CD44 molecule (CD44) is a well-known surface glycoprotein on tumor-initiating cells or cancer stem cells. However, its utility as a therapeutic target for managing metastases remains to be fully evaluated. We previously demonstrated that CD44 mediates homophilic interactions for circulating tumor cell (CTC) cluster formation, which enhances cancer stemness and metastatic potential in association with an unfavorable prognosis. Furthermore, CD44 self-interactions activate the P21-activated kinase 2 (PAK2) signaling pathway. Here, we further examined the biochemical properties of CD44 in homotypic tumor cell aggregation. The standard CD44 form (CD44s) mainly assembled as intercellular homodimers (trans-dimers) in tumor clusters rather than intracellular dimers (cis-dimers) present in single cells. Machine learning-based computational modeling combined with experimental mutagenesis tests revealed that the extracellular Domains I and II of CD44 are essential for its trans-dimerization and predicted high-score residues to be required for dimerization. Substitutions of 10 these residues in Domain I (Ser-45, Glu-48, Phe-74, Cys-77, Arg-78, Tyr-79, Ile-88, Arg-90, Asn-94, and Cys-97) or 5 residues in Domain II (Ile-106, Tyr-155, Val-156, Gln-157, and Lys-158) abolished CD44 dimerization and reduced tumor cell aggregation in vitro Importantly, the substitutions in Domain II dramatically inhibited lung colonization in mice. The CD44 dimer-disrupting substitutions decreased downstream PAK2 activation without affecting the interaction between CD44 and PAK2, suggesting that PAK2 activation in tumor cell clusters is CD44 trans-dimer-dependent. These results shed critical light on the biochemical mechanisms of CD44-mediated tumor cell cluster formation and may help inform the development of therapeutic strategies to prevent tumor cluster formation and block cluster-mediated metastases.

中文翻译：

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细胞表面糖蛋白CD44的胞外域I和II介导其反同型二聚化和肿瘤簇聚集。

CD44分子（CD44）是肿瘤引发细胞或癌症干细胞上的一种众所周知的表面糖蛋白。然而，其作为治疗转移的治疗靶点的效用仍有待充分评估。我们以前证明CD44介导循环肿瘤细胞（CTC）簇形成的同质相互作用，这会增加癌症的干性和转移潜力，并伴有不良的预后。此外，CD44自相互作用激活P21激活的激酶2（PAK2）信号传导途径。在这里，我们进一步检查了CD44在同型肿瘤细胞聚集中的生化特性。标准的CD44形式（CD44s）主要组装为肿瘤簇中的细胞间同型二聚体（反式二聚体），而不是单个细胞中存在的细胞内二聚体（顺式二聚体）。基于机器学习的计算模型与实验诱变测试相结合，揭示了CD44的胞外域I和II对于其反式二聚作用至关重要，并且预测了二聚化所需的高分数残基。域I（Ser-45，Glu-48，Phe-74，Cys-77，Arg-78，Tyr-79，Ile-88，Arg-90，Asn-94和Cys-97）中10个这些残基的取代域II中的5个残基（Ile-106，Tyr-155，Val-156，Gln-157和Lys-158）在体外消除了CD44二聚化作用并减少了肿瘤细胞的聚集。重要的是，域II中的取代显着抑制了小鼠肺部定植老鼠。CD44二聚体破坏性取代降低了下游PAK2活化，而没有影响CD44与PAK2之间的相互作用，这表明肿瘤细胞簇中PAK2活化是CD44反式二聚体依赖性的。