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Background Previous studies by our group have shown that oxidative phosphorylation (OXPHOS) is the main pathway by which pancreatic cancer stem cells (CSCs) meet their energetic requirements; therefore, OXPHOS represents an Achille’s heel of these highly tumorigenic cells. Unfortunately, therapies that target OXPHOS in CSCs are lacking. Methods The safety and anti-CSC activity of a ruthenium complex featuring bipyridine and terpyridine ligands and one coordination labile position (Ru1) were evaluated across primary pancreatic cancer cultures and in vivo, using 8 patient-derived xenografts (PDXs). RNAseq analysis followed by mitochondria-specific molecular assays were used to determine the mechanism of action. Results We show that Ru1 is capable of inhibiting CSC OXPHOS function in vitro, and more importantly, it presents excellent anti-cancer activity, with low toxicity, across a large panel of human pancreatic PDXs, as well as in colorectal cancer and osteosarcoma PDXs. Mechanistic studies suggest that this activity stems from Ru1 binding to the D-loop region of the mitochondrial DNA of CSCs, inhibiting OXPHOS complex-associated transcription, leading to reduced mitochondrial oxygen consumption, membrane potential, and ATP production, all of which are necessary for CSCs, which heavily depend on mitochondrial respiration. Conclusions Overall, the coordination complex Ru1 represents not only an exciting new anti-cancer agent, but also a molecular tool to dissect the role of OXPHOS in CSCs. Results indicating that the compound is safe, non-toxic and highly effective in vivo are extremely exciting, and have allowed us to uncover unprecedented mechanistic possibilities to fight different cancer types based on targeting CSC OXPHOS.

Deregulation of Src kinases is associated with cancer. We previously showed that SrcDN conditional expression in MCF7 cells reduces tumorigenesis and causes tumor regression in mice. However, it remained unclear whether SrcDN affected breast cancer stem cell functionality or it reduced tumor mass. Here, we address this question by isolating an enriched population of Breast Cancer Stem Cells (BCSCs) from MCF7 cells with inducible expression of SrcDN. Induction of SrcDN inhibited self-renewal, and stem-cell marker expression (Nanog, Oct3-4, ALDH1, CD44). Quantitative proteomic analyses of mammospheres from MCF7-Tet-On-SrcDN cells (data are available via ProteomeXchange with identifier PXD017789, project DOI: 10.6019/PXD017789) and subsequent GSEA showed that SrcDN expression inhibited glycolysis. Indeed, induction of SrcDN inhibited expression and activity of hexokinase, pyruvate kinase and lactate dehydrogenase, resulting in diminished glucose consumption and lactate production, which restricted Warburg effect. Thus, c-Src functionality is important for breast cancer stem cell maintenance and renewal, and stem cell transcription factor expression, effects linked to glucose metabolism reduction.

Human iPSC line N44SV.5 was generated from primary normal human dermal fibroblasts belonging to the European mitochondrial haplogroup U. For this purpose, reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using a non-integrative methodology that involves the use of Sendai virus.

Human iPSC line LND554SV.3 was generated from heteroplasmic fibroblasts of a patient with Leigh syndrome carrying a mutation in the MT-ND5 gene (m.13513G. >. A; p.D393N). Reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using a non-integrative methodology that involves the use of Sendai virus.

Human iPSC line PG64SV.2 was generated from fibroblasts of a patient with a defect of intergenomic communication. This patient harbored a homozygous mutation (c.2243G>C; p.Trp748Ser) in the gene encoding the catalytic subunit of the mitochondrial DNA polymerase gamma gene (POLG). Reprogramming factors Oct3/4, Sox2, Klf4, and cMyc were delivered using a non integrative methodology that involves the use of Sendai virus.

Human iPSC line L749.1 was generated from fibroblasts of a patient with Leigh syndrome associated with a heteroplasmic mutation in the MT-ATP6 gene. Reprogramming factors OCT4, SOX2, CMYC and KLF4 were delivered using retroviruses.

Las enfermedades mitocondriales (EM) constituyen un amplio grupo de enfermedades genéticas multisistémicas cuyo nexo de unión es una disfunción en el sistema de fosforilación oxidativa. La falta de modelos experimentales adecuados para el estudio de dichas enfermedades, ha dificultado el avance en el desarrollo de nuevas terapias. En este sentido, la reprogramación de células somáticas a células madre pluripotentes inducidas (iPSC) mediante la expresión ectópica de cuatro factores de transcripción, OCT4, SOX2, KLF4 y c-MYC, ha abierto enormes posibilidades. Por ello, el objetivo general de este trabajo ha consistido en la generación de iPSC a partir de fibroblastos de pacientes con EM para el estudio y tratamiento potencial de las mismas. Para este fin, hemos establecido una colección de fibroblastos procedentes de biopsias de piel de pacientes con EM e individuos sanos. A continuación, se han generado líneas de iPSC a partir de dichos fibroblastos mediante el uso de vectores retrovirales o métodos no integrativos tipo virus Sendai. Así, hemos generado 16 líneas de IPSC portadoras de distintas mutaciones en genes mitocondriales codificados en el DNA mitocondrial o nuclear asociadas a distintas EM: Síndrome de Leigh, atrofia óptica sindrómica y EM producidas por defectos de comunicación intergenómica. Actualmente, estamos realizando la confirmación, molecular y funcional, de la pluripotencia de las líneas generadas, para posteriormente, diferenciarlas y caracterizarlas en los tipos celulares diana. La disponibilidad de iPSC como modelo experimental de las EM nos permitirá mejorar el conocimiento de los mecanismos fisiopatológicos de estas enfermedades y podrá suponer la apertura de nuevas vías para la identificación de tratamientos farmacológicos y de terapia celular.