Global methylation-demethylation status in pituitary neuroendocrine tumors as potential therapeutic target

Borbála Szabó; Kinga Németh; Katalin Mészáros; Nikolette Szücs; Sándor Czirják; Lilla Reiniger; Attila Patócs; Henriett Butz

doi:10.1530/endoabs.70.AEP730

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Endocrine Abstracts (2020) 70 AEP730 | DOI: 10.1530/endoabs.70.AEP730

ECE2020 Audio ePoster Presentations Pituitary and Neuroendocrinology (217 abstracts)

Global methylation-demethylation status in pituitary neuroendocrine tumors as potential therapeutic target

Borbála Szabó ¹ , Kinga Németh ² , Katalin Mészáros ^2,3 , Nikolette Szücs ¹ , Sándor Czirják ⁴ , Lilla Reiniger ⁵ , Attila Patócs ^2,3,6 & Henriett Butz ^2,3,6

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Author affiliations

¹Semmelweis University, 2nd Department of Internal Medicine, Faculty of Medicine, Budapest, Hungary; ²Semmelweis University, Momentum Hereditary Endocrine Tumors Research Group, Budapest, Hungary; ³Semmelweis University, National Bionics Program, Budapest, Hungary; ⁴National Institute of Clinical Neurosciences, Budapest, Hungary; ⁵Semmelweis University, 1st Department of Pathology and Experimental Cancer Research, Budapest, Hungary; ⁶Semmelweis University, Department of Laboratory Medicine, Budapest, Hungary

Background: The altered DNA methylation of certain genes in Pituitary Neuroendocrine Tumors (PitNETs) are well known. However little information is available regarding global methylation changes and the process of demethylation in these tumors. In addition, influencing global methylation-demethylation could be a potential new therapeutic option especially in clinically non-functional PitNETs.

Material and Methods: Overall, 44 fresh frozen pituitary adenoma tissues (29 gonadotroph, 12 somatotroph, 3 corticotroph) were collected and characterized according to the 2017 WHO classification. Decitabine was used to alter global methylation-demethylation status on in vitro GH3 and RC-4B/B cell lines. In tissue samples 5-hydroxymethylcytisone (5 hmC), UHRF1-2 protein and Ki-67 were assessed by immunohistochemistry; gene expression of DNA Methyl-transferase (DNMT1), methyl-cytosine dioxygenases (TET1-3) and ubiquitin-like with PHD and ring finger domain (UHRF1-2) were investigated by RT-qPCR. 5-methylcytosine (5 mC) and 5 hmC level were determined by HPLC-MS/MS method.

Results: Decitabine decreased 5-methylcytosine (5 mC) and increased 5 hmC levels in vitro in both pituitary cell lines. Parallel, cell proliferation and viability were decreased significantly. UHRF1-2 were also altered upon decitabine treatment in vitro. Interestingly, in PitNET tissue samples 5 hmC was gradually decreased in samples with higher Ki-67 index. In samples with different histology UHRF2 showed different expression, while UHRF1 showed gradual increase in adenoma samples with higher Ki-67 index. Additionally, UHRF2 positively correlated with 5 hmC level in pitNET tissues and both UHRF1 and UHRF2 showed significant positive correlation with DNMT1 and TET1-3 expression.

Conclusion: Our results showed that methylation-demethylation process (5 hmC, DNMT1, TET1-3 and UHRF1-2) is closely linked to proliferative behaviour of PitNETs. Altering global 5 mC and 5 hmC level can be a potential, new therapeutic target in therapy resistant pituitary tumors.

Grants and financial support: This work has been funded by the National Program of Bionics (Program Medical Bionics lead by Attila Patócs) and Semmelweis Innovation Found (STIA_19) to Henriett Butz. Henriett Butz is a recipient of Bolyai Research Fellowship of Hungarian Academy of Sciences and ÚNKP-18-4-SE-8 New National Excellence Program of The Ministry of Human Capacities.