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Plasma-derived extracellular vesicles (pEVs) are a potential source of diseased biomarker proteins. However, characterizing the pEV proteome is challenging due to its relatively low abundance and difficulties in enrichment. This study presents a streamlined workflow to identify EV proteins from cancer patient plasma using minimal sample input. Starting with 400 µL of plasma, we generated a comprehensive pEV proteome using size exclusion chromatography (SEC) combined with HiRIEF prefractionation-based mass spectrometry (MS). First, we compared the performance of HiRIEF and long gradient MS workflows using control pEVs, quantifying 2076 proteins with HiRIEF. In a proof-of-concept study, we applied SEC-HiRIEF-MS to a small cohort (12) of metastatic lung adenocarcinoma (LUAD) and malignant melanoma (MM) patients. We also analyzed plasma samples from the same patients to study the relationship between plasma and pEV proteomes. We identified and quantified 1583 proteins in cancer pEVs and 1468 proteins in plasma across all samples. While there was substantial overlap, the pEV proteome included several unique EV markers and cancer-related proteins. Differential analysis revealed 30 DEPs in LUAD vs the MM group, highlighting the potential of pEVs as biomarkers. This work demonstrates the utility of a prefractionation-based MS for comprehensive pEV proteomics and EV biomarker discovery. Data are available via ProteomeXchange with the identifiers PXD039338 and PXD038528.
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PURPOSE OF THE REVIEW: Papillary renal cell carcinoma (pRCC) is the second most frequent renal cancer subtype and represents 15-20% of all RCC. Classification of pRCC is changing because novel tumour entities have been discovered in the last years. In this review, we summarise recent studies relevant for the understanding of the molecular complexity and the broader differential diagnosis of pRCC. RECENT FINDINGS: It has been 25âyears ago, that pRCC was morphologically subdivided into type 1 and type 2. Recently described tumour entities in the 2022 WHO classification challenged this concept and allow a new view on the molecular background in pRCC. Biphasic hyalinizing psammomatous RCC and papillary renal neoplasm with reversed polarity are emerging tumour entities derived from the new concept of molecularly defined RCC subtypes. Immune checkpoint inhibition and tyrosine kinase inhibitors have been introduced as the new backbone in the first-line treatment of advanced pRCCs. To identify novel targeted treatments for patients with pRCC it is crucial to investigate the specific molecular background of pRCC considering emerging pRCC subtypes. SUMMARY: In the future, a deeper understanding of the correlation between molecular aberrations and new pRCC subtypes may improve the classification of pRCC patients and could reveal potential predictive biomarkers for each subgroup.
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Carcinoma de Células Renais , Neoplasias Renais , Carcinoma de Células Renais/diagnóstico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/terapia , Diagnóstico Diferencial , Humanos , Neoplasias Renais/diagnóstico , Neoplasias Renais/genética , Neoplasias Renais/terapiaRESUMO
BACKGROUND: Ankrd2 is a stress responsive protein mainly expressed in muscle cells. Upon the application of oxidative stress, Ankrd2 translocates into the nucleus where it regulates the activity of genes involved in cellular response to stress. Emery-Dreifuss Muscular Dystrophy 2 (EDMD2) is a muscular disorder caused by mutations of the gene encoding lamin A, LMNA. As well as many phenotypic abnormalities, EDMD2 muscle cells also feature a permanent basal stress state, the underlying molecular mechanisms of which are currently unclear. METHODS: Experiments were performed in EDMD2-lamin A overexpressing cell lines and EDMD2-affected human myotubes. Oxidative stress was produced by H2O2 treatment. Co-immunoprecipitation, cellular subfractionation and immunofluorescence analysis were used to validate the relation between Ankrd2 and forms of lamin A; cellular sensibility to stress was monitored by the analysis of Reactive Oxygen Species (ROS) release and cell viability. RESULTS: Our data demonstrate that oxidative stress induces the formation of a complex between Ankrd2 and lamin A. However, EDMD2-lamin A mutants were able to bind and mislocalize Ankrd2 in the nucleus even under basal conditions. Nonetheless, cells co-expressing Ankrd2 and EDMD2-lamin A mutants were more sensitive to oxidative stress than the Ankrd2-wild type lamin A counterpart. CONCLUSIONS: For the first time, we present evidence that in muscle fibers from patients affected by EDMD2, Ankrd2 has an unusual nuclear localization. By introducing a plausible mechanism ruling this accumulation, our data hint at a novel function of Ankrd2 in the pathogenesis of EDMD2-affected cells.
