AXIN genetic analysis in adrenocortical carcinomas updated
A. Guimier1,2, B. Ragazzon1,2, G. Assié1-5, F. Tissier1,2,5-7, B. Dousset1-3,5,8, J. Bertherat1-5, and S. Gaujoux1-3,5,8
1Institut Cochin, Université Paris Descartes, CNRS (UMR 8104); 2Inserm, U1016, Endocrinology, Metabolism & Cancer Department; 3Université Paris Descartes; 4Assistance Publique Hôpitaux de Paris, Hôpital Cochin, Department of Endocrinology, Center for rare adrenal diseases; 5COMETE-INCA- Rare Adrenal Cancer Newtork; 6Assistance Publique Hôpitaux de Paris, Groupe Hospitalier Pitié-Salpêtrière Charles Foix, Department of Pathology; 7Université Pierre et Marie Curie; 8Assistance Publique Hôpitaux de Paris, Hôpital Cochin, Department of Digestive and Endocrine Surgery, Paris, France
ABSTRACT. Background: Wnt/B-catenin signaling pathway activation plays an important role in adrenocortical tumori- genesis, but is only in part related to ß-catenin activating so- matic mutations. Recently, genetic alteration in AXIN2, a key component of the Wnt/B-catenin signaling pathway, has been described in adrenocortical tumors and specifically in adreno- cortical carcinoma (ACC). Aim: To assess frequency and con- sequences of AXIN genes alteration on a large cohort of ACC. Patients and methods: Forty-nine adult sporadic ACC, with expression data available, in addition to both ACC cell lines H295 and H295R were studied. AXIN2 exon 8 hot-spot sequencing was performed on the entire cohort. AXIN1 en- tire coding region was studied on the 8 ACC with nuclear B-catenin staining. Results: The previously described AXIN2 in-frame heterozygous 12bp deletion c2013_2024del12 was found in 1 of the 49 ACC studied (2%), in a tumor with
INTRODUCTION
Recently important advances towards understanding the molecular mechanisms of adrenocortical carcinoma (ACC) development have been made. Activation of the Wnt/ß- catenin signaling pathway plays a key role (1-3) in tumor development, as well as a prognosis marker (4) or a po- tential therapeutic target (5). Additionally, according to gene expression profiling (6), ACC with a ß-catenin nu- clear staining represent a relatively homogeneous group of tumors, exclusively found in a cluster of poor-outcome ACC. Currently, ß-catenin gene (CTNNB1) activating mu- tations are the most frequent genetic defect observed in adrenocortical tumors (1, 3), but they only account for a part of the observed ACC with Wnt/B-catenin signaling pathway activation. Alternative genetic defects, such as APC or WTX mutations (7), able to explain this discrep- ancy, have been explored with disappointing results in sporadic tumors. Overall Wnt/ß-catenin signaling path- way activation remains unexplained in about half of cas- es.
Recently, Chapman et al., observed in a heterogeneous cohort of ACT AXIN2 gene alteration in adrenocortical
pSer45del activating CTNNB1 mutation and nuclear ß-catenin staining. This heterozygous deletion was also found in the patient’s germline DNA, extracted from peripheral blood leukocytes. This genetic alteration was also present in H295 and H295R cell lines. The single-nucleotide polymorphism rs35415678 was found with an allele frequency similar to those found in reference populations. No correlation be- tween AXIN2 expression, AXIN2 genetic variant or nuclear ß- catenin staining was observed. No AXIN1 alterations were found in the 8 ACC studied. Conclusions: AXIN genes do not play a major role in ACC tumorigenesis and Wnt/B-catenin signaling pathway activation. AXIN2 germline variant c2013_2024del12 is likely to be a non-pathogenic polymor- phism.
(J. Endocrinol. Invest. 36: 1000-1003, 2013) @2013, Editrice Kurtis
tumors, suggesting a possible role in Wnt/ß-catenin sig- naling pathway activation (8). In order to analyze the role of AXIN genes in ACC, we assess on a large and homo- geneous cohort, the states of the AXIN genes.
