Targeting the Cx26/NANOG/Focal Adhesion Kinase Complex via Cell Penetrating Peptides in Triple Negative Breast Cancers

Abstract

Purpose Triple-negative breast cancer (TNBC) represents the most lethal and treatment-resistant breast cancer subtype and has limited treatment options. We previously identified a protein complex unique to TNBC cancer stem cells composed of the gap junction protein connexin 26 (Cx26), the pluripotency transcription factor NANOG, and focal adhesion kinase (FAK). We sought to determine whether a peptide mimetic of Cx26 designed to target the complex could attenuate tumor growth in pre-clinical models. Experimental Design Histological assessment was employed to verify expression of complex members. We designed peptides based on Cx26 juxtamembrane domains and performed binding experiments with NANOG and FAK using surface plasmon resonance. Peptides with high affinity were engineered with a cell-penetrating sequence and assessed in functional assays including cell proliferation, self-renewal, and in vivo tumor growth, and downstream signaling changes were measured. Results Binding studies revealed that the Cx26 cytoplasmic C-terminal tail and intracellular loop bound to NANOG and FAK with submicromolar to micromolar affinity and that a 5-amino acid sequence in the C-terminal tail of Cx26 (RYCSG) was sufficient for binding. An antennapedia cell penetrating peptide sequence was engineered to the Cx26 C-terminal tail and confirmed intracellular localization. The cell-penetrating Cx26 C-terminal tail peptide (aCx26-pep) disrupted self-renewal as assessed by the tumorsphere formation assay and inhibited NANOG target gene expression in TNBC cells but not in luminal mammary epithelial cells. In a pre-clinical setting, aCx26-pep reduced tumor growth and proliferation and induced cell death. Conclusions We provide proof-of-concept that a Cx26 peptide-based strategy can inhibit TNBC growth. Translational Relevance Triple-negative breast cancer (TNBC) is the most treatment-refractory breast cancer subtype and has limited targeted therapy options. TNBC contains a cancer stem cell (CSC) population that underlies growth and therapeutic resistance. We leveraged an aberrant intracellular protein complex in TNBC CSCs containing the gap junction subunit connexin 26 (Cx26), focal adhesion kinase (FAK), and NANOG to develop a peptide-based therapeutic strategy. We show that TNBC can be selectively targeted using this approach. Our findings provide a new strategy for treatment of this patient population by disrupting intracellular Cx26 signaling, which is linked to the CSC and epithelial-to-mesenchymal programs. Our results support the development of therapeutics targeting the Cx26/NANOG/FAK complex via peptide- and small molecule-based approaches, and future efforts will also focus on the development of a combinatorial treatment strategy with FDA-approved chemotherapeutics. One Sentence Summary A peptide-based targeting strategy for triple-negative breast cancer was developed by targeting connexin 26-associated signaling partners.