• breast cancer
• drug repurposing
• altered metabolism
• screening tools

• Department of Biotechnology, Ministry of Science and Technology, India
• Indian Institute of Technology Guwahati
• Ministry of Electronics and Information technology
• Ministry of Education, India
• Indian Council of Medical Research

• Drug development
• Drug discovery
• Drug target
• Medicinal research
• WNT/β-catenin

• C. elegans
• DPI
• aging
• antibiotics
• lifespan
• lipofuscin
• metabolism
• mitochondria

• Animals
• Longevity
• Caenorhabditis elegans/metabolism
• Caenorhabditis elegans Proteins/genetics
• Caenorhabditis elegans Proteins/metabolism
• Anti-Bacterial Agents/pharmacology
• Lipofuscin/metabolism
• Mitochondria/metabolism
• Adenosine Triphosphate/metabolism

• Anticancer therapy
• Cancer
• Cardiotoxicity
• Cardiovascular diseases
• Mitochondrial dynamics
• Mitochondrial dysfunction

• Humans
• Cardiovascular Diseases
• Neoplasms/complications
• Heart Diseases
• Carcinogenesis
• Mitochondria

• Associazione Italiana per la Ricerca sul Cancro
• Ministero della Salute
• Ministero dell'Università e della Ricerca

• cancer therapy
• functional materials
• nanomedicine
• precise therapy
• subcellular organelles

• WJ2019H349 Health and Family Planning Commission of Hubei Province
• 81502087 National Natural Science Foundation of China
• 31600692 National Natural Science Foundation of China
• 2023AFB926 the Natural Science Fund of Hubei Province
• WJ2023M138 Hubei Province health and family planning scientific research project

• TPGS
• cancer stem cell
• doxycycline
• micelle
• ovarian cancer

• Humans
• Female
• Micelles
• Doxycycline/pharmacology
• Cell Line, Tumor
• Polyethylene Glycols/chemistry
• Antineoplastic Agents/chemistry
• Ovarian Neoplasms/drug therapy
• Vitamin E/chemistry
• Neoplastic Stem Cells
• Succinates
• Particle Size
• Drug Carriers/chemistry

• ATPase inhibitory factor 1
• OXPHOS
• RNA binding proteins
• Warburg effect
• cancer
• cell death
• metabolic reprogramming
• metastasis
• mitochondrial ATP synthase
• mitohormesis

• PID2019-108674RB-100 Ministry of Economy, Industry and Competitiveness
• CB06/07/0017 Instituto de Salud Carlos III

• Cancer stem cells
• Cellular plasticity
• EMT
• Metabolism
• Signaling pathways
• Solid tumors
• Tumor microenvironment

• Humans
• Tumor Microenvironment
• Neoplasms/pathology
• Neoplastic Stem Cells/metabolism
• Epithelial-Mesenchymal Transition

• Tabriz University of Medical Sciences

• Antibiotics
• Chemoradiation
• Head-and-neck cancer
• Head-and-neck squamous cell carcinoma
• Radiotherapy

• 413517907 Deutsche Forschungsgemeinschaft

• BUB1
• Cancer
• DNA damage
• DNA repair
• Radiosensitization
• Radiotherapy

• Beta cells
• Diabetes
• Hyperglycemia
• Insulin secretion
• Pyruvate kinase
• ROCK1

• Animals
• Mice
• Glucose/metabolism
• Insulin/metabolism
• Insulin Secretion/physiology
• Pyruvate Kinase/antagonists & inhibitors
• Pyruvate Kinase/metabolism
• Pyruvates
• rho-Associated Kinases

• R01 DK129946 NIDDK NIH HHS
• R01 DK123002 NIDDK NIH HHS
• R01 DK083567 NIDDK NIH HHS
• P30 DK036836 NIDDK NIH HHS
• R01 DK067536 NIDDK NIH HHS

