Bela Ozsvari

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/β-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.

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.

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.

Repurposing of FDA-approved drugs against cancer – focus on metastasis

Research on existing FDA-approved drugs is increasing in the field of anti-cancer drug development. Drug repurposing may allow progressing through the drug development process more quickly as well as reducing project costs compared to traditional de novo drug discovery. One of the main reasons why a non-cancer drug may prove effective against cancer is that not all of its biological targets may have been identified when the drug was originally developed. Until recently, the main focus in oncology drug development has been on the tumor cell — first with traditional chemotherapy and then with more targeted agents. But much of the focus has now shifted to targeting the tumor micro-environment, especially its key role in metastasis and drug resistance. Wan L. et al. proposed that initiation of circulating tumor cell adhesion to the local vascular endothelium is an important first step for circulating tumor cells to start the metastatic cascade. They showed that a combination of 3 FDA-approved drugs, the over-the-counter drug aspirin, the well-known antibiotic doxycycline and mifepristone (a progesterone receptor antagonist known as an abortifacient pill) used together with the amino acid lysine could effectively and safely prevent cancer metastasis (Figure ​(Figure1.)1.) [1]. This quadruplet drug combination has successfully inhibited adhesion of mouse melanoma (B16-F10) and human melanoma (M619) cell lines to either endothelial cells or extracellular matrix via down-regulating cell adhesion molecules ICAM-1 and α4-integrin. The drug treatment also inhibited platelet-cloaked aggregation of tumor cells in a dose-dependent manner. In their in vivo experiment, a four-day pre-treatment followed by a 30-day oral administration of the quadruplet drug combination to mice inoculated with melanoma cells produced significant inhibition of cancer metastasis in the lung dose-dependently without any marked side effects. In addition, the authors confirmed that all the four of these drugs were required for inhibition of cancer metastasis. Figure 1 Effects of FDA-approved non-cancer drugs on early events of tumor metastasis Aspirin, a widely used anti-inflammatory drug, a potent COX-2 inhibitor and an antiaggregant has been recently shown that its regular intake might reduce long-term risk of colorectal, oesophageal, gastric, biliary, and breast cancers and the risk of distant metastasis as well [2]. Add-Aspirin, the worlds largest ever phase III clinical trial was launched in the UK in October 2015. Oncologists are looking to assess whether regular aspirin use after standard therapy prevents recurrence and prolongs survival in patients with non-metastatic common solid tumors (breast, colorectal, gastro-oesophageal, and prostate) [3]. We proposed a novel strategy for the treatment of primary tumors and advanced metastases via the selective targeting of cancer stem-like cells and identified a conserved phenotypic weak point - a strict dependence on mitochondrial biogenesis. Given that mitochondria have a bacterial origin, antibiotics also target mitochondrial translation and impair mitochondrial function. In our study we have shown that treatment with 4-5 different classes of FDA-approved antibiotics, especially doxycycline can be used to eradicate cancer stem-like cells in several tumor types (Figure ​(Figure1)1) [4]. Pulvino et al. describe in their paper that a connectivity map (a collection of genome-wide gene expression profiles of cultured human cells treated with various bioactive small molecules) analysis uncovered several tetracycline antibiotics. Among them doxycycline as the most potent inhibitor of NF-κB signaling. Also, doxycycline successfully inhibited the growth of diffuse large B-cell lymphoma in vitro and in a mouse xenograft model. Importantly, this effect may be achievable in human sera with a therapeutic dose of the drug [5]. Hu et al. showed that tigecycline, another tetracycline antibiotic, suppressed cell proliferation, cell invasion and migration in melanoma (Figure ​(Figure1).1). It induced cell cycle arrest at G0/G1 phase through the downregulation of p21 promoted cell cycle progression via CDK2/cyclin E cell kinases. In addition, intraperitoneal treatment with tigecycline significantly reduced tumor growth in vivo in a subcutaneous model of two human melanoma cell lines (A375 and MV3) in nude mice [6]. Cancer is a leading cause of death worldwide and despite the investment of millions of dollars of large pharmaceutical companies and governments into anti-cancer drug development, the current chemotherapeutic approaches are highly costly while their efficacy is not universally effective in all patients. These factors are leading to repurposing of well-known FDA-approved drugs, which can bypass the early stages of drug development, even Phase I clinical trials in some cases, thus reducing project costs and time. Taken together, the previously mentioned studies show that FDA approved drugs can be successfully applied in tumor therapies.

