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BACKGROUND: The previously underestimated effects of commensal gut microbiota on the human body are increasingly being investigated using omics. The discovery of active molecules of interaction between the microbiota and the host may be an important step towards elucidating the mechanisms of symbiosis. RESULTS: Here, we show that in the bloodstream of healthy people, there are over 900 peptides that are fragments of proteins from microorganisms which naturally inhabit human biotopes, including the intestinal microbiota. Absolute quantitation by multiple reaction monitoring has confirmed the presence of bacterial peptides in the blood plasma and serum in the range of approximately 0.1 nM to 1 µM. The abundance of microbiota peptides reaches its maximum about 5 h after a meal. Most of the peptides correlate with the bacterial composition of the small intestine and are likely obtained by hydrolysis of membrane proteins with trypsin, chymotrypsin and pepsin - the main proteases of the gastrointestinal tract. The peptides have physicochemical properties that likely allow them to selectively pass the intestinal mucosal barrier and resist fibrinolysis. CONCLUSIONS: The proposed approach to the identification of microbiota peptides in the blood, after additional validation, may be useful for determining the microbiota composition of hard-to-reach intestinal areas and monitoring the permeability of the intestinal mucosal barrier.
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Microbioma Gastrointestinal , Péptidos , Humanos , Microbioma Gastrointestinal/fisiología , Péptidos/análisis , Masculino , AdultoRESUMEN
Multipotent mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) play important roles in cellular communication and are extensively studied as promising therapeutic agents. While there is a substantial pool of studies on liquid-phase EVs, data on EVs bound to the extracellular matrix (ECM) is lacking. There is also an emerging trend of accumulating and comparing data on characteristics of EVs obtained in different culturing conditions. Aiming to reveal proteomic signatures of EVs obtained from conditioned media and ECM of MSCs cultured in 2D and 3D conditions, we performed liquid chromatography with tandem mass spectrometry. Bioinformatic analysis revealed common patterns in proteomic composition of liquid-phase EVs and matrix-bound vesicles (MBVs), namely extracellular environment organization, immune, and transport pathways enrichment. However, extracellular environmental organization pathways are more enriched in liquid-phase EVs than in MBVs, while MBVs proteins noticeably enrich enzymatic pathways. Furthermore, each type of EVs from 2D and 3D cultures has a unique differential abundance profile. We have also performed comparative functional assays, namely scratch assay to assess EVs effect on cell migration and tubulogenesis assay to evaluate EVs angiogenic potential. We found that both liquid-phase EVs and MBVs enhance cell migration, while angiogenic potential is higher in MBVs. Results of the present study suggest that while both liquid-phase EVs and MBVs have therapeutic potential, some unique features of each subgroup may determine optimal areas of their application.
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Glioblastoma (GBM) is one of the most aggressive human cancers with a median survival of less than two years. A distinguishing pathological feature of GBM is a high degree of inter- and intratumoral heterogeneity. Intertumoral heterogeneity of GBM has been extensively investigated on genomic, methylomic, transcriptomic, proteomic and metabolomics levels, however only a few studies describe intratumoral heterogeneity because of the lack of methods allowing to analyze GBM samples with high spatial resolution. Here, we applied TOF-SIMS (Time-of-flight secondary ion mass spectrometry) for the analysis of single cells and clinical samples such as paraffin and frozen tumor sections obtained from 57 patients. We developed a technique that allows us to simultaneously detect the distribution of proteins and metabolites in glioma tissue with 800 nm spatial resolution. Our results demonstrate that according to TOF-SIMS data glioma samples can be subdivided into clinically relevant groups and distinguished from the normal brain tissue. In addition, TOF-SIMS was able to elucidate differences between morphologically distinct regions of GBM within the same tumor. By staining GBM sections with gold-conjugated antibodies against Caveolin-1 we could visualize border between zones of necrotic and cellular tumor and subdivide glioma samples into groups characterized by different survival of the patients. Finally, we demonstrated that GBM contains cells that are characterized by high levels of Caveolin-1 protein and cholesterol. This population may partly represent a glioma stem cells. Collectively, our results show that the technique described here allows to analyze glioma tissues with a spatial resolution beyond reach of most of other omics approaches and the obtained data may be used to predict clinical behavior of the tumor.
