Downregulation of C3 and C4A/B complement factor fragments in plasma from patients with squamous cell carcinoma of the penis

Purpose To investigate the use of ClinProt technique to identify cancer markers in plasma of patients suffering from squamous cell carcinoma of the penis (SCCP). Materials and Methods Plasma of 36 healthy subjects and 25 patients with penile carcinoma who underwent surgical treatment between June 2010 and June 2011 was collected and analyzed by the ClinProt/MALDI/ToF technique. Then the peptides were identified from the C8 MB eluted fraction of patients' and control subjects' plasma by LIFT MS/MS. Results A cluster of 2 peptides (A=m/z 1897.22 ± 9 Da and B=m/z 2021.99 ± 9 Da) was able to discriminate patients from control subjects. Cross validation analysis using the whole casuistic showed 62.5% and 86.76% sensitivity and specificity, respectively. The cluster also showed very high sensitivity (100%) and specificity (97%) for SCCP patients that died due to the disease. Furthermore, patients with lymph node involvement presented sensitivity and specificity of 80% and 97%, respectively. These two peptides were identified by the proteomic approach based on a MALDI-TOF/TOF as fragments of C3 (m/z 1896.17) and C4a/b (m/z 2021.26) complement proteins. Conclusions The results showed that as the disease progresses, the fragments C3 and C4 A/B are less expressed in comparison with healthy subjects. These results may be useful as prognostic tools

Lineage tracing SPM using <i>Myf5-Cre;R26R</i> in <i>Irf6</i>−/− Embryos.

<p>Coronal sections through anterior, middle and posterior tongue of control and <i>Irf6</i>−/− mice at E13.5, E14.5, and E15.5 performed in triplicate. In the E13.5 control tongues there was a strong contribution of the SPM (<i>Myf5</i>+) to the tongue (blue cells in A, C, E) with striations extending from the midline representing the transversal muscle (black arrow). In the <i>Irf6</i>−/− tongues there was reduced SPM (blue cells in B, D, F) with reduced and poorly organized striations of transversal muscle (black arrow). At E14.5 there was increased SPM in the control tongues (blue cells in G, I, K) and more striations of the transversal muscle (black arrow). In the E14.5 <i>Irf6</i>−/− tongue there was reduced SPM (blue cells in H, J, L) and reduction in striations of the transversal muscle and poor organization (black arrows). In the E15.5 control tongue there was primarily SPM (blue cells in M, O, Q) with increasing striations of the transversal muscle (black arrows). In the E15.5 <i>Irf6</i>−/− tongue there was a significant reduction of SPM (blue cells in N, P, R) with reduced striations of the transversal muscle and poor organization (black arrows). The inter-molar eminence was not populated by SPM and was absent in the <i>Irf6</i>−/− tongue (white arrows in E, K, Q).</p

Cell-Autonomous and Non-Cell-Autonomous Roles for <em>Irf6</em> during Development of the Tongue

<div><p>Interferon regulatory factor 6 (<i>IRF6</i>) encodes a highly conserved helix-turn-helix DNA binding protein and is a member of the interferon regulatory family of DNA transcription factors. Mutations in <i>IRF6</i> lead to isolated and syndromic forms of cleft lip and palate, most notably Van der Woude syndrome (VWS) and Popliteal Ptyerigium Syndrome (PPS). Mice lacking both copies of <i>Irf6</i> have severe limb, skin, palatal and esophageal abnormalities, due to significantly altered and delayed epithelial development. However, a recent report showed that <i>MCS9.7</i>, an enhancer near <i>Irf6</i>, is active in the tongue, suggesting that <i>Irf6</i> may also be expressed in the tongue. Indeed, we detected Irf6 staining in the mesoderm-derived muscle during development of the tongue. Dual labeling experiments demonstrated that Irf6 was expressed only in the <i>Myf5</i>+ cell lineage, which originates from the segmental paraxial mesoderm and gives rise to the muscles of the tongue. Fate mapping of the segmental paraxial mesoderm cells revealed a cell-autonomous <i>Irf6</i> function with reduced and poorly organized <i>Myf5</i>+ cell lineage in the tongue. Molecular analyses showed that the <i>Irf6</i>−/− embryos had aberrant cytoskeletal formation of the segmental paraxial mesoderm in the tongue. Fate mapping of the cranial neural crest cells revealed non-cell-autonomous <i>Irf6</i> function with the loss of the inter-molar eminence. Loss of <i>Irf6</i> function altered <i>Bmp2</i>, <i>Bmp4</i>, <i>Shh</i>, and <i>Fgf10</i> signaling suggesting that these genes are involved in Irf6 signaling. Based on these data, <i>Irf6</i> plays important cell-autonomous and non-cell-autonomous roles in muscular differentiation and cytoskeletal formation in the tongue.</p> </div

