DNA damage and growth hormone hypersecretion in pituitary somatotroph adenomas
Anat Ben-Shlomo, Nan Deng, Evelyn Ding, Masaaki Yamamoto, Adam Mamelak, Vera Chesnokova, Artak Labadzhyan, Shlomo Melmed
Anat Ben-Shlomo, Nan Deng, Evelyn Ding, Masaaki Yamamoto, Adam Mamelak, Vera Chesnokova, Artak Labadzhyan, Shlomo Melmed
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Research Article Endocrinology

DNA damage and growth hormone hypersecretion in pituitary somatotroph adenomas

Abstract

Drivers of sporadic benign pituitary adenoma growth are largely unknown. Whole-exome sequencing of 159 prospectively resected pituitary adenomas showed that somatic copy number alteration (SCNA) rather than mutation is a hallmark of hormone-secreting adenomas and that SCNAs correlate with adenoma phenotype. Using single-gene SCNA pathway analysis, we observed that both cAMP and Fanconi anemia DNA damage repair pathways were affected by SCNAs in growth hormone–secreting (GH-secreting) somatotroph adenomas. As somatotroph differentiation and GH secretion are dependent on cAMP activation and we previously showed DNA damage, aneuploidy, and senescence in somatotroph adenomas, we studied links between cAMP signaling and DNA damage. Stimulation of cAMP in C57BL/6 mouse primary pituitary cultures using forskolin or a long-acting GH-releasing hormone (GHRH) analog increased GH production and DNA damage measured by H2AX phosphorylation and a comet assay. Octreotide, a somatostatin receptor ligand that targets somatotroph adenoma GH secretion in patients with acromegaly, inhibited cAMP and GH and reversed DNA damage induction. In vivo long-acting GHRH treatment also induced pituitary DNA damage in mice. We conclude that cAMP, which induces somatotroph proliferation and GH secretion, may concomitantly induce DNA damage, potentially linking hormone hypersecretion to SCNA and genome instability. These results elucidating somatotroph adenoma pathophysiology identify pathways for targeted treatment.

Authors

Anat Ben-Shlomo, Nan Deng, Evelyn Ding, Masaaki Yamamoto, Adam Mamelak, Vera Chesnokova, Artak Labadzhyan, Shlomo Melmed

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Figure 8

CJC-1295 with and without octreotide actions in C57BL/6 normal mouse primary pituitary cultures.

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CJC-1295 with and without octreotide actions in C57BL/6 normal mouse pri...
(A) GH and (B) PRL concentrations in the supernatant after 16 hours of treatment with vehicle, 10 ng/mL CJC-1295 (CJC 10), cotreatment with 10 ng/mL CJC-1295 and 100 nM octreotide, 50 ng/mL CJC-1295 (CJC 50), or cotreatment with 50 ng/mL CJC-1295 and 100 nM octreotide, normalized to WST1. n = 7 per treatment group. Results are presented as the mean ± SEM. **P ≤ 0.01 and ***P ≤ 0.001 versus vehicle; ##P ≤ 0.01 versus the corresponding CJC-1295 dose; †††P ≤ 0.001 versus 10 ng/mL CJC-1295, by 2-tailed, unpaired t test with Bonferroni’s correction. (C) Western blot showing γH2AX expression compared with total H2AX expression after 16 hours of treatment as above in 3 different experiments: E1, E2, and E3. (D) Quantitative presentation of γH2AX change normalized to total H2AX derived from 2 wells per treatment group. Band intensities were calculated by ImageJ. Results are presented as the mean ± SEM. **P ≤ 0.01, by 2-tailed, unpaired t test with Bonferroni’s correction. (E) Comet assay results depicting Olive tail moment measured after 16 hours of treatment as above. The number of cells analyzed by a blinded observer is indicated. Results are presented as the mean ± SEM (vehicle, 12 ± 0.6; 10 ng/mL CJC-1295, 24.3 ± 1.2; 10 ng/mL CJC-1295 plus octreotide, 14.6 ± 0.9; 50 ng/mL CJC-1295, 33.1 ± 1.2; 50 ng/mL CJC-1295 plus octreotide, 14.8 ± 0.8). ***P ≤ 0.001 versus vehicle; ###P ≤ 0.001 versus the corresponding CJC-1295 dose; †††P ≤ 0.001 versus 50 ng/mL CJC-1295, by 2-tailed, unpaired t test with Bonferroni’s correction. (F) Comet assay results depicting Olive tail moment measured after 16 hours of treatment with 10 nM octreotide and 100 nM octreotide. The number of cells assessed by a blinded observer is indicated. Results are presented as the mean ± SEM (vehicle, 14.7 ± 0.8; 10 nM octreotide, 14.6 ± 0.7; 100 nM octreotide, 16.5 ± 0.8). The experiment was performed once.