Modulation of Lonp1 in Colorectal Cancer Cells: effects on mtDNA Localization, Mitochondrial Function, and Cytokine Production

Lonp1 is a mitochondrial protease encoded by nuclear DNA and expressed in all tissues, although expression levels vary depending on cell type. Lonp1 is a multifunctional enzyme, with proteolytic, chaperone and mitochondrial DNA (mtDNA) binding activities. Lonp1 is involved in maintaining mitochondrial homeostasis, stress response, metabolic and mtDNA copy number regulation. Its expression increases in response to stress stimuli such as hypoxia, nutrient depletion, and oxidative stress. Furthermore, it is frequently overexpressed in various tumours, including melanoma, glioma, breast cancer, prostate cancer and colorectal cancer (CRC). Its increased expression is often associated with lower patient survival, although it is not a prognostic marker. To clarify the role of Lonp1 in CRC and, in particular, the impact of its modulation on the quantity and localization of mtDNA, we evaluated the effects of Lonp1 overexpression and silencing, using siRNA, in three CRC cell lines. mtDNA quantification was performed using droplet digital PCR on different cell fractions. To analyse the morphology and organization of the mitochondrial network, cells were observed by confocal microscopy. The functional status of mitochondria was investigated through complementary assays: the production of reactive oxygen species (ROS) was monitored as an indicator of oxidative stress, the mitochondrial membrane potential was measured to monitor the degree of internal membrane polarization, and cellular energy metabolism was evaluated through oxygen consumption measurement, useful for determining the bioenergetic profiles of cells. At the same time, to explore the possible consequences of Lonp1 modulation on the inflammatory response, the expression of a set of cytokines were quantified by RT-PCR. We observed an increase in mtDNA release into the extracellular environment in the presence of Lonp1 overexpression, and a reduction when Lonp1 is silenced. Furthermore, we observed a different leakage of mtDNA from mitochondria into the cytosol based on Lonp1 expression levels. Different levels of Lonp1 also modify mitochondrial structure and functions, leading to an increase in ROS when overexpressed and a consequent reduction when silenced. The mitochondrial membrane potential shows changes in the different condition analyzed; respiration, on the other hand, decreases in the absence of Lonp1 and increases with elevated protein levels. Finally, we also observed a variation in the production of proinflammatory cytokines based on Lonp1 expression levels. These results confirm that Lonp1 plays a key role in mtDNA maintenance and highlight its key role in mtDNA cellular localization and release into the extracellular environment, and mitochondrial functions, with a significant impact on respiration, ROS production, and even mitochondrial structure. The different localization of mtDNA and its release is also reflected in a different production of proinflammatory cytokines. These changes, together with metabolic changes in CRC cells with different levels of Lonp1, may affect the different levels of lymphocytic infiltrate observed in human CRC tissues with different levels of Lonp1 expression.