Leandro Machado Colli

AIP expression in sporadic somatotropinomas is a predictor of the response to octreotide LAR therapy independent of SSTR2 expression.

mutation and/or a history of medical treatment with SRL before surgery. Biochemical assessment was performed 3 months after surgery by oral glucose tolerance test (OGTT) and evaluations of IGF1 levels. Patients were considered non-cured based on nonsuppressible GH levels on OGTT and plasma IGF1 levels higher than those of age-matched normal subjects. Medical therapy with OCT-LAR was started at a dose of 20 mg every 4 weeks, and the dose was increased to 30 mg in patients with non-controlled disease after 3 months of therapy. The efficacy of medical therapy was evaluated during the last patient visit. Patients were considered to have non-controlled disease if they had plasma IGF1 levels greater than those of age-matched normal subjects or basal GH levels O1.0 ng/ml after at least 6 months of treatment with OCT-LAR at a dosage of 30 mg. The AIP and SSTR2 expressions were analyzed through immunohistochemistry in paraffin-embedded

Sonic Hedgehog pathway is upregulated in adamantinomatous craniopharyngiomas

OBJECTIVES Pituitary stem cells play a role in the oncogenesis of human adamantinomatous craniopharyngiomas (aCPs). We hypothesized that crosstalk between the Wnt/β-catenin and Sonic Hedgehog (SHH) pathways, both of which are important in normal pituitary development, would contribute to the pathogenesis of aCPs. DESIGN To explore the mRNA and protein expression of components of the SHH signaling pathway in aCPs and their relationship with the identification of CTNNB1/β-catenin mutations and patients outcomes. PATIENTS AND METHODS In 18 aCP samples, CTNNB1 was sequenced, and the mRNA expression levels of SHH pathway members (SHH, PTCH1, SMO, GLI1, GLI2, GLI3, and SUFU) and SMO, GLI1, GLI3, SUFU, β-catenin, and Ki67 proteins were evaluated by quantitative real-time PCR and immunohistochemistry respectively. Anterior normal pituitaries were used as controls. Associations between molecular findings and clinical data were analyzed. RESULTS The aCPs presented higher mRNA expression of SHH (+400-fold change (FC); P<0.01), GLI1 (+102-FC; P<0.001), and GLI3 (+5.1-FC; P<0.01) than normal anterior pituitaries. Longer disease-free survival was associated with low SMO and SUFU mRNA expression (P<0.01 and P=0.02 respectively). CTNNB1/β-catenin mutations were found in 47% of the samples. aCPs with identified mutations presented with higher mRNA expression of SMO and GLI1 (+4.3-FC; P=0.02 and +10.2-FC; P=0.03 respectively). SMO, GLI1, GLI3, and SUFU staining was found in 85, 67, 93, and 64% of the samples respectively. Strong GLI1 and GLI3 staining was detected in palisade cells, which also labeled Ki67, a marker of cell proliferation. CONCLUSIONS The upregulation of SHH signaling occurs in aCPs. Thus, activation of Wnt/β-catenin and SHH pathways, both of which are important in pituitary embryogenesis, appears to contribute to the pathogenesis of aCP.

Dopamine receptor subtype 2 expression profile in nonfunctioning pituitary adenomas and in vivo response to cabergoline therapy

To determine the dopamine receptor subtype 2 (DR2) mRNA levels and protein expression and to evaluate the effect of adjuvant cabergoline therapy on tumour volume (TV) in patients with postoperative residual nonfunctioning pituitary adenoma (NFPA).

Regulation of aryl hydrocarbon receptor interacting protein (AIP) protein expression by MiR-34a in sporadic somatotropinomas.

Introduction Patients with germline AIP mutations or low AIP protein expression have large, invasive somatotroph adenomas and poor response to somatostatin analogues (SSA). Methods To study the mechanism of low AIP protein expression 31 sporadic somatotropinomas with low (n = 13) or high (n = 18) AIP protein expression were analyzed for expression of AIP messenger RNA (mRNA) and 11 microRNAs (miRNAs) predicted to bind the 3’UTR of AIP. Luciferase reporter assays of wild-type and deletion constructs of AIP-3’UTR were used to study the effect of the selected miRNAs in GH3 cells. Endogenous AIP protein and mRNA levels were measured after miRNA over- and underexpression in HEK293 and GH3 cells. Results No significant difference was observed in AIP mRNA expression between tumors with low or high AIP protein expression suggesting post-transcriptional regulation. miR-34a was highly expressed in low AIP protein samples compared high AIP protein adenomas and miR-34a levels were inversely correlated with response to SSA therapy. miR-34a inhibited the luciferase-AIP-3’UTR construct, suggesting that miR-34a binds to AIP-3’UTR. Deletion mutants of the 3 different predicted binding sites in AIP-3’UTR identified the c.*6–30 site to be involved in miR-34a’s activity. miR-34a overexpression in HEK293 and GH3 cells resulted in inhibition of endogenous AIP protein expression. Conclusion Low AIP protein expression is associated with high miR-34a expression. miR-34a can down-regulate AIP-protein but not RNA expression in vitro. miR-34a is a negative regulator of AIP-protein expression and could be responsible for the low AIP expression observed in somatotropinomas with an invasive phenotype and resistance to SSA.

