Antibiotics that target mitochondria extend lifespan in C. elegans.

Aging is a continuous degenerative process caused by a progressive decline of cell and tissue functions in an organism. It is induced by the accumulation of damage that affects normal cellular processes, ultimately leading to cell death. It has been speculated for many years that mitochondria play a key role in the aging process. In the aim of characterizing the implications of mitochondria in aging, here we used Caenorhabditis elegans (C. elegans) as an organismal model treated a panel of mitochondrial inhibitors and assessed for survival. In our study, we assessed survival by evaluating worm lifespan, and we assessed aging markers by evaluating the pharyngeal muscle contraction, the accumulation of lipofuscin pigment and ATP levels. Our results show that treatment of worms with either doxycycline, azithromycin (inhibitors of the small and the large mitochondrial ribosomes, respectively), or a combination of both, significantly extended median lifespan of C. elegans, enhanced their pharyngeal pumping rate, reduced their lipofuscin content and their energy consumption (ATP levels), as compared to control untreated worms, suggesting an aging-abrogating effect for these drugs. Similarly, DPI, an inhibitor of mitochondrial complex I and II, was capable of prolonging the median lifespan of treated worms. On the other hand, subjecting worms to vitamin C, a pro-oxidant, failed to extend C. elegans lifespan and upregulated its energy consumption, revealing an increase in ATP level. Therefore, our longevity study reveals that mitochondrial inhibitors (i.e., mitochondria-targeting antibiotics) could abrogate aging and extend lifespan in C. elegans.

Identification of natural products and FDA-approved drugs for targeting cancer stem cell (CSC) propagation.

Here, we report the identification of key compounds that effectively inhibit the anchorage-independent growth and propagation of cancer stem cells (CSCs), as determined via screening using MCF7 cells, a human breast adenocarcinoma cell line. More specifically, we employed the mammosphere assay as an experimental format, which involves the generation of 3D spheroid cultures, using low-attachment plates. These positive hit compounds can be divided into 5 categories: 1) dietary supplements (quercetin and glucosamine); 2) FDA-approved drugs (carvedilol and ciprofloxacin); 3) natural products (aloe emodin, aloin, tannic acid, chlorophyllin copper salt, azelaic acid and adipic acid); 4) flavours (citral and limonene); and 5) vitamins (nicotinamide and nicotinic acid). In addition, for the compounds quercetin, glucosamine and carvedilol, we further assessed their metabolic action, using the Seahorse to conduct metabolic flux analysis. Our results indicate that these treatments can affect glycolytic flux and suppress oxidative mitochondrial metabolism (OXPHOS). Therefore, quercetin, glucosamine and carvedilol can reprogram the metabolic phenotype of breast cancer cells. Despite having diverse chemical structures, these compounds all interfere with mitochondrial metabolism. As these compounds halt CSCs propagation, ultimately, they may have therapeutic potential.

Acid microenvironment promotes cell survival of human bone sarcoma through the activation of cIAP proteins and NF-κB pathway.

Extracellular acidification is a very common cause of stress in tumor microenvironment and of Darwinian pressure. In acid areas of the tumor, most cancer cells are-albeit slowly proliferating-more resistant to cell death than those in well-perfused regions. Tumor acidosis can directly regulate the expression of pro-survival proteins since a low extracellular pH activates the caspase-dependent cell death machinery. This mechanism has never been explored in bone sarcomas. We cultured osteosarcoma and Ewing sarcoma cells under low pH (pH 6.5), and we performed deep-sequencing and protein analysis. Both in in vitro and in vivo models, acidification activity enhanced tumor cells survival. However, we did not observe any change in ERK1 phosphorylation. On the contrary, both at the mRNA and protein level, we found a significant induction of TRAF adaptor proteins and of cIAP proteins (BIRC2 and/or BIRC3). As a consequence, the downstream nuclear transcription factor kappa B (NF-κB) survival pathway was increased. Furthermore, the treatment with the cIAP inhibitor LCL161 reverted the protection from apoptosis under low pH. In vitro results were confirmed both in Ewing sarcoma xenograft and in osteosarcoma patients, since the analysis of tumor tissues demonstrated that the levels of expression of TRAF1 or NF-κB1 significantly correlate with the level of expression of the vacuolar ATPase (V-ATPase), the most important proton pump in eukaryotes. Moreover, in the tissue sections of xenograft model, the nuclear translocation of RelB, a key subunit of the NF-κB transcriptional complex, localized in the tumor region that also corresponded to the acid microenvironment associated with the highest levels of expression of LAMP2 and V-ATPase, in the internal area of the tumor, as revealed by immunohistochemistry. Our data confirm that tumor acid microenvironment activates a stress-regulated switch to promote cell survival of bone sarcoma, and support the hypothesis that this mechanism is mediated by the recruitment of TRAF/cIAP complexes. Altogether, these results suggest that TRAF/cIAP can be considered as a target for anti-cancer therapies.

