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. 2013 Feb 1;19(3):610-9.
doi: 10.1158/1078-0432.CCR-12-2024. Epub 2012 Dec 5.

Dual blockade of HER2 in HER2-overexpressing tumor cells does not completely eliminate HER3 function

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Dual blockade of HER2 in HER2-overexpressing tumor cells does not completely eliminate HER3 function

Joan T Garrett et al. Clin Cancer Res. .

Abstract

Purpose: Dual blockade of HER2 with trastuzumab and lapatinib or with pertuzumab is a superior treatment approach compared with single-agent HER2 inhibitors. However, many HER2-overexpressing breast cancers still escape from this combinatorial approach. Inhibition of HER2 and downstream phosphoinositide 3-kinase (PI3K)/AKT causes a transcriptional and posttranslational upregulation of HER3 which, in turn, counteracts the antitumor action of the HER2-directed therapies. We hypothesized that suppression of HER3 would synergize with dual blockade of HER2 in breast cancer cells sensitive and refractory to HER2 antagonists.

Experimental design: Inhibition of HER2/HER3 in HER2(+) breast cancer cell lines was evaluated by Western blotting. We analyzed drug-induced apoptosis and two- and three-dimensional growth in vitro. Growth inhibition of PI3K was examined in vivo in xenografts treated with combinations of trastuzumab, lapatinib, and the HER3-neutralizing monoclonal antibody U3-1287.

Results: Treatment with U3-1287 blocked the upregulation of total and phosphorylated HER3 that followed treatment with lapatinib and trastuzumab and, in turn, enhanced the antitumor action of the combination against trastuzumab-sensitive and -resistant cells. Mice bearing HER2(+) xenografts treated with lapatinib, trastuzumab, and U3-1287 exhibited fewer recurrences and better survival than mice treated with lapatinib and trastuzumab.

Conclusions: Dual blockade of HER2 with trastuzumab and lapatinib does not eliminate the compensatory upregulation of HER3. Therapeutic inhibitors of HER3 should be considered as part of multidrug combinations aimed at completely and rapidly disabling the HER2 network in HER2-overexpressing breast cancers.