Background and Objectives: In spite of the fact that antibiotics are considered to be the cornerstone of modern medicine, their use in the treatment of cancer remains controversial. In the present study, the main objective was to examine the effects of two antibiotics—tetracycline and ampicillin—on the viability, morphology, migration, and organization and structure of the nuclei and the actin fiber network of pharyngeal carcinoma cells—Detroit-562. Materials and Methods: In order to determine the viability of the cells, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method was applied after the cells were stimulated with five concentrations of tetracycline and ampicillin (10, 25, 50, 75, and 100 μM) for 72 h. A scratch assay was used to assess the migration ability of the cells. For the visualization of the nuclei and actin fibers, 4,6-diamidino-2-phenylindole (Dapi) and Rhodamine-Phalloidin were used. Results: There are different effects of tetracycline and ampicillin. Thus, tetracycline: (i) exhibited a concentration-dependent cytotoxic effect, decreasing cell viability to approximately 46%; (ii) inhibits cellular migration up to 16% compared to 60% for control cells; and (iii) induces changes in cell morphology as well as apoptotic changes in the nucleus and F-actin fibers. In contrast, in the case of ampicillin, an increase in viability up to 113% was observed at 10 μM, while a decrease in viability up to approximately 94% was observed at the highest concentration tested (100 μM). Conclusions: The results indicated a different effect regarding the impact on pharyngeal carcinoma cells. Thus, tetracycline has a concentration-dependent cytotoxic effect, while in the case of ampicillin a slight stimulation of cell viability was observed.

• DAPI staining
• ampicillin
• pharyngeal cells
• rhodamine-phalloidin staining
• tetracycline

• DNA damage
• cancer therapy
• combination therapy
• immunotherapy
• innate immunity
• radiotherapy

• 28724 Cancer Research UK

• ATM
• ataxia-telangiectasia (AT)
• cancer biology
• cell migration
• confined migration
• lamin (A/C)
• mechanobiology
• nuclear mechanics

• R01 CA201246 NCI NIH HHS
• R01 GM137605 NIGMS NIH HHS
• R01 HL082792 NHLBI NIH HHS
• U54 CA210184 NCI NIH HHS
• National Institutes of Health
• National Science Foundation
• Congressionally Directed Medical Research Programs

Nanotherapeutics can enhance the characteristics of drugs, such as rapid systemic clearance and systemic toxicities. Polymeric nanoparticles (PRNPs) depend on dispersion of a drug in an amorphous state in a polymer matrix. PRNPs are capable of delivering drugs and improving their safety. The primary goal of this study is to formulate doxycycline-loaded PRNPs by applying the nanoprecipitation method. Eudragit S100 (ES100) (for DOX-PRNP1) and hydroxypropyl methyl cellulose phthalate HP55 (for DOX-PRNP2) were tested as the drug carrying polymers and the DOX-PRNP2 showed better characteristics and drug release % and was hence selected to be tested in the biological study. Six different experimental groups were formed from sixty male albino mice. 1,2,-Dimethylhydrazine was used for 16 weeks to induce experimental colon cancer. We compared the oral administration of DOX-PRNP2 in doses of 5 and 10 mg/kg with the free drug. Results indicated that DOX-PRNP2 had greater antitumor activity, as evidenced by an improved histopathological picture for colon specimens as well as a decrease in the tumor scores. In addition, when compared to free DOX, the DOX-PRNP2 reduced the angiogenic indicators VEGD and CD31 to a greater extent. Collectively, the findings demonstrated that formulating DOX in PRNPs was useful in enhancing antitumor activity and can be used in other models of cancers to verify their efficacy and compatibility with our study.

• adverse events
• antibiotics
• cancer
• dysbiosis
• microbiota
• patient treatment outcomes
• radiation therapy
• radiation-induced toxicity
• radiosensitization

• Brain metastasis (BM)
• Laser interstitial thermal therapy (LITT)
• Stereotactic laser ablation (SLA)
• Stereotactic radiosurgery (SRS)

• Cancer
• DNA-dependent protein kinase
• Epidermal growth factor receptor
• Estrogen receptors
• Progesterone receptor

• DDR (DNA damage response)
• DNA repair
• PARP inhibition (PARPi)
• PD-1 - PD-L1 axis
• TNBC
• breast cancer
• immunotherapy

Cancer incidence and recurrence, antibiotic resistance, and overuse of antibiotics have become a global concern. The purpose of this study was to identify and isolate bacteria from the skin of the Rana ridibunda, Iranian swamp frog, which has produced antimicrobial compounds, and investigate its cytotoxic activity on the breast (MCF7) and glioblastoma (U87) cancer cell line.

An antibiotic-producing bacterium was isolated from the frog skin. The bacterium was identified based on 16S rDNA sequencing and biochemical and morphological characteristics. Antimicrobial activity of the culture supernatant was examined by disc diffusion and MIC methods. Cytoplasmic and cell wall extracts of bacteria were prepared by sonication. SDS-PAGE was then used to examine protein contents of them. The cancer cell lines were treated with cytoplasmic and cell wall extracts at different concentrations. The effects of cytotoxicity were assessed by MTT assay at 24 and 48 h intervals. Finally, the results were analyzed by SPSS.

