CDK9 and cyclin regulators in Medullary Thyroid Cancer

项目总结/摘要 甲状腺髓样癌（MTC）是一种进行性的、无法治愈的疾病， 转移病例。遗传性MTC是由RET基因的致癌突变引起的。的治疗选择 进行性MTC非常有限，并且对诸如凡德他尼的疗法产生耐药性。因此， 探索针对Ret突变和非突变病例的新疗法，包括对 目前的治疗。散发性形式可由RET的体细胞突变引起，但60%的散发性MTCs 不携带RET突变。小鼠模型表明，在失去了肿瘤细胞的负调节因子后， CDK/RB通路，如Rb、p16、p18和p27。人类MTC样本的IHC显示增加的核 RB水平和阵列CGH数据显示p18和E2 F2基因座杂合性缺失，包括那些 没有RET突变。这些发现表明，CDK/RB通路激活在功能上与细胞凋亡相关。 人MTC。我们的实验室已经使用Dinaciclib，一种CDK 1，2，5和9的抑制剂，靶向CDK/RB通路， MTC细胞系（TT和MZ-CRC-1）表达临床观察到的Ret突变体（C634 W和M918 T）。结果 显示出在低浓度下以剂量和时间依赖性方式显著降低细胞活力和增殖。 浓度（IC 50：TT为5 nM，MZ为30 nM）。TT细胞系对治疗更敏感， 缺乏p18表达。对凡德他尼耐药的MTC系的处理也导致活力降低。 通过PARP裂解证实了细胞凋亡的激活。CDK 1、2、5表达稳定; 然而，CDK 9 mRNA和蛋白表达在细胞活力降低的剂量下显著降低， 受损并激活凋亡。此外，Ret水平也随着CDK 9的丢失而降低， 这表明它们可能有联系然后，我们评估了人类样本中的CDK 9， MTC中CDK 9基因扩增（34%）和IHC CDK 9染色增加（79%），包括RET患者 基因突变CDK 9基因转录的预测调节因子如Stat 3和NFkB亚基是 据报道在人MTC中上调并被Ret信号转导激活。因此，我们假设， CDK 9在MTC及其对Dinaciclib的反应中具有机制重要性，并且p18水平可预测 对MTC中CDK抑制剂的反应。我们的目的是：1）确定Ret介导的增强CDK 9 表达是MTC细胞对Dinaciclib敏感的机制，并确定是否增加CDK 9水平或 基因扩增预测侵袭性MTC肿瘤行为，2）确定Dinaciclib在 体内抗凡德他尼的进行性MTC，和3）确定p18损失在细胞对 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子 对进展性MTC的反应，包括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.