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.

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 Sánchez-Alvarez et al. MFF Reduces CSCs Activity 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.