Regulation of Osteosarcoma Metastasis by Notch and Hes 1 Pathway Signaling

描述（由申请人提供）：转移是癌症的标志性行为之一，了解控制转移的调控过程是改善实体瘤患者结局的关键。然而，影响转移的调节途径通常影响癌症的其他标志性行为，即增殖，使得难以分离转移所必需的机制。骨肉瘤是最常见的原发性骨癌，在青春期发病率最高。骨肉瘤也是研究常见恶性行为的良好模型系统。最近我们发现了Notch信号通路的新作用，特别是Notch靶基因Hes 1，特别是在促进骨肉瘤转移中的作用。我们发现Hes 1的表达对于骨肉瘤的体外侵袭和体内转移是必不可少的，而肿瘤的其他标志性行为不受Hes 1变化的影响。Hes 1的这种转移促进作用不是由MMPs、VEGF、Ezrin或其他公认的转移介质介导的，而是代表了一种对控制转移和侵袭重要的新的调节机制。因此，Hes 1在促进骨肉瘤侵袭和转移中的集中作用代表了一个独特和令人兴奋的机会，以评估该基因的作用仅限于转移的系统中的这种新的调节机制。鉴于Notch通路表达在许多实体瘤中预后较差，我们关于骨肉瘤转移调控的研究结果应广泛适用于许多实体瘤。 我们有数据表明Hes 1通过上调Metadherin的表达促进转移，Metadherin是一种可以介导肿瘤归巢到肺的细胞表面分子。我们的第一个具体目标是确定这种调节关系，并确定Metadherin是否是骨肉瘤转移所必需的。由于我们的初步工作已经验证了在我们的肿瘤模型中使用基质胶侵袭作为转移潜力的替代物，因此我们的大多数实验将依赖于这种转移的体外替代物来有效地评估Hes 1和Metadherin的实验室操作。然而，我们将使用我们实验室开发的两种新的转移异种移植模型评估Metadherin的体内敲低。我们也有初步的数据表明，Notch途径的表观遗传调控-特别是HDAC抑制和/或microRNA 34家族的差异表达-可能是骨肉瘤中这种新的转移促进信号的上游调节因子。具体目标2将定义microRNA和HDAC抑制剂是否影响Notch介导的转移，使用基质胶侵袭作为侵袭性的主要功能测定。最后，我们希望通过识别转移风险最大的患者，将我们的观察结果应用于改善患者的预后。为此，第三个具体目标是使用Q-PCR和免疫组织化学在一组140个来自U. T. M. D.安德森和比较表达的结果，确定标志物和测定具有最大的预后潜力。 公共卫生相关性：当成功完成后，这些研究将确定Notch 1和Hes 1的表达在骨肉瘤中是如何调节的，以及这种表达如何与Metadherin的表达相关，以控制侵袭和转移。这些机制可能不仅对骨肉瘤很重要，而且对广泛的实体瘤也很重要，其中控制转移是延长生存期和提高治愈率的关键。对这些转移的基本机制的更好理解应该导致在这个新途径中鉴定可重复的组分，其将作为阻断转移的新疗法的靶点。

DESCRIPTION (provided by applicant): Metastasis is one of the hallmark behaviors of cancer and understanding the regulatory processes controlling it is key to improving outcomes for solid tumor patients. However, regulatory pathways affecting metastasis often affect other hallmark behaviors of cancer, i.e. proliferation, making it difficult to isolate mechanisms essential to metastasis. One disease in which metastasis is the primary cause of morbidity and mortality is osteosarcoma, most common primary bone cancer, with a peak incidence during adolescence. Osteosarcoma also has served excellent model system for studying common malignant behaviors. Recently we discovered a novel role for Notch signaling, especially the Notch target gene Hes1, specifically in promoting osteosarcoma metastasis. We showed Hes1 expression is essential for in vitro invasion and in vivo metastasis of osteosarcoma, while other hallmark behaviors of cancer were unaffected by Hes1 changes. This metastasis-promoting effect of Hes1 was not mediated by MMPs, VEGF, Ezrin or other recognized mediators of metastasis, but rather represents a novel regulatory mechanism important for controlling metastasis and invasion. Thus the focused role of Hes1 in promoting invasion and metastasis of osteosarcoma represents a unique and exciting opportunity to evaluate this new regulatory mechanism in a system where the effect of the gene is limited to metastasis. Given that Notch pathway expression confers a worse prognosis on many solid tumors, our findings about metastasis regulation in osteosarcoma should be broadly applicable to many solid tumors. We have data suggesting that Hes1 promotes metastasis by upregulating expression of Metadherin, a cell-surface molecule that can mediate homing of tumor to lung. Our first specific aim will define that regulatory relationship and determine if Metadherin is essential for osteosarcoma metastasis. Since our preliminary work has validated the use of matrigel invasion as a surrogate for metastatic potential in our tumor models, most of our experiments will rely on this in vitro surrogate for metastasis to efficiently evaluate laboratory manipulations of Hes1 and Metadherin. However, we will assess Metadherin knock-down in vivo using two novel xenograft models of metastasis developed in our laboratory. We also have preliminary data showing that epigenetic regulation of the Notch pathway - specifically HDAC inhibition and/or the differential expression of the microRNA34 family - may be the upstream regulators of this novel metastasis promoting signal in osteosarcoma. Specific aim 2 will define whether microRNAs and HDAC inhibitors influence Notch-mediated metastasis, using matrigel invasion as the primary functional assay for invasiveness. Finally, we wish to apply our observations to improve outcomes for patients by identifying those at greatest risk for metastasis. To that end, the third specific aim measures the expression of Notch pathway genes and Metadherin using Q-PCR and immunohistochemistry in a panel of 140 archival tumors from patients treated at U. T. M. D. Anderson and compared expression to outcome, identifying the marker and assay with the greatest prognostic potential. PUBLIC HEALTH RELEVANCE: When successfully completed, these studies will determine how expression of Notch1 and Hes1 are regulated in osteosarcoma, and how this expression relates to expression of Metadherin in controlling invasiveness and metastasis. These mechanisms are likely to be important not only for osteosarcoma, but also for a wide range of solid tumors in which control of metastasis is key to prolonging survival and increasing cure rates. Improved understanding of these basic mechanisms of metastasis should lead to identification of inhibitable components within this new pathway that will serve as the targets for novel therapies to block metastasis.