Identification of Genetic Alterations of AXIN2 Gene in Adrenocortical Tumors
Audrey Chapman, Julien Durand, Lydia Ouadi, and Isabelle Bourdeau
Division of Endocrinology, Department of Medicine, Research Centre, Centre Hospitalier de l’Université de Montréal, Hôtel-Dieu, Montréal, Québec, Canada H2W 1T7
Background: Mutations of the -catenin gene (CTNNB1), which lead to constitutive activation of Wnt signaling, have recently been described in adrenocortical adenomas (AA) and carcinomas (ACC). However, somatic CTNNB1 mutations may explain only about 50% of ß-catenin accumula- tion observed in adrenocortical tumors, indicating that other components of the Wnt pathway may be involved.
Objective: The objective of the study was to investigate whether alterations in AXIN2 may be present in adrenocortical tumors.
Methods: We studied 49 human adrenocortical samples: 30 AA, six ACC, five primary pigmented nodular adrenocortical disease five ACTH-independent macronodular adrenal hyperplasias (AIMAH), and three ACTH-dependent hyperplasias in addition to the human ACC cell lines SW13 and H295R. Samples were screened for somatic genetic alterations in exon 3 of CTNNB1 and exons 5, 7, and 9 of AXIN2.
Results: We found an in-frame, 12-bp deletion beginning at coding nucleotide 2013 in exon 7 of the AXIN2 gene, c.2013_2024del12 (p.Arg671_Pro674del), in two of 30 AA (7%), one of six ACC (17%), and the ACC H295R cell line. Immunohistochemistry revealed that tumors with AXIN2 genetic defects showed nuclear/cytoplasmic accumulation of B-catenin, indicating the activation of Wnt signaling. In addition, the ACC and H295R cells with AXIN2 deletion (c.2013_2024del12) harbored p.Ser45del and p.Ser45Pro CTNNB1 mutations, respectively. Two single-nucleotide poly- morphisms were identified in exon 7 of AXIN2, c.2351C>Tin 2 AA, and one AIMAH and c.2342A>G in an AIMAH tissue.
Conclusion: The present study reports, for the first time, that AXIN2 genetic defects may be found in adrenocortical tumors. However, the functional consequence of this genetic alteration remains to be determined. (J Clin Endocrinol Metab 96: E1477-E1481, 2011)
A lthough adrenocortical lesions may be associated with hereditary syndromes, only a few genetic al- terations in sporadic adrenocortical tumors have been de- scribed. B-Catenin (CTNNB1) gene mutations are found in sporadic adrenocortical adenomas (AA) and adreno- cortical carcinomas (ACC) (1-3) and lead to abnormal nuclear and/or cytoplasmic accumulation of ß-catenin protein and constitutive transcriptional activation of Wnt signaling (1, 2). Somatic CTNNB1 mutations may
explain about 50% of Wnt activation in adrenocortical tumors, indicating that other components of the Wnt signaling pathway may be involved in adrenocortical tumorigenesis.
In various cancers, mutations have been reported in other components of the Wnt pathway, such as in APC, AXIN1, and AXIN2 genes (4). AXIN2, also named Axil/ conductin, is a negative regulator of Wnt signaling (5). It works as a scaffold protein and promotes the assembly of
Abbreviations: AA, Adrenocortical adenomas; ACC, adrenocortical carcinomas; AIMAH, ACTH-independent macronodular adrenal hyperplasias; AXIN, Axil/conductin; CTNNB1, ß-catenin gene; GSK, glycogen synthase kinase; PPNAD, primary pigmented nodular ad- renocortical disease.
multiprotein complexes, which include adenomatous poly- posis coli, the serine/threonine glycogen synthase kinase (GSK)-3ß kinase GSK-3B, AXIN1, and ß-catenin. Once as- sociated with this complex, ß-catenin is phosphorylated by GSK-3ß, which culminates in its degradation by protea- some complex. In addition, AXIN2 is itself activated by Wnt/T-cell factor signaling, resulting in a negative-feed- back loop, which regulates the duration and intensity of Wnt/T-cell factor activation (6). Mutations of AXIN2 cause familial tooth agenesis (7) and have been identified in colorectal carcinomas, hepatocellular carcinomas, hepatoblastomas, melanomas, gastric cancer, and ovarian endometrioid adenocarcinomas (8-12). Because CT- NNB1 mutations are observed only in a subgroup of ad- renocortical tumors with ß-catenin accumulation, it is likely that defects in other components of the Wnt/ß- catenin pathway are involved in adrenocortical tumori- genesis. As described previously in other cancers, we hy- pothesized that AXIN2 genetic alterations may be implicated in the pathogenesis of adrenocortical tumors. We report here, for the first time, genetic alterations of the AXIN2 gene in AA and ACC.
