Final Report Summary - FIGHT-CANCER (Long non-coding RNAs of tumor infiltrating lymphocytes as novel anti-cancer therapeutic targets)
Our proposal aimed at understanding the functional features of tumor infiltrating CD4+ Treg cell to better understand their role in tumor escape and allow the identification of new therapeutic targets for the effective modulation of these cells in cancer patients. To this aim we performed RNAseq analysis of both peripheral blood and T cell subsets from two different tumors, non-small cell lung cancer and colo-rectal cancer as well as from the normal tissue adjacent to the tumors, and from peripheral blood samples. We identified sequence specific signatures for tumor infiltrating lymphocytes for both tumor specific coding and non-coding transcripts. In particular through this analysis we defined a subset of signature genes that describe the specific gene expression profile of tumor infiltrating Treg cells with unprecedented resolution (De Simone et al Immunity, 2016). We further investigated the molecular blueprints of tumoral Treg cells by single-cell RNA-sequencing (10x Genomics) coupled to direct TCR sequencing to investigate Treg clonality.
As outlined in the proposal we then focused on long non coding RNA transcripts which have been described to play a crucial role in the control of cell differentiation and in the maintenance of cell identity. Indeed, we obtained the proof of concept that lncRNAs can contribute to lymphocyte differentiation and identity definition, through the functional characterization of linc-MAF-4 a CD4+ Th1 signature lincRNA (Ranzani et al Nat Imm, 2015).For this reason we focused on a specific non-coding RNA whose expression is highly specific for Treg cells: lncFOXP3, whose gene is located on X chromosome in close proximity to the promoter of FOXP3 gene, the master transcription factor or regulatory T cells. Our findings showed that lncFOXP3 down-modulation in Treg cells impair their ability to suppress proliferation of effector cells. To understand the molecular mechanisms underlying lncFOXP3 function we characterized its interaction with USP7 a deubiquitinase known to be involved in the maintenance of FOXP3 stability and we established that lncFOXP3 is required for the stabilization of FOXP3 specifically in Treg cell through USP7. We have also set up with great efforts the protocol to generate conditional Knock-out of linc-FOXP3 in human primary Treg cells by the CRISPR/Cas9 methodology that can in principle be adapted also for the study of other long non-coding RNA expressed by human lymphocytes.
Alongside the characterization of specific lncRNAs, we aimed at improving the approaches to study the functional role of lncRNA. Functional studies of long non-coding RNAs are complicated by their low sequence conservation and by their ability to interact with proteins and nuclei acids both in the cytoplasm and in the nucleus. Accumulating evidence showed that chromatin bound long non-coding RNAs might contribute to transcriptional regulation by the formation of triplex structures between DNA and portion of the RNA molecules. Identification of such structures in vivo is very difficult, we have exploited our experience with SELEX protocols for the identification of aptamers able to recognize specific triplex sequences which we are now validating and we will exploit for the characterization of the biological relevance of these structures in human lymphocytes. We are also developing bioinformatics tools to improve aptamer selection and optimization following the SELEX procedure.
As outlined in the proposal we then focused on long non coding RNA transcripts which have been described to play a crucial role in the control of cell differentiation and in the maintenance of cell identity. Indeed, we obtained the proof of concept that lncRNAs can contribute to lymphocyte differentiation and identity definition, through the functional characterization of linc-MAF-4 a CD4+ Th1 signature lincRNA (Ranzani et al Nat Imm, 2015).For this reason we focused on a specific non-coding RNA whose expression is highly specific for Treg cells: lncFOXP3, whose gene is located on X chromosome in close proximity to the promoter of FOXP3 gene, the master transcription factor or regulatory T cells. Our findings showed that lncFOXP3 down-modulation in Treg cells impair their ability to suppress proliferation of effector cells. To understand the molecular mechanisms underlying lncFOXP3 function we characterized its interaction with USP7 a deubiquitinase known to be involved in the maintenance of FOXP3 stability and we established that lncFOXP3 is required for the stabilization of FOXP3 specifically in Treg cell through USP7. We have also set up with great efforts the protocol to generate conditional Knock-out of linc-FOXP3 in human primary Treg cells by the CRISPR/Cas9 methodology that can in principle be adapted also for the study of other long non-coding RNA expressed by human lymphocytes.
Alongside the characterization of specific lncRNAs, we aimed at improving the approaches to study the functional role of lncRNA. Functional studies of long non-coding RNAs are complicated by their low sequence conservation and by their ability to interact with proteins and nuclei acids both in the cytoplasm and in the nucleus. Accumulating evidence showed that chromatin bound long non-coding RNAs might contribute to transcriptional regulation by the formation of triplex structures between DNA and portion of the RNA molecules. Identification of such structures in vivo is very difficult, we have exploited our experience with SELEX protocols for the identification of aptamers able to recognize specific triplex sequences which we are now validating and we will exploit for the characterization of the biological relevance of these structures in human lymphocytes. We are also developing bioinformatics tools to improve aptamer selection and optimization following the SELEX procedure.