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Núcleo Celular/metabolismo , Lamina Tipo A/metabolismo , Proteínas Musculares/metabolismo , Distrofia Muscular de Emery-Dreifuss/patologia , Proteínas Nucleares/metabolismo , Estresse Oxidativo , Proteínas Repressoras/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células HEK293 , Humanos , Peróxido de Hidrogênio/toxicidade , Imunoprecipitação , Lamina Tipo A/química , Lamina Tipo A/genética , Microscopia de Fluorescência , Proteínas Musculares/química , Proteínas Musculares/genética , Músculo Esquelético/citologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Distrofia Muscular de Emery-Dreifuss/genética , Distrofia Muscular de Emery-Dreifuss/metabolismo , Mioblastos/citologia , Mioblastos/efeitos dos fármacos , Mioblastos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/genética , Estresse Oxidativo/efeitos dos fármacos , Plasmídeos/genética , Plasmídeos/metabolismo , Ligação Proteica , Prenilação de Proteína/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genéticaRESUMO
BACKGROUND: Immune checkpoint inhibitors and antiangiogenic agents are used for first-line treatment of advanced papillary renal cell carcinoma (pRCC) but pRCC response rates to these therapies are low. OBJECTIVE: To generate and characterise a functional ex vivo model to identify novel treatment options in advanced pRCC. DESIGN, SETTING, AND PARTICIPANTS: We established patient-derived cell cultures (PDCs) from seven pRCC samples from patients and characterised them via genomic analysis and drug profiling. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Comprehensive molecular characterisation in terms of copy number analysis and whole-exome sequencing confirmed the concordance of pRCC PDCs with the original tumours. We evaluated their sensitivity to novel drugs by generating drug scores for each PDC. RESULTS AND LIMITATIONS: PDCs confirmed pRCC-specific copy number variations such as gains in chromosomes 7, 16, and 17. Whole-exome sequencing revealed that PDCs retained mutations in pRCC-specific driver genes. We performed drug screening with 526 novel and oncological compounds. Whereas exposure to conventional drugs showed low efficacy, the results highlighted EGFR and BCL2 family inhibition as the most effective targets in our pRCC PDCs. CONCLUSIONS: High-throughput drug testing on newly established pRCC PDCs revealed that inhibition of EGFR and BCL2 family members could be a therapeutic strategy in pRCC. PATIENT SUMMARY: We used a new approach to generate patient-derived cells from a specific type of kidney cancer. We showed that these cells have the same genetic background as the original tumour and can be used as models to study novel treatment options for this type of kidney cancer.
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Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Variações do Número de Cópias de DNA , Receptores ErbB/genética , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Neoplasias Renais/patologia , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
Mutations in the splicing factor SF3B1 are frequently occurring in various cancers and drive tumor progression through the activation of cryptic splice sites in multiple genes. Recent studies also demonstrate a positive correlation between the expression levels of wild-type SF3B1 and tumor malignancy. Here, we demonstrate that SF3B1 is a hypoxia-inducible factor (HIF)-1 target gene that positively regulates HIF1 pathway activity. By physically interacting with HIF1α, SF3B1 facilitates binding of the HIF1 complex to hypoxia response elements (HREs) to activate target gene expression. To further validate the relevance of this mechanism for tumor progression, we show that a reduction in SF3B1 levels via monoallelic deletion of Sf3b1 impedes tumor formation and progression via impaired HIF signaling in a mouse model for pancreatic cancer. Our work uncovers an essential role of SF3B1 in HIF1 signaling, thereby providing a potential explanation for the link between high SF3B1 expression and aggressiveness of solid tumors.
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Neoplasias Pancreáticas , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Hipóxia/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Neoplasias Pancreáticas/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Sítios de Splice de RNA , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Neoplasias PancreáticasRESUMO
Chromophobe renal cell carcinoma (chRCC) patients have good prognosis. Only 5%-10% patients die of metastatic disease after tumorectomy, but tumor progression cannot be predicted by histopathological parameters alone. chRCC are characterized by losses of many chromosomes, whereas gene mutations are rare. In this study, we aim at identifying genes indicating chRCC progression. A bioinformatic approach was used to correlate chromosomal loss and mRNA expression from 15287 genes from The Cancer Genome Atlas (TCGA) database. All genes in TCGA chromophobe renal cancer dataset (KICH) for which a significant correlation between chromosomal loss and mRNA expression was shown, were identified and their associations with outcome was assessed. Genome-wide DNA copy-number alterations were analyzed by Affymetrix OncoScan® CNV FFPE Microarrays in a second cohort of Swiss chRCC. In both cohorts, tumors with loss of chromosomes 2, 6, 10, 13, 17 and 21 had signs of tumor progression. There were 4654 genes located on these chromosomes, and 13 of these genes had reduced mRNA levels, which was associated with poor outcome in chRCC. Decreased CDKN1A expression at mRNA (p = 0.02) and protein levels (p = 0.02) were associated with short overall survival and were independent predictors of prognosis (p <0.01 and <0.05 respectively). CDKN1A expression status is a prognostic biomarker independent of tumor stage. CDKN1A immunohistochemistry may be used to identify chRCC patients at greater risk of disease progression.
