Mitochondria as metabolic markers of melanoma stem cells and targets of delta-tocotrienol

Cancer stem cells (CSCs) are a small subpopulation within the tumor mass, characterized by the ability of self-renewal, differentiate into heterogeneous populations and give rise to tumor when transplanted in vivo. Their aggressive behavior is responsible for metastatic dissemination, chemotherapy resistance and tumor relapse. The identification of specifc markers in order to characterize this cell subpopulation could provide new targets for cancer treatment. Different cell surface markers, such as CD133 and CD271, have been proposed as CSCs markers in melanoma, but is now accepted that they do not represent a reliable method to detect these cells. CSCs have also been reported to be coupled with metabolic alterations. We recently demonstrated that melanoma CSCs deriving from the A375 cell line are characterized by autofl uorescence and by the expression of the ABCG2 transporter; based on this observation we sought to investigate the metabolic profi le of this subpopulation. We propose that higher mitochondrial mass, in addition to ABCG2 expression, could be considered a novel metabolic marker for this cell subpopulation in human melanoma cells. First, by flow cytometry we observed that ABCG2 positive cells are characterized by higher mitochondrial mass compared with ABCG2 negative cells. Western blot analysis confi rmed that A375 derived melanospheres have increased OXPHOS and PGC1-alpha expression levels. Moreover, an increased expression of mitochondria fusion markers OPA1 and MFN2 and a decreased expression of the fission marker DRP1 have been observed in A375 derived melanospheres. Despite this, ABCG2 positive cells showed a lower mitochondrial activity and ROS production. We hypothesize that it could be due to the quiescent state of CSCs within the tumor bulk. Furthermore, we previously reported that delta-tocotrienol exerts anti-cancer effects in A375 melanoma cell line targeting ABGC2 positive cells. We then analyzed whether it might affect stem cell mitochondria. By western blot we observed that delta-tocotrienol signifi cantly decreased the expression levels of ETC complex I, complex IV, PGC1-alpha and proteins involved in mitochondrial dynamics (OPA1, MFN2, DRP1), and it activated the energy stress sensor p-AMPK. Taken together, all these data demonstrate that 1) mitochondrial mass could be considered, in addition to ABCG2 expression, a reliable marker mass could be considered, in addition to ABCG2 expression, a reliable marker mitochondria.