Quantitative proteomics reveals reduction of endocytic machinery components in gliomas.

BACKGROUND: Gliomas are the most frequent and aggressive malignancies of the central nervous system. Decades of molecular analyses have demonstrated that gliomas accumulate genetic alterations that culminate in enhanced activity of receptor tyrosine kinases and downstream mediators. While the genetic alterations, like gene amplification or loss, have been well characterized, little information exists about changes in the proteome of gliomas of different grades. METHODS: We performed unbiased quantitative proteomics of human glioma biopsies by mass spectrometry followed by bioinformatic analysis. FINDINGS: Various pathways were found to be up- or downregulated. In particular, endocytosis as pathway was affected by a vast and concomitant reduction of multiple machinery components involved in initiation, formation, and scission of endocytic carriers. Both clathrin-dependent and -independent endocytosis were changed, since not only clathrin, AP-2 adaptins, and endophilins were downregulated, but also dynamin that is shared by both pathways. The reduction of endocytic machinery components caused increased receptor cell surface levels, a prominent phenotype of defective endocytosis. Analysis of additional biopsies revealed that depletion of endocytic machinery components was a common trait of various glioma grades and subclasses. INTERPRETATION: We propose that impaired endocytosis creates a selective advantage in glioma tumor progression due to prolonged receptor tyrosine kinase signaling from the cell surface. FUND: This work was supported by Grants 316030-164105 (to P. Jenö), 31003A-162643 (to M. Spiess) and PP00P3-176974 (to G. Hutter) from the Swiss National Science Foundation. Further funding was received by the Department of Surgery from the University Hospital Basel.

Regulation of glioma cell invasion by 3q26 gene products PIK3CA, SOX2 and OPA1.

Diffuse gliomas progress by invading neighboring brain tissue to promote postoperative relapse. Transcription factor SOX2 is highly expressed in invasive gliomas and maps to chromosome region 3q26 together with the genes for PI3K/AKT signaling activator PIK3CA and effector molecules of mitochondria fusion and cell invasion, MFN1 and OPA1. Gene copy number analysis at 3q26 from 129 glioma patient biopsies revealed mutually exclusive SOX2 amplifications (26%) and OPA1 losses (19%). Both forced SOX2 expression and OPA1 inactivation increased LN319 glioma cell invasion in vitro and promoted cell dispersion in vivo in xenotransplanted D. rerio embryos. While PI3 kinase activity sustained SOX2 expression, pharmacological PI3K/AKT pathway inhibition decreased invasion and resulted in SOX2 nucleus-to-cytoplasm translocation in an mTORC1-independent manner. Chromatin immunoprecipitation and luciferase reporter gene assays together demonstrated that SOX2 trans-activates PIK3CA and OPA1. Thus, SOX2 activates PI3K/AKT signaling in a positive feedback loop, while OPA1 deletion is interpreted to counteract OPA1 trans-activation. Remarkably, neuroimaging of human gliomas with high SOX2 or low OPA1 genomic imbalances revealed significantly larger necrotic tumor zone volumes, corresponding to higher invasive capacities of tumors, while autologous necrotic cells are capable of inducing higher invasion in SOX2 overexpressing or OPA1 knocked-down relative to parental LN319. We thus propose necrosis volume as a surrogate marker for the assessment of glioma invasive potential. Whereas glioma invasion is activated by a PI3K/AKT-SOX2 loop, it is reduced by a cryptic invasion suppressor SOX2-OPA1 pathway. Thus, PI3K/AKT-SOX2 and mitochondria fission represent connected signaling networks regulating glioma invasion.

Proteomic Landscape of Aldosterone-Producing Adenoma.

