Molecular determinants of the interaction between glioblastoma CD133+ cancer stem cells and the extracellular matrix.

Glioblastoma multiforme (GBM) is the most common primary tumor of the human brain. It is characterized by invasive growth and strong resistance to treatment, and the median survival time of patients is 15 months. The invasive growth of this tumor type is associated with tumor cells with an aggressive phenotype, while its treatment resistance is attributed to cancer stem cells (CSCs). It remains unclear if CSCs have a more invasive nature than differentiated glioblastoma cells (DGCs), and what contribution CSCs make to the aggressive phenotype of GBM. Interaction with the extracellular matrix (ECM) is a key factor in the development of invasion. The aim of the present study was to compare the expression levels of signaling pathway proteins involved in interaction of receptors with the ECM in CSCs and DGCs. The U-87MG GBM cell line was used in the present study CSCs were extracted from gliomaspheres through magnetic-activated cell sorting based on the expression of cluster of differentiation 133 (CD133); CD133-negative DCGs were used as a control. HPLC and mass spectrometry were also used, and biological and molecular functions, signaling pathways and protein-protein interactions were analyzed using publicly available databases. Increased expression levels of the following 10 proteins involved in interaction with the ECM were identified in CSCs, compared with expression levels in DGCs: COL6A1, COL6A3, FN1, ITGA2, ITGA5, ITGAV, ITGB1, ITGB3, LAMB1 and LAMC1. The proteome of CSCs was observed to have >2-fold higher expression of these key proteins, when compared with the DGC proteome. Increased expression levels of four proteins (FERMT2, LOXL2, HDAC2 and FBN1) involved in activating signaling in response to receptor interaction with the ECM was also observed, indicating that CSCs may have highly invasive nature. LOXL2 expression level was >9-fold higher in CSCs compared to DGCs, suggesting that this protein may have potential as an marker for CSCs and as a target for this cell type in GBM.

Proteins of Wnt signaling pathway in cancer stem cells of human glioblastoma.

RATIONALE: Glioblastoma multiforme (GBM) is the most aggressive primary glial brain tumor. The prognosis for GBM patients is not favorable, with the median survival time being 15 months. Its treatment resistance is associated with GBM cell population having cancer stem cells (CSCs). Wnt/ß-catenin signaling pathway is a strategically important molecular mechanism, providing proliferation of stem cells of all types. This study compares the expression levels of signaling pathway proteins in CD133(+) CSCs and CD133(-) differentiated glioblastoma cells (DGCs). MATERIALS AND METHODS: the present study used U-87MG cells of human glioblastoma, the material was tested for mycoplasma contamination. High-performance liquid chromatography (HPLC) mass spectrometry was used for proteome analysis. Biological and molecular functions, signaling pathways and protein-protein interactions were analyzed using free-access databases: PubMed, PANTHER, Gene Ontology, Swiss-Prot and KEGG. Protein-protein interactions (PPIs) were analyzed using the STRING database (version 10). RESULTS: There were identified 589 proteins with significantly changed expression in CD133+ CSCs, as compared with CD133-DGCs (P<0.05). Bioinformatics analysis allowed to attribute 134 differentially expressed proteins to 16 signaling pathways. A significant increase in expression of eight Wnt signaling pathway proteins (APC, CSNK1E, CSNK1A, CSNK2A2, CSNK2B, CTNNB1, DVL1, RUVBL) was detected, as well as four proteins of the non-canonical Wnt pathway-RHOA, ROCK2, RAC2, DAAM1. Special attention should be paid to ß-catenin (CTNNB1) with more than 13.98-fold increase of expression in CSCs and Disheveled-associated activator of morphogenesis 1 (DAAM1) with 6.15-fold higher upregulation level. CONCLUSION: proteins of Wnt/ß-catenin signaling cascade are a prospective target for regulating CSCs activity.

Transforming growth factor-ß mimics the key proteome properties of CD133- differentiated and CD133+ cancer stem cells in glioblastoma.