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Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Análisis de la Célula Individual/métodos , Espectrometría de Masa de Ion Secundario/métodos , Animales , Neoplasias Encefálicas/mortalidad , Neoplasias Encefálicas/patología , Caveolina 1/metabolismo , Colesterol/metabolismo , Femenino , Glioblastoma/mortalidad , Glioblastoma/patología , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Recurrencia Local de Neoplasia , Pronóstico , Análisis Espacial , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Adenosine-to-inosine RNA editing is a system of post-transcriptional modification widely distributed in metazoans which is catalyzed by ADAR enzymes and occurs mostly in double-stranded RNA (dsRNA) before splicing. This type of RNA editing changes the genetic code, as inosine generally pairs with cytosine in contrast to adenosine, and this expectably modulates RNA splicing. We review the interconnections between RNA editing and splicing in the context of human cancer. The editing of transcripts may have various effects on splicing, and resultant alternatively spliced isoforms may be either tumor-suppressive or oncogenic. Dysregulated RNA splicing in cancer often causes the release of excess amounts of dsRNA into cytosol, where specific dsRNA sensors provoke antiviral-like responses, including type I interferon signaling. These responses may arrest cell division, causing apoptosis and, externally, stimulate antitumor immunity. Thus, small-molecule spliceosome inhibitors have been shown to facilitate the antiviral-like signaling and are considered to be potential cancer therapies. In turn, a cytoplasmic isoform of ADAR can deaminate dsRNA in cytosol, thereby decreasing its levels and diminishing antitumor innate immunity. We propose that complete or partial inhibition of ADAR may enhance the proapoptotic and cytotoxic effects of splicing inhibitors and that it may be considered a promising addition to cancer therapies targeting RNA splicing.
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Adenosina Desaminasa , Neoplasias , Adenosina/metabolismo , Adenosina Desaminasa/genética , Adenosina Desaminasa/metabolismo , Antivirales , Humanos , Inosina/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Empalme del ARN , ARN Bicatenario/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Three new Pt(II) complexes [(dpp-DAD)PtCl2] (I), [(Mes-DAD(Me)2)PtCl2] (II) and [(dpp-DAD(Me)2)PtCl2] (III) were synthesized by the direct reaction of [(CH3CN)2PtCl2] and corresponding redox-active 1,4-diaza-1,3-butadienes (DAD). The compounds were isolated in a single crystal form and their molecular structures were determined by X-ray diffraction. The purity of the complexes and their stability in solution was confirmed by NMR analysis. The Pt(II) ions in all compounds are in a square planar environment. The electrochemical reduction of complexes I-III proceeds in two successive cathodic stages. The first quasi-reversible reduction leads to the relatively stable monoanionic complexes; the second cathodic stage is irreversible. The coordination of 1,4-diaza-1,3-butadienes ligands with PtCl2 increases the reduction potential and the electron acceptor ability of the DAD ligands. The synthesized compounds were tested in relation to an adenocarcinoma of the ovary (SKOV3).
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Cisplatino , Femenino , Humanos , Cisplatino/farmacología , Cisplatino/química , Estructura Molecular , Ligandos , Espectroscopía de Resonancia Magnética , Difracción de Rayos XRESUMEN
Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.