Expression of <i>Irf6</i> in Tongue Occurs in <i>Myf5</i>+ Cell Lineage.

<p>Coronal sections through the anterior, middle and posterior tongue during critical stages of tongue differentiation performed in triplicate (E13.5–E15.5). At E13.5 there was moderate <i>Irf6</i> expression in the tongue giving a striated appearance representing the transversal muscle extending from the midline (A, B, C). At E14.5 there was intense <i>Irf6</i> staining throughout the tongue (D, E, F) with more prominent striations of the transversal muscle extending from the midline (white arrow) and an <i>Irf6</i>-free zone in the inter-molar eminence (white asterisk). At E15.5 there was a reduction in <i>Irf6</i> signaling throughout the tongue (G, H, I). Lineage tracing of segmental paraxial mesoderm was performed using <i>Myf5-Cre;R26R</i> mice. <i>Irf6</i> staining occurs in the striations of the transversal muscle of the tongue tissue (J, M, P) and detection of B-Galactosidase activity was found in the same region of the tongue of <i>Myf5-Cre;R262R</i> mice (K, N, Q). Overlay of these images demonstrated that <i>Irf6</i> staining occurred primarily in the <i>Myf5</i>+ cell lineage (L, O, R).</p

Altered Actin Cytoskeleton in <i>Irf6</i>−/− Tongue.

<p>Coronal sections of E14.5 control and <i>Irf6</i>−/− tongue stained with Phalloidin to detect F-actin performed in triplicate. In the <i>Irf6</i>−/− tongue there was reduced Phalloidin staining and poor organization of the stained fibers (B) compared to controls (A). Using confocal imaging on phalloidin-stained images of similar areas of the tongue (denoted by circle in images A, B) we identified reduced actin filament length and poor organization of actin fibers in the <i>Irf6</i>−/− fibers (D) compared to controls (C). Myosin heavy chain expression was greatly reduced in E15.5 <i>Irf6</i>−/− tongue using MF-20 staining (F) compared to controls (E). Myogenin was not effected in <i>Irf6</i>−/− tongue (H) compared to controls (G) * denotes inter-molar eminence.</p

Reduced <i>Shh, Bmp4</i> and <i>Fgf10</i> in the <i>Irf6</i>−/− Tongue.

<p>Quantitative PCR of <i>Irf6</i>−/− tongue tissue was performed on E14.5 mice and compared to controls. The Irf6ko mice showed relative expression changes where <i>Shh</i>, <i>Bmp4</i>, and <i>Fgf10</i> were greatly reduced whereas <i>Bmp2</i> was increased two fold. Columns, median fold change obtained from 5 separate experiments; bars, SEM. * = p<0.05.</p

Reduced Hyaluronic Acid and CD44 staining in the <i>Irf6</i>−/− Tongue.

<p>The inter-molar eminence had Wnt1+ cells however did not contain <i>Irf6</i> expression or <i>Myf5</i>+ cells. Hyaluronic Acid (HA) staining done at E14.5 showed that the intra-molar eminence was comprised of HA (blue staining in A) and was absent in the <i>Irf6</i>−/− mice (B) in triplicate samples. Furthermore, CD44, a receptor for HA signaling, was reduced (red staining) in the <i>Irf6</i>−/− posterior tongue (D) compared to controls (C) in triplicate samples.</p