A mitochondrial based oncology platform for targeting cancer stem cells (CSCs): MITO-ONC-RX.

Here, we wish to propose a new systematic approach to cancer therapy, based on the targeting of mitochondrial metabolism, especially in cancer stem cells (CSCs). In the future, we envision that anti-mitochondrial therapy would ultimately be practiced as an add-on to more conventional therapy, largely for the prevention of tumor recurrence and cancer metastasis. This mitochondrial based oncology platform would require a panel of FDA-approved therapeutics (e.g. Doxycycline) that can safely be used to inhibit mitochondrial OXPHOS and/or biogenesis in CSCs. In addition, new therapeutics that target mitochondria could also be developed, to optimize their ability to eradicate CSCs. Finally, in this context, mitochondrial-based biomarkers (i.e. "Mito-signatures") could be utilized as companion diagnostics, to identify high-risk cancer patients at diagnosis, facilitating the early detection of tumor recurrence and the prevention of treatment failure. In summary, we suggest that new clinical trials are warranted to test and possibly implement this emerging treatment strategy, in a variety of human cancer types. This general approach, using FDA-approved antibiotics to target mitochondria, was effective in killing CSCs originating from many different cancer types, including DCIS, breast (ER(+) and ER(-)), prostate, ovarian, lung and pancreatic cancers, as well as melanoma and glioblastoma, among others. Thus, we propose the term MITO-ONC-RX, to describe this anti-mitochondrial platform for targeting CSCs. The use of re-purposed FDA-approved drugs will undoubtedly help to accelerate the clinical evaluation of this approach, as these drugs can move directly into Phase II clinical trials, saving considerable amounts of time (10-15 y) and billions in financial resources.

Matcha green tea (MGT) inhibits the propagation of cancer stem cells (CSCs), by targeting mitochondrial metabolism, glycolysis and multiple cell signalling pathways.

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.

Mitochondrial fission as a driver of stemness in tumor cells: mDIVI1 inhibits mitochondrial function, cell migration and cancer stem cell (CSC) signalling.

Mitochondria are dynamic organelles frequently undergoing fission and fusion events to maintain their integrity, bioenergetics and spatial distribution, which is fundamental to the processes of cell survival. Disruption in mitochondrial dynamics plays a role in cancer. Therefore, proteins involved in regulating mitochondrial dynamics are potential targets for treatment. mDIVI1 is an inhibitor of the mitochondrial fission protein DRP1, which induces i) mitochondrial oxidative stress and ii) effectively reduces mitochondrial metabolism. We show here that mDIVI1 is able to inhibit 3D tumorsphere forming capacity, cell migration and stemness-related signalling in breast cancer cells, indicating that mDIVI1 can potentially be used for the therapeutic elimination of cancer stem cells (CSCs).

Targeting flavin-containing enzymes eliminates cancer stem cells (CSCs), by inhibiting mitochondrial respiration: Vitamin B2 (Riboflavin) in cancer therapy.

