Periodic Reporting for period 2 - tRNAtoGO (“tRNA actors” heterogeneity: a new identifier of cancer stem cells)
Período documentado: 2022-08-01 hasta 2024-01-31
Cells need to constantly change their proteome in order to respond to external and internal stimuli; this continuous adaptation is regulated by changes in transcription and translation of mRNAs. Transfer RNAs (tRNA) are the most abundant non-coding RNA molecules in cells: their role is to decode mRNAs through the correct pairing of mRNA codons with tRNA anticodon within the ribosome. The degenerative nature of the genomic code (61 mRNA codons code for 20 amino acids) and the fact that different genes encode sequence variants of tRNAs recognizing the same mRNA codon, make tRNAs repertoires extremely heterogeneous among tissues and cellular populations. The tissue specificity of tRNA pools is furthermore increased by the presence of a variety of tRNA modification patterns and by the recently discovered tissue specific expression of tRNA derived fragments. For very long time, the scientific community had underestimated the importance of this tissue-specific expression of tRNA pools considering it void of functional relevance. Our recent work contribute to demonstrate that the presence of a specific set of modified tRNAs, responsible for the decoding fidelity of codons enriched in cancers mRNAs, are necessary to correctly establish onco-proteomes and highlighted the key role of tRNA diversity in sustaining proteome rewiring in cancer.
Cancer stem cells (CSC) are a small portion of cancer cells entitled to initiate and sustain tumours by possessing self-renewal proprieties. Tumour initiation and acquired resistance to therapies are classical cancer stem-cell features; therefore, the capability to discriminate those cells and eradicate them is of great interest for cancer therapy. Consequently, it is not a surprise that a great effort has been put in place from the scientific community to identify CSC in different tumours and to define their molecular characteristics. Nevertheless, cancer stem cell biology remains still unclear at the molecular level. Interestingly, some recent data suggest an association between regulation of mRNA translation and healthy or cancer stem cell status. In particular, the alternative presence of different pools of tRNAs was associated with the proliferative or differentiated status of cells. Recently, we had demonstrated that the loss of mcm5s2 wobble tRNA modification leads to functional impairment of the hematopoietic healthy stem cells and decrease in tumour initiation capability of CSC in the intestine. Moreover, loss of other TM-enzymes (i.e.: PUS7, TRMT6/TRMT61A) have recently been associated with loss of CSC fitness in glioblastoma and hepatocellular carcinoma. Finally, an increasing amount of evidences suggest that tRFs can be involved in healthy and cancer stem cells maintenance. All together these studies provide a first line of evidences associating the CSC establishment with a specific regulation of tRNA molecules- i.e.: modifications status, tRNA expression levels and tRFs formation.
Colon cancer is one of the deadliest forms of cancer worldwide. The genetic events leading to colon cancer insurgence involve in more than 80% of cases the hyper activation of the Wnt-signaling pathway –i.e. loss of the adenomatous polyposis coli (APC) gene, activating mutations of -catenin, etc.- followed by other oncogenic mutations such as KRAS activating mutation (40-50% of cases). Mouse models that mimic the most common genetic alteration of colon cancer have been produced and resemble to some extent to the human disease. Moreover, the in detail characterization of the intestinal architecture as well as the possibility of growing healthy and cancerous ex-vivo organoids, made these models incredibly suitable to study cell population behavior in health and disease.
Here, we want to identify key tRNA actors that are necessary to sustain Wnt-dependent oncogenic transformation of intestinal stem cells in order to identify new therapeutic targets that could improve patients’ prognosis.
The hypothesis of the proposed project is that the expression of a signature of tRNA actors in healthy cells is permissive towards the establishment of oncogene-related proteomes and can be therefore predictive of the healthy cell population of origin for CSC. To prove this hypothesis, we will (a) to identify the changes in tRNA species, tRNA modifications (TM), tRNA modifying enzymes (TM-enzymes) and tRNA derived fragments (tRF) in intestinal healthy and cancer stem cell population; (b) to assess if a specific signature of tRNA species and/or TM-enzymes is permissive to intestinal CSC establishment; and (c) to dissect the functional role of the identified signature in CSC biology
Cancer stem cells (CSC) are a small portion of cancer cells entitled to initiate and sustain tumours by possessing self-renewal proprieties. Tumour initiation and acquired resistance to therapies are classical cancer stem-cell features; therefore, the capability to discriminate those cells and eradicate them is of great interest for cancer therapy. Consequently, it is not a surprise that a great effort has been put in place from the scientific community to identify CSC in different tumours and to define their molecular characteristics. Nevertheless, cancer stem cell biology remains still unclear at the molecular level. Interestingly, some recent data suggest an association between regulation of mRNA translation and healthy or cancer stem cell status. In particular, the alternative presence of different pools of tRNAs was associated with the proliferative or differentiated status of cells. Recently, we had demonstrated that the loss of mcm5s2 wobble tRNA modification leads to functional impairment of the hematopoietic healthy stem cells and decrease in tumour initiation capability of CSC in the intestine. Moreover, loss of other TM-enzymes (i.e.: PUS7, TRMT6/TRMT61A) have recently been associated with loss of CSC fitness in glioblastoma and hepatocellular carcinoma. Finally, an increasing amount of evidences suggest that tRFs can be involved in healthy and cancer stem cells maintenance. All together these studies provide a first line of evidences associating the CSC establishment with a specific regulation of tRNA molecules- i.e.: modifications status, tRNA expression levels and tRFs formation.
