Altered expression of the IGF2­H19 locus and mitochondrial respiratory complexes in adrenocortical carcinoma.

Abnormalities of the insulin­like growth factor 2 (IGF2)­H19 locus with the overexpression of IGF2 are frequent findings in adrenocortical carcinoma (ACC). The present study assessed the expression of RNAs and microRNAs (miRNAs/miRs) from the IGF2­H19 locus using PCR­based methods in ACC and adrenocortical adenoma (ACA). The results were associated with proteomics data. IGF2 was overexpressed in ACC, and its expression correlated with that of miR­483­3p and miR­483­5p hosted by IGF2. The downregulated expression of H19 in ACC compared to ACA correlated with miR­675 expression hosted by H19. Several proteins exhibited an inverse correlation in expression and were predicted as targets of miR­483­3p, miR­483­5p or miR­675. Subsets of these proteins were differentially expressed between ACC and ACA. These included several proteins involved in mitochondrial metabolism. Among the mitochondrial respiratory complexes, complex I and IV were significantly decreased in ACC compared to ACA. The protein expression of NADH:ubiquinone oxidoreductase subunit C1 (NDUFC1), a subunit of mitochondrial respiratory complex I, was further validated as being lower in ACC compared to ACA and normal adrenals. The silencing of miR­483­5p increased NDUFC1 protein expression and reduced both oxygen consumption and glycolysis rates. On the whole, the findings of the present study reveal the dysregulation of the IGF2­H19 locus and mitochondrial respiration in ACC. These findings may provide a basis for the further understanding of the pathogenesis of ACC and may have potential values for diagnostics and treatment.

Proteomics identifies neddylation as a potential therapy target in small intestinal neuroendocrine tumors.

Patients with small intestinal neuroendocrine tumors (SI-NETs) frequently develop spread disease; however, the underlying molecular mechanisms of disease progression are not known and effective preventive treatment strategies are lacking. Here, protein expression profiling was performed by HiRIEF-LC-MS in 14 primary SI-NETs from patients with and without liver metastases detected at the time of surgery and initial treatment. Among differentially expressed proteins, overexpression of the ubiquitin-like protein NEDD8 was identified in samples from patients with liver metastasis. Further, NEDD8 correlation analysis indicated co-expression with RBX1, a key component in cullin-RING ubiquitin ligases (CRLs). In vitro inhibition of neddylation with the therapeutic agent pevonedistat (MLN4924) resulted in a dramatic decrease of proliferation in SI-NET cell lines. Subsequent mass spectrometry-based proteomics analysis of pevonedistat effects and effects of the proteasome inhibitor bortezomib revealed stabilization of multiple targets of CRLs including p27, an established tumor suppressor in SI-NET. Silencing of NEDD8 and RBX1 using siRNA resulted in a stabilization of p27, suggesting that the cellular levels of NEDD8 and RBX1 affect CRL activity. Inhibition of CRL activity, by either NEDD8/RBX1 silencing or pevonedistat treatment of cells resulted in induction of apoptosis that could be partially rescued by siRNA-based silencing of p27. Differential expression of both p27 and NEDD8 was confirmed in a second cohort of SI-NET using immunohistochemistry. Collectively, these findings suggest a role for CRLs and the ubiquitin proteasome system in suppression of p27 in SI-NET, and inhibition of neddylation as a putative therapeutic strategy in SI-NET.

Molecular profiles of oxyphilic and chief cell parathyroid adenoma.

CONTEXT: Parathyroid adenomas may be composed of chief cells (conventional or water-clear), oxyphilic cells or a mixture of both cells. The molecular background is rarely studied. OBJECTIVE: To molecularly characterize parathyroid adenomas of different cell type composition. DESIGN: Chief and oxyphilic cell adenomas were compared in a cohort of 664 sporadic cases. Extensive analyses of parathyroid tissues were performed in subgroup. Gene expressions of known parathyroid-related genes were quantified by qRT-PCR. Protein expression profiles determined by liquid chromatography - tandem mass spectrometry (LC-MS/MS) were compared between each type of parathyroid adenomas. Selected proteins were analysed by Western blot and immunohistochemistry. RESULTS: Patients with oxyphilic cell adenoma were found to be older at the time of operation than chief cell adenoma cases but did not differ in gender, serum calcium or tumor weight. The gene expression of CASR, VDR, FGFR1, CYP27B1, CYP24A1, PTHLH, GCM2, NDUFA13, CDKN1B, MEN1 and CNND1 did not differ between the groups. VDR protein levels were weaker in oxyphilic adenomas. The proteomic studies identified a set of novel dysregulated proteins of interest such as nuclear receptor subfamily 2 group C member 2 (TR4), LIM domain only protein 3 (LMO3) and calcium-binding protein B (S100B). LMO3 and S100B showed higher expression in oxyphilic adenoma and may be involve in parathyroid tumorgenesis through the p53 pathway. TR4 showed different subcellular localisation between adenoma and normal rim. CONCLUSION: Chief and oxyphilic cell parathyroid adenomas have partly overlapping but also distinct molecular profiles. The calmodulin-eEF2K, TR4 and p53 pathways may be involved in the tumor development.

