Investigating the role of ALG3 in the regulation of N-glycosylation by PI3K/AKT signaling in breast and lung cancer.

PI3K/AKT信号通路在癌症中经常失控，控制着关键的细胞过程 例如生存、增殖、新陈代谢和生长。此外，蛋白质的糖基化过程是通过 哪些碳水化合物被添加到氨基酸中，对于正确的蛋白质折叠是必不可少的，而且经常 在癌症方面放松管制。在癌症中，癌细胞以更快的速度生长和扩散，因此需要增加 蛋白质折叠能力，以维持更高的增殖能力。天冬酰胺连接的糖基转移酶 内质网膜上的糖基化3同系物(ALG3)催化加成反应 一旦在多糖过程中将甘露糖单位翻转到内质网管腔，甘露糖单位就变成了葡聚糖前体 生物合成。ALG3功能是多糖生物合成过程中的一个限速步骤，来自我们的初步数据 实验室证明，它是一种新型的AKT基片。值得注意的是，我们发现在肺癌和乳腺癌细胞中， ALG3在PI3K和AKT下游被磷酸化。据我们所知，这是第一个确定的 癌特异性背景下PI3K/AKT致癌信号和蛋白质糖基化之间的联系 发信号。此外，ALG3位于3q26扩增子中PIK3CA基因的近端。因此， PIK3CA和ALG3在、28%和76%的肺鳞状细胞癌和乳腺癌中共扩增 分别为卵巢癌和卵巢癌。推动我提出的项目的假设是ALG3起着关键作用 在PI3K/AKT信号下游蛋白N-糖基化调节中的功能作用 过度激活的PI3K/AKT改变糖基化，导致癌症的功能后果。具体来说，我 假设含有PIK3CA扩增和ALG3上调的细胞增加了糖基化和蛋白质 折叠速度，使细胞能够应对过度活跃的PI3K/AKT增加的蛋白质翻译。 本项目的目标是确定PI3K/AKT信号与N-糖基化的关系。在……里面 目的1我将评估PI3K/AKT信号在通过ALG3磷酸化调节糖基化中的作用 通过蛋白质印迹和信号分析来研究磷酸化模式，糖组分来研究 糖链图谱的变化，以及肽-糖组分和凝集素染色分析，以确定受影响的蛋白质 解除对ALG3的管制。在目标2中，我将确定PIK3CA中解除管制的ALG3的功能后果- 使用依赖图，增殖分析，细胞凋亡分析，通过测量压力- 反应基因的表达，并使用体内模型。这个项目将增进我们对 PI3K/AKT信号对糖基化的调节及其在肿瘤进展中的作用 探索未来针对PI3K/AKT和蛋白质糖基化的组合策略。

The PI3K/AKT signaling pathway, which is frequently dysregulated in cancer, controls key cellular processes such as survival, proliferation, metabolism, and growth. Additionally, protein glycosylation, the process by which carbohydrates are added to amino acids, is essential for proper protein folding and is also frequently deregulated in cancer. In cancer, cancer cells grow and proliferate at faster rates, and thus require increased protein folding capacity to sustain increased proliferation. The glycosyltransferase Asparagine-linked glycosylation 3 homolog (ALG3), which resides in the endoplasmic reticulum membrane, catalyzes the addition of mannose units to a glycan precursor once the glycan is flipped into the ER lumen during glycan biosynthesis. ALG3 function is a rate-limiting step during glycan biosynthesis and preliminary data from our laboratory show that it is a novel AKT substrate. Notably, we find that in both lung and breast cancer cells, ALG3 is phosphorylated downstream of PI3K and AKT. This represents, to our knowledge, the first identified link between PI3K/AKT oncogenic signaling and protein glycosylation in the context of cancer-specific signaling. Additionally, ALG3 resides proximal to the PIK3CA gene in the 3q26 amplicon. Consequently, PIK3CA and ALG3 are co-amplified in 89%, 28% and 76% of lung squamous cell carcinoma and breast and ovarian carcinoma, respectively. The hypothesis driving my proposed project is that ALG3 plays a key functional role in the regulation of protein N-glycosylation downstream of PI3K/AKT signaling, and that hyperactivated PI3K/AKT alters glycosylation, leading to functional consequences in cancer. Specifically, I postulate that cells that harbor PIK3CA amplification and ALG3 upregulation increase glycosylation and protein folding rates, allowing cells to cope with increased protein translation in response to hyperactive PI3K/AKT. The goal of this project is to determine the relationship between PI3K/AKT signaling and N-glycosylation. In Aim 1 I will assess the role of PI3K/AKT signaling in modulating glycosylation through ALG3 phosphorylation by using western blotting and signaling assays to investigate phosphorylation patterns, glycomics to investigate changes in the glycan profile, and peptido-glycomics and lectin staining assays to identify proteins affected by deregulated ALG3. In Aim 2 I will determine the functional consequences of deregulated ALG3 in PIK3CA- driven cancer using dependency mapping, proliferation assays, apoptosis assays, by measuring stress- response gene expression, and by using an in vivo model. This project will advance our understanding of the regulation of glycosylation by PI3K/AKT signaling and its role in cancer progression, and pave the way for exploring future combination strategies targeting PI3K/AKT and protein glycosylation.