Materials and Methods
Patients, adrenocortical samples, and cell lines
Tissue specimens were obtained from 49 patients with dif- ferent types of adrenocortical lesions: 30 AA (one nonsecreting, 14 cortisol-secreting, and 15 aldosterone-secreting), six ACC, five ACTH-independent macronodular adrenal hyperplasias (AIMAH), five primary pigmented nodular adrenocortical dis- ease (PPNAD) tissues, and three ACTH-dependent hyperplasias. All samples were acquired from patients under research proto- cols approved by the Centre Hospitalier de l’Université de Mon- tréal, Montréal, Québec, Canada, and all of them signed in- formed consent forms. Adrenal tissues were obtained at surgery, frozen immediately in liquid nitrogen, and stored. Tissue sections were fixed in formalin and embedded in paraffin for histopatho- logical analysis (13). The human adrenocortical cell lines NCI- H295R and SW13 (American Type Culture Collection, Manas- sas, VA) were cultured as described earlier (1).
DNA extraction, RNA extraction, and cDNA preparation
Genomic DNA was isolated from frozen tissues by standard proteinase K/sodium dodecyl sulfate digestion, followed by phe- nol/chloroform extraction. RNA was obtained from frozen tis- sues with TriZOL reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. cDNA was generated with Molo- ney murine leukemia virus reverse transcriptase (Invitrogen) and random hexamers (Invitrogen).
Mutational analysis of AXIN2 and CTNNB1 genes
Mutational studies of 49 adrenocortical tissues and the hu- man ACC cell lines H295R and SW13 were undertaken. Exons
5, 7, and 9 coding regions of AXIN2 gene according to GenBank accession no. NM_004655 (or exons 6, 8, and 10 of the recently updated sequence GenBank accession no. NM_004655.3) were investigated because all AXIN2 mutations reported earlier were localized in them. AXIN2 primers were as described previously except that primers (exons) 5-1 and 5-2 were combined as 5-3 and 5-4 (8). DNA amplification was performed, using TAQ DNA polymerase (Invitrogen) in a final volume of 25 ul with 200 ng of DNA in a buffer containing 0.3 um of each primer, 0.2 mM deoxynucleotide triphosphate, 2 mM MgCl2, and 0.5 U/ul TAQ DNA polymerase. The PCR program contained a 5-min step at 95 C, followed by 35 amplifications for all primers (at 95 C/45 sec, annealing at 56-58 C/30 sec and 72 C/45 sec) and termi- nated by an extension step of 72 C for 7 min. Amplicons were purified with the QIAquick gel extraction kit (QIAGEN, Valen- cia, CA) and sequenced directly on an automated sequencer in both directions (SUCOF; Université Laval, Québec, Québec, Canada). All mutations were confirmed in at least two tissue extractions and at the cDNA level, with the following primer set: forward 5’-TTATGCTTTGCACTACGTCCCTCCA-3’ and re- verse 5’-CGCAACATGGTCAACCCTCAGAC-3’. cDNA bands were extracted, purified, and sequenced as above. CT- NNB1 mutational analysis of exon 3 was conducted as described elsewhere (1, 14).
Immunohistochemical analysis of ß-catenin
Formalin-fixed, paraffin-embedded tissues from two AA har- boring AXIN2 deletion were studied. Tissue sections were deparaffinized, treated to eliminate endogenous peroxidase ac- tivity, and incubated for 17 min at 95 C in citrate buffer (pH 6.0) for antigen retrieval. Nonspecific antigens were blocked with a protein-blocking, serum-free reagent (Dako Diagnostics Can- ada, Inc., Mississauga, Ontario, Canada). The slides were then incubated for 60 min with specific mouse monoclonal anti-ß- catenin antibody (1:25; BD Transduction Laboratories, BD Bio- sciences, Mississauga, Ontario, Canada). Incubations with sec- ondary biotinylated antibody and streptavidin-horseradish peroxidase (Dako Diagnostics Canada) followed sequentially. Reaction products were developed with diaminobenzidine con- taining 0.3% H2O2 as substrate for peroxidase. Hematoxylin counterstaining was analyzed. The negative controls included substitution of the primary antibody by PBS and an isotype con- trol, IgG1 (Cymbus Biotechnology, Hampshire, UK). Ten con- secutive fields (×400) from two slides for each tumor studied were examined under standard light microscopy by a patholo- gist. Staining was quantified as a percentage of cells stained from 0 to 100%, which represented a mean of the percentage in 10 consecutive fields from two slides per tissue.
