TMZ resistance remains one of the main reasons why treatment of glioblastoma (GBM) fails. In order to investigate the underlying proteins and pathways associated with TMZ resistance, we conducted a cytoplasmic proteome research of U87 cells treated with TMZ for 1 week, followed by differentially expressed proteins (DEPs) screening, KEGG pathway analysis, protein–protein interaction (PPI) network construction, and validation of key candidate proteins in TCGA dataset. A total of 161 DEPs including 65 upregulated proteins and 96 downregulated proteins were identified. Upregulated DEPs were mainly related to regulation in actin cytoskeleton, focal adhesion, and phagosome and PI3K-AKT signaling pathways which were consistent with our previous studies. Further, the most significant module consisted of 28 downregulated proteins that were filtered from the PPI network, and 9 proteins (DHX9, HNRNPR, RPL3, HNRNPA3, SF1, DDX5, EIF5B, BTF3, and RPL8) among them were identified as the key candidate proteins, which were significantly associated with prognosis of GBM patients and mainly involved in ribosome and spliceosome pathway. Taking the above into consideration, we firstly identified candidate proteins and pathways associated with TMZ resistance in GBM using proteomics and bioinformatic analysis, and these proteins could be potential biomarkers for prevention or prediction of TMZ resistance in the future.
Glioblastoma (GBM) remains one of the most lethal cancers for human beings and the prognosis of GBM is still pessimistic [
Over the past years, extensive genomics and proteomics studies have been conducted and also greatly advanced our understanding of the molecular mechanism which underlies the pathogenesis of GBM. However, most of these studies mainly focus on the whole proteome and genome. Subcellular distribution of proteins under different conditions is a major challenge in cell biology indeed; thus subcellular proteome has been developed to address this issue reliably [
Our previous researches indicated that TMZ treatment for 1 week could induce the reconstruction of cytoskeleton [
The human glioblastoma U87 cell line was purchased from the American type culture collection (ATCC, USA) and cultivated in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY) supplemented with 10% FBS and cultured at 37°C in a humidified atmosphere of 5% CO2. TMZ was purchased from Sigma-Aldrich (St. Louis, MO, USA) and diluted in dimethyl sulfoxide (DMSO) (Solarbio Inc., Beijing, China) to a stock solution of 200 mM TMZ. Immediately before use in cell culture, the stock was diluted in media to a concentration of 200
The method of trypan blue staining was described in our previous study [
Digestion of protein (250
The raw MS/MS spectra search was carried out using a freely available software suite, MaxQuant (version. 1.3.0.5). MS data were searched through the UniProtKB database [
The raw data of cytoplasmic proteome expression was analyzed. Differently expressed proteins (DEPs) were identified with classic Student’s
Online database Search Tool for the Retrieval of Interacting Genes (STRING,
For validation of the significant candidate DEPs, The Cancer Genome Atlas (TCGA) Glioblastoma dataset was analyzed using R2: Genomics Analysis and Visualization Platform (
We treated U87 cells with 200
Cytoplasmic proteome research of U87 GBM cells treated with TMZ for 1 week. (a) Morphology and viability change of U87 cells after temozolomide treatment for 1 week; (b) outline of the experimental workflow; (c) verification of the purity of nucleus and cytoplasma extractions; (d) volcano figure of all identified proteins. Red plots mean upregulated proteins in cytoplasma after TMZ treatment, and purple plots mean downregulated proteins.
A total of 161 proteins displayed more than 2-fold quantitative alterations with
List of 161 DEPs identified in the proteome analysis, including 65 upregulated proteins and 96 downregulated proteins in cytoplasma of U87 GBM cells treated with TMZ compared to the control group treated with DMSO.
