Lonp1 isoforms overexpression differentially affects mitochondrial functions, mtDNA localization, and cytokine production in colorectal cancer cells

Lonp1 is a mitochondrial protease encoded by nuclear DNA and expressed in all tissues, with proteolytic, chaperone and mitochondrial DNA (mtDNA) binding activities. Lonp1 is involved in mitochondrial proteostasis, mtDNA copy number regulation and cellular adaptation to stress. Its expression increases in response to stress stimuli such as hypoxia, nutrient depletion, and oxidative stress. Moreover, Lonp1 expression is increased in several tumours, including colorectal cancer (CRC), where hypoxia represents an important feature of the tumour microenvironment (TME). Although three Lonp1 isoforms have been identified, their specific contribution to mitochondrial homeostasis and CRC cell adaptation remains poorly understood. In this study, we investigated the effects of Lonp1 isoform expression on mitochondrial morphology and function, mtDNA localization and release, inflammatory signalling, and metabolic responses in CRC cells under normoxic and hypoxic conditions. The three Lonp1 isoforms were stably overexpressed in four different CRC cell lines (HCT116, RKO, SW480 and SW620) to assess their effects across distinct CRC cellular models. Confocal microscopy confirmed distinct intracellular localization patterns, with ISO1 predominantly mitochondrial, ISO2 distributed between mitochondria and cytosol, and ISO3 showing no mitochondrial localization. Analysis of mitochondrial morphology revealed changes mainly in mitochondrial number and volume, while sphericity, branch length and branch diameter remained unaffected. Lonp1 overexpression was also associated with increased mitochondrial ROS production, particularly in ISO2- and ISO3-expressing cells, whereas an acute hypoxic stimulus reduced ROS levels. Importantly, ISO2 and ISO3 expression was associated with increased mtDNA levels in the cytosolic and extracellular compartments, indicating altered mtDNA distribution and release. To explore the possible consequences of Lonp1 modulation on the inflammatory response, the expression of a set of cytokines were quantified by RT-PCR. Specifically, Lonp1 overexpression was associated with increased TNF-α expression, while IL-1β and IL-18 were not detectable under the experimental conditions. Preliminary analysis of metabolic markers also revealed changes in GLUT1 and LDHA expression, particularly under hypoxia. Overall, these findings suggest that Lonp1 isoforms are not functionally equivalent and may differentially affect mitochondrial homeostasis, mtDNA trafficking, inflammatory signaling and metabolic adaptation in CRC cells. Further experiments, including cGAS-STING pathway and metabolomic analyses, will help clarify the mechanisms underlying these effects and their potential relevance in CRC progression.