Aryl Hydrocarbon Receptor-interacting Protein Expression Research Articles

Aryl-hydrocarbon receptor-interacting protein regulates tumorigenic and metastatic properties of colorectal cancer cells driving liver metastasis.

Liver metastasis is the primary cause of colorectal cancer (CRC)-associated death. Aryl-hydrocarbon receptor-interacting protein (AIP), a putative positive intermediary in aryl-hydrocarbon receptor-mediated signalling, is overexpressed in highly metastatic human KM12SM CRC cells and other highly metastatic CRC cells. Meta-analysis and immunohistochemistry were used to assess the relevance of AIP. Cellular functions and signalling mechanisms mediated by AIP were assessed by gain-of-function experiments and in vitro and in vivo experiments. A significant association of high AIP expression with poor CRC patients' survival was observed. Gain-of-function and quantitative proteomics experiments demonstrated that AIP increased tumorigenic and metastatic properties of isogenic KM12C (poorly metastatic) and KM12SM (highly metastatic to the liver) CRC cells. AIP overexpression dysregulated epithelial-to-mesenchymal (EMT) markers and induced several transcription factors and Cadherin-17 activation. The former induced the signalling activation of AKT, SRC and JNK kinases to increase adhesion, migration and invasion of CRC cells. In vivo, AIP expressing KM12 cells induced tumour growth and liver metastasis. Furthermore, KM12C (poorly metastatic) cells ectopically expressing AIP became metastatic to the liver. Our data reveal new roles for AIP in regulating proteins associated with cancer and metastasis to induce tumorigenic and metastatic properties in colon cancer cells driving liver metastasis.

Transcription factor GTF2B regulates AIP protein expression in growth hormone-secreting pituitary adenomas and influences tumor phenotypes.

Clinically, the low expression of wild-type aryl hydrocarbon receptor-interacting protein (AIP) in patients with sporadic growth hormone (GH)-secreting pituitary adenoma (GHPA) is associated with a more aggressive phenotype. However, the mechanism by which AIP expression is regulated in GHPA remains unclear. Herein, we investigated a transcription factor that regulates AIP expression and explored its role in tumor phenotypes. General transcription factor IIB (GTF2B) was predicted by several bioinformatic tools to regulate AIP expression transcriptionally. Regulation by GTF2B was evaluated using chromatin immunoprecipitation (ChIP), reverse transcription PCR, luciferase reporter, and western blot experiments in SH-SY5Y cells. Furthermore, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, transwell invasive assay, ELISA, western blot, immunohistochemical staining, and terminal deoxynucleotidyl transferase dUTP nick end labeling were performed to investigate the effects of GTF2B and AIP on tumor cell proliferation, apoptosis, growth hormone secretion, and invasiveness in GH3 cells and mouse xenograft models. Moreover, correlations between GTF2B and AIP expression were explored in GHPA cases. ChIP and luciferase reporter studies demonstrated that the regulation of AIP expression by GTF2B was dependent on the intergenic-5' untranslated region element of AIP and the initial residual S65 of GTF2B. In vitro and in vivo experiments indicated that GTF2B regulated AIP expression to impact the GHPA phenotype; this was confirmed by data from 33 GHPA cases. We determined the regulation by GTF2B of AIP transcription in GHPA and its impact on tumor phenotype. Our findings suggest that GTF2B may be a potential therapeutic target for GHPA with low AIP expression.

Aryl hydrocarbon receptor interacting protein (AIP) significantly influences prognosis of pancreatic carcinoma

IntroductionAryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor. Aryl hydrocarbon receptor interacting protein (AIP) in one of AHR ligands. The aim of this study is to analyze the prognostic influence of AIP in pancreatic carcinoma. Material and methodsRetrospective case series with immunohistochemical analysis of AIP. We have estimated a multivariate Cox's model for the outcome (progression free and overall survival). Results204 patients were included in the study. As expected prognosis was poor and 67.8% died of disease. As for AIP 9.8% of the cases showed nuclear staining of the epithelial tumor cells and 59.4% a cytoplasmic one. Stroma was stained in 53.1% of the cases.Univariate survival analysis revealed a significantly worse prognosis of patients with cytoplasmic AIP expression (stroma and epithelium), but nuclear expression was associated to a better prognosis. In the multivariate analysis stromal AIP expression was an independent prognosticator of progression free survival, together with pT stage, histological grade and history of diabetes. DiscussionAIP Is a conserved cochaperone protein binding to many proteins. AIP has been proposed as a potential tumor suppressor gene. To date, no study has analyzed the immunohistochemical expression of AIP in pancreatic carcinoma. Our results indicate that both epithelial and stromal cytoplasmic expression of AIP is associated to bad prognosis, while nuclear translocation seems to improve prognosis. ConclusionAlthough we must deepen into the complex signaling pathways underlying this potential association, our results open a way to inhibiting AHR as a potential target against pancreatic carcinoma.

