PXDNL activates the motility of urothelial bladder carcinoma cells through the Wnt/ß-catenin pathway and has a prognostic value.

AIMS: Although aberrant expression of peroxidasin-like (PXDNL) has been associated with carcinogenesis, its potential role in the Urothelial Carcinoma of the Bladder (UCB) remains unknown. The present study aimed to explore the role of PXDNL in UCB carcinogenesis and its potential clinical value. MAIN METHODS: Based on The Cancer Genome Atlas (TCGA) data, bioinformatics was used to explore the potential clinical value of PXDNL. Wound healing and Transwell invasion assays were employed for the purpose of assessing the cell motility, while the Western Blotting experiments were utilized for investigating the protein expression pattern of PXDNL in UCB and investigating the Epithelial-to-Mesenchymal Transition (EMT) and Wnt/ß-catenin pathways for understanding the probable mechanisms involved. KEY FINDS: PXDNL mRNA was overexpressed in UCB tissues and indicated a poor prognosis. High PXDNL mRNA levels were also associated with advanced clinicopathological features and were regarded as independent prognostic factors for UCB. However, PXDNL showed a weak correlation with immune cell infiltration in UCB. In addition, the findings of the study verified that the existing form of the PXDNL protein was 57-kDa and it was upregulated in the UCB cell lines and tissue samples. Furthermore, silencing PXDNL inhibited, while overexpressing PXDNL promoted EMT and motility of UCB cells in vitro. Mechanistic studies showed that PXDNL activated UCB cell motility via the Wnt/ß-catenin pathway. SIGNIFICANCE: The results reveal a novel molecular target that could be further explored for developing preventive, predictive, and individualized treatment strategies for UCB.

Ubiquitination-mediated molecular pathway alterations in human lung squamous cell carcinomas identified by quantitative ubiquitinomics.

Abnormal ubiquitination is extensively associated with cancers. To investigate human lung cancer ubiquitination and its potential functions, quantitative ubiquitinomics was carried out between human lung squamous cell carcinoma (LSCC) and control tissues, which characterized a total of 627 ubiquitin-modified proteins (UPs) and 1209 ubiquitinated lysine sites. Those UPs were mainly involved in cell adhesion, signal transduction, and regulations of ribosome complex and proteasome complex. Thirty three UPs whose genes were also found in TCGA database were significantly related to overall survival of LSCC. Six significant networks and 234 hub molecules were obtained from the protein-protein interaction (PPI) analysis of those 627 UPs. KEGG pathway analysis of those UPs revealed 47 statistically significant pathways, and most of which were tumor-associated pathways such as mTOR, HIF-1, PI3K-Akt, and Ras signaling pathways, and intracellular protein turnover-related pathways such as ribosome complex, ubiquitin-mediated proteolysis, ER protein processing, and proteasome complex pathways. Further, the relationship analysis of ubiquitination and differentially expressed proteins shows that ubiquitination regulates two aspects of protein turnover - synthesis and degradation. This study provided the first profile of UPs and molecular networks in LSCC tissue, which is the important resource to insight into new mechanisms, and to identify new biomarkers and therapeutic targets/drugs to treat LSCC.

Suberoylanilide Hydroxamic Acid (SAHA) Treatment Reveals Crosstalk Among Proteome, Phosphoproteome, and Acetylome in Nasopharyngeal Carcinoma Cells.

Suberoylanilide hydroxamic acid (SAHA), a famous histone deacetylase (HDAC) inhibitor, has been utilized in clinical treatment for cutaneous T-cell lymphoma. Previously, the mechanisms underlying SAHA anti-tumor activity mainly focused on acetylome. However, the characteristics of SAHA in terms of other protein posttranslational modifications (PTMs) and the crosstalk between various modifications are poorly understood. Our previous work revealed that SAHA had anti-tumor activity in nasopharyngeal carcinoma (NPC) cells as well. Here, we reported the profiles of global proteome, acetylome, and phosphoproteome of 5-8 F cells upon SAHA induction and the crosstalk between these data sets. Overall, we detected and quantified 6,491 proteins, 2,456 phosphorylated proteins, and 228 acetylated proteins in response to SAHA treatment in 5-8 F cells. In addition, we identified 46 proteins exhibiting both acetylation and phosphorylation, such as WSTF and LMNA. With the aid of intensive bioinformatics analyses, multiple cellular processes and signaling pathways involved in tumorigenesis were clustered, including glycolysis, EGFR signaling, and Myc signaling pathways. Taken together, this study highlighted the interconnectivity of acetylation and phosphorylation signaling networks and suggested that SAHA-mediated HDAC inhibition may alter both acetylation and phosphorylation of viral proteins. Subsequently, cellular signaling pathways were reprogrammed and contributed to anti-tumor effects of SAHA in NPC cells.

