Mapping the interactome of OSCC prognostic-associated proteins NDRG1 and PGK1 through proximity labeling using TurboID

Oral squamous cell carcinoma (OSCC) is a prevalent type of head and neck cancer, accounting for more than 90% of all oral malignancies worldwide. The identification of diagnostic and prognostic markers for OSCC is crucial for improving patient outcomes, as early detection and treatment are critical for successful management of this disease. In this regard, our group recently demonstrated that N-myc downstream-regulated gene 1 (NDRG1) and phosphoglycerate kinase 1 (PGK1) are prognostic markers in OSCC, however, little is known about the role of these proteins in OSCC development. To better understand their signaling pathways, we used TurboID-based proximity labeling to identify the interactomes of NDRG1 and PGK1 in HEK293 cells. Here, protein abundance patterns associated with three distinct time-points were utilized for protein clustering, allowing the identification of protein clusters with a “fast” or “slow” response to biotin. Functional enrichment of GO terms revealed processes mostly associated with mRNA processing for both PGK1 and NDRG1 “fast” clusters, while GO processes related to protein localization and catalytic activity were identified in PGK1 and NDRG1 “slow” clusters, respectively. A total of 65 out of 361 proteins from different clusters were also identified in neoplastic islands of OSCC tissues from our previous study. Additionally, 28 of these proteins have their gene expression associated with prognostic features such as death, metastasis and/or relapse in OSCC patients. Ultimately, our data enabled the identification of 17 previously known physical interactions and functional associations among these proteins, as well as 30 new putative interactions, characterizing a prognostic-associated interactome composed of 28 proteins. This interactome enables the prioritization of candidates that can be further explored in OSCC progression.

口腔鳞状细胞癌（oral squamous cell carcinoma, OSCC）是一类常见的头颈部恶性肿瘤，占全球所有口腔恶性肿瘤的90%以上。明确OSCC的诊断与预后标志物对改善患者预后结局至关重要，因为早期检出与干预是该疾病成功管控的关键。既往本团队研究证实，N-myc下游调控基因1（NDRG1）与磷酸甘油酸激酶1（PGK1）可作为OSCC的预后标志物，但目前对这两种蛋白在OSCC发生发展中的作用尚知之甚少。为深入解析二者的信号通路，我们采用基于TurboID的邻近标记技术，在HEK293细胞中鉴定了NDRG1与PGK1的互作组。本研究利用三个不同时间点的蛋白质丰度模式开展蛋白质聚类，成功鉴定出对生物素呈现“快速”或“缓慢”响应的蛋白质簇。基因本体（Gene Ontology, GO）功能富集分析显示，PGK1与NDRG1的“快速”响应簇主要富集于mRNA加工相关生物学过程；而PGK1与NDRG1的“缓慢”响应簇则分别富集于蛋白质定位与催化活性相关的GO生物学过程。我们从前述研究的OSCC组织肿瘤岛中，共鉴定出361个不同簇蛋白中的65个。此外，其中28个蛋白的基因表达与OSCC患者的死亡、转移及/或复发等预后特征显著相关。最终，本研究不仅鉴定出这些蛋白间已报道的17种物理相互作用与功能关联，还发现了30种全新的潜在相互作用，构建了由28个蛋白组成的预后相关互作组。该互作组可优先筛选出可进一步用于OSCC进展研究的候选蛋白。