DNA methylation analysis in malignant pheochromocytoma and paraganglioma

Toshihiro Oishi, Kazumi Iino, Yuta Okawa, Keisuke Kakizawa, Shoko Matsunari, Miho Yamashita, Terumi Taniguchi, Masato Maekawa, Takafumi Suda, Yutaka Oki, Toshihiro Oishi, Kazumi Iino, Yuta Okawa, Keisuke Kakizawa, Shoko Matsunari, Miho Yamashita, Terumi Taniguchi, Masato Maekawa, Takafumi Suda, Yutaka Oki

Abstract

Aims: In recent years, aberrant DNA methylation of specific CpG sites has been detected in many types of malignant tumors, and the epigenetic regulation of promoter CpG sites is considered an important mechanism underlying carcinogenesis. This study aimed to establish the epigenetics of the malignant transformation of malignant pheochromocytoma (PCC) and paraganglioma (PGL) by performing a methylation analysis.

Materials and methods: Based on the results of the Infinium HumanMethylation450 BeadChip array using DNA samples of PCC/PGL patients, candidate CpG sites that were hyper/hypo-methylated in metastatic tumors relative to those in the primary tumors of 2 patients with malignant PCC/PGL were selected. The methylation levels of the chosen candidate CpG sites were evaluated quantitatively.

Results: Twelve CpG sites were selected as hypermethylated candidates, and 16 CpG sites were selected as hypomethylated candidates. Using two quantitative methylation analysis methods, one hypermethylated site (cg02119938) and one hypomethylated site (cg26870725) remained as candidates. These sites were related to ACSBG1 (acyl-CoA synthetase bubblegum family member 1) and MAST1 (microtubule-associated serine-threonine kinase 1), respectively. Immunohistochemical studies of ACSBG1 and MAST1 revealed that epigenetic changes in the malignant transformation of PCC/PGL might be associated with ACSBG1 silencing or MAST1 overexpression.

Conclusions: Here, we report two noteworthy genes, ACSBG1 and MAST1; the aberrant promoter methylation/demethylation of these genes might be involved in their silencing/expression in malignant PCC/PGL. Further investigations are necessary to determine the role of ACSBG1 and/or MAST1 expression in malignant transformation and to establish pathological markers that can evaluate the malignant potential of PCC/PGL.

Keywords: ACSBG1; MAST1; Methylation; Paraganglioma; Pheochromocytoma.

Figures

Fig. 1
Fig. 1
Outline of the study design.
Fig. 2
Fig. 2
Methylation β values of the candidate CpG sites in three benign PCCs. Bar graphs represent Cases 10, 12 and 18, from left to right. The dotted line represents β value = 0.4. We identified hyper/hypo-methylated CpG candidates that were in opposite methylation status to those of benign tumors. We selected them with the β values of the benign tumors were lower/higher than 0.4 respectively. Underlining indicates the candidate CpG sites selected for further analysis.
Fig. 3
Fig. 3
Methylation analysis of candidate CpG sites using PTMR. B: Benign tumor, M: Metastatic lesion. unmethyl: unmethylated, methyl: methylated, PTMR: PCR following treatment with a methylation-dependent restriction enzyme.
Fig. 4
Fig. 4
Hematoxylin-eosin staining and immunohistochemical analysis of Chromogranin A, ACSBG1 and MAST1. Panel A shows the Hematoxylin-eosin staining of normal adrenal tissue. Panels B-M show the immunohistochemical staining of Chromogranin A (B), ACSBG1 (C, F–I) and MAST1 (D, E, J–M). Panels A–E show normal adrenals, and Panels F–K show benign tumors; Panels H and L show primary lesions of malignant tumors, and panels I and M show metastatic lesions. Normal adrenal medulla is shown at a higher magnification in panel E. The magnification of panels A–D is 200x. The magnification of panels E–M is 400x.