N6-methyladenosine-related non-coding RNAs are potential prognostic and immunotherapeutic responsiveness biomarkers for bladder cancer.

BACKGROUND: Bladder cancer (BC) is a commonly occurring malignant tumor of the urinary system, demonstrating high global morbidity and mortality rates. BC currently lacks widely accepted biomarkers and its predictive, preventive, and personalized medicine (PPPM) is still unsatisfactory. N6-methyladenosine (m6A) modification and non-coding RNAs (ncRNAs) have been shown to be effective prognostic and immunotherapeutic responsiveness biomarkers and contribute to PPPM for various tumors. However, their role in BC remains unclear. METHODS: m6A-related ncRNAs (lncRNAs and miRNAs) were identified through a comprehensive analysis of TCGA, starBase, and m6A2Target databases. Using TCGA dataset (training set), univariate and least absolute shrinkage and selection operator (LASSO) regression analyses were performed to develop an m6A-related ncRNA-based prognostic risk model. Kaplan-Meier analysis of overall survival (OS) and receiver operating characteristic (ROC) curves were used to verify the prognostic evaluation power of the risk model in the GSE154261 dataset (testing set) from Gene Expression Omnibus (GEO). A nomogram containing independent prognostic factors was developed. Differences in BC clinical characteristics, m6A regulators, m6A-related ncRNAs, gene expression patterns, and differentially expressed genes (DEGs)-associated molecular networks between the high- and low-risk groups in TCGA dataset were also analyzed. Additionally, the potential applicability of the risk model in the prediction of immunotherapeutic responsiveness was evaluated based on the "IMvigor210CoreBiologies" data set. RESULTS: We identified 183 m6A-related ncRNAs, of which 14 were related to OS. LASSO regression analysis was further used to develop a prognostic risk model that included 10 m6A-related ncRNAs (BAALC-AS1, MIR324, MIR191, MIR25, AC023509.1, AL021707.1, AC026362.1, GATA2-AS1, AC012065.2, and HCP5). The risk model showed an excellent prognostic evaluation performance in both TCGA and GSE154261 datasets, with ROC curve areas under the curve (AUC) of 0.62 and 0.83, respectively. A nomogram containing 3 independent prognostic factors (risk score, age, and clinical stage) was developed and was found to demonstrate high prognostic prediction accuracy (AUC = 0.83). Moreover, the risk model could also predict BC progression. A higher risk score indicated a higher pathological grade and clinical stage. We identified 1058 DEGs between the high- and low-risk groups in TCGA dataset; these DEGs were involved in 3 molecular network systems, i.e., cellular immune response, cell adhesion, and cellular biological metabolism. Furthermore, the expression levels of 8 m6A regulators and 12 m6A-related ncRNAs were significantly different between the two groups. Finally, this risk model could be used to predict immunotherapeutic responses. CONCLUSION: Our study is the first to explore the potential application value of m6A-related ncRNAs in BC. The m6A-related ncRNA-based risk model demonstrated excellent performance in predicting prognosis and immunotherapeutic responsiveness. Based on this model, in addition to identifying high-risk patients early to provide them with focused attention and targeted prevention, we can also select beneficiaries of immunotherapy to deliver personalized medical services. Furthermore, the m6A-related ncRNAs could elucidate the molecular mechanisms of BC and lead to a new direction for the improvement of PPPM for BC. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13167-021-00259-w.

Dissection of Anti-tumor Activity of Histone Deacetylase Inhibitor SAHA in Nasopharyngeal Carcinoma Cells via Quantitative Phosphoproteomics.