SNPs of miR-23b, miR-107 and HMGA2 and their Relations with the Response to Medical Treatment in Acromegaly Patients.

Acromegaly is a chronic disease of increased growth hormone (GH) secretion and elevated insulin-like growth factor-I (IGF-I) levels induced by a pituitary adenoma. HMGA2 (high mobility group A2) and AIP (aryl hydrocarbon receptor-interacting protein) expression levels are related to GH-secreting adenomas, and also a response to Somatostatin Analogs (SSAs). We studied SNPs in miR-107 and miR-23b that related with AIP and HMGA2 genes respectively and control their expression, and also SNP in the 3'UTR of HMGA2 gene. Our aim was to investigate genotype distributions of the studied SNPs, as well as the possible relationship between disease and/or response to SSAs treatment in patients with acromegaly. Genotypes were determined by qRT-PCR method from DNA materials obtained blood samples of acromegaly patients (141) and healthy individuals (99). The genotype distributions of patients and healthy groups, as well as the relationship between the clinical data of the disease and genotypes were statistically compared. In acromegaly patients with T-allele, p53 expression (p=0.049) was significantly higher. In patients with CT+TT genotype and T-allele who were responder to SSA-treatment Ki-67 index (respectively p=0.019, p=0.020 respectively) was higher. We did not observe the genotypes for miR-23b and miR-107 polymorphisms in the patients and control group of Turkish population. The genetic variations of the miRNAs genes related with HMGA2 and AIP genes were not seen in our study. Although there is no relationship between HMGA2-rs1351394 polymorphism and acromegaly disease, T allele was associated with some clinical features related to adenoma in patients with acromegaly.

Prognostic Role of Aryl Hydrocarbon Receptor Interacting Protein (AIP) Immunohistochemical Expression in Patients with Resected Gastric Carcinomas.

Aryl hydrocarbon receptor (AHR) interacting protein (AIP) is a chaperone which binds to inactive AHR in the cell cytoplasm. AHR is best known for mediating the toxicity of halogenated aromatics, but it has also been linked to carcinogenesis and tumor progression in several tumor types. Our aims are to assess the features of AIP immunohistochemical (IHC) staining and to evaluate its possible role as a prognostic marker in gastric cancer (GC). Retrospective study of 147 cases of resected GC. Clinicopathological features were collected, tissue microarrays were constructed for AIP IHC and statistical analysis were performed. AIP staining was observed in 50.3% of tumors. All AIP-positive cases exhibited cytoplasmic or membranous staining, variably associated with nuclear co-staining. 93.2% of AIP-positive tumors showed AIP immunoreactivity in 100% of cells. Staining intensity was mild, moderate and intense in 33.8%, 13.5% and 52.7% of cases. Tumors were stratified according to AIP staining intensity into low expression (no or mild AIP immunoreactivity) and high expression (moderate or intense AIP immunoreactivity). 36.6% of our cases showed high AIP expression. High AIP expression was significantly and independently correlated to tumor progression and cancer death. Tumors with high AIP expression showed lower survival and higher progression rates. AIP expression might be useful for determining GC prognosis. More studies are needed to clarify the role of AHR pathway in GC, AIP expression and its potential use as a surrogate marker for selecting patients for AHR modulation therapy.

SAT-LB056 Is AIP a Tumor Suppressor or an Oncogene? AIP as a Novel Regulator of the Oncogene BCL6 in Diffuse Large B Cell Lymphoma

