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A Novel TP53 Mutation Associated with TWIST1 and SIP1 Expression in an Aggressive Adrenocortical Carcinoma
Daniel Bulzico 1,2 D . Davi Coe Torres3 . Gerson Moura Ferreira 4 . Bruno Ricardo Barreto Pires4 . Paulo Antônio Silvestre de Faria 5 . Rocio Hassan3 .
Eliana Abdelhay4 · Mario Vaisman6 · Leonardo Vieira Neto 2,6
C Springer Science+Business Media New York 2017
Abstract Adrenocortical carcinomas (ACC) are very rare tu- mors related to TP53 mutations mostly in childhood onset cases. Epithelial-mesenchymal transition (EMT) transcription factors TWIST1 and Smad interacting protein 1 (SIP1) are related to poorer outcomes in other malignancies, but their role in ACC is unknown. We describe a case of an advanced metastatic ACC (Weiss-score of 9) in a patient at age 76. After primary tumor resection, mitotane therapy was started as pal- liation to low-volume liver metastasis. After a 2-year period of stable disease, the patient died due to brain metastasis. Somatic gene sequencing revealed a novel TP53 mutation in DNA extracted from paraffin-embedded tissue, a deletion of 8bp in exon 8 (c.811_818del8; GAGGTGCG/-) in homo or hemizygosis causing a subsequent frameshift and premature stop codon at position 302. Immunohistochemistry of P53 and p-Ser-15 P53 showed absent tumoral staining. In addition,
immunohistochemical analysis showed an increased expres- sion of the mesenchymal markers vimentin and fibronectin. At last, EMT transcription factors TWIST1 and SIP1 were also overexpressed in tumoral cells. This case report describes an aggressive ACC with not only a novel somatic mutation, but also a novel International Agency for Research on Cancer database 8 base-pair deletion in TP53 exon 8. In addition, the expression of EMT inducers TWIST1 and SIP1 have been reported for the first time in an ACC case. Further investiga- tion is needed to clarify the biologic significance of this new TP53 mutation and its role in the EMT process.
Keywords Adrenocortical carcinoma . TWIST1 . SIP1 . TP53 · Mutation
☒ Daniel Bulzico danielbulzico@gmail.com
1 Endocrine Oncology Unit, Brazilian National Cancer Institute-INCA, Praça da Cruz Vermelha, 23, 8th floor, Rio de Janeiro/RJ CEP 20.230-130, Brazil
2 Endocrinology Section, Federal Hospital of Lagoa, Rio de Janeiro, Brazil
3 Laboratory of Oncovirology, Center for Bone Marrow Transplants, Brazilian National Cancer Institute-INCA, Rio de Janeiro, Brazil
4 Stem Cell Laboratory, Center for Bone Marrow Transplants, Brazilian National Cancer Institute - INCA, Rio de Janeiro, Brazil
5 Division of Pathology, Brazilian National Cancer Institute - INCA, Rio de Janeiro, Brazil
6 Department of Internal Medicine and Endocrinology Section-Medical School and Clementino Fraga Filho University Hospital, Rio de Janeiro Federal University, Rio de Janeiro, Brazil
Introduction
Adrenocortical carcinoma (ACC) is an extremely rare malig- nancy accounting for 0.2% of all cancer deaths in the USA [1]. ACC patients generally harbor a poor prognosis since most cases are diagnosed with advanced disease and distant metas- tasis [2].
Genetic predisposition due to germline TP53 mutations has been related to around 50% of childhood onset ACC [3], but only to 3-6% of adult cases [4, 5]. Somatic TP53 mutations have also been described in 25-70% of adulthood small series [6, 7].
Epithelial-mesenchymal transition (EMT), a molecular- biological process in which epithelial-derived cells acquire mesenchymal characteristics, has been related to invasiveness, metastatic potential, and chemotherapy resistance in a variety of solid malignancies [8, 9]. Data upon the EMT process in ACC are scarce.
Here we describe an aggressive case of ACC, which har- bored a novel somatic TP53 mutation, associated to the EMT phenotype and a remarkably expression of transcriptional fac- tors TWIST1 and Smad Interacting Protein 1 (SIP1).