PATIENTS AND METHODS Patients and tissue collection
Forty-nine sporadic ACC, with available somatic DNA, from a previously described cohort (6), were included, in addition to human ACC cell line H295 and H295R. Informed signed con- sent for genetic diagnosis, tumor analysis and for access to clin- ical data were obtained from all patients. The study was ap- proved by our Institutional Review Board (Comité Consultatif de Protection des Personnes dans la Recherche Biomédicale, Cochin Hospital, Paris).
Nucleic acid extraction and mutation analysis AXIN2 and AXIN1
All AXIN2 gene mutations previously described were located in exon 8 (GenBank accession no. NM_004655.3, previously known as exon 7 of the previous sequence GenBank no. NM_004655) (9). We therefore focused and screened the coding region with exon-intron boundaries of exon 8 using PCR and direct sequenc- ing on these 49 ACC. Briefly, after tumor DNA extraction and pu- rification as previously described (2), PCR was performed using the following primers: forward 5’-GACAAGTTTCTATTGAAGT- CAC-3’ and reverse 5’-ATTAGCCACAGACCAGGTC-3’ accord- ing to the genomic sequence Genbank no. NC_000017.10 (10). PCR reactions were then performed in 25 ul of final reaction vol- ume using AmpliTaq Gold DNA polymerase (Applied Biosystems)
Correspondence: B. Ragazzon, Institut Cochin - INSERM U1016 - CNRS UMR 8104 - Université Paris 5, Département d’Endocrinologie Métabolisme et Cancer, Faculté de Médecine - 24, rue du Faubourg Saint-Jacques - 75014 Paris, France.
| Characteristics (no .= 49) | No. (%) median (range) |
|---|---|
| Age (yr) | 44 (15-81) |
| Female | 39 (80%) |
| Hormone secretions | 39 (80%) |
| ENSAT stage | |
| I | 3 |
| II | 25 |
| III | 4 |
| IV | 17 |
n
and 50-100 ng of genomic DNA. Denaturation at 95 C for 5 min was followed by 40 cycles (95 C/45sec, annealing at 53 C/30 sec and 72 C/45 sec) then final elongation at 72 C for 10 min. Both strands of the amplified products were directly sequenced on an automated sequencer (ABI 3700; Perkin- Elmer, Boston, MA). Nu- cleotides were numbered in accordance with the reference se- quence.
Mutation analysis of entire coding regions of AXIN1 was per- formed as previously described (11) for ACC of the previous co- hort presenting with an activated Wnt/B-catenin signaling path- way (no .= 8).
All sequence variants identified were confirmed by bidirection- al sequencing in two independent experiments.
Axin2 expression analysis
For the 49 ACC, expression data for AXIN2 was obtained from quantitative reverse transcription-PCR analysis. RT-qPCR was performed in duplicate, using Taq-Man (Applied Biosystems, Foster City, CA). Relative quantification of target cDNA was de- termined by calculating the difference in cross-threshold (CT) values after normalization to PPIA (CYCLO) signals (DDCT method).
RESULTS
Clinical, hormonal and histological characteristics of the patients are shown in Table 1.
AXIN2 genetic analysis
Among the 49 tumors sequenced for AXIN2 exon 8, a so- matic alteration was detected in only one. As shown in Figure 1, this alteration was an in-frame 12-bp heterozygous deletion [c2013_2024del12, now referenced as rs151279728 in the Ensembl database (http://www.ensembl.org)] result- ing in a deletion of codons 671 to 674 (R-T-T-P). Inter- estingly, the same AXIN2 heterozygous mutation was found in the patient’s germline DNA. In this ACC, the c2013_2024del12 variation was associated with a somatic
A
ACC cohort
H295
H295R
CTNNB1 mutation
and IHC
A
S
AXIN2 rs151279728
expression
4
AXIN2
-AA(Ct)
2
O
na
2
-4
B
ref.
