Anti‐cancer Effects of Coumestrol in Triple‐Negative Inflammatory Breast Cancer 3D Models: Opportunities for In Vivo Studies

Abstract

Inflammatory breast cancer (IBC) is the most aggressive subtype of breast cancer. Each molecular subtype is characterized by the presence or absence of two hormonal steroid receptors, estrogen receptor (ERα‐66) and progesterone receptor (PR), and a growth factor receptor called HER2. Particularly, a subtype of breast cancer that lacks these three biomarkers is known as triple‐negative breast cancer (TNBC). However, previous studies showed that TNBC can express alternate estrogen receptors known as ERα‐36 and GPR30, mediating a non‐genomic signaling upon estrogen (E2) treatment. Today, effective targeted therapeutics for triple‐negative inflammatory breast cancer (TN‐IBC) patients, accounting 20‐40% of TN‐IBC cases, is limited. Coumestrol (Cou), a plant derived compound structurally similar to estrogen has shown to induce a cytotoxic effect in TNBC. Till this day, there is no known study of the effects of Cou in three‐dimensional culture models using Matrigel, a solubilized membrane matrix that mimics the tissue microenvironment. Additionally, we want to test Cou’s inhibitory effect on tumor emboli formation, a distinct hallmark of IBC. This tumor cell clusters are known to infiltrate and block the breast lymphatics of patients, presenting a barrier for successful IBC treatment (Arora Etl. 2017). We hypothesize that Cou inhibits cell viability and tumor emboli formation in TN‐IBC cells. First, we performed 3D dose response curves to determine the half‐maximal inhibitory concentration (IC50) of Cou (50µM). Next, 3D proliferationassays were performed using 50µM of Cou. After 10 days, E2 showed a significant increase in colony size in comparison to vehicle control (DMSO). Also, proliferation decreased more than a half after treating the cells with Cou alone and in combination of both treatments. When testing the tumor emboli formation after 72 hours, Cou did not inhibit the formation of emboli, but it decreased the tumor emboli growth rate shown by the decreased in tumor emboli size in both culture conditions. In comparison, E2 promoted a significant increase in the size of the tumor emboli. Lastly, we used Western Blot analysis to validate the decrease in phosphorylation levels of ERK1/2 and AKT, two kinases involved in estrogen non‐genomic signaling, upon Cou treatment. Together, these data demonstrated that Cou decreases cell viability in 3D proliferation assays and decreased growth rate of tumor emboli in TN‐IBC cells. In summary, the anticancer effects of Coumestrol in IBC three‐dimensional models provide the basis for future in vivo studies using animal models.