Periodic Reporting for period 1 - MIEXIA (Mitochondria-Inflammation crosstalk and EXercise-induced pathways in cachexIA)
Reporting period: 2023-07-01 to 2025-08-31
The MSCA Postdoctoral Fellowship MIEXIA (“Mitochondria-inflammation crosstalk and exercise-induced pathways in cachexia”) set out to investigate the molecular mechanisms linking mitochondrial dysfunction, inflammation, and skeletal muscle (SkM) wasting in cancer cachexia (CC). The project focused on the implication of the mitochondrial RNA-binding protein LRPPRC, which is upregulated by exercise but decreased in CC, in promoting SkM health through maintenance of proper mitochondrial function and mitigating inflammation, and its potential as a therapeutic target to preserve SkM in CC.
CC affects up to 80% of patients with metastatic cancer, reducing quality of life, treatment tolerance, and survival. Despite its prevalence, there are no effective pharmacological treatments. Exercise has proven beneficial in improving SkM function and reducing inflammation, but it remains difficult to prescribe to cachectic patients due to extreme fatigue and disease burden. By elucidating the exercise-responsive pathways that protect mitochondrial function and dampen inflammation, MIEXIA aimed to identify molecular entry points for future therapeutic strategies.
CC affects up to 80% of patients with metastatic cancer, reducing quality of life, treatment tolerance, and survival. Despite its prevalence, there are no effective pharmacological treatments. Exercise has proven beneficial in improving SkM function and reducing inflammation, but it remains difficult to prescribe to cachectic patients due to extreme fatigue and disease burden. By elucidating the exercise-responsive pathways that protect mitochondrial function and dampen inflammation, MIEXIA aimed to identify molecular entry points for future therapeutic strategies.
To delineate the molecular link between mitochondrial dysfunction and inflammation in cancer cachexia (CC), analyses were performed across in vitro, in vivo, and human models. In vitro, differentiated skeletal muscle myotubes exposed to conditioned media from cachexia-inducing cancer cells (C26 and LLC) displayed clear signs of atrophy compared to control conditions (GM and MC38), as evidenced by reduced myotube diameter and altered expression of mitochondrial and inflammatory markers. These findings were recapitulated in vivo in the C26 mouse model of CC, where tumor-bearing mice showed progressive body weight loss, impaired muscle strength, and reduced muscle mass relative to PBS-treated controls. Mitochondrial gene and protein expression and functional analyses confirmed considerable mitochondrial dysfunction in all three models, accompanied by increased expression of pro-inflammatory cytokines in both skeletal muscle and plasma. In this regard, IL6 shows a consistent remarkable upregulation in all three models compared to the rest of cytokines assessed. Importantly, analyses of skeletal muscle biopsies from patients with non-small cell lung cancer (NSCLC) further supported these preclinical findings (Fig. 3). Patients exhibited molecular signatures consistent with mitochondrial impairment and enhanced inflammatory activation, mirroring the changes observed in the experimental models. Altogether, these data consistently demonstrate that mitochondrial dysregulation and IL6-mediated inflammation are closely intertwined in CC, and they validate the translational relevance of the MIEXIA experimental framework spanning cell culture, animal, and human contexts.
Gene expression analyses showed no significant changes in LRPPRC mRNA levels across the three CC models examined. However, a modest reduction in LRPPRC protein abundance was observed in skeletal muscle from both C26 tumor-bearing mice and patients with non-small cell lung cancer (NSCLC). These results only partially replicate the preliminary findings from the host laboratory described in the MIEXIA proposal. The discrepancy may stem from the heterogeneity in disease severity among the analyzed samples, as both the C26 mice and NSCLC patients displayed variable degrees of cachexia, likely increasing data variability.
Unexpectedly, imKO mice did not display overt muscle atrophy, mitochondrial dysfunction, or inflammation under basal conditions. Nevertheless, complementary work conducted in parallel by members of the host laboratory—including the researcher—has since demonstrated that these phenotypes emerge in aged imKO mice, confirming that LRPPRC plays a critical role in sustaining skeletal muscle integrity under metabolic stress.
Finally, subcellular interactome analyses revealed that LRPPRC associates with multiple proteins implicated in RNA processing and stability, both within mitochondria and in the cytosol. This provides the first direct experimental evidence that LRPPRC performs mRNA-stabilizing functions beyond mitochondria, extending to the cytosolic compartment. These findings support complementary ongoing research at the host institution, which suggests that LRPPRC may also participate in mitochondria–nucleus communication pathways.
Oxygen consumption analyses performed on skeletal muscle samples from patients with NSCLC demonstrated that exercise training enhanced mitochondrial respiratory capacity, particularly under maximal and uncoupled conditions. This indicates a beneficial effect of exercise on mitochondrial efficiency and overall oxidative metabolism in this patient population. Interestingly, when the data were stratified by sex, this improvement was observed exclusively in female participants, suggesting potential sex-specific adaptations to exercise in the context of cancer and mitochondrial function.
Gene expression analyses showed no significant changes in LRPPRC mRNA levels across the three CC models examined. However, a modest reduction in LRPPRC protein abundance was observed in skeletal muscle from both C26 tumor-bearing mice and patients with non-small cell lung cancer (NSCLC). These results only partially replicate the preliminary findings from the host laboratory described in the MIEXIA proposal. The discrepancy may stem from the heterogeneity in disease severity among the analyzed samples, as both the C26 mice and NSCLC patients displayed variable degrees of cachexia, likely increasing data variability.
