Crosstalk between cancer cells and the niche deeply influences self-renewal, plasticity and metastasis and Extracellular Vesicles (EVs) are considered pivotal in cellular intercommunication also in these contexts.
This project builds on our findings showing that the RNA-Binding-Protein (RBP) SYNCRIP impacts on exosome miRNA content. Furthermore, we identified a miRNA extra-seed motif sufficient to determine SYNCRIP-mediated exosome loading. These results pave the way for novel therapeutic approaches based on the in vivo production of exosomes containing ncRNAs engineered to be loaded into exosomes, to modulate gene expression in a customized way. Moreover, our preliminary results shed light on the role of other RBPs in the regulation of miRNA exosome loading. Thus, we here propose the identification of others RBPs and consensus motifs in the hypothesis that they may control specific miRNA sorting in the EMT, a transdifferentiation process required for the metastasis of epithelial cancers.
The proposed aims are 1-to validate the RBPs impact on cells and tumor niche during the Epithelial-to-Mesenchymal Transition (EMT), by controlling miRNA EVs loading; 2-to reconduct the EVs pro- or anti-EMT activity to specific subsets of miRNAs and to engineer ncRNAs with specific motifs to vehiculate them into EVs; 3-to unveil the epitranscriptomic modifications of differentially exported miRNAs and to explore their functional role.
We expect to dissect the functional role of EVs in cell communication in EMT, in relation to presence/absence of specific RBPs; to identify specific subsets of differentially exported/cell retained miRNAs and conserved sequence motifs as RBPs binding sites; to clarify the role of m6A RNA modification in controlling RBPs-mediated miRNA sorting in EVs.
The progressive understanding of the loading machinery and of the mechanisms involved in ncRNAs compartmentalization within EVs, hold strong promise for further selective miRNA EVs cargo loading.
The body of evidence indicating that exosome miRNAs content does not match the intracellular profile implies the existence of a molecular machinery regulating exosome-loading/ intracellular-retention of a specific repertoire of these molecules. Literature mining highlights, as yet, only few reports shedding light on this mechanisms and in particular, to our knowledge, in only two of them a specific RBP has been shown to play a causal role in this process. Moreover, only in our previous report a hEXO miRNA extra-seed motif was proven sufficient to determine exosomal loading. This latter results pave the way for novel therapeutic approaches based on the production of engineered exosomes containing ncRNAs (miRNAs, shRNAs, antagomiRNAs) subsets able to modulate gene expression in a customized way. Specifically, the identification and the functional characterization of conserved extra-seed sequences shared by subsets of EVs-enriched or cellular-retained miRNAs, in dependence on the function of specific RBPs, opens the way for the possible selective modification of the ncRNAs EVs cargoes that, in turn, could impact cell-to-cell communication.
We here propose a broad systematic attempt to identify RBPs/extra-seed consensus functional interaction in the context of EMT, which features largely impact on cancer onset/progression.
It is expected that the silencing of specific RBPs results in an interference against the cell-to-cell communications driving EMT, thus impacting tumor progression. Furthermore, the progressive understanding of the loading machinery and of the mechanisms involved in ncRNAs compartmentalization within EVs, hold promise for further selective miRNA EVs cargos modifications.
With respect to the specific aims proposed the expected results that should provide conceptual advantagesand progress beyond the state of the art are:
AIM1: it is expected to acquire -the knowledge on the functional role of EVs in cell-to-cell communication triggered by cells in EMT, in relation to presence/absence of specific RBPs able to control EVs miRNAs content. -the identification of specific subsets of differentially exported/cell-retained miRNAs and the identification of conserved sequence motifs as RBPs binding sites. - The merged functional and structural (i.e. miRNA content) knowledge obtained from this analysis, together with the functional correlation of miRNAs with the EMT readout, will allow to move to AIM2.
AIM2: it is expected: -to define the minimal functional repertoire of miRNAs that, being differentially exported in EVs, drives or counteracts EMT. Moreover, specific miRNAs chimeras will be functionally tested; -to demonstrate in vitro the possibility to engineer short ncRNAs to be exported in EV as tumor suppressor tools.
AIM3: clarification of the role of m6A RNAs modification in controlling RBPs-mediated miRNAs sorting in EVs.- to define the impact of epigenetic modifications on RBPs-miRNAs interaction. -to identify novel inhibitors of METTL3/14.
Finally, with respect to contingency plans the AIM1 proposes parallel experimental approaches on various RBPs; pitfalls in one will not interfere with the achievement of the others. Moreover, preliminary results hold promises for success at least with respect to one RBP. With respect to AIM2, we can suggest alternative approaches i.e. the choice of ncRNA molecules (to be loaded into EVs by means of the embedment of EXO or hEXO motifs) selected on the basis of literature data mining. As yet, we propose the use of the following molecules: i) tumor suppressor miRNAs miR-34a and miR200 family chimeras and ii) a chimeric antagomiR for the oncogenic miR-21. Members of the miR-200 family and miR-34a were identified as tumor-suppressive miRNAs. With respect to microRNA-21, it is an oncomiR with a pivotal role in cell proliferation, activation of EMT, immune response and migration/invasion (17). The sorting of the described chimeras in EVs from stably expressing hepatocytes and their functionality will be tested. The output of this approach, therefore, is the validation of alternative strategies for the delivering of therapeutic molecules via EVs. The described molecules are expected to provide innovative and efficient therapeutic tools for anti-EMT and anti-cancer applications.
Overall the proposed research, unavoidably based in its initial stage on in vitro models, on its successfully outcome, is foreseen within next few years to be continued in in vivo animal models.
References 17- RNA Biol 2011;8:706-713.