The Activating Mutation R201C in GNAS Promotes Intestinal Tumourigenesis in Apcmin/ Þ Mice Through Activation of Wnt and ERK1/2 MAPK Pathways

Oncogene (2010) 29, 4567–4575 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE The activating mutation R201C in GNAS promotes intestinal tumourigenesis in ApcMin/ þ mice through activation of Wnt and ERK1/2 MAPK pathways

CH Wilson1,3, RE McIntyre1,3, MJ Arends2,4 and DJ Adams1,4

1Experimental Cancer Genetics, Wellcome Trust, Sanger Institute, Hinxton, UK and 2Department of Pathology, Addenbrooke’s Hospital, University of Cambridge, Cambridge, UK

Somatically acquired, activating mutations of GNAS, the Introduction gene encoding the stimulatory G-protein Gsa subunit, have been identified in kidney, thyroid, pituitary, leydig Genome-wide exon re-sequencing studies of human cell, adrenocortical and, more recently, in colorectal cancers have identified a number of frequently mutated tumours, suggesting that mutations such as R201C may genes that are associated with cancer formation and be oncogenic in these tissues. To study the role of GNAS progression; however, their functional significance is in intestinal tumourigenesis, we placed GNAS R201C unknown (Sjoblom et al., 2006; Wood et al., 2007). One under the control of the A33-antigen promoter (Gpa33), potentially interesting candidate is GNAS, the gene which is almost exclusively expressed in the intestines. The encoding the stimulatory G-protein alpha subunit GNAS R201C mutation has been shown to result in the (Gsa), which is situated on human chromosome constitutive activation of Gsa and adenylate cyclase and 20q13.3. Heterotrimeric G-proteins that are composed to lead to the autonomous synthesis of cyclic adenosine of a-, b- and g-subunits mediate signal transduction monophosphate (cAMP). Gpa33tm1(GnasR201C)Wtsi/ þ mice from a large number of hormone and growth factor- showed significantly elevated cAMP levels and a compen- activated seven-transmembrane receptors to diverse satory upregulation of cAMP-specific phosphodiesterases intracellular signalling pathways (Weinstein et al., in the intestinal epithelium. GNAS R201C alone was not 2004). Ligand-bound G-protein-coupled receptors acti- sufficient to induce tumourigenesis by 12 months, but vate the Gs-protein by promoting the exchange of there was a significant increase in adenoma formation guanosine diphosphate for guanosine triphosphate on when Gpa33tm1(GnasR201C)Wtsi/ þ mice were bred onto an Gsa, which results in dissociation from the receptor and ApcMin/ þ background. GNAS R201C expression was the bg-complex. The free Gsa subunit interacts with associated with elevated expression of Wnt and extra- adenylate cyclase to stimulate the synthesis of cyclic cellular signal-regulated kinase 1/2 mitogen-activated adenosine monophosphate (cAMP) until hydrolysis of protein kinase (ERK1/2 MAPK) pathway target genes, guanosine triphosphate returns it to the inactive increased phosphorylation of ERK1/2 MAPK and in- guanosine diphosphate-bound form, which reassociates creased immunostaining for the proliferation marker with the bg-complex to enter a new cycle (Weinstein Ki67. Furthermore, the effects of GNAS R201C on the et al., 2004). Wnt pathway were additive to the inactivation of Apc. Somatically acquired, activating mutations of GNAS Our data strongly suggest that activating mutations of have been identified in adrenal hyperplasia, ovarian GNAS cooperate with inactivation of APC and are likely cysts, thyroid carcinomas (5%), adrenocortical, pitui- to contribute to colorectal tumourigenesis. tary (22–40%), kidney (17%) and leydig cell tumours Oncogene (2010) 29, 4567–4575; doi:10.1038/onc.2010.202; (67%) (Landis et al., 1989; Fragoso et al., 1998; published online 7 June 2010 Hayward et al., 2001; Kalfa et al., 2006; Palos-Paz et al., 2008; Taboada et al., 2009). Furthermore, several Keywords: cAMP; colorectal cancer; GNAS reports have documented the presence of thyroid, pituitary and adrenocortical tumours in patients with McCune–Albright syndrome, a mosaic disease caused by sporadic, post-zygotic, activating mutations of GNAS (Happle, 1986; Kirk et al., 1999; Yang et al., 1999; Collins et al., 2003; Fragoso et al., 2003; Chen Correspondence: Dr DJ Adams, Experimental Cancer Genetics, et al., 2004; Weinstein et al., 2004). The common Wellcome Trust, Sanger Institute, Wellcome Trust Genome Campus, mutations of GNAS that have been identified in Hinxton CB10 1SA, UK. tumours, including R201C, R201H and Q227R, are E-mail: [email protected] thought to inhibit guanosine triphosphate hydrolysis 3These authors contributed equally to this work. 4These authors are co-senior authors. and result in the constitutive activation of Gsa and its Received 11 December 2009; revised 17 March 2010; accepted 25 April effector adenylate cyclase, leading to autonomous 2010; published online 7 June 2010 synthesis of cAMP (Landis et al., 1989). Collectively, Activating mutations of GNAS cooperate with ApcMin CH Wilson et al 4568 these data suggest that activating mutations of GNAS proliferation, neoplastic transformation, differentiation can modify cell growth and may be oncogenic; however, and survival of many cell types (Barbacid, 1987). how GNAS functions as an oncogene remains unclear. cAMP has been shown to have opposing effects on cell Interestingly, a number of studies have reported an growth: cAMP either inhibits or stimulates ERK1/2 association between McCune–Albright syndrome and MAPK-mediated cell proliferation and/or differentiation