Mode of action of pertuzumab in combination with trastuzumab plus docetaxel therapy in a HER2-positive breast cancer xenograft model

Abstract

In a Phase III trial for HER2-positive breast cancer (the CLEOPATRA study), the triple-drug combination arm of pertuzumab plus trastuzumab plus docetaxel showed significantly longer progression-free survival and overall survival than did the trastuzumab plus docetaxel arm. In this study, we investigated the mechanism of action of the triple-drug combination therapy in vivo. For this purpose, we established a mouse xenograft model using KPL-4, a HER2-positive human breast cancer cell line, in which the triple-drug combination treatment dramatically induced tumor regression compared with double-drug combinations (trastuzumab plus docetaxel, pertuzumab plus docetaxel, or pertuzumab plus trastuzumab). Four days after the triple-drug treatment was started, strong reduction in the phosphorylation of HER2, epidermal growth factor receptor (EGFR), HER3, extracellular signal-regulated kinase (ERK), and AKT in tumor tissues was seen, despite only weak suppression of phosphorylation seen with the single- or double-drug treatments. Histopathological analysis and flow cytometric analysis showed that the triple-drug treatment enhanced apoptosis after mitotic arrest induced by docetaxel. Furthermore, infiltration of mononuclear cells around the tumor cells was strongly induced by the triple-drug combination treatment. These results suggested that the mechanism underlying the synergistic efficacy of the triple-drug combination was attributable, at least in part, to the docetaxel-mediated apoptosis being promoted by enhanced inhibition of HER2-HER3-AKT signaling as well to the intratumor infiltration of mononuclear cells induced by anti-HER2 antibodies being enhanced by docetaxel.

Keywords: HER2; breast cancer; docetaxel; pertuzumab; trastuzumab.

Figure 1.

In vivo efficacy of triple-drug combination. (A) Mice bearing KPL-4 tumors were randomly divided into eight groups (n=6 per group) and were treated with trastuzumab, pertuzumab, docetaxel, trastuzumab + docetaxel, trastuzumab + pertuzumab, pertuzumab + docetaxel, or trastuzumab + docetaxel + pertuzumab. As a control, human IgG and vehicle of docetaxel were administered. (B) Mice bearing KPL-4 tumors were randomly divided into eight groups (n=6 per group) and treated with trastuzumab, pertuzumab, paclitaxel, trastuzumab + paclitaxel, trastuzumab + pertuzumab, pertuzumab + paclitaxel, or trastuzumab + paclitaxel + pertuzumab. As a control, human IgG and vehicle of paclitaxel were administered. Data points are mean + standard deviation of the tumor volume (mm³). Statistically significant differences are shown as ^#P<0.05 and *P<0.025. TRAS, trastuzumab; PER, pertuzumab; DTX, docetaxel; PTX, paclitaxel.

Figure 2.

Tumor growth rates under the triple-drug combination and under trastuzumab + docetaxel containing the same amount of antibodies. Mice bearing KPL-4 tumors were randomly divided into three groups (n=6 per group) and treated with pertuzumab (20 mg/kg) + trastuzumab (10 mg/kg) + docetaxel or with trastuzumab (30 mg/kg) + docetaxel. As a control, human IgG and vehicle of docetaxel were administered. Data points are mean + standard deviation of the tumor growth rate. Statistically significant difference is shown as *P<0.05. TRAS, trastuzumab; PER, pertuzumab; DTX, docetaxel.

Figure 3.

Inhibition of HER2-signal transduction after the triple-drug treatment. Lysates from tumor tissues collected 4 days after starting treatment were used for Western blotting. TRAS, trastuzumab; PER, pertuzumab; DTX, docetaxel (n=4 per group).

Figure 4.

Evaluation of apoptotic and proliferating tumor cells in vivo after the triple-drug treatment. (A) Mice bearing KPL-4 tumors were randomly divided into seven groups (n=6 or 7 per group) and treated with each combination of agents. Four days after starting treatment, apoptotic tumor cells were histopathologically evaluated. The numbers of (B) apoptotic cells, (C) mitotic cells and (D) Ki-67-positive cells in every 1,000 tumor cells were counted. Scale bar indicated 50 µm. Statistically significant differences are shown as ^#P<0.05 and *P<0.025. TRAS, trastuzumab; PER, pertuzumab; DTX, docetaxel.

Figure 5.

Evaluation of cell cycle of tumor cells in vivo and the percentage of mitotic cells in viable tumor cells after the triple-drug treatment. Tumor tissues were collected 4 days after starting treatment and dissociated. Tumor cells were then isolated and the cell cycle distribution was examined by flow cytometry. The percentage of cells in each cell cycle phase was calculated. TRAS, trastuzumab; PER, pertuzumab; DTX, docetaxel (n=3 per group).

Figure 6.

Invasion of MNCs around tumor cells after the triple-drug treatment. (A) Tumor tissues were collected 1 day (Day 2) or 4 days (Day 5) after starting treatment and stained with hematoxylin-eosin stain. Invasion of MNCs were histopathologically evaluated. Arrows show examples of infiltrating MNCs around tumor cells (representative images from Day 5). (B) Infiltration of MNCs was scored as 0: no change; 1: very slight; 2: slight; 3: moderate; or 4: marked, and shown graphically. Scale bar indicated 50 µm. Statistically significant differences are shown as *P<0.05. TRAS, trastuzumab; PER, pertuzumab; DTX, docetaxel (n=6 or 7 per group).