Glioblastoma (GBM) is the most aggressive brain tumor and different efforts have been employed in the search for new drugs and therapeutic protocols for GBM. Epitranscriptomics has shed light on new ...druggable Epigenetic therapies specifically designed to modulate GBM biology and behavior such as Histone Deacetylase inhibitors (iHDAC). Although the effects of iHDAC on GBM have been largely explored, there is a lack of information on the underlaying mechanisms HDAC-dependent that modulate the repertoire of GBM secreted molecules focusing on the set of Extracellular Matrix (ECM) associated proteins, the Matrisome, that may impact the surrounding tumor microenvironment. To acquire a better comprehension of the impacts of HDAC activity on the GBM Matrisome, we studied the alterations on the Matrisome-associated ECM regulators, Core Matrisome ECM glycoproteins, ECM-affiliated proteins and Proteoglycans upon HDAC inhibition in vitro as well as their relationship with glioma pathophysiological/clinical features and angiogenesis. For this, U87MG GBM cells were treated for with iHDAC or vehicle (control) and the whole secretome was processed by Mass Spectrometry NANOLC-MS/MS. In silico analyses revealed that proteins associated to the Angiogenic Matrisome (AngioMatrix), including Decorin, ADAM10, ADAM12 and ADAM15 were differentially regulated in iHDAC versus control secretome. Interestingly, genes coding for the Matrisome proteins differentially regulated were found mutated in patients and were correlated to glioma pathophysiological/clinical features. In vitro functional assays, using HBMEC endothelial cells exposed to the secretome of control or iHDAC treated GBM cells, coupled to 2D and 3D GBM cell culture system, showed impaired migratory capacity of endothelial cells and disrupted tubulogenesis in a Fibronectin and VEGF independent fashion. Collectively, our study provides understanding of epigenetic mechanisms HDAC-dependent to key Matrisomal proteins that may contribute to identify new druggable Epigenetic therapies or gliomagenesis biomarkers with relevant implications to improve therapeutic protocols for this malignancy.
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•HDAC activity modulates the repertoire of GBM-secreted molecules.•HDAC activity inhibition alters the GBM angiogenic Matrisome, the AngioMatrix.•The secretome of GBM cells impairs endothelial cells migration and tubulogenesis.
Glioblastoma (GBM) is the most aggressive brain tumor. Histone deacetylase inhibitors (iHDAC) have emerged as druggable epigenetic therapies for GBM. The effects of iHDAC on the GBM matrisome is currently elusive. In this study, we performed a high throughput proteomic analysis of the GBM matrisome upon pharmacological knockdown of HDAC activity in GBM cells. We characterize the GBM angiogenic matrisome signature, the AngioMatrix, and its impact on endothelial cell behavior, strengthening our comprehension of GBM biology.
Chronic periodontal disease is a chronic inflammatory process affecting tooth supporting tissues in the presence of pathogenic bacterial biofilm. There is some evidence for changes in the protein ...composition of whole saliva from chronic periodontitis patients, but there have been no studies using a proteomic approach. Hence, the aim of this study was to compare the protein profiles of unstimulated whole saliva from patients with periodontitis and healthy subjects by two complementary approaches (2D-gel electrophoresis and liquid chromatography). Protein spots of interest were analyzed by MALDI-TOF-TOF, and the data was complemented by an ESI-Q-TOF experiment. The analyses revealed that subjects with periodontal disease have increased amounts of blood proteins (serum albumin and hemoglobin) and immunoglobulin, and they have a lower abundance of cystatin compared to the control group. A higher number of protein spots were observed in the periodontitis group, of which most were identified as alpha-amylase. This higher number of alpha-amylase variants seems to be caused by hydrolysis by cysteine proteases under such inflammatory conditions. This approach gives novel insights into alterations of salivary protein in presence of periodontal inflammation and may contribute to the improvement of periodontal diagnosis.
Specific spots identified as alpha-amylase (2D-gel of saliva from periodontitis group) were grouped according to MS/MS identification of peptides in catalytic domain (Area I) and C-terminal (Area II) regions of alpha-amylase sequence.
