Background and purpose Radiotherapy (RT) is an integral treatment part for patients with head and neck squamous cell carcinoma (HNSCC), but radioresistance remains a major issue. Here, we use MitoTam, a mitochondrially targeted analogue of tamoxifen, which we aim to stimulate ferroptotic cell death with, and sensitize radioresistant cells to RT. Materials and methods We assessed viability, reactive oxygen species (ROS) production, disruption of mitochondrial membrane potential, and lipid peroxidation in radiosensitive (UT-SCC-40) and radioresistant (UT-SCC-5) HNSCC cells following MitoTam treatment. To assess ferroptosis specificity, we used the ferroptosis inhibitor ferrostatin-1 (fer-1). Also, total antioxidant capacity and sensitivity to tert-butyl hydroperoxide were evaluated to assess ROS-responses. 53BP1 staining was used to assess radiosensitivity after MitoTam treatment. Results Our data revealed increased ROS, cell death, disruption of mitochondrial membrane potential, and lipid peroxidation following MitoTam treatment in both cell lines. Adverse effects of MitoTam on cell death, membrane potential and lipid peroxidation were prevented by fer-1, indicating induction of ferroptosis. Radioresistant HNSCC cells were less sensitive to the effects of MitoTam due to intrinsic higher antioxidant capacity. MitoTam treatment prior to RT led to superadditive residual DNA damage expressed by 53BP1 foci compared to RT or MitoTam alone. Conclusion MitoTam induced ferroptosis in HNSCC cells, which could be used to overcome the elevated antioxidant capacity of radioresistant cells and sensitize such cells to RT. Treatment with MitoTam followed by RT could therefore present a promising effective therapy of radioresistant cancers.Statement of significance.Radiotherapy is applied in the treatment of a majority of cancer patients. Radioresistance due to elevated antioxidant levels can be overcome by promoting ferroptotic cell death combining ROS-inducing drug MitoTam with radiotherapy.

Dear Editor, Cancer is a pathology still on the rise,1 with unmet need for efficient therapy, owing to factors such as considerable differences in mutational signature in the same patient in primary tumours and proximal/distal metastases, shown, for example, for renal cancer.2 What is needed then is an invariant target predominantly only affected by drugs in cancer cells. A thus far untested approach is targeting mitochondrial respiration using compounds from the group of mitocans,3 epitomised by mitochondrially targeted tamoxifen (MitoTam), that is, tamoxifen tagged with the mitochondrial vector triphenylphosphonium (TPP) (Figure S1A; see also Supporting Information for description of synthesis).4,5 This strategy is based on the premise that cancer cells differ from their non-cancerous counterparts,3 making them selectively vulnerable to TPP-tagged anti-cancer agents,6 and on the premise that mitochondrial function is vital for tumour progression.7,8 Wehave recently conducted Phase 1/1bMitoTamclinical trial for metastatic solid tumour patients, with all patients undergoing palliative therapy after exhaustion of established therapeutic regimens (MitoTam-01 trial; EudraCT 2017-004441-25). Although the Phase 1/1b clinical trial will be published in its entirety elsewhere, of the individual types of cancer, the greatest benefit was found for clear cell renal cancer patients represented here by two subjects (Tables S1 and S2). These patients underwent three and four rounds of MitoTam therapy, respectively, at 1 mg/kg three times per week followed by a week of rest, totalling four such cycles, with one patient showing tumour stabilisation and the other partial remission (Figure 1A). The trial revealed excellent safety profile of MitoTam, with only occasional grade 1 toxicity. The high efficiency for renal cancer was found to correlate with the highest level of MitoTam and its metabolites reached in kidneys (Figures 1B, S1B and S1C), being excreted via bile (Figure S1D).

Iron is a crucial nutrient for proliferating cancer cells. Thus, we have synthesized a mitochondrially targeted derivative of the iron chelator deferoxamine (mDFO) and evaluated its ability to selectively eliminate cancer cells both in vitro and in vivo. Viability of human cancer cells treated with mDFO was assessed by crystal violet or annexin V staining. ROS production was assessed through fluorescent probes MitoSOX and DCF. Blue native electrophoresis and Seahorse were used to analyse mitochondrial function. The in vivo effect of mDFO was assessed by xenografting triple negative breast cancer cells in NOD/Scid mice. Our results show that mDFO is at least 100-fold more efficient than DFO in killing cancer cells. In addition, mDFO was shown to reduce respiration and dramatically decrease the amount and composition of mitochondrial respiratory supercomplexes. Both the latter effects might trigger the observed increase in mitochondrial superoxide production, the fragmentation of mitochondria and the activation of mitophagy. Importantly, in vivo results show that mDFO is effective in slowing the tumour growth. mDFO represents an effective drug to inhibit tumour growth in vivo and might represent the lead compound in a new generation of anti-cancer agents based on mitochondrial iron chelation.

The anti-diabetic biguanide metformin may exert health-promoting effects via metabolic regulation of the epigenome. Here we show that metformin promotes global DNA methylation in non-cancerous, cancer-prone and metastatic cancer cells by decreasing S-adenosylhomocysteine (SAH), a strong feedback inhibitor of S-adenosylmethionine (SAM)-dependent DNA methyltransferases, while promoting the accumulation of SAM, the universal methyl donor for cellular methylation. Using metformin and a mitochondria/complex I (mCI)-targeted analog of metformin (norMitoMet) in experimental pairs of wild-type and AMP-activated protein kinase (AMPK)-, serine hydroxymethyltransferase 2 (SHMT2)- and mCI-null cells, we provide evidence that metformin increases the SAM:SAH ratio-related methylation capacity by targeting the coupling between serine mitochondrial one-carbon flux and CI activity. By increasing the contribution of one-carbon units to the SAM from folate stores while decreasing SAH in response to AMPK-sensed energetic crisis, metformin can operate as a metabolo-epigenetic regulator capable of reprogramming one of the key conduits linking cellular metabolism to the DNA methylation machinery.