AIP Inactivation Leads to Pituitary Tumorigenesis Through Defective GΑI-Camp Signaling

ORIGINAL ARTICLE AIP inactivation leads to pituitary tumorigenesis through defective Gαi-cAMP signaling I Tuominen1,6,7, E Heliövaara1,6, A Raitila1, M-R Rautiainen1,8, M Mehine1, R Katainen1, I Donner1, V Aittomäki2, HJ Lehtonen1, M Ahlsten1, L Kivipelto3, C Schalin-Jäntti4, J Arola5, S Hautaniemi2 and A Karhu1

The aryl hydrocarbon receptor interacting protein (AIP) is a tumor-suppressor gene underlying the pituitary adenoma predisposition. Thus far, the exact molecular mechanisms by which inactivated AIP exerts its tumor-promoting action have been unclear. To better understand the role of AIP in pituitary tumorigenesis, we performed gene expression microarray analysis to examine changes between Aip wild-type and knockout mouse embryonic fibroblast (MEF) cell lines. Transcriptional analyses implied that Aip deficiency causes a dysfunction in cyclic adenosine monophosphate (cAMP) signaling, as well as impairments in signaling cascades associated with developmental and immune-inflammatory responses. In vitro experiments showed that AIP deficiency increases intracellular cAMP concentrations in both MEF and murine pituitary adenoma cell lines. Based on knockdown of various G protein α subunits, we concluded that AIP deficiency leads to elevated cAMP concentrations through defective Gαi-2 and Gαi-3 proteins that normally inhibit cAMP synthesis. Furthermore, immunostaining of Gαi-2 revealed that AIP deficiency is associated with a clear reduction in Gαi-2 protein expression levels in human and mouse growth hormone (GH)-secreting pituitary adenomas, thus indicating defective Gαi signaling in these tumors. By contrast, all prolactin-secreting tumors showed prominent Gαi-2 protein levels, irrespective of Aip mutation status. We additionally observed reduced expression of phosphorylated extracellular signal-regulated kinases 1/2 and cAMP response element-binding protein levels in mouse and human AIP-deficient somatotropinomas. This study implies for the first time that a failure to inhibit cAMP synthesis through dysfunctional Gαi signaling underlies the development of GH-secreting pituitary adenomas in AIP mutation carriers.

Oncogene (2015) 34, 1174–1184; doi:10.1038/onc.2014.50; published online 24 March 2014

INTRODUCTION by which inactivated AIP exerts its tumor-promoting action in the The aryl hydrocarbon receptor interacting protein (AIP) is an pituitary have been unclear, and further work is required to immunophilin-like protein found in the cytoplasm as part of a identify the key proteins and pathways underlying the genesis of multiprotein complex. AIP has several well-characterized inter- AIP-associated pituitary adenomas. action partners, through which it has a potential to affect a large The heterotrimeric guanine nucleotide-binding proteins number of different pathways, such as the cyclic adenosine (G proteins) are formed of Gα,Gβ and Gγ subunits that hydrolyze monophosphate (cAMP) and various nuclear receptor signaling guanosine triphosphate to guanosine diphosphate. G proteins are – pathways.1 3 We have previously identiﬁed AIP as a tumor- activated upon ligand-binding to the G protein-coupled receptors suppressor gene associated with pituitary adenomas, neoplasms (GPCRs). The α-subunits, which determine the identity of G 4 of the anterior pituitary gland. Patients with an AIP mutation are proteins, are divided into four families: Gαs,Gαq/Gα11,Gα12/13 and 9 typically diagnosed at a young age (median age 25 years) and Gαi/Gαo. G proteins communicate signals from many hormones with growth hormone (GH)-secreting pituitary tumors (somato- and neurotransmitters, mediated by second messengers, such as tropinomas, ~ 80% of cases) that cause acromegaly/gigantism.5,6 cAMP. Somatic gain-of function mutations in the α subunit of the AIP mutation carriers have also been detected among prolactin stimulatory G protein (Gαs, also known as GNAS) convert the Gαs (PRL) and nonsecreting adenoma patients and, rarely, among gene into an oncogene (termed gsp) that encodes for a protein cases of adrenocorticotropic hormone-secreting tumors. Recent that stimulates adenylyl cyclase to constantly produce cAMP.10 studies have implicated that AIP-associated pituitary tumors have These mutations are observed in 30–40% of somatotropinomas. an aggressive disease proﬁle, with large and invasive adenomas It is not fully established how these defects result in pituitary that poorly respond to available treatments, such as somatostatin tumorigenesis, but it has been suggested that Ser133 phosphory- – analogs used in acromegaly.6 8 The exact molecular mechanisms lated cAMP response element-binding protein (CREB) enhances

