Breast cancer stem cells (BCSCs) play a vital role in tumor progression and metastasis. They are heterogeneous and inherently radio- and chemoresistant. They have the ability to self-renew and differentiate into non-BCSCs. These determinants of BCSCs including the plasticity between the mesenchymal and epithelial phenotypes often leads to minimal residual disease (MRD), tumor relapse, and therapy failure. By studying the resistance mechanisms in BCSCs, a combinatorial therapy can be formulated to co-target BCSCs and bulk tumor cells. This review addresses breast cancer stemness and molecular underpinnings of how the cancer stemness can lead to pharmacological resistance. This might occur through rewiring of signaling pathways and modulated expression of various targets that support survival and self-renewal, clonogenicity, and multi-lineage differentiation into heterogeneous bulk tumor cells following chemotherapy. We explore emerging novel and alternative molecular targets against BC stemness and chemoresistance involving survival, drug efflux, metabolism, proliferation, cell migration, invasion, and metastasis. Strategic targeting of such vulnerabilities in BCSCs may overcome the chemoresistance and increase the longevity of the metastatic breast cancer patients.

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.

Triple-negative breast cancer (TNBC) remains clinically challenging as effective targeted therapies are lacking. In addition, patient mortality mainly results from the metastasized lesions. CXCR4 has been identified to be one of the major chemokine receptors involved in breast cancer metastasis. Previously, our lab had identified LIM and SH3 Protein 1 (LASP1) to be a key mediator in CXCR4-driven invasion. To further investigate the role of LASP1 in this process, a proteomic screen was employed and identified a novel protein-protein interaction between LASP1 and components of eukaryotic initiation 4F complex (eIF4F). We hypothesized that activation of the CXCR4-LASP1-eIF4F axis may contribute to the preferential translation of oncogenic mRNAs leading to breast cancer progression and metastasis. To test this hypothesis, we first confirmed that the gene expression of CXCR4, LASP1, and eIF4A are upregulated in invasive breast cancer. Moreover, we demonstrate that LASP1 associated with eIF4A in a CXCL12-dependent manner via a proximity ligation assay. We then confirmed this finding, and the association of LASP1 with eIF4B via co-immunoprecipitation assays. Furthermore, we show that LASP1 can interact with eIF4A and eIF4B through a GST-pulldown approach. Activation of CXCR4 signaling increased the translation of oncoproteins downstream of eIF4A. Interestingly, genetic silencing of LASP1 interrupted the ability of eIF4A to translate oncogenic mRNAs into oncoproteins. This impaired ability of eIF4A was confirmed by a previously established 5′UTR luciferase reporter assay. Finally, lack of LASP1 sensitizes 231S cells to pharmacological inhibition of eIF4A by Rocaglamide A as evident through BIRC5 expression. Overall, our work identified the CXCR4-LASP1 axis to be a novel mediator in oncogenic protein translation. Thus, our axis of study represents a potential target for future TNBC therapies.

The CXCL12-CXCR4 axis plays a vital role in many steps of breast cancer metastasis, but the molecular mechanisms have not been fully elucidated. We previously reported that activation of CXCR4 by CXCL12 promotes the nuclear localization of LASP1 (LIM and SH3 protein 1). The nuclear LASP1 then interacts with Snail1 in triple-negative breast cancer (TNBC) cell lines. In this study, we report that the nuclear accumulation and retention of Snail1 was dependent on an increase in nuclear LASP1 levels driven by active CXCR4. The CXCR4-LASP1 axis may directly regulate the stabilization of nuclear Snail1, by upregulating nuclear levels of pS473-Akt, pS9-GSK-3β, A20, and LSD1. Furthermore, the activation of CXCR4 induced association of LASP1 with Snail1, A20, GSK-3β, and LSD1 endogenously. Thus, nuclear LASP1 may also regulate protein-protein interactions that facilitate the stability of Snail1. Genetic ablation of LASP1 resulted in the mislocalization of nuclear Snail1, loss of the ability of TNBC cells to invade Matrigel and a dysregulated expression of both epithelial and mesenchymal markers, including an increased expression of ALDH1A1, a marker for epithelial breast cancer stem-like cells. Our findings reveal a novel role for the CXCR4-LASP1 axis in facilitating the stability of nuclear localized Snail1.

The CXCR4-LASP1 axis is an emerging target in the field of breast cancer metastasis. C-X-C chemokine receptor type 4 (CXCR4) mediates directed cell migration when activated by its cognate ligand CXCL12. LIM and SH3 Protein 1 (LASP1) is a critical node in the CXCR4 signaling pathway, as its deficiency blocks CXCR4-dependent Matrigel invasion. The mechanism by which LASP1 facilitates this invasive ability of tumor cells when CXCR4 is activated is unknown. Our previous proteomics work had revealed several components of the RNA interference (RNAi) machinery as being potential LASP1 interacting proteins. Here we report that argonaute 2 (Ago2), a protein with central involvement in RNAi, associates with LASP1 in triple-negative breast cancer (TNBC) cells. We demonstrate that LASP1 co-immunoprecipitates with Ago2 endogenously in a CXCL12-dependent manner, with further confirmation of this interaction by proximity ligation assay. Furthermore, this association is specific to CXCR4 as it can be abrogated by the CXCR4 antagonist, AMD3465. By GST-pulldown approach, we identify that LASP1 directly binds to Ago2 through its LIM and SH3 domains, and that this binding is dictated by the S146 and Y171 phosphorylation sites of LASP1. Additionally, the phosphorylation status of LASP1 affected tumor suppressor microRNA (miRNA) Let-7a-guided Ago2 activity. Levels of several endogenous targets of Let-7a were found to be altered including C-C chemokine receptor type 7 (CCR7), which is another critical chemokine receptor involved in metastasis to lymph nodes. Our results suggest a novel role for the LASP1-Ago2 module in shaping the RNAi landscape, functionally impacting the invasive ability of cancer cells.