Here, we performed high-throughput drug-screening to identify new non-toxic mitochondrial inhibitors. This screening platform was specifically designed to detect compounds that selectively deplete cellular ATP levels, but have little or no toxic side effects on cell viability. Using this approach, we identified DPI (Diphenyleneiodonium chloride) as a new potential therapeutic agent. Mechanistically, DPI potently blocks mitochondrial respiration by inhibiting flavin-containing enzymes (FMN and FAD-dependent), which form part of Complex I and II. Interestingly, DPI induced a chemo-quiescence phenotype that potently inhibited the propagation of CSCs, with an IC-50 of 3.2 nano-molar. Virtually identical results were obtained using CSC markers, such as CD44 and CD24. We further validated the effects of DPI on cellular metabolism. At 10 nM, DPI inhibited oxidative mitochondrial metabolism (OXPHOS), reducing mitochondrial driven ATP production by >90%. This resulted in a purely glycolytic phenotype, with elevated L-lactate production. We show that this metabolic inflexibility could be rapidly-induced, after only 1 hour of DPI treatment. Remarkably, the mitochondrial inhibitory effects of DPI were reversible, and DPI did not induce ROS production. Cells maintained in DPI for 1 month showed little or no mitochondrial activity, but remained viable. Thus, it appears that DPI behaves as a new type of mitochondrial inhibitor, which maintains cells in a state of metabolic-quiescence or "suspended animation".In conclusion, DPI treatment can be used to acutely confer a mitochondrial-deficient phenotype, which we show effectively depletes CSCs from the heterogeneous cancer cell population. These findings have significant therapeutic implications for potently targeting CSCs, while minimizing toxic side effects. We also discuss the possible implications of DPI for the aging process. Interestingly, previous studies in C. elegans have shown that DPI prevents the accumulation of lipofuscin (an aging-associated hallmark), during the response to oxidative stress. Our current results are consistent with data showing that flavins (FAD, FMN and/or Riboflavin) are auto-fluorescent markers of i) increased mitochondrial "power" (OXPHOS) and ii) elevated CSC activity.Finally, we believe that DPI is one of the most potent and highly selective CSC inhibitors discovered to date. Therefore, our current findings suggest a new impetus to create novel analogues of i) DPI (Diphenyleneiodonium chloride) and ii) DPI-related compounds (Diphenyliodonium chloride), using medicinal chemistry, to optimize this very promising and potent anti-CSC activity. We propose to call these new molecules "Mitoflavoscins".For example, DPI is ~30 times more potent than Palbociclib (IC-50 = 100 nM), which is an FDA-approved CDK4/6 inhibitor, that broadly targets proliferation in any cell type, including CSCs.

Vitamin C and Doxycycline: A synthetic lethal combination therapy targeting metabolic flexibility in cancer stem cells (CSCs).

Here, we developed a new synthetic lethal strategy for further optimizing the eradication of cancer stem cells (CSCs). Briefly, we show that chronic treatment with the FDA-approved antibiotic Doxycycline effectively reduces cellular respiration, by targeting mitochondrial protein translation. The expression of four mitochondrial DNA encoded proteins (MT-ND3, MT-CO2, MT-ATP6 and MT-ATP8) is suppressed, by up to 35-fold. This high selection pressure metabolically synchronizes the surviving cancer cell sub-population towards a predominantly glycolytic phenotype, resulting in metabolic inflexibility. We directly validated this Doxycycline-induced glycolytic phenotype, by using metabolic flux analysis and label-free unbiased proteomics. Next, we identified two natural products (Vitamin C and Berberine) and six clinically-approved drugs, for metabolically targeting the Doxycycline-resistant CSC population (Atovaquone, Irinotecan, Sorafenib, Niclosamide, Chloroquine, and Stiripentol). This new combination strategy allows for the more efficacious eradication of CSCs with Doxycycline, and provides a simple pragmatic solution to the possible development of Doxycycline-resistance in cancer cells. In summary, we propose the combined use of i) Doxycycline (Hit-1: targeting mitochondria) and ii) Vitamin C (Hit-2: targeting glycolysis), which represents a new synthetic-lethal metabolic strategy for eradicating CSCs. This type of metabolic Achilles' heel will allow us and others to more effectively "starve" the CSC population.

Mitoriboscins: Mitochondrial-based therapeutics targeting cancer stem cells (CSCs), bacteria and pathogenic yeast.