Suberoylanilide hydroxamic acid (SAHA), a pan HDAC inhibitor, has been approved by the Food and Drug Administration (FDA) to treat cutaneous T cell lymphoma (CTCL). Nevertheless, the mechanisms underlying the therapeutic effects of SAHA on tumors are yet not fully understood. Protein phosphorylation is one of the most important means to regulate key biological processes (BPs), such as cell division, growth, migration, differentiation, and intercellular communication. Thus, investigation on the impacts of SAHA treatment on global cellular phosphorylation covering major signaling pathways deepens our understanding on its anti-tumor mechanisms. Here we comprehensively identified and quantified protein phosphorylation for the first time in nasopharyngeal carcinoma (NPC) cells upon SAHA treatment by combining tandem mass tags (TMTs)-based quantitative proteomics and titanium dioxide (TiO2)-based phosphopeptide enrichment. In total, 7,430 phosphorylation sites on 2,456 phosphoproteins were identified in the NPC cell line 5-8F, of which 1,176 phosphorylation sites on 528 phosphoproteins were significantly elevated upon SAHA treatment. Gene ontology (GO) analysis showed that SAHA influenced several BPs, including mRNA/DNA processing and cell cycle. Furthermore, signaling pathway analysis and immunoblotting demonstrated that SAHA activated tumor suppressors like p53 and Rb1 via phosphorylation and promoted cell apoptosis in NPC cells but inactivated energetic pathways such as AMPK signaling. Overall, our study indicated that SAHA exerted anti-tumor roles in NPC cells, which may serve as novel therapeutic for NPC patients.

Integration of quantitative phosphoproteomics and transcriptomics revealed phosphorylation-mediated molecular events as useful tools for a potential patient stratification and personalized treatment of human nonfunctional pituitary adenomas.

BACKGROUND: Invasiveness is a very challenging clinical problem in nonfunctional pituitary adenomas (NFPAs), and currently, there are no effective invasiveness-related molecular biomarkers. The post-neurosurgery treatment is much different as for invasive and noninvasive NFPAs. The aim of this study was to integrate phosphoproteomics and transcriptomics data to reveal phosphorylation-mediated molecular events for invasive characteristics of NFPAs to achieve a potential tool for patient stratification, and prognostic/predictive assessment to discriminate invasive from noninvasive NFPAs for personalized attitude. METHODS: The 6-plex tandem mass tag (TMT) labeling reagents coupled with TiO2 enrichment of phosphopeptides and liquid chromatography-tandem mass spectrometry (LC-MS/MS) were used to identify and quantify each phosphoprotein and phosphosite in NFPAs and controls. Differentially expressed genes (DEGs) between invasive NFPA and control tissues were obtained from the Gene Expression Omnibus (GEO) database. The overlapping analysis was performed between phosphoprotiens and invasive DEGs. Gene Ontology (GO) enrichment, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and protein-protein interaction (PPI) analyses were used to analyze these overlapped molecules. RESULTS: In total, 1035 phosphoproteins with 2982 phosphorylation sites were identified in NFPAs vs. controls, and 2751 DEGs were identified in invasive NFPAs vs. controls. Overlapping analysis of these phosphoproteins and DEGs exposed 130 overlapped molecules (phosphoproteins; invasive DEGs). GO enrichment and KEGG pathway analyses of 130 overlapped molecules revealed multiple biological processes and signaling pathway network alterations, including cell-cell adhesion, platelet activation, GTPase signaling pathway, protein kinase signaling, calcium signaling pathway, estrogen signaling pathway, glucagon signaling pathway, cGMP-PKG signaling pathway, GnRH signaling pathway, inflammatory mediator regulation of TRP channels, vascular smooth muscle contraction, and Fc gamma R-mediated phagocytosis, which were obviously associated with tumor invasive characteristics. For 130 overlapped molecules, PPI network-based molecular complex detection (MCODE) identified 10 hub molecules, namely SLC2A4, TSC2, AKT1, SCG3, ALB, APOL1, ACACA, SPARCL1, CHGB, and IGFBP5. These hub molecules are involved in multiple signaling pathways and represent potential predictive/prognostic markers in NFPA patients as well as they represent potential therapeutic targets. CONCLUSIONS: This study provided the first large-scale phosphoprotein profiling and phosphorylation-related signaling pathway network alterations in human NFPA tissues. Further, overlapping analysis of phosphoproteins and invasive DEGs revealed the phosphorylation-mediated signaling pathway network changes in invasive NFPAs. These findings are the precious resource for in-depth insight into the molecular mechanisms of NFPAs, as well as for the discovery of effective phosphoprotein biomarkers and therapeutic targets for invasive NFPAs.

Label-free quantitative identification of abnormally ubiquitinated proteins as useful biomarkers for human lung squamous cell carcinomas.

