Li Tao, Fei Yuxiang, Sun Xu, Zhu Jun, Fang Xin, Meng Fanjun, Wang Danyi, Zhang Xu, Liu Chao, Du Qianming
General Clinical Research Center, Nanjing First Hospital, Nanjing Medical University, Nanjing, P.R. China; School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, P.R. China; Nanjing First Hospital, Nanjing University of Chinese Medicine, Nanjing, P.R. China.
School of Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing, P.R. China; Department of Pharmacy, Nanjing First Hospital, Nanjing Medical University, Nanjing, P.R. China; Nanjing First Hospital, Nanjing University of Chinese Medicine, Nanjing, P.R. China.
Cell Mol Gastroenterol Hepatol. 2025 Apr 26;19(9):101524. doi: 10.1016/j.jcmgh.2025.101524.
BACKGROUND & AIMS: Heterogeneity is the malignancy feature of colorectal cancer (CRC), which augments the difficulty of CRC treatment. Consensus molecular subtypes (CMSs) classified CRC into 4 subtypes. CMS4 is the most aggressive subtype, characterized with epithelial mesenchymal transition (EMT) and angiogenesis activation. However, the mechanism of CMS4 formation still remains unclear. We aimed to investigate the role of phospholamban (PLN) on CMS4 formation.
Immunohistochemistry and Western blotting were used to detected PLN expressions. Transwell and wound-healing assays were used to evaluate migration and invasion of HCT116 and HT29. Tube formation assay was used to evaluate angiogenesis of HUVECs. GST pull-down was used to detected the interaction between PLN and AXIN2. The CAF-CT26 spleen co-transplantation mouse model was built to evaluate PLN's effects on CRC metastasis.
PLN knockdown reversed CAFs' conditional medium (CM)-induced EMT, cell migration, and angiogenesis (P < .01). PLN strongly bound to AXIN2, the main component of β-catenin degradation complex, and PLN knockdown increased β-catenin ubiquitination in CAFs (P < .01). PLN knockdown blocked GREM1 secretion (P < .01), and PLN overexpression-induced EMT, cell migration, and angiogenesis were blocked by anti-GREM1 neutralizing antibody (P < .01). Knockdown of BMP2 and VEGFR2 reversed CAFs' CM induced EMT and angiogenesis effects, respectively (P < .01). PLN knockdown attenuated CAFs mediated tumor promoting effects in vivo (P < .01).
PLN competitively binds with AXIN2 to increase β-catenin activity in CAFs. β-catenin signal induces GREM1 secretion, driving EMT and angiogenesis via BMP2 and VEGFR2, respectively. These findings demonstrate that PLN is a driver for CMS4 formation.
异质性是结直肠癌（CRC）的恶性特征，这增加了CRC治疗的难度。共识分子亚型（CMSs）将CRC分为4种亚型。CMS4是最具侵袭性的亚型，其特征为上皮-间质转化（EMT）和血管生成激活。然而，CMS4形成的机制仍不清楚。我们旨在研究受磷蛋白（PLN）在CMS4形成中的作用。
采用免疫组织化学和蛋白质印迹法检测PLN表达。采用Transwell和伤口愈合试验评估HCT116和HT29的迁移和侵袭能力。采用管腔形成试验评估人脐静脉内皮细胞（HUVECs）的血管生成能力。采用谷胱甘肽-S-转移酶沉降试验（GST pull-down）检测PLN与轴抑制蛋白2（AXIN2）之间的相互作用。构建癌相关成纤维细胞（CAF）-CT26脾脏共移植小鼠模型，评估PLN对CRC转移的影响。
敲低PLN可逆转CAF条件培养基（CM）诱导的EMT、细胞迁移和血管生成（P <.01）。PLN与β-连环蛋白降解复合物的主要成分AXIN2紧密结合，敲低PLN可增加CAF中β-连环蛋白的泛素化水平（P <.01）。敲低PLN可阻断生长调节致癌基因1（GREM1）的分泌（P <.01），抗GREM1中和抗体可阻断PLN过表达诱导的EMT、细胞迁移和血管生成（P <.01）。敲低骨形态发生蛋白2（BMP2）和血管内皮生长因子受体2（VEGFR2）可分别逆转CAF的CM诱导的EMT和血管生成作用（P <.01）。敲低PLN可减弱CAF在体内介导的肿瘤促进作用（P <.01）。
PLN与AXIN2竞争性结合，以增加CAF中β-连环蛋白的活性。β-连环蛋白信号诱导GREM1分泌，分别通过BMP2和VEGFR2驱动EMT和血管生成。这些发现表明，PLN是CMS4形成的驱动因素。