DEPs | Protein name |
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Upregulated | CCDC25, HIST1H1E, KIF5C, SNAPIN, VAMP5, SLC33A1, GOLT1B, IL1B, PCNP, MINOS1, ZW10, UPP1, CTTN, FDXR, TOP1, RRM2B, ANXA4, PSMD10, ITGB, CKAP4, S100A13, LPP, ATP5I, TCEA1, TRG14, SLC2A1, TUBA4A, GNG12, hCG, CTSB, MYO1C, SIGMAR1, MAPK1IP1L, MGST1, SEC22B, ITGB1, P4HA2, FTH1, CSTB, ITGAV, GLRX, BAX, PRDX3, PLOD2, DNAJC3, PSAP, S100A11, LEPRE1, GALM, TMSB4X, SOD2, P4HA1, TMSB10, PATL1, TGM2, GNB2, MCFD2, CMBL, TNKS1BP1, GSN, MARCKS, S100A6, FKBP10, FHL2, HRSP12 |
Downregulated | SEC63, DPH5, NONO, MATR3, DDX5, KIAA1524, MYLK, NHP2L1, RPL29, RPL35, HNRNPR, HSD17B11, CCDC134, HSBP1, MSH2, ACTR1B, PITHD1, EEF1A1, SETD3, PRKDC, DHX9, ELAVL1, RPL14, MPDU1, TBC1D23, RPL21, TAGLN3, TOLLIP, TBC1D13, HLA-A, MYO5A, SACS, UCC1, SF1, ENOPH1, KATNAL2, FBXO7, PPP6C, EIF5B, IFI35, FAM213A, PSME3, RPL8, PMVK, NMD3, EFTUD2, SFPQ, HNRPA1, RPS15, VPS4B, CSNK2A2, SDCBP, PPID, GAPD, IPO9, EIF4A1, ARL1, EIF3M, RPS24, FAU, AHNAK2, HNRNPA3, SAR1A, NDUFAB1, PRSS1, CUL4B, TRIM28, MYO18A, ITGB6, SCAMP3, EIF5, BTF3, HDLBP, SYNCRIP, RPL9, KPNA2, G3BP2, MIF, RPL3, FASN, MCM7, AKR1C3, RPS23, PFDN4, SURF4, EEF1B2, RPL14, DDX39A, ANP32A, AHNAK, RPS25, ABCF2, NME2P1, NAP1L1, RRM1, DDX3X |
DEPs: differentially expressed proteins.
Proteins were subjected to signaling pathway enrichment analyses; the pathways enriched by downregulated DEPs were mainly related to ribosome and RNA transport (Figure
Signaling pathway enrichment analyses of down- and upregulated DEPs.
Pathway ID | Name | Count |
|
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ko04810 | Regulation of actin cytoskeleton | 6 |
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ko05410 | Hypertrophic cardiomyopathy (HCM) | 3 |
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ko05412 | Arrhythmogenic right ventricular cardiomyopathy (ARVC) | 3 |
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ko04512 | ECM-receptor interaction | 3 |
|
ko05414 | Dilated cardiomyopathy | 3 |
|
ko05200 | Pathways in cancer | 6 |
|
ko05205 | Proteoglycans in cancer | 5 |
|
ko04151 | PI3K-Akt signaling pathway | 5 |
|
ko04380 | Osteoclast differentiation | 3 |
|
ko05032 | Morphine addiction | 2 |
|
ko04640 | Hematopoietic cell lineage | 2 |
|
ko04919 | Thyroid hormone signaling pathway | 3 |
|
ko05020 | Prion diseases | 2 |
|
ko04727 | GABAergic synapse | 2 |
|
ko04728 | Dopaminergic synapse | 3 |
|
ko04145 | Phagosome | 4 |
|
ko00361 | Chlorocyclohexane and chlorobenzene degradation | 1 |
|
ko00364 | Fluorobenzoate degradation | 1 |
|
ko00623 | Toluene degradation | 1 |
|
ko04510 | Focal adhesion | 4 |
|
ko05100 | Bacterial invasion of epithelial cells | 3 |
|
ko04723 | Retrograde endocannabinoid signaling | 2 |
|
ko04713 | Circadian entrainment | 2 |
|
ko04130 | SNARE interactions in vesicular transport | 2 |
|
ko05140 | Leishmaniasis | 2 |
|
ko04724 | Glutamatergic synapse | 2 |
|
ko04115 | p53 signaling pathway | 2 |
|
ko04514 | Cell adhesion molecules (CAMs) | 2 |
|
ko05133 | Pertussis | 2 |
|
ko05222 | Small cell lung cancer | 2 |
|
ko04726 | Serotonergic synapse | 2 |
|
ko04725 | Cholinergic synapse | 2 |
|
ko04211 | Longevity regulating pathway, mammal | 2 |
|
ko05134 | Legionellosis | 2 |
|
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ko03010 | Ribosome | 13 |
|
ko03008 | Ribosome biogenesis in eukaryotes | 3 |
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ko03013 | RNA transport | 5 |
|
Signaling pathway enrichment analysis of DEPs identified in this research. Significantly enriched KEGG terms of upregulated DEPs (a) and downregulated DEPs (b), identified bar represented the percentage of DEPs enriched in specific KEGG term, and the referenced bar represented the percentage of all identified proteins enriched in this KEGG term, which act as a control.
