A Synthetic Triterpenoid CDDO-Im Inhibits Tumorsphere Formation by Regulating Stem Cell Signaling Pathways in Triple-Negative Breast Cancer

Triple-negative breast cancer is associated with poor prognosis because of a high rate of tumor recurrence and metastasis. Previous studies demonstrated that the synthetic triterpenoid, CDDO-Imidazolide (CDDO-Im) induced cell cycle arrest and apoptosis in triple-negative breast cancer. Since a small subpopulation of cancer stem cells has been suggested to be responsible for drug resistance and metastasis of tumors, our present study determined whether the effects of CDDO-Im in triple-negative breast cancer are due to the inhibition of a cancer stem cell subpopulation. CDDO-Im treatment markedly induced cell cycle arrest at G2/M-phase and apoptosis in the triple-negative breast cancer cell lines, SUM159 and MDA-MB-231. Because SUM159 cells were more sensitive to CDDO-Im than MDA-MB-231 cells, the effects of CDDO-Im on the cancer stem cell subpopulation were further investigated in SUM159 cells. SUM159 cells formed tumorspheres in culture, and the cancer stem cell subpopulation, CD24−/EpCAM+ cells, was markedly enriched in SUM159 tumorspheres. The CD24−/EpCAM+ cells in SUM159 tumorspheres were significantly inhibited by CDDO-Im treatment. CDDO-Im also significantly decreased sphere forming efficiency and tumorsphere size in both primary and secondary sphere cultures. PCR array of stem cell signaling genes showed that expression levels of many key molecules in the stem cell signaling pathways, such as Notch, TGF-β/Smad, Hedgehog and Wnt, were significantly down-regulated by CDDO-Im in SUM159 tumorspheres. Protein levels of Notch receptors (c-Notch1, Notch1 and Notch3), TGF-β/Smad (pSmad2/3) and Hedgehog downstream effectors (GLI1) also were markedly reduced by CDDO-Im. In conclusion, the present study demonstrates that the synthetic triterpenoid, CDDO-Im, is a potent anti-cancer agent against triple-negative breast cancer cells by targeting the cancer stem cell subpopulation.

三阴性乳腺癌（Triple-negative breast cancer）因肿瘤复发与转移发生率高，预后极差。既往研究表明，合成三萜类化合物CDDO-Imidazolide（CDDO-Im）可诱导三阴性乳腺癌发生细胞周期阻滞与细胞凋亡。鉴于少量癌症干细胞（cancer stem cell）亚群被认为与肿瘤耐药及转移密切相关，本研究旨在明确CDDO-Im对三阴性乳腺癌的作用是否通过抑制癌症干细胞亚群实现。CDDO-Im处理可显著诱导三阴性乳腺癌细胞系SUM159与MDA-MB-231发生G2/M期细胞周期阻滞及细胞凋亡。由于SUM159细胞对CDDO-Im的敏感性高于MDA-MB-231细胞，本研究进一步在SUM159细胞中探究CDDO-Im对癌症干细胞亚群的作用。SUM159细胞在培养过程中可形成肿瘤球，且癌症干细胞亚群CD24−/EpCAM+细胞在SUM159肿瘤球中显著富集。CDDO-Im处理可显著抑制SUM159肿瘤球中的CD24−/EpCAM+细胞。此外，CDDO-Im还可显著降低原代及传代肿瘤球培养中的球形成效率与肿瘤球尺寸。干细胞信号通路基因PCR芯片（PCR array）检测结果显示，CDDO-Im可显著下调SUM159肿瘤球中多条干细胞信号通路关键分子的表达水平，包括Notch、TGF-β/Smad、Hedgehog及Wnt通路。CDDO-Im还可显著降低Notch受体（c-Notch1、Notch1及Notch3）、TGF-β/Smad通路下游分子pSmad2/3及Hedgehog通路下游效应蛋白GLI1的蛋白表达水平。综上，本研究证实合成三萜类化合物CDDO-Imidazolide（CDDO-Im）可通过靶向癌症干细胞亚群，成为强效的抗三阴性乳腺癌制剂。