CARP-1/CCAR1 is a novel regulator of triple-negative breast cancer metastasis

CARP-1/CCAR1 regulates proinflammatory, oncogenic, and metastasis pathways in part by activating cell cycle, steroid receptors, NF-κB, and STAT3 pathways. We conducted high throughput proteomics study and identified that eukaryotic translation initiation factor (EIF4A)2 and HNRNPD (aka AU-rich RNA binding factor 1; AUF1) were significantly down-regulated in multiple CARP-1 knockout (KO) cell lines relative to their CARP-1 expressing counterparts. EIF4A proteins have three isoforms (4A1, 4A2, and 4A3) that belong to dead-box RNA helicase family, act as RNA-dependent ATPases, and function for protein translation stages of initiation, mRNA localization, export, and splicing. EIF4A is a therapeutic target in cancer, and inhibitors of EIF4A are being clinically tested for their anti-cancer potential. EIF4A2 regulates stem cell pluripotency during embryogenesis, and AUF1 regulates expression of multiple stem cell and metastasis-promoting transcription factors. Further, RNA-seq analyses revealed significant down-regulation of epithelial mesenchymal transition (EMT), interferon alpha and gamma response, and TNFalpha-NF-κB signaling pathways in CARP-1 KO cells. These observations suggest that CARP-1 likely promotes cancer cells growth, survival and EMT in part by regulating translation, expression, and signaling by EIF4A, AUF1, and NF-κB complexes. Although homozygous deletion of EIF4A1, but not EIF4A2, is embryonic lethal, homozygous deletion of CARP-1 is also embryonic lethal and CARP-1 -/- embryos fail to express EIF4A2 in dermal, mesenchymal and intercostal muscle cells. CARP-1 KO murine TNBC cells express reduced levels of metastasis transducers Vimentin and ICAM1 and form diminished lung and liver metastases in vivo. Co-IP and western blot analyses revealed CARP-1 interactions with EIF4A1, EIF4A2, and AUF1 proteins. Yeast two hybrid analysis revealed CARP-1 (552-654) interacted with EIF4A1 and EIF3D proteins that further support our hypothesis that CARP-1 is a novel component of eukaryotic protein translation complex. Epitope mapping resulted in identification of minimal, 40-50 amino acid epitopes of CARP-1 and EIF4A1 necessary for their mutual binding. As EIF4A structure is resolved, we utilized CARP-1 interacting EIF4A1 epitope to conduct in silico screening of a library of 1.6 million compounds and identified several hits. In vitro testing revealed binding of multiple novel pharmacophores with purified human EIF4A1 protein. Together our studies demonstrate that CARP-1 is a TNBC metastasis regulator and identify novel class of EIF4A1-binding compounds as tools to investigate functions of the eukaryotic protein translation complex for cancer growth and/or metastasis.