Glioblastoma multiforme is the most aggressive type of primary brain tumor in humans. Its invasive growth is associated with cluster of differentiation (CD)133 cancer stem cells (CSCs) and CD133- differentiated glioblastoma cells (DGCs) with aggressive phenotype, which are developed under the influence of transforming growth factor (TGF)-ß. The present study aimed to compare the proteomes of CD133 CSCs and CD133- DGCs stimulated by TGF-ß, as well as the expression levels of the main proteins responsible for activating the signaling pathway of receptor interactions with the extracellular matrix (ECM). The U87MG GBM cell line was used in this study. CSCs were extracted from gliomaspheres through magnetic-activated cell sorting based on the expression of CD133 (CD133); CD133- DCGs served as a control. CD133- DGCs of the U87-MG cell line were treated with 10ng/mL TGF-ß1, and cell proliferation and migration were analyzed via real-time quantitative microscopy. High-performance liquid chromatography mass spectrometry was used for proteome analysis. The results revealed 589 proteins with significantly changes in expression among CD133 CSCs compared with those in CD133- DGCs (P<0.05). Bioinformatics analysis allowed to attribute 134 differentially expressed proteins to 15 signaling pathways; among these proteins, 14 were involved in signaling cascades associated with the interaction between CSCs and the ECM, and were upregulated >twofold, while four proteins activated this signaling cascade. TGF-ß-stimulation increased the mobility, suppressed the proliferation and transformed the proteome profile of CD133- DGCs. Were identified 13 key proteins that activate the signaling pathway of receptor interaction with the ECM and three proteins activating this signaling pathway in CD133- DGCs which had the same values as those of CD133 CSCs. In conclusion, TGF-ß increased the expression of proteins that activate the signaling pathway of receptor interaction with the ECM in CD133- DGCs to the level of those in CD133 CSCs.

Human blood plasma proteome mapping for search of potential markers of the lung squamous cell carcinoma.

Blood plasma proteomes obtained from 77 lung squamous cell carcinoma (LSCC) patients (Stages I-III) and 67 healthy controls (all males) were analyzed by using the label-free liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the search of potential cancer biomarkers. All plasma samples were depleted of 14 highly-abundant plasma proteins by immune-affinity column chromatography before LC-MS/MS. We identified and quantified 809 differential proteins with molecular weights from 6.4 kDa to 3900 kDa using a label-free method. Three hundred and sixty four proteins were identified in all three groups. Changes in levels of an expression of blood plasma proteins associated with LSCC were discovered. Among them, 43 proteins were overexpressed and 39 proteins were down-regulated by more than two-fold between the plasmas of lung cancer patients and healthy men. We focused our attention on proteins whose expression levels increased from control to early stage and then to advanced stage tumor. Each of the 43 unique overexpressed proteins was classified according to its cellular localization, biological processes, molecular function and classes. Many of these proteins are involved in biological pathways pertinent to tumor progression and metastasis and some of these deregulated proteins may be useful clinical markers.

Identificationof potential lung cancer biomarkers by liquid chromatography tandem mass spectrometry-based proteomics analysis of secretomes of two lung cancer cell lines.

A label-free nano-liquid chromatography tandem mass spectrometry proteomics analysis on the conditioned media (CM) of two lung cancer cell lines of different histological backgrounds to identify secreted or membrane-bound proteins as novel lung cancer biomarkers was performed. Five hundred and seventy seven proteins were identified and 38% of them were classified as extracellular or membrane-bound. For the search of potential biomarkers of lung cancer a series of selection criteria were proposed. We detected known or putative lung cancer markers. In addition, 40 novel proteins were identified, whose role as biomarkers of lung cancer should be explored further.

Detection of lung cancer using plasma protein profiling by matrix-assisted laser desorption/ionization mass spectrometry.

There are no satisfactory plasma biomarkers which are available for the early detection and monitoring of lung cancer, one of the most frequent cancers worldwide. The aim of this study is to explore the application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) to plasma proteomic patterns to distinguish lung cancer patients from healthy individuals. The EDTA plasma samples have been pre-fractionated using magnetic bead kits functionalized with weak cation exchange coatings. We compiled MS protein profiles for 90 patients with squamous cell carcinomas (SCC) and compared them with profiles from 187 healthy controls. The MALDI-ToF spectra were analyzed statistically using ClinProTools bioinformatics software. Depending on the sample used, up to 441 peaks/spectrum could be detected in a mass range of 1000-20,000 Da; 33 of these proteins had statistically differential expression levels between SCC and control plasma (P < 0.001). The series of the peaks were automatically chosen as potential biomarker patterns in the training set. They allowed the discrimination of plasma samples from healthy control and samples from SCC patients (sensitivity and specificity >90%) in external validation test. These results suggest that plasma MALDI-ToF MS protein profiling can distinguish patients with SCC and also from healthy individuals with relatively high sensitivity and specificity and that MALDI- ToF MS is a potential tool for the screening of lung cancer.