Background: Heterozygous germline loss-of-function mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene are usually associated with acromegaly or gigantism due to a young-onset somatotropinoma. AIP functions as a tumor suppressor gene in the pituitary, while its role in other tumours is unknown. AIP is highly expressed in patients with Diffuse Large B Cell Lymphoma (DLBCL) compared to any other tumor type. DLBCL arises from germinal centre B cells which characteristically express the transcriptional repressor B cell lymphoma-6 (BCL6), key for germinal centre formation. Increased expression of BCL6 can lead to the development of DLBCL. Despite the obvious importance of BCL6 expression in GC B cells, the mechanisms by which it is regulated are still poorly understood. Aim: The aim of this study was to investigate the relationship of the high AIP expression, BCL6 and the pathobiology of DLBCL. Methods: We generated mice carrying a conditional homozygous deletion of Aip in T and B cells (Aipfl/fl;Rag1Cre/+). Co-localization of AIP and members of the BCL6 ubiquitination/proteasome pathway by immunofluorescence analysis. Ubiquitin-mediated proteasomal degradation study was done by immuno-precipitation (IP). Results: Our study found that AIP is highly expressed in DLBCL. Genetic deletion of Aip revealed that AIP supported BCL6 expression in mice. The ubiquitin E3 ligase FBXO11 has been previously shown to add ubiquitin to BCL6. We found that AIP could bind to the deubiquitinase UCHL1 and that UCHL1 could remove ubiquitin from BCL6 thereby supporting BCL6 expression by preventing FBXO11 mediated degradation of BCL6 recapitulating what we observed in Aip deficient B cells. Conclusions: The data presented here reveal AIP to be a novel positive regulator of BCL6 protein expression which is commonly up-regulated in DLBCL and prevents FBXO11 degradation of BCL6 by enabling the UCHL1 to remove ubiquitin from BCL6. Therefore, AIP is a potential novel therapeutic target to treat DLBCL. It appears that AIP function as a tumor suppressor in the pituitary and as an oncogene to the pathobiology of DLBCL. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Expression of Peroxisome Proliferator-Activated Receptor Alpha (PPARα) in Non-Somatotroph Pituitary Tumours and the Effects of PPARα Agonists on MMQ Cells

Peroxisome proliferator-activated receptor alpha (PPARα) has been involved in the regulation of somatotroph tumour cells and may be targeted by different drugs, some of them are in current clinical use. The aim of this study was to investigate the expression of PPARα in additional phenotypes of pituitary adenomas (PA), the relationship between PPARα and its potential molecular partner aryl hydrocarbon receptor interacting protein (AIP) in these tumours, and the effects of PPARα agonists on lactotroph cells. Seventy-five human PA - 57 non-functioning (NFPA) and 18 prolactinomas (PRL-PA) - were characterised for PPARα and AIP expression by real time RT-PCR and/or immunohistochemistry (IHC), and the effects of fenofibrate and WY 14 643 on MMQ cells were studied in vitro. PPARα was expressed in a majority of PA. PPARα immunostaining was observed in 93.7% PRL-PA vs. 60.6% NFPA (p=0.016), the opposite being found for AIP (83.3% in NFPA vs. 43.7% in PRL-PA, p=0.003). PPARα expression was unrelated to gonadotroph differentiation in NFPA, but positively correlated with tumour volume in PRL-PA. Both drugs significantly reduced MMQ cell growth at high concentrations (100-200 μM). At the same time, despite modest stimulating effects on PRL secretion were observed, these were overcome by the reduction in cell number. In conclusion, PPARα is commonly expressed by PRL-PA and NFPA, regardless of AIP, and may represent a new target of PPARα agonists.

Study of major genetic factors involved in pituitary tumorigenesis and their impact on clinical and biological characteristics of sporadic somatotropinomas and non-functioning pituitary adenomas.

Genetic and functional aberrations of guanine nucleotide-binding protein, alpha stimulating (GNAS), aryl hydrocarbon receptor interacting protein (AIP), and pituitary tumor transforming gene (PTTG) are among the most prominent events in pituitary tumorigenesis. A cohort of Brazilian patients with somatotropinomas (n=41) and non-functioning pituitary adenomas (NFPA, n=21) from a single tertiary-referral center were evaluated for GNAS and AIP mutations and gene expression of AIP and PTTG. Results were compared to the clinical and biological (Ki67 and p53 expression) characteristics of tumors and their response to therapy, if applicable. Genetic analysis revealed that 27% of somatotropinomas and 4.8% of NFPA harbored GNAS mutations (P=0.05). However, no differences were observed in clinical characteristics, tumor extension, response to somatostatin analog therapy, hormonal/surgical remission rates, Ki67 index, and p53 expression between mutated and non-mutated somatotropinomas patients. PTTG overexpression (RQ mean=10.6, min=4.39, max=11.9) and AIP underexpression (RQ mean=0.56, min=0.46-max=0.92) were found in virtually all cases without a statistically significant relationship with clinical and biological tumor features. No patients exhibited somatic or germline pathogenic AIP mutations. In conclusion, mutations in GNAS and abnormal PTTG and AIP expression had no impact on tumor features and treatment outcomes in this cohort. Our data support some previous studies and point to the need for further investigations, probably involving epigenetic and transcriptome analysis, to improve our understanding of pituitary tumor behavior.

