Mechanisms of VEGF-A regulated tumor lymphangiogenesis

描述（由申请人提供）：淋巴转移是许多实体瘤的主要传播途径。这一过程的先决条件是产生新的淋巴管可接近肿瘤细胞。新淋巴管的形成，即，淋巴管生成被认为主要由两种因子VEGF-C或VEGF-D诱导。我们最近发现，针对VEGF家族的另一个成员VEGF-A的抗体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。这一观察结果鉴定了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疗法可用于预防癌症患者的淋巴转移。这一发现也提供了一个独特的机会，以更好地了解淋巴管生成的过程及其相关性转移扩散。我们的体内数据显示，抗VEGF-A治疗抑制三种促淋巴管生成介质的表达：血管生成素-2（Ang-2）、其受体Tie-2和VEGFR-3，VEGFR-3是传递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节的潜在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，其随后通过Tie-2活化增加淋巴内皮中VEGFR-3的表达;和2）已知在功能上增强VEGFR-3信号传导的血管内皮细胞中其自身受体VEGFR-2的自分泌扩增。通过增加VEGF-C受体的数量并增强它们的转导活性，这些VEGF-A和Ang-2依赖性事件使淋巴细胞对VEGF-C预敏感并加速VEGF-C信号的翻译成新淋巴管的稳健形成。为了验证这些假设，我们提出确定：1）VEGF-A对淋巴管内皮细胞中VEGFR-2表达的体外调节作用; 2）Ang-2对淋巴管内皮细胞中Tie-2和VEGFR-3表达的体外调节作用;和3）Ang-2介导的、VEGF-A非依赖性的、乳腺癌模型中肿瘤淋巴管生成的体内调节。解开这些机制的细节是至关重要的优化现有的抗VEGF-A靶向策略，并确定新的目标，以抵消癌症患者的淋巴转移。我们建议在实验确定的体内和体外模型中描述通过抗VEGF-A治疗介导肿瘤抑制的分子事件。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的相互作用的新范式，这将导致对健康和疾病中淋巴管形成的新理解;和2）它们将确定VEGF-A和Ang-2在调节肿瘤淋巴管生成中的特定作用，从而为将VEGF-A和Ang-2抑制剂应用于具有高淋巴转移风险的癌症患者提供了强大的动力。由于转移是癌症死亡的主要原因，这些研究有可能显着改善大量癌症患者的健康结果。

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.