Colon cancer is one of the deadliest forms of cancer worldwide. The genetic events leading to colon cancer insurgence involve in more than 80% of cases the hyper activation of the Wnt-signaling pathway –i.e. loss of the adenomatous polyposis coli (APC) gene, activating mutations of -catenin, etc.- followed by other oncogenic mutations such as KRAS activating mutation (40-50% of cases). Mouse models that mimic the most common genetic alteration of colon cancer have been produced and resemble to some extent to the human disease. Moreover, the in detail characterization of the intestinal architecture as well as the possibility of growing healthy and cancerous ex-vivo organoids, made these models incredibly suitable to study cell population behavior in health and disease.
Here, we want to identify key tRNA actors that are necessary to sustain Wnt-dependent oncogenic transformation of intestinal stem cells in order to identify new therapeutic targets that could improve patients’ prognosis.
The hypothesis of the proposed project is that the expression of a signature of tRNA actors in healthy cells is permissive towards the establishment of oncogene-related proteomes and can be therefore predictive of the healthy cell population of origin for CSC. To prove this hypothesis, we will (a) to identify the changes in tRNA species, tRNA modifications (TM), tRNA modifying enzymes (TM-enzymes) and tRNA derived fragments (tRF) in intestinal healthy and cancer stem cell population; (b) to assess if a specific signature of tRNA species and/or TM-enzymes is permissive to intestinal CSC establishment; and (c) to dissect the functional role of the identified signature in CSC biology
1/ We have defined the changes occurring upon oncogenic transformation of intestinal stem cells in the repertoire of tRNA molecules, tRNA-modifications, tRNA-enzymes and tRNA fragments.
We had characterized the changes in tRNA families, tRNA-modifications, tRNA-enzymes and tRNA fragments (from now on referred as “tRNA actors”) in healthy intestinal stem cells (Lgr5high population) and in intestinal CSC (Lgr5high/Apclox/lox). Then we had correlated these changes with modulation of mRNA translation and protein expression in intestinal stem cells and CSC. Finally, we had validated the specificity of the CSC overexpressed tRNA actors by measuring their levels in Lgr5neg cell population.
2/ We have identified an intestinal CSC tRNA permissive oncogenic signature.
We performed a negative genetic screen (dropout screen) using Lgr5high/Apclox/lox derived murine organoids, treated or not with tamoxifen, to identify the tRNA actors whose loss-of-function affects Wnt-driven tumorigenesis. Then, I generated an engineered mouse models (conditional knockout- cKO) of the identified tRNA actor(s) to assess their role in tumor formation in vivo.
We had characterized the changes in tRNA families, tRNA-modifications, tRNA-enzymes and tRNA fragments (from now on referred as “tRNA actors”) in healthy intestinal stem cells (Lgr5high population) and in intestinal CSC (Lgr5high/Apclox/lox). Then we had correlated these changes with modulation of mRNA translation and protein expression in intestinal stem cells and CSC. Finally, we had validated the specificity of the CSC overexpressed tRNA actors by measuring their levels in Lgr5neg cell population.
2/ We have identified an intestinal CSC tRNA permissive oncogenic signature.
We performed a negative genetic screen (dropout screen) using Lgr5high/Apclox/lox derived murine organoids, treated or not with tamoxifen, to identify the tRNA actors whose loss-of-function affects Wnt-driven tumorigenesis. Then, I generated an engineered mouse models (conditional knockout- cKO) of the identified tRNA actor(s) to assess their role in tumor formation in vivo.
In the tRNAtoGO project, we postulate that the expression of a signature of tRNA molecules in healthy cells is permissive towards the establishment of oncogene-related proteomes. Thereby, we will here test the hypothesis that the differences in tRNA molecules, tRNA modifying enzymes/modifications and tRNA derived fragments (all together “tRNA actors”) between healthy and cancer stem cells are predictive of the capability of cells to rewire their proteome in response to a driving mutation.
The demonstration that a specific signature of tRNA actors in healthy cells can discriminate how permissive a cell population is to an oncogenic driving mutations could represent a unique opportunity to better characterize the molecular features of cancer stem cells.
The demonstration that a specific signature of tRNA actors in healthy cells can discriminate how permissive a cell population is to an oncogenic driving mutations could represent a unique opportunity to better characterize the molecular features of cancer stem cells.