Brain tumor angiogenesis: Integrin ceramide signaling

描述(申请人提供)：脑瘤是癌症相关死亡的重要原因。恶性脑肿瘤是高度促血管生成的肿瘤，其微血管表达整合素细胞表面受体αvbeta3和αvbeta5。使用整合素-αvbeta3/β5抑制剂RGDfV进行的临床试验很有希望。因此，更好地了解α-整合素抑制的分子机制对于在未来的临床试验中提高其治疗效果至关重要。我们在这项提案中的目标是研究神经酰胺作为抑制内皮细胞整合素αvbeta3/Beta5的分子靶点。 抑制整合素Alphavbeta3/Beta5诱导内皮细胞(EC)凋亡，并防止脑肿瘤在小鼠颅内移植模型中的生长。我们以前的工作表明，抑制ECαvbeta3/Beta5会增加细胞内促凋亡脂质第二信使神经酰胺的水平。我们最近的结果表明，这是由于酸性鞘磷脂酶激活而导致的神经鞘磷脂的水解性。重要的是，整合素Alphavbeta3/Beta5和P53共同作用于新生血管，但整合素调控P53的机制尚不清楚。我们的初步数据，EC整合素αvbeta3/Beta5的抑制抑制了促生存激酶PKB/Akt的磷酸化，以及Akt调节P53的活性，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，我们发现合成的维甲酸，芬维甲素，也能诱导神经酰胺介导的内皮细胞凋亡。然而，这种神经酰胺的产生是通过从头合成的，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抑制整合素αvbeta3/Beta5诱导内皮细胞凋亡的机制中起关键作用。由此引出的重要问题是：1)RGDfV诱导的神经酰胺是否是整合素αvbeta3/beta3介导的细胞凋亡所必需的；2)整合素alphavbeta3/Beta5是否通过其对Akt的调节作用来控制p53的活性，以及神经酰胺是否介导了这一功能；3)RGDfV+Fenretinide联合应用是否比单独使用RGDfV对脑肿瘤有更好的疗效；以及4)RGDfV的体内作用针对的是内皮细胞、肿瘤细胞，还是两者兼而有之。 我们研究这些问题并验证我们的假设的具体目的是：1.确定神经酰胺、整合素αvbeta3/αvbeta5与内皮细胞凋亡之间的分子相互作用。 2.阐明αvbeta3/alphavbeta5整合素阻断神经酰胺生成下游的信号机制。 3.观察芬维甲素+RGDfV联合应用于小鼠脑内肿瘤模型的疗效。这些研究将为α-β-3/β-5整合素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，并将对设计恶性脑瘤患者的改进治疗方案至关重要。

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.