Unexpectedly, imKO mice did not display overt muscle atrophy, mitochondrial dysfunction, or inflammation under basal conditions. Nevertheless, complementary work conducted in parallel by members of the host laboratory—including the researcher—has since demonstrated that these phenotypes emerge in aged imKO mice, confirming that LRPPRC plays a critical role in sustaining skeletal muscle integrity under metabolic stress.
Finally, subcellular interactome analyses revealed that LRPPRC associates with multiple proteins implicated in RNA processing and stability, both within mitochondria and in the cytosol. This provides the first direct experimental evidence that LRPPRC performs mRNA-stabilizing functions beyond mitochondria, extending to the cytosolic compartment. These findings support complementary ongoing research at the host institution, which suggests that LRPPRC may also participate in mitochondria–nucleus communication pathways.
Oxygen consumption analyses performed on skeletal muscle samples from patients with NSCLC demonstrated that exercise training enhanced mitochondrial respiratory capacity, particularly under maximal and uncoupled conditions. This indicates a beneficial effect of exercise on mitochondrial efficiency and overall oxidative metabolism in this patient population. Interestingly, when the data were stratified by sex, this improvement was observed exclusively in female participants, suggesting potential sex-specific adaptations to exercise in the context of cancer and mitochondrial function.
All experimental models were successfully established, and the majority of planned analyses have been completed, yielding robust preliminary findings that meet the main objectives of this work package. Some complementary molecular analyses are ongoing to strengthen mechanistic interpretations.
Despite multiple attempts using different strategies, it was not possible to generate LRPPRC-deficient muscle cells in vitro due to the exceptional stability and long half-life of both LRPPRC’s mRNA and protein. Consequently, the detailed phenotypic characterization performed in the skeletal muscle-specific knockout (imKO) mice could not be replicated in cultured muscle cells.
In parallel, the experimental plan designed to elucidate the downstream molecular pathways regulated by LRPPRC was adapted to overcome technical constraints. While the original proposal envisioned performing protein and RNA interactomics in LRPPRC-FLAG-tagged mouse muscles (immunoprecipitation-mass spectrometry [IP-MS] and RNA immunoprecipitation-sequencing [RIP-Seq], respectively), a revised approach was implemented. Specifically, a GFP-TOM20 and FLAG-LRPPRC double-tagged HeLa cell line was generated to establish a cost-effective and high-yield IP-based method for isolating intact mitochondria and pure cytosol, surpassing the efficiency of traditional subcellular fractionation techniques.
The combination of this optimized IP-based method with MS enabled the identification of a robust set of LRPPRC interactors, distributed across mitochondrial and cytosolic compartments. The project concluded at the stage of interactomics data analysis, and therefore, experimental validation of these candidate interactors remains ongoing. Members of the host lab are currently pursuing this line of research, highlighting the scientific value and continuity of the results generated during MIEXIA.
Following significant project amendments approved approximately 6 months after the start of MIEXIA, the scope of work included in this work package was adjusted accordingly. Due to these changes and the resulting time constraints, follow-up analyses on the collected muscle biopsies, such as protein and gene expression studies, could not be completed within the fellowship period. Nonetheless, the primary objective—assessing the impact of exercise training on mitochondrial respiratory function in patients with NSCLC—was successfully achieved, generating valuable and novel data. The remaining molecular analyses are currently being pursued by members of the host lab to complement and extend these findings.
Despite multiple attempts using different strategies, it was not possible to generate LRPPRC-deficient muscle cells in vitro due to the exceptional stability and long half-life of both LRPPRC’s mRNA and protein. Consequently, the detailed phenotypic characterization performed in the skeletal muscle-specific knockout (imKO) mice could not be replicated in cultured muscle cells.
In parallel, the experimental plan designed to elucidate the downstream molecular pathways regulated by LRPPRC was adapted to overcome technical constraints. While the original proposal envisioned performing protein and RNA interactomics in LRPPRC-FLAG-tagged mouse muscles (immunoprecipitation-mass spectrometry [IP-MS] and RNA immunoprecipitation-sequencing [RIP-Seq], respectively), a revised approach was implemented. Specifically, a GFP-TOM20 and FLAG-LRPPRC double-tagged HeLa cell line was generated to establish a cost-effective and high-yield IP-based method for isolating intact mitochondria and pure cytosol, surpassing the efficiency of traditional subcellular fractionation techniques.
The combination of this optimized IP-based method with MS enabled the identification of a robust set of LRPPRC interactors, distributed across mitochondrial and cytosolic compartments. The project concluded at the stage of interactomics data analysis, and therefore, experimental validation of these candidate interactors remains ongoing. Members of the host lab are currently pursuing this line of research, highlighting the scientific value and continuity of the results generated during MIEXIA.
Following significant project amendments approved approximately 6 months after the start of MIEXIA, the scope of work included in this work package was adjusted accordingly. Due to these changes and the resulting time constraints, follow-up analyses on the collected muscle biopsies, such as protein and gene expression studies, could not be completed within the fellowship period. Nonetheless, the primary objective—assessing the impact of exercise training on mitochondrial respiratory function in patients with NSCLC—was successfully achieved, generating valuable and novel data. The remaining molecular analyses are currently being pursued by members of the host lab to complement and extend these findings.