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Hongos/genética , Proteínas Luminiscentes/genética , Secuencia de Aminoácidos , Animales , Vías Biosintéticas/genética , Ácidos Cafeicos , Línea Celular , Línea Celular Tumoral , Femenino , Duplicación de Gen/genética , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Endogámicos BALB C , Alineación de Secuencia , Xenopus laevisRESUMEN
The malignant tumor is a complex heterogeneous set of cells functioning in a no less heterogeneous microenvironment. Like any dynamic system, cancerous tumors evolve and undergo changes in response to external influences, including therapy. Initially, most tumors are susceptible to treatment. However, remaining cancer cells may rapidly reestablish the tumor after a temporary remission. These new populations of malignant cells usually have increased resistance not only to the first-line agent, but also to the second- and third-line drugs, leading to a significant decrease in patient survival. Multiple studies describe the mechanism of acquired therapy resistance. In past decades, it became clear that, in addition to the simple selection of pre-existing resistant clones, therapy induces a highly complicated and tightly regulated molecular response that allows tumors to adapt to current and even subsequent therapeutic interventions. This review summarizes mechanisms of acquired resistance, such as secondary genetic alterations, impaired function of drug transporters, and autophagy. Moreover, we describe less obvious molecular aspects of therapy resistance in cancers, including epithelial-to-mesenchymal transition, cell cycle alterations, and the role of intercellular communication. Understanding these molecular mechanisms will be beneficial in finding novel therapeutic approaches for cancer therapy.
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Resistencia a Antineoplásicos , Redes Reguladoras de Genes , Neoplasias/genética , Autofagia , Ciclo Celular , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , HumanosRESUMEN
Acute inflammatory demyelinating polyneuropathy (AIDP) - the main form of Guillain-Barre syndrome-is a rare and severe disorder of the peripheral nervous system with an unknown etiology. One of the hallmarks of the AIDP pathogenesis is a significantly elevated cerebrospinal fluid (CSF) protein level. In this paper CSF peptidome and proteome in AIDP were analyzed and compared with multiple sclerosis and control patients. A total protein concentration increase was shown to be because of even changes in all proteins rather than some specific response, supporting the hypothesis of protein leakage from blood through the blood-nerve barrier. The elevated CSF protein level in AIDP was complemented by activization of protein degradation and much higher peptidome diversity. Because of the studies of the acute motor axonal form, Guillain-Barre syndrome as a whole is thought to be associated with autoimmune response against neurospecific molecules. Thus, in AIDP, autoantibodies against cell adhesion proteins localized at Ranvier's nodes were suggested as possible targets in AIDP. Indeed, AIDP CSF peptidome analysis revealed cell adhesion proteins degradation, however no reliable dependence on the corresponding autoantibodies levels was found. Proteome analysis revealed overrepresentation of Gene Ontology groups related to responses to bacteria and virus infections, which were earlier suggested as possible AIDP triggers. Immunoglobulin blood serum analysis against most common neuronal viruses did not reveal any specific pathogen; however, AIDP patients were more immunopositive in average and often had polyinfections. Cytokine analysis of both AIDP CSF and blood did not show a systemic adaptive immune response or general inflammation, whereas innate immunity cytokines were up-regulated. To supplement the widely-accepted though still unproven autoimmunity-based AIDP mechanism we propose a hypothesis of the primary peripheral nervous system damaging initiated as an innate immunity-associated local inflammation following neurotropic viruses egress, whereas the autoantibody production might be an optional complementary secondary process.
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Autoanticuerpos/líquido cefalorraquídeo , Citocinas/sangre , Síndrome de Guillain-Barré/inmunología , Esclerosis Múltiple/inmunología , Proteómica/métodos , Adhesión Celular , Cromatografía Liquida , Femenino , Humanos , Inmunidad Innata , Masculino , Espectrometría de Masas en Tándem , Regulación hacia ArribaRESUMEN
Ovarian cancer ascites is a native medium for cancer cells that allows investigation of their secretome in a natural environment. This medium is of interest as a promising source of potential biomarkers, and also as a medium for cell-cell communication. The aim of this study was to elucidate specific features of the malignant ascites metabolome and proteome. In order to omit components of the systemic response to ascites formation, we compared malignant ascites with cirrhosis ascites. Metabolome analysis revealed 41 components that differed significantly between malignant and cirrhosis ascites. Most of the identified cancer-specific metabolites are known to be important signaling molecules. Proteomic analysis identified 2096 and 1855 proteins in the ovarian cancer and cirrhosis ascites, respectively; 424 proteins were specific for the malignant ascites. Functional analysis of the proteome demonstrated that the major differences between cirrhosis and malignant ascites were observed for the cluster of spliceosomal proteins. Additionally, we demonstrate that several splicing RNAs were exclusively detected in malignant ascites, where they probably existed within protein complexes. This result was confirmed in vitro using an ovarian cancer cell line. Identification of spliceosomal proteins and RNAs in an extracellular medium is of particular interest; the finding suggests that they might play a role in the communication between cancer cells. In addition, malignant ascites contains a high number of exosomes that are known to play an important role in signal transduction. Thus our study reveals the specific features of malignant ascites that are associated with its function as a medium of intercellular communication.