The "endo-symbiotic theory of mitochondrial evolution" states that mitochondrial organelles evolved from engulfed aerobic bacteria, after millions of years of symbiosis and adaptation. Here, we have exploited this premise to design new antibiotics and novel anti-cancer therapies, using a convergent approach. First, virtual high-throughput screening (vHTS) and computational chemistry were used to identify novel compounds binding to the 3D structure of the mammalian mitochondrial ribosome. The resulting library of ∼880 compounds was then subjected to phenotypic drug screening on human cancer cells, to identify which compounds functionally induce ATP-depletion, which is characteristic of mitochondrial inhibition. Notably, the top ten "hit" compounds define four new classes of mitochondrial inhibitors. Next, we further validated that these novel mitochondrial inhibitors metabolically target mitochondrial respiration in cancer cells and effectively inhibit the propagation of cancer stem-like cells in vitro. Finally, we show that these mitochondrial inhibitors possess broad-spectrum antibiotic activity, preventing the growth of both gram-positive and gram-negative bacteria, as well as C. albicans - a pathogenic yeast. Remarkably, these novel antibiotics also were effective against methicillin-resistant Staphylococcus aureus (MRSA). Thus, this simple, yet systematic, approach to the discovery of mitochondrial ribosome inhibitors could provide a plethora of anti-microbials and anti-cancer therapies, to target drug-resistance that is characteristic of both i) tumor recurrence and ii) infectious disease. In summary, we have successfully used vHTS combined with phenotypic drug screening of human cancer cells to identify several new classes of broad-spectrum antibiotics that target both bacteria and pathogenic yeast. We propose the new term "mitoriboscins" to describe these novel mitochondrial-related antibiotics. Thus far, we have identified four different classes of mitoriboscins, such as: 1) mitoribocyclines, 2) mitoribomycins, 3) mitoribosporins and 4) mitoribofloxins. However, we broadly define mitoriboscins as any small molecule(s) or peptide(s) that bind to the mitoribosome (large or small subunits) and, as a consequence, inhibit mitochondrial function, i.e., mitoribosome inhibitors.

NADH autofluorescence, a new metabolic biomarker for cancer stem cells: Identification of Vitamin C and CAPE as natural products targeting "stemness".

Here, we assembled a broad molecular "tool-kit" to interrogate the role of metabolic heterogeneity in the propagation of cancer stem-like cells (CSCs). First, we subjected MCF7 cells to "metabolic fractionation" by flow cytometry, using fluorescent mitochondrial probes to detect PCG1α activity, as well ROS and hydrogen-peroxide (H2O2) production; NADH levels were also monitored by auto-fluorescence. Then, the various cell populations were functionally assessed for "stem cell activity", using the mammosphere assay (3D-spheroids). Our results indicate that a sub-population of MCF7 cells, with increased PGC1α activity, high mitochondrial ROS/H2O2 production and high NADH levels, all form mammospheres with a higher efficiency. Thus, it appears that mitochondrial oxidative stress and the anti-oxidant response both contribute to the promotion of mitochondrial biogenesis and oxidative metabolism in CSCs. Further validation was provided by using specific inhibitors to target metabolic processes (the NAD+ salvage pathway, glycolysis, mitochondrial protein synthesis and OXPHOS), significantly reducing CSC propagation. As a consequence, we have now identified a variety of clinically-approved drugs (stiripentol), natural products (caffeic acid phenyl ester (CAPE), ascorbic acid, silibinin) and experimental pharmaceuticals (actinonin, FK866, 2-DG), that can be used to effectively inhibit CSC activity. We discuss the use of CAPE (derived from honey-bee propolis) and Vitamin C, as potential natural therapeutic modalities. In this context, Vitamin C was ~10 times more potent than 2-DG for the targeting of CSCs. Similarly, stiripentol was between 50 to 100 times more potent than 2-DG.

Targeting cancer stem cell propagation with palbociclib, a CDK4/6 inhibitor: Telomerase drives tumor cell heterogeneity.