Primary aldosteronism is a disease of excessive production of adrenal steroid hormones and the most common cause of endocrine hypertension. Primary aldosteronism results mainly from bilateral adrenal hyperplasia or unilateral aldosterone-producing adenoma (APA). Primary aldosteronism cause at the molecular level is incompletely understood and a targeted treatment preventing excessive adrenal steroid production is not available. Here, we perform deep quantitative proteomic and phosphoproteomic profiling of 6 pairs of APA and adjacent nontumoral adrenal cortex. We show that increased steroidogenesis in APA is accompanied by upregulation of steroidogenic enzymes (HSD3B2, CYP21A2, CYP11B2) and of proteins involved in cholesterol uptake (LSR). We demonstrate that HSD3B2 is phosphorylated at Ser95 or 96 and identify a novel phosphorylation site, Ser489, in CYP21A2, suggesting that steroidogenic enzymes are regulated by phosphorylation. Our analysis also reveals altered ECM (extracellular matrix) composition in APA that affects ECM-cell surface interactions and actin cytoskeleton rearrangements. We show that RHOC, a GTPase controlling actin organization in response to extracellular stimuli, is upregulated in APA and promotes expression of the aldosterone synthase gene CYP11B2. Our data also indicate deregulation of protein N-glycosylation and GABAergic signaling in APAs. Finally, we find that mTORC1 (mammalian target of rapamycin complex 1) signaling is the major pathway deregulated in APA. Our study provides a rich resource for future research on the molecular mechanisms of primary aldosteronism.

Tear Film Proteomics Reveal Important Differences Between Patients With and Without Ocular GvHD After Allogeneic Hematopoietic Cell Transplantation.

Purpose: To date, no biomarkers for ocular graft versus host disease (GvHD), a frequent complication following allogeneic hematopoietic cell transplantation (HCT), exist. In this prospective study, we evaluated the potential of human tear proteins as biomarkers for ocular GvHD. Methods: Tears from 10 patients with moderate-to-severe ocular GvHD were compared to 10 patients without ocular GvHD. After a full ocular surface clinical examination, tears were collected onto Schirmer strips and protein composition was analyzed by liquid chromatography tandem mass spectrometry. Statistical evaluation was performed using the Mann-Whitney U test to compare means and the false discovery rate method to adjust for multiple comparisons. Functional annotation of differentially expressed proteins was done with the PANTHER classification system. Results: We identified 282 proteins in tryptic digests of Schirmer strips; 79 proteins were significantly differentially expressed between the two groups, from which 54 were up- and 25 downregulated. The most upregulated proteins were classified as nucleic acid binding and cytoskeletal proteins, while the most extensively downregulated proteins belong to an array of classes including transfer and receptor proteins, enzyme modulators, and hydrolases. In addition to proteins already confirmed as differentially expressed in dry eye disease, we report changes in 36 novel proteins. Conclusions: This study reports the proteomic profile of tears in ocular GvHD for the first time and identifies a number of unique differentially expressed proteins. Further studies with a higher number of participants are necessary to confirm these results and to evaluate the reliability of these expression patterns in longitudinal studies.

The protein histidine phosphatase LHPP is a tumour suppressor.

Histidine phosphorylation, the so-called hidden phosphoproteome, is a poorly characterized post-translational modification of proteins. Here we describe a role of histidine phosphorylation in tumorigenesis. Proteomic analysis of 12 tumours from an mTOR-driven hepatocellular carcinoma mouse model revealed that NME1 and NME2, the only known mammalian histidine kinases, were upregulated. Conversely, expression of the putative histidine phosphatase LHPP was downregulated specifically in the tumours. We demonstrate that LHPP is indeed a protein histidine phosphatase. Consistent with these observations, global histidine phosphorylation was significantly upregulated in the liver tumours. Sustained, hepatic expression of LHPP in the hepatocellular carcinoma mouse model reduced tumour burden and prevented the loss of liver function. Finally, in patients with hepatocellular carcinoma, low expression of LHPP correlated with increased tumour severity and reduced overall survival. Thus, LHPP is a protein histidine phosphatase and tumour suppressor, suggesting that deregulated histidine phosphorylation is oncogenic.

Insulin resistance causes inflammation in adipose tissue.

Obesity is a major risk factor for insulin resistance and type 2 diabetes. In adipose tissue, obesity-mediated insulin resistance correlates with the accumulation of proinflammatory macrophages and inflammation. However, the causal relationship of these events is unclear. Here, we report that obesity-induced insulin resistance in mice precedes macrophage accumulation and inflammation in adipose tissue. Using a mouse model that combines genetically induced, adipose-specific insulin resistance (mTORC2-knockout) and diet-induced obesity, we found that insulin resistance causes local accumulation of proinflammatory macrophages. Mechanistically, insulin resistance in adipocytes results in production of the chemokine monocyte chemoattractant protein 1 (MCP1), which recruits monocytes and activates proinflammatory macrophages. Finally, insulin resistance (high homeostatic model assessment of insulin resistance [HOMA-IR]) correlated with reduced insulin/mTORC2 signaling and elevated MCP1 production in visceral adipose tissue from obese human subjects. Our findings suggest that insulin resistance in adipose tissue leads to inflammation rather than vice versa.

mTORC2 Promotes Tumorigenesis via Lipid Synthesis.

