Discrimination between Adenocarcinoma and Normal Pancreatic Ductal Fluid by Proteomic and Glycomic Analysis

Sensitive and specific biomarkers for pancreatic cancer are currently unavailable. The high mortality associated with adenocarcinoma of the pancreatic epithelium justifies the broadest possible search for new biomarkers that can facilitate early detection or monitor treatment efficacy. Protein glycosylation is altered in many cancers, leading many to propose that glycoproteomic changes may provide suitable biomarkers. In order to assess this possibility for pancreatic cancer, we have performed an in-depth LC–MS/MS analysis of the proteome and MSⁿ-based characterization of the N-linked glycome of a small set of pancreatic ductal fluid obtained from normal, pancreatitis, intraductal papillary mucinous neoplasm (IPMN), and pancreatic adenocarcinoma patients. Our results identify a set of seven proteins that were consistently increased in cancer ductal fluid compared to normal (AMYP, PRSS1, GP2-1, CCDC132, REG1A, REG1B, and REG3A) and one protein that was consistently decreased (LIPR2). These proteins are all directly or indirectly associated with the secretory pathway in normal pancreatic cells. Validation of these changes in abundance by Western blotting revealed increased REG protein glycoform diversity in cancer. Characterization of the total N-linked glycome of normal, IPMN, and adenocarcinoma ductal fluid clustered samples into three discrete groups based on the prevalence of six dominant glycans. Within each group, the profiles of less prevalent glycans were able to distinguish normal from cancer on this small set of samples. Our results emphasize that individual variation in protein glycosylation must be considered when assessing the value of a glycoproteomic marker, but also indicate that glycosylation diversity across human subjects can be reduced to simpler clusters of individuals whose N-linked glycans share structural features

Box plots display examples of genes significantly hyper or hypomethylated in ACC.

<p><i>EPHX3</i> and <i>MEIS</i> genes are significantly hypermethylated in ACC compared with <i>TMEM132D</i> and <i>ADCY2</i> which are significantly hypomethylated.</p

Unsupervised clustering analysis of normal and ACC samples using DML.

<p>Clustering analysis revealed separation of ACC and normal samples. Samples are on the horizontal axis: normal samples are shown with a yellow bar and ACC samples are shown with a blue bar.</p

Significantly regulated genes in the WNT pathway.

<p>Significantly regulated genes in the WNT pathway.</p

Methylation/expression correlations according to discretization method.

<p>For each gene, the upper heatmap represents the log2 methylation values for 14 ACC samples each normalized to the average of 6 normal adrenal samples. Log2 methylation ratios >0 represent hypermethylation and <0 represent hypomethylation. The lower heat map shows expression of z-transformed expression levels, where a value 0 indicates no expression change compared to average expression level of 14 ACC samples. Three genes with negative correlations were selected for visualization: <i>CCDC8</i> (TP53 pathway), <i>TBX3</i> (WNT pathway), and <i>PAX8</i> (other known cancer gene, involved in invasion and migration). Samples with higher methylation (peach-maroon) had lower expression (green). Samples with lower methylation (blue) had higher expression (red). Only the correlated methylation loci and expression probes are shown, and samples are organized by their discretization classification of M or U for each gene.</p

Frequency of DML by chromosome after normalization to chromosome length.