Results
AXIN2 genetic alterations occur in human adrenocortical tumors and the human ACC H295R cell line
We observed genetic changes of AXIN2 gene in two of 30 AA (7%) and one of six ACC (17%). The genetic defect is an in-frame 12-bp deletion in exon 7 of AXIN2 begin- ning at coding nucleotide 2013, c.2013_2024del12 (p.Arg671_Pro674del). The deletion results in heterozy-
gous loss of codons 671-674 (Arg-Thr-Thr-Pro) contain- ing two potential sites of phosphorylation (Fig. 1A). We did not find AXIN2 genetic alterations in PPNAD, AIMAH, and ACTH-dependent hyperplasias. However, we noted the same deletion (c.2013_2024del12) in DNA from the human ACC cell line NCI-H295R but not in SW13 (Table 1). Somatic mutations in exon 3 of the CTNNB1 gene were detected in six of 30 AA (p.Ser37Cys, p.Tyr41Ala, p.Ser45Phe, c.26943del55, c.27127del6, c.26995del271) (1), one of six ACC (p.Ser45del), and one of five PPNAD (p.Ser45Pro) (14). Interestingly, the ACC and H295R cells with AXIN2 deletion harbored, in ad- dition, the activating CTNNB1 mutations p.Ser45del and p.Ser45Pro, respectively (Table 1), which affects a site of B-catenin phosphorylation by GSK3B. Genetic changes were confirmed by repeat DNA extraction and sequenced in both the sense and antisense orientation and at the RNA level by RT-PCR and cDNA sequencing (Fig. 1A). Two single-nucleotide polymorphisms were identified in exon 7 of AXIN2, c.2351C>Tin two AA, and one AIMAH and c.2342A>G in an AIMAH tissue. In addition, we noted the presence of two single-nucleotide polymorphisms de- scribed previously in exon 5 of AXIN2, c.1654A>G (25 of 30 AA, six of six ACC, five of five AIMAH, five of five PPNAD, and three of three ACTH dependent adrenal hy- perplasias) and c.1675C>T (23 of 30 AA, five of six ACC,
A
c.2013_2024del12
Normal DNA
a
3
Tumoral DNA
G
A
Tumoral cDNA
B
C
four of five AIMAH, three of five PPNAD, and three of three ACTH dependent adrenal hyperplasias) in the ma- jority of samples. We did not find any genetic alterations in exon 9 of AXIN2.
B-Catenin protein is aberrantly expressed in adrenocortical tumors harboring genetic alterations of AXIN2
We observed nuclear (0.7 and 7.8% of cells) and cyto- plasmic (87 and 91% of cells) ß-catenin immunostaining in the two AA harboring AXIN2 genetic alterations avail- able for immunohistochemical studies, indicating activa- tion of Wnt signaling (Fig. 1B), as described previously in tumors harboring CTNNB1 mutations (1, 14).
Discussion
Previous studies have demonstrated that CTNNB1 somatic mutations are frequent in AA and ACC but could not explain all cases of aberrant cytoplasmic and/or nuclear accumula- tion of ß-catenin in adrenocortical tumors (2), suggesting that other components of the Wnt signaling pathway may also be involved in adrenocortical tumorigenesis.
We identified a 12-bp in-frame deletion in exon 7 of AXIN2 (c.2013_2024del12) in two of 30 AA (7%), one of six ACC (17%), and the ACC H295R cell line that has never been described in adrenocortical tumors. The prev- alence of AXIN2 genetic alterations may be underesti- mated in our work because we concentrated our initial study on the three exons that were reported as being mu- tated in cancers.
In accordance with our study, the same 12-bp deletion (c.2013_2024del12) was described previously in a hepa- tocellular carcinoma (10), a colorectal cancer, a colorectal cell line (9), and a melanoma cell line (15). Unfortunately, we did not have access to the lymphocyte DNA of stud- ied patients to ascertain the germline or somatic origin of the deletion. However, in 2008, Castiglia et al. (15) reported that the c.2013_2024del12 genetic change was present in the peripheral blood mononuclear cells and tumoral cells of a patient with melanoma from whom a melanoma cell line was established, suggesting a germ- line genetic alteration. These authors screened 93 con- trol individuals to exclude a gene polymorphism. The deletion was not detected in any of their subjects. Very recently, Pedace et al. (16) found the same genetic varia- tion in familial melanoma subjects, which was not de- tected in 153 healthy controls. The functional conse- quence of c.2013_2024del12 has not been investigated, but it is localized in the tracts of exon 7, which is known to be a common site for AXIN2 mutations. Exon 7 of the
| Age (yr) | Sex | Diagnosis | AXIN2 genetic alterations | CTNNB1 mutations | ß-Catenin immunostaining | |
|---|---|---|---|---|---|---|
| E123 | 38 | F | Aldosterone-secreting adenoma | c.2013_2024del12 | No | N: 7.8%; C: 91% |
| E128 | 40 | M | Cortisol-secreting adenoma | c.2013_2024del12 | No | N: 0.7%; C: 87% |
| E193 | 49 | F | Cortisol/androgen-secreting carcinoma | c.2013_2024del12 | p.Ser45del | NA |
| H295R | Human adrenocortical carcinoma cell line | c.2013_2024del12 | p.Ser45Pro | NA |
F, Female; M, male; N, nuclear; C, cytoplasmic; NA, not available.
