performed the biochemical data; K.J.C. efficacy by enhancing T-DM1 internalization. As cellular caveolin-1 was suppressed by shRNA, the effect of metformin-enhanced T-DM1 cytotoxicity was decreased. This study demonstrated that metformin can be applied prior to T-DM1 treatment to improve the clinical efficacy of T-DM1 by enhancing caveolin-1-mediated endocytosis. Introduction Trastuzumab emtansine (trastuzumab-DM1; T-DM1) was recently developed as a new-generation target drug for breast cancer. T-DM1 is a trastuzumab (Herceptin)-based antibody drug conjugate (ADC) that is conjugated to emtansine, which can prevent microtubule assembly. The trastuzumab in T-DM1 can bind to HER-2 receptors, followed by internalization of T-DM1 into cells; emtansine is then released, resulting in cell toxicity1,2. T-DM1 significantly prolonged the progression-free and overall survival in metastatic breast cancer patients, with less toxicity, in phase III of the EMILIA trial3. The drug efficacy of T-DM1 is dependent on cell PROTAC ERRα Degrader-2 endocytosis4C7. Recent studies demonstrated that caveolin-1, a 21KD membrane protein that plays a role in endocytosis and vesicle trafficking, is co-localized with trastuzumab to promote T-DM1 internalization and enhance drug efficacy8,9. The expression level of caveolin-1 is varied in patients, and is not correlated with HER-2 expression9. Therefore, T-DM1 may not work well in HER-2-positive patients with low caveolin-1 expression. T-DM1 was reported to exert a significant benefit in terms of survival in patients with HER-2-positive advanced breast cancer previously treated with trastuzumab and a taxane in a second-line study3. However, in the recent MARIANNE trial10, the progression-free survival (PFS) under T-DM1 treatment was found to be non-inferior, but not superior, to trastuzumab plus a taxane as the first-line treatment for local advanced or metastatic breast cancer. Although T-DM1 treatment did not result in significant improvement of PFS, subgroup analyses showed a numerical trend of an increasing beneficial effect of T-DM1 in patients who had received HER-2-directed therapy or taxanes during early treatment10. Similar results were also obtained in the TH3RESA trials11. However, the mechanism of T-DM1 in improving the clinical outcome in patients previously treated with trastuzumab or taxanes remains elusive. Furthermore, whether expression of caveolin-1 could be induced by treatment PROTAC ERRα Degrader-2 with trastuzumab or taxanes in breast cancer cells to obtain a greater beneficial effect of T-DM1 treatment was also assessed in the current study. In previous studies, the first-line diabetes drug metformin, which can inhibit mitochondria ATP production through up-regulation of AMPK, was demonstrated to induce caveolin-1 expression in lung and breast cancer cells12,13, and caveolin-1 is required for AMPK activity when metformin is applied. Therefore, whether metformin-mediated caveolin-1 overexpression can improve T-DM1 efficacy in breast cancer cells was examined in this study. Our study assessed the caveolin-1 expression upon treatment with metformin, and the drug efficacy of Rabbit Polyclonal to CD3 zeta (phospho-Tyr142) T-DM1 after caveolin-1 induction was also determined. Results Pretreatment with trastuzumab improves T-DM1 efficacy BT-474 cells are HER-2-positive breast cancer cells. The results of our previous study demonstrated that caveolin-1 expression is necessary for T-DM1 uptake in BT-474 cells9. In this study, BT-474 cells were treated with trastuzumab, which up-regulated the overexpression of caveolin-1 peaking at around 12C24?hours PROTAC ERRα Degrader-2 (Fig.?1A). To investigate whether induced caveolin-1 can promote T-DM1 efficacy, the pretreatment strategy was applied in this study. When T-DM1 was applied to trastuzumab-pretreated BT-474 cells, the cytotoxic effect was improved significantly as compared with trastuzumab or the T-DM1 treatment (Fig.?1B). These results suggested that trastuzumab-induced caveolin-1 expression may be critical prior to T-DM1 treatment. Open in a separate window Figure 1 Pretreatment with trastuzumab in HER-2-positive cells up-regulated caveolin-1 expression and improved T-DM1 drug efficacy. (A) Treatment of BT-474 cells with trastuzumab (10?g/ml) induced caveolin-1 overexpression at 12 and 24?hours. (B) BT-474 cells pretreated with trastuzumab (TRA) for 24?hours were then treated with trastuzumab or T-DM1 for an additional 72?hours, and the cell viability was significantly decreased. Ctr: control group without treatment; TRA: trastuzumab. Statistical values with different letters were significantly different ( em P /em ? ?0.01). At least three independent experiments were performed.