Proteomics Suggests a Role for APC-Survivin in Response to Somatostatin Analog Treatment of Neuroendocrine Tumors.

CONTEXT: Somatostatin analogs are established in the treatment of neuroendocrine tumors (NETs) including small intestinal NET; however, the molecular mechanisms are not well known. Here, we examined the direct effects of lanreotide in NET cell line models. SETTING AND DESIGN: The cell lines HC45 and H727 were treated with 10nM lanreotide for different time periods and alterations of the proteome were analyzed by in-depth high-resolution isoelectric focusing tandem liquid chromatography-mass spectrometry. We next investigated whether the observed suppression of survivin was mediated by adenomatous polyposis coli (APC) and possible effects on tumor proliferation in vitro. Expression of survivin was assessed by immunohistochemistry in 112 NET cases and compared with patient outcome. RESULTS: We quantified 6451 and 7801 proteins in HC45 and H727, respectively. After short time lanreotide treatment APC was increased and survivin reduced. Overexpression of APC in H727 cells decreased, and APC knock-down elevated the survivin level. The lanreotide regulation of APC-survivin could be suppressed by small interfering RNA against somatostatin receptor 2. Although lanreotide only gave slight inhibition of proliferation, targeting of survivin with the small molecule YM155 dramatically reduced proliferation. Moderate or high as compared with low or absent total survivin expression was associated with shorter progression-free survival, independent of tumor stage, grade, and localization. CONCLUSIONS: We report a proteome-wide analysis of changes in response to lanreotide in NET cell lines. This analysis suggests a connection between somatostatin analog, APC, and survivin levels. Survivin is a possible prognostic factor and a new potential therapeutic target in NETs.

Alterations in the membrane-associated proteome fraction of alveolar macrophages in sarcoidosis.

BACKGROUND: Alveolar macrophages are implicated in the pathogenesis of lung sarcoidosis. Their interaction with T-cells leads to an inflammatory response that may either resolve within 2 years, or become chronic with an increased risk to develop lung fibrosis. OBJECTIVE: To perform quantitative profiling of the membrane-associated proteome of alveolar macrophages in sarcoidosis patients and healthy individuals to identify specific proteins and pathways involved in sarcoidosis pathology. METHODS: Differential proteomic analysis was performed on iTRAQ (isobaric Tag for Relative and Absolute Quantitation) labeled samples using tandem mass spectrometry. Subsequently, uni- and multivariate statistical analyses and pathway- and network analyses were performed. RESULTS: Eighty proteins were differentially expressed between healthy and sarcoidosis patients. Down-stream pathway analysis confirmed our recent reports of up-regulation of two phagocytotic pathways: Fcγ receptor-mediated phagocytosis and clathrin-mediated endocytosis signaling. An additional pathway, pyruvate metabolism, was found to be up-regulated in sarcoidosis patients. The oxidative phosphorylation pathway was differentially expressed in subgroups of sarcoidosis, with up-regulation in Löfgren's patients and down-regulation in non-Löfgren's patients. CONCLUSION: This unprecedented proteome profiling of the membrane-associated fraction of alveolar macrophages confirmed previous findings of alterations in phagocytotic pathways due to sarcoidosis, as well as indicated a differential dysregulation of the oxidative phosphorylation pathway related to disease outcome in sarcoidosis.

Genetic and epigenetic background and protein expression profiles in relation to telomerase activation in medullary thyroid carcinoma.

