oligomycins and Breast-Neoplasms

Energy imbalance is one of the key properties of tumour cells, which in certain cases supports fast cancer progression and resistance to therapy. The simultaneous blocking of glycolytic processes and oxidative phosphorylation pathways seems to be a promising strategy for antitumor therapies. The study aimed to evaluate the effect of glucose starvation on the antiproliferative and antiestrogenic potency of oligomycin A against hormone-dependent breast cancer cells. Cell viability was assessed by the MTT test. Estrogen receptor alpha (ERα) activity was evaluated by reporter assay. mTOR, AMPK, Akt, and S6 kinase expression was assessed by immunoblotting. Glucose starvation caused multiple increases in the antiproliferative potency of oligomycin A in the hormone-dependent breast cancer MCF-7 cells, while its effect on the sensitivity of the second hormone-dependent cancer cell line, named T47D, was weak and limited. Glycolytic inhibitors, 3-bromopyruvate and 2-deoxyglucose, greatly enhanced the antiproliferative potency of oligomycin A in MCF-7 cells. Glucose starvation leads to remarkable activation of Akt in MCF-7 cells, whereas oligomycin A enhances its effect. The mTOR, S6 kinase, and AMPK signalling pathways are significantly modulated by oligomycin A under glucose starvation. Oligomycin A demonstrates more pronounced antiestrogenic effects under glucose starvation. Thus, glucose starvation and pharmacological inhibition of glycolysis are of interest for revealing the antitumor potential of macrolide oligomycin A against hormone-dependent breast cancers.

Topics: Breast Neoplasms; Cell Proliferation; Estrogen Receptor Modulators; Female; Glucose; Humans; MCF-7 Cells; Oligomycins

Acquired resistance to HER2-targeted therapies occurs frequently in HER2

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Cell Proliferation; Drug Resistance, Neoplasm; Enzyme Inhibitors; Female; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Mitochondrial Proton-Translocating ATPases; Oligomycins; Receptor, ErbB-2; Trastuzumab; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Here, we show that new mitochondrial biogenesis is required for the anchorage independent survival and propagation of cancer stem-like cells (CSCs). More specifically, we used the drug XCT790 as an investigational tool, as it functions as a specific inhibitor of the ERRα-PGC1 signaling pathway, which governs mitochondrial biogenesis. Interestingly, our results directly demonstrate that XCT790 efficiently blocks both the survival and propagation of tumor initiating stem-like cells (TICs), using the MCF7 cell line as a model system. Mechanistically, we show that XCT790 suppresses the activity of several independent signaling pathways that are normally required for the survival of CSCs, such as Sonic hedgehog, TGFβ-SMAD, STAT3, and Wnt signaling. We also show that XCT790 markedly reduces oxidative mitochondrial metabolism (OXPHOS) and that XCT790-mediated inhibition of CSC propagation can be prevented or reversed by Acetyl-L-Carnitine (ALCAR), a mitochondrial fuel. Consistent with our findings, over-expression of ERRα significantly enhances the efficiency of mammosphere formation, which can be blocked by treatment with mitochondrial inhibitors. Similarly, mammosphere formation augmented by FOXM1, a downstream target of Wnt/β-catenin signaling, can also be blocked by treatment with three different classes of mitochondrial inhibitors (XCT790, oligomycin A, or doxycycline). In this context, our unbiased proteomics analysis reveals that FOXM1 drives the expression of >90 protein targets associated with mitochondrial biogenesis, glycolysis, the EMT and protein synthesis in MCF7 cells, processes which are characteristic of an anabolic CSC phenotype. Finally, doxycycline is an FDA-approved antibiotic, which is very well-tolerated in patients. As such, doxycycline could be re-purposed clinically as a 'safe' mitochondrial inhibitor, to target FOXM1 and mitochondrial biogenesis in CSCs, to prevent tumor recurrence and distant metastasis, thereby avoiding patient relapse.

