Morphological and molecular characterization of breast cancer spheroids after treatment with Mitochondrial Sirtuins modulators

Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer, characterized by the absence of estrogen, progesterone, and HER2 receptors, making it unresponsive to conventional hormonal therapies. As one of the most difficult forms of breast cancer, there is an urgent need for innovative therapeutic approaches. Recent studies have highlighted the significant role of sirtuins, particularly mitochondrial sirtuins, in the pathogenesis of TNBC. These proteins modulate key cellular processes, including metabolism, oxidative stress, autophagy, and cancer cell survival, through the post-translational modification of key proteins. In this study, we examined the ultrastructural and molecular changes following the modulation of SIRT3 and SIRT5 in MDA-MB-231 cells, a well-known TNBC cell line, using 2D cell cultures and 3D spheroid models. The selected modulators, a SIRT5 activator (MC3138) and a SIRT3 inhibitor (3 TyP), were chosen based on their inhibitory effect on glutamine metabolism, a key process for cancer cell survival. Initial results showed a significant reduction in cell proliferation in both 2D cultures and 3D spheroids treated with the modulators, with more pronounced effects when the two modulators were combined. This reduction in proliferation was confirmed through clonogenic assays and spheroid size measurements, without compromising overall cell viability. Additionally, ultrastructural analyses revealed significant alterations, such as cytoplasmic disorganization, mitochondrial damage, and the formation of lipid droplets, particularly evident in treated cells. Molecular analysis also revealed a reduction in the levels of HIF-1α, a factor that regulates cancer cell adaptation to low oxygen conditions, BNIP3, a key mediator of mitophagy, and YAP and TAZ, factors related to stemness and cancer cell proliferation. One of the most surprising results of the study was the accumulation of alpha-lactalbumin, a protein typically found in human breast milk, in both treated cells and spheroids. The presence of this protein in lipid droplets suggests that sirtuin modulation induces cancer cell differentiation, potentially causing the cells to revert to a more differentiated state, typical of milk-producing cells. The results of this study suggest that mitochondrial sirtuin modulation acts on key metabolic and proliferative pathways of the cancer cells, inducing morphological changes consistent with cancer cell differentiation.