Mechanisms of VEGF-A regulated tumor lymphangiogenesis

描述（申请人提供）：淋巴转移是许多实体瘤的主要传播途径。这个过程的先决条件是产生新的淋巴管，使肿瘤细胞能够进入淋巴管。新淋巴管的形成，即淋巴管生成，被认为主要由两个因素诱导，VEGF-C或VEGF-D。我们最近发现，一种针对VEGF家族另一成员VEGF- a的抗体可以抑制肿瘤淋巴管生成和淋巴转移，而不影响VEGF- c或VEGF- d的表达。这一观察结果确定了VEGF-A，一种有效的血管生成因子，作为肿瘤淋巴管生成的调节因子，表明抗VEGF-A治疗可能有助于预防癌症患者的淋巴转移。这一发现也为更好地理解淋巴管生成过程及其与转移性扩散的相关性提供了一个独特的机会。我们的体内数据显示，抗vegf - a治疗抑制了三种促淋巴管生成介质的表达：血管生成素-2 （ang2）、其受体Tie-2和VEGFR-3（一种传递VEGF-C信号的主要受体）。这些介质之间的功能关联及其对VEGF-A的依赖性得到了文献报道和我们的初步数据的有力支持。然而，VEGF-A诱导和Ang-2介导的淋巴管生成调控的机制在很大程度上是未知的。我们假设调节淋巴管生成的主要vegf - a依赖性事件是：1)Ang-2的转录上调，随后通过Tie-2激活增加淋巴管内皮中VEGFR-3的表达；2)淋巴管中自身受体VEGFR-2的自分泌扩增，已知其能增强VEGFR-3信号传导。通过增加VEGF-C受体的数量并增强其转导活性，这些VEGF-A和Ang-2依赖事件使淋巴管对VEGF-C预敏感，并加速VEGF-C信号的翻译，促进新淋巴管的稳健形成。为了验证这些假设，我们建议确定：1)体外淋巴内皮细胞中VEGF-A对VEGFR-2表达的调节作用；2) Ang-2对淋巴内皮细胞Tie-2和VEGFR-3表达的调控作用；3) Ang-2介导的、不依赖vegf - a的乳腺癌模型体内肿瘤淋巴管生成调控。揭示这些机制细节对于优化现有的抗vegf - a靶向策略以及确定对抗癌症患者淋巴转移的新靶点至关重要。我们建议在实验定义的体内和体外模型中描述抗vegf - a治疗介导肿瘤淋巴抑制的分子事件。这些研究的意义是双重的：1)它们将建立血管生成和淋巴管生成介质之间相互作用的新范式，这将导致对健康和疾病中淋巴管形成的新理解；2)明确VEGF-A和Ang-2在肿瘤淋巴管生成调控中的具体作用，为VEGF-A和Ang-2抑制剂在淋巴转移高危肿瘤患者中的应用提供强有力的推动。由于转移是癌症死亡的主要原因，这些研究有可能显著改善大量癌症患者的健康状况。

DESCRIPTION (provided by applicant): Lymphatic metastasis is the main dissemination pathway in many solid tumors. The prerequisite for this process is generation of new lymphatic vessels accessible to tumor cells. The formation of new lymphatic vessels, i.e., lymphangiogenesis, is thought to be induced primarily by two factors, VEGF-C or VEGF-D. We recently discovered that an antibody against another member of the VEGF family, VEGF-A, inhibits both tumor lymphangiogenesis and lymphatic metastasis without affecting expression of VEGF-C or VEGF-D. This observation identifies VEGF-A, a potent angiogenic factor, as a regulator of tumor lymphangiogenesis, suggesting that anti-VEGF-A therapies could be useful for preventing lymphatic metastasis in cancer patients. This finding also provides a unique opportunity to better understand the process of lymphangiogenesis and its relevance to metastatic spread. Our in vivo data showed that anti-VEGF-A treatment suppressed expression of three pro- lymphangiogenic mediators: angiopoietin-2 (Ang-2), its receptor Tie-2 and VEGFR-3, a main receptor transmitting VEGF-C signals. The functional association among these mediators and their dependence on VEGF-A is strongly supported by both literature reports and our preliminary data. However, the mechanisms underlying VEGF-A induced and Ang-2 mediated regulation of lymphangiogenesis are largely unknown. We hypothesize that the main VEGF-A-dependent events that regulate lymphangiogenesis are: 1) transcriptional up- regulation of Ang-2, which subsequently increases expression of VEGFR-3 in lymphatic endothelium through Tie-2 activation; and 2) autocrine amplification of its own receptor in lymphatics, VEGFR-2, known to functionally enhance VEGFR-3 signaling. By increasing a number of VEGF-C receptors and enhancing their transduction activity, these VEGF-A and Ang-2 dependent events pre-sensitize lymphatics to VEGF-C and accelerate the translation of VEGF-C signals into robust formation of the new lymphatic vessels. To test these hypotheses, we propose to determine: 1) a regulatory effect of VEGF-A on the expression of VEGFR-2 in lymphatic endothelial cells in vitro; 2) a regulatory effect of Ang-2 on the expression of Tie-2 and VEGFR-3 in lymphatic endothelial cells in vitro; and 3) Ang-2 mediated, VEGF-A-independent, regulation of tumor lymphangiogenesis in breast carcinoma model in vivo. Unraveling these mechanistic details is crucially important for optimizing the existing anti-VEGF-A targeted strategy and for identifying new targets to counteract lymphatic metastasis in cancer patients. We propose to delineate the molecular events mediating inhibition of tumor lymphatics by anti-VEGF-A therapy in experimentally defined in vivo and in vitro models. The significance of these studies is two-fold: 1) they will establish a new paradigm of cross- talk among angiogenic and lymphangiogenic mediators, which would lead to a novel understanding of the formation of the lymphatic vessels in health and disease; and 2) they will define specific roles of VEGF-A and Ang-2 in the regulation of tumor lymphangiogenesis, thus providing a strong impetus for applying VEGF-A and Ang-2 inhibitors to cancer patients with a high risk of lymphatic metastasis. Because metastasis is a primary cause of mortality from cancer, these studies have the potential to significantly improve health outcomes in a large number of cancer patients.