1Department of Medical Genetics, Genome-Scale Biology Research Program, Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki , Helsinki, Finland; 2Systems Biology Laboratory, Genome-Scale Biology Research Program, Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland; 3Department of Neurosurgery, University of Helsinki and Helsinki University Hospital, Helsinki, Finland; 4Division of Endocrinology, Department of Medicine, University of Helsinki and Helsinki University Hospital, Helsinki, Finland and 5Department of Pathology, HUSLAB, Helsinki University Central Hospital and Haartman Institute, University of Helsinki, Helsinki, Finland. Correspondence: Dr A Karhu, Department of Medical Genetics, Genome-Scale Biology Research Program, Institute of Biomedicine, Biomedicum Helsinki, University of Helsinki, Helsinki 00014, Finland. E-mail: auli.karhu@helsinki.fi 6These authors contributed equally to this work. 7Current address: Division of Digestive Diseases, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA. 8Current address: Public Health Genomics Unit and Institute for Molecular Medicine FIMM, National Institute for Health and Welfare, Helsinki, Finland. Received 14 February 2013; revised 16 December 2013; accepted 1 January 2014; published online 24 March 2014 AIP deficiency leads to elevated cAMP levels I Tuominen et al 1175 mitogenic signaling in GH-secreting cells (somatotrophs).11–13 Along with the well-known somatic gsp mutations, elevated cAMP levels have been linked to McCune–Albright syndrome and Carney complex, both of which are associated with pituitary tumors.11,12,14–17 In addition, the role of Gα proteins in tumorigenesis of other tumor types has recently emerged from studies 18,19 of Van Raamsdonk et al. showing that Gαq and Gα11 are targets of somatic mutations in melanocytic neoplasms. Gα11 have been associated also with regulation of calcium signaling.20 Recent studies have suggested that AIP might also be involved in regulating cAMP signaling. It was found that AIP interacts with 21 two Gα proteins, namely Gα13 and Gαq. Intriguingly, it has been demonstrated that Gα13 is capable of regulating cAMP concentra- 22,23 tions by cooperating with Gαs. As cAMP is a mitogenic factor selectively in somatotrophs,11,13 these AIP interactions provide a conceivable mechanism of how inactivation of this tumor suppressor contributes to pituitary tumor development. To date, however, the exact role of AIP in cAMP signaling is incompletely defined. To characterize the role of AIP in pituitary tumorigenesis, we have previously created an Aip mouse model.24 Heterozygous mice were extremely prone to pituitary adenomas, in particular to somatotropinomas, and the total lack of Aip resulted in embryonic lethality. Here, we performed a microarray analysis to examine changes in expression levels between Aip wild-type (WT) and knockout (KO) mouse embryonic fibroblast (MEF) cell lines obtained from Aip mouse model. Pathway analysis suggested that Aip deficiency causes a dysfunction of cAMP signaling, as well as impairments in pathways associated with developmental and immune-inflammatory responses. In vitro experiments further revealed that the lack of AIP leads to increased accumulation of cAMP through defective Gαi signaling and to the subsequent downregulation of phosphorylated extracellular signal-regulated kinases 1/2 (p-ERK1/2) and p-CREB. These data, for the first time, provide evidence that constitutive activation of cAMP production through dysfunctional Gαi signaling is a major contributor in AIP-associated GH-secreting pituitary adenoma development.

RESULTS GPCR and cAMP signaling pathways are transcriptionally altered in Aip KO vs WT MEFs We used gene expression data to understand the molecular effects of Aip deficiency. The data were obtained from seven Aip KO and nine WT MEF cell lines (GSE37927). A total of 608 differentially expressed genes were identified (Supplementary Table S1). Consistent with the earlier work showing that Aip is not transcribed in Aip KO MEFs,25 Aip was the most significantly Figure 1. Aip KO and WT MEF cell line pathway analysis based on the downregulated transcript in Aip KO cell lines (fold change –2.2, primary MEF cell line exon array. IPA pathway analysis (www. P =2×10À9). ingenuity.com) showing canonical pathways involved with Aip deficiency. The vertical bar denotes Po0.05 threshold (1.3 –log). The To understand the biological relevance of the differentially fi expressed genes during Aip deficiency, we performed the ratio: number of signi cantly differentially expressed genes in pathway meeting cutoff criteria divided by total number of genes in ingenuity pathway analysis (IPA) (Ingenuity Systems, Redwood pathway. City, CA, USA, www.ingenuity.com). The top biological function categories that emerged were associated with developmental functions, immune-inflammatory responses, cancer, cellular move- (Figure 2). Furthermore, the microarray data revealed that ment, growth and proliferation (Supplementary Table S2). The IPA phosphodiesterase enzymes, involved in the deactivation of pathway analysis highlighted pathways associated with cAMP cAMP,26 were overexpressed in the Aip KO cells (Table 1). The signaling, immune-inflammatory response, cardiovascular signal- expression profiling hence underscore the potential involvement ing, cancer and cell proliferation (Figure 1 and Supplementary of Aip in the regulation of GPCR-cAMP signaling, and in a variety Table S3). Two of the pathways were related to cAMP production, of physiological events such as developmental processes, namely GPCR-mediated (P = 1.8 × 10À5) and cAMP-mediated immune-inflammatory responses, and cellular proliferation and signaling (P = 5.4 × 10À5). In the GPCR pathway, receptor sub- differentiation. classes Gq and Gi showed aberrant expression. Upregulation of To investigate Aip-mediated inhibition of cell proliferation, Aip A kinase anchor protein 5 (Akap5) and dual specificity protein KO and WT MEF cell lines were analyzed by the 3-(4,5- phosphatase 1 (Dusp1) suggested involvement of protein kinase A dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophe- and mitogen-activated protein kinase signaling in Aip deficiency nyl)-2H-tetrazolium) (MTS) assay. Aip KO MEF cells showed higher

Figure 2. Defective GPCR pathway involved with Aip deﬁciency. Colored genes were identiﬁed by microarray analysis as differentially upregulated or downregulated in Aip KO cells. The genes with aberrant expression in GPCR pathway, their fold changes and P-values are listed in Table 1. Genes are linked by their subcellular location. Red label, upregulated; green label, downregulated.

