Yes-Associated Protein 1 Is a Novel Calcium Sensing Receptor Target in Human Parathyroid Tumors

Abstract

The Hippo pathway is involved in human tumorigenesis and tissue repair. Here, we investigated the Hippo coactivator Yes-associated protein 1 (YAP1) and the kinase large tumor suppressor 1/2 (LATS1/2) in tumors of the parathyroid glands, which are almost invariably associated with primary hyperparathyroidism. Compared with normal parathyroid glands, parathyroid adenomas (PAds) and carcinomas show variably but reduced nuclear YAP1 expression. The kinase LATS1/2, which phosphorylates YAP1 thus promoting its degradation, was also variably reduced in PAds. Further, YAP1 silencing reduces the expression of the key parathyroid oncosuppressor multiple endocrine neoplasia type 1(MEN1), while MEN1 silencing increases YAP1 expression. Treatment of patient-derived PAds-primary cell cultures and Human embryonic kidney 293A (HEK293A) cells expressing the calcium-sensing receptor (CASR) with the CASR agonist R568 induces YAP1 nuclear accumulation. This effect was prevented by the incubation of the cells with RhoA/Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitors Y27632 and H1152. Lastly, CASR activation increased the expression of the YAP1 gene targets CYR61, CTGF, and WNT5A, and this effect was blunted by YAP1 silencing. Concluding, here we provide preliminary evidence of the involvement of the Hippo pathway in human tumor parathyroid cells and of the existence of a CASR-ROCK-YAP1 axis. We propose a tumor suppressor role for YAP1 and LATS1/2 in parathyroid tumors.

The Core Stem Genes SOX2, POU5F1/OCT4, and NANOG Are Expressed in Human Parathyroid Tumors and Modulated by MEN1, YAP1, and β-catenin Pathways Activation

“…The transcriptional co-activator YAP1 is involved in the modulation of core stem genes [19][20][21]. A recent report showed that YAP1 accumulation at the nuclear level is induced by calcium-sensing receptor (CASR) activation in PAds-derived cells [18]. In PAds-derived cells (n = 3), R568-mediated CASR activation increased SOX2 mRNA levels, while the stimulatory effect was abolished by YAP1 silencing (Figure 2F,G).…”

“…In PAdsderived primary cell cultures, CASR activation by the selective agonist R568 increased the expression of SOX2, while POU5F1/OCT4 and NANOG were unaffected. Reduction of YAP1 nuclear accumulation by silencing YAP1 [18] abolished the stimulatory effect of CASR activation on SOX2 expression levels, thus suggesting that it is mediated by YAP1 signaling activation. Considering that both CASR and YAP1 feature as tumor suppressors in parathyroid adenomas, the induction of SOX2 expression may promote cell resting more than cell proliferation [36].…”

“…We further investigated regulation of stem core genes expression in sporadic PAds. To this aim, we tested the hypothesis that regulatory molecules critical in parathyroid tumorigenesis, namely menin, encoded by the MEN1 gene [30], calcium sensing receptor (CASR) [31], YAP1 [18], and WNT/β-catenin [6], may be involved in modulation of the stem core genes. Chromosome 11 loss of heterozygosity (LOH) can be detected in at least 40% of parathyroid tumors [32] and MEN1, which maps on chromosome 11, is an important oncosuppressor involved in parathyroid tumorigenesis [33].…”

“…Recently, it has been reported that CASR activation induces nuclear yes-associated protein 1 (YAP1) accumulation and transcription of YAP1 target genes in PAds-derived cells [18]. The transcription factor YAP1, a major effector of the tumor suppressive Hippo signaling pathway, is necessary to maintain pluripotency in embryonic stem cells.…”

“…Moreover, we hypothesized that the known tumor suppressor menin, encoded by the MEN1 gene, whose expression is lost in multiple endocrine neoplasia (MEN1)-related parathyroid tumors [17], may be involved in the modulation of the core stem genes. Similarly, we investigated the potential role of the Hippo pathway-related YAP1 co-factor, whose nuclear expression has been recently found to be downregulated in parathyroid cancers [18], and the role of the WNT/β-catenin signaling. YAP1 [19][20][21] and active β-catenin [22] have been reported to modulate the expression of the core stem cell genes.…”