Biology subjects

D'Agostino, V. G.

Publications and source records attributed to D'Agostino, V. G..

4 recordsLinked to original sources

bioRxiv2024

Small molecule inhibitors of hnRNPA2B1-RNA interactions reveal a predictable sorting of RNA subsets into extracellular vesicles

Extracellular vesicles (EVs) are cell-secreted membranous particles contributing to intercellular communication. Coding and non-coding RNAs are widely detected EV cargo, and RNA-binding proteins (RBPs), such as hnRNPA2B1, have been circumstantially implicated in sorting vesicular RNAs. However, the contribution of competitive RBP-RNA interactions responsible for RNA-sorting outcomes still needs to be deciphered, especially for EV-RNA interference and predictability. We conducted a reverse proteomic analysis that prioritized heterogeneous nuclear ribonucleoproteins recognizing purine-rich RNA sequences representing a subset of previously identified EXO motifs. A screening campaign using a full-length human hnRNPA2B1 protein and artificial purine-rich RNA brought to small molecule inhibitors orthogonally validated through biochemical and cell-based approaches. Selected drugs effectively interfered with a post-transcriptional layer impacting secreted EV- RNAs, reducing the vesicular pro-inflammatory miR-221 while counteracting the hnRNPA2B1- or TDP43Q331K-dependent paracrine activation of NF-{kappa}B in EV-recipient cells. This study demonstrates the possibility of predicting the EV-RNA quality for developing innovative strategies targeting discrete paracrine functions. SummaryExtracellular vesicles (EVs) are cell-released, heterogeneous lipid particles conceived as vehicles for intercellular communication. RNA is a widely detected cargo, and the comprehension of EV-sorting mechanisms represents a step forward in predicting EV quality and associated paracrine effects. While it is known that specific RNA-binding proteins (RBPs) play a role in EV-RNA sorting, the quantitative contribution of competing RBP-RNA interactions and the predictability of RNA-sorting outcomes are poorly understood. Here, we show that a core of hnRNPs compete for the binding to the heterogeneous EV-RNA in vitro. Given prioritized interactions with purine-rich RNA motifs, we set up a pharmacological screen platform to find inhibitors of protein-RNA interactions. Our results suggest that selected small molecules can interfere with EV-RNA quality, altering the distribution of specific miRNA cargoes and associating with a discriminant NF-kB activation in EV-recipient cells. This work highlights the role of RBP-RNA interactions in influencing the EV-RNA quality and paracrine functions.

molecular biology↗

bioRxiv2024

CD81-guided heterologous EVs present heterogeneous interactions with breast cancer cells

Extracellular vesicles (EVs) are cell-secreted particles conceived as natural vehicles for intercellular communication. The intrinsic biocompatibility, stability in biofluids, and heterogeneous molecular cargo of EVs promise advancements in targeted therapy applications. However, predicting cell-targeting spectrum and cargo delivery are fundamental challenges for exploiting EVs or hybrid formulations. In this work, we combined cell-based and biochemical approaches to understand if secreted EVs show predictable EV-cell interactions and consequent cargo delivery. We exploited the tetraspanin CD81 to encode full-length recombinant proteins with a C-terminal GFP reporter encompassing or not Trastuzumab light chains targeting the HER2 receptor. These fusion proteins participated in vesicular trafficking dynamics and accumulated on secreted EVs when transiently over-expressed in HEK293T cells. Despite the presence of GFP, secreted EV populations retained a HER2 receptor-binding capacity and were used in EV-cell interaction assays. In time-frames where the global GFP spot distribution did not change between HER2-positive (SK-BR-3) or -negative (MDA-MB-231) breast cancer cell lines, the HER2 manipulation in isogenic cells remarkably affected the tropism of heterologous EVs. In this line, secreted doxorubicin-EVs, which showed improved efficacy compared to the free drug, had a reduced cell-killing activity on SK-BR-3 with a knocked-out HER2 receptor. Interestingly, the fusion protein-corresponding transcripts also present as full-length mRNAs in recombinant EVs could reach orthotopic breast tumors in JIMT-1-xenografted mice, as detected by ddPCR in tissue biopsies, improving our sensitivity in detecting bioavailable cargoes. These data show multiple mechanisms underlying EV-cell interactions and prioritize the profiling of surfaceomes for better comprehension of cell engagement and design new generations of EV-based nanovehicles.