Dysregulated mammalian target of rapamycin (mTOR) promotes cancer, but underlying mechanisms are poorly understood. We describe an mTOR-driven mouse model that displays hepatosteatosis progressing to hepatocellular carcinoma (HCC). Longitudinal proteomic, lipidomics, and metabolomic analyses revealed that hepatic mTORC2 promotes de novo fatty acid and lipid synthesis, leading to steatosis and tumor development. In particular, mTORC2 stimulated sphingolipid (glucosylceramide) and glycerophospholipid (cardiolipin) synthesis. Inhibition of fatty acid or sphingolipid synthesis prevented tumor development, indicating a causal effect in tumorigenesis. Increased levels of cardiolipin were associated with tubular mitochondria and enhanced oxidative phosphorylation. Furthermore, increased lipogenesis correlated with elevated mTORC2 activity and HCC in human patients. Thus, mTORC2 promotes cancer via formation of lipids essential for growth and energy production.

Syrosingopine sensitizes cancer cells to killing by metformin.

We report that the anticancer activity of the widely used diabetic drug metformin is strongly potentiated by syrosingopine. Synthetic lethality elicited by combining the two drugs is synergistic and specific to transformed cells. This effect is unrelated to syrosingopine's known role as an inhibitor of the vesicular monoamine transporters. Syrosingopine binds to the glycolytic enzyme α-enolase in vitro, and the expression of the γ-enolase isoform correlates with nonresponsiveness to the drug combination. Syrosingopine sensitized cancer cells to metformin and its more potent derivative phenformin far below the individual toxic threshold of each compound. Thus, combining syrosingopine and codrugs is a promising therapeutic strategy for clinical application for the treatment of cancer.

Quantitative proteomics and phosphoproteomics on serial tumor biopsies from a sorafenib-treated HCC patient.

Compensatory signaling pathways in tumors confer resistance to targeted therapy, but the pathways and their mechanisms of activation remain largely unknown. We describe a procedure for quantitative proteomics and phosphoproteomics on snap-frozen biopsies of hepatocellular carcinoma (HCC) and matched nontumor liver tissue. We applied this procedure to monitor signaling pathways in serial biopsies taken from an HCC patient before and during treatment with the multikinase inhibitor sorafenib. At diagnosis, the patient had an advanced HCC. At the time of the second biopsy, abdominal imaging revealed progressive disease despite sorafenib treatment. Sorafenib was confirmed to inhibit MAPK signaling in the tumor, as measured by reduced ribosomal protein S6 kinase phosphorylation. Hierarchical clustering and enrichment analysis revealed pathways broadly implicated in tumor progression and resistance, such as epithelial-to-mesenchymal transition and cell adhesion pathways. Thus, we describe a protocol for quantitative analysis of oncogenic pathways in HCC biopsies and obtained first insights into the effect of sorafenib in vivo. This protocol will allow elucidation of mechanisms of resistance and enable precision medicine.

Cytokine-induced macropinocytosis in macrophages is regulated by 14-3-3ζ through its interaction with serine-phosphorylated coronin 1.

The induction of macropinocytosis in macrophages during an inflammatory response is important for clearance of pathogenic microbes as well as the generation of appropriate immune responses. Recent data suggest that cytokine stimulation of macrophages induces macropinocytosis through phosphorylation of the protein coronin 1, thereby redistributing coronin 1 from the cell cortex to the cytoplasm followed by the activation of phosphoinositol-3 (PI-3) kinase. However, how coronin 1 phosphorylation regulates these processes remains unclear. We here define an essential role for 14-3-3ζ in cytokine-induced and coronin-1-dependent macropinocytosis in macrophages. We found that, upon stimulation, phosphorylated coronin 1 transiently associated with 14-3-3ζ and receptor of activated C kinase 1 (RACK1). Importantly, downregulation of 14-3-3ζ, but not RACK1, prevented relocation of coronin 1, as well as the induction of PI-3 kinase activity and thereby macropinocytosis upon cytokine stimulation. Together these data define an essential role for 14-3-3ζ in the regulation of macropinocytosis in macrophages upon cytokine stimulation through modulation of the localization of coronin 1.

