Abstract 5279: Molecular analysis of AMBRA1 as a candidate tumor suppressor in sporadic parathyroid adenomas

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Abstract Primary hyperparathyroidism is a common endocrine disorder that is most often caused by a sporadic single-gland parathyroid adenoma. Currently, the only known and experimentally validated oncoprotein for parathyroid adenomas is cyclin D1. Overexpression of cyclin D1 has been reported in 20-40% of cases; about 8 percent of cases harbor a DNA rearrangement involving the cyclin D1 (CCND1) locus. Other molecular mechanisms underlying this cyclin D1 overexpression have yet to be identified. Here, we explored a potential parathyroid tumorigenic mechanism that could increase cyclin D1 stability through a defect in the AMBRA1 E3 ligase adaptor, a molecule responsible for its degradation. Evidence that AMBRA1 may be a tumor suppressor gene includes: accumulation of cyclin D with hyperproliferation in AMBRA1-deficient cells, context-dependent growth of tumors in Ambra1-deficient mice, and the correlation between low levels of AMBRA1 and poor clinical outcomes in The Cancer Genome Atlas. Therefore, we examined AMBRA1 for evidence of tumor suppressor-type inactivation in a cohort of 98 typical parathyroid adenomas. Tumor genomic DNA was subjected to PCR-amplification of all 18 coding exons of AMBRA1, followed by Sanger sequencing. Sequences were analyzed by comparison to the normal reference sequence (ENST00000683756.1), seeking to assess any observed intragenic or splice-site variants as potential or likely loss-of-function mutations. Thus far, with 97 percent of the coding region for this 1298-amino acid protein fully sequenced, we have identified an inactivating mutation in 1 of 98 tumors (1%): c.126G>A, resulting in an early stop codon p.Trp42. This mutation showed loss of heterozygosity and was confirmed to be somatic by sequencing of the patient’s matched germline DNA. We also identified likely non-pathogenic variants in 5 tumors: missense variant c.3385G>T (p.Ala1129Ser), 3’ UTR variants c.*576A>T and c.*491T>C and synonymous variants c.579C>T and c.2776C>T, each in a single tumor. 4 of 5 were found as germline variants in the normal population and the one missense scored as benign by in silico criteria. To conclude, our observations suggest that AMBRA1 may function as a classical tumor suppressor gene in sporadic parathyroid adenomas at very low frequency. Further study may include protein expression analysis of AMBRA1 to investigate influences on gene expression. To further elucidate the mechanisms behind cyclin D1 overexpression in parathyroid adenomas, other molecules that participate in its degradation should be explored as potential tumor suppressors. Citation Format: Stephanie Chinwo, Justin Bellizzi, Jessica Costa-Guda, Andrew Arnold. Molecular analysis of AMBRA1 as a candidate tumor suppressor in sporadic parathyroid adenomas. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5279.