proliferation rates compared with WT MEFs (Figure and 97% in both cell lines, as measured by quantitative-PCR 3a; Supplementary Figure S1). This finding is consistent with the (Supplementary Figure S1). No statistically significant differences work of Heliövaara et al.25 that showed increased cell proliferation in silencing efficiencies/locus were observed between the cell in GH3 pituitary adenoma cells after Aip silencing. lines. The silencing of Gαs, Gα12, Gα13, Gα11 and Gα14 decreased intracellular cAMP concentrations in both cell lines when Aip deficiency elevates cAMP concentrations in MEF and pituitary compared with mock siRNA-treated cells (Table 2 and Figure 4a). adenoma cell lines The cAMP concentration reductions were generally more pro- nounced in the WT cells, but a statistically significant difference As it is known that cAMP promotes proliferation of somatotrophs was observed only in the Gα (Po0.05) and Gα (Po0.01) genes. in the pituitary and contributes to the development of a subset of s 13 Gα is reported to regulate cAMP concentrations by cooperating somatotropinomas,11,17 we measured intracellular cAMP concen- 13 with Gα .22,23 Therefore, we simultaneously silenced both Gα and trations in primary and immortalized WT and Aip KO MEFs (two WT s s Gα . The combination knockdown reduced cAMP concentrations and two KO cell lines). When concentrations (pmol/ml) were 13 by 43% in WT and 8% in KO cells. The reductions were thus normalized against the total protein concentrations, we found that comparable to that of Gα alone (Table 2). Altogether, changes in cAMP levels were two- to threefold higher in Aip KO cells s cAMP concentration after silencing of these Gα genes were compared with the WT cells (Figure 3b). To determine whether the relatively small in the KO cells, and hence insufficient to explain effect of Aip deficiency on cAMP concentrations is limited to MEF the two- to threefold higher cAMP concentrations observed in cells, we performed small interfering RNA (siRNA)-mediated Aip Aip-deficient cells. knockdown in a rat GH/PRL-secreting pituitary adenoma cell line Proteins of the inhibitory Gα (Gα ) subfamily, Gα ,Gα and GH3 (Figure 3c). Partial silencing of Aip (56–73%, three indepen- i i-1 i-2 Gα , mediate receptor-dependent inhibition of various types of dent experiments) induced 20–30% higher cAMP concentrations i-3 adenylyl cyclases, leading to a decrease in cAMP levels. Knock- compared with the mock siRNA treated GH3 cells; Po0.05 down of Gα did not significantly alter cAMP concentrations in (percentual change of cAMP levels between ctrl and Aip siRNA- i-1 either WT or Aip KO cell lines. In WT cells, silencing of Gα and treated GH3 cells; three independent experiments with duplicates) i-2 Gα led to a marked increase in cAMP levels. Silencing of (Figure 3d). i-3 Gαi-2 elevated cAMP levels by 77% and that of Gαi-3 by 115%. Strikingly, silencing of these genes in Aip KO cells caused only α Defective G i signaling underlies elevated intracellular cAMP slight reductions of cAMP concentrations (Table 2 and Figure 4b), concentrations suggesting that both Gαi-2 (KO vs WT; Po0.02) and Gαi-3 In an attempt to clarify the role of Gα proteins in the dysregulation (Po0.01) were dysfunctional in Aip KO cells already before the of cAMP synthesis induced by Aip deficiency, we examined siRNA treatments. siRNA/cAMP experiments of the Gαi loci were whether siRNA silencing of Gα12/13,Gαq/11, GαS and Gαi/o subfamily performed four times in Aip KO and three times in WT MEFs. α protein genes (Gα12, Gα13; Gα11, Gαq, Gα14,Gα15, GαS, Gαi-1, Gαi-2, To examine the role of G i signaling in AIP-mediated pituitary α Gαi-3) has an effect on cAMP levels in immortalized Aip WT and KO tumorigenesis, G i expression levels were immunohistochemically MEFs. cAMP concentrations were measured after silencing one (IHC) analyzed in AIP mutation positive (AIPmut+) and negative gene at a time. The knockdown efficiencies were between 60 (AIPmut–) mouse and human pituitary tumors. Gαi-1 expression