Topics: Acetylcarnitine; Breast Neoplasms; Cell Adhesion; Cell Proliferation; Cell Survival; Chromatography, Liquid; Doxycycline; ERRalpha Estrogen-Related Receptor; Forkhead Box Protein M1; Forkhead Transcription Factors; Glycolysis; Humans; MCF-7 Cells; Mitochondria; Neoplastic Stem Cells; Nitriles; Oligomycins; Organelle Biogenesis; Oxidative Phosphorylation; Proteomics; Receptors, Estrogen; Signal Transduction; Spheroids, Cellular; Tandem Mass Spectrometry; Thiazoles; Wnt Signaling Pathway

Mitochondria are emerging as important players in the transformation process of cells, maintaining the biosynthetic and energetic capacities of cancer cells and serving as one of the primary sites of apoptosis and autophagy regulation. Although several avenues of cancer therapy have focused on mitochondria, progress in developing mitochondria-targeting anticancer drugs nonetheless has been slow, owing to the limited number of known mitochondrial target proteins that link metabolism with autophagy or cell death. Recent studies have demonstrated that two members of the newly discovered family of NEET proteins, NAF-1 (CISD2) and mitoNEET (mNT; CISD1), could play such a role in cancer cells. NAF-1 was shown to be a key player in regulating autophagy, and mNT was proposed to mediate iron and reactive oxygen homeostasis in mitochondria. Here we show that the protein levels of NAF-1 and mNT are elevated in human epithelial breast cancer cells, and that suppressing the level of these proteins using shRNA results in significantly reduced cell proliferation and tumor growth, decreased mitochondrial performance, uncontrolled accumulation of iron and reactive oxygen in mitochondria, and activation of autophagy. Our findings highlight NEET proteins as promising mitochondrial targets for cancer therapy.

Topics: Animals; Breast Neoplasms; Carcinogenesis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Glycolysis; Homeostasis; Humans; Immunoblotting; MCF-7 Cells; Membrane Potential, Mitochondrial; Membrane Proteins; Mice; Mice, Nude; Microscopy, Electron, Transmission; Mitochondria; Mitochondrial Proteins; Oligomycins; Pioglitazone; Reactive Oxygen Species; RNA Interference; Thiazolidinediones; Transplantation, Heterologous; Tumor Burden

During multi-cellular tumor spheroid growth, oxygen and nutrient gradients develop inducing specific genetic and metabolic changes in the proliferative and quiescent cellular layers. An integral analysis of proteomics, metabolomics, kinetomics and fluxomics revealed that both proliferative- (PRL) and quiescent-enriched (QS) cellular layers of mature breast tumor MCF-7 multi-cellular spheroids maintained similar glycolytic rates (3-5 nmol/min/10(6) cells), correlating with similar GLUT1, GLUT3, PFK-1, and HKII contents, and HK and LDH activities. Enhanced glycolytic fluxes in both cell layer fractions also correlated with higher HIF-1α content, compared to MCF-7 monolayer cultures. On the contrary, the contents of the mitochondrial proteins GA-K, ND1, COXIV, PDH-E1α, 2-OGDH, SDH and F1-ATP synthase (20 times) and the oxidative phosphorylation (OxPhos) flux (2-times) were higher in PRL vs. QS. Enhanced mitochondrial metabolism in the PRL layers correlated with an increase in the oncogenes h-Ras and c-Myc, and transcription factors p32 and PGC-1α, which are involved in the OxPhos activation. On the other hand, the lower mitochondrial function in QS was associated with an increase in Beclin, LC3B, Bnip3 and LAMP protein levels, indicating active mitophagy and lysosome biosynthesis processes. Although a substantial increase in glycolysis was developed, OxPhos was the predominant ATP supplier in both QS and PRL layers. Therefore, targeted anti-mitochondrial therapy by using oligomycin (IC(50)=11 nM), Casiopeina II-gly (IC(50)=40 nM) or Mitoves (IC(50)=7 nM) was effective to arrest MCF-7 spheroid growth without apparent effect on normal epithelial breast tissue at similar doses; canonical anti-neoplastic drugs such as cisplatin and tamoxifen were significantly less potent.