Benzene and 2-ethyl-phthalate induce proliferation in normal rat pituitary cells

PurposeEndocrine disruptors are known to modulate a variety of endocrine functions and increase the risk for neoplasia. Epidemiological data reported increased prevalence of pituitary tumors in high industrial areas while genotyping studies showed that mutations in the aryl hydrocarbon receptor (AhR) interacting protein (AIP)—chaperone to the dioxin ligand AhR—gene are linked to predisposition to pituitary tumor development. Aim of the present study was to establish whether endocrine pollutants can induce cell proliferation in normal rat pituitary cells.MethodsPituitary primary cultures were incubated with 250, 650 and 1250 pM benzene or 2-ethyl-phthalate for up to 96 h and viability, energy content and cell proliferation assessed. Expression of pituitary tumor transforming gene (PTTG), cyclin D1 (Ccnd1), AhR and AIP was quantified by RT-qPCR.ResultsIncubation with benzene or 2-ethyl-phthalate increased viability and energy content in pituitary cells. The endocrine disruptors also increased cell proliferation as well as Ccnd1 and PTTG expression. Increased AhR and AIP expression was observed after incubation with the two pollutants.ConclusionsOur findings indicate that benzene and 2-ethyl-phthalate activate AhR/AIP expression and stimulate proliferation in normal rat pituitary cells. This study is the first demonstration that pollutants can induce normal pituitary cells to proliferate and provides a link between epidemiological and genomic findings in pituitary tumors.

AIP mutations impair AhR signaling in pituitary adenoma patients fibroblasts and in GH3 cells.

Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene predispose humans to pituitary adenomas through unknown molecular mechanisms. The best-known interacting partner of AIP is the aryl hydrocarbon receptor (AhR), a transcription factor that mediates the effects of xenobiotics implicated in carcinogenesis. As 75% of AIP mutations disrupt the physical and/or functional interaction with AhR, we postulated that the tumorigenic potential of AIP mutations might result from altered AhR signaling. We evaluated the impact of AIP mutations on the AhR signaling pathway, first in fibroblasts from AIP-mutated patients with pituitary adenomas, by comparison with fibroblasts from healthy subjects, then in transfected pituitary GH3 cells. The AIP protein level in mutated fibroblasts was about half of that in cells from healthy subjects, but AhR expression was unaffected. Gene expression analyses showed significant modifications in the expression of the AhR target genes CYP1B1 and AHRR in AIP-mutated fibroblasts, both before and after stimulation with the endogenous AhR ligand kynurenine. Kynurenine increased Cyp1b1 expression to a greater extent in GH3 cells overexpressing wild type compared with cells expressing mutant AIP Knockdown of endogenous Aip in these cells attenuated Cyp1b1 induction by the AhR ligand. Both mutant AIP expression and knockdown of endogenous Aip affected the kynurenine-dependent GH secretion of GH3 cells. This study of human fibroblasts bearing endogenous heterozygous AIP mutations and transfected pituitary GH3 cells shows that AIP mutations affect the AIP protein level and alter AhR transcriptional activity in a gene- and tissue-dependent manner.

Expression of Peroxisome Proliferator-Activated Receptor alpha (PPARα) in somatotropinomas: Relationship with Aryl hydrocarbon receptor Interacting Protein (AIP) and in vitro effects of fenofibrate in GH3 cells

PurposeTo search for a possible role of Peroxisome Proliferator-Activated Receptor α (PPARα), a molecular partner of the Aryl hydrocarbon receptor Interacting Protein (AIP), in somatotropinomas. MethodsTumours from 51 acromegalic patients were characterized for PPARα and AIP expression by immunohistochemistry (IHC) and/or Real Time RT-PCR. Data were analysed according to tumour characteristics and pre-operative treatment with somatostatin analogues (SSA). The effects of fenofibrate were studied in GH3 cells in vitro. ResultsPPARα was expressed in most somatotropinomas. A modest relationship was found between PPARα and AIP expression, both being significantly higher in the presence of pre-operative SSA. However, only AIP expression was influenced by the response to treatment. Dual effects of fenofibrate were observed in GH3 cells, consisting of cell growth inhibition and an increase in GH secretion inhibited by octreotide. ConclusionsPPARα is a new player in somatotropinomas. Potential interactions between PPARα agonists and SSA may deserve further investigation.