Table 1. GPCR pathway enriched genes in Aip knockout cell lines

Symbol Entrez gene name Fold change FDR P-value Type

ADRA1D Adrenergic, alpha-1D-, receptor 1.261 1.05E–05 GPCR C3AR1 Complement component 3a receptor 1 –1.613 9.52E–07 GPCR CHRM2 Cholinergic receptor, muscarinic 2 1.293 8.35E–07 GPCR CMKLR1 Chemokine-like receptor 1 –1.279 2.51E–06 GPCR CX3CR1 Chemokine (C-X3-C motif) receptor 1 –1.232 3.50E–03 GPCR CXCR7 Chemokine (C-X-C motif) receptor 7 1.224 2.62E–05 GPCR CYSLTR1 Cysteinyl leukotriene receptor 1 1.325 7.47E–06 GPCR DUSP1 Dual specificity phosphatase 1 1.216 1.10E–10 Phosphatase ELTD1 EGF, latrophilin and seven transmembrane domain containing 1 1.204 6.69E–06 GPCR F2RL1 Coagulation factor II (thrombin) receptor-like 1 –1.304 1.27E–05 GPCR GPR39 G protein-coupled receptor 39 1.319 2.40E–08 GPCR GPR98 G protein-coupled receptor 98 –1.262 4.37E–06 GPCR GPR126 G protein-coupled receptor 126 1.235 1.76E–06 GPCR GPRC5A G protein-coupled receptor, family C, group 5, member A 1.655 5.89E–11 GPCR GRM3 Glutamate receptor, metabotropic 3 –1.258 3.60E–04 GPCR HTR1D 5-Hydroxytryptamine (serotonin) receptor 1D 1.239 1.10E–03 GPCR NPR3 Natriuretic peptide receptor C/guanylate cyclase C 1.262 2.68E–07 GPCR NPY1R Neuropeptide Y receptor Y1 1.265 1.86E–05 GPCR P2RY14 Purinergic receptor P2Y, G protein-coupled, 14 2.036 1.60E–11 GPCR PDE1B Phosphodiesterase 1B, calmodulin-dependent –1.285 1.73E–08 Enzyme PDE3A Phosphodiesterase 3 A, cGMP-inhibited 1.265 1.25E–07 Enzyme PDE4B Phosphodiesterase 4B, cAMP-specific 1.745 5.24E–12 Enzyme PDE4D Phosphodiesterase 4D, cAMP-specific 1.333 2.74E–05 Enzyme PDE7B Phosphodiesterase 7B 1.545 5.68E–11 Enzyme PDE8A Phosphodiesterase 8 A 1.540 2.48E–08 Enzyme PDE8B Phosphodiesterase 8B 1.769 5.24E–12 Enzyme PDE9A Phosphodiesterase 9 A –1.203 2.73E–07 Enzyme PROKR1 Prokineticin receptor 1 –1.268 1.13E–05 GPCR RASGRP1 RAS guanyl releasing protein 1 (calcium and DAG-regulated) –1.268 3.25E–06 Other RGS16 Regulator of G-protein signaling 16 –1.283 1.73E–08 Other S1PR1 Sphingosine-1-phosphate receptor 1 –1.210 4.69E–06 GPCR GNG11 Guanine nucleotide-binding protein (G protein), gamma 11 –1.204 4.40E–03 Enzyme GNG4 Guanine nucleotide-binding protein (G protein), gamma 4 1.206 2.51E–05 Enzyme Abbreviations: AIP, aryl hydrocarbon receptor interacting protein; FDR, false discovery rate; GPCR, G protein-coupled receptor.

was negative in the mouse normal anterior pituitary and either through ERK1/2 activation.29–32 We therefore investigated the negative or weak in Aipmut+ (17 GH and 9 PRL) and Aipmut– (11 phosphorylation status of ERK1/2 (Thr202/Tyr204) in pituitary PRL) tumors (Supplementary Table S4). Consistent with previous tumors and cell lines. Even relative intensities of the two bands (44 reports suggesting that Gαi-2 represents the quantitatively and 42 kDa) were seen in immortalized Aip KO and WT MEFs with 27,28 predominant Gαi isoform, Gαi-2 was prominently expressed immunoblotting (Figure 6a). No significant reduction of p-ERK1/2 in the cytoplasm of mouse normal anterior pituitary (Figure 5a). was detected in Aip siRNA-treated GH3 cells (Figure 6b). Epidermal Prominent cytoplasmic expression of Gαi-2 was also seen in growth factor (EGF) regulates hormone secretion and cell growth Aipmut+ (n = 11) and Aipmut– (n = 13) PRL tumors. On the in pituitary cells through activation of EGF receptor (EGFR). EGF contrary, Aipmut+ GH-secreting tumors (n = 21) showed a clear has shown to enhance ERK/mitogen-activated protein kinase 33 reduction of Gαi-2 with weak-to-moderate expression (tumor vs phosphorylation. To characterize the ability of EGF to stimulate À6 normal, P =3×10 ; Figure 5a). The Gαi-3 isoform was weakly ERK1/2 activation during Aip deficiency, the ERK1/2 levels were expressed in the normal anterior pituitary and similarly studied in MEF and GH3 cell lines after EGF treatment. In MEF cells, overexpressed in mouse Aipmut+ (15 GH and 10 PRL) and 5nM EGF did not enhance p-ERK1/2 levels (data not shown), while Aipmut– (10 PRL) tumors (Figure 5b). 20 nM EGF treatment induced p-ERK1/2 levels within 15 min Consistent with the observations in mouse tumors, AIPmut+ similarly in Aip KO and WT MEFs (Figure 6c). In GH3 cells, 5 nM EGF human somatotropinomas showed a reduction in Gαi-2 with weak- elevated p-ERK1/2 levels similarly in Aip (66–73% knockdown) and to-moderate expression (three GH and one GH/PRL tumors). All control siRNA-treated GH3 cell line. Of note, the discrepancy AIPmut– GH tumors displayed strong Gαi-2 staining (three GH and between basal p-ERK1/2 levels in Figures 6a and b vs Figure 6c one GH/PRL tumors) (Figure 5c; Supplementary Table S5). Again, arise from different exposure times used. Gαi-3 was found to be evenly expressed between AIPmut+ and In mouse, normal anterior pituitary p-ERK1/2 showed strong AIPmut– human somatotropinomas. Taken together, our findings nuclear and weak-to-intermediate cytoplasmic immunostaining suggest that impaired Gαi signaling and reduction of Gαi-2 protein (Figure 7a). Aipmut+ mouse somatotropinomas (n = 14), on the is involved in tumorigenesis of AIP-associated somatotropinomas. contrary, were completely negative for p-ERK1/2 (P =1×10À6), whereas PRL tumors displayed weak-to-moderate p-ERK1/2 staining (8 Aipmut+ and 12 Aipmut–) (Supplementary Table S4). Effect of AIP-mediated activation of cAMP synthesis on p-ERK1/2 IHC staining of human pituitary adenomas confirmed the absence and p-CREB levels of p-ERK1/2 in AIPmut+ somatotropinomas (Figure 7a). In contrast, ERK1/2 is one of the downstream effectors known to be regulated AIP-proficient somatotropinomas displayed moderate-to-strong by the G proteins. Furthermore, it has been suggested that cAMP nuclear and weak cytoplasmic p-ERK1/2 staining (Supplementary can exert its mitogenic effect, selectively on GH-secreting cells, Table S5). To test if the absence of AIP has an effect on activation

