Differentially Expressed Proteins in Malignant and Benign Adrenocortical Tumors

<div><p>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.</p></div

Enrichment analysis.

<p>Panel A shows a pie chart depicting the number of transmembrane segments the 1902 identified and quantified proteins are predicted to contain (from ProteinCenter). Panel B shows the GOrilla results performed on the 1902 proteins, ranked by t-test p-value. Shown are enriched GO terms in “cellular component”.</p

Correlation between protein expression levels and tumor size.

<p>Protein expression levels of the 26 proteins that overlapped in the t-test and OPLS analyses correlate with the size of the tumors. Two proteins have increased expression levels (light grey dots/lines), the rest have decreased expression levels (black dots/lines). Corresponding protein names can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087951#pone.0087951.s005" target="_blank">Table S4</a>.</p

Clinical data of analyzed cases.

a<p><i>Time between surgery and follow-up.</i></p>b<p><i>Dead of disease.</i></p

Western blot and immunohistochemical analyses.

<p>A–D) Expression levels of aldolase A and GRIM-19. To the left are the expression levels from the MS data and to the right are the western blot analyses. Ku70 and actin are loading controls. Western blot analyses were performed on tissue samples that showed the most significant differences in the iTRAQ experiments (two ACAs and two ACCs). E–F) Immunohistochemical analyses with anti-GRIM-19 (panels E and F). GRIM-19 staining in ACAs had a grain-like pattern, suggesting mitochondrial localization (panel E). In ACCs there was a more cytoplasmic staining (panel F).</p

Visualization of the data analysis workflow.

<p>A) Both univariate (Student's t-test) and multivariate (OPLS) analyses were performed. B) The top canonical pathway identified by Ingenuity Pathway Analysis was mitochondrial dysfunction. Many proteins in this pathway were found to be downregulated in the malignant samples. C) Hierarchical clustering of the overlapping proteins (t-test and OPLS). Class 1: ACA, class 2: ACC.</p

Top 10 proteins identified by LC-MS/MS with significantly different expression in cyst fluids from cPTCs (n = 6) compared to benign lesions (n = 7) after depletion.

<p>(*) Selected for further analyses</p><p>Top 10 proteins identified by LC-MS/MS with significantly different expression in cyst fluids from cPTCs (n = 6) compared to benign lesions (n = 7) after depletion.</p

Differential Protein Expression Profiles of Cyst Fluid from Papillary Thyroid Carcinoma and Benign Thyroid Lesions

<div><p>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.</p></div

Unsupervised hierarchical clustering showing the 59 proteins in the OPLS model.

<p>Up-regulation and down-regulation of proteins in cPTC vs. benign lesions are indicated in red and green, respectively. Proteins selected for further verification are indicated by (*).</p

Expression of CK-19 in cPTC, benign cystic lesions and references.

<p>(A) Western blot analyses of CK-19 with depleted cyst fluids of cPTCs and benign lesions from the LC-MS/MS screening samples. Depleted cyst fluids of a parathyroid adenoma (PHPT), anaplastic thyroid carcinoma (ATC) and total protein extract from A549 cells are shown as reference. Representative transfer of proteins to nitrocellulose membrane stained with 0.1% Ponceau S solution used as loading control is shown below. (<b>B</b>) Immunohistochemistry showing CK-19 expression in cPTC tissue tumor cells but not in surrounding normal thyroid cells (cPTC—normal) or multinodular goiter (Benign).</p