The activating mutation R201C in GNAS promotes intestinal tumourigenesis in Apc(Min/+) mice through activation of Wnt and ERK1/2 MAPK pathways

Abstract

Somatically acquired, activating mutations of GNAS, the gene encoding the stimulatory G-protein Gsalpha subunit, have been identified in kidney, thyroid, pituitary, leydig cell, adrenocortical and, more recently, in colorectal tumours, suggesting that mutations such as R201C may be oncogenic in these tissues. To study the role of GNAS in intestinal tumourigenesis, we placed GNAS R201C under the control of the A33-antigen promoter (Gpa33), which is almost exclusively expressed in the intestines. The GNAS R201C mutation has been shown to result in the constitutive activation of Gsalpha and adenylate cyclase and to lead to the autonomous synthesis of cyclic adenosine monophosphate (cAMP). Gpa33(tm1(GnasR201C)Wtsi/+) mice showed significantly elevated cAMP levels and a compensatory upregulation of cAMP-specific phosphodiesterases in the intestinal epithelium. GNAS R201C alone was not sufficient to induce tumourigenesis by 12 months, but there was a significant increase in adenoma formation when Gpa33(tm1(GnasR201C)Wtsi/+) mice were bred onto an Apc(Min/+) background. GNAS R201C expression was associated with elevated expression of Wnt and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase (ERK1/2 MAPK) pathway target genes, increased phosphorylation of ERK1/2 MAPK and increased immunostaining for the proliferation marker Ki67. Furthermore, the effects of GNAS R201C on the Wnt pathway were additive to the inactivation of Apc. Our data strongly suggest that activating mutations of GNAS cooperate with inactivation of APC and are likely to contribute to colorectal tumourigenesis.

Figure 1

Generation and validation of the intestine specific GNAS R201C allele. A, Schematic illustration of the Gpa33 wildtype locus (Gpa33, upper panel), Gpa33^{tm1(GnasR201C)Wtsi} conditional targeted locus (C, middle panel) and the Gpa33^{tm1(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 Gpa33^{tm1(GnasR201C)Wtsi/+} (R201C) and control (WT) ES cells. C, RT-PCR expression analysis of GNAS R201C transcripts (Gpa33-GNAS) and β-Actin (Actb) expression in tissues taken from Gpa33^{tm1(GnasR201C)Wtsi/+} (R201C) mice and wildtype 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. Gpa33^{tm1(GnasR201C)Wtsi/+} (R201C) and wildtype (WT) tissues were analyzed. E, Quantitative RT-PCR analysis of cAMP-specific phosphodiesterases 4a, 4b, 7a, 8a and 8b in the intestine of Gpa33^{tm1(GnasR201C)Wtsi/+} (R201C) and wildtype littermate control mice (WT). *P<0.05.

Figure 2

Gpa33^{tm1(GnasR201C)Wtsi} (R201C) promotes intestinal adenoma formation in Apc^Min/+ mice when compared to Apc^Min/+ controls but alone does not promote tumor formation. A, Macroscopic counts of intestinal tumours (≥1.5 mm in size) at 16 weeks in Gpa33^{tm1(GnasR201C)Wtsi/+} Apc^Min/+ mice and Apc^Min/+ littermates. No tumours were found in Gpa33^{tm1(GnasR201C)Wtsi/+} or wildtype littermate controls (WT) aged to 12 months. B, Representative photograph of small intestines from Gpa33^{tm1(GnasR201C)Wtsi/+}Apc^Min/+ and Apc^Min/+ littermate (arrows indicate adenomas). C, Representative H&E stained section of a Swiss roll from an Gpa33^{tm1(GnasR201C)Wtsi/+}Apc^Min and Apc^Min/+ littermate (arrows indicate adenomas, boxes enlarged to right). Histological analysis revealed no difference in the dysplastic grade of adenoma between Gpa33^{tm1(GnasR201C)Wtsi/+}Apc^Min and Apc^Min/+ littermates.

Figure 3

Activation of Wnt and ERK1/2 MAPK pathways in the intestines of Gpa33^{tm1(GnasR201C)Wtsi/+} 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 Gpa33^{tm1(GnasR201C)Wtsi/+} mice (R201C) when compared to control mice littermate controls (WT). B, Western blot analysis of intestinal tissue from Gpa33^{tm1(GnasR201C)Wtsi/+} (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 Gpa33^{tm1(GnasR201C)Wtsi/+} (R201C) And littermate control mice (WT).