Proteomic Approaches Identify Members of Cofilin Pathway Involved in Oral Tumorigenesis

<div><p>The prediction of tumor behavior for patients with oral carcinomas remains a challenge for clinicians. The presence of lymph node metastasis is the most important prognostic factor but it is limited in predicting local relapse or survival. This highlights the need for identifying biomarkers that may effectively contribute to prediction of recurrence and tumor spread. In this study, we used one- and two-dimensional gel electrophoresis, mass spectrometry and immunodetection methods to analyze protein expression in oral squamous cell carcinomas. Using a refinement for classifying oral carcinomas in regard to prognosis, we analyzed small but lymph node metastasis-positive versus large, lymph node metastasis-negative tumors in order to contribute to the molecular characterization of subgroups with risk of dissemination. Specific protein patterns favoring metastasis were observed in the “more-aggressive” group defined by the present study. This group displayed upregulation of proteins involved in migration, adhesion, angiogenesis, cell cycle regulation, anti-apoptosis and epithelial to mesenchymal transition, whereas the “less-aggressive” group was engaged in keratinocyte differentiation, epidermis development, inflammation and immune response. Besides the identification of several proteins not yet described as deregulated in oral carcinomas, the present study demonstrated for the first time the role of cofilin-1 in modulating cell invasion in oral carcinomas.</p> </div

Genomic analysis of head and neck cancer cases from two high incidence regions

<div><p>We investigated how somatic changes in HNSCC interact with environmental and host risk factors and whether they influence the risk of HNSCC occurrence and outcome. 180-paired samples diagnosed as HNSCC in two high incidence regions of Europe and South America underwent targeted sequencing (14 genes) and evaluation of copy number alterations (SCNAs). <i>TP53</i>, <i>PIK3CA</i>, <i>NOTCH1</i>, <i>TP63</i> and <i>CDKN2A</i> were the most frequently mutated genes. Cases were characterized by a low copy number burden with recurrent focal amplification in 11q13.3 and deletion in 15q22. Cases with low SCNAs showed an improved overall survival. We found significant correlations with decreased overall survival between focal amplified regions 4p16, 10q22 and 22q11, and losses in 12p12, 15q14 and 15q22. The mutational landscape in our cases showed an association to both environmental exposures and clinical characteristics. We confirmed that somatic copy number alterations are an important predictor of HNSCC overall survival.</p></div

Immunodetection of keratin 4 expression in OSCC samples. Immunohistochemistry analysis:

<p>pattern of keratin 4 immunostaining in (A) superficial layers of epithelium in margin showing intense positivity in stratum corneum (A, insert); (B) absence of keratin 4 immunostaining in nests of well differentiated and (C) poorly differentiated areas of OSCC. Scale bar indicates 100 µm. <b>Western blot</b>: (D) tumor samples (lanes 1, 3, 5, 7) and matched margins (lanes 2, 4, 6, 8) from patients with T1N0, T4N2, T4N1 and T4N1 carcinomas, respectively; (E) Surgical margin (lane 1) and tumor samples (lanes 2, 3, 4, 5) from patients with T4N2, T4N2, T4N2, T1N0 and T2N2, respectively. β-actin was used as an internal control. MW, PageRuler™ Prestained Protein Ladder.</p

Immunodetection <i>of</i> cofilin-1 and p-cofilin in OSCC samples.

<p>Immunostaining for total cofilin-1 and p-cofilin in FFPE sections of (A and B, respectively) surgical margins and (C and D, respectively) OSCC samples. Note the low positivity of total cofilin (A) and the nuclear staining for p-cofilin (B) in the more basal layers of epithelium in margins, and (C and D, inserts) the more intense staining of tumor cell nuclei for p-cofilin than for total cofilin. Figures and inserts = 100X and 400X magnification, respectively.</p

siRNA-mediated knockdown of cofilin-1 resulted in decreased invasive ability of oral cancer cells.

