Kim Seung-Su, Park Issac, Kim Jeesoo, Ka Na-Lee, Lim Ga Young, Park Mi-Ye, Hwang Sewon, Kim Ji-Eun, Park So Yeon, Kim Jong-Seo, Rhee Hyun-Woo, Lee Mi-Ock
College of Pharmacy, Seoul National University, Seoul, 08826, South Korea.
Department of Chemistry, Seoul National University, Seoul, Republic of Korea.
Breast Cancer Res. 2025 Jan 9;27(1):4. doi: 10.1186/s13058-024-01958-8.
Patients with estrogen receptor (ER)-positive breast cancer (BC) can be treated with endocrine therapy targeting ER, however, metastatic recurrence occurs in 25% of the patients who have initially been treated. Secreted proteins from tumors play important roles in cancer metastasis but previous methods for isolating secretory proteins had limitations in identifying novel targets.
We applied an in situ secretory protein labeling technique using TurboID to analyze secretome from tamoxifen-resistant (TAMR) BC. The increased expression of LGALS3BP was validated using western blotting, qPCR, ELISA, and IF. Chromatin immunoprecipitation was applied to analyze estrogen-dependent regulation of LGALS3BP transcription. The adhesive and angiogenic functions of LGALS3BP were evaluated by abrogating LGALS3BP expression using either shRNA-mediated knockdown or a neutralizing antibody. Xenograft mouse experiments were employed to assess the in vivo metastatic potential of TAMR cells and the LGALS3BP protein. Clinical evaluation of LGALS3BP risk was carried out with refractory clinical specimens from tamoxifen-treated ER-positive BC patients and publicly available databases.
TAMR secretome analysis revealed that 176 proteins were secreted at least 2-fold more from MCF7/TAMR cells than from sensitive cells, and biological processes such as cell adhesion and angiogenesis were associated with the TAMR secretome. Galectin-3 binding protein (LGALS3BP) was one of the top 10 most highly secreted proteins in the TAMR secretome. The expression level of LGALS3BP was suppressed by estrogen signaling, which involves direct ERα binding to its promoter region. Secreted LGALS3BP in the TAMR secretome helped BC cells adhere to the extracellular matrix and promoted the tube formation of human umbilical vein endothelial cells. Compared with sensitive cells, xenograft animal experiments with MCF7/TAMR cells showed increased pulmonary metastasis, which completely disappeared in LGALS3BP-knockdown TAMR cells. Finally, higher levels of LGALS3BP were associated with poor prognosis in ER-positive BC patients treated with adjuvant tamoxifen in the clinic.
TAMR secretome analysis identified secretory proteins, such as LGALS3BP, that are involved in biological processes closely related to metastasis. Secreted LGALS3BP from the TAMR cells promoted adhesion of the cells to the extracellular matrix and vasculature formation, which may support metastasis of TAMR cells.
雌激素受体（ER）阳性乳腺癌（BC）患者可采用靶向ER的内分泌治疗，但在初始接受治疗的患者中，有25%会发生转移性复发。肿瘤分泌的蛋白质在癌症转移中起重要作用，但以往分离分泌蛋白的方法在识别新靶点方面存在局限性。
我们应用TurboID原位分泌蛋白标记技术分析他莫昔芬耐药（TAMR）BC的分泌组。通过蛋白质免疫印迹法、定量聚合酶链反应、酶联免疫吸附测定和免疫荧光法验证了LGALS3BP的表达增加。应用染色质免疫沉淀法分析LGALS3BP转录的雌激素依赖性调控。通过短发夹RNA介导的敲低或中和抗体消除LGALS3BP表达，评估LGALS3BP的黏附及血管生成功能。采用异种移植小鼠实验评估TAMR细胞和LGALS3BP蛋白的体内转移潜能。利用他莫昔芬治疗的ER阳性BC患者的难治性临床标本和公开可用数据库对LGALS3BP风险进行临床评估。
TAMR分泌组分析显示，MCF7/TAMR细胞分泌的176种蛋白质比敏感细胞至少多2倍，细胞黏附、血管生成等生物学过程与TAMR分泌组相关。半乳糖凝集素-3结合蛋白（LGALS3BP）是TAMR分泌组中分泌量最高的前10种蛋白质之一。LGALS3BP的表达水平受雌激素信号抑制，雌激素信号涉及雌激素受体α直接结合到其启动子区域。TAMR分泌组中分泌的LGALS3BP有助于BC细胞黏附于细胞外基质，并促进人脐静脉内皮细胞的管腔形成。与敏感细胞相比，用MCF7/TAMR细胞进行的异种移植动物实验显示肺转移增加，而在LGALS3BP敲低的TAMR细胞中肺转移完全消失。最后，在临床上，接受辅助他莫昔芬治疗的ER阳性BC患者中，较高水平的LGALS3BP与不良预后相关。
TAMR分泌组分析鉴定出了参与与转移密切相关的生物学过程的分泌蛋白，如LGALS3BP。TAMR细胞分泌的LGALS3BP促进细胞与细胞外基质的黏附以及脉管系统形成，这可能支持TAMR细胞的转移。