multiple gastrointestinal polyps (MacMahon, 1971; in a cell-type-specific manner (reviewed in Stork and Weinstein et al., 1991). More recently GNAS R201C Schmitt, 2002). Whether elevated levels of cAMP result in mutations were identified in 9% of colorectal tumours the activation of the ERK1/2 MAPK pathway and (3/35; Sjoblom et al., 2006; Wood et al., 2007). The most growth of intestinal cells is not known. frequent early event in 480% of sporadic colorectal Here, we have generated mice that specifically express carcinomas is loss-of-function mutations of the adeno- GNAS R201C in the intestinal stem cells and all matous polyposis coli (APC) gene, which is also epithelial cell lineages from E14.5 into adulthood (Abud mutated in the germline of patients with familial et al., 2000; Johnstone et al., 2000) to assess whether this adenomatous polyposis (Nishisho et al., 1991). APC mutation participates in the formation and/or progres- forms a complex with axin and glycogen synthase kinase sion of colorectal cancer in vivo. 3b, which results in the phosphorylation of b-catenin and consequently its degradation through the ubiquitin– proteasome system (Castellone et al., 2005). ApcMin/ þ mice represent a valuable model of intestinal tumouri- Results genesis, as sporadic loss of heterozygosity of the Generation of an intestine-specific conditional wild-type allele of Apc recapitulates the initiation of tm1(GnasR201C)Wtsi allele adenomagenesis observed in humans (Levy et al., Gpa33 Full-length human GNAS complementary DNA was 1994). A number of groups have shown that loss of cyclooxygenase-1 or -2 (COX) dramatically reduces synthesized to introduce the putative oncogenic mutation R201C. The mutation R201C was chosen over tumour formation in ApcMin/ þ mice (reviewed in Taketo, R201H and Q227R because it is found more frequently 2006). Accordingly, the expression of PTGS1 and 2 (COX-1 and 2, respectively) has been shown to be in cancers; however, both mutations have been shown to have a similar affect on adenylyl cyclase stimulation upregulated in approximately 80% of colorectal adeno- (Landis et al., 1989). To express the mutant GNAS mas and carcinomas and COX inhibition represents a R201C cDNA specifically in the intestinal epithelium, valuable therapeutic target (Eberhart et al., 1994). COX- we obtained and modified a Lox-Stop-Lox targeting 1 and -2 synthesize the pro-inflammatory metabolite vector to place it under the control of the endogenous prostaglandin E2, which activates prostaglandin recep- Gpa33-antigen gene promoter (Figure 1a; Orner et al., tors 2 and 4 (EP2 and EP4), resulting in the activation of Gsa, adenylate cyclase and cAMP synthesis. The effects 2002). This approach was used to avoid complications that may arise from imprinting at the endogenous Gnas of COX-2 on intestinal tumour formation have been locus and extra-intestinal phenotypes such as those shown to be mediated by EP2 receptor activation (Castellone et al., 2005), suggesting that both the spatial observed in patients with McCune–Albright syndrome (Plagge et al., 2008). Upon Cre-mediated recombination and temporal production of cAMP is important in of the loxP sites, GNAS R201C cDNA is predicted to be intestinal tumourigenesis. The promotion of intestinal expressed bicistronically from the Gpa33 gene locus tumourigenesis by COX-2 is thought to be due to the (Figure 1a). In adult mice Gpa33 is exclusively expressed direct binding of activated Gsa to the regulator of throughout the epithelium of the intestinal tract, thus G-protein signalling domain of axin, which promotes directing mutant GNAS R201C expression exclusively to the release of glycogen synthase kinase 3b from proliferating and differentiating intestinal epithelial cells the complex and its inactivation. Furthermore, on the and crypt stem cells (Abud et al., 2000). The linearized loss of APC, the threshold of Wnt pathway activation conditional Gpa33tm1(GnasR201C)Wtsi vector was electropo- by constitutively active Gsa is thought to be lowered (Castellone et al., 2005). Therefore, loss of APC or rated into Bruce4 C57BL/6J mouse embryonic stem cells and correctly targeted clones were identified by South- activation of Gsa is predicted to lead to reduced ern blot analysis (Figure 1b) and polymerase chain degradation and increased nuclear translocation of Gpa33tm1(GnasR201C)Wtsi/ þ b-catenin. Constitutively active b-catenin/T-cell factor/ reaction. The F1 conditional males generated were crossed with CMV-CreÀ/À females. lymphocyte enhancer factor (TCF-LEF)-mediated tran- A total of 69 progeny were obtained from 10 litters: 34 scription leads to the expression of growth-promoting were Gpa33tm1(GnasR201C)Wtsi/ þ , 35 were Gpa33 þ / þ , and all genes and the transformation of normal crypts into the were heterozygous for CMV-Cre. This is the expected earliest colorectal cancer precursor lesions, called dys- 1:1 Mendelian ratio; therefore, expression of mutant plastic aberrant crypt foci or monocryptal/oligocryptal GNAS R201C from the Gpa33 locus does not cause adenomas (reviewed in Radtke and Clevers, 2005). embryonic lethality. Activating mutations of either KRAS or BRAF are found in 40–50% of colorectal cancers (Bos, 1989; Brink et al., 2003) and lead to activation of the extracellular Expression of GNAS R201C from the Gpa33 locus signal-regulated kinase 1/2 mitogen-activated protein RT–PCR expression analysis was performed on RNA, kinase (ERK1/2 MAPK) pathway, which enhances extracted from a range of tissues from Gpa33tm1(GnasR201C)Wtsi/ þ