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Copy-number alterations Yielding Cancer Liabilities Owing to Partial losS (CYCLOPS) genes have been recently identified as the most enriched class of copy-number associated gene dependencies in human cancer. These genes are cell essential and render tumor cells highly sensitive to the expression of the remaining copy. Chromophobe renal cell carcinoma (chRCC) is characterized by frequent chromosomal deletions, but the relevance of CYCLOPS genes in this tumor subtype is unclear. We found 39 (31%) of 124 recently published candidate CYCLOPS genes (B. Paolella et al., eLife 2017;6:e23268) located on 7 autosomes that are frequently lost in chRCC. GISTIC and RNA-seq data obtained from the TCGA-KICH database showed that 62% of these CYCLOPS genes had significantly lower expression levels in samples with deletion of the respective gene. As copy number (CN) loss of the CYCLOPS gene SF3B1 (Splicing factor 3B subunit 1) has been recently reported in 71% chRCC, we explored the relevance of SF3B1 CN alteration and SF3B1 expression in a set of chRCC and additional oncocytic renal neoplasms. The frequency of SF3B1 CN loss (65%) was similar to that obtained from the TCGA-KICH database and correlated significantly with both lower SF3B1 mRNA (Pâ¯<â¯.05) and protein expression (Pâ¯<â¯.001). Other tumor subtypes with oncocytic cytoplasm had normal SF3B1 CN and displayed strong SF3B1 protein expression. These results suggest that CN loss of CYCLOPS genes is a characteristic feature in chRCC. Since many CYCLOPS genes code for components of proteasomes and transcriptional regulation, their alteration could make chRCC vulnerable to targeted drugs.
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Chromophobe renal cell carcinoma (chRCC) is a renal tumor subtype with a good prognosis, characterized by multiple chromosomal copy number variations (CNV). The World Health Organization (WHO) chRCC classification guidelines define a classic and an eosinophilic variant. Large cells with reticular cytoplasm and prominent cell membranes (pale cells) are characteristic for classic chRCC. Classic and eosinophilic variants were defined in 42 Swiss chRCCs, 119 Japanese chRCCs and in whole-slide digital images of 66 chRCCs from the Cancer Genome Atlas (TCGA) kidney chromophobe (KICH) dataset. 32 of 42 (76.2%) Swiss chRCCs, 90 of 119 (75.6%) Japanese chRCCs and 53 of 66 (80.3%) TCGA-KICH were classic chRCCs. There was no survival difference between eosinophilic and classic chRCC in all three cohorts. To identify a genotype/phenotype correlation, we performed a genome-wide CNV analysis using Affymetrix OncoScan® CNV Assay (Affymetrix/Thermo Fisher Scientific, Waltham, MA, USA) in 33 Swiss chRCCs. TCGA-KICH subtypes were compared with TCGA CNV data. In the combined Swiss and TCGA-KICH cohorts, losses of chromosome 1, 2, 6, 10, 13, and 17 were significantly more frequent in classic chRCC (p < 0.05, each), suggesting that classic chRCC are characterized by higher chromosomal instability. This molecular difference justifies the definition of two chRCC variants. Absence of pale cells could be used as main histological criterion to define the eosinophilic variant of chRCC.
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Thrombocytopenia 2 (THC2) is an inherited disorder caused by monoallelic single nucleotide substitutions in the 5'UTR of the ANKRD26 gene. Patients have thrombocytopenia and increased risk of myeloid malignancies, in particular, acute myeloid leukemia (AML). Given the association of variants in the ANKRD26 5'UTR with myeloid neoplasms, we investigated whether, and to what extent, mutations in this region contribute to apparently sporadic AML. To this end, we studied 250 consecutive, non-familial, adult AML patients and screened the first exon of ANKRD26 including the 5'UTR. We found variants in four patients. One patient had the c.-125T>G substitution in the 5'UTR, while three patients carried two different variants in the 5' end of the ANKRD26 coding region (c.3G>A or c.105C>G). Review of medical history showed that the patient carrying the c.-125T>G was actually affected by typical but unrecognized THC2, highlighting that some apparently sporadic AML cases represent the evolution of a well-characterized familial predisposition disorder. As regards the c.3G>A and the c.105C>G, we found that both variants result in the synthesis of N-terminal truncated ANKRD26 isoforms, which are stable and functional in cells, in particular, have a strong ability to activate the MAPK/ERK signaling pathway. Moreover, investigation of one patient with the c.3G>A showed that mutation was associated with strong ANKRD26 overexpression in vivo, which is the proposed mechanism for predisposition to AML in THC2 patients. These data provide evidence that N-terminal ANKRD26 truncating mutations play a potential pathogenetic role in AML. Recognition of AML patients with germline ANKRD26 pathogenetic variants is mandatory for selection of donors for bone marrow transplantation.