The isolated bacterium was identified as a new strain of Bacillus atrophaeus. MIC and disc diffusion methods showed that the Bacillus atrophaeus antimicrobial activity was broad spectrum. MTT assay showed IC50 values 30 μg/ml and 20 μg/ml for U87 and MCF7 cells after 24–48 h exposure, respectively.

The cytoplasmic extracts of Bacillus atrophaeus has anticancer potential and can be used as an alternative or complementary candidate in the treatment of cancer. Further in vivo and in vitro mechanistic studies are suggested to confirm the biological activities.

• Anti-microbial
• Anticancer effect
• Bacillus atrophaeus
• Bacterial extract

• Doxycycline
• antagonistic interaction
• azithromycin
• combination treatment
• glioblastoma multiforme

• Anticancer
• doxycycline
• drug resistance
• gemcitabine
• pancreatic cancer
• synergism

• anthelmintic agents
• antibiotics
• antifungals
• antimalarial drugs
• antivirals
• off-targets
• oncology
• repurposing drugs
• resistance

• Antioxidants
• Cancer cell lines
• Dietary Supplements
• Healthy nutrition
• Redox status

• chromatin
• genomic states affecting HR
• homologous recombination
• rAAV

• Chromatin/genetics
• Dependovirus/genetics
• Gene Targeting/methods
• Gene Transfer Techniques/statistics & numerical data
• Genetic Vectors/administration & dosage
• Genetic Vectors/genetics
• Homologous Recombination
• Humans
• K562 Cells
• Transgenes

• U01 HG009431 NHGRI NIH HHS
• R01 HL064274 NHLBI NIH HHS
• T15 LM007033 NLM NIH HHS
• T32 HG000044 NHGRI NIH HHS
• K22 HG000044 NHGRI NIH HHS
• S10 OD018220 NIH HHS

• Cancer
• Metabolic pathways
• Metabolism
• Mitochondria
• mtDNA

Elevated mitochondrial biogenesis and metabolism represent key features of breast cancer stem cells (CSCs), whose propagation is conducive to disease onset and progression. Therefore, interfering with mitochondria biology and function may be regarded as a useful approach to eradicate CSCs. Here, we used the breast cancer cell line MCF7 as a model system to interrogate how mitochondrial fission contributes to the development of mitochondrial dysfunction toward the inhibition of metabolic flux and stemness. We generated an isogenic MCF7 cell line transduced with Mitochondrial Fission Factor (MCF7-MFF), which is primarily involved in mitochondrial fission. We evaluated the biochemical, molecular and functional properties of MCF7-MFF cells, as compared to control MCF7 cells transduced with the empty vector (MCF7-Control). We observed that MFF over-expression reduces both mitochondrial mass and activity, as evaluated using the mitochondrial probes MitroTracker Red and MitoTracker Orange, respectively. The analysis of metabolic flux using the Seahorse XFe96 revealed the inhibition of OXPHOS and glycolysis in MCF7-MFF cells, suggesting that increased mitochondrial fission may impair the biochemical properties of these organelles. Notably, CSCs activity, assessed by 3D-tumorsphere assays, was reduced in MCF7-MFF cells. A similar trend was observed for the activity of ALDH, a well-established marker of stemness. We conclude that enhanced mitochondrial fission may compromise CSCs propagation, through the impairment of mitochondrial function, possibly leading to a quiescent cell phenotype. Unbiased proteomic analysis revealed that proteins involved in mitochondrial dysfunction, oxidative stress-response, fatty acid metabolism and hypoxia signaling are among the most highly up-regulated in MCF7-MFF cells. Of note, integrated analysis of top regulatory networks obtained from unbiased proteomics in MCF7-MFF cells predicts that this cell phenotype activates signaling systems and effectors involved in the inhibition of cell survival and adhesion, together with the activation of specific breast cancer cell death programs. Overall, our study shows that unbalanced and abnormal activation of mitochondrial fission may drive the impairment of mitochondrial metabolic function, leading to inhibition of CSC propagation, and the activation of quiescence programs. Exploiting the potential of mitochondria to control pivotal events in tumor biology may, therefore, represent a useful tool to prevent disease progression.

