Moreover, recent studies have demonstrated that activated signaling by the TGF-b superfamily, such as TGF-b, Nodal and Activin, increases the subpopulation of cancer stem cells in breast and pancreatic cancers. CDDO-Im inhibits TGF-b-stimulated cell migration by altering the trafficking and turnover of TGF-b receptors. The present study also demonstrated the inhibitory effect of CDDO-Im on TGF-b/Smad signaling as shown by significantly reduced mRNA levels of Activin and TGF-b receptors as well as decreased protein levels of pSmad2/3. Investigation at to how inhibition of TGFb signaling by CDDO-Im affects EMT and cancer stem cells in triple negative breast cancer will be an interesting area for future study. The Hedgehog signaling pathway is involved in patterning and growth of various cells during embryonic development, and its deregulation has been implicated in cancer. The high expression level of GLI1, the key downstream effector of the Hedgehog signaling pathway, is associated with unfavorable overall survival in cancer patients. Moreover, a recent study demonstrated that GLI1 was a central regulator of cancer stem cells in triple-negative breast cancer. In the present study, CDDO-Im decreased both mRNA and protein level of GLI1 in tumorspheres of triple-negative breast cancer. Since GLI1 is the essential effector of Hedgehog signaling, this result suggests that CDDO-Im inhibits Hedgehog signaling. However, CDDO-Im also reduced SUFU, a negative regulator of Hedgehog signaling, which can activate Hedgehog signaling. Further investigation is necessary in order to understand the direct and feedback mechanisms of CDDO-Im on the Hedgehog signaling pathway. CDDO and its derivatives, including CDDO-Im, have been shown to induce diverse biological functions in a concentration or cell-type dependent manner. At low concentrations, CDDOIm high throughput screening inhibitor showed cytoprotective effects by inducing Heme Oxygenase-1 and Nrf2/ARE signaling. On the other hand, high concentrations of CDDO-Im induced apoptosis in pancreatic cancer cells by rapid depletion of mitochondrial glutathione, causing accumulation of reactive oxygen species. A recent study also showed that high concentrations of CDDO-Im induced apoptosis in BRCA-mutated breast cancer cells by increasing reactive oxygen species and DNA damage. However, the same dose of CDDO-Im did not induce ROS in untransformed human breast epithelial cells or mouse fibroblasts. Interestingly, triple-negative breast cancer cells with mutated BRCA1 or with wild-type BRCA1 showed high sensitivity to oxidative DNA damage because of their genomic instability and defective DNA-repair system. In the present study, CDDO-Im significantly induced apoptosis in SUM159 and MDA-MB-231 cells which are triple-negative breast cancer cells with wild-type BRCA1. MDA-MB231 cells were less sensitive to CDDO-Im than SUM159 cells. This might be because of a gain-of-function p53 mutation in MDA-MB-231 cells, which has been shown to promote cancer cell survival.