Oncogene Activating mutations of GNAS cooperate with ApcMin CH Wilson et al 4569

Figure 1 Generation and validation of the intestine- speciﬁc GNAS R201C allele. (a) Schematic illustration of the Gpa33 wild-type locus (Gpa33, upper panel), Gpa33tm1(GnasR201C)Wtsi conditional targeted locus (c, middle panel) and the Gpa33tm1(GnasR201C)Wtsi knock-in locus (KI, lower panel). Exons (numbered black rectangles), the positions of relevant PstI sites and the predicted sizes of fragments (thin lines with arrows) and probes (small black rectangle) are shown. (b) Southern blot analysis of PstI digested DNA from targeted Gpa33tm1(GnasR201C)Wtsi/ þ (R201C) and control (WT) embryonic cells. (c) RT–PCR expression analysis of GNAS R201C transcripts (Gpa33-GNAS) and b-Actin (Actb) expression in tissues taken from Gpa33tm1(GnasR201C)Wtsi/ þ (R201C) mice and wild-type littermate controls (WT). I—small intestine, B—brain, H—heart, Lu—lung, Li—liver, K—kidney and N—blank water control. (d) Flow cytometric analysis of cAMP levels in intestinal epithelial cells. Gpa33tm1(GnasR201C)Wtsi/ þ (R201C) and wild-type (WT) tissues were analysed. (e) Quantitative RT–PCR analysis of cAMP-speciﬁc phosphodiesterases 4a, 4b, 8a and 8b in the intestine of Gpa33tm1(GnasR201C)Wtsi/ þ (R201C) and wild-type littermate control mice (WT). *Po0.05.

and wild-type (Gpa33 þ / þ ) littermates using primers specific the expression of the cAMP-specific phosphodiesterases for the Gpa33 locus (exon 5) and the mutant GNAS 4a, 4b, 7a, 8a and 8b by real-time reverse trans- cDNA. As expected, a 1.1 kb product was only generated criptase–PCR. Expression of Pde4a and Pde8b was from the intestinal tissue of Gpa33tm1(GnasR201C)Wtsi/ þ mice upregulated by 2.5-fold in the intestinal epithelium of (Figure 1c). The transcript was not present in other non- Gpa33tm1(GnasR201C)Wtsi/ þ mice relative to control mice intestinal tissues of Gpa33tm1(GnasR201C)Wtsi/ þ mice or in the (P ¼ 0.05), whereas expression levels of Pde4b and intestinal tissue of wild-type littermate controls. Cloning Pde8a were unchanged (Figure 1e). Expression of Pde7a and sequencing of the product generated from the was negligible in this tissue. intestines of Gpa33tm1(GnasR201C)Wtsi/ þ mice revealed that the expected spliced transcript contained Gpa33 exons 5, Gpa33tm1(GnasR201C)Wtsi/ þ mice do not develop intestinal Lox - -GNAS 6 and 7 followed by P IRES R201C cDNA polyps by 12 months of age sequences. To establish the phenotypic effect of expression of mutant GNAS R201C in the intestine, we aged a cohort Gpa33tm1(GnasR201C)Wtsi/ þ Enhanced cAMP levels and upregulation of of 21 and 20 wild-type littermate cAMP-specific phosphodiesterases in the intestinal control mice to 12 months. Macroscopic and histological analysis did not reveal any polyp formation in the epithelium of Gpa33tm1(GnasR201C)Wtsi/ þ mice intestines, suggesting that by itself the activating GNAS The GNAS R201C mutation is thought to constitutively R201C mutation is not sufficient to generate intestinal activate Gsa and adenylate cyclase and to lead to neoplasms (Figure 2a). autonomous production of cAMP (Landis et al., 1989). We used immunofluorescence and flow cytometry to show that expression of GNAS R201C leads to a 1.5- The GNAS R201C mutation promotes intestinal adenoma fold increase in cAMP levels in the intestinal epithelium formation in ApcMin/ þ mice of Gpa33tm1(GnasR201C)Wtsi/ þ mice relative to wild-type To further explore the role of the GNAS R201C littermate controls (Po0.05; Figure 1d). In the long mutation in intestinal adenoma formation, we crossed term, high levels of cAMP are subjected to negative Gpa33tm1(GnasR201C)Wtsi/ þ mice with ApcMin/ þ mice. Mice feedback control by upregulation of phosphodiesterases were killed at 16 weeks to determine the number, size (Conti and Beavo, 2007). We therefore determined and location of intestinal adenomas in the small and