AXIN2 gene is the hypothetical binding site for the protein phosphatase 2A, which acts as a positive regulator of the Wnt pathway by dephosphorylation of AXIN1 and, to some ex- tent, AXIN2. The c.2013_2024del12 deletion leads to the removal of two threonine amino acid residues that could be the target of protein phosphatase 2A (17, 18).
We observed increased phosphatase PP2A cytoplasmic and nuclear accumulation in tumoral cells of AA harbor- ing the 12-bp deletion of AXIN2, supporting activation of the Wnt signaling pathway in these tumors. Our findings are consistent with other studies in which somatic AXIN2 mutations were associated with the nuclear translocation of ß-catenin (8, 10-12, 19).
AXIN2 genetic alterations were seen in one cortisol- secreting AA, one aldosterone-secreting AA, one cortisol- androgen-secreting ACC, and the human ACC H295R cell line. Interestingly, the ACC and H295R cells har- bored, in addition, a CTNNB1 mutation. The presence of genetic changes in two components of Wnt signaling was somehow surprising. However, in colorectal cancer, AXIN2 mutations were encountered in a subgroup of tu- mors also containing either APC or CTNNB1, contribut- ing to further up-regulation of Wnt activity compared with tumors containing only APC or CTNNB1 genetic defects (4). Thus, mutational activation of CTNNB1 and simultaneous abrogation of the negative feedback of AXIN2 may both result in activation of Wnt/ß-catenin signaling.
In conclusion, this is the first study to report the pres- ence of AXIN2 genetic alterations in AA, ACC, and the H295R cell line with abnormal accumulation of ß-catenin protein, further supporting the role of Wnt/B-catenin sig- naling in adrenocortical tumorigenesis. However, studies are required to investigate the functional consequence and the germline or somatic origin of c.2013_2024del12 AXIN2 deletion in adrenocortical tumors.
Acknowledgments
We are grateful to Dr. André Lacroix (Centre Hospitalier de l’Université de Montréal, Montréal, Québec, Canada) for pro- viding adrenocortical samples. We thank Dr. Anne-Marie Mess-
Masson and members of her laboratory for their assistance in immunohistochemistry as well as the MacDonald Stewart Foun- dation for photographic support. We also thank Mimi Tadjine, who performed mutational analysis of adrenocortical tumors at the beginning of the project, and Dr. Sophie Vallette for her helpful discussion. The editing of our manuscript by Mr. Ovid Da Silva through the Research Support Office, Centre Hospita- lier de l’Université de Montréal, is acknowledged.
Address all correspondence and requests for reprints to: Isa- belle Bourdeau, M.D., Division of Endocrinology, Department of Medicine, Centre Hospitalier de l’Université de Montréal, Hôtel-Dieu, 3850 Saint Urbain Street, Montréal, Québec, Can- ada H2W 1T7. E-mail: isabelle.bourdeau@umontreal.ca.
This work was supported by Grant FRSQ-6519/5360 from Fonds de la Recherche en Santé du Québec (principal investiga- tor: I.B.) and The Cancer Research Society (principal investiga- tor: I.B.).
Disclosure Summary: The authors have nothing to disclose.