• CSCs
• breast cancer
• metabo-stemness
• mitochondrial dynamics
• mitochondrial fission factor
• mitochondrial mass
• mitochondrial metabolism
• oxidative metabolism

Here, we describe the chemical synthesis and biological activity of a new Doxycycline derivative, designed specifically to more effectively target cancer stem cells (CSCs). In this analog, a myristic acid (14 carbon) moiety is covalently attached to the free amino group of 9-amino-Doxycycline. First, we determined the IC₅₀ of Doxy-Myr using the 3D-mammosphere assay, to assess its ability to inhibit the anchorage-independent growth of breast CSCs, using MCF7 cells as a model system. Our results indicate that Doxy-Myr is >5-fold more potent than Doxycycline, as it appears to be better retained in cells, within a peri-nuclear membranous compartment. Moreover, Doxy-Myr did not affect the viability of the total MCF7 cancer cell population or normal fibroblasts grown as 2D-monolayers, showing remarkable selectivity for CSCs. Using both gram-negative and gram-positive bacterial strains, we also demonstrated that Doxy-Myr did not show antibiotic activity, against Escherichia coli and Staphylococcus aureus. Interestingly, other complementary Doxycycline amide derivatives, with longer (16 carbon; palmitic acid) or shorter (12 carbon; lauric acid) fatty acid chain lengths, were both less potent than Doxy-Myr for the targeting of CSCs. Finally, using MDA-MB-231 cells, we also demonstrate that Doxy-Myr has no appreciable effect on tumor growth, but potently inhibits tumor cell metastasis in vivo, with little or no toxicity. In summary, by using 9-amino-Doxycycline as a scaffold, here we have designed new chemical entities for their further development as anti-cancer agents. These compounds selectively target CSCs, e.g., Doxy-Myr, while effectively minimizing the risk of driving antibiotic resistance. Taken together, our current studies provide proof-of-principle, that existing FDA-approved drugs can be further modified and optimized, to successfully target the anchorage-independent growth of CSCs and to prevent the process of spontaneous tumor cell metastasis.

• 9-amino-Doxycycline
• Doxycycline
• antimitoscins
• cancer cell metastasis
• cancer stem-like cells (CSCs)
• fatty acylation
• myristic acid
• prophylaxis of metastasis

Using proteomics analysis, we previously compared MCF7 breast cancer cells grown as 3D tumor spheres, with the same cell line grown as monolayers. Our results indicated that during 3D anchorage‐independent growth, the cellular machinery associated with i) mitochondrial biogenesis and ii) ribosomal biogenesis, were both significantly increased. Here, for simplicity, we refer to these two new oncogenic hallmarks as “mito‐stemness” and “ribo‐stemness” features. We have now applied this same type of strategy to begin to understand how fibroblasts and MCF7 breast cancer cells change their molecular phenotype, when they are co‐cultured together. We have previously shown that MCF7‐fibroblast co‐cultures are a valuable model of resistance to apoptosis induced by hormonal therapies, such as Tamoxifen and Fulvestrant. Here, we directly show that these mixed co‐cultures demonstrate the induction of mito‐stemness and ribo‐stemness features, likely reflecting a mechanism for cancer cells to increase their capacity for accumulating biomass. In accordance with the onset of a stem‐like phenotype, KRT19 (keratin 19) was induced by ~6‐fold during co‐culture. KRT19 is a well‐established epithelial CSC marker that is used clinically to identify metastatic breast cancer cells in sentinel lymph node biopsies. The potential molecular therapeutic targets that we identified by label‐free proteomics of MCF7‐fibroblast co‐cultures were then independently validated using a bioinformatics approach. More specifically, we employed publically‐available transcriptional profiling data derived from primary tumor samples from breast cancer patients, which were previously subjected to laser‐capture micro‐dissection, to physically separate breast cancer cells from adjacent tumor stroma. This allowed us to directly validate that the proteins up‐regulated in this co‐culture model were also transcriptionally elevated in patient‐derived breast cancer cells in vivo. This powerful approach for target identification and translational validation, including the use of patient outcome data, can now be applied to other tumor types as well, to validate new therapeutic targets that are more clinically relevant, for patient benefit. Moreover, we discuss the therapeutic implications of these findings for new drug development, drug repurposing and Tamoxifen‐resistance, to effectively target mito‐stemness and ribo‐stemness features in breast cancer patients. We also discuss the broad implications of this “organelle biogenesis” approach to cancer therapy.

