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

研究 ALG3 在乳腺癌和肺癌中通过 PI3K/AKT 信号传导调节 N-糖基化的作用。

• 批准号: 10116159
• 负责人: Adrija Navarro
• 金额: $ 3.93万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10116159
• 关键词: 3q26; AKT Signaling Pathway; Affect; Amino Acids; Anabolism; Apoptosis; Asparagine; Automobile Driving; Biological Assay; Breast Cancer Cell; Breast Cancer Patient; Breast Carcinoma; Cancer Control; Cancer cell line; Carbohydrates; Cause of Death; Cell physiology; Cells; Combined Modality Therapy; Data; Dependence; Endoplasmic Reticulum; Enzymes; Future; Gene Expression; Genetic; Goals; Growth; Homeostasis; Homologous Gene; Hyperactivity; Laboratories; Lectin; Link; Malignant Neoplasms; Malignant neoplasm of lung; Mannose; Measures; Membrane; Metabolism; Molecular; Nature; Oncogenic; Ovarian Carcinoma; PI3K/AKT; PIK3CA gene; Pathway interactions; Patients; Pattern; Phosphorylation; Play; Polysaccharides; Post-Translational Protein Processing; Process; Proliferating; Protein Glycosylation; Proteins; Proto-Oncogene Proteins c-akt; Regulation; Role; Signal Pathway; Signal Transduction; Signaling Protein; Squamous Cell Lung Carcinoma; Stains; Technology; Therapeutic; Translations; United States; Up-Regulation; Western Blotting; Woman; biological adaptation to stress; cancer cell; cancer therapy; carbohydrate metabolism; effective therapy; endoplasmic reticulum stress; glycosylation; glycosyltransferase; improved; in vivo; in vivo Model; lung cancer cell; malignant breast neoplasm; novel; protein folding; response; targeted treatment; tool; tumor growth; tumor progression; tumorigenesis

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.

PI 3 K/AKT信号通路在癌症中经常失调，控制着关键的细胞过程 例如存活、增殖、代谢和生长。此外，蛋白质糖基化， 这些碳水化合物被添加到氨基酸中，对于蛋白质的正确折叠是必不可少的， 在癌症中失调。在癌症中，癌细胞以更快的速度生长和增殖，因此需要增加的免疫力。 蛋白质折叠能力以维持增加的增殖。天冬酰胺连接的糖基转移酶 糖基化3同源物（ALG 3），其存在于内质网膜中，催化添加 一旦聚糖在聚糖形成过程中翻转到ER腔中， 生物合成ALG 3功能是聚糖生物合成过程中的限速步骤，我们的初步数据显示， 实验表明它是一种新型AKT底物。值得注意的是，我们发现在肺癌和乳腺癌细胞中， ALG 3在PI 3 K和AKT下游磷酸化。据我们所知，这是第一个被发现的 PI 3 K/AKT致癌信号传导和蛋白质糖基化之间的联系在癌症特异性 发信号。此外，ALG 3位于3q 26扩增子中PIK 3CA基因的近端。因此，委员会认为， PIK 3CA和ALG 3在89%、28%和76%的肺鳞状细胞癌和乳腺癌中共扩增， 卵巢癌，分别。推动我提出的项目的假设是ALG 3发挥着关键作用 在调节PI 3 K/AKT信号传导下游的蛋白质N-糖基化中的功能作用， 过度活化的PI 3 K/AKT改变糖基化，导致癌症中的功能后果。我特别 假设具有PIK 3CA扩增和ALG 3上调的细胞增加糖基化和蛋白质合成， 折叠速率，允许细胞科普响应于过度活跃的PI 3 K/AKT而增加的蛋白质翻译。 本项目的目标是确定PI 3 K/AKT信号传导和N-糖基化之间的关系。在 目的1研究PI 3 K/AKT信号通路在通过ALG 3磷酸化调节糖基化中的作用 通过使用蛋白质印迹和信号传导分析来研究磷酸化模式，糖组学来研究 聚糖谱的变化以及肽-糖组学和凝集素染色测定，以鉴定受 去调节ALG 3。在目标2中，我将确定PIK 3CA中ALG 3失调的功能后果。 使用依赖性作图、增殖测定、凋亡测定，通过测量压力来驱动癌症， 反应基因表达，并通过使用体内模型。这个项目将促进我们对 通过PI 3 K/AKT信号调节糖基化及其在癌症进展中的作用，并为 探索靶向PI 3 K/AKT和蛋白糖基化的未来组合策略。