Triple‐negative breast cancer (TNBC), the most refractory subtype of breast cancer to current treatments, accounts disproportionately for the majority of breast cancer‐related deaths. This is largely due to cancer plasticity and the development of cancer stem cells (CSCs). Recently, distinct yet interconvertible mesenchymal‐like and epithelial‐like states have been revealed in breast CSCs. Thus, strategies capable of simultaneously inhibiting bulk and CSC populations in both mesenchymal and epithelial states have yet to be developed. Wnt/β‐catenin and Hippo/YAP pathways are crucial in tumorigenesis, but importantly also possess tumor suppressor functions in certain contexts. One possibility is that TNBC cells in epithelial or mesenchymal state may differently affect Wnt/β‐catenin and Hippo/YAP signaling and CSC phenotypes. In this report, we found that YAP signaling and CD44high/CD24−/low CSCs were upregulated while Wnt/β‐catenin signaling and ALDH+ CSCs were downregulated in mesenchymal‐like TNBC cells, and vice versa in their epithelial‐like counterparts. Dual knockdown of YAP and Wnt/β‐catenin, but neither alone, was required for effective suppression of both CD44high/CD24−/low and ALDH+ CSC populations in mesenchymal and epithelial TNBC cells. These observations were confirmed with cultured tumor fragments prepared from patients with TNBC after treatment with Wnt inhibitor ICG‐001 and YAP inhibitor simvastatin. In addition, a clinical database showed that decreased gene expression of Wnt and YAP was positively correlated with decreased ALDH and CD44 expression in patients’ samples while increased patient survival. Furthermore, tumor growth of TNBC cells in either epithelial or mesenchymal state was retarded, and both CD44high/CD24−/low and ALDH+ CSC subpopulations were diminished in a human xenograft model after dual administration of ICG‐001 and simvastatin. Tumorigenicity was also hampered after secondary transplantation. These data suggest a new therapeutic strategy for TNBC via dual Wnt and YAP inhibition.

Triple-negative breast cancer (TNBC) is the most refractory subtype of breast cancer. It causes the majority of breast cancer-related deaths, which has been largely associated with the plasticity of tumor cells and persistence of cancer stem cells (CSCs). Conventional chemotherapeutics enrich CSCs and lead to drug resistance and disease relapse. Development of a strategy capable of inhibiting both bulk and CSC populations is an unmet medical need. Inhibitors against estrogen receptor 1, HDACs, or mTOR have been studied in the treatment of TNBC; however, the results are inconsistent. In this work, we found that patient TNBC samples expressed high levels of mTORC1 and HDAC genes in comparison to luminal breast cancer samples. Furthermore, co-inhibition of mTORC1 and HDAC with rapamycin and valproic acid, but neither alone, reproducibly promoted ESR1 expression in TNBC cells. In combination with tamoxifen (inhibiting ESR1), both S6RP phosphorylation and rapamycin-induced 4E-BP1 upregulation in TNBC bulk cells was inhibited. We further showed that fractionated CSCs expressed higher levels of mTORC1 and HDAC than non-CSCs. As a result, co-inhibition of mTORC1, HDAC, and ESR1 was capable of reducing both bulk and CSC subpopulations as well as the conversion of fractionated non-CSC to CSCs in TNBC cells. These observations were partially recapitulated with the cultured tumor fragments from TNBC patients. Furthermore, co-administration of rapamycin, valproic acid, and tamoxifen retarded tumor growth and reduced CD44high/+/CD24low/− CSCs in a human TNBC xenograft model and hampered tumorigenesis after secondary transplantation. Since the drugs tested are commonly used in clinic, this study provides a new therapeutic strategy and a strong rationale for clinical evaluation of these combinations for the treatment of patients with TNBC.

Development of targeted therapies for triple-negative breast cancer (TNBC, a more aggressive subtype) is an unmet medical need. We analyzed data from 887 patients with invasive breast cancer and observed that increased Wnt and histone deacetylase (HDAC) activities are associated with estrogen receptor 1 (ESR1) and progesterone receptor (PGR) repression, poor survival, and increased relapse. The inverse correlation between Wnt signaling and repression of ESR1 and PGR expression was found to be magnified in cancer stem cell (CSC) subpopulations in TNBC cell lines. Cosuppression of Wnt, HDAC, and ESR1 using clinically relevant low-dose inhibitors effectively repressed both bulk and CSC subpopulations and converted CSCs to non-CSCs in TNBC cells without affecting MCF-10A mammary epithelial cells.

Unlike other breast cancer subtypes, triple-negative breast cancer (TNBC) has no specific targets and is characterized as one of the most aggressive subtypes of breast cancer that disproportionately accounts for the majority of breast cancer-related deaths. Current conventional chemotherapeutics target the bulk tumor population, but not the cancer stem cells (CSCs) that are capable of initiating new tumors to cause disease relapse. Recent studies have identified distinct epithelial-like (E) ALDH+ CSCs, mesenchymal-like (M) CD44+/CD24− CSCs, and hybrid E/M ALDH+/CD44+/CD24− CSCs. These subtypes of CSCs exhibit differential signal pathway regulations, possess plasticity, and respond differently to treatment. As such, co-inhibition of different subtypes of CSCs is key to viable therapy. This review serves to highlight different pathway regulations in E and M CSCs in TNBC, and to further describe their role in disease progression. Potential inhibitors targeting E and/or M CSCs based on clinical trials are summarized for further investigation. Since future research needs to adopt suitable tumor models and take into account the divergence of E and M CSCs for the development of effective treatments, TNBC models for clinically translatable studies are further discussed.