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Proliferative verrucous leukoplakia (PVL) is an oral potentially malignant disorder associated with high risk of malignant transformation. Currently, there is no treatment available, and restrictive ...follow-up of patients is crucial for a better prognosis. Oral leukoplakia (OL) shares some clinical and microscopic features with PVL but exhibits different clinical manifestations and a lower rate of malignant transformation. This study aimed to investigate the proteomic profile of PVL in tissue and saliva samples to identify potential diagnostic biomarkers with therapeutic implications. Tissue and saliva samples obtained from patients with PVL were compared with those from patients with oral OL and controls. Label-free liquid chromatography with tandem mass spectrometry was employed, followed by qualitative and quantitative analyses, to identify differentially expressed proteins. Potential biomarkers were identified and further validated using immunohistochemistry. Staining intensity scan analyses were performed on tissue samples from patients with PVL, patients with OL, and controls from Brazil, Spain, and Finland. The study revealed differences in the immune system, cell cycle, DNA regulation, apoptosis pathways, and the whole proteome of PVL samples. In addition, liquid chromatography with tandem mass spectrometry analyses showed that calreticulin (CALR), receptor of activated protein C kinase 1 (RACK1), and 14-3-3 Tau-protein (YWHAQ) were highly expressed in PVL samples. Immunohistochemistry validation confirmed increased CARL expression in PVL compared with OL. Conversely, RACK1 and YWHA were highly expressed in oral potentially malignant disorder compared to the control group. Furthermore, significant differences in CALR and RACK1 expression were observed in the OL group when comparing samples with and without oral epithelial dysplasia, unlike the PVL. This research provides insights into the molecular mechanisms underlying these conditions and highlights potential targets for future diagnostic and therapeutic approaches.
Human T-cell lymphotropic virus type-1 (HTLV-1) is the etiological agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The endothelial breakdown and migration of leukocytes, ...including monocytes, to the spinal cord are involved in HAM/TSP development. Monocytes from HTLV-1-infected individuals exhibit important functional differences when compared to cells from uninfected donors. Using proteomic shot gun strategy, performed by nanoACQUITY-UPLC system, we analyzed monocytes isolated from peripheral blood of asymptomatic carriers (AC), HAM/TSP and uninfected individuals. 534 proteins were identified among which 376 were quantified by Expression
software. Our study revealed a panel of changes in protein expression linked to HTLV-1 infection. Upregulation of heat shock proteins and downregulation of canonical histone expression were observed in monocytes from HTLV-1-infected patients. Moreover, expression of cytoskeleton proteins was increased in monocytes from HTLV-1-infected patients, mainly in those from HAM/TSP, which was confirmed by flow cytometry and fluorescence microscopy. Importantly, functional assays demonstrated that monocytes from HAM/TSP patients present higher ability for adhesion and transmigration thought endothelium than those from AC and uninfected individuals. The major changes on monocyte protein profile were detected in HAM/TSP patients, suggesting that these alterations exert a relevant role in the establishment of HAM/TSP.
Abstract Objective The aim of this study was to evaluate the differentially secreted protein profile in the urine from patients with clear cell renal cell carcinoma (ccRCC) using mass ...spectrometry–based methods. Urine composition can reflect kidney physiology and can be used to detect markers for renal diseases. Moreover, characterization of the secretome is likely to assist in the investigation of new drugs for biological targets and diagnose the ccRCC at an early stage. Methods and materials Urine samples from patients were divided according to Fuhrman degree (FI-IV), which was associated with the cellular differentiation as good prognosis (GP) and poor prognosis (PP). Healthy individuals were used as the control group (CG). We used both qualitative and quantitative mass spectrometry–based analyses that involved the following approaches: 1-dimensional gel electrophoresis combined with liquid chromatography mass spectrometry in tandem (1DE LC-MS/MS), in-solution digestion combined with label-free 1-dimensional LC-MSE (1D LC-MSE ), and bidimensional gel electrophoresis combined with matrix-assisted laser desorption/ionization time of flight in tandem (2DE MALDI-TOF/TOF) or combined with LC-MS/MS. Results All the strategies allowed the identification of 354 proteins from the CG, GP, and PP groups. Qualitative experiments using 1DE LC-MS/MS analysis detected different protein profiles, and 224 proteins were identified in all groups. The label-free MSE quantitative analysis identified 113 proteins and generated novel information on secreted protein profiles, including 49 up-secreted proteins in the urine from patients with ccRCC and 40 down-secreted proteins related to the CG. Proteins such as kininogen-1, uromodulin, apolipoprotein D, polyubiquitin, and CD59 glycoprotein were down secreted according to the groups CG>GP>PP. In contrast, apolipoprotein A, fibrinogen, and haptoglobin were up secreted in patient groups. The same expression profile observed for kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin was corroborated by 2DE LC-MS/MS or 2DE MALDI-TOF/TOF analyses. These 2 strategies also showed 13 differentially secreted proteins among the 3 groups. Conclusions The proteins kininogen-1, apolipoprotein D, fibrinogen, and haptoglobin presented similar quantitative protein profiles according to MSE and 2DE approaches. The latter proteins were up secreted and the former ones were down-regulated. The strategies used proved to be valuable in identifying proteins that were differentially secreted in urine from patients with RCC.