References

    1. Waguespack S.G., Rich T., Grubbs E., Ying A.K., Perrier N.D., Ayala-Ramirez M. A current review of the etiology, diagnosis, and treatment of pediatric pheochromocytoma and paraganglioma. J Clin Endocrinol Metab. 2010;95:2023–2037.
    1. Ayala-Ramirez M., Feng L., Habra M.A., Rich T., Dickson P.V., Perrier N. Clinical benefits of systemic chemotherapy for patients with metastatic pheochromocytomas or sympathetic extra-adrenal paragangliomas: insights from the largest single-institutional experience. Cancer. 2012;118:2804–2812.
    1. Edstrom Elder E., Hjelm Skog A.L., Hoog A., Hamberger B. The management of benign and malignant pheochromocytoma and abdominal paraganglioma. Eur J Surg Oncol. 2003;29:278–283.
    1. Huang H., Abraham J., Hung E., Averbuch S., Merino M., Steinberg S.M. Treatment of malignant pheochromocytoma/paraganglioma with cyclophosphamide, vincristine, and dacarbazine: recommendation from a 22-year follow-up of 18 patients. Cancer. 2008;113:2020–2028.
    1. Khorram-Manesh A., Ahlman H., Nilsson O., Friberg P., Oden A., Stenstrom G. Long-term outcome of a large series of patients surgically treated for pheochromocytoma. J Intern Med. 2005;258:55–66.
    1. Chen Y.A., Lemire M., Choufani S., Butcher D.T., Grafodatskaya D., Zanke B.W. Discovery of cross-reactive probes and polymorphic CpGs in the Illumina Infinium HumanMethylation450 microarray. Epigenetics. 2013;8:203–209.
    1. Shigematsu Y., Niwa T., Yamashita S., Taniguchi H., Kushima R., Katai H. Identification of a DNA methylation marker that detects the presence of lymph node metastases of gastric cancers. Oncol Lett. 2012;4:268–274.
    1. Cohen-Karni D., Xu D., Apone L., Fomenkov A., Sun Z., Davis P.J. The MspJI family of modification-dependent restriction endonucleases for epigenetic studies. Proc Natl Acad Sci USA. 2011;108:11040–11045.
    1. Zheng Y., Cohen-Karni D., Xu D., Chin H.G., Wilson G., Pradhan S. A unique family of Mrr-like modification-dependent restriction endonucleases. Nucleic Acids Res. 2010;38:5527–5534.
    1. Horton J.R., Mabuchi M.Y., Cohen-Karni D., Zhang X., Griggs R.M., Samaranayake M. Structure and cleavage activity of the tetrameric MspJI DNA modification-dependent restriction endonuclease. Nucleic Acids Res. 2012;40:9763–9773.
    1. Garland P., Quraishe S., French P., O'Connor V. Expression of the MAST family of serine/threonine kinases. Brain Res. 2008;1195:12–19.
    1. Baylin S.B., Jones P.A. A decade of exploring the cancer epigenome – biological and translational implications. Nat Rev Cancer. 2011;11:726–734.
    1. Jiang M., Zhang Y., Fei J., Chang X., Fan W., Qian X. Rapid quantification of DNA methylation by measuring relative peak heights in direct bisulfite-PCR sequencing traces. Lab Invest. 2010;90:282–290.
    1. Kanwal R., Gupta S. Epigenetic modifications in cancer. Clin Genet. 2012;81:303–311.
    1. Cui H. Loss of imprinting of IGF2 as an epigenetic marker for the risk of human cancer. Dis Markers. 2007;23:105–112.
    1. Fishbein L., Nathanson K.L. Pheochromocytoma and paraganglioma: understanding the complexities of the genetic background. Cancer Genet. 2012;205:1–11.
    1. Gimm O., DeMicco C., Perren A., Giammarile F., Walz M.K., Brunaud L. Malignant pheochromocytomas and paragangliomas: a diagnostic challenge. Langenbecks Arch Surg. 2012;397:155–177.
    1. Kolackov K., Tupikowski K., Bednarek-Tupikowska G. Genetic aspects of pheochromocytoma. Adv Clin Exp Med. 2012;21:821–829.
    1. Letouze E., Martinelli C., Loriot C., Burnichon N., Abermil N., Ottolenghi C. SDH mutations establish a hypermethylator phenotype in paraganglioma. Cancer Cell. 2013;23:739–752.
    1. de Cubas A.A., Korpershoek E., Inglada-Perez L., Letouze E., Curras-Freixes M., Fernandez A.F. DNA methylation profiling in pheochromocytoma and paraganglioma reveals diagnostic and prognostic markers. Clin Cancer Res. 2015;21:3020–3030.
    1. Richter S., Klink B., Nacke B., de Cubas A.A., Mangelis A., Rapizzi E. Epigenetic mutation of the succinate dehydrogenase C promoter in a patient with two paragangliomas. J Clin Endocrinol Metab. 2016;101:359–363.
    1. Bollati V., Schwartz J., Wright R., Litonjua A., Tarantini L., Suh H. Decline in genomic DNA methylation through aging in a cohort of elderly subjects. Mech Ageing Dev. 2009;130:234–239.
    1. Fraga M.F., Esteller M. Epigenetics and aging: the targets and the marks. Trends Genet. 2007;23:413–418.
    1. Conesa-Zamora P., Garcia-Solano J., Turpin Mdel C., Sebastian-Leon P., Torres-Moreno D., Estrada E. Methylome profiling reveals functions and genes which are differentially methylated in serrated compared to conventional colorectal carcinoma. Clin Epigenetics. 2015;7:101.
    1. Gerlinger M., Rowan A.J., Horswell S., Larkin J., Endesfelder D., Gronroos E. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med. 2012;366:883–892.
    1. Pribluda A., de la Cruz C.C., Jackson E.L. Intratumoral heterogeneity: from diversity comes resistance. Clin Cancer Res. 2015;21:2916–2923.
    1. Grunau C., Clark S.J., Rosenthal A. Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res. 2001;29 [E65-5]
    1. Mashek D.G., Li L.O., Coleman R.A. Long-chain acyl-CoA synthetases and fatty acid channeling. Future Lipidol. 2007;2:465–476.
    1. Ohkuni A., Ohno Y., Kihara A. Identification of acyl-CoA synthetases involved in the mammalian sphingosine 1-phosphate metabolic pathway. Biochem Biophys Res Commun. 2013;442:195–201.
    1. Wang X., Fredericksen Z.S., Vierkant R.A., Kosel M.L., Pankratz V.S., Cerhan J.R. Association of genetic variation in mitotic kinases with breast cancer risk. Breast Cancer Res Treat. 2010;119:453–462.
    1. Robinson D.R., Kalyana-Sundaram S., Wu Y.M., Shankar S., Cao X., Ateeq B. Functionally recurrent rearrangements of the MAST kinase and Notch gene families in breast cancer. Nat Med. 2011;17:1646–1651.

Source: PubMed

Sponsors et collaborateurs

Les conditions médicales

Interventions en matière de drogue

3
S'abonner