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Figures

Figure 1
Figure 1. U3-1287 downregulates HER3 and blocks HER3 phosphorylation following inhibition of HER2
A. BT474, SKBR3 and MDA453 were treated with 20 μg/ml of U3-1287 over the indicated time course. Whole cell lysates were prepared and separated in a 7% SDS gel followed by immunoblot analysis with HER3 and β-actin antibodies. B. Cells were treated with U3-1287 (20 μg/ml), trastuzumab (20 μg/ml), lapatinib (1 μM) or the indicated combinations for 24 h and then biotinylated on their cell surface as described in Methods. Cell lysates were precipitated with immobilized Neutravidin gel; eluates were separated by SDS-PAGE and subjected to HER3 immunoblot analysis. C. Cells were treated with U3-1287, trastuzumab, lapatinib or the indicated combinations for 24 h. Whole cell lysates were prepared and separated in a 7% SDS gel followed by immunoblot analysis with the indicated antibodies.
Figure 2
Figure 2. Neutralizing HER3 antibody sensitizes to combination of HER2 inhibitors
A. BT474, SKBR3 and MDA453 cells were seeded in 60-mm plates in growth medium containing 10% FCS. The following day, cells were changed to serum-free media and treated with lapatinib (1 μM), trastuzumab (20 μg/ml) and U3-1287 (20 μg/ml) as indicated. Forty-eight h later, adherent and detached cells were collected and subjected to TUNEL assay using the APOBrdU kit. Each bar represents the mean ± SEM of cells with apoptotic nuclei (n=2-4; *, p<0.05, t test). B. Cells were seeded in 6-well plates in triplicate and treated with DMSO, 20 μg/ml trastuzumab, 0.1 μM lapatinib, 20 μg/ml U3-1287 or the indicated combinations. Media and inhibitors were replenished every 3-4 days. The monolayers were stained with crystal violet when the untreated cells became confluent after 14-21 days. Quantification of integrated intensity (% control) is shown (*, p<0.05, t test). C. BT474, SKBR3 and MDA453 cells were seeded in Matrigel and allowed to grow in the absence or presence of lapatinib (1 μM), trastuzumab (20 μg/ml) and/or U3-1287 (20 μg/ml). Media and inhibitors were replenished every 3 days. Mean total colony volume was quantified using the GelCount system. Each bar graph represents the mean ± S.E.M. of triplicate samples (*, p<0.05, t test).
Figure 3
Figure 3. Trastuzumab resistant cells remain sensitive to HER2-HER3 blockade
A. Trastuzumab-resistant HR6 cells were treated with U3-1287, trastuzumab, lapatinib or the indicated combinations for 24 h. Whole cell lysates were prepared and separated by 7% SDS-PAGE followed by immunoblot analysis with the indicated antibodies. B. HR6 cells were seeded in Matrigel and allowed to grow in the absence or presence of lapatinib, trastuzumab and/or U3-1287 as indicated. Medium and inhibitors were replenished every 3 days. Images shown were recorded 15 days after cell seeding. Each bar graph represents the mean ± S.E.M. of triplicate wells (*, p<0.05, t test). C. Cells were seeded in triplicate and treated with DMSO, 20 μg/ml trastuzumab, 0.1μM lapatinib, 20 μg/ml U3-1287 or the indicated combinations. Media and inhibitors were replenished every 3-4 days. The monolayers were stained with crystal violet when the untreated cells became confluent after 14-21 days. Quantification of integrated intensity (% control) was measured as describe in Methods (*, p<0.05, t test). D. HR6 cells were seeded in 60-mm plates in growth medium containing 10% FCS. The following day, cells were changed to serum-free media and treated with lapatinib (1 μM), trastuzumab (20 μg/ml) and U3-1287 (20 μg/ml) as indicated. Forty-eight h later, adherent and detached cells were collected and subjected to TUNEL assay using the APO-BrdU kit. Each bar represents the mean ± SEM of cells with apoptotic nuclei for each treatment group (n=3; *, p<0.05, t test). E. Female athymic mice were injected with HR6 cells as indicated in Methods; once tumors reached a volume ≥200 mm3, mice were randomized to the indicated treatments. Tumor volumes were measured overtime. Each data point represents the mean tumor volume + SEM(*, p<0.05, trastuzumab versus trastuzumab + U3-1287).
Figure 4
Figure 4. Addition of U3-1287 to the combination of trastuzumab and pertuzumab enhances inhibition of HER3/PI3K signaling and tumor growth
A. Cells were treated with U3-1287 (20 μg/ml), trastuzumab (20 μg/ml), pertuzumab (20 μg/ml) or the indicated combinations for 24 h. Whole cell lysates were prepared and separated in a 7% SDS gel followed by transfer to nitrocellulose and immunoblot analysis with the indicated antibodies. B. Cells were seeded in in triplicate and treated with vehicle, U3-1287 (20 μg/ml), trastuzumab (20 μg/ml), pertuzumab (20 μg/ml) or the indicated combinations. Media and inhibitors were replenished every 3-4 days. The monolayers were stained with crystal violet when the untreated cells became confluent after 14-21 days. Quantification of integrated intensity (% control) is shown (*, p<0.05, t test).
Figure 5
Figure 5. Dual blockade of HER2 in combination with HER3 inhibitor reduces tumor growth
A. Female athymic mice were injected with BT474 cells; once xenografts reached a volume of 350-400 mm3, they were randomized to the indicated treatments. Each data point represents the mean tumor volume in mm3 + SEM. B. Some mice were sacrificed after 5 days of treatment, receiving the last dose of lapatinib at 1h and/or trastuzumab and/or U3-1287 at 24 h before sacrifice. IHC analysis of S473 P-Akt in formalin-fixed, paraffin-embedded tumor sections from mice on treatment for 5 days (n=4-6). Top: Quantitative comparison of P-Akt histoscore for intensity of cytoplasmic staining. Bottom: Representative images from tumor sections are shown.
Figure 6
Figure 6. Dual HER2 blockade in combination with HER3 antibody improves survival
Female athymic mice were injected with BT474 cells as described in Methods. Once tumors reached a volume ≥350 mm3, mice were randomized to lapatinib and trastuzumab or lapatinib, trastuzumab and U3-1287. Treatment was administered for 21 days after which time treatment stopped and mice were monitored for tumor regrowth. The x-axis indicates weeks after drug treatment stopped. Mice were sacrificed once recurrent tumors were ≥2000 mm3.

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