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

Introduction Canonical and non-canonical Wnt pathways are involved in the genesis of multiple tumors; however, their role in pituitary tumorigenesis is mostly unknown. Objective This study evaluated gene and protein expression of Wnt pathways in pituitary tumors and whether these expression correlate to clinical outcome. Materials and Methods Genes of the Wnt canonical pathway: activating ligands (WNT11, WNT4, WNT5A), binding inhibitors (DKK3, sFRP1), β-catenin (CTNNB1), β-catenin degradation complex (APC, AXIN1, GSK3β), inhibitor of β-catenin degradation complex (AKT1), sequester of β-catenin (CDH1), pathway effectors (TCF7, MAPK8, NFAT5), pathway mediators (DVL-1, DVL-2, DVL-3, PRICKLE, VANGL1), target genes (MYB, MYC, WISP2, SPRY1, TP53, CCND1); calcium dependent pathway (PLCB1, CAMK2A, PRKCA, CHP); and planar cell polarity pathway (PTK7, DAAM1, RHOA) 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. Results 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 WISP2, 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. Conclusions 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.

ZAC1 and SSTR2 Are Downregulated in Non-Functioning Pituitary Adenomas but Not in somatotropinomas

Introduction There are few data regarding ZAC1 expression in clinically non-functioning pituitary adenomas (NFPA). Because somatotropinomas and NFPA behave differently with respect to tumor shrinkage during somatostatin analogs (SA) therapy, we sought to compare the ZAC1 and somatostatin receptor (sstr) types 1, 2, 3 and 5 mRNA expression in these two pituitary adenoma subtypes and in normal human pituitaries. Methods ZAC1 and SSTR mRNA expression levels were evaluated using real-time RT-PCR (TaqMan) in 20 NFPA and compared with the expression levels in 23 somatotropinomas and five normal pituitaries. The NFPA invasiveness was evaluated using magnetic resonance imaging with Hardy’s modified criteria. Ki-67 and p53 were evaluated using immunohistochemistry. Results A total of 20 patients with NFPA [6 males, median age 56 years (range: 30-78)], 23 with acromegaly [12 males, median age 43 years (range: 24–57)] and five normal pituitaries [4 males, median age 48 years (range: 36–54)] were included. Four of the patients (20%) had Hardy’s grade 2 tumors; all of the others had Hardy’s grade 3 tumors. The Ki-67 median expression was 2.35 (range: 0.2–9.23), and only four of the tumors (20%) were positive for p53. The ZAC1 mRNA expression was significantly lower in NFPA than in somatotropinomas and in normal pituitaries (p<0.001 for both), as well as the SSTR2 (p=0.001 and 0.01, respectively). The SSTR3 expression was higher in the NFPA than in the somatotropinomas and in the normal pituitaries (p=0.03 and 0.02, respectively). No correlation was found between the ZAC1 mRNA expression and the tumor invasiveness, Ki-67 and p53. Conclusion ZAC1 and SSTR2 are underexpressed and SSTR3 is overexpressed in NFPA compared to those in somatotropinomas and in normal pituitaries, which might explain the lack of tumor shrinkage that is observed in response to commercially available SA therapy in patients with NFPA.

Resistance to octreotide LAR in acromegalic patients with high SSTR2 expression: analysis of AIP expression.

We present here the clinical and molecular data of two patients with acromegaly treated with octreotide LAR after non-curative surgery, and who presented different responses to therapy. Somatostatin receptor type 2 and 5 (SSTR2 and SSTR5), and aryl hydrocarbon receptor-interacting protein (AIP) expression levels were analyzed by qPCR. In both cases, high SSTR2 and low SSTR5 expression levels were detected; however, only one of the patients achieved disease control after octreotide LAR therapy. When we analyzed AIP expression levels of both cases, the patient whose disease was controlled after therapy exhibited AIP expression levels that were two times higher than the patient whose disease was still active. These two cases illustrate that, although the currently available somatostatin analogs bind preferentially to SSTR2, some patients are not responsive to therapy despite high expression of this receptor. This difference could be explained by differences in post-receptor signaling pathways, including the recently described involvement of AIP.

HPA axis dysregulation, NR3C1 polymorphisms and glucocorticoid receptor isoforms imbalance in metabolic syndrome

Metabolic syndrome (MetS) shares several similarities with hypercortisolism.

Altered expression of noncanonical Wnt pathway genes in paediatric and adult adrenocortical tumours

The role of planar cell polarity (Wnt/PCP) and calcium‐dependent (Wnt/Ca) noncanonical Wnt pathways in adrenocortical tumours (ACTs) is unknown.

SAGE analysis highlights the putative role of underexpression of ribosomal proteins in GH-secreting pituitary adenomas.

BACKGROUND Although the molecular pathogenesis of pituitary adenomas has been assessed by several different techniques, it still remains partially unclear. Ribosomal proteins (RPs) have been recently related to human tumorigenesis, but they have not yet been evaluated in pituitary tumorigenesis. OBJECTIVE The aim of this study was to introduce serial analysis of gene expression (SAGE), a high-throughput method, in pituitary research in order to compare differential gene expression. METHODS Two SAGE cDNA libraries were constructed, one using a pool of mRNA obtained from five GH-secreting pituitary tumors and another from three normal pituitaries. Genes differentially expressed between the libraries were further validated by real-time PCR in 22 GH-secreting pituitary tumors and in 15 normal pituitaries. RESULTS Computer-generated genomic analysis tools identified 13,722 and 14,993 exclusive genes in normal and adenoma libraries respectively. Both shared 6497 genes, 2188 were underexpressed and 4309 overexpressed in tumoral library. In adenoma library, 33 genes encoding RPs were underexpressed. Among these, RPSA, RPS3, RPS14, and RPS29 were validated by real-time PCR. CONCLUSION We report the first SAGE library from normal pituitary tissue and GH-secreting pituitary tumor, which provide quantitative assessment of cellular transcriptome. We also validated some downregulated genes encoding RPs. Altogether, the present data suggest that the underexpression of the studied RP genes possibly collaborates directly or indirectly with other genes to modify cell cycle arrest, DNA repair, and apoptosis, leading to an environment that might have a putative role in the tumorigenesis, introducing new perspectives for further studies on molecular genesis of somatotrophinomas.