Markers of proliferation and invasiveness in somatotropinomas.

Introduction In the search for markers of invasiveness of pituitary adenomas, we studied the expression of Ki-67 antigen, TOPO 2A (topoisomerase 2 alpha), AIP (Aryl Hydrocarbon Receptor-Interacting Protein) and VEGF (Vascular Endothelial Growth Factor) in somatotropinomas. Material and Methods We retrospectively studied a group of 31 patients who underwent pituitary tumour surgery. Expression of Ki-67, TOPO 2A, AIP and VEGF in surgical specimens was determined by immunohistochemistry. Relations between quantitatively determined markers and clinical symptoms, tumour features, and MR imaging, were analysed. Acromegaly was confirmed by hormonal tests in all patients studied. Local invasiveness (cavernous sinus penetration, optic chiasm compression or suprasellar extension) was observed in 18/31 patients (58,1%). Results Ki-67 was expressed in 77.4%, TOPO 2A in 87.1%, AIP in 83.8%, and VEGF in 87.1% of 31 cases of somatropinoma. Median values of Ki-67, TOPO 2A, AIP and cytoplasmic VEGF indices were 1.2% [IQR=2.2], 1.5% [IQR=1.6], 21.26% [IQR=20.1] and 20.4% [IQR=15.4], respectively. Ki-67, TOPO 2A, AIP and VEGF expression was not correlated with age nor with patient gender (p > 0.05). Only Ki-67 and TOPO 2A correlated with tumour size (for Ki-67: r=0.42, p=0.025; for TOPO 2A: r=0.53, p=0.003). Ki-67 and TOPO 2A levels were significantly higher in invasive compared to noninvasive somatropinomas (Ki67 mean values: 1.85±1.33% vs. 0.95±1.07%, p=0.024; TOPO 2A mean values: 2.19±1.63% vs. 1.45±1.23%, , p=0.011). Conclusions Ki-67, TOPO 2A, AIP and VEGF were expressed in over 70% of all somatotropinomas. Only Ki-67 and TOPO 2A expression correlated with tumour size and tumour invasiveness.

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

IntroductionPatients with germline AIP mutations or low AIP protein expression have large, invasive somatotroph adenomas and poor response to somatostatin analogues (SSA).MethodsTo 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.ResultsNo 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.ConclusionLow 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.

AHR over-expression in papillary thyroid carcinoma: clinical and molecular assessments in a series of Italian acromegalic patients with a long-term follow-up.

AimAcromegaly reportedly carries an increased risk of malignant and benign thyroid tumors, with a prevalence of thyroid cancer of around 3–7%. Germline mutations in the aryl-hydrocarbon receptor (AHR) interacting protein (AIP) have been identified in familial forms of acromegaly. The molecular and endocrine relationships between follicular thyroid growth and GH-secreting pituitary adenoma have yet to be fully established. Our aim was to study the prevalence of differentiated thyroid cancer (DTC) in acromegaly, focusing on the role of genetic events responsible for the onset of thyroid cancer.MethodsGermline mutations in the AIP gene were assessed in all patients; BRAF and H-N-K RAS status was analyzed by direct sequencing in thyroid specimens, while immunohistochemistry was used to analyze the protein expression of AIP and AHR. A set of PTCs unrelated to acromegaly was also studied.Results12 DTCs (10 papillary and 2 follicular carcinomas) were identified in a cohort of 113 acromegalic patients. No differences in GH/IGF-1 levels or disease activity emerged between patients with and without DTC, but the former were older and more often female. BRAF V600E was found in 70% of the papillary thyroid cancers; there were no RAS mutations. AIP protein expression was similar in neoplastic and normal cells, while AHR protein was expressed more in PTCs carrying BRAF mutations than in normal tissue, irrespective of acromegaly status.ConclusionsThe prevalence of DTC in acromegaly is around 11% and endocrinologists should bear this in mind, especially when examining elderly female patients with uninodular goiter. The DTC risk does not seem to correlate with GH/IGF-1 levels, while it may be associated with BRAF mutations and AHR over-expression. Genetic or epigenetic events probably play a part in promoting thyroid carcinoma.

