Triple negative breast cancer (TNBC) (ER-, PR-, Her2-), constituting 10-20% of all breast cancers, is a heterogeneous disease with limited treatment options and poor prognosis. TNBCs exhibit rapid progression with the duration of response to first line palliative chemotherapy typically less than 12 weeks, and overall five year survival of patients with metastatic TNBC of 22%. The cancer stem cell (CSC) model provides an attractive explanation for relapse of TNBC after primary therapy since these cells demonstrate resistance to conventional chemotherapy. CSCs which survive primary treatments, such as docetaxel, may self-renew and differentiate into the heterogeneous tumor bulk resulting in local recurrence and distant metastasis. Docetaxel has been demonstrated to not only fail to eliminate CSCs but expands this population in preclinical models. Further, docetaxel increases circulating IL-6 in patients following therapy, a cytokine reported to expand breast CSCs. Therefore, we sought to combine docetaxel with a small molecule CSC inhibitor capable of reducing IL-6 production (sulforaphane, SF) for the effective treatment of TNBCs.
Our results in vitro demonstrate that docetaxel treatment (5 nM) increases the proportion of CSCs in TNBC cell lines (SUM149 and SUM159) as evident by flow cytometry analysis using the ALDEFLUOR assay (70.6±22.0%) and cells which are CD44+/CD24-/EpCAM+ (2.9 fold). Mammosphere formation assay reveals 1 nM docetaxel increases secondary sphere formation rate by 75.8±29%. As determined by ELISA, 5 nM docetaxel treatment for 72 hours induces 3.5 fold increase in IL-6 production. Conversely, SF (2.5 μM) selectively reduces the proportion of ALDEFLUOR positive cells (51.5±15.0%) and mammosphere formation (39.2±3.8%) while reducing IL-6 (55.6±5.0%) through regulation of NF-kB activity. In combination docetaxel and SF synergize to effectively reduce bulk cell line proliferation (combination index range 1-0.093). Further, SF prevents docetaxel mediated CSC expansion and IL-6 production. Using a mouse xenograft model docetaxel (10 mg/kg weekly) reduces tumor growth of established tumors by 83.2±6.0% whereas SF (50 mg/kg daily) inhibits primary tumor growth by 37.4±14.6%. In addition, secondary reimplantation assays with limiting dilution analysis reveals docetaxel increases the frequency of the tumor initiating CSCs (1/1514 control cells vs 1/330 docetaxel treated cells) while SF reduces the frequency to 1/3181 cells. In vivo, the combination of docetaxel and SF exhibits a greater reduction in primary tumor volume (92.5±2.1% reduction relative to control), and synergistically inhibit the CSC population (1 in 4245 cells). These results suggest that SF mediated inhibition of breast CSCs and IL-6 provide a scientific rationale for using this agent in combination with docetaxel for TNBC.
Citation Format: Joseph P. Burnett, Ronack B. Shah, Hayley J. Paholak, Sean P. McDermott, Yasuhiro Tsume, Max W. Wicha, Duxin Sun. Combination of docetaxel with sulforaphane synergistically inhibits triple negative breast cancer and cancer stem cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4076. doi:10.1158/1538-7445.AM2015-4076