Abstract 4381: Role of Kindlin-2 in the regulation of integrins and TBR1 signaling in TNBC
Authors: Neelum A. Yousaf Zai, Lamyae El Khalki, Wei Wang, Justin Szpendyk, Khalid Sossey-Alaoui
Journal: Cancer Research (2024)

Abstract Background: Kindlins are a small gene family of FERM domain-containing adaptor proteins that function as essential drivers of integrin activation. Kindlin-2 (K2) is the most widely expressed member of the Kindlin family; its homozygous deletion in mice is embryonically lethal, K2 expression is also dysregulated in several human cancers, including the breast. We established K2 as a major driver of TNBC tumors progression and metastasis through the regulation of several hallmarks of cancer. A well-established pathway whereby K2 regulates TNBC oncogenic behavior is through the regulation of integrin inside-out signaling by directly binding to the cytoplasmic tail of integrin β subunit. Interestingly, our recent studies found K2 to play a major role in activating the TGF-β-mediated regulation of the CSF1/EGF signaling through macrophage polarization to the M2 tumorigenic state and their increased tumor infiltration, as well as inhibiting tumor infiltration of cytotoxic lymphocytes, therefore, driving tumor immune evasion. Here we present novel findings involving K2 in the stabilization of the β1-Integrin:TβRI complexes by establishing a physical bridge that links β1-Integrin to TβRI. Loss of K2 results in the degradation of this protein complex and therefore, the inhibition of the oncogenic pathways downstream of these two major regulators of several hallmarks of cancer. Methods: We applied a combination of in vitro assays; CRISPR/Cas9 gene editing, cell migration, 3D tumor-sphere, solid binding, co-immunoprecipitation, cell adhesion and spreading, as well as western blot and flow cytometry analyses with MDA-MB-231 and 4T1 TNBC cell lines. We also used preclinical in vivo mouse models of TNBC tumor progression and metastasis to support or investigations. Results: We found that the K2/β1-Integrin direct interaction is mediated through the C-terminal F3 domain of K2, while the K2/TβRI direct interaction is mediated through the F2 domain of K2. Disruption of this bridge, via CRISPR/Cas9-mediated knockout of K2, leads to β1-Integrin and TβRI degradation, and inhibition of the oncogenic pathways downstream of both β1-Integrin and TβRI, and inhibited tumor growth and metastasis in levels comparable to loss of expression of either β1-Integrin and TβRI. Treatment of the K2-deficent cells with the proteasome inhibitor MG-132 restored expression of both β1-Integrin and TβRI, confirming that K2 is required for the stabilization of the β1-integrin/TβR1 complexes at the protein level. Rescue of K2 expression in the K2-KO cells restored their oncogenic activities both in vitro and in vivo. Conclusion: We identified K2 to play a novel and major in the stabilization of the β1-integrin/TβR1 complexes and the regulation of their downstream oncogenic signaling. These studies have a significant translational impact as they may identify new therapeutically targeted pathways that are desperately needed for the treatment of TNBC tumors. Citation Format: Neelum A. Yousaf Zai, Lamyae El Khalki, Wei Wang, Justin Szpendyk, Khalid Sossey-Alaoui. Role of Kindlin-2 in the regulation of integrins and TBR1 signaling in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4381.