Functional determinants of metastatic dormancy

描述(申请人提供)：我们的长期目标是确定控制转移性生长和休眠的机制。由于转移性疾病的发病率和死亡率，找到通过诱导休眠甚至根除播散的休眠细胞来阻止转移性生长的方法，将大大减少癌症造成的死亡。 我们以前发现，处于休眠状态的癌细胞有高度激活的p38，它可以阻止ERK的激活，从而产生有利于维持休眠状态的平衡。基于这一观察结果，我们对p38信号的下游靶点进行了蛋白质组学分析。这项研究得到了两个主要发现，一个是整合素信号和p38的激活与两个内质网受体PERK和IRE1α的激活有关，后者诱导了整合的应激反应(ISR)；另一个发现表明hnRNPA1的亚细胞定位发生了变化，这种变化影响了mRNA的剪接和/或稳定性。 我们现在提出三个特定的目标：在特定的目标1中，我们将确定整合素信号、p38激活和作为休眠细胞ISR诱导剂的perk和！RE1cc激活之间的因果联系。我们将验证我们的假设，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-&gt；perk/IRE1α通路的组成部分，并使用异种移植瘤裸鼠模型测试它们对转移生长的影响。 在特定目标2中，我们将研究p38高活性与hnRNPA1在休眠细胞胞浆中定位之间的联系，作为调节ISR调节基因转录后基因表达的机制，我们已经发现的8个肿瘤抑制基因是由p38诱导的。使用免疫沉淀、RT-PCR和基因阵列(核基因组学)相结合的方法，我们将在高p38(休眠)和低p38(增殖)细胞中鉴定受ISR和p38调控的mRNAs，这些mRNAs与hnRNPA1相关。 在具体目标3中，我们将使用一种新的方法，即用GFP标记ERK或p38信号通路。通过这样做，我们能够监测体内原发和转移癌细胞中ERK或p38的激活状态。我们将使用工程设计的表达ERK或p38诱导的GFP的致瘤和休眠细胞来从原发肿瘤中分离，当p38处于休眠状态时激活p38的细胞。这些细胞将被用来获得总的mRNA表达谱。这些信号将与p38信号相关联，并将提供休眠基因表达的信号。我们将使用这个休眠信号来测试它是否存在于裸鼠的增殖性转移中(高ERK/低p38)，并确定它是否影响它们的行为。 我们的建议将揭示两条新的途径在肿瘤休眠中的功能贡献：一条是连接休眠细胞中的整合素、p38和内质网应激信号，另一条是p38调节hnRNPA1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第一个p38调节的休眠细胞的基因表达特征，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疗和/或延长生命的治疗方法提供途径。

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.