Regulation of Osteosarcoma Metastasis by Notch and Hes 1 Pathway Signaling

描述(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预后的关键。然而，影响转移的调控通路经常影响肿瘤的其他标志性行为，即增殖，这使得分离转移的关键机制变得困难。转移是导致发病率和死亡率的主要原因的一种疾病是骨肉瘤，这是最常见的原发骨癌，在青春期发病率最高。骨肉瘤也为研究常见的恶性行为提供了极好的模型系统。最近我们发现了Notch信号的一个新的作用，特别是Notch靶基因Hes1，特别是在促进骨肉瘤转移方面。我们发现Hes1的表达对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Hes1变化的影响。Hes1的这种促转移作用不是由MMPs、VEGF、Ezrin或其他公认的转移介质介导的，而是代表了一种新的调控机制，对控制转移和侵袭具有重要意义。因此，Hes1在促进骨肉瘤侵袭和转移中的集中作用代表了一个独特而令人兴奋的机会，可以在该基因的作用仅限于转移的系统中评估这一新的调节机制。鉴于Notch通路的表达在许多实体肿瘤中的预后较差，我们关于骨肉瘤转移调控的研究结果应该广泛适用于许多实体肿瘤。我们有数据表明，Hes1通过上调Metherin的表达来促进转移，Metherin是一种细胞表面分子，可以介导肿瘤向肺的归巢。我们的第一个具体目标将定义这种调控关系，并确定Metherin是否对骨肉瘤转移是必需的。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Matrigel侵袭作为转移潜能的替代物，我们的大多数实验将依赖于这种体外转移替代物来有效地评估Hes1和Metherin的实验室操作。然而，我们将使用我们实验室开发的两个新的转移异种移植模型来评估体内Metherin的下调。我们也有初步的数据表明，Notch通路的表观遗传调控--特别是HDAC抑制和/或microRNA34家族的差异表达--可能是骨肉瘤中这一新的促进转移信号的上游调控因素。具体目标2将定义microRNAs和HDAC抑制剂是否影响Notch介导的转移，将基质侵袭作为侵袭性的主要功能测试。最后，我们希望通过识别转移风险最高的患者来应用我们的观察结果来改善患者的预后。为此，第三个特定的目的是使用Q-PCR和免疫组织化学方法测量来自安德森大学安德森分校治疗的140例存档肿瘤患者的Notch通路基因和Metherin的表达，并将表达与结果进行比较，确定具有最大预后潜力的标记物和分析。 公共卫生相关性：这些研究成功完成后，将确定Notch1和Hes1在骨肉瘤中的表达是如何调节的，以及这种表达与Metherin在控制侵袭和转移中的表达如何相关。这些机制可能不仅对骨肉瘤很重要，而且对范围广泛的实体肿瘤也很重要，在这些实体肿瘤中，控制转移是延长生存和提高治愈率的关键。对这些转移的基本机制的更好的理解将导致识别这一新途径中的可抑制成分，这些成分将作为新的治疗方法的靶点来阻断转移。

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.