Department of Immunology, School of Basic Medical Sciences, Anhui Medical University, Hefei, 230032, Anhui, China.
School of Biomedical Sciences and Pharmacy, The University of Newcastle, Newcastle, NSW, 2308, Australia.
J Exp Clin Cancer Res. 2024 Jan 11;43(1):17. doi: 10.1186/s13046-023-02937-1.
Prohibitin 2 (PHB2) exhibits opposite functions of promoting or inhibiting tumour across various cancer types. In this study, we aim to investigate its functions and underlying mechanisms in the context of gastric cancer (GC).
PHB2 protein expression levels in GC and normal tissues were examined using western blot and immunohistochemistry. PHB2 expression level associations with patient outcomes were examined through Kaplan-Meier plotter analysis utilizing GEO datasets (GSE14210 and GSE29272). The biological role of PHB2 and its subsequent regulatory mechanisms were elucidated in vitro and in vivo. GC cell viability and proliferation were assessed using MTT cell viability analysis, clonogenic assays, and BrdU incorporation assays, while the growth of GC xenografted tumours was measured via IHC staining of Ki67. The interaction among PHB2 and SHIP2, as well as between SHIP2 and NEDD4, was identified through co-immunoprecipitation, GST pull-down assays, and deletion-mapping experiments. SHIP2 ubiquitination and degradation were assessed using cycloheximide treatment, plasmid transfection and co-immunoprecipitation, followed by western blot analysis.
Our analysis revealed a substantial increase in PHB2 expression in GC tissues compared to adjacent normal tissues. Notably, higher PHB2 levels correlated with poorer patient outcomes, suggesting its clinical relevance. Functionally, silencing PHB2 in GC cells significantly reduced cell proliferation and retarded GC tumour growth, whereas overexpression of PHB2 further enhanced GC cell proliferation. Mechanistically, PHB2 physically interacted with Src homology 2-containing inositol 5-phosphatase 2 (SHIP2) in the cytoplasm of GC cells, thus leading to SHIP2 degradation via its novel E3 ligase NEDD4. It subsequently activated the PI3K/Akt signaling pathway and thus promoted GC cell proliferation.
Our findings highlight the importance of PHB2 upregulation in driving GC progression and its association with adverse patient outcomes. Understanding the functional impact of PHB2 on GC growth contributes valuable insights into the molecular underpinnings of GC and may pave the way for the development of targeted therapies to improve patient outcomes.
在各种癌症类型中，抑素 2（PHB2）表现出促进或抑制肿瘤的相反功能。在这项研究中，我们旨在研究其在胃癌（GC）中的功能和潜在机制。
使用 Western blot 和免疫组织化学检测 GC 和正常组织中的 PHB2 蛋白表达水平。通过利用 GEO 数据集（GSE14210 和 GSE29272）中的 Kaplan-Meier plotter 分析检查 PHB2 表达水平与患者结局的关联。在体外和体内阐明 PHB2 的生物学作用及其后续调节机制。通过 MTT 细胞活力分析、集落形成测定和 BrdU 掺入测定评估 GC 细胞活力和增殖，通过 Ki67 的 IHC 染色测量 GC 异种移植瘤的生长。通过共免疫沉淀、GST 下拉测定和缺失作图实验鉴定 PHB2 与 SHIP2 之间以及 SHIP2 与 NEDD4 之间的相互作用。通过使用环己酰亚胺处理、质粒转染和共免疫沉淀，随后进行 Western blot 分析来评估 SHIP2 的泛素化和降解。
我们的分析表明，GC 组织中的 PHB2 表达明显高于相邻正常组织。值得注意的是，较高的 PHB2 水平与较差的患者结局相关，提示其临床相关性。功能上，GC 细胞中 PHB2 的沉默显著降低了细胞增殖并延缓了 GC 肿瘤的生长，而 PHB2 的过表达进一步增强了 GC 细胞的增殖。从机制上讲，PHB2 在 GC 细胞质中与 Src 同源 2 包含的肌醇 5-磷酸酶 2（SHIP2）相互作用，从而通过其新型 E3 连接酶 NEDD4 导致 SHIP2 降解。它随后激活了 PI3K/Akt 信号通路，从而促进了 GC 细胞的增殖。
我们的研究结果强调了 PHB2 上调在推动 GC 进展中的重要性及其与不良患者结局的关联。了解 PHB2 对 GC 生长的功能影响为 GC 的分子基础提供了有价值的见解，并为开发改善患者结局的靶向治疗铺平了道路。
