A Combined SIRT5 Activation and SIRT3 Inhibition Prevents Breast Cancer Spheroids Growth by Reducing HIF-1α and Mitophagy

<b>Background/Objectives:</b> Metabolic reprogramming is an essential feature of tumors. Mitochondrial sirtuins SIRT3 and SIRT5 differently regulate glutamine metabolism with SIRT5 inhibiting glutaminase (GLS) and SIRT3 increasing glutamate dehydrogenase (GDH). Considering the important and interconnected role of glutamine, SIRT3 and SIRT5 for cancer growth and progression, our hypothesis is that a simultaneous modulation of SIRT3 and SIRT5 could represent a valid anti-tumoral strategy. <b>Methods:</b> wt and GLS1-silenced triple negative breast cancer spheroids were treated with 3-TYP, a selective SIRT3 inhibitor, and with MC3138, a new selective SIRT5 activator, both alone and in combination. The effects of such treatments on hypoxia, autophagy and mitophagy markers were determined by immunofluorescence and Western blot. Mitochondria morphology was studied by transmission electron microscopy (TEM) and mitochondrial ROS production by confocal analysis. <b>Results:</b> We observed that 3-TYP+MC3138 treatment decreased the size of spheroids by affecting HIF-1α, c-Myc, glutamine transporter SLC1A5 and autophagy (LC3II) and mitophagy (BNIP3) markers. Moreover, such treatments altered the morphology and conformation of the mitochondria. Finally, we also documented an increase in mitochondria reactive oxygen species (mtROS). <b>Conclusions:</b> The combined inhibition of SIRT3 and activation of SIRT5 greatly reduces the size of spheroids through the inhibition of hypoxic response, which is then followed by the alteration of the autophagic and mitophagic process and the toxic accumulation of mitochondrial ROS, representing a new anti-tumoral strategy.