Background: Mitochondrial citrate has been shown to serve as a central substrate for many cytosolic processes critical for tumor progression. Experiments with stable isotopes and mass spectrometry have been used for this conclusion, but fixed pools of metabolites yielding from those studies do not help us understand kinetics of subcellular metabolic fluxes. New methods are necessary for such measurements. In clinical setting, luminal-A subtype of breast cancer progresses slower than triple negative subtype and we hypothesize that mitochondrial citrate efflux in more aggressive tumors must be greater than that in slow-growing tumors to support the faster progression.
Methods: New permeabilization-based method for quantifying time-dependent mitochondrial fluxes was developed. Luminal-A type MCF7 and triple negative MDA-MB-231 cell lines were used; excised primary tumors were used from human subjects (luminal-A n=21, triple negative n=10). Mitochondrial substrate-to-citrate efflux was measured using glutamate (GM), pyruvate (PV) or palmitoylcarnitine (PC) as substrates. Finally, samples were analyzed using HPLC or UV-spectroscopy.
Summary: Mitochondrial GM-to-citrate efflux in less aggressive MCF7 cells was 91.5% lower than that in the faster growing MDA-MB-231 cells (4.1 vs. 48.4 ng/min*106cell, respectively). Difference in PV-to-citrate efflux was smaller, yielding 25.0% lower result for MCF7 compared to MDA-MB-231. An opposite result was observed for PC-to-citrate efflux as the conversion of PC was 20.1% higher in MCF7 cells compared to MDA-MB-231. Significant amount of cytosolic GM-to-citrate was measured only for MDA-MB-231, possibly a result of reverse IDH1 action. In primary tumor samples from breast cancer patients, mitochondrial GM-to-citrate efflux in luminal-A subtype was 27.4% lower than that in more aggressive triple-negative subtype (92.9 vs. 127.9 ng/h*mg, respectively). The respective differences were 24.7% for PV-to-citrate efflux and 8.2% for PC-to-citrate.
Conclusions: Here we showed for the first time that temporal mitochondrial fluxes can be directly quantified in cell lines and clinical tumors. Results from the new method showed that less aggressive breast cancer samples have significantly lower mitochondrial citrate efflux and GM-to-citrate increases the most as aggressiveness increases. Clinical cancer samples showed heterogeneity in citrate profile and it can be an addition in predicting individual disease progression as standard subtype is often not sufficient for it.
Further studies are warranted to expand the use of this method to other mitochondrial targets (e.g. fluxes of folate or glutamine metabolism) and to conduct studies in tumor types other than breast cancer where usable aggressiveness markers are missing. In addition, suitability of this method for predicting tumor response to mitochondrial cancer drugs will be evaluated.
Citation Format: Andre Koit, Natalja Timohhina, Vladimir Chekulayev, Vahur Valvere, Tuuli Kaambre. Rate of temporal citrate efflux from malignant mitochondria predicts clinical aggressiveness in breast tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-268. doi:10.1158/1538-7445.AM2017-LB-268