Oncogene Activating mutations of GNAS cooperate with ApcMin CH Wilson et al 4570

Figure 2 Gpa33tm1(GnasR201C)Wtsi (R201C) promotes intestinal adenoma formation in ApcMin/ þ mice when compared with ApcMin/ þ controls but alone does not promote tumour formation. (a) Macroscopic counts of intestinal tumours (X1.5 mm in size) at 16 weeks in Gpa33tm1(GnasR201C)Wtsi/ þ ApcMin/ þ mice and ApcMin/ þ littermates. No tumours were found in Gpa33tm1(GnasR201C)Wtsi/ þ or wild-type littermate controls (WT) aged to 12 months. (b) Representative photograph of small intestines from Gpa33tm1(GnasR201C)Wtsi/ þ ApcMin/ þ and ApcMin/ þ littermate (arrows indicate adenomas). (c) Representative haematoxylin and eosin-stained section of a Swiss roll from an Gpa33tm1(GnasR201C)Wtsi/ þ ApcMin and ApcMin/ þ littermate (arrows indicate adenomas, boxes enlarged to the right). Histological analysis revealed no difference in the dysplastic grade of adenoma between Gpa33tm1(GnasR201C)Wtsi/ þ ApcMin and ApcMin/ þ littermates.

large intestines. The mean number of adenomas in Activation of Wnt and ERK1/2 MAPK pathways ApcMin/ þ mice (C57BL6) was 30±2.9 (mean±s.e., and enhanced proliferation in the intestines n ¼ 12), which is comparable to data obtained in other of Gpa33tm1(GnasR201C)Wtsi/ þ mice studies (Figure 2a; Su et al., 1992). The activating To identify a mechanism for the enhanced formation of R201C mutation of GNAS resulted in a mean adenoma adenomas in ApcMin/ þ mice carrying the mutant GNAS number of 62±5.2 (n ¼ 11), a twofold increase R201C allele, we examined two of the most important (P ¼ 0.0001; Figures 2a and b). The majority of signalling pathways in intestinal tumourigenesis, the intestinal adenomas were in the small intestine and ERK1/2 MAPK and Wnt pathways. cAMP signalling showed a distribution very similar to that of ApcMin mice has been shown to activate ERK1/2 MAPKs in a (Supplementary Figure 1). There was no difference in cell-type-speciﬁc manner (Stork and Schmitt, 2002; the dysplastic grade of adenoma between the two McCubrey et al., 2007). Immunohistochemical analysis cohorts of mice—all were low-grade, non-invasive, of the small intestines revealed a signiﬁcant increase in tubular or tubulovillous adenomas typical of ApcMin the number of nuclei that stained positively for mice (Figures 2b and c). Collectively these data show phosphorylated-ERK1/2 MAPK within the crypt region that activation of GNAS cooperates with inactivation of (P ¼ 0.0453; Figure 3a) of Gpa33tm1(GnasR201C)Wtsi/ þ relative Apc in the formation of intestinal adenomas. to wild-type littermate control mice. In agreement with