AXIN2 rs151279728
*
*- L
H295
H295R
activating CTNNB1 mutation (pSer45del) and nuclear ß- catenin immunohistochemical staining was observed. The same AXIN2 genetic variant was also found, on a homo or hemizygous state, in addition to an activating CTNNB1 mu- tation (pSer45Pro) in the H295 and H295R cell lines. This patient was a 26-yr-old man, with secreting ACC (Weiss score of 7).
Additionally, the previously described single-nucleotide polymorphism rs35415678 (synonymous variation), was found with an allele frequency similar to those found in reference populations (http://www.ncbi.nlm.nih.gov/snp). No other variation was observed.
AXIN2 expression
As shown in Figure 1, regarding expression data from RT-qPCR analysis, we did not find any correlation be- tween AXIN2 expression and AXIN2 genetic variant. AX- IN2 expression level was not associated with abnormal nuclear ß-catenin staining. In tumors with ß-catenin nu- clear staining, as expected, increased AXIN2 expression was observed.
AXIN1 genetic analysis
In view of AXIN2 results, we therefore investigate the en- tire coding region of AXIN1 in the 8 ACC for which Wnt/B-catenin signaling pathway was activated based on B-catenin nuclear staining. No mutation for the entire coding region of AXIN1 was observed. Frequent single nucleotide polymorphisms (SNP) reported as non- pathogenic in the Ensembl database were found with a similar frequency to the one of the general population.
DISCUSSION
Wnt/B-catenin signaling pathway appears to be one of the promising field of research in both understanding and treatment of ACC. Nevertheless, in the homoge- neous group of ACC harboring Wnt/B-catenin signaling pathway activation, ß-catenin gene (CTNNB1) activating mutations are only found in about half of tumors. Since screening of alternative genetic defects, such as APC or WTX mutations has been disappointing (7), AXIN genes could represent interesting candidates. Indeed, AXIN genes are key components and regulator of the Wnt/B- catenin signaling pathway (12) and have previously been involved as tumor suppressor genes in other neoplasm including colorectal cancer (13), hepatocellular carcinoma (14), or melanoma (15, 16). Recently, AXIN2 alteration (8) has been described in adrenocortical tumors including ACC and ACA, suggesting its possible role in adreno- cortical tumorigenesis through Wnt/B-catenin signaling pathway activation.
In the present series, only one AXIN2 genetic alteration (c2013_2024del12) was found among the 49 (2%) ACC studied. We were able to assess the germline origin of this variant, finding the same heterozygous deletion in the patient’s germline DNA. It is important to note that no ACC familial history was present in this family. Inter- estingly, this alteration was the same as the one de- scribed by Chapman et col. (8), who did not get access to germline DNA. Additionally, this genetic variant was,
as also previously described by Chapman et al. (8), found in a tumor with an activating CTNNB1 mutation (pSer45del), sufficient by it-self to explain Wnt/B-catenin signaling pathway activation and nuclear ß-catenin stain- ing. If this germline variant has been already described in different types of tumor including familial melanoma (16), Pedace et al. latter demonstrated in familial melanoma that the specific c2013_2024del12 variant do not segre- gate with the disease and is consequently very unlikely to be involved in heritable predisposition (15). Finally, the c2013_2024del12 variant is now recorded as a polymor- phism according to recent genomic database update. In the Ensembl database (http://www.ensembl.org) the rs151279728 deletion is found with an overall allele fre- quency of 2%, and up to 9% in the African population, that is not known to be at risk for ACC. We also find the c2013_2024del12 in the H295 and H295R cell lines at a homo or hemizygous status. As for the expression data, no correlation between AXIN2 expression and this AX- IN2 genetic status was found, suggesting no major role in Wnt/B-catenin signaling pathway regulation in ACC. The sequence analysis of AXIN2 exon 8 revealed a previ- ously described SNP rs35415678 (L688L, CCT>CTT) found in 3 ACC (one homozygous for the minor allele, and two heterozygous). This SNP has been presented as a poten- tial risk factor for tooth agenesis (17) and was found with a higher frequency in colorectal cancer in Kashmiri popula- tion (18). Nevertheless, in this study, rs35415678 was found with allele frequencies similar to reference population ac- cording to Single Nucleotide Polymorphism database (db- SNP) (http://www.ncbi.nlm.nih.gov/SNP/). Additionally, this polymorphism was neither associated with nuclear ß- catenin staining or a specific AXIN2 expression, that over- all make unlikely its role in ACC ß-catenin-mediated tu- morigenesis.