© 2015 Macmillan Publishers Limited Oncogene (2015) 1174 – 1184 AIP deficiency leads to elevated cAMP levels I Tuominen et al 1178 of other MAP kinases, the levels of phosphorylated p38 (p-p38) differences in p-p38 and p-JNK1+JNK2 protein levels were and JNK1+JNK2 (p-JNK1+JNK2) were studied in Aip KO and WT observed in Aip KO vs WT MEF cells (Supplementary Figure S2). MEF cells and in mouse and human somatotropinomas. No Mouse Aipmut+ somatotropinomas showed moderate cytoplasmic and moderate-to-strong nuclear staining of p-p38, and weak cytoplasmic and moderate-to-strong nuclear expression of p-JNK1 +JNK2. Human AIPmut+ and AIPmut– somatotropinomas showed moderate-to-strong nuclear and occasionally weak cytoplasmic staining of p-p38 and p-JNK1+JNK2 (Figure 7a). These results hence do not support a general downregulation of MAP kinases in AIP-deficient somatotropinomas. The nuclear response of cAMP is mediated by transcription factors including the CREB. A moderate, although not statistically significant, reduction of p-CREB (Ser133) protein was observed in immortalized Aip KO MEF (Po0.06). A slight reduction of p-CREB was detected in Aip siRNA-treated GH3 cell lines (Figures 6a and 6b). As it has been suggested that elevated p-CREB enhances cell cycle progression and GH secretion in pituitary tumors,11–13,34 we immunostained p-CREB in mouse and human pituitary tumors. Strong nuclear and occasionally weak cytoplasmic staining of p-CREB was observed in normal mouse anterior pituitary, whereas GH-secreting (n = 13) Aipmut+ adenomas showed negative to moderate immunoreactivity (normal vs tumor P =1×10À5) (Figure 7b; Supplementary Table S4). Aipmut+ (n = 9) and Aipmut– (n = 10) PRL tumors also displayed reduced p-CREB

Table 2. Effects of siRNA-mediated Gα-gene silencing on cAMP concentrations in Aip KO- and WT-cell lines

Gene KO WT

Mock Target Mock Target

Gαs*1± 0.1 0.73 ± 0.1 (–27%) 1 ± 0.1 0.46 ± 0(–46%) Gα12 1 ± 0.1 0.70 ± 0(–30%) 1 ± 0.09 0.70 ± 0.04 (–30%) Gα13** 1 ± 0.03 0.85 ± 0.02 (–15%) 1 ± 0.1 0.70 ± 0.04 (–30%) Gαs/Gα13*1± 0.04 0.92 ± 0.03 (–8%) 1 ± 0 0.57 ± 0.15 (–43%) Gαq 1 ± 0.03 1 ± 0.03 (0%) 1 ± 0.1 0.94 ± 0.06 (–6%) Gα11 1 ± 0 0.87 ± 0.02 (–13%) 1 ± 0.05 0.79 ± 0.07 (–21%) Gα14 1 ± 0.03 0.78 ± 0.04 (–22%) 1 ± 0 0.75 ± 0.01 (–25%) Gα15 1 ± 0.04 0.98 ± 0.04 (–2%) 1 ± 0.06 1 ± 0.06 (0%) Gαi-1 1 ± 0.04 0.97 ± 0.03 (–3%) 1 ± 0 0.88 ± 0.1 (–12%) Gαi-2*1± 0.04 0.86 ± 0.03 (–14%) 1 ± 0 1.77 ± 0.01 (+77%) Gαi-3** 1 ± 0.04 0.91 ± 0.09 (–9%) 1 ± 0 2.15 ± 0.09 (+115%) Abbreviations: AIP, aryl hydrocarbon receptor interacting protein; cAMP, cyclic adenosine monophosphate; KO, knockout; siRNA, small interfering RNA; WT, wild type. cAMP concentrations in control siRNA-treated cells were set as 1 and relative cAMP concentrations ± s.d. are shown. Also percentual changes (– or +) in cAMP concentrations after siRNA treatments are shown. Mock = control siRNA pool-treated cells, target = target gene siRNA pool-treated cells. The Student`s t-test was used to compare the difference in cAMP concentration changes between Aip KO and WT cells. *Po0.05, **Po0.01.

Figure 3. Aip deﬁciency increases proliferation and elevates cAMP concentrations. (a) Cell proliferation rates of Aip KO and WT MEF cells at different time points. Error bars represent the mean ± s.d. of four parallel wells. (b) Intracellular cAMP concentrations of primary and immortalized Aip KO and WT MEF cell lines. Data from two WT and two KO MEF cell lines (mean ± s.d. of three independent experiments with duplicates; six samples per cell line). (c) Effect of Aip siRNA treatment in GH3 cell line. Data represent the percent (means ± s.d. of two independent technical replicates) of AIP protein intensity in Aip and mock (ctrl) siRNA-treated and -untreated GH3 cell line. (d) cAMP concentrations measured from GH3 cell line after ctrl and Aip siRNA treatments. Representative assay with 64% Aip silencing efﬁciency and 21% higher cAMP concentration (mean ± s.d. of duplicates).