Medullary thyroid carcinomas (MTCs) exhibit telomerase activation in strong association with shorter patient survival. To understand the background of telomerase activation we quantified TERT copy numbers and TERT promoter methylation in 42 MTCs and normal thyroid references. Gain of TERT was demonstrated by quantitative PCR in 5/39 sporadic MTC. Increased methylation index (MetI) for CpG methylation at the TERT promoter was found in sporadic MTCs (P < 0.0001) and in MEN 2 associated MTCs (P = 0.011) vs. normal thyroid tissues. MetI correlated positively with TERT gene expression (r = 0.432, P = 0.006) and negatively with telomere length (r = -0.343, P = 0.032). MTC cases with MetI above the median of 52% had shorter survival as compared to cases with lower MetI (P = 0.005 for overall survival and P = 0.007 for disease-related survival).Protein expression profiles obtained by mass spectrometry were then studied in relation to telomerase activation in MTCs. Comparing protein levels between tumors defined by telomerase activity status, 240 proteins were associated with telomerase activity. Among telomerase activation positive cases a set of proteins was found to discriminate between MTCs with high and low TERT gene expression with enrichment for proteins involved in telomerase regulation. XRCC5 mRNA expression was found increased in MTCs vs. normal thyroid (P = 0.007). In conclusion the findings suggest a role for TERT copy number gain, TERT promoter methylation and XRCC5 expression in telomerase activation and telomere maintenance of MTC.

Differential protein expression profiles of cyst fluid from papillary thyroid carcinoma and benign thyroid lesions.

Cystic papillary thyroid carcinoma (cPTC) is a subgroup of PTC presenting a diagnostic challenge at fine needle aspiration biopsy (FNAB). To further investigate this entity we aimed to characterize protein profiles of cyst fluids from cPTC and benign thyroid cystic lesions. In total, 20 cPTCs and 56 benign thyroid cystic lesions were studied. Profiling by liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed on cyst fluids from a subset of cases after depletion, and selected proteins were further analyzed by Western blot (WB), immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). A total of 1,581 proteins were detected in cyst fluids, of which 841 were quantified in all samples using LC-MS/MS. Proteins with different expression levels between cPTCs and benign lesions were identified by univariate analysis (41 proteins) and multivariate analysis (59 proteins in an orthogonal partial least squares model). WB analyses of cyst fluid and IHC on corresponding tissue samples confirmed a significant up-regulation of cytokeratin 19 (CK-19/CYFRA 21-1) and S100A13 in cPTC vs. benign lesions. These findings were further confirmed by ELISA in an extended material of non-depleted cyst fluids from cPTCs (n = 17) and benign lesions (n = 55) (p<0.05). Applying a cut-off at >55 ng/ml for CK-19 resulted in 82% specificity and sensitivity. For S100A13 a cut-off at >230 pg/ml revealed a 94% sensitivity, but only 35% specificity. This is the first comprehensive catalogue of the protein content in fluid from thyroid cysts. The up-regulations of CK-19 and S100A13 suggest their possible use in FNAB based preoperative diagnostics of cystic thyroid lesions.

RIFINs are adhesins implicated in severe Plasmodium falciparum malaria.

Rosetting is a virulent Plasmodium falciparum phenomenon associated with severe malaria. Here we demonstrate that P. falciparum-encoded repetitive interspersed families of polypeptides (RIFINs) are expressed on the surface of infected red blood cells (iRBCs), where they bind to RBCs--preferentially of blood group A--to form large rosettes and mediate microvascular binding of iRBCs. We suggest that RIFINs have a fundamental role in the development of severe malaria and thereby contribute to the varying global distribution of ABO blood groups in the human population.

Differentially expressed proteins in malignant and benign adrenocortical tumors.

We have compared the microsomal protein composition of eight malignant and six benign adrenocortical tumors with proteomic methods. IGF2 had increased level in the malignant tumors, confirming previous microarray studies on the same material. Aldolase A, a glycolytic enzyme, also showed increased levels in the malignant tissue compared to the benign. Additionally, several proteins belonging to complex I in the mitochondrial respiration chain showed decreased levels in the malignant tissue. Taken together, this may indicate a shift in energy metabolism where glycolysis may be favored over tight coupling of glycolysis and mitochondrial respiration, a phenomenon known as the Warburg effect. One of the complex I proteins that showed decreased levels in the malignant tissue was GRIM-19. This protein has been suggested as a tumor suppressive protein by being a negative regulator of STAT3. In summary, an analysis of the microsomal proteome in adrenocortical tumors identifies groups of proteins as well as specific proteins differentially expressed in the benign and malignant forms. These proteins shed light on the biology behind malignancy and could delineate future drug targets.