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Ascitis/genética , Regulación Neoplásica de la Expresión Génica , Metaboloma/genética , Proteínas de Neoplasias/genética , Neoplasias Ováricas/genética , Proteoma/genética , ARN Neoplásico/genética , Empalme Alternativo , Ascitis/metabolismo , Ascitis/patología , Comunicación Celular , Línea Celular Tumoral , Exosomas/química , Exosomas/metabolismo , Femenino , Fibrosis/genética , Fibrosis/metabolismo , Fibrosis/patología , Humanos , Proteínas de Neoplasias/metabolismo , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Proteoma/metabolismo , ARN Neoplásico/metabolismo , Transducción de Señal , Empalmosomas/química , Empalmosomas/metabolismo , Vesículas Transportadoras/química , Vesículas Transportadoras/metabolismoRESUMEN
Splicing factor polyglutamine binding protein-1 (PQBP1) is abundantly expressed in the central nervous system during development, and mutations in the gene cause intellectual disability. However, the roles of PQBP1 in cancer progression remain largely unknown. Here, it is shown that PQBP1 overexpression promotes tumor progression and indicates worse prognosis in ovarian cancer. Integrative analysis of spyCLIP-seq and RNA-seq data reveals that PQBP1 preferentially binds to exon regions and modulates exon skipping. Mechanistically, it is shown that PQBP1 regulates the splicing of genes related to the apoptotic signaling pathway, including BAX. PQBP1 promotes BAX exon 2 skipping to generate a truncated isoform that undergoes degradation by nonsense-mediated mRNA decay, thus making cancer cells resistant to apoptosis. In contrast, PQBP1 depletion or splice-switching antisense oligonucleotides promote exon 2 inclusion and thus increase BAX expression, leading to inhibition of tumor growth. Together, the results demonstrate an oncogenic role of PQBP1 in ovarian cancer and suggest that targeting the aberrant splicing mediated by PQBP1 has therapeutic potential in cancer treatment.
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Discapacidad Intelectual , Neoplasias Ováricas , Femenino , Humanos , Proteína X Asociada a bcl-2/genética , Proteínas de Unión al ADN/genética , Discapacidad Intelectual/genética , Discapacidad Intelectual/patología , Neoplasias Ováricas/genética , Empalme del ARN/genética , Factores de Empalme de ARN/genéticaRESUMEN
Introduction: Among the various stromal cell types within the tumor microenvironment, cancer-associated fibroblasts (CAFs) emerge as the predominant constituent, exhibiting a diverse array of oncogenic functions not intrinsic to normal fibroblasts. Their involvement spans across all stages of tumorigenesis, encompassing initiation, progression, and metastasis. Current understanding posits the coexistence of distinct subpopulations of CAFs within the tumor microenvironment across a spectrum of solid tumors, showcasing both pro- and antitumor activities. Recent advancements in single-cell transcriptomics have revolutionized our ability to meticulously dissect the heterogeneity inherent to CAF populations. Furthermore, accumulating evidence underscores the pivotal role of CAFs in conferring therapeutic resistance to tumors against various drug modalities. Consequently, efforts are underway to develop pharmacological agents specifically targeting CAFs. Methods: This review embarks on a comprehensive analysis, consolidating data from 36 independent single-cell RNA sequencing investigations spanning 17 distinct human malignant tumor types. Results: Our exploration centers on elucidating CAF population markers, discerning their prognostic relevance, delineating their functional contributions, and elucidating the underlying mechanisms orchestrating chemoresistance. Discussion: Finally, we deliberate on the therapeutic potential of harnessing CAFs as promising targets for intervention strategies in clinical oncology.