In this report, we systematically examined the role of telomerase activity in lung and ovarian cancer stem cell (CSC) propagation. For this purpose, we indirectly gauged telomerase activity, by linking the hTERT-promoter to eGFP. Using lung (A549) and ovarian (SKOV3) cancer cells, transduced with the hTERT-GFP reporter, we then employed GFP-expression levels to fractionate these cell lines into GFP-high and GFP-low populations. We functionally compared the phenotype of these GFP-high and GFP-low populations. More specifically, we now show that the cancer cells with higher telomerase activity (GFP-high) are more energetically activated, with increased mitochondrial mass and function, as well as increased glycolytic activity. This was further validated and confirmed by unbiased proteomics analysis. Cells with high telomerase activity also showed an increased capacity for stem cell activity (as measured using the 3D-spheroid assay) and cell migration (as measured using a Boyden chamber approach). These enhanced biological phenotypes were effectively inhibited by classical modulators of energy metabolism, which target either i) mitochondrial metabolism (i.e., oligomycin) or ii) glycolysis (i.e., 2-deoxy-glucose), or iii) by using the FDA-approved antibiotic doxycycline, which inhibits mitochondrial biogenesis. Finally, the level of telomerase activity also determined the ability of hTERT-high cells to proliferate, as assessed by measuring DNA synthesis via EdU incorporation. Consistent with these observations, treatment with an FDA-approved CDK4/6 inhibitor (PD-0332991/palbociclib) specifically blocked the propagation of both lung and ovarian CSCs. Virtually identical results were obtained with breast CSCs, which were also highly sensitive to palbociclib at concentrations in the nanomolar range. In summary, CSCs with high telomerase activity are among the most energetically activated, migratory and proliferative cell sub-populations. These observations may provide a mechanistic explanation for tumor metabolic heterogeneity, based on telomerase activity. FDA-approved drugs, such as doxycycline and palbociclib, were both effective at curtailing CSC propagation. Thus, these FDA-approved drugs could be used to target telomerase-high proliferative CSCs, in multiple cancer types. Finally, our experiments also allowed us to distinguish two different cellular populations of hTERT-high cells, one that was proliferative (i.e., replicative immortality) and the other that was non-proliferative (i.e., quiescent). We speculate that the non-proliferative population of hTERT-high cells that we identified could be mechanistically involved in tumor dormancy.

Tumor-Activated Mesenchymal Stromal Cells Promote Osteosarcoma Stemness and Migratory Potential via IL-6 Secretion.

Osteosarcoma (OS) is an aggressive bone malignancy with a high relapse rate despite combined treatment with surgery and multiagent chemotherapy. As for other cancers, OS-associated microenvironment may contribute to tumor initiation, growth, and metastasis. We consider mesenchymal stromal cells (MSC) as a relevant cellular component of OS microenvironment, and have previously found that the interaction between MSC and tumor cells is bidirectional: tumor cells can modulate their peripheral environment that in turn becomes more favorable to tumor growth through metabolic reprogramming. Here, we determined the effects of MSC on OS stemness and migration, two major features associated with recurrence and chemoresistance. The presence of stromal cells enhanced the number of floating spheres enriched in cancer stem cells (CSC) of the OS cell population. Furthermore, the co-culturing with MSC stimulated the migratory capacity of OS via TGFß1 and IL-6 secretion, and the neutralizing antibody anti-IL-6 impaired this effect. Thus, stromal cells in combination with OS spheres exploit a vicious cycle where the presence of CSC stimulates mesenchymal cytokine secretion, which in turn increases stemness, proliferation, migration, and metastatic potential of CSC, also through the increase of expression of adhesion molecules like ICAM-1. Altogether, our data corroborate the concept that a comprehensive knowledge of the interplay between tumor and stroma that also includes the stem-like fraction of tumor cells is needed to develop novel and effective anti-cancer therapies.

Tumour-specific metabolic adaptation to acidosis is coupled to epigenetic stability in osteosarcoma cells.

The glycolytic-based metabolism of cancers promotes an acidic microenvironment that is responsible for increased aggressiveness. However, the effects of acidosis on tumour metabolism have been almost unexplored. By using capillary electrophoresis with time-of-flight mass spectrometry, we observed a significant metabolic difference associated with glycolysis repression (dihydroxyacetone phosphate), increase of amino acid catabolism (phosphocreatine and glutamate) and urea cycle enhancement (arginino succinic acid) in osteosarcoma (OS) cells compared with normal fibroblasts. Noteworthy, metabolites associated with chromatin modification, like UDP-glucose and N(8)-acetylspermidine, decreased more in OS cells than in fibroblasts. COBRA assay and acetyl-H3 immunoblotting indicated an epigenetic stability in OS cells than in normal cells, and OS cells were more sensitive to an HDAC inhibitor under acidosis than under neutral pH. Since our data suggest that acidosis promotes a metabolic reprogramming that can contribute to the epigenetic maintenance under acidosis only in tumour cells, the acidic microenvironment should be considered for future therapies.

