TY - GEN A1 - Heß, Markus Simon UR - https://archiv.ub.uni-heidelberg.de/volltextserver/35035/ N2 - Cellular senescence describes the state of a stable cell proliferation arrest. The cell cycle arrest is triggered by various endogenous and exogenous stressors including telomere shortening, DNA damage, oxidative stress, cytotoxic drugs, and oncogene activation among others. Replicative senescence (RS) occurs in ageing cells to impede unlimited proliferation. Further, in normal somatic cells, the activation of oncogenes can induce transformation to cancerous cells. However, by entering the premature senescent state cells avoid to undergo transformation. Hence, cellular senescence functions as a barrier to transformation and is a failsafe mechanism to protect the organism from potentially damaged and dangerous cells by enforcing their cell cycle arrest. The type of senescence initiated through oncogenes such as RASG12V and BRAFV600E is defined as oncogene-induced senescence (OIS). Following, senescent cells evoke the senescence-associated secretory phenotype (SASP) composed of signalling molecules including cytokines and lipid mediators. In contrast to the anti-inflammatory molecules, the pro-inflammatory side of the SASP can promote cell transformation and tumour growth. In order to further understand metabolic alterations associated with senescence induction and to find vulnerabilities of OIS cells, I aimed to investigate the transcriptome, metabolome, and lipidome of BRAFV600E-expressing human diploid fibroblasts in a time-resolved manner. First, I could describe increased tricarboxylic acid (TCA) cycle activity as well as pyruvate anaplerosis in oncogenic BRAFV600E-induced senescent cells. Furthermore, these data revealed that the serine synthesis pathway (SSP) was impaired upon senescence. Especially, the rate limiting enzyme of the SSP phosphoglycerate dehydrogenase (PHGDH) was strongly downregulated. Overexpression of PHGDH in BRAFV600E-expressing fibroblasts resulted in reduced senescence capacity, while PHGDH expression itself was not sufficient to overcome OIS. Next, the exposure of a panel of melanoma cell lines to serine and glycine deprived medium and its combination with a PHGDH inhibitor treatment revealed different dependencies on exogenous serine or de novo serine synthesis in melanoma. Secondly, comprehensive metabolic and transcriptomic profiling exposed the lipid metabolism as widely altered metabolic process upon OIS. Therefore, I conducted a full lipidome profile of OIS cells in a time-resolved manner. Intriguingly, the neutral lipid class of triacylglycerides (TAGs) was highly accumulated in senescent cells over time. TAGs are fatty acid (FA)-containing lipids stored in lipid droplets (LDs) and can be utilised as main energy source. TAG synthesis is mainly conducted by diacylglycerol O-acyltransferase 1 and 2 (DGAT1/2), latter one was upregulated upon OIS. LDs have been discussed to protect against oxidative stress and lipid peroxidation by sequestering polyunsaturated FAs (PUFAs) in form of TAGs. The accumulation of lipid peroxides in PUFA-phospholipids in cellular membranes can drive ferroptosis, an iron-dependent form of non-apoptotic cell death. Reduced lipid peroxidation and increased levels of specific long chain-PUFA-containing TAGs explained the observed ferroptosis resistance in OIS. DGAT inhibition increased ferroptosis susceptibility and lipid peroxidation in senescent cells. Additionally, the abundance of TAGs consisting of highly unsaturated long acyl-chains was reduced upon DGAT treatment, whereas the abundance of PUFAs in the membrane phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE) was elevated. Overall, I discovered that OIS cells have an elevated TAG synthesis which protects senescent cells from ferroptosis. Also, inhibition of DGAT prevents TAG formation and restores ferroptosis sensitivity by channelling PUFAs into membrane lipids. Concluding, I could demonstrate that remodelling of the cellular lipidome contributes to ferroptosis resistance of senescent cells. Additionally, by generating a total FA profile I identified the ?-6 PUFA arachidonic acid (AA) as strongly diminished in OIS cells. AA serves as a substrate for eicosanoid biosynthesis. Eicosanoids are diverse signalling molecules which are important modulators of inflammation and immune response. AA for eicosanoid synthesis can be generated by cleavage from membrane phospholipids. Further analysis revealed augmented activity of the AA metabolism including elevated expression of the phospholipase A2 group IVA (PLA2G4A) and cyclooxygenase 2 (COX2; PTGS2) in senescent cells in order to drive increased secretion of oxylipins. It has been shown that inhibition of DGAT can redirect PUFAs from TAGs into phospholipids. Thereby, inhibition of DGAT further induced oxylipin production in OIS cells. In addition, DGAT inhibition influenced the senescence phenotype and reduced cell cycle arrest markers but did not drive proliferation of senescent cells. Overall, my findings demonstrate the importance of understanding metabolic mechanisms, especially lipid metabolism, in a highly metabolically active system as senescence. They also suggest new combinatorial treatment strategies to antagonise the harmful state of senescence and to open up new concepts to combat cancerous cells. CY - Heidelberg AV - restricted Y1 - 2025/// TI - Investigation of Metabolic Vulnerabilities in Oncogene-Induced Senescence KW - Seneszenz KW - Senescence KW - Oncogene-induced senescence KW - OIS KW - BRAF KW - BRAFV600E KW - Zellzyklusarrest KW - cell cycle arrest KW - Ferroptose KW - Ferroptosis KW - TAG KW - Triacylglyceride KW - Oxylipin KW - Eicosanoid KW - Arachidonsäure KW - Arachidonic acid KW - PHGDH KW - phosphoglycerate dehydrogenase KW - DGAT KW - diacylglycerol O-acyltransferase KW - PUFA KW - polyunsaturated fatty acid KW - tricarboxylix acid cycle KW - TCA cycle KW - Melanoma KW - Phospholipase KW - transsulfuration pathway ID - heidok35035 ER -