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，影响转移的调控途径经常影响癌症的其他标志性行为，即增殖，这使得很难分离转移的必要机制。骨肉瘤是一种转移是发病和死亡的主要原因的疾病，它是最常见的原发性骨癌，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良好的模型系统。最近，我们发现Notch信号，特别是Notch靶基因Hes1在促进骨肉瘤转移中的新作用。我们发现Hes1的表达对于骨肉瘤的体外侵袭和体内转移至关重要，而癌症的其他标志性行为不受Hes1变化的影响。Hes1的这种促进转移的作用不是由MMPs、VEGF、Ezrin或其他公认的转移介质介导的，而是一种控制转移和侵袭的重要新调控机制。因此，Hes1在促进骨肉瘤侵袭和转移中的作用是一个独特而令人兴奋的机会，可以在一个基因作用仅限于转移的系统中评估这种新的调控机制。鉴于Notch通路的表达在许多实体瘤中导致较差的预后，我们关于骨肉瘤转移调控的发现应该广泛适用于许多实体瘤。我们有数据表明Hes1通过上调Metadherin的表达来促进转移，Metadherin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是确定这种调节关系，并确定Metadherin是否对骨肉瘤转移至关重要。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侵袭作为转移潜力的替代物，我们的大多数实验将依赖于这种体外转移替代物来有效评估Hes1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型转移瘤模型来评估Metadherin在体内的敲除。我们也有初步的数据显示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-可能是骨肉瘤中这种新型转移促进信号的上游调控。特异性目的2将确定microrna和HDAC抑制剂是否影响notch介导的转移，使用基质侵袭作为侵袭性的主要功能测定。最后，我们希望通过识别那些转移风险最大的患者来应用我们的观察结果来改善患者的预后。为此，第三个特定目的是使用Q-PCR和免疫组织化学方法在140个来自安德森大学治疗的患者的档案肿瘤中测量Notch通路基因和metadherin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检测方法。

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.