BACKGROUND: Ubiquitination is an important molecular event in lung squamous cell carcinoma (LSCC), which currently is mainly studied in nonsmall cell lung carcinoma cell models but lacking of ubiquitination studies on LSCC tissues. Here, we presented the ubiquitinated protein profiles of LSCC tissues to explore ubiquitination-involved molecular network alterations and identify abnormally ubiquitinated proteins as useful biomarkers for predictive, preventive, and personalized medicine (PPPM) in LSCC. METHODS: Anti-ubiquitin antibody-based enrichment coupled with LC-MS/MS was used to identify differentially ubiquitinated proteins (DUPs) between LSCC and control tissues, followed by integrative omics analyses to identify abnormally ubiquitinated protein biomarkers for LSCC. RESULTS: Totally, 400 DUPs with 654 ubiquitination sites were identified,, and motifs A-X (1/2/3)-K* were prone to be ubiquitinated in LSCC tissues. Those DUPs were involved in multiple molecular network systems, including the ubiquitin-proteasome system (UPS), cell metabolism, cell adhesion, and signal transduction. Totally, 44 hub molecules were revealed by protein-protein interaction network analysis, followed by survival analysis in TCGA database (494 LSCC patients and 20,530 genes) to obtain 18 prognosis-related mRNAs, of which the highly expressed mRNAs VIM and IGF1R were correlated with poorer prognosis, while the highly expressed mRNA ABCC1 was correlated with better prognosis. VIM-encoded protein vimentin and ABCC1-encoded protein MRP1 were increased in LSCC, which were all associated with poor prognosis. Proteasome-inhibited experiments demonstrated that vimentin and MRP1 were degraded through UPS. Quantitative ubiquitinomics found ubiquitination level was decreased in vimentin and increased in MRP1 in LSCC. These findings showed that the increased vimentin in LSCC might be derived from its decreased ubiquitination level and that the increased MRP1 in LSCC might be derived from its protein synthesis > degradation. GSEA and co-expression gene analyses revealed that VIM and MRP1 were involved in multiple crucial biological processes and pathways. Further, TRIM2 and NEDD4L were predicted as E3 ligases to regulate ubiquitination of vimentin and MRP1, respectively. CONCLUSION: These findings revealed ubiquitinomic variations and molecular network alterations in LSCC, which is in combination with multiomics analysis to identify ubiquitination-related biomarkers for in-depth insight into the molecular mechanism and therapeutic targets and for prediction, diagnosis, and prognostic assessment of LSCC.

A20 targets PFKL and glycolysis to inhibit the progression of hepatocellular carcinoma.

Abnormal expression of the E3 ubiquitin ligase A20 has been found in some malignant cancers, including hepatocellular carcinoma (HCC). Here, we discovered that A20 is an E3 ubiquitin ligase for phosphofructokinase, liver type (PFKL) in HCC A20 interacts with PFKL and promotes its degradation, therefore inhibiting glycolysis in HCC cell lines. Downregulation of A20 in HCC cells promotes proliferation, migration, and glycolysis, all of which can be inhibited by targeting PFKL with RNA interference. Importantly, A20 is downregulated in advanced HCC tissues and inversely correlated with PFKL expression. Thus, our findings establish A20 as a critical regulator of glycolysis and reveal a novel mechanism for A20 in tumor suppression and PFKL regulation. Given that an increased level of glycolysis is linked with HCC, this study also identifies potential therapeutic targets for HCC treatment.

TMT-based quantitative proteomics revealed follicle-stimulating hormone (FSH)-related molecular characterizations for potentially prognostic assessment and personalized treatment of FSH-positive non-functional pituitary adenomas.