Mitochondrial mass, a new metabolic biomarker for stem-like cancer cells: Understanding WNT/FGF-driven anabolic signaling.

Here, we developed an isogenic cell model of "stemness" to facilitate protein biomarker discovery in breast cancer. For this purpose, we used knowledge gained previously from the study of the mouse mammary tumor virus (MMTV). MMTV initiates mammary tumorigenesis in mice by promoter insertion adjacent to two main integration sites, namely Int-1 (Wnt1) and Int-2 (Fgf3), which ultimately activates Wnt/ß-catenin signaling, driving the propagation of mammary cancer stem cells (CSCs). Thus, to develop a humanized model of MMTV signaling, we over-expressed WNT1 and FGF3 in MCF7 cells, an ER(+) human breast cancer cell line. We then validated that MCF7 cells over-expressing both WNT1 and FGF3 show a 3.5-fold increase in mammosphere formation, and that conditioned media from these cells is also sufficient to promote stem cell activity in untransfected parental MCF7 and T47D cells, as WNT1 and FGF3 are secreted factors. Proteomic analysis of this model system revealed the induction of i) EMT markers, ii) mitochondrial proteins, iii) glycolytic enzymes and iv) protein synthesis machinery, consistent with an anabolic CSC phenotype. MitoTracker staining validated the expected WNT1/FGF3-induced increase in mitochondrial mass and activity, which presumably reflects increased mitochondrial biogenesis. Importantly, many of the proteins that were up-regulated by WNT/FGF-signaling in MCF7 cells, were also transcriptionally over-expressed in human breast cancer cells in vivo, based on the bioinformatic analysis of public gene expression datasets of laser-captured patient samples. As such, this isogenic cell model should accelerate the discovery of new biomarkers to predict clinical outcome in breast cancer, facilitating the development of personalized medicine.Finally, we used mitochondrial mass as a surrogate marker for increased mitochondrial biogenesis in untransfected MCF7 cells. As predicted, metabolic fractionation of parental MCF7 cells, via MitoTracker staining, indicated that high mitochondrial mass is a new metabolic biomarker for the enrichment of anabolic CSCs, as functionally assessed by mammosphere-forming activity. This observation has broad implications for understanding the role of mitochondrial biogenesis in the propagation of stem-like cancer cells. Technically, this general metabolic approach could be applied to any cancer type, to identify and target the mitochondrial-rich CSC population.The implications of our work for understanding the role of mitochondrial metabolism in viral oncogenesis driven by random promoter insertions are also discussed, in the context of MMTV and ALV infections.

Dissecting tumor metabolic heterogeneity: Telomerase and large cell size metabolically define a sub-population of stem-like, mitochondrial-rich, cancer cells.

Tumor cell metabolic heterogeneity is thought to contribute to tumor recurrence, distant metastasis and chemo-resistance in cancer patients, driving poor clinical outcome. To better understand tumor metabolic heterogeneity, here we used the MCF7 breast cancer line as a model system to metabolically fractionate a cancer cell population. First, MCF7 cells were stably transfected with an hTERT-promoter construct driving GFP expression, as a surrogate marker of telomerase transcriptional activity. To enrich for immortal stem-like cancer cells, MCF7 cells expressing the highest levels of GFP (top 5%) were then isolated by FACS analysis. Notably, hTERT-GFP(+) MCF7 cells were significantly more efficient at forming mammospheres (i.e., stem cell activity) and showed increased mitochondrial mass and mitochondrial functional activity, all relative to hTERT-GFP(-) cells. Unbiased proteomics analysis of hTERT-GFP(+) MCF7 cells directly demonstrated the over-expression of 33 key mitochondrial proteins, 17 glycolytic enzymes, 34 ribosome-related proteins and 17 EMT markers, consistent with an anabolic cancer stem-like phenotype. Interestingly, MT-CO2 (cytochrome c oxidase subunit 2; Complex IV) expression was increased by >20-fold. As MT-CO2 is encoded by mt-DNA, this finding is indicative of increased mitochondrial biogenesis in hTERT-GFP(+) MCF7 cells. Importantly, most of these candidate biomarkers were transcriptionally over-expressed in human breast cancer epithelial cells in vivo. Similar results were obtained using cell size (forward/side scatter) to fractionate MCF7 cells. Larger stem-like cells also showed increased hTERT-GFP levels, as well as increased mitochondrial mass and function. Thus, this simple and rapid approach for the enrichment of immortal anabolic stem-like cancer cells will allow us and others to develop new prognostic biomarkers and novel anti-cancer therapies, by specifically and selectively targeting this metabolic sub-population of aggressive cancer cells. Based on our proteomics and functional analysis, FDA-approved inhibitors of protein synthesis and/or mitochondrial biogenesis, may represent novel treatment options for targeting these anabolic stem-like cancer cells.

