The Novel Triterpenoid C-28 Methyl Ester of 2-Cyano-3, 12-Dioxoolen-1, 9-Dien-28-Oic Acid Inhibits Metastatic Murine Breast Tumor Growth through Inactivation of STAT3 Signaling Xiaoyang Ling, 1 Marina Konopleva, 1 Zhihong Zeng, 1 Vivian Ruvolo, 1 L. Clifton Stephens, 2 Wendy Schober, 1 Teresa McQueen, 1 Martin Dietrich, 1 Timothy L. Madden, 3 and Michael Andreeff 1 1 Section of Molecular Hematology and Therapy, Department of Stem Cell Transplantation and Cellular Therapy, 2 Department of Veterinary Medicine and Surgery, and 3 Department of Experimental Therapeutics, The University of Texas M. D. Anderson Cancer Center, Houston, Texas Abstract We and others have reported that C-28 methyl ester of 2- cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me) effec- tively inhibits the growth of multiple cancer cell types. Our previous studies indicated that prolonged CDDO-Me treat- ment inactivated extracellular signal-regulated kinase signal- ing in acute myelogenous leukemia cells. Whether treatment with CDDO-Me has an earlier effect on other proteins that are important for either signal transduction or oncogenesis is unknown. Constitutively activated signal transducer and activator of transcription 3 (STAT3) is frequently found in human breast cancer samples. Constitutively activated STAT3 was shown to up-regulate c-Myc in several types of cancer and has a feedback effect on Src and Akt. To examine the effects of CDDO-Me on STAT3 signaling in breast cancer, we used the murine 4T1 breast tumor model, which is largely resistant to chemotherapy. In vitro , after treatment of 4T1 cells with 500 nmol/L CDDO-Me for 2 h, we found (a ) inactivation of STAT3, (b) inactivation of Src and Akt, (c ) 4-fold reduction of c-Myc mRNA levels, (d ) accumulation of cells in G 2 -M cell cycle phase, (e ) abrogation of invasive growth of 4T1 cells, and ( f ) lack of apoptosis induction. In in vivo studies, CDDO-Me completely eliminated 4T1 breast cancer growth and lung metastases induced by 4T1 cells in mice when treatment started 1 day after tumor implantation and significantly inhibited tumor growth when started after 5 days. In vivo studies also indicated that splenic mature dendritic cells were restored after CDDO-Me treatment. In summary, these data suggest that CDDO-Me may have therapeutic potential in breast cancer therapy, in part, through inactivation of STAT3. [Cancer Res 2007;67(9):4210–8] Introduction Signal transducer and activator of transcription 3 (STAT3) has frequently been found in human breast cancer samples (1). STAT3- mediated up-regulation of c-Myc levels was proposed to be one of the important mediators of STAT3 oncogenesis (2). c-Myc is a potent oncoprotein that has been found to play a critical role in tumorigenesis (3). The use of an inducible c-Myc model showed tumor growth or suppression, respectively, by controlling c-Myc expression in a murine hepatocarcinoma model (4). Our previous studies and that of others have shown that c-Myc expression is regulated by constitutive activation of STAT3 in mouse breast cancer and other types of cancer cells (5–7). As the immediate upstream regulator of STAT3, Src has been considered to play an important role in breast carcinogenesis. However, we found that Src can be inactivated by STAT3 knockdown in 4T1 cells (5). We have also shown that Akt can be inactivated following STAT3 knockdown in 4T1 cells (5), suggesting that STAT3 is required for maintaining the activation status of Src and Akt. As a downstream target of Src, Akt plays an important role in regulating antiapoptotic proteins and proteins that regulate cell cycle progression (8). However, it is unclear how these intracellular signal transduction proteins interact with each other, and the biological consequences of these interactions remain uncertain. Recent reports provided convincing evidence that constitutively activated STAT3 in breast cancer cells is associated with impaired cell-mediated immunity (9) and that STAT3 activation in cancer cells may allow them to evade host immune surveillance (10). In agreement with these observations, we reported that knockdown of STAT3 completely abolished breast tumor formation in an immunocompetent mouse model (5). The 4T1 cell line was originally derived from a spontaneous mammary carcinoma in BALB/c mice (11). It has been reported that 4T1 cells mimic the effects of human mammary carcinoma in that morbidity is due to the outgrowth of spontaneous micrometastatic tumor cells that migrate to distant organs relatively early during primary tumor growth (12, 13) and are notoriously chemoresistant (14). Therefore, 4T1-driven breast cancer in BALB/c mice constitutes an appropriate in vivo system for modeling human breast cancer with regard to tumor growth and metastasis. As such, the 4T1/BALB/c mouse system also provides a useful experimental model for exploring the biological effects in mammary tumorigenesis and for studying the effects of small molecules that target signal transduction pathways in vivo . 2-Cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO) and its derivative CDDO-Me are synthetic triterpenoids proven to be effective agents in controlling cancer cell growth in preclinical models of leukemias, multiple myeloma, lymphomas, and solid tumors, such as breast, pancreatic, and colon cancer (15–18). CDDO-Me has been shown by us to (a ) promote apoptosis in several leukemic cell lines, (b) induce proapoptotic Bax protein as a Requests for reprints: Michael Andreeff, Department of Blood and Marrow Transplantation, Unit #448, 1515 Holcombe Boulevard, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030-4009. Phone: 713-792-7260; Fax: 713-794- 4747; E-mail: mandreef@mdanderson.org. I2007 American Association for Cancer Research. doi:10.1158/0008-5472.CAN-06-3629 Cancer Res 2007; 67: (9). May 1, 2007 4210 www.aacrjournals.org Research Article Research. on April 13, 2017. © 2007 American Association for Cancer cancerres.aacrjournals.org Downloaded from