<p>Frequency of DML by chromosome after normalization to chromosome length.</p

<i>ZNF367</i> Inhibits Cancer Progression and Is Targeted by miR-195

<div><p>Background</p><p>Several members of the zinc finger protein family have been recently shown to have a role in cancer initiation and progression. Zinc finger protein 367 (<i>ZNF367</i>) is a member of the zinc finger protein family and is expressed in embryonic or fetal erythroid tissue but is absent in normal adult tissue.</p><p>Methodology/Principal Findings</p><p>We show that <i>ZNF367</i> is overexpressed in adrenocortical carcinoma, malignant pheochromocytoma/paraganglioma and thyroid cancer as compared to normal tissue and benign tumors. Using both functional knockdown and ectopic overexpression in multiple cell lines, we show that <i>ZNF367</i> inhibits cellular proliferation, invasion, migration, and adhesion to extracellular proteins <i>in vitro</i> and <i>in vivo</i>. Integrated gene and microRNA expression analyses showed an inverse correlation between <i>ZNF367</i> and miR-195 expression. Luciferase assays demonstrated that miR-195 directly regulates <i>ZNF367</i> expression and that miR-195 regulates cellular invasion. Moreover, integrin alpha 3 (<i>ITGA3</i>) expression was regulated by <i>ZNF367</i>.</p><p>Conclusions/Significance</p><p>Our findings taken together suggest that <i>ZNF367</i> regulates cancer progression.</p></div

<i>ZNF367</i> regulates <i>ITGA3</i> expression.

<p>(<b>A</b>) <i>ZNF367</i> knockdown upregulates <i>ITGA3</i> expression in SW13 cells. Error bars represent ± SEM. (<b>B</b>) The correlation between <i>ITGA3</i> and <i>ZNF367</i> mRNA expression in adrenocortical tumor samples. X and Y axes represent log 2–transformed values. (<b>C</b>) Western blot quantification of ITGA3 protein expression with <i>ZNF367</i> knockdown. (<b>D</b>–<b>E</b>) ITGA3 expression with ZNF367 overexpression. Error bars represent ± SEM.</p

Effect of <i>ZNF367</i> knockdown and overexpression.

<p><i>ZNF367</i> overexpression decreases cellular invasion and migration. (<b>A</b>) SW13, (<b>B</b>) BD104A, (<b>C</b>) TPC-1, and (<b>D</b>) HEK293 cell lines. After transfection, cells were plated inside a Boyden chamber for 48 hours. Cells were stained and counted in 4 fields. The left panel shows the representative image (12.5X) from the siRNA knockdown and negative control groups. The right panel indicates the quantitative measurement of invaded and migrated cells in knockdown and the negative control. *p<0.05 and error bars indicate ± SD. <i>ZNF367</i> overexpression decreases colony number, cellular invasion, and migration in HEK293 cells. (<b>E</b>) Western blot of <i>ZNF367</i> overexpression in HEK293 and SW13 cells (Empty vector, XL4). GAPDH was used as a loading control. (<b>F</b>) Representative clonogenic image for cells with ectopic <i>ZNF367</i> expression and its corresponding control (Empty vector, XL4). (<b>G</b>) Cellular invasion and migration decreased with <i>ZNF367</i> overexpression in HEK293 cells. The quantitative measurement of invaded and migrated cells per group (HEK293-HEK293-Empty vector or <i>ZNF367</i>) is represented in the bar graph on the right panel. (<b>H</b>) The extracellular protein attachment of SW13 cells with <i>ZNF367</i> knockdown. Cells were transfected with <i>ZNF367</i> siRNA and negative control siRNA, and were plated into adhesion plates and incubated for 90 minutes. The Y axis represents absorbance at 540 nM of cells adherent to each protein. Error bars represent ± SEM. *p<0.05, ***p<0.001.</p

<i>ZNF367</i> mRNA and protein levels in adrenocortical carcinoma, papillary thyroid cancer, and pheochromocytoma/paraganglioma compared to benign and normal tissue samples for each cancer type.

<p>(A and B) Expression level in the normal adrenal cortex, benign adrenocortical adenomas, and adrenocortical carcinomas; (C and D) normal adrenal medulla, benign and malignant pheochromocytoma/paraganglioma tissue samples; and (E and F) normal thyroid and papillary thyroid cancer tissue samples. The Y axis on each graph represents the percentage of mRNA expression using the 2^∧-ΔCt*100% method ± SEM. *p<0.05, **p<0.001, ***p<0.001 (Kruskal-Wallis test). Representative immunohistochemistry images are from normal, benign, and malignant tumor samples at 20X magnification.</p