PO.TB03.05 · 肿瘤生物学
骨肉瘤细胞中LRRC15-整合素β1的联系：将黏附与侵袭相连接
LRRC15-Integrin beta1 connection in osteosarcoma cells: Linking adhesion to invasion
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
骨肉瘤(OSA)是儿童和青少年中最常见的原发性骨癌，通常在骨骼快速生长期间发生。OSA的一个常见特征是伴有异常骨形成的成骨细胞表型。富含亮氨酸重复序列15(LRRC15)是一种在多种实体瘤(尤其是OSA)中高表达的细胞表面蛋白。我们此前的工作将LRRC15鉴定为调控OSA细胞迁移、侵袭和细胞-细胞外基质(ECM)相互作用的关键黏附分子。LRRC15敲低的时程单细胞转录组学揭示了若干整合素的失调。整合素是αβ异二聚体跨膜受体，对细胞-ECM黏附和双向信号传导至关重要。β1亚基(ITGB1)在癌症中尤为重要，它与多个α亚基配对以结合胶原、纤连蛋白和层粘连蛋白。LRRC15耗竭在基因和蛋白水平上均适度降低了ITGB1。胶原结合整合素(ITGA1、ITGA10)和RGD结合整合素(ITGAV、ITGB5)同样下调。鉴于LRRC15缺失所影响的通路与整合素生物学之间的重叠，我们假设LRRC15和ITGB1可能功能性相互作用以调控OSA细胞的黏附和迁移。
我们在OSA细胞系中生成了LRRC15和ITGB1的CRISPR-Cas9敲除(KO)。LRRC15和ITGB1 KO细胞在增殖、迁移和球体侵袭方面均表现出相当的缺陷，提示这些分子之间存在强烈的功能联系。定量质谱和Western印迹表明ITGB1 KO细胞中LRRC15表达降低，LRRC15 KO细胞中ITGB1表达降低。值得注意的是，在KO细胞中挽救LRRC15导致ITGB1表达升高。此外，为检测物理相互作用，我们使用亲本和LRRC15 KO细胞的提取物进行了LRRC15免疫沉淀，随后用ITGB1抗体进行免疫印迹。在LRRC15 KO细胞中检测到显著更低的ITGB1信号。同样，当用ITGB1抗体进行免疫沉淀并用LRRC15抗体进行免疫印迹检测时，我们观察到ITGB1 KO细胞中LRRC15表达降低。这些发现表明ITGB1和LRRC15可能彼此物理相互作用。总之，我们的发现表明LRRC15可能调节OSA中整合素介导的黏附和信号传导。具体而言，LRRC15与整合素β1之间的相互作用对于维持驱动细胞增殖、迁移和侵袭的整合素依赖通路似乎至关重要。将需要进一步的机制研究来阐明这种相互作用的分子基础及其对肿瘤进展和转移的贡献。
查看英文原文 English abstract
Osteosarcoma (OSA) is the most common primary bone cancer in children and adolescents, typically arising during rapid skeletal growth. A frequent feature of OSA is an osteoblastic phenotype with abnormal bone formation. Leucine-rich repeat containing 15 (LRRC15) is a cell-surface protein highly expressed in several solid tumors, particularly OSA. Our previous work identified LRRC15 as a key adhesion molecule regulating migration, invasion, and cell-extracellular matrix (ECM) interactions in OSA cells. Time-course single-cell transcriptomics of LRRC15 knockdown revealed dysregulation of several integrins. Integrins are alphabeta heterodimeric transmembrane receptors essential for cell-ECM adhesion and bidirectional signaling. The beta1 subunit (ITGB1) is especially important in cancer, pairing with several alpha subunits to bind collagen, fibronectin, and laminin. LRRC15 depletion modestly reduced ITGB1 at both gene and protein levels. Collagen-binding integrins (ITGA1, ITGA10) and RGD-binding integrins (ITGAV, ITGB5) were similarly downregulated. Given the overlap between pathways affected by LRRC15 loss and integrin biology, we hypothesized that LRRC15 and ITGB1 may functionally interact to regulate OSA cell adhesion and migration.
We generated CRISPR-Cas9 knockouts (KO) of LRRC15 and ITGB1 in OSA cell lines. Both LRRC15 and ITGB1 KO cells exhibited comparable defects in proliferation, migration, and spheroid invasion, suggesting a strong functional connection between these molecules. Quantitative mass spectrometry and western blots demonstrated decreased LRRC15 expression in ITGB1 KO cells, and reduced ITGB1 expression in LRRC15 KO cells. Notably, rescue of LRRC15 in KO cells resulted in elevated ITGB1 expression. Furthermore, to detect physical interaction, we performed immunoprecipitation with LRRC15 using extracts from parental and LRRC15 KO cells, followed by immunoblotting with an ITGB1 antibody. A significantly lower ITGB1 signal was detected in LRRC15 KO cells. Similarly, when immunoprecipitation was performed with an ITGB1 antibody and detected by immunoblotting with an LRRC15 antibody, we observed reduced LRRC15 expression in ITGB1 KO cells. These findings indicate that ITGB1 and LRRC15 may physically interact with each other. Taken together, our findings indicate that LRRC15 may modulate integrin-mediated adhesion and signaling in OSA. Specifically, the interaction between LRRC15 and integrin beta1 appears critical for maintaining integrin-dependent pathways that drive cell proliferation, migration, and invasion. Further mechanistic studies will be required to delineate the molecular basis of this interaction and its contribution to tumor progression and metastasis
利益披露 Disclosure
S. Mukherjee, None..
W. Chen, None..
L. M. Jenkins, None..
Y. Zhu, None..
M. Pineda, None..
R. Walker, None..
P. S. Meltzer, None.