Case Description
In 2009, a 76-year-old female patient discovered a large mass in the right adrenal during the investigation of abdominal pain. No clinical findings of virilization and/or hypercortisolism were noticed. An abdominal computed tomography (CT) scan revealed a 14.6 × 12.6 × 9.5 cm right adrenal tumor with high spontaneous density (48 Hounsfield units). A metastatic 4.1 cm nodule in the liver was also described. Hypertension, diabetes, obesity, or previous cancer history were not noticed. There was also no family history of cancer.
In January 2010, she was submitted to a conventional ap- proach en bloc adrenalectomy (tumor, right kidney, and ureter resection), with incomplete resection of the liver metastasis (R2). The pathology study revealed a 26 × 22 × 8 cm highly pleomorphic ACC with a Weiss-score of 9 (Fig. 1). Liver, vascular, and soft-tissue invasion were evident, with no kid- ney invasion. After surgery, she was referred to our institution for follow-up. A 2-month post-operative abdominal magnetic resonance imaging (MRI) showed metastatic foci in IVa (5.0 × 5.0 cm) and VI (2.0 × 2.0 cm) liver segments. She started on mitotane therapy, titrated up to 4.5 g/day with good tolerance. Stabilization of disease was confirmed by subse- quent abdominal MRI. In January 2012, a 2.7 cm bone me- tastasis in D6 vertebra was diagnosed due to back pain and treated with palliative 20 Gy external beam radiotherapy. In September 2012, additional progression of disease was evi- dent with multiple subcentimeter lung metastases. Mitotane therapy was maintained until October 2013 when significant increase of vertebral and lung metastases were noticed. The patient was then referred to palliative care. In February 2014, she was hospitalized due to disorientation, dying a few days later after the confirmation of brain metastasis.
In 2016, pathology species were reviewed confirming the ACC diagnosis (Weiss index of 9). Ki-67 immunohistochem- istry (1:600, MIB-1-Dako, Glostrup, Denmark) revealed an index of 15% (Fig. 1i). EMT epithelial marker e-cadherin (1:1200, SPM471-Santa Cruz, Dallas, USA), and mesen- chymal markers vimentin (1:100, sc-7557-Santa Cruz, Dallas, USA) and fibronectin (1:100, P1H11-Santa Cruz, Dallas, USA) showed, respectively, negative, weak focal cy- toplasmic, and intense diffuse cytoplasmic immunohisto- chemical intensity staining patterns (Fig. 2a-c). Finally, im- munohistochemistry of EMT-transcription factors TWIST1 (1:50, Twist2Cla-Santa Cruz, Dallas, USA) and SIP1 (1:200, sc-48789-Santa Cruz, Dallas, USA) revealed a strong nuclear and cytoplasmic diffuse staining for both
(Fig. 2d-h). All immunohistochemistry reactions were per- formed applying the NovoLink Polymer Detection System (Leica, Newcastle Upon Tyne, UK) except for vimentin, which was performed with Dako LSAB System (Dako, Glostrup, Denmark). For epitope retrieval, a high- temperature technique with steamer and Trilogy buffer (Cell mark, Rocklin, USA) was employed in all cases. Positive controls were performed with different human breast cancer sections (TWIST1, SIP1, Ki-67, and p-Ser-15-P53); endome- trial carcinoma (latent P53); normal appendix (vimentin and fibronectin), and normal mammary ducts (e-cadherin). Negative controls were performed by omitting the application of primary antibody.
