CDK9 and cyclin regulators in Medullary Thyroid Cancer

项目摘要/摘要 甲状腺髓样癌(MTC)是一种进展性的、无法治愈的疾病，其10年生存率为40%。 转移性病例。遗传性MTC是由RET基因的致癌突变引起的。治疗方案的选择 进展性MTC非常有限，并且对Vandetanib等疗法产生耐药性。因此，它是至关重要的 探索针对Ret突变和非突变病例的新疗法，包括那些对 目前的治疗方法。散发性形式可由RET的体细胞突变引起，但60%的散发性MTC 不要怀有RET突变。小鼠模型显示，MTC可以在失去负调控因子后发生 CDK/Rb通路如Rb、p16、p18、p27等。人类MTC样本的IHC显示细胞核增加 Rb水平和阵列CGH数据显示p18和E2F2基因座的杂合性丢失，包括那些 而且没有RET突变。这些发现表明CDK/Rb通路的激活在功能上与糖尿病相关。 人类MTC。我们的实验室已经使用了CDK 1、2、5和9的抑制剂diaciclib来靶向CDK/Rb通路。 临床发现表达Ret突变体(C634W和M918T)的MTC细胞系(TT和MZ-CRC-1)。结果 在低浓度下，细胞活力和增殖显著降低，且呈剂量和时间依赖性。 浓度(IC50：TT为5 nm，MZ为30 nm)。对这种治疗更敏感的TT细胞系， 缺乏p18的表达。对对Vandetanib耐药的MTC株的处理也导致存活率下降。 PARP裂解证实了细胞凋亡的激活。CDK1、CDK2、CDK5表达稳定； 然而，CDK9mRNA和蛋白的表达在细胞存活率降低的剂量下显著降低。 受损并激活了细胞凋亡。此外，Ret水平也随着CDK9的丢失而降低， 这表明它们可能有关联。然后我们对人体样本中的CDK9进行了评估，发现CDK9出现的频率很高 CDK9基因扩增(34%)和IHC CDK9染色增加(79%)在包括RET在内的MTC中 基因突变。CDK9基因转录的预测调控因子，如STAT3和NFkB亚基 据报道，在人类MTC中上调，并被Ret信号激活。因此，我们假设 CDK9在MTC及其对Daciclib和p18水平预测反应中具有重要的机械作用 MTC对CDK抑制剂的反应。我们的目标是1)确定Ret介导的增强型CDK9 表达是MTC细胞对地奈西利敏感的机制，并决定CDK9水平升高或 基因扩增预测侵袭性MTC肿瘤行为，2)确定地奈西利对肿瘤侵袭性的影响 3)确定p18缺失在细胞对药物敏感性中的作用。 CDK抑制剂治疗。这项研究的长期目标是揭示新的治疗靶点和预测因素。 进展性MTC的反应，包括对Vandetanib耐药肿瘤。

PROJECT SUMMARY/ABSTRACT Medullary Thyroid Cancer (MTC) is a progressive, incurable disease with a 40% 10-year survival rate for metastatic cases. Hereditary MTC is caused by oncogenic mutations in the RET gene. Treatment options for progressive MTC are very limited and resistance develops to therapies such as Vandetanib. Thus, it is crucial to explore new therapies for Ret mutant and non-mutant cases, including those who develop resistance to current treatments. Sporadic forms can be caused by somatic mutations in RET, but 60% of sporadic MTCs do not harbor RET mutations. Mouse models show that MTC can arise after loss of negative regulators of the CDK/RB pathway such as Rb, p16, p18, and p27. IHC of human MTC samples reveal increased nuclear levels of RB, and array CGH data show loss of heterozygosity at the p18 and E2F2 loci, including those with and without RET mutations. These findings indicate that CDK/RB pathway activation is functionally relevant in human MTC. Our lab has used Dinaciclib, an inhibitor of CDK 1, 2, 5, and 9, to target the CDK/RB pathway in MTC cell lines (TT and MZ-CRC-1) expressing Ret mutants (C634W and M918T) seen with clinically. Results showed a remarkable reduction in cell viability and proliferation in a dose and time-dependent manner at low concentrations (IC50: 5nM for TT and 30nM for MZ). The TT cell line, which is more sensitive to the treatment, lacks p18 expression. Treatment of Vandetanib-resistant MTC lines also resulted in decreased viability. Activation of apoptosis was evidenced by PARP cleavage. Expression of CDK 1, 2, and 5 was stable; however, CDK9 mRNA and protein expression remarkably decreased at doses where cell viability was compromised and apoptosis activated. In addition, Ret levels were also reduced in concert with loss of CDK9, suggesting they may be linked. We then evaluated CDK9 in human samples and discovered a high frequency of CDK9 gene amplification (34%) and increased IHC CDK9 staining (79%) in MTC, including those with RET gene mutations. Predicted regulators of CDK9 gene transcription such as Stat3 and NFkB subunits are reported to be upregulated in human MTC and are activated by Ret signaling. We therefore hypothesize that CDK9 is mechanistically important in MTC and in its response to Dinaciclib and that p18 levels predict response to CDK inhibitors in MTC. Our aims are 1) To determine if Ret-mediated enhanced CDK9 expression is the mechanism for MTC cell sensitivity to Dinaciclib and determine if increased CDK9 levels or gene amplification predict aggressive MTC tumor behavior, 2) to determine the effect of Dinaciclib in Vandetanib-resistant progressive MTC in-vivo, and 3) to determine the role of p18 loss in cell sensitivity to CDK inhibitor treatment. The long term goal of this research is to reveal new therapeutic targets and predictors of response for progressive MTC, including Vandetanib-resistant tumors.