molecular biology↗

bioRxiv2024

The DNA-repair protein APE1 participates with hnRNPA2B1 to motif-enriched and prognostic miRNA secretion

The base excision repair (BER) Apurinic/apyrimidinic endonuclease 1 (APE1) enzyme is endowed with several non-repair activities including miRNAs processing. APE1 is overexpressed in many cancers but its causal role in the tumorigenic processes is largely unknown. We recently described that APE1 can be actively secreted by mammalian cells through exosomes. However, APE1 role in EVs or exosomes is still unknown, especially regarding a putative regulatory function on small non-coding RNAs vesicular secretion. Through dedicated transcriptomic analysis on cellular and vesicular small RNAs of different APE1-depleted cancer cell lines, we found that miRNAs loading into EVs is a regulated process, dependent on APE1, distinctly conveying RNA subsets into vesicles. We identified APE1-dependent secreted miRNAs characterized by enriched sequence motifs and possible binding sites for APE1. In 33 out of 34 APE1-dependent-miRNA precursors, we surprisingly found EXO-motifs and proved that APE1 cooperates with hnRNPA2B1 for the EV-sorting of a subset of miRNAs, including miR-1246, through direct binding to GGAG stretches. Using TCGA-datasets, we showed that these miRNAs identify a signature with high prognostic significance in cancer. In summary, we provided evidence that APE1 is part of the protein cargo of secreted EVs, suggesting a novel post-transcriptional role for this ubiquitous DNA-repair enzyme that could explain its role in cancer progression.

molecular biology↗

bioRxiv2023

Integrating extracellular vesicle and circulating cell-free DNA analysis on a single plasma aliquot from breast cancer patients improves the detection of HER2 positivity

BackgroundMulti-analyte liquid biopsies represents an emerging opportunity for non-invasive cancer assessment. We developed ONCE (ONe Aliquot for Circulating Elements), a novel multi-analytes liquid biopsy approach for the isolation of extracellular vesicles (EVs) and cell-free DNA (cfDNA) from a single aliquot of blood. MethodsWe assessed ONCE performance to classify HER2-positive early-stage breast cancer (BrCa) patients by combining RNA and DNA signals on n=64 healthy donors (HD) and non-metastatic BrCa patients. Specifically, we investigated EVs-derived RNA (EV-RNA) and cfDNA by next-generation sequencing (NGS) and by digital droplet PCR (ddPCR). Additionally, we utilized imaging flow cytometry to evaluate EVs as potential carriers of the HER2 protein. ResultsWestern blot analysis and immunocapture assay revealed that EVs-enriched proteins were detected at similar levels among the HER2+ and HER2- subtypes. Sequencing of cfDNA and EV-RNA from HER2- and HER2+ patients demonstrated concordance with in situ molecular analyses of matched tissues. Combined analysis of the two circulating analytes by ddPCR showed increased sensitivity in ERBB2/HER2 detection compared to single nucleic acid components. Multi-analyte liquid biopsy prediction performance was comparable to tissue-based sequencing results from TCGA. Also, we observed HER2 protein on the surface of EVs isolated from the HER2+ BrCa plasma, thus corroborating the potential relevance of studying EVs as companion analyte to cfDNA. ConclusionsThis data confirms the relevance of combining cfDNA and EV-RNA analytes for cancer assessment and supports the ONCE approach as a valuable tool for multi-analytes liquid biopsies clinical implementation.

cancer biology↗