Joshi P P, Kulkarni M V, Yu B K, Smith K R, Norton D L, van Veelen W, Höppener J W M, Franklin D S
Department of Biological Sciences, Purdue Cancer Center, Purdue University, West Lafayette, IN, USA.
Oncogene. 2007 Jan 25;26(4):554-70. doi: 10.1038/sj.onc.1209811. Epub 2006 Sep 4.
Multiple endocrine neoplasia type 2A (MEN2A) is predisposed by mutations in the RET proto-oncogene. Low expression of the cyclin-dependent kinase inhibitor (CDKI) p27(Kip1) is present in thyroid tumors, and recent evidence demonstrates p27 downregulation by the active RET mutant, RET/PTC1, found in papillary thyroid carcinoma. This implicates decreased p27 activity as an important event during thyroid tumorigenesis. However, p27(-/-) mice develop MEN-like tumors only in combination with loss of another CDKI, p18(Ink4c). This suggests that p18 and p27 functionally collaborate in suppression of tumorigenesis, that loss of both is critical in the development of MEN tumors and that both p18 and p27 are regulated by RET. We report that induction of the constitutively active MEN2A-specific RET mutant, RET2A(C634R), correlates with reduced p18/p27, and elevated cyclin D protein levels, leading to increased CDK activity, increased pRb phosphorylation and proliferation under growth arrest conditions. Mechanistically, RET2A represses p18/p27 mRNA levels while elevating cyclin D1 mRNA levels. RET2A expression also correlates with decreased p27 protein stability. RET2A-mediated regulation of p18 and p27, but not of cyclins D1 and D2, requires functional mitogen-activated protein kinase signaling. Additionally, RET2A-dependent p18 repression is required and sufficient to increase cell proliferation. Perhaps most significantly, MEN2A adrenal tumors also display these changes in cell cycle expression profile, demonstrating the biological relevance of our cell culture studies. Our results demonstrate for the first time that RET2A regulates p18, and suggest that loss of not only p27 but also of p18 expression is a key step in MEN tumorigenesis.
2A型多发性内分泌腺瘤（MEN2A）由RET原癌基因突变引发。细胞周期蛋白依赖性激酶抑制剂（CDKI）p27（Kip1）在甲状腺肿瘤中表达较低，最近有证据表明，甲状腺乳头状癌中发现的活性RET突变体RET/PTC1可下调p27。这表明p27活性降低是甲状腺肿瘤发生过程中的一个重要事件。然而，p27基因敲除小鼠仅在与另一种CDKI p18（Ink4c）缺失共同作用时才会发生类MEN肿瘤。这表明p18和p27在抑制肿瘤发生方面存在功能协同作用，二者缺失对MEN肿瘤的发生至关重要，且p18和p27均受RET调控。我们报告称，组成型活性MEN2A特异性RET突变体RET2A（C634R）的诱导与p18/p27水平降低、细胞周期蛋白D蛋白水平升高相关，导致细胞周期蛋白依赖性激酶（CDK）活性增加、视网膜母细胞瘤蛋白（pRb）磷酸化增加以及在生长停滞条件下细胞增殖增加。从机制上讲，RET2A可抑制p18/p27 mRNA水平，同时提高细胞周期蛋白D1 mRNA水平。RET2A的表达还与p27蛋白稳定性降低相关。RET2A介导的对p18和p27的调控，但不是对细胞周期蛋白D1和D2的调控，需要功能性丝裂原活化蛋白激酶信号传导。此外，RET2A依赖性的p18抑制对于增加细胞增殖是必需的且足够的。也许最显著的是，MEN2A肾上腺肿瘤在细胞周期表达谱中也显示出这些变化，证明了我们细胞培养研究的生物学相关性。我们的结果首次证明RET2A可调节p18，并表明不仅p27表达缺失，p18表达缺失也是MEN肿瘤发生的关键步骤。