Oncogene Activating mutations of GNAS cooperate with ApcMin CH Wilson et al 4571

Figure 3 Activation of Wnt and ERK1/2 MAPK pathways in the intestines of Gpa33tm1(GnasR201C)Wtsi/ þ mice. (a) Immunohistochemical analysis of Ki67 and phosphorylated ERK1/2 MAPK showed an increase in the number of nuclei that stained positively within the crypt region of Gpa33tm1(GnasR201C)Wtsi/ þ mice (R201C) when compared with control mice littermate controls (WT). (b) Western blot analysis of intestinal tissue from Gpa33tm1(GnasR201C)Wtsi/ þ (R201C) and littermate controls mice (WT) showed an increase in phosphorylated ERK1/2 MAPK. (c) Quantitative RT–PCR analysis of Myc, Birc5, Fos and Pgts2 in the intestine of Gpa33tm1(GnasR201C)Wtsi/ þ (R201C) and littermate control mice (WT). this, western blot analysis showed a twofold increase in Gpa33tm1(GnasR201C)Wtsi/ þ intestines (Figure 3c), indicating the amount of phosphorylated-ERK1/2 MAPK relative that the effects of GNAS R201C occur independently to total ERK1/2 MAPK (P ¼ 0.05; Figure 3b). Phos- and downstream of COX-2. phorylated-ERK1/2 MAPK activates transcription fac- c-Myc and c-Fos transcription factors coordinate tors such as c-Myc and consequently leads to enhanced the expression of genes that drive cellular proliferation expression of the transcription factor c-Fos (McCubrey and/or differentiation. There was a significant increase et al., 2007). Expression of Fos was increased twofold in in the number of nuclei that stained positively for Ki67 Gpa33tm1(GnasR201C)Wtsi/ þ mice relative to wild-type litter- within the crypt region of Gpa33tm1(GnasR201C)Wtsi/ þ mice mate control mice (P ¼ 0.02; Figure 3c). relative to Gpa33 þ / þ (P ¼ 0.005; Figure 3a), suggesting Gsa has been implicated in the aberrant activation that activation of GNAS R201C within the intestine of Wnt signalling by COX2 and prostaglandin E2 augments proliferation. Immunohistochemical and his- (Castellone et al., 2005); therefore, we examined the tological analysis of the differentiated cells of the expression of two Wnt target genes, Myc and Birc5 intestinal tissue revealed no difference in the number (survivin), by real-time reverse transcriptase–PCR (Giles of enteroendocrine (P ¼ 0.1282), Paneth (P ¼ 0.8102), et al., 2003). Both Myc and Birc5 were upregulated goblet (P ¼ 0.1735), mitotic cells (P ¼ 0.1735) and twofold, suggesting significant activation of the Wnt apoptotic fragments (P ¼ 1.0; Supplementary Figure 3), pathway in Gpa33tm1(GnasR201C)Wtsi/ þ intestines when suggesting that the activating R201C mutation of GNAS compared with control mice (P ¼ 0.05; Figure 3c). To has no effect on the differentiation or apoptosis of test whether the effects of loss of Apc and activation of intestinal cells. Gnas on the Wnt pathway were additive, we determined the expression of Myc and Birc5 in intestinal polyps. Myc was upregulated 1.5-fold and Birc5 was upregulated 2-fold in intestinal polyps from ApcMin/ þ Discussion Gpa33tm1(GnasR201C)Wtsi/ þ mice compared with polyps from ApcMin/ þ mice (Po0.05 for both; Supplementary Somatically acquired, activating mutations of GNAS Figure 2). Pgts2 (COX-2) expression has been reported have been identified in kidney, thyroid, leydig cell, to be upregulated by both the Wnt and ERK1/2 MAPK pituitary and adrenocortical tumours (Landis et al., pathways (Araki et al., 2003); however, we found no 1989; Fragoso et al., 1998; Hayward et al., 2001; Kalfa evidence to suggest that Pgts2 is upregulated in et al., 2006; Palos-Paz et al., 2008; Taboada et al., 2009).