Figure 4. The absence of AIP results in defective Gαi signaling. (a) Effects of Gαs, Gα12/13 and Gαq/11 subfamily gene siRNA knockdowns (24 h) on cAMP concentrations in immortalized Aip WT and KO cells. (b) cAMP concentrations after siRNA-mediated silencing of Gαi-1, Gαi-2 and Gαi-3 loci in Aip WT and KO cells. cAMP levels in mock siRNA-treated WT and KO cells are set as 1 and relative concentrations are shown. Bars represent the means ± s.d. of at least two independent experiments with duplicates. For additional information related to the Gα loci siRNA knockdown, see Table 2. *Po0.05, **Po0.01.

Figure 5. Reduction of Gαi-2 protein expression in AIP-deﬁcient somatotropinomas. (a)Gαi-2 IHC in mouse Aip mutation positive (Aipmut+) and negative (Aipmut–) pituitary adenomas. (b)Gαi-3 IHC analysis of mouse Aipmut+ and Aipmut– pituitary adenomas. (c)Gαi-2 and (d) Gαi-3 IHC analysis of human AIPmut+ and Aipmut– somatotropinomas. Scale bars = 20 μm; GH, growth hormone-secreting adenoma; PRL, prolactin- secreting adenoma; N, normal pituitary tissue; T, tumor tissue. Mouse tumors are depicted by white circles.

staining. Consistent with these observations, human GH-secreting inhibition of cAMP synthesis, but Gαi-mediated signaling has AIPmut+ adenomas showed reduced nuclear p-CREB been implicated also in the regulation of various pathological staining compared with AIPmut– somatotropinomas (Figure 7b; responses and developmental pathways.36,38,39 It is currently Supplementary Table S5). Overall, our results suggest that the unclear why the absence of AIP results in defective Gαi signaling. downstream events in AIP-mutated somatotropinomas differ from Recently, AIP was shown to be involved in the regulation of cAMP 21 those seen in sporadic GH tumors. synthesis by interacting with Gα13 and Gαq proteins. It is therefore possible that Gαi-2 and Gαi-3 proteins need either a direct protein–protein interaction or interaction via one or more DISCUSSION bridging molecules with AIP for proper biological function. Our results provide novel evidence that AIP has an important role We have previously reported that AIP germline mutations in the regulation of cAMP synthesis. cAMP signaling is known to predispose to pituitary adenomas, mainly somatotropinomas regulate many pivotal cellular and biological functions, including (~80%) with an aggressive disease phenotype.4,6,40 Thus far, the various developmental processes, immune-inflammatory molecular pathogenesis of these tumors has been unclear but the responses, endocrine functions, Ca2+ signaling, as well as cell previously postulated role of AIP in the cAMP signaling has made proliferation and differentiation.12,26,35,36 Notably, these same this pathway a promising candidate in explaining the develop- functions and pathways were highly enriched also in our analyses ment of these tumors.21,41,42 Although we were not able to (Figure 1 and Supplementary Tables S2 and S3). Gα silencing measure cAMP concentrations in AIP-mutated somatotropinomas experiments revealed that although many of the Gαs,Gα12/13 and because of the lack of fresh tumor material, we demonstrated that Gαq/11 proteins appear to have a role in the regulation of cAMP Aip silencing elevates cAMP concentration in a GH/PRL-secreting levels, they do not clearly contribute to the constitutive activation pituitary adenoma cell line GH3. This result is consistent with the of cAMP during Aip deficiency. Instead, our findings suggest that work of Formosa et al.43 showing that silencing of Aip in GH3 the absence of AIP causes elevated cAMP levels through deficient induces elevated cAMP levels.43 In addition, we were able to show α Gαi signaling and the subsequent disruption of many cellular a reduction of the G i-2 protein selectively in AIP-mutated fi processes (summarized in Figure 7c). The Gαi subfamily com- somatotropinomas. This is a very intriguing nding since previous 37 prises three highly related members, Gαi-1,Gαi-2 and Gαi-3. These studies have already shown that cAMP is a mitogenic factor in isoforms are most notably involved in receptor-dependent somatotrophs as well as in adrenal cells, although AIP germline