<p>Western blot analysis showed reduced levels of (A) cofilin-1 in <b><i>SCC-9 cells</i></b><i> t</i>ransfected with different concentrations of siRNA (siCofilin I) for 48 h and of (B) cofilin-1 and p-cofilin in SCC-9 cells transfected with 20 nM siCofilin I for 48 h. (C) Immunofluorescence analysis of cofilin-1 knockdown SCC-9 cells (siCofilin I) using anti-p-cofilin antibody (green). (D) Invasion assays using Matrigel-coated filters were performed on SCC-9 and Cal 27 cells (cofilin-1 knockdown cells and controls). Bar graph represents the mean ± S.E. of the number cells that invaded through the Matrigel from three independent experiments (Student’s <i>t</i> test, * = p<0.01).</p

Workflow of processing and analysis of HNSCC samples from the three different studies.

<p>QC for copy number evaluation: Quality control of samples based on signal to noise ratio>5.0. Maps show estimated age-standardized incidence rates for HNSCC (other pharynx sites) in Europe and South America. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191701#pone.0191701.ref031" target="_blank">31</a>].</p

Cofilin pathway.

<p>Microenvironmental stimuli signal through Rho-GTPases and their regulating kinases (ROCK1 and Pak-1), stimulating LIMK to phosphorylate and inactivate cofilin-1. Otherwise, SSH phosphatases dephosphorylate cofilin. Rap proteins may increase the enzymatic activity of SSHs, possibly by promoting their release from 14-3-3 proteins. Cofilin is sequestered by PIP2 and released after hydrolysis of PIP2 by phosphorylated PLC to IP3 and DAG. The active cofilin severs “old” actin filaments to generate free actin barbed ends. ATP-actin assembles into these barbed ends and ADP-actin subunits are, in turn, dissociated from the pointed end. Free actin monomers exchange ADP to ATP, frequently with the help of profilin and CAP proteins. ARP2/3 complex binds to F-actin and nucleates the growth of daughter filaments, generating a dendritic network at the leading edge of migratory cells. Other members of this pathway include Hsp90, which promotes stability of LIMK, and CAPZ, which interacts with barbed ends and inhibits filament assembly. ARP = actin-related protein 2/3 complex; CAP = adenylyl cyclase-associated protein 1; CAPZ = F-actin-capping protein subunit alpha-1; CFL = cofilin-1; F-actin = filamentous actin; DAG =  diacylglycerol; G-actin = globular actin; GF = growth factor; HSP90 = heat shock protein HSP 90-alpha; IP3 = inositoltrisphosphate; LIMK = LIM kinases; Pak-1 = serine/threonine-protein kinase PAK 1; PIP2 = phosphatidylinositol-4-5-biphosphate; PLC = phospholipase C; RAP = Ras-related protein; ROCK-1 = Rho-associated protein kinase 1; SSH = slingshot phosphatase.</p

Kaplan-Meier curves showing overall survival outcome for nodal status, significant focal copy number alterations in 22q11.2,15q22 and 12p12 regions associated to smoking and advanced stage, amplification in 4p16.3 and for the three SCNAs clusters.

<p>Kaplan-Meier curves showing overall survival outcome for nodal status, significant focal copy number alterations in 22q11.2,15q22 and 12p12 regions associated to smoking and advanced stage, amplification in 4p16.3 and for the three SCNAs clusters.</p

Integrative cluster analysis plot.

<p>Cases are grouped by mutation and SCNA status. Top panel: only significant clustering genes are shown (0 = non-mutated, 1 = mutated), middle panel: SCNAs. Amplified (red) and deleted (blue) chromosomal regions. Altered regions are arranged vertically and sorted by genomic locus, with chromosome 1 at the top of the panel and chromosome 22 at the bottom, lower panel: colour coded clinical and epidemiological characteristics.</p

OncoPrint diagram of mutational frequencies and types of alterations of the 14 genes sequenced.

<p>Only altered samples are shown. Rows are sorted based on the frequency of the alterations in all samples and columns are sorted to visualize the mutual exclusivity across genes. Frequency of mutations for the following Head and Neck cancer publications are shown: Head & Neck (TCGA)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191701#pone.0191701.ref010" target="_blank">10</a>], Head & Neck (JHU)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191701#pone.0191701.ref039" target="_blank">39</a>], Head & Neck (Broad)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191701#pone.0191701.ref032" target="_blank">32</a>], Head & Neck (MDA)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191701#pone.0191701.ref040" target="_blank">40</a>], Head & Neck (MSKCC)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191701#pone.0191701.ref041" target="_blank">41</a>]. NA: Not available.</p