Oncogene Activating mutations of GNAS cooperate with ApcMin CH Wilson et al 4572 However, a recent mutational analysis of 35 colorectal ERKs. For Rap-1 to activate ERKs, the Raf isoform cancers by Sjoblom et al. (Sjoblom et al., 2006; Wood B-Raf must be expressed, as is the case in intestinal cells. et al., 2007) was the first study to suggest that the R201 cAMP has also been shown to activate ERKs indepen- mutations of GNAS occur frequently (9%) in colorectal dently of PKA, for example in the crypt-like intestinal cancers. Furthermore, a number of studies have cell-line T84 (Nishihara et al., 2004; Rudolph et al., reported an association between McCune–Albright 2004), and this is thought to occur through activation of syndrome, a mosaic disease caused by sporadic, post- cAMP–GTPase exchange factors, which then directly zygotic, activating mutations of GNAS, and multiple activate the Rap-1/B-raf/MEK cascade. Furthermore, gastrointestinal polyps (MacMahon, 1971; Weinstein cAMP-mediated activation of ERK1/2 MAPK can et al., 1991). However, how GNAS functions in this stimulate proliferation and/or differentiation depending context remains unclear. on the stimulus and cell type. In Gpa33tm1(GnasR201C)Wtsi/ þ To examine the role of GNAS R201C in intestinal mice we saw both enhanced levels of activated phospho- tumourigenesis, we generated mice expressing GNAS ERK1/2 MAPK and increased levels of proliferation, as R201C under the control of the Gpa33-antigen shown by an increased number of nuclei that stained promoter, which is almost exclusively expressed by positively for activated phospho-ERK1/2 MAPK and intestinal stem cells and all epithelial cell lineages from the proliferative marker Ki67 in the crypt region of the E14.5 into adulthood (Abud et al., 2000; Johnstone intestines. We found no changes in the markers for et al., 2000). Gpa33tm1(GnasR201C)Wtsi/ þ mice were shown to enteroendocrine, Paneth and goblet cells or apoptotic express mutant GNAS R201C in the intestine, which bodies, suggesting that cAMP may stimulate the led to a significant increase in cAMP levels in the proliferation of intestinal cell types in an ERK1/2 intestinal mucosa, in agreement with an earlier report MAPK-dependent manner, but that it does not affect indicating that the R201C mutation leads to constitutive the differentiation or apoptosis of these cells. Although activation of Gsa protein (Landis et al., 1989). We we have not established the exact mechanism of cAMP- show that GNAS R201C alone was not sufficient to mediated activation of ERK1/2 MAPK following induce tumourigenesis by 12 months, but there was a constitutive activation of Gsa in intestinal cells, activa- two-fold increase in adenoma formation when tion of ERK1/2 MAPK is a significant contributory Gpa33tm1(GnasR201C)Wtsi/ þ mice were crossed with ApcMin/ þ factor in the genesis of colorectal cancer. ERK1/2 mice. Furthermore, we have shown that constitutive MAPK activation is the result of activating mutations in activation of Gsa in intestinal cells results in increased either KRAS or BRAF in 40–50% of colorectal cancers activation of two of the most important signalling (Bos, 1989; Brink et al., 2003). pathways in the development of colorectal cancers: the Expression of activated K-Ras from its endogenous Wnt and ERK1/2 MAPK pathways. locus or a transgene, and therefore activation of ERK1/2, Several reports have suggested that both the spatial by itself has been shown to be insufficient to induce and temporal production of cAMP are important in the neoplasia within the murine intestine (Sansom et al., 2006; promotion of intestinal tumourigenesis by COX-2 Haigis et al., 2008; Luo et al., 2007, 2009). However, in (reviewed by Taketo, 2006). cAMP differentially reg- combination with inactivation of Apc, tumourigenesis is ulates cell growth through the compartmental organiza- promoted (Sansom et al., 2006; Haigis et al., 2008; Luo tion of cAMP signalling, which is predominantly the et al., 2007, 2009). In support, our data suggest that the result of cell-type-specific isoform expression of phos- intestinal expression of mutationally activated GNAS phodiesterases, adenylate cyclases, protein kinase A and the subsequent increase in activated ERK1/2 within (PKA) and cAMP effectors such as Rap-1 and B-raf the intestine were insufficient (by 12 months) to induce (Houslay and Milligan, 1997; Stork and Schmitt, 2002; tumourigenesis alone, but that in combination with Conti and Beavo, 2007). We found that the 1.5-fold inactivation of Apc, the tumour number doubled. increase in cAMP was accompanied by a 2.5-fold Aberrant activation of the Wnt/b-catenin pathway is upregulation of the cAMP-specific phosphodiesterases an initiating event in the vast majority of colorectal Pde4a and Pde8b, but there was no change in the adenomas and cancers. The upregulation of c-Myc expression of Pde4b and Pde8a. As high levels of cAMP following activation of b-catenin is thought to be a are subjected to negative feedback control by upregula- critical mediator of the phenotypes of APC inactivation tion of phosphodiesterases (Conti and Beavo, 2007), our in the intestine (reviewed by Wilkins and Sansom, 2008), data suggest that Gsa R201C is localized to the same which include cytoskeleton organization, apoptosis, cell cellular compartment(s) as Pde4a and Pde8b. In many cycle control and cell adhesion (Sieber et al., 2000). cells cAMP serves to inhibit cell growth through Other models predict increased stem cell survival after inhibition of ERK1/2 MAPK; however, activation of loss of function of APC, which can be explained by ERK1/2 MAPK by cAMP has been shown to occur in a enhanced expression of the Wnt target genes (Zhang cell-type-specific manner through a number of mechan- et al., 2001). Upregulation of the Wnt target genes Myc isms (Stork and Schmitt, 2002). Briefly, cAMP has been and Birc5 (survivin) in Gpa33tm1(GnasR201C)Wtsi/ þ mice is shown to activate ERKs in a PKA-dependent manner: likely to be because of the binding of constitutively by stimulation of the PKA/Src/Rap-1/B-raf/MEK activated Gsa to the regulator of G-protein signalling cascade, by stimulation of a PKA/Ras/B-raf or Raf-1/ domain of Axin, thereby promoting the release of MEK cascade, or through PKA-mediated inhibition of glycogen synthase kinase 3b from the APC/Axin specific protein tyrosine phosphatases that inhibit complex and leading to reduced degradation of