• coculture
• mito-stemness
• mitochondria
• organelle biogenesis
• protein synthesis
• proteomics
• ribo-stemness
• tumor microenvironment

Objectives: The aim of this study was to prepare doxycycline polymeric nanoparticles (DOXY-PNPs) with hope to enhance its chemotherapeutic potential against solid Ehrlich carcinoma (SEC). Methods: Three DOXY-PNPs were formulated by nanoprecipitation method using hydroxypropyl methyl cellulose (HPMC) as a polymer. The prepared DOXY-PNPs were evaluated for the encapsulation efficiency (EE%), the drug loading capacity, particle size, zeta potential (ZP) and the in-vitro release for selection of the best formulation. PNP number 3 was selected for further biological testing based on the best pharmaceutical characters. PNP3 (5 and 10 mg/kg) was evaluated for the antitumor potential against SEC grown in female mice by measuring the tumor mass as well as the expression and immunohistochemical staining for the apoptosis markers; caspase 3 and BAX. Results: The biological study documented the greatest reduction in tumor mass in mice treated with PNP3. Importantly, treatment with 5 mg/kg of DOXY-PNPs produced a similar chemotherapeutic effect to that produced by 10 mg/kg of free DOXY. Further, a significant elevation in mRNA expression and immunostaining for caspase 3 and BAX was detected in mice group treated with DOXY-PNPs. Conclusions: The DOXY-PNPs showed greater antitumor potential against SEC grown in mice and greater values for Spearman’s correlation coefficients were detected when correlation with tumor mass or apoptosis markers was examined; this is in comparison to free DOXY. Hence, DOXY-PNPs should be tested in other tumor types to further determine the utility of the current technique in preparing chemotherapeutic agents and enhancing their properties.

• apoptosis
• doxycycline
• female mice
• hydroxypropyl methyl cellulose (HPMC), polymeric nanoparticles
• solid Ehrlich carcinoma

• Autophagy
• Cancer stem cells
• Epithelial-to-mesenchymal transition
• Metastatic potential
• Therapeutic resistance

• Autophagy
• Cell Plasticity
• Drug Resistance, Neoplasm
• Epithelial Cells/cytology
• Epithelial-Mesenchymal Transition
• Humans
• Neoplasm Metastasis/pathology

Here, we devised a new strategy for eradicating cancer stem cells (CSCs), via a “synthetic-metabolic” approach, involving two FDA-approved antibiotics and a dietary vitamin supplement. This approach was designed to induce a “rho-zero-like” phenotype in cancer cells. This strategy effectively results in the synergistic eradication of CSCs, using vanishingly small quantities of two antibiotics. The 2 metabolic targets are i) the large mitochondrial ribosome and ii) the small mitochondrial ribosome. Azithromycin inhibits the large mitochondrial ribosome as an off-target side-effect. In addition, Doxycycline inhibits the small mitochondrial ribosome as an off-target side-effect. Vitamin C acts as a mild pro-oxidant, which can produce free radicals and, as a consequence, induces mitochondrial biogenesis. Remarkably, treatment with a combination of Doxycycline (1 μM), Azithromycin (1 μM) plus Vitamin C (250 μM) very potently inhibited CSC propagation by >90%, using the MCF7 ER(+) breast cancer cell line as a model system. The strong inhibitory effects of this DAV triple combination therapy on mitochondrial oxygen consumption and ATP production were directly validated using metabolic flux analysis. Therefore, the induction of mitochondrial biogenesis due to mild oxidative stress, coupled with inhibition of mitochondrial protein translation, may be a new promising therapeutic anti-cancer strategy. Consistent with these assertions, Vitamin C is known to be highly concentrated within mitochondria, by a specific transporter, namely SVCT2, in a sodium-coupled manner. Also, the concentrations of antibiotics used here represent sub-antimicrobial levels of Doxycycline and Azithromycin, thereby avoiding the potential problems associated with antibiotic resistance. Finally, we also discuss possible implications for improving health-span and life-span, as Azithromycin is an anti-aging drug that behaves as a senolytic, which selectively kills and removes senescent fibroblasts.