Carbonic anhydrase IX inhibition is an effective strategy for osteosarcoma treatment.

OBJECTIVE: Hypoxia-inducible factor 1, a regulator of CA IX activity, is often overexpressed in human osteosarcoma (OS) but not in normal tissues, and its expression levels correlate with prognosis. In this study, we investigated the therapeutic potential of newly synthesized CA IX sulfonamide inhibitors in OS. METHODS: CA IX expression was evaluated in OS cell lines and bone marrow stromal cells (BMSC). After treatment with CA IX inhibitors, cell proliferation, apoptosis, cell cycle, extracellular and cytosolic pH changes were evaluated both in vitro and in mouse OS xenografts. RESULTS: CA IX expression levels were significantly higher in OS than in BMSC. Accordingly, CA IX inhibitor 3 induced remarkable cytotoxicity on OS cells without affecting BMSC proliferation. This activity was increased under hypoxia, and was mediated by cell cycle arrest and by the modulation of cytosolic and extracellular pH. In vivo, CA IX inhibitor 3 reduced tumor growth by inducing significant necrosis. CONCLUSIONS: Our results provide a strong rationale for the clinical use of the newly synthesized CA IX inhibitor 3 in human OS.

Impairment of lysosomal activity as a therapeutic modality targeting cancer stem cells of embryonal rhabdomyosarcoma cell line RD.

Rhabdomyosarcoma is the most frequent soft tissue sarcoma in children and adolescents, with a high rate of relapse that dramatically affects the clinical outcome. Multiagent chemotherapy, in combination with surgery and/or radiation therapy, is the treatment of choice. However, the relapse rate is disappointingly high and identification of new therapeutic tools is urgently needed. Under this respect, the selective block of key features of cancer stem cells (CSC) appears particularly promising. In this study, we isolated rhabdomyosarcoma CSC with stem-like features (high expression of NANOG and OCT3/4, self-renewal ability, multipotency). Rhabdomyosarcoma CSC showed higher invasive ability and a reduced cytotoxicity to doxorubicin in comparison to native cells, through a mechanism unrelated to the classical multidrug resistance process. This was dependent on a high level of lysosome acidity mediated by a high expression of vacuolar ATPase (V-ATPase). Since it was not associated with other paediatric cancers, like Ewing's sarcoma and neuroblastoma, V-ATPase higher expression in CSC was rhabdomyosarcoma specific. Inhibition of lysosomal acidification by the V-ATPase inhibitor omeprazole, or by specific siRNA silencing, significantly enhanced doxorubicin cytoxicity. Unexpectedly, lysosomal targeting also blocked cell growth and reduced the invasive potential of rhabdomyosarcoma CSC, even at very low doses of omeprazole (10 and 50 µM, respectively). Based on these observations, we propose lysosome acidity as a valuable target to enhance chemosensitivity of rhabdomyosarcoma CSC, and suggest the use of anti-V-ATPase agents in combination with standard regimens as a promising tool for the eradication of minimal residual disease or the prevention of metastatic disease.

Role of mesenchymal stem cells in osteosarcoma and metabolic reprogramming of tumor cells.

