Ephrin-A1 in lipid metabolism and breast cancer

项目摘要 肿瘤细胞的增殖率增加，需要更多的能量和生物量 要求。增殖的肿瘤细胞通常通过移位支持大分子复制 生物能量学从氧化磷酸化，如在非增殖细胞中观察到的有氧 糖酵解作为这种新陈代谢的形式，会产生关键的积木和能量。肿瘤细胞脂质 新陈代谢与有氧糖酵解密切相关，最近的研究结果表明，脂肪在代谢中起着关键作用 代谢在肿瘤发生和恶性进展中的作用。脂肪酸合成酶(FASN)，关键 内源性脂肪合成中的酶在乳腺癌中过度表达，并与不良有关 预后。然而，尽管脂类代谢在乳腺癌中很重要，但对它的研究还很少。 区域。调节脂质代谢的一种方式是通过受体酪氨酸激酶(RTK)信号。 RTK信号的失调可以促进异常代谢，从而促进细胞 转型。因此，有必要阐明促进肿瘤发生和发展的机制。 代谢重新编程。 我们最近发现，EphA2 RTK的原型配体Ephin-A1抑制乳腺 增生症。接下来，我们进行了两个单独的实验，以评估这种生长表型 这是由于上皮细胞或周围间质中的eaffin-A1的内在功能所致。 首先，我们从WT和Ephen-A1基因敲除的小鼠中分离出乳腺上皮细胞，并进行了 三维有机体培养。接下来，我们进行了乳腺移植手术， 我们将乳腺移植到相反基因的小鼠的透明脂肪垫中。两个都是 这些实验表明，正是上皮细胞内的ephin-A1的功能发挥了 在这一生长表型中的最大作用。由于脂肪代谢会影响生长，我们测试了 Eaffin-A1是否调节细胞脂质沉积水平。在人类癌细胞系中，我们应用了 利用shRNA技术敲除eaffin-A1(EFNA1)并观察到eaffin-A1基因敲除细胞 不仅有高水平的脂肪沉积，而且还获得了侵袭性特征和增强 组织球体形成。由于FASN有助于生长，因此与乳腺癌的进展有关 和是内源性脂肪合成的关键酶，我们研究了FASN蛋白在 来自少数WT和ephin-A1基因敲除小鼠的增生腺/肿瘤。我们能够 为了证明与WT相比，ePhin-A1基因敲除小鼠的FASN蛋白水平升高 纸巾。这些初步数据表明，eaffin-A1可能通过以下方式调节乳腺肿瘤的恶性程度 调节脂类代谢。为了在人类疾病的背景下进一步研究这一点，侵入性 对导管癌和淋巴转移组织微阵列数据集进行分析，以确定ephin- A1和FASN蛋白表达。我们观察到，低表达的ephin-A1与 FASN蛋白表达上调。根据我们的初步数据，我们假设ephin-A1 抑制乳腺上皮细胞脂代谢以抑制乳腺癌的发展和 进步。 阐明ePhin-A1、脂代谢与乳腺癌的机制联系 成长我们提出了两个具体目标的实验。在目标1中，我们将确定机制 来调节肿瘤细胞中的脂代谢，并将决定它是否通过 EphA2/HER2集群或Akt-mTOR-SREBP信令。基于先前的研究，我们假设 EPhin-A1与EphA2 RTK结合可能阻止EphA2/HER聚集或可能促进其 降解，从而抑制脂质的生物合成。另一种可能性是ePhin-A1抑制SREBP 激活，因为我们已经证明eaffin-A1抑制乳腺上皮细胞中的Akt活性。在AIM 2 我们将系统地分析eaffin-A1在乳腺肿瘤发生发展中的作用。 MMTV-Neu小鼠。我们将从这些动物身上评估肿瘤内的蛋白质表达，并将 能够进一步评估目标1中确定的目标。这些目标将共同确定 Ephin-A1与乳腺癌的发生、发展和代谢。此外，这些研究 将具有更广泛的影响，因为它可能揭示一种新的代谢调节机制， 潜在地将eaffin-A1与其他癌症和具有异常脂代谢的人类疾病联系起来。

Project Summary Tumor cells have increased rates of proliferation that demand enhanced energy and biomass requirements. Proliferating tumor cells commonly support macromolecule duplication by shifting bioenergetics from oxidative phosphorylation, as observed in non-proliferating cells, to aerobic glycolysis as this form of metabolism produces critical building blocks and energy. Tumor cell lipid metabolism is intimately linked to aerobic glycolysis and recent findings suggest a critical role for lipid metabolism in tumorigenesis and malignant progression. Fatty acid synthase (FASN), the key enzyme in endogenous lipid synthesis, is overexpressed in breast cancer and is linked to poor prognosis. Yet, despite the importance of lipid metabolism in breast cancer, it is an understudied area. One way that lipid metabolism is regulated is by receptor tyrosine kinase (RTK) signaling. Dysregulation of RTK signaling can promote aberrant metabolism that contributes to cellular transformation. Therefore, it is necessary to elucidate the mechanisms that promote malignancy and metabolic reprogramming. We recently found that ephrin-A1, a prototypic ligand of EphA2 RTK, suppresses mammary gland hyperplasia. We next performed two separate experiments to evaluate whether this growth phenotype was due to the intrinsic function of ephrin-A1 within the epithelial cells or the surrounding stroma. First, we isolated mammary epithelial cells from both