Using the STRING online database (
Protein–protein interaction (PPI) network analysis of DEPs. Based on STRING online database, a total of 161 DEPs constructed the PPI network. The nodes included in the pink region had strong interactions with other nodes, which indicated significant importance for the screening of key candidate proteins.
Protein–protein interaction (PPI) network analysis of DEPs. (a) Subnetwork analysis of DEPs for key candidate proteins identification; (b) a total of 28 key candidate proteins whose degree
To validate the reliability of the 28 key candidate proteins, we used R2 Platform to confirm the predictive capability for overall survival or progress-free survival probability in TCGA 540 GBM dataset. We found that all the 28 key candidate proteins (Figure
Validation of the key candidate proteins in TCGA-GBM-540 dataset (a total of 504 patients; 36 patients lacking survival data were omitted from the analysis). Nine special proteins (DHX9, HNRNPR, RPL3, HNRNPA3, SF1, DDX5, EIF5B, BTF3, and RPL8), which were downregulated after TMZ treatment and related to both overall survival (a) and progress-free survival (b) probability of GBM patients.
Although the use of TMZ has improved the OS of GBM patients from 12.6 to 14.6 months [
The upregulated DEPs were mainly enriched in regulation of actin cytoskeleton, pathways in cancer, PI3K-Akt pathway, and phagosome and focal adhesion signaling pathways. Consistent with our previous researches, we have already found that treatment of TMZ in U87 GBM cells could induce reconstruction of cytoskeleton [
In order to screen the key candidate proteins responsible for TMZ resistance, we conducted PPI-network analysis, and 28 DEPs were identified finally. Interestingly, all these 28 proteins were downregulated DEPs, and all of them were related to the prognosis of GBM patients (either OS or PFS). After further investigating and validating TCGA-GBM dataset, a total of 9 proteins (DHX9, HNRNPR, RPL3, HNRNPA3, SF1, DDX5, EIF5B, BTF3, and RPL8) among them were identified as the key candidate proteins which were related to TMZ therapy. All these 9 proteins were downregulated in the cytoplasma of TMZ-treated U87 cells, and lower expression of these proteins indicated worse prognosis of GBM patients (both OS and PFS) after analyzing the TCGA-GBM-540 database. For the survival analysis, the results could be more robust if we have done a survival analysis of subgroup only including GBM cases treated with TMZ. All these 9 proteins identified in this study were significantly associated with ribosome and spliceosome signaling pathways.
Ribosome, consisting of nucleic acids and proteins, catalyzes protein synthesis according to the genetic instructions in all organisms [
Alternative splicing of mRNA precursors enables one gene to produce multiple protein isoforms with differing functions, and aberrant splicing of mRNA precursors leads to production of aberrant proteins that contribute to tumorigenesis [
We have identified 161 DEPs related to TMZ therapy in GBM through cytoplasmic proteome research and finally found 9 mostly changed hub proteins which were significantly enriched in ribosome and spliceosome signaling pathway after performing further bioinformatics analysis. These findings of our study may contribute to the understanding of the underlying molecular mechanisms of TMZ resistance in GBM cells, and the candidate proteins and pathways can be used as potential biomarkers for the prevention or prediction of TMZ resistance in the future.
The funders had no role in study design, data collection, data analysis, decision to publish, or preparation of the manuscript.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
Guo-zhong Yi and Wei Xiang have contributed equally to this work.
This study was funded by the National Natural Science Foundation of China (Grant nos. 81773290, 81472315, and 81302229), Natural Science Foundation of Guangdong Province (2014A030313167 and 2017A030313497), National Key Technology Research and Development Program of the Ministry of Science and Technology of China (2014BAI04B01), and President Foundation of Nanfang Hospital, Southern Medical University (2017C025).