MetroHealth System, Cleveland, OH, USA.
Case Western Reserve University, Cleveland, OH, USA.
Oncogene. 2024 Nov;43(45):3291-3305. doi: 10.1038/s41388-024-03166-2. Epub 2024 Sep 19.
Kindlin-2, an adapter 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. Loss of Kindlin-2 results in the degradation of this protein complex, leading to the inhibition of downstream oncogenic pathways. We used 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. Our studies established the direct interaction between Kindlin-2 and β1-Integrin and between Kindlin-2 and TβRI. Disruption of these interactions, 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 in vitro and in vivo, while Kindlin-2 lacking domains involved in the interaction of Kindlin-2 with β1-Integrin or TβRI did not. This study identifies a novel function of Kindlin-2 in stabilizing the β1-Integrin:TβRI 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.
连接蛋白-2（Kindlin-2）是一种衔接蛋白，在多种人类癌症中失调，包括三阴性乳腺癌（TNBC），在该癌症中，它通过影响几种癌症特征来驱动肿瘤进展和转移。Kindlin-2 的一个成熟作用涉及整合素信号的调节，通过直接结合整合素β亚基的细胞质尾巴来实现。在这项研究中，我们提供了有关 Kindlin-2 参与稳定 β1-整合素：转化生长因子-β 型 1 受体（TβRI）复合物的新见解，作为连接 β1-整合素和 TβRI 的物理桥。Kindlin-2 的缺失导致该蛋白复合物的降解，从而抑制下游致癌途径。我们使用了多种体外测定法，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、共免疫沉淀、细胞黏附和铺展测定以及 Western blot 和流式细胞术分析，利用 MDA-MB-231 和 4T1 TNBC 细胞系。此外，还使用 TNBC 肿瘤进展和转移的临床前体内小鼠模型来证实我们的发现。我们的研究确立了 Kindlin-2 与 β1-整合素之间以及 Kindlin-2 与 TβRI 之间的直接相互作用。通过 CRISPR/Cas9 介导的 Kindlin-2 基因敲除破坏这些相互作用，导致 β1-整合素和 TβRI 的降解，从而抑制这两种蛋白下游的致癌途径，随后阻碍肿瘤生长和转移。用蛋白酶体抑制剂 MG-132 处理缺乏 Kindlin-2 的细胞可恢复 β1-整合素和 TβRI 的表达。此外，在体外和体内恢复 Kindlin-2 的表达可恢复其致癌活性，而缺乏与 Kindlin-2 与 β1-整合素或 TβRI 的相互作用相关的结构域的 Kindlin-2 则没有。这项研究确定了 Kindlin-2 在稳定 β1-整合素：TβRI 复合物及其调节其下游致癌信号中的新功能。这些发现的转化意义重大，可能揭示了新的治疗靶点途径，对 TNBC 肿瘤的治疗至关重要。
