Contribution of Lonp1 to the suppression of the immune response in the tumor microenvironment

Background Cancer progression relies on cell proliferation and interaction with tumour microenvironment. Lonp1 is a mitochondrial protease, crucial for adaptation to hypoxia and proteotoxic stress, often present in cancer. Lonp1 degrades damaged/oxidized proteins, assists protein folding, binds mtDNA, regulates mitophagy and several metabolic pathways. Lonp1 silencing causes mitochondrial impairment and cell death, while higher expression is correlated to enhanced tumor aggressiveness. Lonp1 promotes metastasis by increasing ROS production or via GSK-3β/β-ctn, but this only partially explains how Lonp1 leads to higher proliferation/metastasis. Lonp1 exists in three isoforms with differential subcellular distribution. Their differential upregulation in CRC could affect cell metabolism, mtDNA misplacement, the production of immunomodulating metabolites, and of pro- and anti-inflammatory cytokines. Hypothesis The hypoxic microenvironment in cancer causes an upregulation of Lonp1 as a whole, and a differential upregulation of its isoforms, which leads to: i) mtDNA leakage to the cytosol, where activates a proinflammatory response; ii) an imbalance of cell metabolism, and the release of secondary metabolites with immunomodulatory properties. These mechanisms inhibit the immune response in Tumour-infiltrating macrophages and lymphocytes, favouring cancer survival. Aims 1. To determine whether Lonp1 overexpression causes mtDNA release from mitochondria and the activation of pro-inflammatory pathways in CRC; 2. To determine which Lonp1 isoform(s) is responsible for that; 3. To evaluate how Lonp1 alters the metabolome of CRC cells in vitro and in vivo; 4. To determine which metabolites modulated by Lonp1 suppress the immune response; 5. To characterize immune cell infiltration in CRC with differential Lonp1 expression; 6. To determine the impact of these difference on the clinical outcome of CRC patients. Experimental Design The project is organized in five work packages. In WP1, we will analyze the effects of silencing or overexpression of Lonp1 isoforms 1, 2, or 3 in the mtDNA leakage to the cytosol, and the activation of inflammatory pathways that lead to the release of proinflammatory cytokines, using different CRC cells with functional or non-functional cGAS/STING pathway. In WP2, we will identify metabolites differentially produced by cells and mice where Lonp1 is silenced or overexpressed, using an untargeted metabolomic approach. In WP3 we will use conditioned media from cells treated as in WP1/WP2 to evaluate its capability to modulate macrophages and tissue-resident T cells ex vivo. In WP4, we will analyze the single-cell transcriptional profile of immune and cancer cells from CRC samples with high or low Lonp1 levels. In WP5 we will evaluate the impact of immune modulation due to Lonp1 differential expression on clinical outcome (mortality, therapy efficacy) of CRC patients. Expected Results At the end of this project, we will know: i) the role of Lonp1 isoforms in regulating mtDNA leakage and activation of inflammatory pathways in cancer; ii) the metabolites with immunomodulating properties whose production is influenced by Lonp1; iii) the consequences of Lonp1 overexpression on the immune response in CRC and on the clinical outcome. Impact On Cancer Lonp1 plays a crucial role in cancer proliferation, at multiple levels. The comprehension of its role in regulating the immune response in the tumor microenvironment will help a more rationale use of immunotherapy