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Serine-threonine protein kinases of the DYRK and CLK families regulate a variety of vital cellular functions. In particular, these enzymes phosphorylate proteins involved in pre-mRNA splicing. Targeting splicing with pharmacological DYRK/CLK inhibitors emerged as a promising anticancer strategy. Investigation of the pyrido[3,4-g]quinazoline scaffold led to the discovery of DYRK/CLK binders with differential potency against individual enzyme isoforms. Exploring the structure-activity relationship within this chemotype, we demonstrated that two structurally close compounds, pyrido[3,4-g]quinazoline-2,10-diamine 1 and 10-nitro pyrido[3,4-g]quinazoline-2-amine 2, differentially inhibited DYRK1-4 and CLK1-3 protein kinases in vitro. Unlike compound 1, compound 2 efficiently inhibited DYRK3 and CLK4 isoenzymes at nanomolar concentrations. Quantum chemical calculations, docking and molecular dynamic simulations of complexes of 1 and 2 with DYRK3 and CLK4 identified a dramatic difference in electron donor-acceptor properties critical for preferential interaction of 2 with these targets. Subsequent transcriptome and proteome analyses of patient-derived glioblastoma (GBM) neurospheres treated with 2 revealed that this compound impaired CLK4 interactions with spliceosomal proteins, thereby altering RNA splicing. Importantly, 2 affected the genes that perform critical functions for cancer cells including DNA damage response, p53 signaling and transcription. Altogether, these results provide a mechanistic basis for the therapeutic efficacy of 2 previously demonstrated in in vivo GBM models.
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Ovarian cancer often develops resistance to conventional therapies, hampering their effectiveness. Here, using ex vivo paired ovarian cancer ascites obtained before and after chemotherapy and in vitro therapy-induced secretomes, we show that molecules secreted by ovarian cancer cells upon therapy promote cisplatin resistance and enhance DNA damage repair in recipient cancer cells. Even a short-term incubation of chemonaive ovarian cancer cells with therapy-induced secretomes induces changes resembling those that are observed in chemoresistant patient-derived tumor cells after long-term therapy. Using integrative omics techniques, we find that both ex vivo and in vitro therapy-induced secretomes are enriched with spliceosomal components, which relocalize from the nucleus to the cytoplasm and subsequently into the extracellular vesicles upon treatment. We demonstrate that these molecules substantially contribute to the phenotypic effects of therapy-induced secretomes. Thus, SNU13 and SYNCRIP spliceosomal proteins promote therapy resistance, while the exogenous U12 and U6atac snRNAs stimulate tumor growth. These findings demonstrate the significance of spliceosomal network perturbation during therapy and further highlight that extracellular signaling might be a key factor contributing to the emergence of ovarian cancer therapy resistance.
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Cisplatino , Resistencia a Antineoplásicos , Neoplasias Ováricas , Empalmosomas , Femenino , Humanos , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Neoplasias Ováricas/genética , Neoplasias Ováricas/tratamiento farmacológico , Empalmosomas/metabolismo , Cisplatino/farmacología , Línea Celular Tumoral , Animales , Ratones , Vesículas Extracelulares/metabolismo , Supervivencia Celular/efectos de los fármacos , Antineoplásicos/farmacología , ARN Nuclear Pequeño/metabolismo , ARN Nuclear Pequeño/genética , Reparación del ADNRESUMEN
Dysregulation of pre-mRNA splicing is a common hallmark of cancer cells and it is associated with altered expression, localization, and mutations of the components of the splicing machinery. In the last few years, it has been elucidated that spliceosome components can also influence cellular processes in a splicing-independent manner. Here, we analyze open source data to understand the effect of the knockdown of splicing factors in human cells on the expression and splicing of genes relevant to cell proliferation, migration, cell cycle regulation, DNA repair, and cell death. We supplement this information with a comprehensive literature review of non-canonical functions of splicing factors linked to cancer progression. We also specifically discuss the involvement of splicing factors in intercellular communication and known autoregulatory mechanisms in restoring their levels in cells. Finally, we discuss strategies to target components of the spliceosome machinery that are promising for anticancer therapy. Altogether, this review greatly expands understanding of the role of spliceosome proteins in cancer progression.
