INSIGHTS INTO THE NON-CODING GENOME OF PARATHYROID TUMORS

Recently, long non-coding RNAs (lncRNAs) have been implicated in the regulation of several physiological processes such as cell growth, differentiation and proliferation. Although lncRNAs functions in human diseases have not been completely disclosed, some lncRNAs have already been identified as prognostic and diagnostic biomarkers in different tumors. LncRNAs have also a crucial role in normal development of endocrine organs and their role in endocrine cancer pathogenesis is emerging. Parathyroid tumors are rare and heterogeneous diseases characterized by genetic and epigenetic alterations resulting in aberrant expression of both protein coding and non-coding genes. Tumors of the parathyroid glands show a great variability in clinical features such as parathormone (PTH) secretion, in the pattern of cell proliferation and in the genetic background. Mutations in the oncosuppressor CDC73 are key events in most carcinomas whereas alterations in the tumor suppressor Multiple Endocrine Neoplasia 1 (MEN1, located at 11q13.1) occur in up to a third of sporadic adenomas. Although lncRNAs play a regulatory role in endocrine cancer pathogenesis, a lncRNAs profiling in human parathyroid tumors is missing. Therefore, we investigated known lncRNAs expression in a series of normal (PaN) and pathological (adenomatous, PAd, and carcinomatous, PCa) parathyroid glands and correlated their expression with cytogenetic aberration, CDC73 status and MEN1 level. Then, we modulated epigenetic complexes and MEN1 expression to get preliminary insights into HAR1B ncRNA functions in parathyroid pathobiology. We found that a nine-lncRNAs signature distinguishes normal parathyroid glands from parathyroid tumors and also more aggressive tumors from indolent ones. Specifically, KCNQ1OT1 and SNHG6 are enriched in PaNs, HAR1B, HOXA3as, MEG3 and NEAT1 expression characterize PAds, whereas BC200 and HOXA6as are significantly upregulated in PCas compared to PaNs. When this signature was analyzed in a second set of samples, that included also atypical PAds (aPAds), unsupervised analysis showed 3 main samples clusters in which PaNs, all grouped in cluster 2, are distinctly separated from PCas and aPAds, both in cluster 1 and 3. Notably, samples with lncRNAs overexpression, grouped in cluster 3, are characterized by a lower age of tumor onset. Of note, validation analysis highlights a different lncRNAs expression pattern in PCas according to CDC73 gene mutation status. Specifically, CDC73-mutated carcinomas are grouped in cluster 3 and overexpress the majority of the lncRNAs. Moreover, CDC73-mutated PCas display an increment in PTH, calcium serum and ionized calcium levels. Conversely, lncRNAs expression in adenomas is highly heterogeneous. Therefore, we further characterized this tumor type by performing array Comparative Genomic Hybridization (aCGH). This analysis revealed that chromosome 11 loss of heterozygosity (Chr11-LOH) is the main chromosomal aberration among PAds (10 out of 24 PAds; 42%). Interestingly, Chr11-LOH is associated to a significative HAR1B upregulation. Based on these observations, we analyzed possible regulators of HAR1B expression and we found that HAR1B promoter is a target of EZH2, the catalytic subunit of the Polycomb Repressive Complex 2. Selective inhibition of EZH2 with Tazemetostat in HEK293 cells restored HAR1B expression levels. In line with this, PAds with Chr-11 LOH show a downregulation of H3K27me3, the direct target of EZH2 activity, compared to PAds without Chr11-LOH. Moreover, HAR1B levels increase after silencing of MEN1 in primary parathyroid adenoma cultures. Overall, these data shed light on lncRNAs deregulation in human parathyroid tumors and suggest an important role of HAR1B in parathyroid pathobiology.