Kindlin-2 Regulates the Oncogenic Activities of Integrins and TGF-β In Triple Negative Breast Cancer Progression and Metastasis

Abstract

Abstract Background 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. Methods 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. Results 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. Conclusions 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.