Genomic DNA was purified from 10 um thick sections of formalin-fixed paraffin embedded tissues from patient’s adre- nal tumor (two different fragments of the primary tumor) and normal kidney using the GeneReed DNA FFPE Kit (Qiagen, Hilden, Germany), according to manufacture’s instructions which included an enzymatic treatment with uracil-DNA- glycosylase to avoid production of cytosine deamination arti- facts. Mutation analysis of TP53 (exons 4-10, including splic- ing sites; RefSeq NM_000546.5) was performed by PCR am- plification and direct sequencing using an ABI PRISM 3130xl Genetic Analyzer (Applied, Foster City, USA). Sequences were compared with the corresponding germline RefSeq se- quence using BioEdit v7.2.5 and GLASS v0.2.9 softwares. Mutations were confirmed on both strands on independent PCR products and annotated by the International Agency for Research on Cancer (IARC) TP53 Mutation Database R18 (http://www-p53.iarc.fr). DNA sequencing revealed a novel tumoral somatic deletion of 8bp in exon 8 (c.811_818del8; GAGGTGCG/-) in homo or hemizygosis causing a subsequent frameshift and premature stop codon at position 302 (Fig. 2j). The mutation was not detected in the normal kidney tissue (Fig. 2l). Immunohistochemistry of P53 (1:100, DO-7-Dako, Glostrup, Denmark) and p-Ser-15 P53 (1:100, 16G8-Cell Signaling, Danvers, USA) showed absent tumor- al staining in both studies (Fig. 2f, i).
Discussion
Here we describe for the first time not only a novel somatic mutation in ACC, but also a novel TP53 mutation in the IARC P53 Mutation Database. TP53 is a tumor suppressor gene and the most frequent genetic impairment among human cancers [10]. The role of TP53 mutation in the oncogenesis of ACC has been extensively addressed, especially in the pediatric population where exon 10 R337H germline mutation is relat- ed to 50-80% of cases [3, 11]. Among adults, TP53 germline mutations are found in up to 6% of cases [4, 5], while IGF-2 overexpression (due to paternal isodisomy or loss of imprint- ing) is the most common genetic aberration [12, 13]. Barzon
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et al. [6] found TP53 somatic mutations in 7/10 adult onset ACC but immunohistochemical reactivity was observed in only one of the mutated cases [6]. Previous studies have re- ported the same phenomena in bladder cancer [14, 15]. We believe that the observed lack of protein immunohistochemi- cal reactivity [both to latent and Serine-15 phophorylated (active) P53 isoforms] is possibly related to a mutation driven unstable protein with conformational disarrangement and im- paired antigen-antibody reaction in some cases, and/or ubiquitination of an aberrant non-functional protein [15, 16].
EMT process is postulated as an essential step in cancer invasion and progression [8, 9]. Basic helix-loop-helix tran- scriptional factor TWIST1 and zinc-finger transcription factor SIP1 have been extensively described as EMT-inducing genes [17, 18]. By reducing the expression of adhesion molecules such as e-cadherin, and overexpressing mesenchymal proteins as vimentin and fibronectin, tumoral cells acquire migration, invasive, and apoptosis resistance properties [8, 9]. EMT has been described in different types of cancer from endodermal, mesodermal, and ectodermal derived organs [18-24]. However the role of EMT, as well as TWIST1 and SIP1 in
adrenocortical tumors has yet to be determined. In fact, using microarray gene expression methodology, Giordano et al. [25] originally found that TWIST1 was upregulated in ACC when compared to adrenocortical adenomas and normal adrenal tis- sue. However, to date, no study has assessed the expression of both TWIST1 and SIP1 at protein level in ACC.