Topics: Antineoplastic Agents; Blotting, Western; Breast; Breast Neoplasms; Cell Proliferation; Copper; Energy Metabolism; Female; Glycolysis; Humans; Immunoenzyme Techniques; Mitochondria; Mitochondrial Proteins; Mitophagy; Oligomycins; Organometallic Compounds; Oxidative Phosphorylation; Proteome; Spheroids, Cellular; Uncoupling Agents

The extracellular pH in malignant tumors is known to be lower than in normal tissues and may therefore facilitate extracellular activation of secreted lysosomal cathepsins. We have tested the capability of human mammary cells (continuous cell lines and primary culture) to acidify their extracellular environment, using two techniques. By measuring pH changes through alterations of phenolsulfone phthaleine absorbance, we found that the more aggressive MDA-MB-231 human breast cancer cells were more active in acidifying a non-buffered balanced salt solution than the estrogen receptor positive MCF7 and ZR75 cell lines and than normal mammary epithelial cells in primary culture. Metastatic breast cancer cells from pleural effusions were up to 200-fold more active in acidifying their extracellular milieu than non-malignant mammary cells cultured in the same conditions, strongly suggesting that this difference also occurs in vivo. The use of inhibitors in the presence or absence of glucose showed that both lactate and an ATP-driven proton pump sharing some characteristics of the vacuolar H+ pump were involved. Bafilomycin A1, a specific inhibitor of the vacuolar (V-type) ATP-H+ pump inhibited part of the acidification by MCF7 cells, but not by MDA-MB-231 cells. We also used microelectrodes to measure extracellular pH, in close contact to the MCF7 breast cancer cells. The pH at the free surface of MCF7 cells was lower by 0.33 +/- 0.14 unit than that of the surrounding medium, while insertion of the microelectrode tip beneath the attached surface of the cells showed a greater lowering of pH from 0.3 to 1.7 pH unit as long as cell attachment on the substrate prevented H+ diffusion. We conclude that breast carcinoma cells have a higher capacity for acidifying their extracellular milieu than normal mammary cells, and that both a plasma membrane H(+)-ATPase, and lactic acid production are involved in this acidification. It is therefore possible that the aspartyl and cysteinyl pro-cathepsins secreted in excess by tumor cells may be activated extracellularly in vivo close to the basement membrane.

Topics: Acids; Amiloride; Anti-Bacterial Agents; Breast; Breast Neoplasms; Cells, Cultured; Extracellular Space; Glucose; Humans; Hydrogen-Ion Concentration; Lactic Acid; Macrolides; Macrophages; Microelectrodes; Oligomycins; Proton Pump Inhibitors; Proton-Translocating ATPases; Tumor Cells, Cultured

Gastric cancer is a leading cause of cancer death in many parts of the world. None of the tumor markers available to date provide for a reliable screening/diagnostic test. By using differential display technology on gastric cancer nonmetastatic (RF1) and metastatic (RF48) cell lines, we have isolated six novel cDNA clones. Five of them have not been previously identified. However, one of them appears to be the human homolog of the bovine oligomycin sensitivity conferral protein (oscp). Northern blot analysis showed that this clone is expressed at a much higher level in the metastatic (RF48) than the nonmetastatic (RF1) cancer cell line from the same patient. High level expression of the same gene is also observed in the breast cancer cell lines BT20 and T47D. The other five novel clones isolated showed either low or no expression in the RF1 and RF48 gastric cell lines, but variable levels of expression were detectable in breast cancers cell lines and normal tissues.

Topics: Adenosine Triphosphatases; Amino Acid Sequence; Animals; Base Sequence; Biomarkers, Tumor; Breast Neoplasms; Carrier Proteins; Cattle; DNA, Neoplasm; Humans; Membrane Proteins; Mitochondrial Proton-Translocating ATPases; Molecular Sequence Data; Neoplasm Metastasis; Oligomycins; Polymerase Chain Reaction; RNA, Messenger; Stomach Neoplasms; Tumor Cells, Cultured

Topics: Adenosine Triphosphatases; Adolescent; Adult; Aged; Antigens, Neoplasm; Breast Neoplasms; Dinitrophenols; Female; Gastrointestinal Neoplasms; Humans; Lectins; Lung Neoplasms; Lymphocyte Activation; Lymphocytes; Male; Middle Aged; Oligomycins; Ouabain; Stimulation, Chemical