Brain tumor angiogenesis: Integrin ceramide signaling

描述（由申请人提供）：脑肿瘤是癌症相关死亡的重要原因。恶性脑肿瘤是高度血管生成的，并且它们的微血管表达整合素细胞表面受体α v β 3和α v β 5。使用RGDfV（一种整合素-α v β 3/β 5抑制剂）的临床试验很有希望。因此，更好地理解α v-整联蛋白抑制的分子机制以在未来的临床试验中提高其治疗功效是至关重要的。我们的目标是研究神经酰胺作为抑制内皮整合素α v β 3/β 5的分子靶点。 在颅内小鼠异种移植模型中，抑制整合素α v β 3/β 5诱导内皮细胞（EC）凋亡并防止脑肿瘤生长。我们以前的工作表明，EC α v β 3/β 5的抑制增加了细胞内促凋亡脂质第二信使神经酰胺的水平。我们最近的研究结果表明，这是由于酸性鞘磷脂酶的激活而导致鞘磷脂水解。重要的是，整合素α v β 3/β 5和p53在新生血管形成中协同作用，但整合素调节p53的机制尚不清楚。我们的初步数据，即EC整合素α v β 3/β 5的抑制抑制了促存活激酶PKB/Akt的磷酸化，并且Akt调节p53活性，在这方面非常有趣，特别是因为Akt是神经酰胺抑制的潜在靶点。最后，我们已经表明，合成类维生素A，芬维A胺，也诱导神经酰胺介导的EC凋亡。然而，这种神经酰胺的产生是通过从头合成，这是一种不同于RGDfV的机制。基于这些数据，我们假设神经酰胺在通过抑制整合素α v β 3/β 5诱导的内皮细胞凋亡机制中是关键的。由此产生的重要问题是：1）整合素α v β 3/β 3介导的细胞凋亡是否需要RGDfV诱导的神经酰胺; 2）整合素α v β 3/β 5是否通过其对Akt的调节作用控制p53活性，以及神经酰胺是否介导该功能; 3）与单独的每种相比，RGDfV +芬维A胺的组合是否对脑肿瘤具有更好的治疗功效;最后，4）RGDfV的体内作用靶向内皮细胞、肿瘤细胞还是两者。 本研究的具体目的是：1.探讨神经酰胺、整合素α v β 3/α v β 5与内皮细胞凋亡的分子相互作用。 2.阐明神经酰胺生成下游α v β 3/α v β 5整联蛋白阻断的信号传导机制。 3.研究芬维A胺+ RGDfV联合应用对小鼠颅内肿瘤模型的治疗作用。这些研究将为α v β 3/β 5整联蛋白抑制在抗血管生成癌症治疗中的作用提供关键的机制数据，并将对恶性脑肿瘤患者的改进治疗设计至关重要。

DESCRIPTION (provided by applicant): Brain tumors are an important cause of cancer-related death. Malignant brain tumors are highly angiogenic and their microvessels express the integrin cell surface receptors alphavbeta3 and alphavbeta5. Clinical trials using RGDfV, an integrin-alphavbeta3/beta5 inhibitor, are promising. It is therefore critical to better understand the molecular mechanism of alphav-integrin inhibition in order to improve its therapeutic efficacy in future clinical trials. Our goal in this proposal is to examine ceramide as a molecular target in inhibition of endothelial integrins alphavbeta3/beta5. Inhibition of integrins alphavbeta3/beta5 induces apoptosis of endothelial cells (EC) and prevents brain tumor growth in an intracranial mouse xenograft model. Our previous work shows that inhibition of EC alphavbeta3/beta5 increases levels of the intracellular pro-apoptotic lipid second messenger ceramide. Our more recent results suggest this is by hydrolysis of sphingomyelin due to activation of acid sphingomyelinase. Importantly, integrins alphavbeta3/beta5 and p53 act in concert in neovascularization, but the mechanism by which integrins regulate p53 is not known. Our preliminary data, that inhibition of EC integrins alphavbeta3/beta5 suppressed phosphorylation of the pro-survival kinase PKB/Akt, and that Akt regulated p53 activity, are very interesting in this regard, especially since Akt is a potential target for inhibition by ceramide. And lastly, we have shown that the synthetic retinoid, fenretinide, also induces ceramide-mediated EC apoptosis. However, this ceramide generation is by de novo synthesis, a mechanism that is different from RGDfV. Based on these data we hypothesize that ceramide is critical in the mechanism of endothelial cell apoptosis that is induced by inhibition of integrins alphavbeta3/beta5. Important questions stemming from this are: 1) Is RGDfV-induced ceramide required for integrin alphavbeta3/beta3-mediated apoptosis; 2) Do integrins alphavbeta3/beta5 control p53 activity via their regulatory effect on Akt, and does ceramide mediate this function; 3) Does combination of RGDfV + fenretinide have better therapeutic efficacy against brain tumors, compared to each alone; and last, 4) Does the in vivo effect of RGDfV target the endothelial cells, the tumor cells, or both. Our Specific Aims to investigate these questions and examine our hypothesis are: 1.To determine molecular interactions between ceramide, integrins alphavbeta3/alphavbeta5 and endothelial cell apoptosis. 2. To elucidate the signaling mechanism of alphavbeta3/alphavbeta5 integrin blockade downstream of ceramide generation. 3.To examine the combination of fenretinide + RGDfV in an intracranial mouse brain tumor model. These studies will provide critical mechanistic data on the function of alphavbeta3/beta5 integrin inhibition in antiangiogenic cancer therapy, and will be essential for design of improved treatments for patients with malignant brain tumors.