BACKGROUND: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors. New drug targets and proteins that would assist sensitive PPGL imagining could improve therapy and quality of life of patients with PPGL, namely those with recurrent or metastatic disease. Using a combined proteomic strategy, we looked for such clinically relevant targets among integral membrane proteins (IMPs) upregulated on the surface of tumor cells and non-membrane druggable enzymes in PPGL. METHODS: We conducted a detailed proteomic analysis of 22 well-characterized human PPGL samples and normal chromaffin tissue from adrenal medulla. A standard quantitative proteomic analysis of tumor lysate, which provides information largely on non-membrane proteins, was accompanied by specific membrane proteome-aimed methods, namely glycopeptide enrichment using lectin-affinity, glycopeptide capture by hydrazide chemistry, and enrichment of membrane-embedded hydrophobic transmembrane segments. RESULTS: The study identified 67 cell surface integral membrane proteins strongly upregulated in PPGL compared to control chromaffin tissue. We prioritized the proteins based on their already documented direct role in cancer cell growth or progression. Increased expression of the seven most promising drug targets (CD146, CD171, ANO1, CD39, ATP8A1, ACE and SLC7A1) were confirmed using specific antibodies. Our experimental strategy also provided expression data for soluble proteins. Among the druggable non-membrane enzymes upregulated in PPGL, we identified three potential drug targets (SHMT2, ARG2 and autotaxin) and verified their upregulated expression. CONCLUSIONS: Application of a combined proteomic strategy recently presented as "Pitchfork" enabled quantitative analysis of both, membrane and non-membrane proteome, and resulted in identification of 10 potential drug targets in human PPGL. Seven membrane proteins localized on the cell surface and three non-membrane druggable enzymes proteins were identified and verified as significantly upregulated in PPGL. All the proteins have been previously shown to be upregulated in several human cancers, and play direct role in cancer progression. Marked upregulation of these proteins along with their localization and established direct roles in tumor progression make these molecules promising candidates as drug targets or proteins for sensitive PPGL imaging.
- Publikační typ
- časopisecké články MeSH
Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors arising from chromaffin cells of adrenal medulla or sympathetic or parasympathetic paraganglia, respectively. To identify new therapeutic targets, we performed a detailed membrane-focused proteomic analysis of five human paraganglioma (PGL) samples. Using the Pitchfork strategy, which combines specific enrichments of glycopeptides, hydrophobic transmembrane segments, and non-glycosylated extra-membrane peptides, we identified over 1800 integral membrane proteins (IMPs). We found 45 "tumor enriched" proteins, i.e., proteins identified in all five PGLs but not found in control chromaffin tissue. Among them, 18 IMPs were predicted to be localized on the cell surface, a preferred drug targeting site, including prostate-specific membrane antigen (PSMA), a well-established target for nuclear imaging and therapy of advanced prostate cancer. Using specific antibodies, we verified PSMA expression in 22 well-characterized human PPGL samples. Compared to control chromaffin tissue, PSMA was markedly overexpressed in high-risk PPGLs belonging to the established Cluster 1, which is characterized by worse clinical outcomes, pseudohypoxia, multiplicity, recurrence, and metastasis, specifically including SDHB, VHL, and EPAS1 mutations. Using immunohistochemistry, we localized PSMA expression to tumor vasculature. Our study provides the first direct evidence of PSMA overexpression in PPGLs which could translate to therapeutic and diagnostic applications of anti-PSMA radio-conjugates in high-risk PPGLs.
- MeSH
- antigeny povrchové genetika MeSH
- feochromocytom diagnóza genetika MeSH
- glutamátkarboxypeptidasa II genetika MeSH
- lidé MeSH
- nádory nadledvin diagnóza genetika MeSH
- paragangliom diagnóza genetika MeSH
- proteom genetika MeSH
- teranostická nanomedicína MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Integral membrane proteins are under-represented in standard proteomic analyses, mostly because of their low expression and absence of trypsin-cleavage sites in their hydrophobic transmembrane segments. Novel and effective strategies for membrane proteomic analysis aim at soluble N-glycosylated segments of integral membrane proteins (CSC, SPEG, N-glyco-FASP) or selectively target the hydrophobic transmembrane alpha-helical segments employing chemical peptide cleavage by CNBr (hpTC). We combined a solid phase enrichment of glycopeptides (SPEG) with a transmembrane segment-oriented hpTC method and a standard "detergent and trypsin" approach into a three-pronged "Pitchfork" strategy to maximize the membrane proteome coverage in human lymphoma cells. This strategy enabled the identification of >1200 integral membrane proteins from all cellular compartments, including 105 CD antigens, 24 G protein-coupled receptors, and 141 solute carrier transporters. The advantage of the combination lies in the complementarity of the methods. SPEG and hpTC target different sets of membrane proteins. HpTC provided identifications of proteins and peptides with significantly higher hydrophobicity compared to SPEG and detergent-trypsin approaches. Among all identified proteins, we observed 32 so-called "missing proteins". The Pitchfork strategy presented here is universally applicable and enables deep and fast description of membrane proteomes in only 3 LC-MS/MS runs per replicate. SIGNIFICANCE: Integral membrane proteins (IMPs) are encoded by roughly a quarter of human coding genes. Their functions and their specific localization makes IMPs highly attractive drug targets. In fact, roughly half of the currently approved drugs in medicine target IMPs. Our knowledge of membrane proteomes is, however, limited. We present a new strategy for the membrane proteome analysis that combines three complementary methods targeting different features of IMPs. Using the combined strategy, we identified over 1200 IMPs in human lymphoma tissue from all sub-cellular compartments in only 3 LC-MS/MS runs per replicate. The three-pronged "Pitchfork" strategy is universally applicable, and offers a fast way toward a reasonably concise description of membrane proteomes in multiple samples.
