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. 2017 Aug;24(8):379-392.
doi: 10.1530/ERC-17-0131. Epub 2017 May 22.

Loss-of-function mutations in the CABLES1 gene are a novel cause of Cushing's disease

Laura C Hernández-Ramírez  1 , Ryhem Gam  2 , Nuria Valdés  1   3 , Maya B Lodish  1 , Nathan Pankratz  4 , Aurelio Balsalobre  2 , Yves Gauthier  2 , Fabio R Faucz  1 , Giampaolo Trivellin  1 , Prashant Chittiboina  5 , John Lane  4 , Denise M Kay  6 , Aggeliki Dimopoulos  7 , Stephan Gaillard  8   9 , Mario Neou  8 , Jérôme Bertherat  8   10 , Guillaume Assié  8   10 , Chiara Villa  8   11   12 , James L Mills  7 , Jacques Drouin  2 , Constantine A Stratakis  13
Affiliations

Loss-of-function mutations in the CABLES1 gene are a novel cause of Cushing's disease

Laura C Hernández-Ramírez et al. Endocr Relat Cancer. 2017 Aug.

Abstract

The CABLES1 cell cycle regulator participates in the adrenal-pituitary negative feedback, and its expression is reduced in corticotropinomas, pituitary tumors with a largely unexplained genetic basis. We investigated the presence of CABLES1 mutations/copy number variations (CNVs) and their associated clinical, histopathological and molecular features in patients with Cushing's disease (CD). Samples from 146 pediatric (118 germline DNA only/28 germline and tumor DNA) and 35 adult (tumor DNA) CD patients were screened for CABLES1 mutations. CNVs were assessed in 116 pediatric CD patients (87 germline DNA only/29 germline and tumor DNA). Four potentially pathogenic missense variants in CABLES1 were identified, two in young adults (c.532G > A, p.E178K and c.718C > T, p.L240F) and two in children (c.935G > A, p.G312D and c.1388A > G, and p.D463G) with CD; no CNVs were found. The four variants affected residues within or close to the predicted cyclin-dependent kinase-3 (CDK3)-binding region of the CABLES1 protein and impaired its ability to block cell growth in a mouse corticotropinoma cell line (AtT20/D16v-F2). The four patients had macroadenomas. We provide evidence for a role of CABLES1 as a novel pituitary tumor-predisposing gene. Its function might link two of the main molecular mechanisms altered in corticotropinomas: the cyclin-dependent kinase/cyclin group of cell cycle regulators and the epidermal growth factor receptor signaling pathway. Further studies are needed to assess the prevalence of CABLES1 mutations among patients with other types of pituitary adenomas and to elucidate the pituitary-specific functions of this gene.

Keywords: Cushing’s disease; corticotropinoma; germline mutation; whole-exome sequencing.

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Figures

Figure 1
Figure 1
Missense CABLES1 variants of interest: effects on CABLES1 transcripts and proteins. (A) Three transcripts of CABLES1, NM_001100619.2, NM_138375.2 and NM_001256438.1, which differ in their first exon, are translated into proteins of 633, 368 and 306 amino acids, respectively. The former (NM_001100619.2,) is the ‘canonical’ sequence (UniProt Q8TDN4-1). For each transcript, mRNA is schematized in blue and the coding sequence in green. The CABLES1 variants identified in Patients 1 and 2 (p.E178K and p.L240F), respectively, affect exon 1 of the reference transcript, but not the other transcripts. The variant detected in Patient 3 is located in exon 3 (p.G312D) and the variant from Patient 4 (p.D463G) affects exon 7. There is no information in the literature about the expression of these transcripts in the pituitary gland. (B) The domains of the CABLES1 protein are incompletely characterized and no 3-D structure is available that represents the majority of the protein sequence. The 633 amino acid (65.7 kDa) isoform of the protein contains an N-terminal site for interaction with TDRD7 and a central large sequence necessary for interacting with CDK3. Four residues are targets for phosphorylation: S168, S287, S313 and T415 (Shi et al. 2015, Uniprot Consortium 2015, Yamochi et al. 2001). The C-terminal end of the protein contains a cyclin-like domain, and for this reason, CABLES1 has been included in the cyclin superfamily, although it differs in functions with cyclins. The alterations found in Patients 1–4 are included in (p.L240F, p.G312D, p.D463G) or very close to (p.E178K) the motif that interacts with CDK3. (C) The CABLES1 gene is highly conserved among species, and the variants found in our patients affect relatively (p.E178K, p.L240F) and highly (p.G312D, p.D463G) conserved residues.
Figure 2
Figure 2
ACTH, CABLES1 and CDKN1B expression in corticotropinomas. We compared ACTH, CABLES1 and CDKN1B immunostaining in samples from the four patients with putative CABLES1 mutations with samples from two patients negative for such alterations (one representative example is presented). ACTH immunostaining was observed in the vast majority of the cells in all the cases, except for Patient 1, for whom only 50% of the cells were immunoreactive (immunoreactivity for other pituitary hormones was ruled out). All the samples displayed positive CABLES1 cytoplasmic and, predominantly, nuclear immunoreactivity. In specimens that contained areas of non-tumoral pituitary, reduced CABLES1 staining was observed in the corticotropinomas, compared with the surrounding tissue (Supplementary Fig. 1), in concordance with previous data (Roussel-Gervais et al. 2016). Nevertheless, we did not observe differences in the CABLES1 immunostaining between samples with and without putative CABLES1 mutations. In contrast, while in controls CDKN1B staining was moderately intense in the cytoplasm of the great majority of the cells and in the nucleus of 50–60% of them, only weak cytoplasmic staining and very few cells with nuclear staining were observed in the cases with putative CABLES1 mutations (<10% cells with nuclear staining for Patients 1 and 2 and 30–40% cells with weak nuclear staining for Patients 3 and 4). Magnification in all the images: 10×, inserts: 20×.
Figure 3
Figure 3
CABLES1 mutant proteins have lost their growth inhibition activity. (A) Schematic representation of tamoxifen-inducible chimeric CABLES1 proteins fused to the ERtam ligand-binding domain. Upon stable transduction of AtT-20 cells, wild-type (WT) and mutant ERtam-CABLES1 proteins are expressed at similar levels as revealed by Western blot against the Flag epitope. (B) Growth curves of AtT20/D16v-F2 cells expressing WT ERtam-CABLES1 treated with vehicle (ethanol), tamoxifen, dexamethasone or both, as indicated. All the conditions rendered statistically significant differences in cell counts at all time points. To analyze exclusively the effect of CABLES1 on cell growth, only the tamoxifen treatment was used for experiments with the mutant proteins. (C, D, E and F) Growth curves for AtT20/D16v-F2 cells expressing CABLES1 p.E178K (C), p.L240F (D), p.G312D (E) or p.D463G (F) mutant protein in vehicle and tamoxifen-treated cells. There were no statistically significant differences in cell count at the different time points, meaning that these variants have lost the ability to suppress cell growth.
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