Abstract 3844: EZH2 engages TGF-β signaling to promote breast cancer bone metastasis

Abstract

Abstract Metastasis, the dissemination of cancer cells to second organs, is an important hallmark of cancer. About 90% of cancer patients die of metastasis and approximately 55% of breast cancer patients develop bone metastases. However, prevention or treatment of bone metastasis remain challenging. To improve the clinical outcome and the quality of life of breast cancer patients with bone metastasis, the mechanisms of bone metastasis needs to be better understood. Enhancer of zeste homolog 2 (EZH2) is the functional enzymatic component of the Polycomb Repressive Complex 2 (PRC2). EZH2 is a histone methyl transferase that generally suppresses downstream target genes through tri-methylation of H3K27, but it also can work as a transcription co-activator. It is frequently over expressed in breast cancer and in bone metastasis of prostate cancer. To explore the function of EZH2 in breast cancer bone metastasis, we knocked down EZH2 by shRNAs in a MDA-MB-231 bone-seeking subline (231.1566. shEZH#3) and intra-cardiaclly injected into nude mice with 231.1566.shEZH#3 and its control cells (231.1566.shCtrl). Mice injected with 231.1566.shEZH#3 have longer bone metastasis free survival than the control mice. Furthermore, we generated a MDA.MB.231 subline that had CRISPR/Cas9-mediated EZH2-knockout (231.KO) and stably re-expressed in the 231.KO cells the wild-type EZH2 (231.KO.EZH2), or the pLenti control vector (231.KO.pLenti). These two derived sublines were intracardiaclly injected into nude mice, and the mice injected with 231.KO.EZH2 had poorer bone metastasis free survival than the control mice (231.KO.pLenti). Both loss-of-function and gain-of-function experiments demonstrated that EZH2 promotes breast cancer bone metastasis. To gain initial insight on the underlying mechanism of EZH2-induced bone metastasis, we co-cultured MDA-MB-231 cells or 231.KO with pre-osteoclasts (RAW264.7) and osteoblasts (MC3T3) under TGF-β treatment (triple co-culture) to mimic bone metastasis tumor microenvironment. We examined osteoclast maturation and breast cancer cell proliferation because osteolysis induced by mature osteoclasts is critical for breast cancer cell growth in the bone metastasis. In triple co-culture of MDA.MB.231 cells, more RAW264.7 pre-osteoclasts were differentiated to mature osteoclasts, as measured by TRAP+ staining, a marker of mature osteoclasts, and MDA.MB.231 cells grew faster than the 231.KO cells in triple co-culture. In addition, we found that the mRNA level of PTHrP, a master regulator of the “vicious cycle” of bone destruction, was expressed at significantly higher level in MDA.MB.231 cells than that in 231.KO cells under TGF-β stimulation. We also detected higher p-Smad2, the downstream of TGF-β in MDA.MB.231 cells than that in 231.KO cells. The data indicated that EZH2 promotes breast cancer bone metastasis through TGF-β/p-Smad2/PTHrP pathway. Currently, we are exploring how EZH2 regulates p-Smad2 in breast cancer cells to promote bone metastasis. Our studies may reveal novel biomarkers and/or therapeutic targets for bone metastasis. Citation Format: Yu-Wen Huang, Lin Zhang, Jingkun Qu, Zhifen Zhou, Dihua Yu. EZH2 engages TGF-β signaling to promote breast cancer bone metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3844.