• Azithromycin
• Doxycycline
• Vitamin C
• combination therapy
• glycolysis
• mitochondrial ATP depletion

• cell biology
• genetics research

• Cell Cycle Checkpoints/radiation effects
• DNA Breaks, Double-Stranded/radiation effects
• DNA Repair/radiation effects
• Humans
• Neoplasm Proteins/metabolism
• Neoplasms/metabolism
• Neoplasms/pathology
• Neoplasms/radiotherapy
• Radiation Tolerance
• Radiotherapy
• Signal Transduction/radiation effects

Doxycycline has anti-tumour effects in a range of tumour systems. The aims of this study were to define the role mitochondria play in this process and examine the potential of doxycycline in combination with gemcitabine. We studied the adenocarcinoma cell line A549, its mitochondrial DNA-less derivative A549 ρ° and cultured fibroblasts. Treatment with doxycycline for 5 days resulted in a decrease of mitochondrial-encoded proteins, respiration and membrane potential, and an increase of reactive oxygen species in A549 cells and fibroblasts, but fibroblasts were less affected. Doxycycline slowed proliferation of A549 cells by 35%. Cellular ATP levels did not change. Doxycycline alone had no effect on apoptosis; however, in combination with gemcitabine given during the last 2 days of treatment, doxycycline increased caspase 9 and 3/7 activities, resulting in a further decrease of surviving A549 cells by 59% and of fibroblasts by 24% compared to gemcitabine treatment alone. A549 ρ° cells were not affected by doxycycline. Key effects of doxycycline observed in A549 cells, such as the decrease of mitochondrial-encoded proteins and surviving cells were also seen in the cancer cell lines COLO357 and HT29. Our results suggest that doxycycline suppresses cancer cell proliferation and primes cells for apoptosis by gemcitabine.

• drug
• repositioning
• repurposing
• oncology

• Antineoplastic Agents/therapeutic use
• Clinical Trials as Topic
• Drug Repositioning
• Humans
• Neoplasms/drug therapy

• Cancer stem cell
• HIF
• Myc
• metabolic reprogramming
• p53

• Animals
• Humans
• Mitochondria/metabolism
• Models, Biological
• Neoplastic Stem Cells/metabolism
• Oxidative Phosphorylation

• antibiotics
• drug action
• erythromycin
• josamycin
• mitochondria
• mitochondrial translation
• natural product
• shRNA

• Aerobic mitochondrial energy metabolism
• Antibiotic
• Antidiabetic
• Chemotherapeutic
• Oxidative phosphorylation complex

Naturally-occurring somatic mutations in the estrogen receptor gene (ESR1) have been previously implicated in the clinical development of resistance to hormonal therapies, such as Tamoxifen. For example, the somatic mutation Y537S has been specifically associated with acquired endocrine resistance. Briefly, we recombinantly-transduced MCF7 cells with a lentiviral vector encoding ESR1 (Y537S). As a first step, we confirmed that MCF7-Y537S cells are indeed functionally resistant to Tamoxifen, as compared with vector alone controls. Importantly, further phenotypic characterization of Y537S cells revealed that they show increased resistance to Tamoxifen-induced apoptosis, allowing them to form mammospheres with higher efficiency, in the presence of Tamoxifen. Similarly, Y537S cells had elevated basal levels of ALDH activity, a marker of “stemness”, which was also Tamoxifen-resistant. Metabolic flux analysis of Y537S cells revealed a hyper-metabolic phenotype, with significantly increased mitochondrial respiration and high ATP production, as well as enhanced aerobic glycolysis. Finally, to understand which molecular signaling pathways that may be hyper-activated in Y537S cells, we performed unbiased label-free proteomics analysis. Our results indicate that TIGAR over-expression and the Rho-GDI/PTEN signaling pathway appear to be selectively activated by the Y537S mutation. Remarkably, this profile is nearly identical in MCF7-TAMR cells; these cells were independently-generated in vitro, suggesting a highly conserved mechanism underlying Tamoxifen-resistance. Importantly, we show that the Y537S mutation is specifically associated with the over-expression of a number of protein markers of poor clinical outcome (COL6A3, ERBB2, STAT3, AFP, TFF1, CDK4 and CD44). In summary, we have uncovered a novel metabolic mechanism leading to endocrine resistance, which may have important clinical implications for improving patient outcomes.

