Breast cancer stem cells (BCSCs) are intrinsically chemoresistant and capable of self-renewal. Following chemotherapy, patients can develop minimal residual disease due to BCSCs which can repopulate into a relapsed tumor. Therefore, it is imperative to co-target BCSCs along with the bulk tumor cells to achieve therapeutic success and prevent recurrence. So, it is vital to identify actionable molecular targets against both BCSCs and bulk tumor cells. Previous findings from our lab and others have demonstrated that inhibition of the emerging drug target eIF4A with Rocaglamide A (RocA) was efficacious against triple-negative breast cancer cells (TNBC). RocA specifically targets the pool of eIF4A bound to the oncogenic mRNAs that requires its helicase activity for their translation. This property enables specific targeting of tumor cells. The efficacy of RocA against BCSCs is unknown. In this study, we postulated that eIF4A could be a vulnerable node in BCSCs. In order to test this, we generated a paclitaxel-resistant TNBC cell line which demonstrated an elevated level of eIF4A along with increased levels of cancer stemness markers (ALDH activity and CD44), pluripotency transcription factors (SOX2, OCT4, and NANOG) and drug transporters (ABCB1, ABCG2, and ABCC1). Furthermore, genetic ablation of eIF4A resulted in reduced expression of ALDH1A1, pluripotency transcription factors and drug transporters. This pointed out that eIF4A is likely associated with selected set of proteins that are critical to BCSCs, and hence targeting eIF4A may eliminate BCSCs. Therefore, we isolated BCSCs from two TNBC cell lines: MDA-Bone-Un and SUM-159PT. Following RocA treatment, the self-renewal ability of the BCSCs was significantly reduced as determined by the efficiency of the formation of primary and secondary mammospheres. This was accompanied by a reduction in the levels of NANOG, OCT4, and drug transporters. Exposure to RocA also induced cell death of the BCSCs as evaluated by DRAQ7 and cell viability assays. RocA treatment induced apoptosis with increased levels of cleaved caspase-3. Overall, we identified that RocA is effective in targeting BCSCs, and eIF4A is an actionable molecular target in both BCSCs and bulk tumor cells. Therefore, anti-eIF4A inhibitors could potentially be combined synergistically with existing chemo-, radio- and/or immunotherapies.

Chemoresistance is a clinically significant issue in triple-negative breast cancer (TNBC) patients, leading to minimal residual disease. The surviving breast cancer stem cells (BCSCs) undergo multilineage differentiation and repopulate a more aggressive primary tumor leading to metastasis and subsequent mortality. Not only are BCSCs innately chemo/radioresistant but also contribute to therapy failure through interconversion between BCSCs and bulk tumor cells. Therefore, it is imperative to co-target BCSCs and bulk tumor cells simultaneously. Identifying novel molecular targets is key to eliminate both bulk tumor cells and BCSCs. The eukaryotic protein translation initiation machinery, the eIF4F complex, is a convergence point for many oncogenic signaling pathways. This complex plays a vital role in translation of many oncogenic mRNAs implicated in cell cycle, tumor progression, chemoresistance and metastasis. We have previously demonstrated that the mRNA helicase eIF4A1, of the eIF4F complex, might serve as a vulnerable node to kill TNBC cells. In the current study, we hypothesized that targeting of eIF4A1 would reduce/eliminate both bulk tumor cells and BCSCs and overcome chemoresistance. In order to evaluate the effects of targeting eIF4A1 in BCSCs, we isolated BCSCs based on aldehyde dehydrogenase activity and CD44 expression by Fluorescence Activated Cell Sorting and characterized for stemness. Through pharmacological targeting (Rocaglamide A) and genetic ablation (CRISPR-Cas9) of eIF4A1, we demonstrated that there was a statistically significant reduction in the self-renewal ability indicated by the reduction in primary and secondary mammosphere formation efficiency (MFE) of BCSCs, levels of the pluripotency transcription factors (SOX2, OCT4 and NANOG), expression of drug transporters (ABCG2, ABCB1 and ABCC1) and increased induction of apoptotic cell death. Overall, we demonstrated that targeting eIF4A1 is effective in eliminating BCSCs through reduction in the levels of stemness factors and drug transporters. This sets up a stage to employ small molecule inhibitors against eIF4A1 in co-targeting strategic therapies. Citation Format: Sangita Sridharan, Megan Robeson, Diwakar Tukaramrao Bastihalli, Cory Howard, Boopathi Subramaniyan, Augustus Tilley, Dayanidhi Raman. Targeting of the eukaryotic translation initiation factor 4A against breast cancer stemness [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4959.