Somatostatin analogues increase AIP expression in somatotropinomas, irrespective of Gsp mutations

Germline aryl hydrocarbon receptor interacting protein (AIP) gene mutations confer a predisposition to pituitary adenoma (PA), predominantly GH-secreting (GH-PA). As recent data suggest a role for AIP in the pathogenesis of sporadic GH-PA and their response to somatostatin analogues (SSA), the expression of AIP and its partner, aryl hydrocarbon receptor (AHR), was determined by semiquantitative immunohistochemistry scoring in 62 sporadic GH-PA (37 treated with SSA preoperatively). The influence of Gsp status was studied in a subset of tumours (n=39, 14 Gsp(+)) and six GH-PA were available for primary cultures. AIP and AHR were detected in most cases, with a positive correlation between AIP and cytoplasmic AHR (P=0.012). Low AIP expression was significantly more frequent in untreated vs SSA-treated tumours (44.0 vs 20.5%, P=0.016). AHR expression or localisation did not differ between the two groups. Similarly, in vitro octreotide induced a median twofold increase in AIP expression (range 1.2-13.9, P=0.027) in GH-PA. In SSA-treated tumours, the AIP score was significantly higher in the presence of preoperative IGF1 decrease or tumour shrinkage (P=0.008 and P=0.014 respectively). In untreated tumours, low AIP expression was significantly associated with invasiveness (P=0.028) and suprasellar extension (P=0.019). The only effect of Gsp status was a significantly lower nuclear AHR score in Gsp(+) vs Gsp(-) tumours (P=0.025), irrespective of SSA. In conclusion, AIP is involved in the aggressiveness of sporadic GH-PA, regardless of Gsp status, and AIP up-regulation in SSA-treated tumours is associated with a better preoperative response, with no clear role for AHR.

Altered subcellular localization of heat shock protein 90 is associated with impaired expression of the aryl hydrocarbon receptor pathway in dogs.

The aryl hydrocarbon receptor (AHR) mediates biological responses to toxic chemicals. An unexpected role for AHR in vascularization was suggested when mice lacking AHR displayed impaired closure of the ductus venosus after birth, as did knockout mice for aryl hydrocarbon receptor interacting protein (AIP) and aryl hydrocarbon receptor nuclear translocator (ARNT). The resulting intrahepatic portosystemic shunts (IHPSS) are frequently diagnosed in specific dog breeds, such as the Irish wolfhound. We compared the expression of components of the AHR pathway in healthy Irish wolfhounds and dogs with IHPSS. To this end, we analyzed the mRNA expression in the liver of AHR,AIP, ARNT, and other genes involved in this pathway, namely, those for aryl hydrocarbon receptor nuclear translocator 2 (ARNT2), hypoxia inducible factor 1alpha (HIF1A), heat shock protein 90AA1 (HSP90AA1), cytochromes P450 (CYP1A1, CYP1A2, and CYP1B1), vascular endothelial growth factor A (VEGFA), nitric oxide synthesase 3 (NOS3), and endothelin (EDN1). The observed low expression of AHR mRNA in the Irish wolfhounds is in associated with a LINE-1 insertion in intron 2, for which these dogs were homozygous. Down regulation in Irish wolfhounds was observed for AIP, ARNT2, CYP1A2, CYP1B1 and HSP90AA1 expression, whereas the expression of HIF1A was increased. Immunohistochemistry revealed lower levels of AHR, HIF1A, and VEGFA protein in the nucleus and lower levels of ARNT and HSP90AA1 protein in the cytoplasm of the liver cells of Irish wolfhounds. The impaired expression of HSP90AA1 could trigger the observed differences in mRNA and protein levels and therefore explain the link between two very different functions of AHR: regulation of the closure of the ductus venosus and the response to toxins.

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.