WT and ephrn-A1 knockout mice and performed 3-dimensional organoid cultures. Next, we performed mammary gland transplant procedures where we transplanted mammary glands into the cleared fat pads of mice of opposite genotype. Both of these experiments indicated that it is the function of ephrin-A1 within epithelial cells that plays the greatest role in this growth phenotype. Since lipid metabolism can influence growth, we tested whether ephrin-A1 regulated levels of cellular lipid deposits. In human cancer cell lines, we applied shRNA techniques to knockdown ephrin-A1 (EFNA1) and observed that ephrin-A1 knockdown cells not only had elevated levels of lipid deposits but also acquired invasive properties and enhanced tissue spheroid formation. Since FASN contributes to growth, is linked to breast cancer progression and is the key enzyme in endogenous lipid synthesis, we investigated FASN protein expression in hyperplastic glands/tumors from a small number of WT and ephrin-A1 knockout mice. We were able to demonstrate that ephrin-A1 knockout mice have elevated levels of FASN protein compared to WT tissues. These preliminary data suggest that ephrin-A1 may regulate breast tumor malignancy by regulating lipid metabolism. To further investigate this in the context of human disease, invasive ductal carcinoma and lymph node metastases tissue microarray datasets were analyzed for ephrin- A1 and FASN protein expression. We observed that low ephrin-A1 expression correlated with elevated FASN protein expression. Based on our preliminary data, we hypothesize that ephrin-A1 suppresses mammary epithelial cell lipid metabolism to inhibit breast cancer development and progression. To elucidate the mechanistic connection between ephrin-A1, lipid metabolism and breast cancer growth we have proposed experiments in 2 Specific Aims. In Aim 1, we will determine the mechanism for ephrin-A1 regulation of lipid metabolism in tumor cells and will determine whether it is through EphA2/HER2 clustering or the Akt-mTOR-SREBP signaling. Based on previous studies, we postulate that ephrin-A1 binding to EphA2 RTK may prevent EPHA2/HER clustering or may promote its degradation thus inhibiting lipid biosynthesis. Another possibility is the ephrin-A1 suppresses SREBP activation, since we have shown that ephrin-A1 inhibits Akt activity in breast epithelial cells. In Aim 2 we will systematically analyze ephrin-A1 function in mammary tumor development and progression in MMTV-Neu mice. From these animals we will evaluate protein expression within tumors and will be able to further evaluate targets identified in aims 1. Together these aims will identify the role of ephrin-A1 in breast cancer development, progression and metabolism. Furthermore, these studies will have broader implications since it may reveal a novel mechanism of metabolic regulation, potentially linking ephrin-A1 to other cancers and human diseases with aberrant lipid metabolism.