Figure 6. Effect of Aip deficiency on ERK1/2 and CREB protein levels in MEFs and GH3 cells. (a) Representative immunoblots of phosphorylated- and total-ERK1/2 and -CREB levels in immortalized Aip WT and KO MEF cells (mean ± s.d. of three independent technical replicates with duplicates). (b) Representative western blots of ERK1/2 and CREB protein levels in Aip and control siRNA-treated GH3 cells (mean ± s.d. of two independent siRNA experiments with duplicates). (c) Representative western blots of EGF-mediated ERK1/2 activation in MEF cell lines (20 nM EGF treatment; mean ± s.d. of two independent technical replicates with duplicates) and in Aip and control siRNA-treated GH3 cells, and non-transfected GH3 cells (5 nM EGF treatment; mean ± s.d. of two independent siRNA experiments with duplicates) at different time points. The graphs show the quantification of p-ERK1/2 and p-CREB proteins normalized to the tot-ERK1/2 and tot-CREB, respectively. Immunoblots were quantified with ImageJ software (http://imagej.nih.gov/ij/). mutations do not predispose to adrenal lesion.11,12,16,30,44 reduced in AIP-mutated somatotropinomas, the poor response of Hence, our results provide an explanation why AIP germline AIP mutation carriers to somatostatin analogs may be caused by 4,6,7 defects predispose their carriers mainly to somatotropinomas. aberrant SSTR function arising from impaired Gαi signaling. It is possible that cell-type-specific regulation cAMP signaling by It has been reported that cAMP exerts its mitogenic effect external GPCR ligands explains why Aip-mutated PRL-secreting through ERK1/2 and CREB activation in gsp mutated and many gsp 28,31,32 13,30,34,52 tumors do not show downregulation of Gαi-2 and p-ERK1/2. mutation negative somatotropinomas. It is also sug- Although we cannot exclude the possibility that cAMP accumula- gested that Carney complex-associated mutations in the protein tion is also involved in the pathogenesis of other AIP-associated kinase A type I-a regulatory subunit (PRKAR1A) increase ERK1/2 and pituitary tumors, it is possible that their development requires CREB activation.53 Our finding that accumulation of cAMP does some additional signaling defects.45 not increase p-ERK1/2 and p-CREB levels in AIPmut+ somatotro- A clinically important aspect of our study is that the Gαi proteins pinomas is therefore somewhat unexpected. This study suggests a mediate somatostatin signaling. In the anterior pituitary, soma- role of ERK1/2 also in the development of AIP-related GH-secreting tostatin inhibits GH- and PRL secretion and basal cAMP production tumors, as p-ERK1/2, unlike two other closely related MAP kinases through five different somatostatin receptor subtypes (SSTR1–5) p38 and JNK, was downregulated in AIP-deficient somatotropino- 46,47 coupled to Gαi proteins. Somatostatin analogs have been the mas. We also observed that EGF-induced ERK1/2 activation seems medical therapy of choice for the treatment of GH-secreting to be independent of AIP. It has been proposed that Gαs-mediated pituitary adenomas.48,49 Patients with AIP-associated somatotro- cAMP synthesis and the subsequent activation of the Ras/Raf/ pinomas generally have a poor response to the somatostatin Mitogen-activated protein kinase (RAS-RAF-MEK) pathway is a analog therapy.6,7 A recent study by Chahal et al.50 reported that primary reason for activation of ERK1/2 in AIP mutation negative SSTR protein expression levels are not reduced in AIPmut+ somatotropinomas.30 This study, however, suggests that defective somatotropinomas. Nevertheless, the stable expression of SSTRs in Gαi signaling underlies the activation of cAMP in AIP-associated AIP-deficient somatotropinomas does not exclude the possibility somatotropinomas. Hence, it is possible that signaling events of defective receptor function. Interestingly, it has been reported occurring downstream of cAMP are differentially regulated in that depletion of Gαi-2 blocks the inhibition of basal cAMP levels AIPmut+ somatotropinomas resulting in downregulation of ERK1/2. 51 by somatostatin receptors. As Gαi-2 protein levels were markedly Similarly, cAMP-CREB cross-talk during AIP deficiency may also be

© 2015 Macmillan Publishers Limited Oncogene (2015) 1174 – 1184 AIP deﬁciency leads to elevated cAMP levels I Tuominen et al 1182 silencing of Aip in this GH/PRL-secreting cell line does not necessarily lead to the same downstream events seen in AIPmut+ somatotropinomas.43 In addition, despite reduction of p-CREB in Aip KO MEFs, overexpression of CREB-target genes was observed; for example, Nr4a2, Id1 and Dusp. As our p-CREB analyses were made utilizing an antibody that recognizes the phosphorylated Ser133, we cannot exclude the possibility that in Aip KO MEFs phosphorylation of p-CREB at residues other than Ser133 may have occurred.61 Furthermore, it is possible that other transcription factors are also involved in CREB-targeted gene expression. In summary, this study provides evidence that defective Gαi signaling resulting in constitutive activation of cAMP synthesis seems to be a major contributor to the development of AIP-associated somatotropinomas. Although further studies are necessary to clarify the exact molecular basis of the impaired Gαi pathway, the new insights into the signaling cascades altered by AIP deﬁciency are important—especially considering the severe clinical phenotype of pituitary adenoma predisposition patients.