- MeSH
- chromatografie kapalinová MeSH
- heterografty MeSH
- lidé MeSH
- lymfom z plášťových buněk metabolismus MeSH
- membránové proteiny metabolismus MeSH
- myši MeSH
- nádorové proteiny metabolismus MeSH
- proteom metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- transplantace nádorů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mantle cell lymphoma (MCL) is a chronically relapsing aggressive type of B-cell non-Hodgkin lymphoma considered incurable by currently used treatment approaches. Fludarabine is a purine analog clinically still widely used in the therapy of relapsed MCL. Molecular mechanisms of fludarabine resistance have not, however, been studied in the setting of MCL so far. We therefore derived fludarabine-resistant MCL cells (Mino/FR) and performed their detailed functional and proteomic characterization compared to the original fludarabine sensitive cells (Mino). We demonstrated that Mino/FR were highly cross-resistant to other antinucleosides (cytarabine, cladribine, gemcitabine) and to an inhibitor of Bruton tyrosine kinase (BTK) ibrutinib. Sensitivity to other types of anti-lymphoma agents was altered only mildly (methotrexate, doxorubicin, bortezomib) or remained unaffacted (cisplatin, bendamustine). The detailed proteomic analysis of Mino/FR compared to Mino cells unveiled over 300 differentially expressed proteins. Mino/FR were characterized by the marked downregulation of deoxycytidine kinase (dCK) and BTK (thus explaining the observed crossresistance to antinucleosides and ibrutinib), but also by the upregulation of several enzymes of de novo nucleotide synthesis, as well as the up-regulation of the numerous proteins of DNA repair and replication. The significant upregulation of the key antiapoptotic protein Bcl-2 in Mino/FR cells was associated with the markedly increased sensitivity of the fludarabine-resistant MCL cells to Bcl-2-specific inhibitor ABT199 compared to fludarabine-sensitive cells. Our data thus demonstrate that a detailed molecular analysis of drug-resistant tumor cells can indeed open a way to personalized therapy of resistant malignancies.
- MeSH
- antitumorózní látky farmakologie MeSH
- chemorezistence * MeSH
- chromatografie kapalinová metody MeSH
- izotopové značení metody MeSH
- lidé MeSH
- lymfom z plášťových buněk farmakoterapie metabolismus MeSH
- nádorové biomarkery metabolismus MeSH
- nádorové buňky kultivované MeSH
- proteomika metody MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vidarabin analogy a deriváty farmakologie MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Mantle cell lymphoma (MCL) is an aggressive type of B-cell non-Hodgkin lymphoma associated with poor prognosis. Implementation of high-dose cytarabine (araC) into induction therapy became standard-of-care for all newly diagnosed younger MCL patients. However, many patients relapse even after araC-based regimen. Molecular mechanisms responsible for araC resistance in MCL are unknown and optimal treatment strategy for relapsed/refractory MCL patients remains elusive. METHODS: Five araC-resistant (R) clones were derived by long-term culture of five MCL cell lines (CTRL) with increasing doses of araC up to 50 microM. Illumina BeadChip and 2-DE proteomic analysis were used to identify gene and protein expression changes associated with araC resistance in MCL. In vitro cytotoxicity assays and experimental therapy of MCL xenografts in immunodeficient mice were used to analyze their relative responsiveness to a set of clinically used anti-MCL drugs. Primary MCL samples were obtained from patients at diagnosis and after failure of araC-based therapies. RESULTS: Marked downregulation of deoxycytidine-kinase (DCK) mRNA and protein expression was identified as the single most important molecular event associated with araC-resistance in all tested MCL cell lines and in 50% primary MCL samples. All R clones were highly (20-1000x) cross-resistant to all tested nucleoside analogs including gemcitabine, fludarabine and cladribine. In vitro sensitivity of R clones to other classes of clinically used anti-MCL agents including genotoxic drugs (cisplatin, doxorubicin, bendamustine) and targeted agents (bortezomib, temsirolimus, rituximab) remained unaffected, or was even increased (ibrutinib). Experimental therapy of immunodeficient mice confirmed the anticipated loss of anti-tumor activity (as determined by overall survival) of the nucleoside analogs gemcitabine and fludarabine in mice transplanted with R clone compared to mice transplanted with CTRL cells, while the anti-tumor activity of cisplatin, temsirolimus, bortezomib, bendamustine, cyclophosphamide and rituximab remained comparable between the two cohorts. CONCLUSIONS: Acquired resistance of MCL cells to araC is associated with downregulation of DCK, enzyme of the nucleotide salvage pathway responsible for the first phosphorylation (=activation) of most nucleoside analogs used in anti-cancer therapy. The data suggest that nucleoside analogs should not be used in the therapy of MCL patients, who relapse after failure of araC-based therapies.
