Combined trastuzumab and paclitaxel treatment better inhibits ErbB-2-mediated angiogenesis in breast carcinoma through a more effective inhibition of Akt than either treatment alone

BACKGROUND. Trastuzumab (Herceptin; Genentech, South San Francisco, CA) is a humanized anti-ErbB-2 monoclonal antibody that has demonstrated antitumor function, especially in combination with other chemotherapies such as paclitaxel (Taxol; Bristol Myers-Squibb, Princeton, NJ), in patients with tumors that overexpress ErbB-2. Because the repeated administration of low-dose chemotherapy, such as paclitaxel, endorsed an antiangiogenic effect in vitro, and because trastuzumab was shown to inhibit angiogenesis in tumor xenografts, the authors investigated whether ErbB-2-mediated angiogenic responses would be inhibited more effectively by the combined treatment of paclitaxel plus trastuzumab. METHODS. Tumor xenografts were established in 37 severe combined immunodeficiency mice by injecting the mammary fat pad with ErbB-2-overexpressing human breast carcinoma cells. Mice then were treated with an immunoglobulin (IgG) control, trastuzumab, paclitaxel, or a combination treatment of trastuzumab plus paclitaxel. Tumorigenicity, lung metastasis, and tumor microvessel density (MVD) were evaluated. Vascular endothelial growth factor (VEGF) secretion, endothelial cell migration after treatment, and the status of phosphorylated Akt were evaluated in vitro to determine mechanisms underlying the inhibition of ErbB-2-induced angiogenesis. RESULTS. Mice treated with the trastuzumab plus paclitaxel combination exhibited significantly reduced mean tumor volumes compared with mice treated with the IgG control (419.5 mm³ vs. 786.6 mm³, P < 0.0001). Mice treated with trastuzumab had a mean tumor volume of 543.9 mm³, and mice treated with paclitaxel had a mean tumor volume of 574.9 mm³. Tumors from the trastuzumab-plus-paclitaxel group also had significantly decreased mean MVD compared with the control (30 ± 8 MVD vs. 44 ± 12 MVD, P < 0.05). The trastuzumab group had tumors with a MVD of 35 ± 7, similar to the paclitaxel-treated group (35 ± 9). Forty-four percent of the mice in the trastuzumab-plus-paclitaxel group had metastases to the lungs compared with 50%, 63%, and 75% of the mice in the paclitaxel, trastuzumab, and control groups, respectively. In vitro, the ErbB-2-overexpressing cells treated with combined trastuzumab plus paclitaxel secreted less VEGF than the cells treated with trastuzumab, paclitaxel, or control (185.9 pg/mL vs. 233.2 pg/mL, 261.3 pg/mL, and 286.4 pg/mL, respectively). In addition, the conditioned media from the combination group stimulated less mean endothelial cell migration (31. 0 cells vs. 47.0 cells, 39.2 cells, and 67.5 cells, respectively). Furthermore, Akt phosphorylation contributed to VEGF up-regulation and Akt phosphorylation was reduced more effectively by combined trastuzumab plus paclitaxel treatment compared with the other treatments. CONCLUSIONS. Combined trastuzumab plus paclitaxel treatment more effectively inhibited ErbB-2-mediated angiogenesis than either treatment alone, which resulted in more pronounced tumoricidal effects. This effect may be mediated via the reduction of phosphorylated Akt.