Functional determinants of metastatic dormancy

描述（由申请人提供）：我们的长期目标是确定控制转移生长和休眠的机制。由于与转移性疾病相关的发病率和死亡率，找到通过诱导休眠或甚至根除散布的休眠细胞来阻止转移性生长的方法将大大减少癌症引起的死亡。 我们以前发现休眠的癌细胞具有高度活化的p38，其阻断ERK活化，产生有利于休眠维持的平衡。基于这一观察结果，我们启动了p38信号转导下游靶点的蛋白质组学分析。这项研究产生了两个主要发现，一个是将整合素信号传导和p38激活与两种内质网受体PERK和IRE 1 α的激活联系起来，这两种受体诱导了整合的应激反应（ISR），另一个发现表明hnRNPA 1的亚细胞定位发生了改变，这是一种影响mRNA剪接和/或稳定性的变化。 我们现在提出3个具体目标：在具体目标1中，我们将确定整合素信号传导，p38激活和PERK激活之间的因果关系，RE 1cc作为休眠细胞中ISR的诱导物。我们将测试我们的假设，即激活这一途径可以迫使恶性细胞进入长期休眠状态。我们将使用遗传学或药理学方法抑制或激活整合素->p38->PERK/IRE 1 α通路的组分，并使用异种移植裸鼠模型测试它们对转移生长的影响。 在具体目标2中，我们将研究高p38活性和hnRNPA 1定位到休眠细胞中的胞质溶胶之间的联系，作为调节ISR调节基因的转录后基因表达的机制，我们发现，在休眠细胞中由p38诱导的8个肿瘤抑制基因。使用免疫沉淀、RT-PCR和基因阵列（核糖体组学）的组合，我们将鉴定在高p38（休眠）和低p38（增殖）细胞中由ISR和p38调节的与hnRNPA 1相关的mRNA。 在Specific Aim 3中，我们将使用一种新的方法，用GFP标记ERK或p38信号通路。通过这样做，我们能够在体内监测原发性和转移性癌细胞中ERK或p38活化的状态。我们将使用致瘤和休眠细胞工程表达ERK或p38诱导的GFP分离原发性肿瘤，细胞激活p38，因为他们成为休眠。这些细胞将用于获得总mRNA表达谱。这些将与p38信号传导相关联，并将提供休眠基因表达标记。我们将使用这种休眠特征来测试它是否存在于裸鼠的增殖转移（高ERK/低p38）中，并确定它是否对它们的行为有影响。 我们的提议将揭示两种新途径对肿瘤休眠的功能贡献：一种是在休眠细胞中连接整联蛋白、p38和ER应激信号传导，第二种是p38调节hnRNPA 1调节ISR调节基因和已知肿瘤抑制因子的转录后基因表达。最后，我们的第三个目标将提供休眠细胞的第一个p38调节的基因表达特征，并将告诉我们，如果这个特征在不断增长的转移中丢失。这些结果可能为设计癌症的治愈性和/或延长生命的疗法提供途径。

DESCRIPTION (provided by applicant): Our long-term goal is to identify the mechanisms that govern metastatic growth and dormancy. Due to the morbidity and mortality associated with metastatic disease, finding ways to stop metastatic growth either by inducing dormancy or even eradicating disseminated dormant cells, would greatly reduce deaths caused by cancer. We previously showed that dormant cancer cells have highly activated p38 which blocks ERK activation generating a balance that favors the maintenance of dormancy. Based on this observation we initiated a proteomic analysis of down-stream targets of p38 signaling. This search yielded two major findings, one that linked integrin signaling and p38 activation to the activation of two endoplasmic reticulum receptors, PERK and IRE1alpha, which induced an integrated stress response (ISR) and a second that indicated an altered sub-cellular localization of hnRNPAl, a change that affects mRNA splicing and/or stability. We now propose 3 specific aims: In Specific Aim 1 we will determine the causal link between integrin signaling, p38 activation and the activation of PERK and !RE1cc as inducers of an ISR in dormant cells. We will test our hypothesis that activation of this pathway can force malignant cells into a state of protracted dormancy. We will inhibit or activate components of the integrin->p38->PERK/IRE1alpha pathways using genetic or pharmacological approaches and test their effect on metastatic growth using the xenograft nude mice model. In Specific Aim 2, we will investigate the link between high p38 activity and localization of hnRNPAl to the cytosol in dormant cells as a mechanism that regulates post-transcriptional gene expression of ISR regulated genes and 8 tumor suppressors genes that we have found, are induced by p38 in dormant cells. Using a combination of immunoprecipitation, RT-PCR and gene arrays (ribonomics) we will identify the mRNAs regulated by the ISR and p38 that are associated with hnRNPAl in high p38 (dormant) and low p38 (proliferative) cells. In Specific Aim 3 we will use a novel approach in which we tag ERK or p38 signaling pathways with GFP. In so doing we are able to monitor the state of ERK or p38 activation in primary and metastatic cancer cells in vivo. We will use tumorigenic and dormant cells engineered to express an ERK- or p38-induced GFP to isolate from primary tumors, cells that activate p38 as they become dormant. These cells will be used to obtain total mRNA expression profiles. These will be linked to p38 signaling and will provide a dormancy gene expression signature. We will use this dormancy signature to test if it is present in proliferating metastasis (high ERK/low p38) in nude mice and determine if it informs on their behavior. Our proposal will reveal the functional contribution of two novel pathways to tumor dormancy: one linking integrin, p38 and ER-stress signaling in dormant cells and a second one where p38 regulation of hnRNPAl regulates post-transcriptional gene expression of ISR regulated genes and known tumor suppressors. Finally, our third aim will provide the first p38-regulated gene expression signature of dormant cells and will teach us if this signature is lost in growing metastasis These results may provide avenues for designing curative and/or life prolonging therapies for cancer.