Abstract A94: Epigallocatechin-3-gallate inhibits stem-like inflammatory breast cancer cells

Abstract

Abstract Inflammatory Breast Cancer (IBC) is a highly aggressive form of cancer characterized by an unusual growth pattern with nests of tumor emboli rather than a solid mass, rapid spread via lymphatic dissemination and high proliferative rates. Multi-modality therapies have improved survival but the outcome still remains poorer for these patients. Given the potent anti-cancer properties of green tea polyphenols, including epigallocatechin-3-gallate (EGCG), here we tested the hypothesis that EGCG inhibits IBC cells. Two IBC cell lines were studied. SUM-149 cells have a triple negative receptor status (ER-, PR-, Her-2-), while SUM-190 cells are ER-, PR- and Her-2 amplified. Doses of EGCG used ranged from 5 μg/ml (10.9 μM) to 160 μg/ml (349.1 μM), depending upon the presence of serum which inactivates the polyphenol. Treatment with 40 μg/ml (87.3 μM) EGCG decreased mRNA expression of genes that promote migration and invasion (RHOC, FN1, CDH1 and VIM), lymphangiogenesis (VEGF-D), proliferation (CCND1), and survival (BCL-XL) in both lines. Consistently, EGCG treatment resulted in decreased invasive phenotype, ability for anchorage independent growth and viability, as judged by Matrigel outgrowth, soft agar colony assays and ATP assays. Substantial apoptosis was detected starting at 80 μg/ml (174.5 μM) and 160 μg/ml (349.1 μM) of EGCG for SUM-149 and SUM-190 cells, respectively. Conditioned media isolated from EGCG-treated IBC cells displayed decreased secretion of the pro-lymphangiogenic factor VEGF-D and reduced ability to promote cultured hTERT-HDLEC lymphatic endothelial cell migration in wound healing assays and tube formation in Matrigel, processes critical for lymphangiogenesis. The poor clinical outcome of IBC patients has been attributed to a stem-like cell compartment, which is characterized by increased aldehyde dehydrogenase (ALDH) activity. The SUM-149, but not the SUM-190, line has been reported to contain such a stem-like compartment. EGCG treatment inhibited SUM-149 cell spheroid formation and decreased the size of pre-formed spheroids, suggesting efficacy on the cancer stem-like cells. ALDH-positive SUM-149 cells were isolated using an ALDEFUOR Fluorescence Activated Cell Sorting-based assay. EGCG treatment of these cells led to a significant dose-dependent decrease in ATP levels. Furthermore, doses of 80 and 160 μg/ml EGCG induced apoptosis, as judged by increased Caspase-3 and -7 activities. ALDH-positive SUM-149 cancer stem-like cells were used in an orthotopic mammary fat pad mouse model (n = 6/ group). After 25 days, palpable tumors were detected in all of the mice. The experimental group was given a 0.1 ml intraperitoneal injection of 16.5 mg/kg EGCG while the control group was injected with vehicle 1X PBS. Treatment was administered five times a week for five weeks and then daily for one last week for a total of 43 days. EGCG significantly reduced the growth of pre-existing tumors derived from ALDH-positive stem-like SUM-149 cells such that at the end of the experiment a 37.7 ± 4.4% decrease in tumor volume was seen in the EGCG-treated vs control groups. RT-PCR and immunoblot analyses revealed substantially reduced expression of VEGF-D RNA and VEGF-D protein within tumor tissues of EGCG-treated vs control animals, which correlated with a trend toward decreased peritumoral lymphatic vessel density. Thus, EGCG has pleiotropic inhibitory effects on IBC cells, including the cancer stem-like cell compartment believed to be responsible for recurrence. Recent clinical trials using various green tea polyphenol preparations have shown efficacy in treatment of prostate cancer and lymphocytic leukemia, and low toxicity. Our studies suggest the use of EGCG or green tea in combination with standard therapeutic modalities for IBC patients warrants further exploration. Citation Format: Nora D. Mineva, K. Eric Paulson, Stephen P. Naber, Amy S. Yee, Gail E. Sonenshein. Epigallocatechin-3-gallate inhibits stem-like inflammatory breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A94.