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Neoplasias , Empalmosomas , Humanos , Empalmosomas/genética , Empalmosomas/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Empalme del ARN/genética , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Puntos de Control del Ciclo Celular , Precursores del ARN/genética , Precursores del ARN/metabolismoRESUMEN
Ovarian cancer is known to be the most lethal malignancy among all gynecological cancers affecting a large number of women worldwide. The treatment of ovarian cancer is challenging due to the high recurrence rate of the disease and is further complicated by acquired chemoresistance. Most ovarian cancer deaths are the result of the metastatic spread of drug-resistant cells. The theory of cancer stem cells (CSC) suggests that both tumor initiation and progression are driven by a population of undifferentiated capable of self-renewal, tumor initiation and development of chemoresistance. The CD117 mast/stem cell growth factor receptor (KIT) is the most commonly used marker for ovarian CSCs. Here, we analyze the correlation between CD117 expression and histological tumor type in ovarian cancer cell lines (SK-OV-3 and MES-OV) and in small/medium extracellular vesicles (EVs) isolated from the urine of ovarian cancer patients. We have demonstrated that the abundance of CD117 on cells and EVs is correlated with tumor grade and therapy resistance status. Moreover, using small EVs isolated from ovarian cancer ascites, it was shown that recurrent disease is characterized by a much higher abundance of CD117 on EVs than primary tumor.
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DNA-intercalated motifs (iMs) are facile scaffolds for the design of various pH-responsive nanomachines, including biocompatible pH sensors. First, DNA pH sensors relied on complex intermolecular scaffolds. Here, we used a simple unimolecular dual-labeled iM scaffold and minimized it by replacing the redundant loop nucleosides with abasic or alkyl linkers. These modifications improved the thermal stability of the iM and increased the rates of its pH-induced conformational transitions. The best effects were obtained upon the replacement of all three native loops with short and flexible linkers, such as the propyl one. The resulting sensor showed a pH transition value equal to 6.9 ± 0.1 and responded rapidly to minor acidification (tau1/2 <1 s for 7.2 â 6.6 pH jump). We demonstrated the applicability of this sensor for pH measurements in the nuclei of human lung adenocarcinoma cells (pH = 7.4 ± 0.2) and immortalized embryonic kidney cells (pH = 7.0 ± 0.2). The sensor stained diffusely the nucleoplasm and piled up in interchromatin granules. These findings highlight the prospects of iMs in the studies of normal and pathological pH-dependent processes in the nucleus, including the formation of biomolecular condensates.
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Núcleo Celular , ADN , Humanos , Concentración de Iones de Hidrógeno , ADN/química , Cuerpos NuclearesRESUMEN
Dysregulated expression of splicing factors has important roles in cancer development and progression. However, it remains a challenge to identify the cancer-specific splicing variants. Here we demonstrate that spliceosome component BUD31 is increased in ovarian cancer, and its higher expression predicts worse prognosis. We characterize the BUD31-binding motif and find that BUD31 preferentially binds exon-intron regions near splicing sites. Further analysis reveals that BUD31 inhibition results in extensive exon skipping and a reduced production of long isoforms containing full coding sequence. In particular, we identify BCL2L12, an anti-apoptotic BCL2 family member, as one of the functional splicing targets of BUD31. BUD31 stimulates the inclusion of exon 3 to generate full-length BCL2L12 and promotes ovarian cancer progression. Knockdown of BUD31 or splice-switching antisense oligonucleotide treatment promotes exon 3 skipping and results in a truncated isoform of BCL2L12 that undergoes nonsense-mediated mRNA decay, and the cells subsequently undergo apoptosis. Our findings reveal BUD31-regulated exon inclusion as a critical factor for ovarian cancer cell survival and cancer progression.