BACKGROUND: Non-functional pituitary adenoma (NFPA) is highly heterogeneous with different hormone expression subtypes. Of them, follicle-stimulating hormone (FSH)-positive expression is an important subtype of NFPAs. It is well-known that FSH exerted its functions through binding its receptor. However, the expression rate of FSH receptor was significantly higher in aggressive pituitary adenomas. This study aimed to investigate the molecular characteristics of FSH-positive NFPAs for effective stratification of patient, target treatment, prognostic assessment, and personalized treatment of FSH-positive NFPAs. METHODS: Tandem mass tag (TMT)-based quantitative proteomics was used to investigate differentially expressed proteins (DEPs) between FSH-positive and negative NFPAs. Gene ontology and KEGG pathway enrichment analyses were used to analyze the DEPs. Differentially expressed genes (DEGs) between invasive and non-invasive NFPAs from GEO database were analyzed with pathway enrichment analysis. Protein-protein interaction (PPI) networks were constructed based on DEPs in excetral cellular matrix (ECM)-receptor interaction, focal adhesion, and PI3K-Akt pathways. Cytoscape was used to obtain most significant modules. Western blot was used to validate the expressions of upregulated proteins (ITGA1, ITGA6, and ITGB4), the expression and phosphorylated status of Akt in PI3K-Akt pathway, and the expression of FSH receptors in FSH-positive relative to negative NFPAs. RESULTS: A total of 594 DEPs (374 upregulated and 220 downregulated) were identified between FSH-positive and negative NFPAs. Nineteen KEGG pathway networks were identified to involve DEPs, and reveal molecular differences between FSH-positive and negative NFPAs, including three important pathways that were significantly associated with tumor invasiveness and aggressiveness: ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling pathways. Further, focal adhesion pathway was also confirmed with invasiveness-related NFPA DEG data that were derived from GEO database. Moreover, the significantly upregulated DEPs (ITGA1, ITGA6, and ITGB4) that were associated with tumor invasiveness and aggressiveness were confirmed by immunoaffinity analysis in FSH-positive vs. negative NFPAs. Also, the phosphorylation level but not its expression level of AKT in PI3K-AKT signaling was significantly increased, and the expression level of FSH receptor was significantly increased in FSH-positive relative to negative NFPAs. Also, overlapping analysis of 594 DEPs and 898 DEGs revealed 45 invasiveness-related DEPs, including 11 upregulated DEPs (ITGA6, FARP1, PALLD, PPBP, LIMA1, SCD, UACA, BAG3, CLU, PLEC, and GATM) that were also upregulated genes in invasive NFPAs, and 8 downregulated DEPs (ALCAM, HP, FSTL4, IL13RA2, NPTX2, DPP6, CRABP2, and SLC27A2) that were also downregulated genes in invasive NFPAs. CONCLUSIONS: FSH-positive expression was an important NFPA subtype. It was the first time for this study to reveal FSH-related proteomic variations and the corresponding molecular network alterations in FSH-positive relative to negative NFPAs. Also, three signaling pathways (ECM-receptor interaction, focal adhesion, and PI3K-Akt signaling pathways) and involved upregulated proteins (ITGA1, ITGA6, ITGB4, pAKT, and FSHR) were significantly associated with tumor invasiveness and aggressiveness, and a set of invasiveness-related DEPs were identified with overlapping analysis of 594 DEPs in FSH-positive vs. negative NFPAs and 898 DEGs in invasive vs. non-invasive NFPAs. These findings offered the scientific evidence to in-depth understand molecular characteristics of FSH-positive NFPAs, and effectively stratify the post-surgery patients for personalized prognostic assessment and targeted treatment of FSH-positive NFPAs.

Quantitative Analysis of Ubiquitinated Proteins in Human Pituitary and Pituitary Adenoma Tissues.

Protein ubiquitination is an important post-translational modification that is associated with multiple diseases, including pituitary adenomas (PAs). Protein ubiquitination profiling in human pituitary and PAs remains unknown. Here, we performed the first ubiquitination analysis with an anti-ubiquitin antibody (specific to K-ε-GG)-based label-free quantitative proteomics method and bioinformatics to investigate protein ubiquitination profiling between PA and control tissues. A total of 158 ubiquitinated sites and 142 ubiquitinated peptides in 108 proteins were identified, and five ubiquitination motifs were found. KEGG pathway network analysis of 108 ubiquitinated proteins identified four statistically significant signaling pathways, including PI3K-AKT signaling pathway, hippo signaling pathway, ribosome, and nucleotide excision repair. R software Gene Ontology (GO) analysis of 108 ubiquitinated proteins revealed that protein ubiquitination was involved in multiple biological processes, cellular components, and molecule functions. The randomly selected ubiquitinated 14-3-3 zeta/delta protein was further analyzed with Western blot, and it was found that upregulated 14-3-3 zeta/delta protein in nonfunctional PAs might be derived from the significantly decreased level of its ubiquitination compared to control pituitaries, which indicated a contribution of 14-3-3 zeta/delta protein to pituitary tumorigenesis. These findings provided the first ubiquitinated proteomic profiling and ubiquitination-involved signaling pathway networks in human PAs. This study offers new scientific evidence and basic data to elucidate the biological functions of ubiquitination in PAs, insights into its novel molecular mechanisms of pituitary tumorigenesis, and discovery of novel biomarkers and therapeutic targets for effective treatment of PAs.

