Components of the Canonical and Non-Canonical Wnt Pathways Are Not Mis-Expressed in Pituitary Tumors

<div><p>Introduction</p><p>Canonical and non-canonical Wnt pathways are involved in the genesis of multiple tumors; however, their role in pituitary tumorigenesis is mostly unknown.</p> <p>Objective</p><p>This study evaluated gene and protein expression of Wnt pathways in pituitary tumors and whether these expression correlate to clinical outcome.</p> <p>Materials and Methods</p><p>Genes of the Wnt canonical pathway: activating ligands (<i>WNT11, WNT4, WNT5A</i>), binding inhibitors (<i>DKK3, sFRP1</i>), β-catenin (<i>CTNNB1</i>), β-catenin degradation complex (<i>APC, AXIN1, GSK3β</i>), inhibitor of β-catenin degradation complex (<i>AKT1</i>), sequester of β-catenin (<i>CDH1</i>), pathway effectors (<i>TCF7, MAPK8, NFAT5</i>), pathway mediators (<i>DVL-1, DVL-2, DVL-3, PRICKLE, VANGL1</i>), target genes (<i>MYB, MYC, WISP2, SPRY1, TP53, CCND1</i>); calcium dependent pathway (<i>PLCB1, CAMK2A, PRKCA, CHP</i>); and planar cell polarity pathway (<i>PTK7, DAAM1, RHOA</i>) were evaluated by QPCR, in 19 GH-, 18 ACTH-secreting, 21 non-secreting (NS) pituitary tumors, and 5 normal pituitaries. Also, the main effectors of canonical (β-catenin), planar cell polarity (JNK), and calcium dependent (NFAT5) Wnt pathways were evaluated by immunohistochemistry.</p> <p>Results</p><p>There are no differences in gene expression of canonical and non-canonical Wnt pathways between all studied subtypes of pituitary tumors and normal pituitaries, except for <i>WISP2,</i> which was over-expressed in ACTH-secreting tumors compared to normal pituitaries (4.8x; p = 0.02), NS pituitary tumors (7.7x; p = 0.004) and GH-secreting tumors (5.0x; p = 0.05). β-catenin, NFAT5 and JNK proteins showed no expression in normal pituitaries and in any of the pituitary tumor subtypes. Furthermore, no association of the studied gene or protein expression was observed with tumor size, recurrence, and progressive disease. The hierarchical clustering showed a regular pattern of genes of the canonical and non-canonical Wnt pathways randomly distributed throughout the dendrogram.</p> <p>Conclusions</p><p>Our data reinforce previous reports suggesting no activation of canonical Wnt pathway in pituitary tumorigenesis. Moreover, we describe, for the first time, evidence that non-canonical Wnt pathways are also not mis-expressed in the pituitary tumors.</p> </div

Immunocytochemistry for β-catenin, JNK, and NFAT5 in normal pituitaries, ACTH-secreting pituitary tumor, GH-secreting pituitary tumor, and non-secreting pituitary tumor (x40).

<p>ACTH and GH immune positivity are shown in the region of the tumor sample immunostained for β-catenin, JNK and NFAT. Craniopharyngioma tissue and two positive samples from GH-secreting pituitary tumors were used as positive controls for β-catenin, NFAT5, and JNK antibodies, respectively.</p

Wnt Pathway gene expression in Pituitary Tumors.

<p>Wnt Pathway gene expression in Pituitary Tumors.</p

Hierarchical clustering for canonical and non-canonical Wnt pathway genes in normal pituitary and in different subtypes of the pituitary tumors.

<p>HN: normal pituitary; ACTH: ACTH-secreting pituitary tumor; GH: GH-secreting pituitary tumor; NS: non-secreting pituitary tumor.</p

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