Immunohistochemical analysis of PDK1, PHD3 and HIF-1α expression defines the hypoxic status of neuroblastoma tumors

<div><p>Neuroblastoma (NB) is the most common solid tumor during infancy and the first cause of death among the preschool age diseases. The availability of several NB genomic profiles improves the prognostic ability, but the outcome prediction for this pathology remains imperfect. We previously produced a novel prognostic gene signature based on the response of NB cells to hypoxia, a condition of tumor microenvironment strictly connected with cancer aggressiveness. Here we attempted to further define the expression of hypoxia-modulated specific genes, looking at their protein level in NB specimens, considering in particular the hypoxia inducible factor-1α (HIF-1α), the mitochondrial pyruvate dehydrogenase kinase 1 (PDK1), and the HIF-prolyl hydroxylase domain 3 (PHD3). The evaluation of expression was performed by Western blot and immunocytochemistry on NB cell lines and by immunohistochemistry on tumor specimens. Stimulation of both HIF-1α and PDK1 and inhibition of PHD3 expression were observed in NB cell lines cultured under prolonged hypoxic conditions as well as in most of the tumors with poor outcome. Our results indicate that the immunohistochemistry analysis of the protein expression of PDK1, PHD3, and HIF-1α defines the hypoxic status of NB tumors and can be used as a simple and relevant tool to stratify high-risk patients.</p></div

Immunocytochemical analysis of NB cells cultured in low oxygen concentration.

<p>ACN, IMR-32, and LAN-1 cells, cultured in normoxic (Normo) or in hypoxic conditions for <b>(A)</b> 18 (Hypo 18h) or <b>(B)</b> 96 (Hypo 96h) hr, were cytospinned on Polysine™ slides, fixed and treated with anti-HIF-1α, anti-PDK1, anti-PHD3, anti-PFKFB4, and anti-VEGFA antibodies (60x magnification). Ctr = negative controls (20x magnification).</p

Evaluation of immunohistochemistry staining intensity of the tumor samples examined and listed in Table 1.

<p><b>(A)</b> Each group of protein expression (L = low percentage of positive cells; M = medium percentage of positive cells; H = high percentage of positive cells) was further subdivided in strong, moderate or weak staining degree. <b>(B)</b> To calculate the total number of tumors with high or low positivity for each gene, the samples identified as M were considered H or L on the basis of staining intensity. The bioinformatics analysis were then performed as reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187206#pone.0187206.t002" target="_blank">Table 2</a>.</p

Immunohistochemical analysis of NB specimens for expression of NB-hypo selected genes.

<p>Tumor samples from the normoxic cluster <b>(A, B)</b> or from the hypoxic cluster <b>(C, D)</b> were fixed and paraffin embedded. <b>(A)</b> adrenal gland, stage 1 (Tumor number 22, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187206#pone.0187206.t001" target="_blank">Table 1</a>); <b>(B)</b> mediastinal mass, stage 2B (Tumor number 15, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187206#pone.0187206.t001" target="_blank">Table 1</a>); <b>(C)</b> abdominal mass, stage 4 (Tumor number 2, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187206#pone.0187206.t001" target="_blank">Table 1</a>); <b>(D)</b> adrenal gland, stage 4 (Tumor number 3, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187206#pone.0187206.t001" target="_blank">Table 1</a>). For histological analysis, sections were stained with hematoxylin and eosin (H/E). Serial sections were treated with anti-HIF-1α, anti-PDK1, anti-PHD3, and anti-N-Myc antibodies. Tumors with favorable prognosis <b>(A, B)</b> show negative staining for HIF-1α and PDK1, and positive staining for PHD3. Tumors with poor prognosis <b>(C, D)</b> display a positive staining for HIF-1α and PDK1, while they are almost negative for PHD3. N-Myc negativity in normoxic samples and N-Myc positivity in hypoxic ones is considered as a control for the accuracy of the immunohistochemical procedure. Ctr = negative controls. (20x magnification).</p

List of the 25 NB pediatric patients analyzed.

<p>List of the 25 NB pediatric patients analyzed.</p

Expression levels and localization of N-Myc protein in NB cell lines.

<p><b>(A)</b> Protein lysates from ACN, IMR-32, and LAN-1 cells were subjected to Western blot and probed with anti-N-Myc antibody. The blot was reprobed with anti-actin antibody as loading control. <b>(B)</b> ACN, IMR-32, and LAN-1 cells were cytospinned on Polysine™ slides and fixed. N-Myc protein nuclear localization was visualized by immunocytochemistry with anti-N-Myc antibody (60x magnification). Ctr = negative controls (20x magnification).</p

Hypoxia modulates expression of NB-hypo selected genes in NB cell lines.

<p>Protein lysates from ACN, IMR-32, and LAN-1 cells, cultured in normoxic (N) or in hypoxic (H) conditions for <b>(A)</b> 18, <b>(B)</b> 72, or <b>(C)</b> 96 hr, were subjected to Western blot and probed with anti-HIF-1α, anti-PDK1, anti-PHD3, anti-PFKFB4, and anti-VEGFA antibodies. The blot was reprobed with anti-actin antibody as loading control. Levels of HIF-1α, PDK1, PHD3, PFKFB4, and VEGFA in NB cells cultured in normoxic or in hypoxic conditions for <b>(D)</b> 18, <b>(E)</b> 72, or <b>(F)</b> 96 hr were quantified by densitometry and normalized to the content of the loading control protein. The optical density of the scanned film was measured with Quantity One v. 2–3 Image software (Versa Doc, Bio-Rad, Hercules, CA, USA). Results represent the mean values ± S.D. from three independent experiments.</p

Dbl oncogene expression in MCF-10 A epithelial cells disrupts mammary acinar architecture, induces EMT and angiogenic factor secretion

<p>The proteins of the Dbl family are guanine nucleotide exchange factors (GEFs) of Rho GTPases and are known to be involved in cell growth regulation. Alterations of the normal function of these proteins lead to pathological processes such as developmental disorders, neoplastic transformation, and tumor metastasis. We have previously demonstrated that expression of Dbl oncogene in lens epithelial cells modulates genes encoding proteins involved in epithelial-mesenchymal-transition (EMT) and induces angiogenesis in the lens. Our present study was undertaken to investigate the role of Dbl oncogene in epithelial cells transformation, providing new insights into carcinoma progression.To assess how Dbl oncogene can modulate EMT, cell migration, morphogenesis, and expression of pro-apoptotic and angiogenic factors we utilized bi- and 3-dimensional cultures of MCF-10 A cells. We show that upon Dbl expression MCF-10 A cells undergo EMT. In addition, we found that Dbl overexpression sustains Cdc42 and Rac activation inducing morphological alterations, characterized by the presence of lamellipodia and conferring a high migratory capacity to the cells. Moreover, Dbl expressing MCF-10 A cells form altered 3D structures and can induce angiogenesis by producing proangiogenic factors such as CCL2. These results support a role for Dbl oncogene in epithelial cell differentiation and transformation and suggest the relevance of GEF deregulation in tumor onset and progression.</p