The MAPK Pathway-Based Drug Therapeutic Targets in Pituitary Adenomas.

Mitogen-activated protein kinases (MAPKs) include ERK, p38, and JNK MAPK subfamilies, which are crucial regulators of cellular physiology, cell pathology, and many diseases including cancers. For the MAPK signaling system in pituitary adenomas (PAs), the activation of ERK signaling is generally thought to promote cell proliferation and growth; whereas the activations of p38 and JNK signaling are generally thought to promote cell apoptosis. The role of MAPK in treatment of PAs is demonstrated through the effects of currently used medications such as somatostatin analogs such as SOM230 and OCT, dopamine agonists such as cabergoline and bromocriptine, and retinoic acid which inhibit the MAPK pathway. Further, there are potential novel therapies based on putative molecular targets of the MAPK pathway, including 18beta-glycyrrhetinic acid (GA), dopamine-somatostatin chimeric compound (BIM-23A760), ursolic acid (UA), fulvestrant, Raf kinase inhibitory protein (RKIP), epidermal growth factor pathway substrate number 8 (Eps8), transmembrane protein with EGF-like and two follistatin-like domains (TMEFF2), cold inducible RNA-binding protein (CIRP), miR-16, and mammaliansterile-20-like kinase (MST4). The combined use of ERK inhibitor (e.g., SOM230, OCT, or dopamine) plus p38 activator (e.g., cabergoline, bromocriptine, and fulvestrant) and/or JNK activator (e.g., UA), or the development of single drug (e.g., BIM-23A760) to target both ERK and p38 or JNK pathways, might produce better anti-tumor effects on PAs. This article reviews the advances in understanding the role of MAPK signaling in pituitary tumorigenesis, and the MAPK pathway-based potential therapeutic drugs for PAs.

Multiomics-Based Signaling Pathway Network Alterations in Human Non-functional Pituitary Adenomas.

Non-functional pituitary adenoma (NFPA) seriously affects hypothanamus-pituitary-target organ axis system, with a series of molecule alterations in the multiple levels of genome, transcriptome, proteome, and post-translational modifications, and those molecules mutually interact in a molecular-network system. Meta analysis coupled with IPA pathway-network program was used to comprehensively analyze nine sets of documented NFPA omics data, including NFPA quantitative transcriptomics data [280 differentially expressed genes (DEGs)], NFPA quantitative proteomics data [50 differentially expressed proteins (DEPs)], NFPA mapping protein data (218 proteins), NFPA mapping protein nitration data (9 nitroproteins and 3 non-nitrated proteins), invasive NFPA quantitative transriptomics data (346 DEGs), invasive NFPA quantitative proteomics data (57 DEPs), control mapping protein data (1469 proteins), control mapping protein nitration data (8 nitroproteins), and control mapping phosphorylation data (28 phosphoproteins). A total of 62 molecular-networks with 861 hub-molecules and 519 canonical-pathways including 54 cancer-related canonical pathways were revealed. A total of 42 hub-molecule panels and 9 canonical-pathway panels were identified to significantly associate with tumorigenesis. Four important molecular-network systems, including PI3K/AKT, mTOR, Wnt, and ERK/MAPK pathway-systems, were confirmed in NFPAs by PTMScan experiments with altered expression-patterns and phosphorylations. Nineteen high-frequency hub-molecules were also validated in NFPAs with PTMScan experiment with at least 2.5-fold changes in expression or phosphorylation, including ERK, ERK1/2, Jnk, MAPK, Mek, p38 MAPK, AKT, PI3K complex, p85, PKC, FAK, Rac, Shc, HSP90, NFκB Complex, histone H3, AP1, calmodulin, and PLC. Furthermore, mTOR and Wnt pathway-systems were confirmed in NFPAs by immunoaffinity Western blot analysis, with significantly decreased expression of PRAS40 and increased phosphorylation levels of p-PRAS40 (Thr246) in mTOR pathway in NFPAs compared to controls, and with the decreased protein expressions of GSK-3ß and GSK-3ß, significantly increased phosphorylation levels of p-GSK3α (Ser21) and p-GSK3ß (Ser9), and increased expression level of ß-catenin in Wnt pathway in NFPAs compared to controls. Those findings provided a comphrensive and large-scale pathway network data for NFPAs, and offer the scientific evidence for insights into the accurate molecular mechanisms of NFPA and discovery of the effective biomarkers for diagnosis, prognosis, and determination of therapeutic targets.

