Upstream regulation of TAZ and YAP in sarcomas: Towards combinatorial therapytargeting the Hippo pathway

肉瘤是发生在骨和软组织中的癌症，是最难治疗的癌症之一。 保肢治疗降低了毁容截肢的发生率，尽管许多肉瘤涉及 关键的解剖结构排除了这种方法。此外，转移性肉瘤的五年存活率 只有16%，这表明需要新的治疗靶点。河马途径是一条高度保守的 丝氨酸/苏氨酸激酶级联负性调节TAZ和YAP转录共激活因子。Taz 和YAP已成为许多癌症的重要癌基因，包括乳腺癌、结肠癌、肝癌、肺癌、 胰腺癌和甲状腺癌。我们已经证明TAZ/YAP在大多数肉瘤中被激活(66%)， 然而，对TAZ和YAP的上游调控机制知之甚少，存在着显著的差距 在知识上。我们在这项提案中的目标是剖析激活TAZ的信号转导级联 还有肉瘤里的YAP。我们的中心假设是核心调控区域的组蛋白去乙酰化 河马蛋白和MAP4蛋白与PI3蛋白一起激活TAZ/YAP。这 这一假说得到了一些研究的支持，这些研究表明河马蛋白激酶的表达缺失在这两种疾病中都很常见 肉瘤临床标本和细胞系以及药理学研究表明，组蛋白的抑制作用 脱乙酰酶(HDAC)导致河马蛋白激酶的重组，从而抑制TAZ/YAP。一组平行的 利用基因工程和异种移植的小鼠模型和肉瘤细胞系的研究表明 TAZ和YAP在未分化的多形性肉瘤和儿童中不同地介导PI3激酶信号 横纹肌肉瘤。我们计划用以下具体目标来检验我们的中心假设： 目的1)明确TAZ和YAP在PI3激酶介导的肉瘤发生中的不同作用。 我们将在野生型、TAZ基因敲除、YAP的横纹肌肉瘤细胞系中刺激PI3K信号转导 和TAZ/YAP基因敲除，并确定TAZ和YAP对PI3激酶的不同贡献 转录组采用RNA-Seq基因表达分析。我们还将刺激肌肉中的PI3K信号 肉瘤易感小鼠Trp53fl/flPtenfl/fl和Tazfl/flYapfl/fl、Tazfl/fl、Yapfl/fl或Taz/Yap野生型和 比较肿瘤潜伏期和基因表达谱。 目的2)确定MST1/2和MAP4的组蛋白去乙酰化是否激活了TAZ和YAP。 我们将抑制HDAC并评估H3K27ac在MST1/2和H3K27ac启动子/增强子区的占有率 横纹肌肉瘤细胞系中MAP4蛋白的表达要测试MM激酶在 体内肿瘤进展，我们将删除编码LATS1/2激酶(位于MM下游)的基因 在有肉瘤倾向的小鼠中)。为了实现这一点，我们将给Trp53fl/fl小鼠注射Lats1fl/flLats2fl/fl或 肌肉注射腺病毒的Lats1/Lats2野生型。 目的3)确定抑制HDACs和PI3K-Akt轴是否能减少细胞的增殖和转移。 我们将确定组蛋白脱乙酰酶和PI3激酶途径的联合抑制是否会失活 塔兹/耶普。这将在体外通过增殖和细胞周期分析进行测试。组合的效果 对自发转移瘤的治疗将在肉瘤异种移植小鼠模型中进行测试。 这项提议是创新的，因为它针对的是上游两条尚未完全理解的平行通道 TAZ/YAP，将该领域的重点扩大到针对TAZ/YAP本身，从而解决了 在知识上有很大的差距。该项目意义重大，因为它确定了上游调控途径 TAZ/YAP可被药物抑制，提供了一种使TAZ成为治疗靶点的机制 YAP在临床上对肉瘤和潜在的其他癌症是可行的。

Sarcomas are cancers arising within bone and soft tissue which rank among the most difficult cancers to treat. Limb-salvage therapy has reduced the incidence of disfiguring amputations, however many sarcomas involve crucial anatomical structures precluding this approach. Additionally, five year survival for metastatic sarcomas is only 16%, underscoring the need for new therapeutic targets. The Hippo pathway is a highly conserved serine/threonine kinase cascade which negatively regulates the TAZ and YAP transcriptional coactivators. TAZ and YAP have emerged as important oncogenes in a number of cancers including breast, colon, liver, lung, pancreas, and thyroid cancers. We have shown that TAZ/YAP are activated in the majority of sarcomas (66%), however upstream mechanisms regulating TAZ and YAP are poorly understood, representing a significant gap in knowledge. Our objective in this proposal is to dissect the signal transduction cascades that activate TAZ and YAP in sarcomas. Our central hypothesis is that histone deacetylation of the regulatory regions of the core Hippo kinases and MAP4 kinases works in tandem with PI3 kinase signaling to activate TAZ/YAP. This hypothesis is supported by studies showing that loss of expression of the Hippo kinases is common in both sarcoma clinical samples and cell lines as well as pharmacological studies showing that inhibition of histone deacetylases (HDACs) lead to reconstitution of the Hippo kinases thus inhibiting TAZ/YAP. A parallel set of studies utilizing genetically engineered and xenograft mouse models and sarcoma cell lines have shown that TAZ and YAP differentially mediate PI3 kinase signaling in undifferentiated pleomorphic sarcoma and pediatric rhabdomyosarcomas. We plan to test our central hypothesis with the following specific aims: Aim 1) Identify how TAZ and YAP differentially contribute to PI3 kinase-mediated sarcomagenesis. We will stimulate PI3K signaling in rhabdomyosarcoma cell lines that are wild-type, TAZ knockout, YAP knockout, and TAZ/YAP knockout and determine how TAZ and YAP differentially contribute to the PI3 kinase transcriptome by RNA-Seq gene expression analysis. We will also stimulate PI3K signaling in the muscle of sarcoma-prone mice that are Trp53fl/flPtenfl/fl and that are Tazfl/flYapfl/fl, Tazfl/fl, Yapfl/fl, or Taz/Yap wild type and compare tumor latency and gene expression profiles. Aim 2) Determine if histone deacetylation of MST1/2 and the MAP4 kinases activates TAZ and YAP. We will inhibit HDACs and assess occupancy of H3K27ac at promoter/enhancer regions of MST1/2 and the MAP4 kinases (MM kinases) in rhabdomyosarcoma cell lines. To test the importance of the MM kinases in tumor progression in vivo, we will delete genes encoding the LATS1/2 kinases (that are downstream of the MM kinases) in sarcoma-prone mice. To achieve this we will inject Trp53fl/fl mice that are Lats1fl/flLats2fl/fl or are Lats1/Lats2 wild-type with intramuscular adeno-Cre virus. Aim 3) Determine if inhibition of HDACs and the PI3K-Akt axis diminish proliferation and metastasis. We will determine if combinatorial inhibition of histone deacetylases and the PI3 kinase pathway will inactivate TAZ/YAP. This will be tested in vitro with proliferation and cell cycle analysis assays. The effect of combination therapy on spontaneous metastases will be tested in a sarcoma xenograft mouse model. The proposal is innovative because it targets two incompletely understood parallel pathways upstream of TAZ/YAP, expanding the focus of the field beyond targeting TAZ/YAP themselves, and thus addressing a significant gap in knowledge. The project is significant because it identifies upstream pathways regulating TAZ/YAP that can be inhibited pharmacologically, providing a mechanism to make therapeutic targeting of TAZ and YAP clinically feasible for sarcomas and potentially other cancers.