Overexpression of human epidermal growth factor receptor-2 (HER-2) occurs in 20% of all breast cancer subtypes, especially those that present the worst prognostic outcome through a very invasive and ...aggressive tumour. HCC-1954 (HER-2+) is a highly invasive, metastatic cell line, whereas MCF-7 is mildly aggressive and non-invasive. We investigated membrane proteins from both cell lines that could have a pivotal biological significance in metastasis. Membrane protein enrichment for HCC-1954 and MCF-7 proteomic analysis was performed. The samples were analysed and quantified by mass spectrometry. High abundance membrane proteins were confirmed by Western blot, immunofluorescence, and flow cytometry. Protein interaction prediction and correlations with the Cancer Genome Atlas (TCGA) patient data were conducted by bioinformatic analysis. In addition, β1 integrin expression was analysed by Western blot in cells upon trastuzumab treatment. The comparison between HCC-1954 and MCF-7 membrane-enriched proteins revealed that proteins involved in cytoskeleton organisation, such as HER-2, αv and β1 integrins, E-cadherin, and CD166 were more abundant in HCC-1954. β1 integrin membrane expression was higher in the HCC-1954 cell line resistant after trastuzumab treatment. TCGA data analysis showed a trend toward a positive correlation between HER-2 and β1 integrin in HER-2+ breast cancer patients. Differences in protein profile and abundance reflected distinctive capabilities for aggressiveness and invasiveness between HCC-1954 and MCF-7 cell line phenotypes. The higher membrane β1 integrin expression after trastuzumab treatment in the HCC-1954 cell line emphasised the need for investigating the contribution of β1 integrin modulation and its effect on the mechanism of trastuzumab resistance.
HER2 amplification can be present in ductal carcinoma in situ (DCIS). The aim of the present study was to test the feasibility of measuring soluble HER2 in the saliva of patients at risk of breast ...cancer towards early diagnosis and prognosis. Women with lesions classified as 4 according to BIRADS and women with spontaneous nipple discharge (NAF) were recruited for this study. Quantification of soluble HER2 in saliva was performed using the enzyme immunoassay ELISA. Median values of HER2 were quantified in saliva of the control groups and in the patient groups. The statistical test nonparametric Mann–Whitney was applied for the evaluation of median differences. Although the medians increased with the severity of the clinical status, no significant difference was found in all possibilities (
p
> 0.05) when comparing the medians among the patients groups. Interestingly, inter-individual HER2 quantity variations in the saliva were detected in this study in some subjects from each group. Considering possible inter-individual variations, research on saliva-based circulating HER2 has to be reinforced to ensure its correct application in diagnosis, treatment and in follow-up of breast cancer patients. Older and current issues surrounding the controversy about the appropriate methods for HER2 evaluation are discussed.
The aim of this study was to identify possible protein biomarkers and/or candidates for therapeutic targets in tissues of patients with SCCP, infected by HPV, applying one dimensional electrophoresis ...(1DE), followed by direct mass spectrometry (MS) analysis.