Quantitative Analysis of Proteome in Non-functional Pituitary Adenomas: Clinical Relevance and Potential Benefits for the Patients.

Background: Non-functional pituitary adenoma (NFPA) is a common tumor that occurs in the pituitary gland, and generally without any symptoms at its early stage and without clinical elevation of hormones, which is commonly diagnosed when it grows up to compress its surrounding tissues and organs. Currently, the pathogenesis of NFPA has not been clarified yet. It is necessary to investigate molecular alterations in NFPA, and identify reliable biomarkers and drug therapeutic targets for effective treatments. Methods: Tandem mass tags (TMT)-based quantitative proteomics was used to identify and quantify proteins in NFPAs. GO and KEGG enrichment analyses were used to analyze the identified proteins. Differentially expressed genes (DEGs) between NFPA and control tissues were obtained from GEO datasets. These two sets of protein and gene data were analyzed to obtain overlapped molecules (genes; proteins), followed by further GO and KEGG pathway analyses of these overlapped molecules, and molecular network analysis to obtain the hub molecules with Cytoscape. Two hub molecules (SRC and AKT1) were verified with Western blotting. Results: Totally 6076 proteins in NFPA tissues were identified, and 3598 DEGs between NFPA and control tissues were identified from GEO database. Overlapping analysis of 6076 proteins and 3598 DEGs obtained 1088 overlapped molecules (DEGs; proteins). KEGG pathway analysis of 6076 proteins obtained 114 statistically significant pathways, including endocytosis, and spliceosome signaling pathways. KEGG pathway analysis of 1088 overlapped molecules obtained 52 statistically significant pathways, including focal adhesion, cGMP-PKG pathway, and platelet activation signaling pathways. These pathways play important roles in cell energy supply, adhesion, and maintenance of the tumor microenvironment. According to the association degree in Cytoscape, ten hub molecules (DEGs; proteins) were identified, including GAPDH, ALB, ACACA, SRC, ENO2, CALM1, POTEE, HSPA8, DECR1, and AKT1. Western-blotting analysis confirmed the upregulated expressions of SRC and PTMScan experiment confirmed the increased levels of pAKT1, in NFPAs compared to controls. Conclusions: This study established the large-scale quantitative protein profiling of NFPA tissue proteome. It offers a basis for subsequent in-depth proteomics analysis of NFPAs, and insight into the molecular mechanism of NFPAs. It also provided the basic data to discover reliable biomarkers and therapeutic targets for NFPA patients.

The crucial role of multiomic approach in cancer research and clinically relevant outcomes.

Cancer with heavily economic and social burden is the hot point in the field of medical research. Some remarkable achievements have been made; however, the exact mechanisms of tumor initiation and development remain unclear. Cancer is a complex, whole-body disease that involves multiple abnormalities in the levels of DNA, RNA, protein, metabolite and medical imaging. Biological omics including genomics, transcriptomics, proteomics, metabolomics and radiomics aims to systematically understand carcinogenesis in different biological levels, which is driving the shift of cancer research paradigm from single parameter model to multi-parameter systematical model. The rapid development of various omics technologies is driving one to conveniently get multi-omics data, which accelerates predictive, preventive and personalized medicine (PPPM) practice allowing prediction of response with substantially increased accuracy, stratification of particular patients and eventual personalization of medicine. This review article describes the methodology, advances, and clinically relevant outcomes of different "omics" technologies in cancer research, and especially emphasizes the importance and scientific merit of integrating multi-omics in cancer research and clinically relevant outcomes.

Exploration of variations in proteome and metabolome for predictive diagnostics and personalized treatment algorithms: Innovative approach and examples for potential clinical application.

