFalpha) when stimulated by lipopolysaccharide/interferon gamma (LPS/IFNgamma); 3) ability to induce MDA-MB-231 apoptosis. To explore the molecular mechanisms involved, cytokine profiles of conditioned media from MDA-MB-231, Mphi and the coculture were characterised by an antibody cytokine array. All experiments were carried out both in presence and in absence of TM.. Incubation of Mphi with MDA-MB-231 induced a pro-angiogenic effect in the CAM. It emerged that the angiogenic activity of the coculture is due to the capacity of Mphi to switch from M1 Mphi towards M2, probably due to an increase in Macrophage Colony Stimulating Factor. This M1-M2 switch was shown by a decreased expression of CD80 upon LPS/IFNgamma stimulation, an increased secretion of IL-10, a decreased secretion of TNFalpha in response to LPS/IFNgamma and an inability to potentiate apoptosis. At the molecular level, the angiogenic activity of the coculture medium can be explained by the secretion of CXC chemokines/ELR+ and CC chemokines. Although TM did not modify either the M2 phenotype in the coculture or the profile of the secreted chemokines, it did decrease the angiogenic activity of the coculture medium, suggesting that TM inhibited angiogenic activity by interfering with the endothelial cell signalling induced by these chemokines.. Cooperation between Mphi and MDA-MB-231 transformed M1 Mphi to an angiogenic, M2 phenotype, attested by secretion of CXC chemokines/ELR+ and CC chemokines. TM inhibited this coculture-induced increase in angiogenic activity, without affecting either Mphi phenotype or cytokine secretion profiles.

Topics: Angiogenesis Inhibitors; Animals; Apoptosis; Breast Neoplasms; Cells, Cultured; Chemokines; Chick Embryo; Coculture Techniques; Culture Media, Conditioned; Cytokines; Enzyme-Linked Immunosorbent Assay; Female; Fluorescent Antibody Technique; Humans; Interferon-gamma; Lipopolysaccharides; Macrophages; Molybdenum; Neovascularization, Pathologic; Recombinant Proteins

Tetrathiomolybdate (TM), a specific copper chelator, has been shown to be a potent antiangiogenic and antimetastatic compound possibly through suppression of the NFkappaB signaling cascade. To further delineate the molecular mechanism of the anticancer effect of TM, we investigated whether TM has antineoplastic activity in the setting of genetic NFkappaB inhibition. In this study, SUM149 inflammatory breast carcinoma cells were transfected with a dominant-negative IkappaBalpha (S32AS36A) expression vector. Similar to TM-treated SUM149 cells, SUM149-IkappaBalphaMut clones secreted lower amounts of proangiogenic mediators, vascular endothelial growth factor, interleukin-1alpha, and interleukin-8 and exhibited a less invasive and motile phenotype. The reduction in the angiogenic and metastatic potential of SUM149-IkappaBalphaMut clones was not further affected by TM in vitro. SUM149-IkappaBalphaMut xenografts grew substantially slower and had less lung metastasis than SUM149 and SUM149-empty vector xenografts. The growth and metastatic potential of SUM149 and SUM149-empty tumors was significantly inhibited with systemic TM treatment, whereas TM had no further antitumor effect on the SUM149-IkappaBalphaMut tumors. Additionally, nuclear proteins isolated from TM-treated SUM149 tumors had lower NFkappaB binding activity, while AP1 and SP1 binding activities were unchanged. Taken together, these results strongly support that suppression of NFkappaB is the major mechanism used by TM to inhibit angiogenesis and metastasis.

Topics: Angiogenesis Inhibitors; Animals; Breast Neoplasms; Cell Division; Cell Line, Tumor; Female; I-kappa B Proteins; Lung Neoplasms; Mice; Molybdenum; Neoplasm Metastasis; Neovascularization, Pathologic; NF-kappa B; NF-KappaB Inhibitor alpha; Transcription, Genetic; Transfection; Xenograft Model Antitumor Assays

Copper plays an essential role in promoting angiogenesis. Tumors that become angiogenic acquire the ability to enter a phase of rapid growth and exhibit increased metastatic potential, the major cause of morbidity in cancer patients. We report that copper deficiency induced by tetrathiomolybdate (TM) significantly impairs tumor growth and angiogenesis in two animal models of breast cancer: an inflammatory breast cancer xenograft in nude mice and Her2/neu cancer-prone transgenic mice. In vitro, TM decreases the production of five proangiogenic mediators: (a) vascular endothelial growth factor; (b) fibroblast growth factor 2/basic fibroblast growth factor; (c) interleukin (IL)-1alpha; (d) IL-6; and (e) IL-8. In addition, TM inhibits vessel network formation and suppresses nuclear factor (NF)kappaB levels and transcriptional activity. Our study suggests that a major mechanism of the antiangiogenic effect of copper deficiency induced by TM is suppression of NFkappaB, contributing to a global inhibition of NFkappaB-mediated transcription of proangiogenic factors.

Topics: Angiogenesis Inhibitors; Animals; Breast Neoplasms; Cell Division; Copper; Disease Models, Animal; Female; Genes, erbB-2; Humans; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Mice, Transgenic; Molybdenum; Neovascularization, Pathologic; NF-kappa B; Rats; Rats, Sprague-Dawley; Transcription, Genetic; Xenograft Model Antitumor Assays