Dodecyl-TPP Targets Mitochondria and Potently Eradicates Cancer Stem Cells (CSCs): Synergy With FDA-Approved Drugs and Natural Compounds (Vitamin C and Berberine).

Elevated mitochondrial biogenesis and/or metabolism are distinguishing features of cancer cells, as well as Cancer Stem Cells (CSCs), which are involved in tumor initiation, metastatic dissemination, and therapy resistance. In fact, mitochondria-impairing agents can be used to hamper CSCs maintenance and propagation, toward better control of neoplastic disease. Tri-Phenyl-Phosphonium (TPP)-based mitochondrially-targeted compounds are small non-toxic and biologically active molecules that are delivered to and accumulated within the mitochondria of living cells. Therefore, TPP-derivatives may represent potentially "powerful" candidates to block CSCs. Here, we evaluate the metabolic and biological effects induced by the TPP-derivative, termed Dodecyl-TPP (d-TPP) on breast cancer cells. By employing the 3D mammosphere assay in MCF-7 cells, we demonstrate that treatment with d-TPP dose-dependently inhibits the propagation of breast CSCs in suspension. Also, d-TPP targets adherent "bulk" cancer cells, by decreasing MCF-7 cell viability. The analysis of metabolic flux using Seahorse Xfe96 revealed that d-TPP potently inhibits the mitochondrial oxygen consumption rate (OCR), while simultaneously shifting cell metabolism toward glycolysis. Thereafter, we exploited this ATP depletion phenotype and strict metabolic dependency on glycolysis to eradicate the residual glycolytic CSC population, by using additional metabolic stressors. More specifically, we applied a combination strategy based on treatment with d-TPP, in the presence of a selected panel of natural and synthetic compounds, some of which are FDA-approved, that are known to behave as glycolysis (Vitamin C, 2-Deoxy-Glucose) and OXPHOS (Doxycyline, Niclosamide, Berberine) inhibitors. This two-hit scheme effectively decreased CSC propagation, at concentrations of d-TPP toxic only for cancer cells, but not for normal cells, as evidenced using normal human fibroblasts (hTERT-BJ1) as a reference point. Taken together, d-TPP halts CSCs propagation and targets "bulk" cancer cells, without eliciting the relevant undesirable off-target effects in normal cells. These observations pave the way for further exploring the potential of TPP-based derivatives in cancer therapy. Moreover, TPP-based compounds should be investigated for their potential to discriminate between "normal" and "malignant" mitochondria, suggesting that distinct biochemical, and metabolic changes in these organelles could precede specific normal or pathological phenotypes. Lastly, our data validate the manipulation of the energetic machinery as useful tool to eradicate CSCs

Mitochondrial and ribosomal biogenesis are new hallmarks of stemness, oncometabolism and biomass accumulation in cancer : mito-stemness and ribo-stemness features

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

Editorial: metabolic reprogramming in breast cancer

Metabolic reprogramming is an emerging hallmark of breast cancer. A common characteristic of tumor cells is the ability to obtain nutrients from a nutrient-deprived environment and to use them to sustain cancer progression, within crucial metabolic pathways including altered metabolism of glucose, lipids, and amino acids. Also, altered metabolism has been recognized as one of the major mechanisms of resistance to therapies. Important advances have been made to elucidate key mechanisms of metabolic reprogramming which will make possible novel strategies for overcoming breast cancer. However, for metabolic therapy to be effective there is a need to clearly understand the metabolic underpinnings of the different subtypes of breast cancer as well as the role the standard-of-care therapies play in targeting the metabolic phenotype

A myristoyl amide derivative of doxycycline potently targets cancer stem cells (CSCs) and prevents metastasis, without retaining antibiotic activity

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 analogue, 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 developed 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 the process of tumor cell metastasis. Based on these findings, we propose that it may be more appropriate to refer to tumor-spheres as metasta-spheres, to better reflect the close relationship between 3D anchorage-independent growth and metastasis

Cytoprotection by the NO-donor SNAP against ischemia/reoxygenation injury in mouse embryonic stem cell-derived cardiomyocytes

Embryonic stem cell (ESC)-derived cardiomyocytes are a promising cell source for the screening for potential cytoprotective molecules against ischemia/reperfusion injury, however, little is known on their behavior in hypoxia/reoxygenation conditions. Here we tested the cytoprotective effect of the NO-donor SNAP and its downstream cellular pathway. Mouse ESC-derived cardiomyocytes were subjected to 150-min simulated ischemia (SI) followed by 120-min reoxygenation or corresponding non-ischemic conditions. The following treatments were applied during SI or normoxia: the NO-donor S-Nitroso-N-acetyl-D,L-penicillamine (SNAP), the protein kinase G (PKG) inhibitor, the KATP channel blocker glibenclamide, the particulate guanylate cyclase activator brain type natriuretic peptide (BNP), and a non- specific NO synthase inhibitor (N-Nitro-L-arginine, L-NNA) alone or in different combinations. Viability of cells was assayed by propidium iodide staining. SNAP attenuated SI-induced cell death in a concentration-dependent manner, and this protection was attenuated by inhibition of either PKG or KATP channels. However, SI-induced cell death was not affected by BNP or by L- NNA. We conclude that SNAP protects mESC-derived cardiomyocytes against SI/R injury and that soluble guanylate-cyclase, PKG, and KATP channels play a role in the downstream pathway of SNAP- induced cytoprotection. The present mESC-derived cardiomyocyte- based screening platform is a useful tool for discovery of cytoprotective molecules