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Screening of exosome-derived proteins and their potential as biomarkers to diagnostic and prognostic for pancreatic cancer

ABSTRACT: This study aimed to investigate the proteomic landscape of plasma exosomes from patients diagnosed with PDAC and IPMN, compared to healthy controls (CT), in order to identify potential biomarkers with prognostic and diagnostic values. Additionally, the study aimed to understand the functional role of the exosome's proteome in pancreatic neoplasia. We isolated the plasma exosomes and analyzed its proteome by nano electrospray tandem mass spectrometry (nanoLC-MS/MS), and performed downstream statistical and bioinformatics analysis, using the Perseus (v. 6.2.2), MSigDB (v. 2023.1), STRING (v. 11.5), and NCG (v. 7.1) databases. A total of 319 differentially expressed proteins (DEPs) were identified among the exosome samples, but our analyses focused on the 135 proteins that were expressed in at least 70% of each group's samples to ensure their representative proteomes. Comparing PDAC and IPMN exosome proteomes to controls, we identified 24 and 33 differentially expressed proteins, respectively. Some of the most upregulated proteins in PDAC exosomes were CPN1, IGHV2-26, ITIH3, and CLU, while some of the most downregulated were C4BPB, APOB, CFH, and C1QB. In IPMN exosomes, KLKB1, LBP, CFB, and SERPINA1 were the most upregulated proteins while C5, APOD, C3, and C1QA were downregulated. In general, the protein-protein networks and enrichment analyses revealed interesting interaction clusters and pathways related to cancer development, such as the immune system, complement cascade, clotting-related and vesicle-mediated processes, regulation of insulin-like growth factor transport, platelet activation signaling, and G-protein signaling. Our analyses also highlighted candidate cancer drivers, including the upregulated IGLL, LBP, SERPINA1, SERPINA4, SERPING1, and the downregulated APOB, and C3, which were identified in these pathways and may have potential roles in pancreatic tumorigenesis.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

DISEASE(S): Pancreatic Cancer

SUBMITTER: Michel Batista

LAB HEAD: Mateus Nobrega Aoki

PROVIDER: PXD042543 | Pride | 2023-09-04

REPOSITORIES: Pride

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Project description:Intraductal Papillary Mucinous Neoplasms (IPMNs) of the pancreas are cyst-like precursor lesions that give rise to 25% of pancreatic ductal adenocarcinomas (PDAC). While ~90% of cases are diagnosed before cancer forms, metrics and markers by which to determine if an IPMN will progress are currently lacking. Overall, 96% of IPMNs harbor KRAS (~80%) and/or GNAS (~66%) driver mutations and IPMN may be classified as either gastric, intestinal, or pancreatobiliary type. Recently, we identified a shared program of pyloric type metaplasia between pancreatic and gastric injury. Here, we combined immunostaining, RNA-sequencing, molecular biology, and analysis of patient samples to identify major drivers of this program in IPMN. Methods Single cell RNA-sequencing datasets of human IPMN were assayed for markers of pyloric-type metaplasia. 37 IPMN patient samples were immunostained using MxIHC for MUC5AC, CD44v9, and AQP5. KrasG12D and GnasR201C expression was modified in IPMN cell lines to identify transcriptomic programs driven by each oncogene, and in combination. Gene sets were compared to murine and human gastric cell types. Transcriptomic drivers were identified and manipulated in vitro. Candidate driver expression was evaluated by immunostaining of patient IPMN and graded. Results Analysis of published bulk and scRNA-seq IPMN datasets revealed expression of previously described markers of pyloric type metaplasia. Marker expression was confirmed in patient samples. Manipulation of KrasG12D and GnasR201C expression in IPMN cell lines identified the relative contributions of each oncogene to this gastric phenotype. Regulon analysis suggests that transcription factors SPDEF, CREB3L1, and CREB3L4 amplify this program in response to GnasR201C expression. All transcription factors were expressed in patient IPMN samples. Conclusions In response to oncogenic mutation(s), IPMN form in the pancreas and express markers of pyloric type metaplasia. KrasG12D and GnasR201C expression drive this program through independent and synergistic mechanisms that result in amplified expression of mucins and gastrokines, consistent with the phenotype identified in vivo.

Project description:RET/GDNF and ET3/EDNRB regulate cell survival, differentiation and migration of neural crest-derived cells. Many signalling mediators of RET have been characterized but the target genes at the end of the signalling cascade are largely unknown. Since the RET/EDNRB crosstalk has been previously shown, we used a Caenorhabditis elegans knockout strain of Nep-1, a homologue of human ECE1 (endothelin-converting enzyme-1), to identify new target genes. Transcriptome comparison between wild-type and Nep-1 strains at different stages identified vit-3 as a differentially expressed gene. Molecular studies of the vit3 mammalian homologue, Apoliporotein B (APOB), were performed in the murine Neuro2a cell line, a model of ENS development. Apob expression in Neuro2a is specifically activated by the RET/GDNF signalling pathway, since Ret silencing abolished Apob increase, and this effect is induced by MAPK P38 kinase activation. Mouse Apob promoter study revealed that there is a p53-dependent repressor element in the promoter region which blocks Apob expression and we show that actually p53 binds to this region. We demonstrated that RET/GDNF and EDNRB/endothelin 3 (ET-3) cooperate in inducing neuronal differentiation resulting in Apob activation. We also show that Apob expression is downregulated in mouse embryos homozygous for the mutation RetC620R and presenting a severe HSCR phenotype, whereas heterozygous mice, phenotypically normal, present a significant increase in Apob expression. These data suggest that Apob has an important role in RET-mediated neuronal development and APOB decrease may have an impact in human disorders where RET absence has been already identified, such as HSCR and Parkinson disease. Gene expression analysis using Affymetrix GeneChip C. elegans arrays in order to identify genes up- or down-regulated in nep-1 strains, homologue of human ECE1 (endothelin-converting enzyme 1). Comparison of the transcriptomes between wt and nep-1 strains in larval stage L3 and adult C. elegans.

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Screening of exosome-derived proteins and their potential as biomarkers to diagnostic and prognostic for pancreatic cancer

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