Representative mass spectrometry data from quantitative analysis of HSPA1-Lys561 methylation status.

<p>(A)-(D) Methylation status of Lys561 in HSPA1 in tumor sample # 14. Left panels, chromatograms were generated by gating for mass-to-charge ratios of the (A) unmethylated (me0), (B) monomethylated (me1), (C) dimethylated (me2) and (D) trimethylated (me3) state of the AspN-generated proteolytic peptide encompassing Asp555-Ala565 in HSPA1. The elution time (arrow) and the area under each curve (in brackets), as well as the calculated relative abundance (as percentage) of the various lysine methylation states, are indicated. Right panels, annotated tandem mass spectra supporting the identity of analyzed peptides.</p

Assessment of HSPA1-Lys561 methylation status in HGSC and breast carcinoma effusions.

<p>(A) Quantitative mass spectrometry analysis of HSPA1-Lys561 methylation. Samples were analyzed as exemplified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140168#pone.0140168.g001" target="_blank">Fig 1</a>. The relative abundance of the various methylation states (me0, me1, me2 and me3) of Lys561 in HSPA1 in 17 breast carcinoma (cases # 1–17) and 53 HGSC (cases # 18–70) samples is shown. The data are grouped by cancer type and sorted by increasing relative abundance of me3. (B) Reproducibility of the analysis. A second analysis of 2 randomly chosen breast cancer samples and 5 randomly chosen HGSC samples was performed, and the results were plotted versus the original data set. Data points for the various lysine methylation states are color-coded as in (A) and each tumor sample is represented by a unique geometrical figure.</p

HSPA1 methylation and patient survival in HGSC.

<p>^aNumber of methyl groups on Lys-561 in HSPA1, calculated from the data in the three previous columns according to the following formula: (Me1 + 2∙Me2 + 3∙Me3)/100</p><p>^bPatient was alive with disease at the end of the follow-up period (110 mo), and was therefore censored in OS curve (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140168#pone.0140168.g003" target="_blank">Fig 3A</a>)</p><p>^cPatient was alive without disease after the end of the follow-up period (132 mo for OS and 127 mo for PFS), and was therefore censored in OS and PFS curves (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140168#pone.0140168.g003" target="_blank">Fig 3</a>)</p><p>HSPA1 methylation and patient survival in HGSC.</p

ESRRA and C11orf20 exon reads in high-throughput data files.

<p>ESRRA and C11orf20 exon reads in high-throughput data files.</p

Putative <i>ESRRA-C11orf20</i> fusion transcript in ovarian carcinomas.

<p>(A) Schematic orientation of the <i>ESRRA</i> and <i>C11orf20</i> genes in the genome (the size of each exon is not in scale). (B) Representation of the three forms of the putative fusion with involvement of <i>ESRRA</i> exon 2 with <i>C11orf20</i> exon 3, 4, or 5. Arrows indicate position of the primers for the first PCR (F1 and R1) and the NESTED PCR (F2 and R2). Primer details are given in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001784#s3" target="_blank">Materials and Methods</a>. (C) Image of the gel electrophoresis for the synthetic transcript containing the fusion (internal positive control). A plasmid containing exon 2 of <i>ESRRA</i> and exon 5 of <i>C11orf20</i> was used as a positive control for our PCR experiments. Three different concentrations of the plasmid were tested using the <i>ESRRA</i> and <i>C11orf20</i> PCR primers. Lane 1, 1 kb DNA ladder; lanes 2 and 6, 2 fg plasmid (∼30 copies of the plasmid); lanes 3 and 7, 20 fg plasmid (∼300 copies of the plasmid); lane 4 and 8, 200 fg plasmid (∼3,000 copies of the plasmid). Lanes 2–5, primers G1P1-FWD (<i>ESRRA</i>) and REV_pair3 (<i>C11orf20</i>) used; lanes 6–9, inner (nested) primers G1P2-FWD (<i>ESRRA</i>) and F1-REV (<i>C11orf20</i>) used. Lanes 5 and 9 are negative controls with no plasmid template.</p