- MeSH
- 2D gelová elektroforéza MeSH
- antitumorózní látky farmakologie terapeutické užití MeSH
- buněčné klony MeSH
- chemorezistence účinky léků MeSH
- cytarabin farmakologie MeSH
- deoxycytidin analogy a deriváty farmakologie MeSH
- deoxycytidinkinasa metabolismus MeSH
- down regulace účinky léků MeSH
- hmotnostní spektrometrie MeSH
- kladribin farmakologie MeSH
- lidé MeSH
- lymfom z plášťových buněk farmakoterapie enzymologie genetika MeSH
- myší monoklonální protilátky farmakologie terapeutické užití MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- proteomika MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- stanovení celkové genové exprese MeSH
- vidarabin analogy a deriváty farmakologie MeSH
- western blotting MeSH
- xenogenní modely - testy antitumorózní aktivity MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mantle cell lymphoma (MCL) is a rare aggressive type of B-cell non-Hodgkin's lymphoma. Response to chemotherapy tends to be short and virtually all patients sooner or later relapse. The prognosis of relapsed patients is extremely poor. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is considered one of the novel experimental molecules with strong antitumor effects. TRAIL triggers extrinsic apoptotis in tumor cells by binding to TRAIL 'death receptors' on the cell surface. Recombinant TRAIL has shown promising pro-apoptotic effects in a variety of malignancies including lymphoma. However, as with other drugs, lymphoma cells can develop resistance to TRAIL. Therefore, the aim of this study was to identify the molecular mechanisms responsible for, and associated with TRAIL resistance in MCL cells. If identified, these features may be used as molecular targets for the effective elimination of TRAIL-resistant lymphoma cells. From an established TRAIL-sensitive mantle cell lymphoma cell line (HBL-2) we derived a TRAIL-resistant HBL-2/R subclone. By TRAIL receptor analysis and differential proteomic analysis of HBL-2 and HBL-2/R cells we revealed a marked downregulation of all TRAIL receptors and, among others, the decreased expression of 3 key enzymes of purine nucleotide metabolism, namely purine nucleoside phosphorylase, adenine phosphoribosyltransferase and inosine-5'-monophosphate dehydrogenase 2, in the resistant HBL-2/R cells. The downregulation of the 3 key enzymes of purine metabolism can have profound effects on nucleotide homeostasis in TRAIL-resistant lymphoma cells and can render such cells vulnerable to any further disruption of purine nucleotide metabolism. This pathway represents a 'weakness' of the TRAIL-resistant MCL cells and has potential as a therapeutic target for the selective elimination of such cells.
- MeSH
- 2D gelová elektroforéza MeSH
- buněčná membrána metabolismus MeSH
- buněčná smrt účinky léků MeSH
- chemorezistence účinky léků MeSH
- lidé MeSH
- lymfom z plášťových buněk enzymologie patologie MeSH
- nádorové buněčné linie MeSH
- nádorové proteiny metabolismus MeSH
- protein TRAIL farmakologie MeSH
- proteomika MeSH
- průtoková cytometrie MeSH
- puriny metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- TRAIL receptory metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Chronic hemodynamic overloading leads to heart failure (HF) due to incompletely understood mechanisms. To gain deeper insight into the molecular pathophysiology of volume overload-induced HF and to identify potential markers and targets for novel therapies, we performed proteomic and mRNA expression analysis comparing myocardium from Wistar rats with HF induced by a chronic aorto-caval fistula (ACF) and sham-operated rats harvested at the advanced, decompensated stage of HF. METHODS: We analyzed control and failing myocardium employing iTRAQ labeling, two-dimensional peptide separation combining peptide IEF and nano-HPLC with MALDI-MS/MS. For the transcriptomic analysis we employed Illumina RatRef-12v1 Expression BeadChip. RESULTS: In the proteomic analysis we identified 2030 myocardial proteins, of which 66 proteins were differentially expressed. The mRNA expression analysis identified 851 differentially expressed mRNAs. CONCLUSIONS: The differentially expressed proteins confirm a switch in the substrate preference from fatty acids to other sources in the failing heart. Failing hearts showed downregulation of the major calcium transporters SERCA2 and ryanodine receptor 2 and altered expression of creatine kinases. Decreased expression of two NADPH producing proteins suggests a decreased redox reserve. Overexpression of annexins supports their possible potential as HF biomarkers. Most importantly, among the most up-regulated proteins in ACF hearts were monoamine oxidase A and transglutaminase 2 that are both potential attractive targets of low molecular weight inhibitors in future HF therapy.
- Publikační typ
- časopisecké články MeSH