Mitoketoscins: Novel mitochondrial inhibitors for targeting ketone metabolism in cancer stem cells (CSCs)

Previous studies have now well-established that epithelial cancer cells can utilize ketone bodies (3-hydroxybutyrate and aceto-acetate) as mitochondrial fuels, to actively promote tumor growth and metastatic dissemination. The two critical metabolic enzymes implicated in this process are OXCT1 and ACAT1, which are both mitochondrial proteins. Importantly, over-expression of OXCT1 or ACAT1 in human breast cancer cells is sufficient to genetically drive tumorigenesis and/or lung metastasis, validating that they indeed behave as metabolic “tumor promoters”. Here, we decided to target these two enzymes, which give cancer cells the ability to recycle ketone bodies into Acetyl-CoA and, therefore, to produce increased ATP. Briefly, we used computational chemistry (in silico drug design) to select a sub-set of potentially promising compounds that spatially fit within the active site of these enzymes, based on their known 3D crystal structures. These libraries of compounds were then phenotypically screened for their effects on total cellular ATP levels. Positive hits were further validated by metabolic flux analysis. Our results indicated that four of these compounds effectively inhibited mitochondrial oxygen consumption. Two of these compounds also induced a reactive glycolytic phenotype in cancer cells. Most importantly, using the mammosphere assay, we showed that these compounds can be used to functionally inhibit cancer stem cell (CSC) activity and propagation. Finally, our molecular modeling studies directly show how these novel compounds are predicted to bind to the active catalytic sites of OXCT1 and ACAT1, within their Coenzyme A binding site. As such, we speculate that these mitochondrial inhibitors are partially mimicking the structure of Coenzyme A. Thus, we conclude that OXCT1 and ACAT1 are important new therapeutic targets for further drug development and optimization. We propose that this new class of drugs should be termed “mitoketoscins”, to reflect that they were designed to target ketone re-utilization and mitochondrial function.

A new mutation-independent approach to cancer therapy : inhibiting oncogenic RAS and MYC, by targeting mitochondrial biogenesis.

Here, we used MCF7 cells as a model system to interrogate how MYC/RAS co-operativity contributes to metabolic flux and stemness in breast cancer cells. We compared the behavior of isogenic MCF7 cell lines transduced with c-Myc or H-Ras (G12V), either individually or in combination. Cancer stem cell (CSC) activity was measured using the mammosphere assay. c-Myc augmented both mammosphere formation and mitochondrial respiration, without any effects on glycolytic flux. In contrast, H-Ras (G12V) synergistically augmented both mammosphere formation and glycolysis, but only in combination with c-Myc, directly demonstrating MYC/RAS co-operativity. As c-Myc is known to exert its effects, in part, by stimulating mitochondrial biogenesis, we next examined the effects of another stimulus known to affect mitochondrial biogenesis, i.e. ROS production. To pharmacologically induce oxidative stress, we used Rotenone (a mitochondrial inhibitor) to target mitochondrial complex I. Treatment with Rotenone showed bi-phasic effects; low-dose Rotenone (1 to 2.5 nM) elevated mammosphere formation, while higher doses (10 to 100 nM) were inhibitory. Importantly, the stimulatory effects of Rotenone on CSC propagation were blocked using a mitochondrial-specific anti-oxidant, namely Mito-tempo. Thus, “mild” mitochondrial oxidative stress, originating at Complex I, was sufficient to pheno-copy the effects of c-Myc, effectively promoting CSC propagation. To validate the idea that mitochondrial biogenesis is required to stimulate CSC propagation, we employed Doxycycline, a well-established inhibitor of mitochondrial protein translation. Treatment with Doxycycline was indeed sufficient to block the stimulatory effects of H-Ras (G12V), c-Myc, and Rotenone on CSC propagation. As such, Doxycycline provides a strong rationale for designing new therapeutics to target mitochondrial biogenesis, suggesting a new “mutation-independent” approach to cancer therapy. In support of this notion, most currently successful anti-cancer agents therapeutically target “cell phenotypes”, such as increased cell proliferation, rather than specific genetic mutations. Remarkably, we demonstrated that Doxycycline inhibits the effects of diverse oncogenic stimuli, of both i) genetic (MYC/RAS) and ii) environmental (Rotenone) origins. Finally, we discuss the advantages of our “Proteomics-to-Genomics (PTG)” approach for in silico validation of new biomarkers and novel drug targets. In this context, we developed a new Myc-based Mito-Signature consisting of 3 mitochondrial genes (HSPD1; COX5B; TIMM44) for effectively predicting tumor recurrence (HR=4.69; p=2.4e-08) and distant metastasis (HR=4.94; p=2.8e-07), in ER(+) in breast cancer patients. This gene signature could serve as a new companion diagnostic for the early prediction of treatment failure in patients receiving hormonal therapy.

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.

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

ABSTRACT 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.

Exploiting mitochondrial targeting signal(s), TPP and bis-TPP, for eradicating cancer stem cells (CSCs)

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.