The tumor microenvironment plays an important role in cancer progression. Here, we focused on the role of reactive mesenchymal stem cells (MSC) in osteosarcoma (OS), and used human adipose MSC and a panel of OS cell lines (Saos-2, HOS, and 143B) to investigate the mutual effect of normal-cancer cell metabolic programming. Our results showed that MSC are driven by oxidative stress induced by OS cells to undergo Warburg metabolism, with increased lactate production. Therefore, we analyzed the expression of lactate monocarboxylate transporters. By real time PCR and immunofluorescence, in MSC we detected the expression of MCT-4, the transporter for lactate efflux, whereas MCT-1, responsible for lactate uptake, was expressed in OS cells. In agreement, silencing of MCT-1 by siRNA significantly affected the ATP production in OS cancer cells. Thus, cancer cells directly increase their mitochondrial biogenesis using this energy-rich metabolite that is abundantly provided by MSC as an effect of the altered microenvironmental conditions induced by OS cells. We also showed that lactate produced by MSC promotes the migratory ability of OS cells. These data provide novel information to be exploited for cancer therapies targeting the mutual metabolic reprogramming of cancer cells and their stroma.

Sphere-forming cell subsets with cancer stem cell properties in human musculoskeletal sarcomas.

Musculoskeletal sarcomas are aggressive malignancies often characterized by an adverse prognosis despite the use of intense multiagent chemotherapy or molecular targeted therapy in combination to surgery and radiotherapy. Stem-like cells identified within solid tumors have been recently implicated in drug resistance, metastasis and local relapse. Here, we report the identification of putative cancer stem cells (CSCs) in sarcomas using a sphere culture system. These sarcospheres, able to grow in anchorage-independent and serum-starved conditions, express the pluripotent embryonic stem cell marker genes OCT3/4, Nanog and SOX2. Expression levels of these genes were greater in sarcospheres than in the parental tumor cultures. Importantly, the isolated tumor spheres transplanted into mice were tumorigenic and capable of recapitulating the human disease. Finally, we demonstrated that low (1%) O2 conditions, reproducing those found within the tumor microenvironment, significantly increase the number and the size of sarcospheres. The sphere formation assay is, therefore, a valuable method for the isolation of putative CSCs from human sarcomas and its efficiency is improved by controlling oxygen availability. This method provides a reliable preclinical model that can be used for future studies aimed at investigating crucial aspects of sarcoma biology, such as resistance to treatments and relapse.

Bortezomib (PS-341) treatment decreases inflammation and partially rescues the expression of the dystrophin-glycoprotein complex in GRMD dogs.

Golden retriever muscular dystrophy (GRMD) is a genetic myopathy corresponding to Duchenne muscular dystrophy (DMD) in humans. Muscle atrophy is known to be associated with degradation of the dystrophin-glycoprotein complex (DGC) via the ubiquitin-proteasome pathway. In the present study, we investigated the effect of bortezomib treatment on the muscle fibers of GRMD dogs. Five GRMD dogs were examined; two were treated (TD- Treated dogs) with the proteasome inhibitor bortezomib, and three were control dogs (CD). Dogs were treated with bortezomib using the same treatment regimen used for multiple myeloma. Pharmacodynamics were evaluated by measuring the inhibition of 20S proteasome activity in whole blood after treatment and comparing it to that in CD. We performed immunohistochemical studies on muscle biopsy specimens to evaluate the rescue of dystrophin and dystrophin-associated proteins in the muscles of GRMD dogs treated with bortezomib. Skeletal tissue from TD had lower levels of connective tissue deposition and inflammatory cell infiltration than CD as determined by histology, collagen morphometry and ultrastructural analysis. The CD showed higher expression of phospho-NFκB and TGF-ß1, suggesting a more pronounced activation of anti-apoptotic factors and inflammatory molecules and greater connective tissue deposition, respectively. Immunohistochemical analysis demonstrated that dystrophin was not present in the sarcoplasmic membrane of either group. However, bortezomib-TD showed higher expression of α- and ß-dystroglycan, indicating an improved disease histopathology phenotype. Significant inhibition of 20S proteasome activity was observed 1 hour after bortezomib administration in the last cycle when the dose was higher. Proteasome inhibitors may thus improve the appearance of GRMD muscle fibers, lessen connective tissue deposition and reduce the infiltration of inflammatory cells. In addition, proteasome inhibitors may rescue some dystrophin-associated proteins in the muscle fiber membrane.