Tissues from 10 HPV positive patients with SCCP and from 10 patients with HPV negative non-tumorous penile foreskins were analyzed applying 1D electrophoresis, followed by analysis with direct mass spectrometry (MS).
Sixty-three different proteins were identified in the first group and 50 in the second group. Recognition was possible for 28 proteins exclusively detected in Group 1 and 21 proteins presented only in Group 2.
Some proteins in the first group are directly involved in the development of other types of cancer, and therefore, suitable for analysis. Complement C3 protein is a strong candidate for evaluating SCCP patients.
Oral leukoplakia is the most common potentially malignant disorder in the oral cavity and can precede carcinoma. This study aimed to identify possible oral leukoplakia salivary biomarkers.
...Unstimulated saliva was collected from participants and protein concentration was determined. Proteins were then precipitated with cold acetone and separated using 2DE over a pH range of 3–10. Spot demarcation and matching were performed and protein identification was done through MS analysis. Oral leukoplakia tissues were submitted to immunohistochemistry analysis for keratin 10 (CK10). A complementary analysis of oral leukoplakias that were not included previously was performed in addition.
226±10 spots were identified in oral leukoplakia 2DE gels, and 262±12 spots were identified in volunteers. Twenty-two spots were highly abundant in oral leukoplakias or not detected in the control group, such as apolipoprotein A1, alpha amylase, cystatins, keratin 10, and lysozyme precursor. All were identified. All oral leukoplakia cases were immunopositive for CK10, mainly in the superficial epithelial layers.
The 2DE salivary protein profiles of individuals with and without oral leukoplakia were observably different. CK10 appears to be an interesting protein and should be further studied in oral carcinogenesis.
MS-based proteomics enables large-scale analysis of proteins. Proteomics can provide detailed descriptions of proteomes of cells and tissues, including body fluids, and appears as a powerful tool to study human disorders. Saliva is readily accessible through non invasive collection and can mirror diverse disease states. Saliva from both diseased and healthy subjects can be analyzed through 2DE and differences between groups could be found. Routine immunohistochemistry analysis confirmed one of these findings, with CK10 being positive tissues from individuals with oral leukoplakia. Therefore, the present study allows insights into development of an important potential oral cancer precursor, named oral leukoplakia. However, the results can be extrapolated and tested in other precancer states, such as proliferative verrucous leukoplakia, patients at risk of oral cancer due to lifestyle behavior and/or cancer history in the family or even those who are under surveillance after a treated primary oral cancer.
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•2DE is suitable to identify differences in salivary proteins from OL and controls.•Common proteins, such as alpha amylase, immunoglobulin and albumin were identified.•Keratin 10, apolipoprotein A1, lysozyme precursor deserve further investigation.•Keratin 10 was present in all tissues from two different set of patients.•Areas with orthokeratin dysplasia were more prone to be CK10 positive.
Glioblastoma (GBM) is a grade IV glioma highly aggressive and refractory to the therapeutic approaches currently in use. O-GlcNAcylation plays a key role for tumor aggressiveness and progression in ...different types of cancer; however, experimental evidence of its involvement in GBM are still lacking. Here, we show that O-GlcNAcylation plays a critical role in maintaining the composition of the GBM secretome, whereas inhibition of OGA activity disrupts the intercellular signaling via microvesicles. Using a label-free quantitative proteomics methodology, we identified 51 proteins in the GBM secretome whose abundance was significantly altered by activity inhibition of O-GlcNAcase (iOGA). Among these proteins, we observed that proteins related to proteasome activity and to regulation of immune response in the tumor microenvironment were consistently downregulated in GBM cells upon iOGA. While the proteins IGFBP3, IL-6 and HSPA5 were downregulated in GBM iOGA cells, the protein SQSTM1/p62 was exclusively found in GBM cells under iOGA. These findings were in line with literature evidence on the role of p62/IL-6 signaling axis in suppressing tumor aggressiveness and our experimental evidence showing a decrease in radioresistance potential of these cells. Taken together, our findings provide evidence that OGA activity may regulate the p62 and IL-6 abundance in the GBM secretome. We propose that the assessment of tumor status from the main proteins present in its secretome may contribute to the advancement of diagnostic, prognostic and even therapeutic tools to approach this relevant malignancy.
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