• TIGAR
• Y537S mutation
• drug resistance
• estrogen receptor
• hormone therapy
• mitochondria

Background: Resistance to endocrine treatments is a major clinical challenge in the management of estrogen receptor positive breast cancers. Although multiple mechanisms leading to endocrine resistance have been proposed, the poor outcome of this subgroup of patients demands additional studies. Methods: FoxO3a involvement in the acquisition and reversion of tamoxifen resistance was assessed in vitro in three parental ER+ breast cancer cells, MCF-7, T47D and ZR-75-1, in the deriving Tamoxifen resistant models (TamR) and in Tet-inducible TamR/FoxO3a stable cell lines, by growth curves, PLA, siRNA, RT-PCR, Western blot, Immunofluorescence, Transmission Electron Microscopy, TUNEL, cell cycle, proteomics analyses and animal models. FoxO3a clinical relevance was validated in silico by Kaplan–Meier survival curves. Results: Here, we show that tamoxifen resistant breast cancer cells (TamR) express low FoxO3a levels. The hyperactive growth factors signaling, characterizing these cells, leads to FoxO3a hyper-phosphorylation and subsequent proteasomal degradation. FoxO3a re-expression by using TamR tetracycline inducible cells or by treating TamR with the anticonvulsant lamotrigine (LTG), restored the sensitivity to the antiestrogen and strongly reduced tumor mass in TamR-derived mouse xenografts. Proteomics data unveiled novel potential mediators of FoxO3a anti-proliferative and pro-apoptotic activity, while the Kaplan–Meier analysis showed that FoxO3a is predictive of a positive response to tamoxifen therapy in Luminal A breast cancer patients. Conclusions: Altogether, our data indicate that FoxO3a is a key target to be exploited in endocrine-resistant tumors. In this context, LTG, being able to induce FoxO3a, might represent a valid candidate in combination therapy to prevent resistance to tamoxifen in patients at risk.

• FoxO3a
• breast cancer
• growth factors signaling
• tamoxifen resistance

Matcha green tea (MGT) is a natural product that is currently used as a dietary supplement and may have significant anti-cancer properties. However, the molecular mechanism(s) underpinning its potential health benefits remain largely unknown. Here, we used MCF7 cells (an ER(+) human breast cancer cell line) as a model system, to systematically dissect the effects of MGT at the cellular level, via i) metabolic phenotyping and ii) unbiased proteomics analysis. Our results indicate that MGT is indeed sufficient to inhibit the propagation of breast cancer stem cells (CSCs), with an IC-50 of ~0.2 mg/ml, in tissue culture. Interestingly, metabolic phenotyping revealed that treatment with MGT is sufficient to suppress both oxidative mitochondrial metabolism (OXPHOS) and glycolytic flux, shifting cancer cells towards a more quiescent metabolic state. Unbiased label-free proteomics analysis identified the specific mitochondrial proteins and glycolytic enzymes that were down-regulated by MGT treatment. Moreover, to discover the underlying signalling pathways involved in this metabolic shift, we subjected our proteomics data sets to bio-informatics interrogation via Ingenuity Pathway Analysis (IPA) software. Our results indicate that MGT strongly affected mTOR signalling, specifically down-regulating many components of the 40S ribosome. This raises the intriguing possibility that MGT can be used as inhibitor of mTOR, instead of chemical compounds, such as rapamycin. In addition, other key pathways were affected, including the anti-oxidant response, cell cycle regulation, as well as interleukin signalling. Our results are consistent with the idea that MGT may have significant therapeutic potential, by mediating the metabolic reprogramming of cancer cells.

• Matcha green tea
• cancer stem-like cells (CSCs)
• glycolysis
• metabolism
• mitochondrial OXPHOS
• proteomics analysis

Tri-phenyl-phosphonium (TPP) is a non-toxic chemical moiety that functionally behaves as a mitochondrial targeting signal (MTS) in living cells. Here, we explored the hypothesis that TPP-related compounds could be utilized to inhibit mitochondria in cancer stem cells (CSCs). We randomly selected 9 TPP-related compounds for screening, using an ATP depletion assay. Based on this approach, five compounds were identified as “positive hits”; two had no detectable effect on ATP production. Remarkably, this represents a >50% hit rate. We validated that the five positive hit compounds all inhibited oxygen consumption rates (OCR), using the Seahorse XFe96 metabolic flux analyzer. Interestingly, these TPP-related compounds were non-toxic and had little or no effect on ATP production in normal human fibroblasts, but selectively targeted adherent “bulk” cancer cells. Finally, these positive hit compounds also inhibited the propagation of CSCs in suspension, as measured functionally using the 3D mammosphere assay. Therefore, these TPP-related compounds successfully inhibited anchorage-independent growth, which is normally associated with a metastatic phenotype. Interestingly, the most effective molecule that we identified contained two TPP moieties (i.e., bis-TPP). More specifically, 2-butene-1,4-bis-TPP potently and selectively inhibited CSC propagation, with an IC-50 < 500 nM. Thus, we conclude that the use of bis-TPP, a “dimeric” mitochondrial targeting signal, may be a promising new approach for the chemical eradication of CSCs. Future studies on the efficacy of 2-butene-1,4-bis-TPP and its derivatives are warranted. In summary, we show that TPP-related compounds provide a novel chemical strategy for effectively killing both i) “bulk” cancer cells and ii) CSCs, while specifically minimizing or avoiding off-target side-effects in normal cells. These results provide the necessary evidence that “normal” mitochondria and “malignant” mitochondria are truly biochemically distinct, removing a significant barrier to therapeutically targeting cancer metabolism.

