背景Kindlin-2,一种衔接蛋白,在各种人类癌症中失调,包括三阴性乳腺癌(TNBC),它通过影响几种癌症标志来驱动肿瘤进展和转移。Kindlin-2的一个公认的作用涉及整合素信号的调节,通过直接结合整合素β亚基的细胞质尾部来实现。在这项研究中,我们对Kindlin-2参与稳定β1-整合素:TGF-β1型受体(TβRI)复合物的新见解,充当连接β1-整合素和TβRI的物理桥梁。Kindlin-2的丢失导致这种蛋白质复合物的降解,导致下游致癌途径的抑制。方法我们的方法包括不同范围的体外测定,包括CRISPR/Cas9基因编辑,细胞迁移,3D肿瘤球形成和侵袭,固体结合,免疫共沉淀,细胞粘附和铺展试验,以及蛋白质印迹和流式细胞术分析,利用MDA-MB-231和4T1TNBC细胞系。此外,TNBC肿瘤进展和转移的临床前体内小鼠模型被用来证实我们的发现。结果研究表明,Kindlin-2与β1-整合素之间的直接相互作用是通过Kindlin-2的C端F3结构域介导的,而Kindlin-2与TβRI之间的相互作用是通过Kindlin-2的F2结构域促进的。这座桥的中断,通过CRISPR/Cas9介导的Kindlin-2基因敲除,导致β1-整合素和TβRI的降解,导致两种蛋白质下游的致癌途径受到抑制,随后阻碍肿瘤的生长和转移。用蛋白酶体抑制剂MG-132处理Kindlin-2缺陷细胞恢复了β1-整合素和TβRI的表达。此外,Kindlin-2表达的挽救在体外和体内恢复了它们的致癌活性。结论本研究鉴定了Kindlin-2在稳定β1-整合素:TβR1复合物和调节其下游致癌信号传导中的新功能。这些发现的转化意义是巨大的,可能揭示对TNBC肿瘤治疗至关重要的新的治疗靶向途径。
UNASSIGNED: Kindlin-2, an adaptor protein, is dysregulated in various human cancers, including triple negative breast cancer (TNBC), where it drives tumor progression and metastasis by influencing several cancer hallmarks. One well-established role of Kindlin-2 involves the regulation of integrin signaling, achieved by directly binding to the cytoplasmic tail of the integrin β subunit. In this study, we present novel insights into Kindlin-2\'s involvement in stabilizing the β1-Integrin:TGF-β type 1 receptor (TβRI) complexes, acting as a physical bridge that links β1-Integrin to TβRI. The loss of Kindlin-2 results in the degradation of this protein complex, leading to the inhibition of downstream oncogenic pathways.
UNASSIGNED: Our methodology encompassed a diverse range of in vitro assays, including CRISPR/Cas9 gene editing, cell migration, 3D tumorsphere formation and invasion, solid binding, co-immunoprecipitation, cell adhesion and spreading assays, as well as western blot and flow cytometry analyses, utilizing MDA-MB-231 and 4T1 TNBC cell lines. Additionally, preclinical in vivo mouse models of TNBC tumor progression and metastasis were employed to substantiate our findings.
UNASSIGNED: The investigation revealed that the direct interaction between Kindlin-2 and β1-Integrin is mediated through the C-terminal F3 domain of Kindlin-2, while the interaction between Kindlin-2 and TβRI is facilitated through the F2 domain of Kindlin-2. Disruption of this bridge, achieved via CRISPR/Cas9-mediated knockout of Kindlin-2, led to the degradation of β1-Integrin and TβRI, resulting in the inhibition of oncogenic pathways downstream of both proteins, subsequently hindering tumor growth and metastasis. Treatment of Kindlin-2-deficient cells with the proteasome inhibitor MG-132 restored the expression of both β1-Integrin and TβRI. Furthermore, the rescue of Kindlin-2 expression reinstated their oncogenic activities both in vitro and in vivo.
UNASSIGNED: This study identifies a novel function of Kindlin-2 in stabilizing the β1-Integrin:TβR1 complexes and regulating their downstream oncogenic signaling. The translational implications of these findings are substantial, potentially unveiling new therapeutically targeted pathways crucial for the treatment of TNBC tumors.