Regarding AXIN1 gene, no genetic alteration was found in 8 ACC with a nuclear ß-catenin staining. AXIN1 is a multidomain scaffold protein implicated in the Wnt/ß- catenin signaling pathway but also in various signaling pathways including JNK, TGFB, p53 pathways. Its role in the Wnt/B-catenin signaling pathway activation is still un- clear. If some advocated its possible implication as a tu- mor suppressor gene (19) in relation with Wnt/B-catenin signaling pathway, others suggested that AXIN1 inacti- vating mutations are not equivalent to CTNNB1 gain of function, and could be related to a non-Wnt pathway (20). We are aware of some limitations of this study. First, on- ly AXIN2 exon 8 was studied, and AXIN1 mutations were only investigated in a small cohort based on the hy- pothesis of mutations involved in a Wnt/B-catenin path- way activation. Nevertheless, all AXIN2 mutations pre- viously described were found in exon 8 hot-spot (9), and AXIN1 alterations were investigated in a sub-group of patients with ACC harboring ß-catenin nuclear staining, i.e. with a maximum likelihood of Wnt/B-catenin path- way activation or related event. Indeed, abnormal nu- clear and/or cytoplasmic ß-catenin localization by im- munohistochemistry, accurately reflect the activation of the Wnt/B-catenin pathway. Nevertheless, abnormal ß- catenin localization as determined by immunohisto- chemistry can currently be explained by somatic ß- catenin activating mutation only in a quarter to a third
of ACC. This strongly suggests the existence of alterna- tive genetic defects. We previously explored the role of APC mutation in the subset of patients with abnormal ß-catenin localization as determined by immunohisto- chemistry and no ß-catenin mutation, and found that, at least in patients with Familial Adenomatous Polyposis (FAP), APC could be involved in adrenal cortex tumori- genesis through Wnt/B-catenin pathway activation. AX- IN 1 and AXIN 2 are expected to play a major role in Wnt/B-catenin pathway regulation as shown in several other tumors. We consequently decided to use the same methodology, i.e. only focused on patients with abnor- mal ß-catenin localization determined by immunohisto- chemistry and currently not explained by somatic ß- catenin activating mutations. Additionally, the limited number of patients studied, due to the rarity of the dis- ease and available biological material, do not allow a statistical analysis between genetic and clinical charac- teristics. Nevertheless, it is clear that a definitive answer will only come from functional studies.
Altogether these results strongly suggest that the AXIN2 germline variant c2013_2024del12 is likely to be a non- pathogenic polymorphism, and that AXIN genes muta- tions play a very little role in ACC development, and Wnt/ß-catenin signaling pathway activation. Clearer an- swers will definitely come from whole genome-exome sequencing of large ACC cohort, and additional func- tional studies.
ACKNOWLEDGMENTS
We thank Fernande René-Corail and Karine Perlemoine for excellent tech- nical assistance. We also thank for helpful discussions Rossela Libé, and Lionel Groussin. We thank Franck Letourneur (Plate-forme sequencage et génomique, of Cochin Institute) for help in sequencing. None of the authors has any conflict of interest.
Grants or fellowships supports
This work was supported in part by the Contrat d’Initiation à la Recherche Clinique (Grant CIRC 05045 - AP-HP), the Plan Hospitalier de Recherche Clinique (AOM06179) to the COMETE Network and the Recherche Trans- lationnelle DHOS/INCA 2009 (RTD09024).
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