MicroRNA miR-107 is overexpressed in pituitary adenomas and inhibits the expression of aryl hydrocarbon receptor-interacting protein in vitro

Abnormal microRNA (miRNA) expression profiles have recently been associated with sporadic pituitary adenomas, suggesting that miRNAs can contribute to tumor formation; miRNAs are small noncoding RNAs that inhibit posttranscriptional expression of target mRNAs by binding to target sequences usually located in the 3'-UTR. In this study, we investigated the role played by miR-107, a miRNA associated with different human cancers, in sporadic pituitary adenomas and its interaction with the pituitary tumor suppressor gene aryl hydrocarbon receptor-interacting protein (AIP). miR-107 expression was evaluated in pituitary adenoma and normal pituitary samples using microRNA screen TLDA (TaqMan Low-Density Array) and RT-qPCR assays. We show that miR-107 expression was significantly upregulated in GH-secreting and nonfunctioning pituitary adenomas. We found that human AIP-3'-UTR is a target of miR-107 since miR-107 inhibited in vitro AIP expression to 53.9 ± 2% of the miRNA control in a luciferase assay and reduced endogenous AIP mRNA expression to 53 ± 22% of the miRNA control in human cells. However, we did not observe a negative correlation between AIP and miR-107 expression in the human tumor samples. Furthermore, we show that miR-107 overexpression inhibited cell proliferation in human neuroblastoma and rat pituitary adenoma cells. In conclusion, miR-107 is overexpressed in pituitary adenomas and may act as a tumor suppressor. We have identified and confirmed AIP as a miR-107 target gene. Expression data in human samples suggest that the expression of AIP and miR-107 could be influenced by a combination of tumorigenic factors as well as compensatory mechanisms stimulated by the tumorigenic process.

Somatostatin Analogs Modulate AIP in Somatotroph Adenomas: The Role of the ZAC1 Pathway

Somatotroph adenomas harboring aryl hydrocarbon receptor interacting protein (AIP) mutations respond less well to somatostatin analogs, suggesting that the effects of somatostatin analogs may be mediated by AIP. The objective of the investigation was to study the involvement of AIP in the mechanism of effect of somatostatin analogs. In the human study, a 16-wk somatostatin analog pretreatment compared with no pretreatment. In the in vitro cell line study, the effect of somatostatin analog treatment or small interfering RNA (siRNA)/plasmid transfection were studied. The study was conducted at a university hospital. Thirty-nine sporadic and 10 familial acromegaly patients participated in the study. Interventions included preoperative lanreotide treatment and pituitary surgery. For the human study, GH and IGF-I levels, AIP, and somatostatin receptor staining were measured. For the cell line, AIP and ZAC1 (zinc finger regulator of apoptosis and cell cycle arrest) expression, metabolic activity, and clone formation were measured. Lanreotide pretreatment reduced GH and IGF-I levels and tumor volume (all P < 0.0001). AIP immunostaining was stronger in the lanreotide-pretreated group vs. the surgery-only group (P < 0.001). After lanreotide pretreatment, the AIP score correlated to IGF-I changes in females (R = 0.68, P < 0.05). Somatostatin receptor staining was not reduced in samples with AIP mutations. In GH3 cells, 1 nm octreotide increased AIP mRNA and protein (both P < 0.01) and ZAC1 mRNA expression (P < 0.05). Overexpression of wild-type (but not mutant) AIP increased ZAC1 mRNA expression, whereas AIP siRNA knockdown reduced ZAC1 mRNA (both P < 0.05). The siRNA-mediated knockdown of AIP led to an increased metabolic activity and clonogenic ability of GH3 cells compared with cells transfected with a nontargeting control (both P < 0.001). These results suggest that AIP may play a role in the mechanism of action of somatostatin analogs via ZAC1 in sporadic somatotroph tumors and may explain their lack of effectiveness in patients with AIP mutations.

Growth of an aggressive tumor during pregnancy in an acromegalic patient

Pregnancy in acromegalic patients is a rare event, but is usually uneventful, with stable GH and IGF-I levels and no tumor enlargement. Medical treatment can usually be withdrawn without problems and although no major adverse event has been reported, the suspension of drug treatments is generally recommended. No case report exists in the literature regarding evolution of a somatotropinoma with invasiveness markers throughout pregnancy. We report a case of an acromegalic patient who was submitted to surgery and treated with octreotide LAR maintaining a stable residual tumor and an IGF-I close to the normal levels. Her tumor presented with a high Ki-67 (11.6%) and a low aryl hydrocarbon receptor-interacting protein (AIP) expression. When she became pregnant, octreotide LAR was withdrawn, and despite remaining asymptomatic during pregnancy, tumor growth occurred with compression of surrounding structures. In conclusion, pregnancy in acromegalic patients has usually a favorable prognosis with no tumor growth. However, in the presence of high Ki-67 labeling index and low AIP expression, tumor enlargement may occur and somatostatin analogue treatment throughout the pregnancy should be considered.