Genome mutually interacts with internal and external environmental factors to result in different phenome that contains two important elements of proteins and metabolites, which link genome to predictive, preventive and personalized medicine (PPPM) or precision medicine (PM). Proteomic variations are the final presentation of the genomic and transcriptomic variations, and are involved in a wide range of variations including copy number of protein, splicing, post-translational modifications, translocation/re-distribution, spatial conformation, and pathway-network systems. Metabolomic variations are the comprehensive results originated from all types of in vivo substances, and are involved in a wide range of alterations of metabolites generated from sugars, lipids, proteins, and nucleic acids, and metabolic network systems. Currently the studies on variations in proteome and in metabolome are much insufficient in the width and depth in the fields of proteomics and metabolomics. The development of high-throughput, high-sensitivity, and especially high-reproducibility approaches is necessary to maximize the coverage of variations in proteome and in metabolome. The studies of proteomic and metabolomic variations directly result in the discovery of effective biomarkers to clarify molecular mechanisms of a disease, determine reliable therapeutic targets, and benefit precise prediction, diagnosis, and prognosis assessment. It has more important scientific values in PPPM or PM. BIOLOGICAL SIGNIFICANCE.

How many proteins can be identified in a 2DE gel spot within an analysis of a complex human cancer tissue proteome?

Two-dimensional gel electrophoresis (2DE) in proteomics is traditionally assumed to contain only one or two proteins in each 2DE spot. However, 2DE resolution is being complemented by the rapid development of high sensitivity mass spectrometers. Here we compared MALDI-MS, LC-Q-TOF MS and LC-Orbitrap Velos MS for the identification of proteins within one spot. With LC-Orbitrap Velos MS each Coomassie Blue-stained 2DE spot contained an average of at least 42 and 63 proteins/spot in an analysis of a human glioblastoma proteome and a human pituitary adenoma proteome, respectively, if a single gel spot was analyzed. If a pool of three matched gel spots was analyzed this number further increased up to an average of 230 and 118 proteins/spot for glioblastoma and pituitary adenoma proteome, respectively. Multiple proteins per spot confirm the necessity of isotopic labeling in large-scale quantification of different protein species in a proteome. Furthermore, a protein abundance analysis revealed that most of the identified proteins in each analyzed 2DE spot were low-abundance proteins. Many proteins were present in several of the analyzed spots showing the ability of 2DE-MS to separate at the protein species level. Therefore, 2DE coupled with high-sensitivity LC-MS has a clearly higher sensitivity as expected until now to detect, identify and quantify low abundance proteins in a complex human proteome with an estimated resolution of about 500 000 protein species. This clearly exceeds the resolution power of bottom-up LC-MS investigations.

Laparoendoscopic Single-Site Retroperitoneoscopic Adrenalectomy Versus Conventional Retroperitoneoscopic Adrenalectomy in Obese Patients.

OBJECTIVE: To compare the operative outcomes of obese patients undergoing laparoendoscopic single-site (LESS) retroperitoneoscopic adrenalectomy or standard laparoscopic (LAP) retroperitoneoscopic adrenalectomy. METHODS: Between September 2011 and April 2015, 51 obese patients underwent LESS retroperitoneoscopic adrenalectomy and their operative outcomes were compared with 65 obese patients who underwent standard retroperitoneoscopic adrenalectomy by the same surgeon. In the LESS group, a single-port access was positioned in the lumbar incision. The standard retroperitoneal adrenalectomy technique was performed with a combination of conventional and curved LAP instruments. The following parameters were adopted: patient demographics, surgical details, perioperative complications, postoperative outcome data, and short-term outcomes. RESULTS: The LESS group was comparable with the LAP group in terms of total operative time (70.4 ± 21.3 vs 65.5 ± 24.8 minutes, p = 0.26), hospital length of stay (5.7 ± 1.2 vs 6.1 ± 1.5 days, p = 0.12), and incidence of complications (4/51 vs 5/65, p = 0.98) for patients with similar baseline demographics. The LESS group had significantly shorter surgical incisions (2.8 ± 0.3 vs 5.3 ± 0.6 cm, p < 0.0001), lower in-hospital analgesic requirement (7.6 ± 3.3 mg morphine equivalent vs 10.5 ± 6.2 mg, p = 0.003), and significantly superior scar satisfaction score (9.3 ± 0.7 vs 7.6 ± 1.4, p < 0.0001) than the LAP group. Although estimated blood loss was greater in the LESS group (28.1 ± 10.6 vs 16.9 ± 7.2 mL in the LAP group, p < 0.0001), <50 mL overall blood loss was not clinically significant. During a mean follow-up of 20.2 months, no recurrences or deaths were documented in either group. CONCLUSIONS: In properly selected patients, LESS retroperitoneoscopic adrenalectomy for obese individuals is technically feasible and safe in experienced hands, offering perioperative outcomes comparable with those of the conventional multiport approach, but with a superior cosmetic outcome.