| We have previously shown that the selective estrogen receptor modulator, Arzoxifene (Arz), and the rexinoid, LG100268 (268) synergistically prevent breast cancer in an ER+ breast cancer model. This study was designed to determine the mechanism of action of these drugs in estrogen receptor positive (ER+) breast cancer. We demonstrate here that the combination of Arz and 268 synergistically increases apoptosis in ER+ breast cancer cell lines and in ER+ breast tumors while the individual agents have no effect. In addition, we present a new model to explain the induction of apoptosis by these agents. We show that Arz induces TGF-beta-mediated apoptosis while 268 inhibits the NF-kappaB and PI3K pro-survival pathways. We have also determined that Arz and 268 cooperate to prevent colon cancer and estrogen receptor negative (ER-) breast cancer in addition to ER+ breast cancer. For the colon cancer experiments, F344 male rats were injected with 15 mg/kg azoxymethane and subsequently fed control diet or diets containing Arz (6 mg/kg of diet), 268 (50 mg/kg of diet), or the combination. While Arz and 268 decreased tumor formation by 17.0% and 20.8% respectively, the combination inhibited tumor formation by 87.3% as compared with controls. For the ER- breast cancer experiments, MMTV-neu transgenic mice were fed control diets or diets containing Arz (6 mg/kg of diet), 268 (30 mg/kg diet), or the combination for 52 weeks. When 100% of control mice developed tumors, only 75% of mice receiving 268 alone and 37.5% mice receiving Arz alone developed tumors. Unexpectedly, mice receiving the combination diet did not develop any tumors. In addition to the in vivo results, the combination of Arz and 268 was found to synergize to promote apoptosis in both colon cancer and ER- breast cancer cell lines. Moreover, we demonstrate that Arz and 268 induce this apoptosis by a similar mechanism as in ER+ breast cancer; the induction of TGF-betamediated apoptosis by Arz and the inhibition of the NF-kappaB and PI3K pro-survival pathways by 268. This study further emphasizes the role of TGFbeta and apoptotic agents in cancer chemoprevention and the importance of combination chemoprevention. Moreover, these data suggest that Arz and 268 may prevent several of the common epithelial cancers found clinically. As a result, these compounds should be considered for chemoprevention trials in humans as soon as possible. |
