Periodic Reporting for period 1 - METAREPAIR (INVESTIGATING THE IMPACT OF NUTRITIONAL METABOLISM ON DNA DAMAGE REPAIR IN CANCER)
Período documentado: 2021-07-01 hasta 2023-06-30
In prostate cancer (PCa), loss of genomic fidelity is one of the drivers of tumorigenesis, but little is known on the mechanisms contributing to genomic instability during cancer progression. A growing amount of epidemiological and preclinical data is showing that diet plays a key role in cancer aetiology. Preliminary data indicates that those diets, such as western-style diet, that favour tumorigenesis exert their tumorigenic potential by generating inside the cells a metabolic environment that promotes genomic instability and, therefore, favours the structural genomic rearrangements necessary to promote PCa progression. I hypothesize that this can occur in part through a breach in the surveillance systems that normally guard genomic integrity, and in part through a loss of efficiency in the DNA damage repair machinery, resulting in incomplete or defective repair of DNA lesions. With METAREPAIR, I set out to investigate one specific set of cellular metabolites, part of the one carbon metabolism pathway, which contribute to the regulation of the epigenetic and epitranscriptomic landscape of the cells and can therefore indirectly affect the DNA repair capacity of cancer cells. The results of METAREPAIR can contribute to better understand the complex link between cancer and diet. This, in turn, can be used to better design cancer-protective diets, or diets that could be used as adjuvant-therapy during radio or chemotherapies. The overarching scientific goal of METAREPAIR was to deconvolute the link between nutritional metabolic alterations and genomic instability in PCa cells and leverage this link as a precision nutrition approach to sensitize PCa tumours to DNA-damaging therapies. The scientific aims were articulated into 3 independent objectives: 1) Define the effect of one carbon metabolites modulation on DNA damage repair (DDR) through the regulation of histone methylation; 2) Define the effect of one carbon metabolites modulation on DDR through the regulation of RNA methylation; 3) Exploit the impact of 1Cmet modulation on DDR to sensitize PCa tumours to DNA-damaging therapies. The results of the DDR time course showed that methionine starvation resulted in decreased 53BP1 foci formation, but this was not coupled with any change in DNA damage load. In line with these observations, the GFP reporter-assays indicated that DNA repair pathway choice was unaffected by 1Cmet manipulation. The same negative results were observed when PCa cells were treated with the PARP inhibitor Olaparib, which was used as an alternative mean to induce DNA damage. Taken together, these results suggest that 1C met manipulation does not sensitize PCa cells to IR-induced DNA damage.
In the first part of METAREPAIR, I set out to define the effect of one carbon metabolites modulation on DDR through the regulation of histone methylation. To modulate intracellular one carbon metabolism, known to regulate methyltransferases activity, PCa cells were cultured for 24 hr in a modified cell culture media formulation in which methionine was depleted or enriched. Cells were then treated with ionizing radiation (IR) and processed and analyzed to understand how methionine manipulation affected 1) cellular metabolite levels; 2) histone marks known to be involved in DDR, and 3), DDR factors themselves. The work performed to achieve Objective 1 let to the observation that methionine manipulation impacted intracellular levels of metabolites that are involved in methylation processes, and the methylation status of specific histone marks. However, no effect was observed on DNA damage load, and only one DDR factor was affected by methionine manipulation. In the second part of METAREPAIR, we investigated the effect of one carbon metabolites modulation on DDR through the regulation of RNA methylation. We observed a transient decrease in some epitranscriptomic marks in PCa cells deprived of methionine, and this observation correlated with altered expression of one DDR factor after DNA damage. When we genetically or pharmacologically inhibited the levels of this epitranscriptomic mark, the levels of some DDR factors were affected, demonstrating the specificity of the effect. However, no impact on the overall DNA damage load of the cells was observed when PCa cells were subjected to methionine starvation, possibly because of the methionine dependency of cells in general.
Altogether, the results of METAREPAIR provided further evidence that metabolite modulation impacts the epigenetic and epitranscriptomic landscape of cells and that, under some conditions, can influence DDR. However, the link between metabolism and DDR is very complex and is regulated by other mechanisms that were not the object of our investigation. Further studies should be necessary to fully deconvolute these mechanisms and be able to exploit them for therapeutic purposes.
During the course of the action, we regularly presented updates on the progress of the project during meetings held at IFOM. However, many of the results obtained in this project were negative, as we did not observe any significant impact of one carbon metabolism modulation on DDR. For this reason, it was not possible to present our data at conferences or international meetings. Nevertheless, we strongly believe that also negative results are valuable to the scientific community, because they can help other researcher directing their effort to other unexplored directions: therefore, we will try and make our work available to the community.
Altogether, the results of METAREPAIR provided further evidence that metabolite modulation impacts the epigenetic and epitranscriptomic landscape of cells and that, under some conditions, can influence DDR. However, the link between metabolism and DDR is very complex and is regulated by other mechanisms that were not the object of our investigation. Further studies should be necessary to fully deconvolute these mechanisms and be able to exploit them for therapeutic purposes.
During the course of the action, we regularly presented updates on the progress of the project during meetings held at IFOM. However, many of the results obtained in this project were negative, as we did not observe any significant impact of one carbon metabolism modulation on DDR. For this reason, it was not possible to present our data at conferences or international meetings. Nevertheless, we strongly believe that also negative results are valuable to the scientific community, because they can help other researcher directing their effort to other unexplored directions: therefore, we will try and make our work available to the community.
From a scientific standpoint, this project on a whole helped to shed light on the molecular mechanism regulating the interaction between metabolism, epigenetics and DNA damage repair, therefore contributing to the understanding of the link between lifestyle and cancer. Considering the pandemic of obesity and diet-associated metabolic diseases, combined with the high frequency of newly diagnosed cancers in developed countries, a better understanding of the mechanistic underpinnings of this link is of significant importance.
From a methodological point of view, we optimised an immunofluorescent protocol to detect and quantify m6A in fixed cells. Given the ever-growing interest in the study of this RNA modification, we believe this method could be of use to the scientific community.
From a methodological point of view, we optimised an immunofluorescent protocol to detect and quantify m6A in fixed cells. Given the ever-growing interest in the study of this RNA modification, we believe this method could be of use to the scientific community.