MATERIALS AND METHODS Gene expression profiling and pathway analyses Nine WT and seven Aip KO MEF cell lines were established from 10.5-day-postcoitum embryos and cultured in 95% air, 5% CO2 at 37 °C in high glucose Dulbecco’s modified Eagle’s medium (4.5 g/l glucose) supplemented with glutamine, 10% fetal bovine serum and antibiotics. Cells were collected at second passage (p2). RNA was extracted with RNeasy Mini Kit (Qiagen, Hilden, Germany). Embryos were genotyped as described.24 Gene expression levels were studied utilizing GeneChip Mouse Exon 1.0 ST Arrays (Affymetrix, Santa Clara, CA, USA) expression arrays. Comple- mentary DNA synthesis, labeling and hybridization was performed according to the manufacturer’s instructions. Quality control, normal- ization and analysis of data were carried out using Partek Genomic Suite v. 6.5 (Partek Incorporated, St Louis, MO, USA). All the arrays passed the quality control metrics cutoffs recommended by Affymetrix. Samples were quantile normalized by the RMA method (Robust Multichip Average) using re-mapped gene annotations from Brainarray Custom CDF files (MoEx10stv1, version 14.1.0, http://brainarray.mbni.med.umich.edu/ Brainarray/default.asp). To examine gene expression, the CEL files were using Affymetrix core metaprobesets and Affymetrix annotation (release 32). A two-way analysis of variance was constructed to identify genes that Figure 7. p-ERK1/2, p-p38, p-JNK1+JNK2 and p-CREB immuno- differentially expressed between KO and WT samples. False discovery rate reaction in mouse and human somatotropinomas. (a) p-ERK1/2, control with Benjamini and Hochberg method was used to correct for p-p38 and p-JNK1+JNK2 IHCs in mouse Aipmut+ and human multiple testing.62 Genes with an false discovery rate o5% and fold AIPmut+ and AIPmut– GH-secreting adenomas. (b) p-CREB change À1.2 ⩾ x ⩾ 1.2 were considered significant. immunostaining in mouse Aipmut+ and human AIPmut+ and The pathway data were generated with IPA (www.ingen.com) software. AIPmut– GH-secreting adenomas. Scale bars = 10 μm; N, normal A data set derived from expression arrays were uploaded into IPA along pituitary tissue; T, tumor tissue. Mouse tumor normal borderlines are with the corresponding fold change and P-values. The expression data depicted by white lines. (c) Schematic summary of how the lack of were mapped into relevant pathways based on their functional annotation the AIP causes elevated intracellular cAMP concentrations through and known molecular interactions in Ingenuity’s Knowledge Base. The –log deficient Gαi signaling. Red color indicates defected molecules and of P-value were calculated by Fisher's exact test. The IPA functional analysis biological functions associated with AIP deficiency. identified the biological functions that were most significant to the data set (right-tailed Fisher’s exact test).

regulated in Gα -dependent manner. All in all, it seems that AIP- i Immortalization of MEF cell lines mutated adenomas, although similarly driven by elevated cAMP levels as the sporadic GH adenomas, differ in terms of the E12.5 MEF cells (p2) were immortalized by transduction of a p53 c-terminal domain (amino acids 302–390) construct with puromycin resistance downstream mediators. However, more research is still needed to (pBABE-puro). Transduction was performed using ecotropic retrovirus evaluate the cAMP-ERK1/2 and -CREB cross-talks in AIP-mediated (Biomedicum Virus Core, Helsinki, Finland), facilitated with polybrene (8 μg/ tumorigenesis, including studies with more extensive collection of ml). MEFs were plated on six-well plates 24 h before transduction, at a human somatotropinomas. density of 400 000 cells per well, to obtain about 60% confluence. At 8 h No clear reduction of p-ERK1/2 was observed in Aip KO MEFs. after the transduction, the media were replaced and 24 h afterward with Neither did we find evidence for downregulation of p-ERK1/2 nor selection media containing 2.5 μg/ml of puromycin. p-CREB in Aip siRNA-treated GH3 cells. This discrepancy between AIP-deficient somatotropinomas vs cell lines might be explained siRNA gene silencing by cell-type-specific regulation of mitogen-activated protein 54–60 Immortalized MEF cells were plated on six-well plates at a density of 100 kinase and CREB pathways. Furthermore, the pituitary 000–200 000 cells per well 24 h prior the siRNA transfections. Transfection adenoma from which the GH3 cell line originates has developed with 20 nM duplex siRNA strands was performed to 80% confluent cells through its own unique tumor evolution, and hence partial using 250 nM Dharma FECT 1 reagent (Dharmacon, Thermo Fisher

Oncogene (2015) 1174 – 1184 © 2015 Macmillan Publishers Limited AIP deficiency leads to elevated cAMP levels I Tuominen et al 1183 Scientific, Waltham, MA, USA). GH3 siRNA transfections were performed as ACKNOWLEDGEMENTS 25 described. Cells were harvested at 24 h time points after the transfection. We thank Inga-Lill Svedberg, Mairi Kuris, Alison Ollikainen and Iina Vuoristo for See Supplementary Information for details of the used siRNA pools. technical assistance. The Biomedicum Imaging Unit is acknowledged for the microscopy service and the Biomedicum Functional Genomics Unit (FuGU) for Proliferation assay and EGF treatments expression array and virus core services. This study was supported by the Academy of Finland (250345), the Cancer Society of Finland (4700325), the Novo Nordisk Aip WT and KO MEFs were plated in four parallel wells (40 000 cells Foundation (A14582) and the Association for International Cancer Research per well) to a six-well plate. The proliferation rates were determined by the (13–1075). This work was supported in part by the Novo Nordisk Foundation (AK). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium) (MTS) assay (G3580; Promega, Madison, WI, USA). EGF treatments were carried out in media containing 5 nM (GH3 and MEF cell lines) or 20 nM (MEFs) of EGF (Sigma, St Louis, MO, USA) for 0, 15 and 60 min at 37 °C.33 In siRNA-treated GH3 cells, EGF treatment was performed REFERENCES 24 h after the transfection. 1 Meyer BK, Perdew GH. Characterization of the AhR-hsp90-XAP2 core complex and the role of the immunophil-related protein XAP2 in AhR stabilization. Biochemistry cAMP immunoassay 1999; 38: 8907–8917. Primary and immortalized KO and WT MEF cells were plated on six-well 2 Kazlauskas A, Poellinger L, Pongratz I. 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Supplementary Information accompanies this paper on the Oncogene website (http://www.nature.com/onc)

AIP Inactivation Leads to Pituitary Tumorigenesis Through Defective G&Alpha;I-Camp Signaling

AIP Inactivation Leads to Pituitary Tumorigenesis Through Defective GΑI-Camp Signaling