Supplementary data Bodoni AF.docx

   Background: Nicotinamide nucleotide transhydrogenase (NNT) acts as an antioxidant defense mechanism. NNT mutations cause familial glucocorticoid deficiency (FGD). How impaired oxidative stress disrupts adrenal steroidogenesis remains poorly understood. Objective: To ascertain the role played by NNT in adrenal steroidogenesis.  Methods: The genotype-phenotype association of a novel pathogenic NNT variant was evaluated in a boy with FGD. Under basal and oxidative stress (OS) induced conditions, transient cell cultures of the patient’s and controls wild type (WT) mononuclear blood cells were used to evaluate antioxidant mechanisms and mitochondrial parameters [reactive oxygen species (ROS) production, reduced glutathione (GSH), and mitochondrial mass]. Using CRISPR/Cas9, a stable NNT gene knockdown model was built in H295R adrenocortical carcinoma cells to determine the role played by NNT in mitochondrial parameters and steroidogenesis. NNT immunohistochemistry was assessed in fetal and post-natal human adrenals. Results: The homozygous NNT p.G866D variant segregated with the FGD phenotype. Under basal and OS conditions, p.G866D homozygous mononuclear blood cells exhibited increased ROS production, and decreased GSH levels and mitochondrial mass when compared to WT NNT cells. In line, H295R NNT knocked-down cells presented impaired NNT protein expression, increased ROS production, decreased the mitochondrial mass, as well as the size and the density of cholesterol lipid droplets. NNT knockdown affected steroidogenic enzyme expression, impairing cortisol and aldosterone secretion. In human adrenals, NNT is abundantly expressed in the transition fetal zone and in zona fasciculata. Conclusion: Together, these studies demonstrate the essential role of NNT in adrenal redox homeostasis and steroidogenesis.</p

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

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

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

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