Oncogene Activating mutations of GNAS cooperate with ApcMin CH Wilson et al 4573 b-catenin (Castellone et al., 2005). Our data suggest data suggest that genes such as GNAS, which are that a two fold upregulation of Myc and Birc5 is not mutated at a modest frequency in colorectal cancer, may sufficient to induce intestinal tumourigenesis in also represent ‘drivers’ of tumourigenesis, making Gpa33tm1(GnasR201C)Wtsi/ þ mice, by 12 months at least. functional validation critical for establishing their However, clinical evidence from patients with diagnostic and therapeutic potential. Our data show McCune–Albright syndrome suggests that constitutive that the R201C activating mutation of GNAS causes activation of Gsa is sufficient to induce intestinal augmentation of both the Wnt and ERK1/2 MAPK tumour formation, although the APC status of these pathways in the intestinal epithelium of mice and that lesions is unknown (MacMahon, 1971; Weinstein et al., the mutation co-operates with inactivation of Apc in 1991). APC and Gsa are both reported to bind to the intestinal tumour formation in vivo. Presumably, the regulator of G-protein signalling domain of axin and it activating R201H mutation of GNAS that has been is thought that APC hinders the activation of the Wnt identified in other human cancers contributes to tumour pathway by Gsa. On the loss of APC, the threshold of formation through the same mechanism. Taken together Wnt pathway activation by constitutively active Gsa is with previous human colorectal cancer mutational thought to be lowered (Castellone et al., 2005). In analysis (Sjoblom et al., 2006; Wood et al., 2007), our agreement, we found that the effects of loss of Apc and data strongly suggest that activating mutations of activation of Gnas were additive, as expression levels GNAS are likely, together with inactivation of APC, of the Wnt targets Myc and Birc5 were increased further to contribute to colorectal tumourigenesis. in polyps from double-mutant mice when compared with those from ApcMin/ þ mice (Supplementary Figure 2). These findings may explain the increase in tumour Materials and methods formation, accompanied by no increase in tumour severity in Gpa33tm1(GnasR201C)Wtsi/ þ ApcMin/ þ mice and Generation of conditional Gpa33tm1(GnasR201C)Wtsi/ þ mice why GNAS R201C and APC mutations have been The human full-length GNAS cDNA was synthesized to found to co-occur in intestinal tumours (Sjoblom et al., contain the codon change R201C. This cDNA was inserted 2006; Wood et al., 2007). into the pGpa33LSL targeting vector (Figure 1; Orner et al., Interestingly, cortisol-producing adrenocortical 2002) and targeted to the Gpa33 locus in C57BL/6J Bruce4 embryonic cells . Correctly targeted clones were identified by tumours have been associated with a number of 0 syndromes other than McCune–Albright that feature Southern blotting (probe primers: F: 5 -TGAGTTAGGGCTG CTTGCTT-30 and R: 50-ATGGGTTCTGAGGATGATG the abnormalities of cAMP signalling. Examples are C-30)onPstI digested genomic DNA. Cushing’s syndrome due to mutations in GNAS, primary pigmented nodular adrenocortical disease Mice (Carney Complex) due to mutations of the PKA Targeted embryonic cells were injected into albino C57BL/6J regulatory subunit type 1A (PRKAR1A), macronodular blastocysts and chimeras bred to C57BL/6J mice for germline adrenocortical disease associated with aberrant expres- transmission. All procedures were carried out in accordance sion of G-protein-coupled receptors in the tumour tissue with Home Office guidelines. Conditional Gpa33tm1(GnasR201C)Wtsi/ þ and, more recently, micronodular adrenocortical hyper- and CMV-CreÀ/À mice (Su et al., 2002) were crossed to produce plasia with inactivating mutations of phosphodiesterases Gpa33tm1(GnasR201C)Wtsi/ þ and Gpa33 þ / þ littermates (heterozygous PDE11A and PDE8B (Kirschner et al., 2000; Stratakis, for CMV-Cre) for phenotyping. Gpa33tm1(GnasR201C)Wtsi/ þ 2003; Horvath et al., 2006, 2008). Moreover, somatic Cre þ /À mice were crossed with ApcMin/ þ mice to produce tm1(GnasR201C)Wtsi/ Min/ activating mutations of the b-catenin gene (CTNNB1), Gpa33 þ Apc þ mice and littermate controls. which cause activation of the Wnt pathway, are the most Mice were genotyped by polymerase chain reaction for the Gpa33tm1(GnasR201C)Wtsi/ þ allele using the primers (1) 50-CGAGG frequent genetic defects in adrenocortical tumours and GAGGGCTAACTTTCT-3 0; (2) 50-AAGAAGTGCTCCACC activating mutations of BRAF, KRAS, NRAS or EGFR, AATGC-30 and (3) 50-CGTCCTGACCTCTGGAATCT-30, all of which result in activation of ERK1/2 MAPK, are which when multiplexed, detect the targeted and wild-type also frequently found in this type of tumour (Kotoula alleles of Gpa33. Expression of GNAS R201C was confirmed et al., 2009). Given the involvement of the phospho- by RT–PCR using primers forward: 50-ACATCACCGTGGC diesterase Pde8b, Wnt and ERK1/2 MAPK pathways in ACCCAGACCTCCCTC-30 and reverse: 50-ATCTTTTTGTT intestinal tumourigenesis that is driven by activating GGCCTCACG-30. Mice were genotyped by polymerase chain mutations of Gsa in mice, it may be hypothesized that reaction for ApcMin using standard methods (Dietrich et al., the above signalling molecules are localized to the same 1993). For assessment of adenoma formation the small and cAMP-signalling compartment in these two cell types large (colon and rectum) intestines were collected. The small intestine was subdivided into three equal segments—denomi- and are involved in the development of both colorectal nated duodenum, jejunum and ileum. All were examined and adrenocortical cancers. macroscopically for number, size and location of adenomas With the advancement of next-generation sequencing and embedded in paraffin wax. technology, genome-wide profiling of somatic mutations in human cancers is becoming a reality; therefore, a Flow cytometric analysis of cAMP levels number of potentially interesting cancer genes are being The intestinal epithelial cells were scraped away from the identified. Data further suggests that only a few genes muscular wall of the ileum (n ¼ 7, Gpa33tm1(GnasR201C)Wtsi/ þ are commonly mutated ‘mountains’ and a much larger and n ¼ 5, Gpa33 þ / þ ) using a scalpel and sieved (70 mm) number of gene ‘hills’ are mutated at low frequency. Our with ice-cold 4% formaldehyde in Ca2 þ - and Mg2 þ -free