Ezrin Is Associated with Disease Progression in Ovarian Carcinoma

<div><p>Objective</p><p>Ezrin and p130Cas are structural proteins with an important role in signaling pathways and have been shown to promote cancer dissemination. We previously reported on overexpression of both ezrin and p130Cas in breast carcinoma effusions compared to primary carcinomas. Since ovarian and breast carcinomas share the ability to disseminate by forming malignant effusions, we sought to study the role of these molecules in ovarian carcinoma (OC).</p><p>Methods</p><p>OC cell lines were cultured in two different 3-dimensional conditions, on alginate scaffolds and as spheroids, which served as models for solid tumor and malignant effusions, respectively. shRNA was used to reduce protein expression in the cells. The malignant potential was evaluated by chemo-invasion assay, branching capacity on Matrigel and rate of proliferation. Subsequently, clinical specimens of high-grade serous carcinoma effusions, ovarian tumors and solid metastases were analyzed for ezrin and p130Cas expression.</p><p>Results</p><p>Higher ezrin expression was found in cells composing the spheroids compared to their counterparts cultured on alginate scaffold and in clinical samples of malignant effusions compared to solid tumors. In addition, reduced Ezrin expression impaired the invasion ability and the branching capacity of OC cells to a greater extent than reduced p130Cas expression. However, ezrin and p130Cas expression in effusions was unrelated to clinical outcome.</p><p>Conclusions</p><p>The 3-dimensional cell cultures were found to mimic the different tumor sites and be applicable as a model. The <i>in vitro</i> results concur with the clinical specimen analysis, suggesting that in OC, the role of ezrin in disease progression is more pronounced than that of p130Cas.</p></div

No change in spheroid morphology following reduction in ezrin or p130Cas expression.

<p>Following 24h incubation on rocking culture dish, (A) ES2 cells formed organized round-shaped spheroids that sometimes were fused to form large clusters: ES2-C1 and ES2-C2, cells transfected with no plasmid and non-transfected cells, respectively; (B) OVCAR3 cells formed less organized cell clusters that varied in shape and size: OVCAR3-C1 and OVCAR3-C2, cells transfected with no plasmid and non-transfected cells, respectively. The transfected cells formed clusters similarly to the control cells. Ezrin reduced expression: ES2-T2 in A and OVCAR3-T2 in B. p130Cas reduced expression: ES2-T4 in A, OVCAR3-T4 in B (Magnification X40).</p

Clinicopathologic parameters of the study cohort (93 patients).

<p>Clinicopathologic parameters of the study cohort (93 patients).</p

Expression of ezrin and p130Cas in cell lines and in clinical samples.

<p>(A) Two human OC cell lines, ES2 and OVCAR3, were cultured on alginate scaffolds and as spheroids, as models for solid tumors and malignant effusions, respectively. Ezrin protein level was higher in cells composing the spheroid compared to their counterparts on scaffolds, though not significantly. Sample distribution is presented for mRNA levels of ezrin and p130Cas (B) and protein levels of ezrin, p-ERM and p130Cas (C) at 3 different tumor sites: the ovary, solid metastases and malignant effusions. Representative blot is shown for the measured proteins and the corresponding reference protein (GAPDH) in ovarian tumors (P), metastases (M) and malignant effusions (E). Ezrin was significantly elevated at both the mRNA (B, p<0.001) and protein (C, p = 0.008) level in malignant effusions compared to the solid tumors. p130cas mRNA and protein and p-ERM protein levels were comparable at the 3 anatomic sites (B and C, p>0.05). Phosphorylated p130Cas was hardly detected, regardless of tumor site (not shown).</p

Significant associations between Ezrin and p130cas expression and clinicopathologic parameters.

<p>Significant associations between Ezrin and p130cas expression and clinicopathologic parameters.</p