Implications of the E-selectin S128R mutation for drug discovery.

The C-type lectin E-selectin mediates the rolling of circulating leukocytes on vascular endothelial cells during the inflammatory process. In numerous studies, the S128R mutation of the E-selectin was associated with cardiovascular and autoimmune diseases. There is evidence that the S128R E-selectin mutation leads to a loss in ligand specificity, thus increasing leukocyte recruitment. Apart from the natural tetrasaccharide ligand sialyl Lewis(x) (sLe(x)), it has previously been proposed that non-fucosylated carbohydrates also bind to S128R E-selectin. To evaluate the therapeutic potential of the antagonism of the E-selectin mutant, ligand specificity was reinvestigated on a molecular basis. We determined the ligand specificity of wild-type and S128R E-selectin in a target-based competitive assay, a glycan array screen and cell-based binding assays under static and flow conditions. Regarding ligand-specificity, the binding properties of S128R E-selectin were identical to those of wt E-selectin, i.e., no mutant-specific binding of 3'-sialyl-N-acetyllactosamine, heparin, fetuin and K562 cells was observed. Additionally, the binding affinities of glycomimetic E-selectin antagonists were identical for wt and S128R E-selectin. Overall, the previous reports on carbohydrate ligand promiscuity of S128R E-selectin could not be confirmed.

Integrated epigenetics of human breast cancer: synoptic investigation of targeted genes, microRNAs and proteins upon demethylation treatment.

BACKGROUND: The contribution of aberrant DNA methylation in silencing of tumor suppressor genes (TSGs) and microRNAs has been investigated. Since these epigenetic alterations are reversible, it became of interest to determine the effects of the 5-aza-2'-deoxycytidine (DAC) demethylation therapy in breast cancer at different molecular levels. METHODS AND FINDINGS: Here we investigate a synoptic model to predict complete DAC treatment effects at the level of genes, microRNAs and proteins for several human breast cancer lines. The present study assessed an effective treatment dosage based on the cell viability, cytotoxicity, apoptosis and methylation assays for the investigated cell lines. A highly aggressive and a non-aggressive cell line were investigated using omics approaches such as MALDI-TOF MS, mRNA- and microRNA expression arrays, 2-D gel electrophoresis and LC-MS-MS. Complete molecular profiles including the biological interaction and possible early and late systematic stable or transient effects of the methylation inhibition were determined. Beside the activation of several epigenetically suppressed TSGs, we also showed significant dysregulation of some important oncogenes, oncomiRs and oncosuppressors miRNAs as well as drug tolerance genes/miRNAs/proteins. CONCLUSIONS: In the present study, the results denote some new molecular DAC targets and pathways based on the chemical modification of DNA methylation in breast cancer. The outlined approach might prove to be useful as an epigenetic treatment model also for other human solid tumors in the management of cancer patients.

The genome and surface proteome of Capnocytophaga canimorsus reveal a key role of glycan foraging systems in host glycoproteins deglycosylation.

Capnocytophaga canimorsus are commensal Gram-negative bacteria from dog's mouth that cause rare but dramatic septicaemia in humans. C. canimorsus have the unusual property to feed on cultured mammalian cells, including phagocytes, by harvesting the glycan moiety of cellular glycoproteins. To understand the mechanism behind this unusual property, the genome of strain Cc5 was sequenced and analysed. In addition, Cc5 bacteria were cultivated onto HEK 293 cells and the surface proteome was determined. The genome was found to encode many lipoproteins encoded within 13 polysaccharide utilization loci (PULs) typical of the Flavobacteria-Bacteroides group. PULs encode surface exposed feeding complexes resembling the archetypal starch utilization system (Sus). The products of at least nine PULs were detected among the surface proteome and eight of them represented more than half of the total peptides detected from the surface proteome. Systematic deletions of the 13 PULs revealed that half of these Sus-like complexes contributed to growth on animal cells. The complex encoded by PUL5, one of the most abundant ones, was involved in foraging glycans from glycoproteins. It was essential for growth on cells and contributed to survival in mice. It thus represents a fitness factor during infection.

