Abstract LB469: CARP-1/CCAR1: A novel transducer of triple-negative breast cancer metastasis

Abstract TNBC chemotherapeutics activate proinflammatory and oncogenic NF-κB pathways that involve CARP-1 interactions with NEMO, RIPK1, and STAT3 [Venkatesh JA et al. JBC 295 (11): 3532-3552, 2020; Venkatesh JA et al. Front. Oncol. 14:1376666, 2024]. To further investigate tumor growth, survival and metastasis potential of CARP-1, we generated multiple, human and murine cancer cells with homozygous CARP-1 knock-out (KO) by CRISPR-Cas9 technology. We next conducted high throughput proteomics analyses to identify proteins that were significantly up- or down-regulated in the CARP-1 KO cell lines relative to their CARP-1 expressing counterparts. We noted a consistent, and statistically significant reduction in levels of eukaryotic translation initiation factor (EIF4A)2 and HNRNPD (aka AU-rich RNA binding factor 1; AUF1), while Nestin levels were elevated in all CARP-1 KO cells. Our co-IP and western blot analyses revealed CARP-1 interactions with eIF4A and AUF1. EIF4A, the catalytic subunit of the eIF complex, functions to promote translation of different oncogenic, invasion, and metastasis-inducing factors, and is often hyper activated in many cancers including TNBCs. eIF4A is a potential therapeutic target in cancer, and inhibitors of eIF4A could be used in antiviral therapies and 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. CARP-1 KO cells also have diminished levels of genotoxic drug-induced EMT and metastasis-promoter Oct4. Further, RNA-seq analyses revealed significant down-regulation of epithelial mesenchymal transition, interferon α and γ response, and TNFα-NF-κB signaling pathways in all CARP-1 KO cells relative to their WT counterparts. These observations strongly suggest that CARP-1 promotes cancer cells growth, survival and EMT in part by regulating translation, expression, and signaling by EIF4A, AUF1, and NF-κB complexes. Homozygous deletion of CARP-1 is embryonic lethal at day 13.5 of embryogenesis, and CARP-1 -/- embryos fail to express EIF4A2 in dermal, mesenchymal and intercostal muscle cells. Although CARP-1 KO murine TNBC 4T1 and EO771 cells form tumors similar to their CARP-1 WT counterparts when subcutaneously xenografted in the female syngeneic mice, we interestingly noted reduced lung metastases in animals that had CARP-1 KO tumors when compared with animals with respective CARP-1 WT expressing tumors. Together with our findings that CARP-1 loss resulted in attenuation of EMT signaling, diminished EIF4A2 expression in mouse embryos as well as TNBC cells, our data underscore a novel pluripotency and tumor metastasis promoting function of CARP-1. Citation Format: Magesh Muthu, Sijana Dzinic, Hunter Dlugas, Jaganathan Venkatesh, Lisa A. Polin, Seongho Kim, Arun K. Rishi. CARP-1/CCAR1: A novel transducer of triple-negative breast cancer metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr LB469.