References
1. Tadjine M, Lampron A, Ouadi L, Bourdeau I 2008 Frequent mu- tations of ß-catenin gene in sporadic secreting adrenocortical ade- nomas. Clin Endocrinol (Oxf) 68:264-270
2. Tissier F, Cavard C, Groussin L, Perlemoine K, Fumey G, Hagneré AM, René-Corail F, Jullian E, Gicquel C, Bertagna X, Vacher- Lavenu MC, Perret C, Bertherat J 2005 Mutations of ß-catenin in adrenocortical tumors: activation of the Wnt signaling pathway is a frequent event in both benign and malignant adrenocortical tumors. Cancer Res 65:7622-7627
3. Masi G, Lavezzo E, Iacobone M, Favia G, Palù G, Barzon L 2009 Investigation of BRAF and CTNNB1 activating mutations in adre- nocortical tumors. J Endocrinol Invest 32:597-600
4. Polakis P 2007 The many ways of Wnt in cancer. Curr Opin Genet Dev 17:45-51
5. Behrens J, Lustig B 2004 The Wnt connection to tumorigenesis. Int J Dev Biol 48:477-487
6. Jho EH, Zhang T, Domon C, Joo CK, Freund JN, Costantini F 2002 Wnt/ß-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway. Mol Cell Biol 22:1172- 1183
7. Lammi L, Arte S, Somer M, Jarvinen H, Lahermo P, Thesleff I, Pirinen S, Nieminen P 2004 Mutations in AXIN2 cause familial tooth agenesis and predispose to colorectal cancer. Am J Hum Genet 74:1043-1050
8. Liu W, Dong X, Mai M, Seelan RS, Taniguchi K, Krishnadath KK, Halling KC, Cunningham JM, Boardman LA, Qian C, Christensen E, Schmidt SS, Roche PC, Smith DI, Thibodeau SN 2000 Mutations
in AXIN2 cause colorectal cancer with defective mismatch repair by activating B-catenin/TCF signalling. Nat Genet 26:146-147
9. Suraweera N, Robinson J, Volikos E, Guenther T, Talbot I, Tom- linson I, Silver A 2006 Mutations within Wnt pathway genes in sporadic colorectal cancers and cell lines. Int J Cancer 119:1837- 1842
10. Taniguchi K, Roberts LR, Aderca IN, Dong X, Qian C, Murphy LM, Nagorney DM, Burgart LJ, Roche PC, Smith DI, Ross JA, Liu W 2002 Mutational spectrum of B-catenin, AXIN1, and AXIN2 in hepatocellular carcinomas and hepatoblastomas. Oncogene 21: 4863-4871
11. Kim MS, Kim SS, Ahn CH, Yoo NJ, Lee SH 2009 Frameshift mu- tations of Wnt pathway genes AXIN2 and TCF7L2 in gastric car- cinomas with high microsatellite instability. Hum Pathol 40:58-64
12. Wu R, Zhai Y, Fearon ER, Cho KR 2001 Diverse mechanisms of ß-catenin deregulation in ovarian endometrioid adenocarcinomas. Cancer Res 61:8247-8255
13. Schteingart DE, Doherty GM, Gauger PG, Giordano TJ, Hammer GD, Korobkin M, Worden FP 2005 Management of patients with adrenal cancer: recommendations of an international consensus conference. Endocr Relat Cancer 12:667-680
14. Tadjine M, Lampron A, Ouadi L, Horvath A, Stratakis CA, Bour- deau I 2008 Detection of somatic ß-catenin mutations in primary
pigmented nodular adrenocortical disease (PPNAD). Clin Endocri- nol (Oxf) 69:367-373
15. Castiglia D, Bernardini S, Alvino E, Pagani E, De Luca N, Falcinelli S, Pacchiarotti A, Bonmassar E, Zambruno G, D’Atri S 2008 Con- comitant activation of Wnt pathway and loss of mismatch repair function in human melanoma. Genes Chromosomes Cancer 47: 614-624
16. Pedace L, Castiglia D, De Simone P, Castori M, De Luca N, Amantea A, Binni F, Majore S, Cozzlino AM, De Bernardo C, Zambruno G, Catricalà C, Grammatico P 2011 AXIN2 germline mutations are rare in familial melanoma. Genes Chromosomes Cancer 50:370- 373
17. Willert K, Shibamoto S, Nusse R 1999 Wnt-induced dephosphor- ylation of axin releases ß-catenin from the axin complex. Genes Dev 13:1768-1773
18. Ikeda S, Kishida S, Yamamoto H, Murai H, Koyama S, Kikuchi A 1998 Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3ß and ß-catenin and promotes GSK-3ß-de- pendent phosphorylation of B-catenin. EMBO J 17:1371-1384
19. Koch A, Hrychyk A, Hartmann W, Waha A, Mikeska T, Waha A, Schüller U, Sörensen N, Berthold F, Goodyer CG, Wiestler OD, Birchmeier W, Behrens J, Pietsch T 2007 Mutations of the Wnt antagonist AXIN2 (Conductin) result in TCF-dependent transcrip- tion in medulloblastomas. Int J Cancer 121:284-291
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