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Empalme Alternativo , Neoplasias Ováricas , Humanos , Femenino , Factores de Empalme de ARN/genética , Empalme del ARN/genética , Neoplasias Ováricas/genética , Isoformas de Proteínas/genética , Carcinoma Epitelial de Ovario , Proteínas Proto-Oncogénicas c-bcl-2/genética , Oligonucleótidos Antisentido , Proteínas Musculares/genética , Proteínas Nucleares/genéticaRESUMEN
Extracellular vesicles (EVs) are promising agents for liquid biopsy-a non-invasive approach for the diagnosis of cancer and evaluation of therapy response. However, EV potential is limited by the lack of sufficiently sensitive, time-, and cost-efficient methods for their registration. This research aimed at developing a highly sensitive and easy-to-use immunochromatographic tool based on magnetic nanoparticles for EV quantification. The tool is demonstrated by detection of EVs isolated from cell culture supernatants and various body fluids using characteristic biomarkers, CD9 and CD81, and a tumor-associated marker-epithelial cell adhesion molecules. The detection limit of 3.7 × 105 EV/µL is one to two orders better than the most sensitive traditional lateral flow system and commercial ELISA kits. The detection specificity is ensured by an isotype control line on the test strip. The tool's advantages are due to the spatial quantification of EV-bound magnetic nanolabels within the strip volume by an original electronic technique. The inexpensive tool, promising for liquid biopsy in daily clinical routines, can be extended to other relevant biomarkers.
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Cancer-associated fibroblasts (CAFs) have long been known as one of the most important players in tumor initiation and progression. Even so, there is an incomplete understanding of the identification of CAFs among tumor microenvironment cells as the list of CAF marker genes varies greatly in the literature, therefore it is imperative to find a better way to identify reliable markers of CAFs. To this end, we summarized a large number of single-cell RNA-sequencing data of multiple tumor types and corresponding normal tissues. As a result, for 9 different types of cancer, we identified CAF-specific gene expression signatures and found 10 protein markers that showed strongly positive staining of tumor stroma according to the analysis of IHC images from the Human Protein Atlas database. Our results give an insight into selecting the most appropriate combination of cancer-associated fibroblast markers. Furthermore, comparison of different approaches for studying differences between cancer-associated and normal fibroblasts (NFs) illustrates the superiority of transcriptome analysis of fibroblasts obtained from fresh tissue samples. Using single-cell RNA sequencing data, we identified common differences in gene expression patterns between normal and cancer-associated fibroblasts, which do not depend on the type of tumor.
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Glioblastoma (GBM) is characterized by exceptionally high intratumoral heterogeneity. However, the molecular mechanisms underlying the origin of different GBM cell populations remain unclear. Here, we found that the compositions of ribosomes of GBM cells in the tumour core and edge differ due to alternative RNA splicing. The acidic pH in the core switches before messenger RNA splicing of the ribosomal gene RPL22L1 towards the RPL22L1b isoform. This allows cells to survive acidosis, increases stemness and correlates with worse patient outcome. Mechanistically, RPL22L1b promotes RNA splicing by interacting with lncMALAT1 in the nucleus and inducing its degradation. Contrarily, in the tumour edge region, RPL22L1a interacts with ribosomes in the cytoplasm and upregulates the translation of multiple messenger RNAs including TP53. We found that the RPL22L1 isoform switch is regulated by SRSF4 and identified a compound that inhibits this process and decreases tumour growth. These findings demonstrate how distinct GBM cell populations arise during tumour growth. Targeting this mechanism may decrease GBM heterogeneity and facilitate therapy.