Periodic Reporting for period 1 - NONCO-MICROTIS (Exploiting single cell long non-coding RNA biology to target therapy-induced senescence in lung cancer)
Période du rapport: 2022-05-01 au 2024-04-30
Senescence is a permanent state of growth arrest that can be induced by different types of stress, such as shortening of telomeres due to extensive replication, DNA damage, oxidative stress, chemotherapy treatment or oncogene overexpression. Senescent cells present characteristic morphological and biochemical features such as enlarged size, halted proliferation, activation of senescence-associated (SA) β-galactosidase activity and increased expression of cell cycle inhibitors. Furthermore, in several types of senescence the cells secrete a combination of interleukins, metalloproteases and growth factors, collectively known as the senescence-associated secretory phenotype (SASP). While the SASP reinforces senescence, its chronic activation can have pro-tumorigenic effects and contribute to several aging related pathologies . In fact, the persistence of therapy-induced senescent cancer cells in the tumor has been linked to resistance to chemotherapy. There is, therefore, a critical need to develop combinatorial approaches to not only stop proliferation, but also induce the elimination of the senescent tumor cells.
Despite their decreased proliferative potential, senescent cells are metabolically active in order to cope with the energetic demands of the senescence program. Indeed, metabolic reprogramming is considered a hallmark of senescence. In general, senescent cells accumulate dysfunctional mitochondria resulting in an increase of reactive oxygen species (ROS). However, different types of senescence can lead to different types of metabolic changes. For instance, OIS cells display a significant increase in tricarboxylic acid (TCA) cycle intermediates, oxidative phosphorylation (OXPHOS) 16 and mitochondrial fatty acid oxidation, while fibroblasts undergoing replicative senescence show increased glycolysis and reduced OXPHOS 18-20. Although different triggers of senescence result in broad metabolic differences, the interplay between senescent cells and metabolism is highly dynamic and context dependent, and the underlying mechanisms remain largely unexplored. In this context, long noncoding RNAs (lncRNAs) represent a relatively uncharted territory for investigation.
LncRNAs are transcripts recently defined as longer than 500 nucleotides with exquisite cell-type specific expression patterns that lack protein-coding potential. They are tightly regulated during development or in response to signaling pathways. Although it is still unclear how many of the thousands of annotated lncRNAs have a significant biological role, several have been found to be essential for the regulation of key cellular processes, such as proliferation and differentiation, as well as a broad range of diseases including cancer . Notably, our previous work and other laboratories’ have reported that specific lncRNAs are differentially expressed during senescence induction, regulating multiple senescence aspects, from transcriptional response to SASP production. Intriguingly, recent studies have involved a number of lncRNAs in the regulation of cellular metabolism, and ncRNAs are emerging as interactors and regulators of metabolic enzymes. However, the contribution of this functional diverse class of molecules to the metabolic reprogramming of senescent cells remains unknown.
Here we report the identification of the senescence-specific lncRNA, sin-lncRNA, which is induced in senescence to prevent uncontrolled metabolic alterations, by regulating the function of a key metabolic enzyme and the transcription of metabolic genes. Together, our results provide evidence of an RNA-dependent metabolic network specific of the senescent cellular state.
Despite their decreased proliferative potential, senescent cells are metabolically active in order to cope with the energetic demands of the senescence program. Indeed, metabolic reprogramming is considered a hallmark of senescence. In general, senescent cells accumulate dysfunctional mitochondria resulting in an increase of reactive oxygen species (ROS). However, different types of senescence can lead to different types of metabolic changes. For instance, OIS cells display a significant increase in tricarboxylic acid (TCA) cycle intermediates, oxidative phosphorylation (OXPHOS) 16 and mitochondrial fatty acid oxidation, while fibroblasts undergoing replicative senescence show increased glycolysis and reduced OXPHOS 18-20. Although different triggers of senescence result in broad metabolic differences, the interplay between senescent cells and metabolism is highly dynamic and context dependent, and the underlying mechanisms remain largely unexplored. In this context, long noncoding RNAs (lncRNAs) represent a relatively uncharted territory for investigation.
