Direct regulation of the cancer stemness by eIF4A1 in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is very aggressive with relapses after chemotherapy and high mortality due to metastases. Though there is an initial response to neoadjuvant chemotherapy (NACT) it frequently recurs leading to chemoresistance. Prior evidence points to a small population of chemoresistant tumor-initiating cells termed breast cancer stem-like cells (BCSCs). These cells exhibit intrinsic drug resistance and self-renewal capacity driven by pluripotency transcription factors (PTFs) such as SOX2, OCT4, NANOG, and KLF4. BCSCs survive following NACT, resulting in minimal residual disease (MRD), a clinically significant state of tumor persistence that seeds relapse, aggressive regrowth and metastasis. Our prior studies identified eukaryotic translation initiation factor 4A1 (eIF4A1) as an important regulator of cancer stemness in TNBC. Here, we investigate the mechanistic basis by which eIF4A1 helicase activity regulates cancer stemness that promotes MRD. To establish clinical relevance, we analyzed human TNBC biospecimens and matched adjacent normal tissues for PTFs expression. To demonstrate a causative role for eIF4A1 in sustaining cancer stemness in vitro , eIF4A1-knockout (KO) TNBC cells generated by CRISPR/Cas9 were rescued by ectopic expression of wild type (WT)-eIF4A1 and its helicase-defective mutants. Expression of PTFs was assessed by immunoblotting and qPCR, while measures of cancer stemness were evaluated by ALDH activity, tumorsphere formation, anchorage-independent growth, and 3D growth assays. To directly determine translational regulation of PTFs by eIF4A1, we employed a luciferase reporter system to measure eIF4A1-dependent translation through mutational analysis of 5′ untranslated regions (UTRs) of SOX2, OCT4, NANOG and KLF4 mRNAs. A significantly higher eIF4A1 and PTF protein levels were observed in TNBC but lower in adjacent normal area. Consistent with this, ectopic WT-eIF4A1 in the eIF4A1-KO background robustly significantly rescued PTF expression, tumorsphere formation efficiency, 3D-colony growth on Matrigel, and anchorage-independent growth. The helicase-defective mutants failed to restore PTF levels and stemness traits. Furthermore, total ALDH activity and expression of some ALDH isoforms followed a similar trend. Cloning of WT or mutant 5′-UTRs of PTFs and analysis of reporter luciferase activity confirmed that eIF4A1 can directly regulate the translation of PTF mRNAs and hence the PTF-driven cancer stemness program. Our findings demonstrate for the first time that eIF4A1 helicase activity directly governs a cancer stemness program in TNBC by promoting translation of PTF mRNAs harboring structured 5′ UTRs. Ongoing studies are assessing tumor initiation frequency in vivo . Thus, targeting the helicase activity of eIF4A1 may suppress cancer stemness, restore sensitivity to therapy, reduce relapses and improve longevity in TNBC.