Despite fragmented, a body of evidences point to the asso- ciation between EMT and ACT biological behavior. Some potential mechanisms related to the adrenal tumorigenesis are highlighted by transcriptome studies. Early microarray data as the aforementioned study by Giodano et al. [25] have described consistent impairment in different signaling path- ways implicated in the tumorigenesis of benign and malignant ACT. Among these, IGF-2 overexpression has consistently been reported as the most important mechanism underlying ACC pathogenesis [12, 13]. Data from the The Cancer Genome Atlas (TCGA ACC) has recently confirmed that IGF-2 expression was higher in 86% of ACC cases, than in benign tissues [13]. Additionally, the TCGA ACC study de- scribed that CTNNB1 mutations, and consequent Wnt/ß- catenin pathway disarrangement, are frequent events in both
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DNA sequence alignment of TP53 exon 8
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Tumor 1
TGGTAATCTA CTGGGACGGA ACAGCTTT-
TGTT TGTGCCTGTC
Tumor 2
TGGTAATCTA CTGGGACGGA ACAGCTTT —
Kidney wt
TGTT TGTGCCTGTC
TGGTAATCTA CTGGGACGGA ACAGCTTTGA GGTGCGTGTT TGTGCCTGTC
TGGTAATCTA CTGGGACGGA ACAGCTTTGA GGTGCGTGTT TGTGCCTGTC
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. 1
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Tumor 1
CTGGGAGAGA CCGGCGCACA GAGGAAGAGA ATCTCCGCAA GAAAGGGGAG
Tumor 2
CTGGGAGAGA CCGGCGCACA GAGGAAGAGA ATCTCCGCAA GAAAGGGGAG
Kidney wt
CTGGGAGAGA CCGGCGCACA GAGGAAGAGA ATCTCCGCAA GAAAGGGGAG
CTGGGAGAGA CCGGCGCACA GAGGAAGAGA ATCTCCGCAA GAAAGGGGAG
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Amino Acid Alignment of TP53 exon 8
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Tumor 1
CCTCACCACG AGCTGCCCCC AGGGAGCACT AAGCGAG
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45
Tumor 2
CCTCACCACG AGCTGCCCCC AGGGAGCACT AAGCGAG
Tumor
GNLLGRNSF- - CLCLSWERP AHRGRESPQE RGASPRAAPR EH*
wt
E VRV. ACPG. D RRTEE . NLRK K. EPHHEL. P GSTKR
Kidney wt
CCTCACCACG AGCTGCCCCC AGGGAGCACT AAGCGAG
CCTCACCACG AGCTGCCCCC AGGGAGCACT AAGCGAG
malignant and benign ACT [13], in line with previous studies [26-29]. Further studies have reported other genes and
cytoplasmic diffuse staining of EMT-transcription factor TWIST1 (20x)-breast cancer negative control at the upper right corner. h Strong nuclear and cytoplasmic diffuse staining of EMT-transcription factor SIP1 (20x)-breast cancer negative control at the upper right cor- ner. i Negative staining of Serine-15 phosphorylated P53 in tumor cells (×10)-breast cancer positive control at the upper right corner. j Sanger sequencing of somatic DNA extracted from two different tumoral frag- ments showing the 8bp deletion in exon 8 (c.811_818del8). k Amino acid sequence alignment of TP53 exon 8 depicting abnormal tumor sequence due to the 8bp deletion (p.E271CfsTer32), followed by precocious stop codon at position 302 (*). l TP53 exon 8 DNA sequence alignment comparing samples from two different tumor fragments and patient’s normal tissue (not invaded kidney) to the wild-type reference sequence
signaling pathways, such as PRKAR1A [13], Sonic-hedgehog (SHH) [30], Notch1 [31], and MEN-1 as potential drivers to
adrenal tumorigenesis. Interestingly, all mentioned genes or signaling pathways have been related to the activation of the EMT process in different types of cancer, although not by common mechanism [32-37]. Finally, mutant TP53 has been shown to induce EMT [38, 39]. In fact, Iwanicki et al. [40] recently demonstrated that mutant TP53 can induce increased tumoral survival and mobility through a dependent TWIST1 and fibronectin secretion mechanism.
In conclusion, this is the first description of a novel TP53 mutation associated to the EMT phenotype with TWIST1 and SIP1 protein overexpression in an aggressive case of ACC. In the light of the available evidences, the results from the pres- ent case report allow us to speculate on the association of this novel TP53 mutation with the observed EMT phenotype and overexpression of the EMT activators TWIST1 and SIP1. However, further studies are needed to elucidate the biologic effect of this novel TP53 mutation, as well as the role of EMT, TWIST1, and SIP1 in ACC tumorigenesis.
Compliance with Ethical Standards
Conflict of Interest The authors declare that they have no conflict of interest.
Funding This research did not receive any specific grant from any funding agency in the public, commercial, or not-for-profit sector.
Ethical Approval This study was approved by INCA’s independent institutional advisory committee in September 24th, 2014 (protocol 33847514.4.0000.5274). All procedures performed in the study involving the reported case were in accordance with the ethical standards of the institutional and national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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