LncRNAs are transcripts recently defined as longer than 500 nucleotides with exquisite cell-type specific expression patterns that lack protein-coding potential. They are tightly regulated during development or in response to signaling pathways. Although it is still unclear how many of the thousands of annotated lncRNAs have a significant biological role, several have been found to be essential for the regulation of key cellular processes, such as proliferation and differentiation, as well as a broad range of diseases including cancer . Notably, our previous work and other laboratories’ have reported that specific lncRNAs are differentially expressed during senescence induction, regulating multiple senescence aspects, from transcriptional response to SASP production. Intriguingly, recent studies have involved a number of lncRNAs in the regulation of cellular metabolism, and ncRNAs are emerging as interactors and regulators of metabolic enzymes. However, the contribution of this functional diverse class of molecules to the metabolic reprogramming of senescent cells remains unknown.
Here we report the identification of the senescence-specific lncRNA, sin-lncRNA, which is induced in senescence to prevent uncontrolled metabolic alterations, by regulating the function of a key metabolic enzyme and the transcription of metabolic genes. Together, our results provide evidence of an RNA-dependent metabolic network specific of the senescent cellular state.
1- To identify novel regulators of this process, we analyzed RNA-seq data from a cellular system that recapitulates senescence induction in vitro thanks to the controlled activation of an oncogene-
Achievement: Oncogene-induced senescence leads to broad changes in gene expression, encompassing alterations in lncRNAs and we have we have characterized RIPOR3-as1 as one of the most significantly and strongly upregulated genes. It is the senescence driver C/EBPβ taht controls sin-lncRNA expression at later stages of senescence
2- To explore the function of sin-lncRNA, we depleted it in senescent IMR90 ER:RAS fibroblasts and analyzed several senescence markers and parameters.
Achievement: We have determine that sin-lncRNA loss contributes to the senescent phenotype through increase of oxidative stress
3- Reasoning that sin-lncRNA function could be mediated through interacting proteins, we set to unbiasedly identify its protein partners by RNA pulldown followed by MS analysis were
Achievement: sin-lncRNA interacts with Dihydrolipoamide S-succinyltransferase (DLST) and controls its subcellular localization
4- We compare the phenotypes of DLST depletion with the phenotypic alterations observed upon sin-lncRNA knockdown in senescent cells.
Achievement:We have demonstrate that sin-lncRNA rewires metabolic activity in senescent cells
5-To gain insight into the potential significance of sin-lncRNA function in a physio-pathological context, we interrogated gene expression data of tumor samples available through The Cancer Genome Atlas (TCGA).
Achievement: sin-lncRNA is lowly expressed across cancer types in line with the upregulation of sin-lncRNA upon OIS
6-To investigate the potential significance of sin-lncRNA in response to cancer therapy, we analyzed the expression of the lncRNA in cisplatin-treated ovarian cancer cells, given their ability to undergo senescence upon treatment with platinum-based agents.
Achievements: sin-lncRNA regulates OXPHOS in ovarian cancer cells.
Achievement: Oncogene-induced senescence leads to broad changes in gene expression, encompassing alterations in lncRNAs and we have we have characterized RIPOR3-as1 as one of the most significantly and strongly upregulated genes. It is the senescence driver C/EBPβ taht controls sin-lncRNA expression at later stages of senescence
2- To explore the function of sin-lncRNA, we depleted it in senescent IMR90 ER:RAS fibroblasts and analyzed several senescence markers and parameters.
Achievement: We have determine that sin-lncRNA loss contributes to the senescent phenotype through increase of oxidative stress
3- Reasoning that sin-lncRNA function could be mediated through interacting proteins, we set to unbiasedly identify its protein partners by RNA pulldown followed by MS analysis were
Achievement: sin-lncRNA interacts with Dihydrolipoamide S-succinyltransferase (DLST) and controls its subcellular localization
4- We compare the phenotypes of DLST depletion with the phenotypic alterations observed upon sin-lncRNA knockdown in senescent cells.
Achievement:We have demonstrate that sin-lncRNA rewires metabolic activity in senescent cells
5-To gain insight into the potential significance of sin-lncRNA function in a physio-pathological context, we interrogated gene expression data of tumor samples available through The Cancer Genome Atlas (TCGA).
Achievement: sin-lncRNA is lowly expressed across cancer types in line with the upregulation of sin-lncRNA upon OIS
6-To investigate the potential significance of sin-lncRNA in response to cancer therapy, we analyzed the expression of the lncRNA in cisplatin-treated ovarian cancer cells, given their ability to undergo senescence upon treatment with platinum-based agents.
Achievements: sin-lncRNA regulates OXPHOS in ovarian cancer cells.