• cancer stem cells (CSCs)
• cancer therapy
• mitochondrial targeting signal
• tri-phenyl-phosphonium (TPP)

Doxycycline (DXC) is a tetracycline antibiotic which has been proposed as a breast cancer radiosensitizer by specifically reducing the expression of the DNA repair enzyme DNA PK in breast cancer initiating cells. Since DXC presents favorable pharmacokinetics properties including the capacity to cross the blood-brain barrier, it has been hypothesized that it could radiosensitize brain tumors as well. We have investigated the radiosensitizing capacity of DXC towards human glioma initiating cells (GIC)-driven orthotopic glioblastomas (GB) in NOD/SCID mice that faithfully mimic the growth properties of the clinical tumors of origin. DXC at 10 mg/Kg body weight was subcutaneously delivered daily, 5 days/week for 4 weeks. At the same time, radiotherapeutic fractions of 0.25 Gy to the head were delivered every 3–4 days (twice/week) for 15 weeks. No survival advantage was observed in DXC-treated mice as compared to vehicle-treated mice by this radiosensitizing protocol. On the contrary, skin damage with hair loss and deep ulcers were observed after 4 weeks in DXC-treated mice leading to discontinuation of drug treatment. These results do not support the use of DXC as a radiosensitizer for brain tumors and indicate skin damage as an important side effect of DXC.

• doxycycline
• glioma animal models
• radiosensitization

• DNA cleavage
• EPR spectroscopy
• doxycycline
• hydrogen sulfide
• hydroxyl radical
• oxytetracycline
• polysulfides
• superoxide
• tetracycline
• •cPTIO radical

• DNA Cleavage/drug effects
• Doxycycline/chemistry
• Doxycycline/pharmacology
• Drug Interactions
• Escherichia coli/drug effects
• Escherichia coli/genetics
• Escherichia coli/metabolism
• Hydroxyl Radical/chemistry
• Spectrum Analysis
• Sulfides/chemistry
• Sulfides/pharmacology
• Superoxides/chemistry

Cancer heterogeneity constitutes the major source of disease progression and therapy failure. Tumors comprise functionally diverse subpopulations, with cancer stem cells (CSCs) as the source of this heterogeneity. Since these cells bear in vivo tumorigenicity and metastatic potential, survive chemotherapy and drive relapse, its elimination may be the only way to achieve long-term survival in patients. Thanks to the great advances in the field over the last few years, we know now that cellular metabolism and stemness are highly intertwined in normal development and cancer. Indeed, CSCs show distinct metabolic features as compared with their more differentiated progenies, though their dominant metabolic phenotype varies across tumor entities, patients and even subclones within a tumor. Following initial works focused on glucose metabolism, current studies have unveiled particularities of CSC metabolism in terms of redox state, lipid metabolism and use of alternative fuels, such as amino acids or ketone bodies. In this review, we describe the different metabolic phenotypes attributed to CSCs with special focus on metabolism-based therapeutic strategies tested in preclinical and clinical settings.

• cancer stem cells
• lipid metabolism
• metabolism
• mitochondria
• oxidative phosphorylation
• redox regulation

• Cancer
• Cancer stem cells
• Clinical outcome
• Drug resistance
• Targeted therapy

• Mito-signatures
• Mito-therapeutics
• Mitochondria
• Mitoflavoscins
• Mitoketoscins
• Mitoriboscins
• drug discovery
• mito-biomarkers
• oncology platform

• DNA damage response
• DNA repair
• HIV-1 latency
• double strand breaks
• telomere