Oncogene Activating mutations of GNAS cooperate with ApcMin CH Wilson et al 4574 phosphate-buffered saline. The cells were fixed for 10 min, Quantitative RT–PCR (qRT–PCR) washed in phosphate-buffered saline, resuspended in phos- Epithelial cells of the normal intestinal mucosa were scraped phate-buffer saline containing 0.2% Tween-20 (Sigma, Gilling- away from the muscular wall of the small intestine (n ¼ 11 of ham, UK) for 10 min, washed again and resuspended in 1 ml each genotype) using a scalpel. They were snap frozen in liquid 3% bovine serum albumin (Sigma: A3803). In all, 2 mg/ml nitrogen and total RNA was isolated using TRIzol (Invitro- anti-cAMP antibody (Abcam, Cambridge, MA, USA: ab24851) gen, Carlsbad, CA, USA). Polyps of 3 mm in diameter were was added to the test samples and incubated for 3 h. The cells dissected away from the muscular wall of the small intestine were washed and incubated in 3% bovine serum albumin (n ¼ 6 of each genotype), snap frozen in liquid nitrogen and containing 40 mg/ml fluorescein isothiocyanate-immunoglobulin total RNA was isolated using the RNeasy Mini Kit (Qiagen, G (Abcam: ab6785) for 1 h. The median fluorescence was Crawley, UK). RNA was DNase treated using Turbo DNase determined by flow cytometry (FC-500, Beckman Coulter, High (Ambion, Austin, TX, USA) and 1–2 mg total RNA was Wycombe, UK) and data were analysed using FlowJo Software reverse transcribed using the BD Sprint kit with random (version7). Each test sample was normalized to the correspond- hexamers (ABI, Foster City, CA, USA) according to the ing no primary antibody control and data were statistically manufacturers’ protocols. Quantitative PCRs were performed analysed using two-tailed Student’s t-test. with SYBR Green (ABI) on the ABI 7900HT sequence detection system in accordance with the manufacturer’s Immunohistochemistry and immunoblotting instructions, using cDNA obtained from 20 ng total RNA Immunostaining was performed using the DAKO Autostainer (see Supplementary Table 1 for primer sequences). The final Plus with the rabbit VECTASTAIN ELITE ABC horseradish quantitation was determined relative to the average CT of the peroxidase kit (Vector Laboratories, Burlingame, CA, USA). house-keeping genes Gapdh and Actb (Livak and Schmittgen, The primary antibodies used were anti-Ki67 (DCS—Innova- 2001). Data were statistically analysed using two-tailed tive Diagnostik-Systeme, Hamburg, Germany), anti-chromo- Student’s t-test. granin A (Abcam: ab15160) and anti-phospho-Thr202/Tyr204 p44/42 ERK1/2 MAPK (Cell Signalling Technology, Danvers, MA, USA: 20G11). Goblet cells were counted from Alcian Conflict of interest blue-stained sections. Paneth cells, mitotic cells and apoptotic fragments were counted from haematoxylin and eosin stained The authors declare no conflict of interest. sections. In all cases, a total of 30 crypts/villus were counted from the small intestine of six Gpa33tm1(GnasR201C)Wtsi/ þ and six Gpa33 þ / þ mice and compared using the Mann–Whitney U test. Western blotting was performed using standard methods. Acknowledgements Primary antibodies used were anti-p44/42 ERK1/2 MAPK (Cell Signalling Technology: 137F5) and anti-phospho- Work in the DJ Adams Laboratory and the MJ Arends Thr202/Tyr204 p44/42 ERK1/2 MAPK. Band density was Laboratory is funded by Cancer Research UK (CR-UK) and quantified using Image J software (NIH), normalized against the Wellcome Trust. We thank Dr Mattias Ernst for providing b-actin and analysed using two-tailed Student’s t-test. the Gpa33 targeting vector.

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