Novel drug therapies for ER- basal-like breast cancer; preclinical evaluation in a xenograft model.

Abstract

Basal-like breast cancers are a subgroup of breast lesions that are triple-negative for the estrogen receptor (ER), progesterone receptor and human epidermal growth factor receptor 2. There is currently no effective treatment for basal-like breast cancer and patients with this disease have a particularly poor prognosis. Previous work has shown that combinations of epigallocatechin gallate (EGCG) and selective estrogen receptor modulators (SERMs) are synergistically cytotoxic to MDA-MB-231 cells, a commonly used in vitro model of basal-like breast cancer. Therefore, the in vivo efficacy of EGCG and SERM combinations was investigated, using an MDA-MB-231 mouse xenograft model. MDA-MB-231 tumor volume was significantly suppressed 71% in mice treated with a combination of EGCG (25 mg/kg, i.p.) and tamoxifen (75 μg/kg, p.o.) daily for 10 weeks, whereas individual treatments failed to suppress tumor growth. Using Western blotting, EGCG and tamoxifen co-therapy was found to significantly reduce tumoral CYP1B1 protein levels 72%, which may be crucial for tumor suppression by preventing CYP1B1-mediated inactivation of 4-hydroxytamoxifen, the active tamoxifen metabolite. Contrary to expectations, raloxifene (850 μg/kg, p.o.) was far more effective at suppressing MDA-MB-231 xenograft growth individually than when used in combination with EGCG (25 mg/kg, i.p.), and tumor volume was reduced 79% and 54% by individual raloxifene and combination therapies, respectively, after 8 weeks of treatment. This finding was unexpected and suggested that EGCG may interfere with raloxifene’s metabolism. However, the activities of hepatic uridine diphospho-glucuronosyl transferase (UGT) enzymes, which metabolize raloxifene, were not altered by EGCG treatment. Because basal-like breast cancers appear dependent on epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) signaling for survival and proliferation, Western blotting was used to investigate the expression and phosphorylation status of various signaling proteins in the MDA-MB-231 tumors. EGCG + tamoxifen combination therapy was associated with a marked reduction in tumoral EGFR, Akt, mTOR and NFκB expression compared with individual treatments, whereas raloxifene therapy did not significantly influence EGFR signaling or VEGFR expression. Therefore, modulation of growth factor signaling contributes to the growth suppression of MDA-MB-231 tumors by EGCG + tamoxifen, which shows therapeutic potential for basal-like breast cancer. Although the tumor suppression elicited by raloxifene is encouraging, further investigation is required to establish the mechanism of action.

Curcumin is another natural product that has received much attention for its anticarcinogenic properties, and previous work in our laboratory has shown that curcumin displays activity toward basal-like breast cancer cells in vitro, but fails in vivo, probably due to poor bioavailability. In an attempt to improve curcumin’s bioavailability, a panel of derivatives had been developed and tested for in vitro cytotoxic activity equal to, or greater than, curcumin (Somers-Edgar, 2010). Among the derivatives tested, three promising cyclohexanone curcumin derivatives were identified. Therefore, the current study investigated the oral bioavailability of these novel curcumin derivatives in mice. Among these, RL-101 was the most bioavailable at low doses (8.5 mg/kg, p.o.) and was not associated with any acute toxicity following 7 days of treatment. Therefore, RL-101 shows the best potential for future studies.