The rapamycin-sensitive phosphoproteome reveals that TOR controls protein kinase A toward some but not all substrates.

Regulation of cell growth requires extensive coordination of several processes including transcription, ribosome biogenesis, translation, nutrient metabolism, and autophagy. In yeast, the protein kinases Target of Rapamycin (TOR) and protein kinase A (PKA) regulate these processes and are thereby the main activators of cell growth in response to nutrients. How TOR, PKA, and their corresponding signaling pathways are coordinated to control the same cellular processes is not understood. Quantitative analysis of the rapamycin-sensitive phosphoproteome combined with targeted analysis of PKA substrates suggests that TOR complex 1 (TORC1) activates PKA but only toward a subset of substrates. Furthermore, we show that TORC1 signaling impinges on BCY1, the negative regulatory subunit of PKA. Inhibition of TORC1 with rapamycin leads to BCY1 phosphorylation on several sites including T129. Phosphorylation of BCY1 T129 results in BCY1 activation and inhibition of PKA. TORC1 inhibits BCY1 T129 phosphorylation by phosphorylating and activating the S6K homolog SCH9 that in turn inhibits the MAP kinase MPK1. MPK1 phosphorylates BCY1 T129 directly. Thus, TORC1 activates PKA toward some substrates by preventing MPK1-mediated activation of BCY1.

A modified KESTREL search reveals a basophilic substrate consensus for the Saccharomyces cerevisiae Npr1 protein kinase.

The Saccharomyces cerevisiae nitrogen permease reactivator Npr1 is a hyperphosphorylated protein that belongs to a family of Ser/Thr protein kinases dedicated to the regulation of plasma membrane transporters. Its activity is regulated by the Tor (target of rapamycin) signaling pathway. Inhibition of the Tor proteins by treating yeast cells with the immunosuppressant drug rapamycin promotes rapid dephosphorylation of Npr1. As an alternative to peptide arrays, the substrate requirement of Npr1 was probed with a peptide library that was generated by cleaving yeast cell extracts with CNBr, and after reverse-phase chromatography, the individual fractions were phosphorylated in vitro with recombinant Npr1. In this way, the ribosomal protein Rpl24a was found to be an excellent in vitro substrate for Npr1. Synthetic peptides tailored around the phosphorylation site of Rpl24a show that Npr1 is a Ser/Thr protein kinase with an absolute requirement for a basic residue at the P-3 position and a strong preference for basic P + 1 residues, whereas proline at P + 1 is strongly disfavored. The results obtained with synthetic peptides suggest a (K/R)-X-X-S-(K/R) consensus sequence for Npr1. The availability of a consensus sequence allows a targeted search for physiologically relevant Npr1 substrates involved in the regulation of yeast amino acid permeases.

Proteins induced by telomere dysfunction and DNA damage represent biomarkers of human aging and disease.

Telomere dysfunction limits the proliferative capacity of human cells by activation of DNA damage responses, inducing senescence or apoptosis. In humans, telomere shortening occurs in the vast majority of tissues during aging, and telomere shortening is accelerated in chronic diseases that increase the rate of cell turnover. Yet, the functional role of telomere dysfunction and DNA damage in human aging and diseases remains under debate. Here, we identified marker proteins (i.e., CRAMP, stathmin, EF-1alpha, and chitinase) that are secreted from telomere-dysfunctional bone-marrow cells of late generation telomerase knockout mice (G4mTerc(-/-)). The expression levels of these proteins increase in blood and in various tissues of aging G4mTerc(-/-) mice but not in aging mice with long telomere reserves. Orthologs of these proteins are up-regulated in late-passage presenescent human fibroblasts and in early passage human cells in response to gamma-irradiation. The study shows that the expression level of these marker proteins increases in the blood plasma of aging humans and shows a further increase in geriatric patients with aging-associated diseases. Moreover, there was a significant increase in the expression of the biomarkers in the blood plasma of patients with chronic diseases that are associated with increased rates of cell turnover and telomere shortening, such as cirrhosis and myelodysplastic syndromes (MDS). Analysis of blinded test samples validated the effectiveness of the biomarkers to discriminate between young and old, and between disease groups (MDS, cirrhosis) and healthy controls. These results support the concept that telomere dysfunction and DNA damage are interconnected pathways that are activated during human aging and disease.