Class III PI3K as an Autophagy Reactivation Switch in Malignant Transformation

项目摘要 巨自噬（Macroautophagy，以下简称自噬）对细胞内稳态至关重要。在健康细胞中， 基础水平的自噬对于适当的体内平衡是必需的，并且对于抗癌是必需的。 免疫监视当自噬受损时，可能会发生恶性转化。自噬 活性很低，直到肿瘤超过其可用的营养供应。随着肿瘤的生长超过血液 供应不足，环境变得缺氧，自噬再激活是肿瘤发生所必需的。 扩散和入侵。抗疟药氯喹先导物对自噬的药物抑制作用 然而，对于肿瘤消退，这种药物不是癌细胞特异性的，并且具有许多脱靶途径。 自噬在癌症中的这种双重作用使我们在治疗上靶向它的能力变得复杂。尚不清楚 自噬功能是如何恢复的，这是该领域的核心问题。 所有的自噬激活机制都涉及脂质激酶III类磷脂酰肌醇3-激酶 含有肿瘤抑制蛋白BECN 1的复合物I（PI 3 KC 3-C1）。PI 3 KC 3-C1是中心引发剂， 然而，它的激活机制是未知的，这使得它成为一个具有挑战性的酶靶向 治疗上在原子水平上发现这种中心脂质激酶的激活机制 复杂的冷冻电子显微镜将大大有助于合理设计治疗的能力， 在癌症转化或增殖的不同阶段选择性地激活或抑制PI 3 KC 3-C1。 为了鉴定SILAC中自噬再激活过程中蛋白质组的大规模变化（稳定 细胞培养物中氨基酸的同位素标记）与质谱联用， 执行。这里的目的是确定增殖肿瘤中是否存在独特的靶点， 依赖于特定的自噬激活机制或选择性自噬途径。 理想情况下，这项研究将产生新的候选蛋白质，以治疗为目标， 比羟氯喹更有针对性，毒性更低，用于治疗胰腺癌。

Project Summary Macroautophagy (hereafter autophagy) is crucial to cellular homeostasis. In healthy cells, high levels of basal autophagy are necessary for proper homeostasis and are required for anticancer immunosurveillance. Malignant transformations can arise when autophagy is impaired. Autophagic activity is low until the tumor outpaces its available nutrient supply. As the tumor outgrows its blood supply, the environment become hypoxic, and autophagy re-activation is necessary for tumor proliferation and invasion. Pharmological inhibition of autophagy by antimalarial drug chloroquine leads to tumor regression, however, this drug is not specific to cancerous cells and has many off-target paths. This dual role of autophagy in cancer complicates our ability to target it therapeutically. It is unknown how autophagic function is restored, and this is a central question in the field. All autophagy activation mechanisms involve the lipid kinase class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) containing tumor suppressor protein BECN1. PI3KC3-C1 is a central initiator, however, its activation mechanism is unknown and this makes it a challenging enzyme to target therapeutically. Discovery of the activation mechanism, at the atomic level, of this central lipid kinase complex through cryo-electron microscopy will greatly aid the ability to rationally design therapeutics to selectively activate or inhibit PI3KC3-C1 at various stages in cancer transformation or proliferation. To identify large-scale changes in the proteome during autophagy reactivation in SILAC (Stable Isotope Labeling with Amino acids in Cell culture) coupled with mass spectrometry will be performed. The objective here is to determine if there is a unique target in proliferating tumors, dependent on a specific autophagy activation mechanism or on a selective autophagy pathway. Ideally, this study would produce new protein candidates to target therapeutically, which would be more specific and less toxic than hydroxychloroquine to treat pancreatic cancers.