The anti-erbB3 antibody MM-121/SAR256212 in combination with trastuzumab exerts potent antitumor activity against trastuzumab-resistant breast cancer cells

To explore whether the anti-erbB3 Ab MM-121 may enhance the activity of trastuzumab against erbB2+ breast cancers, we investigated the combinatorial effects of MM-121 and trastuzumab on erbB3 signaling and cell proliferation in two erbB2+ breast cancer cell lines (SKBR3 and BT474). The cells were treated with either MM-121 or trastuzumab alone, or their combinations for 24 hrs, and then subjected to western blot analysis. We found that treatment with trastuzumab mainly reduced the levels of phosphorylated erbB3 (P-erbB3) and phosphorylated Akt (P-Akt) in both SKBR3 and BT474 cell lines, whereas MM-121 had no obvious effects on P-erbB3 and P-Akt. However, the combinations of MM-121 and trastuzumab more potently decreased P-erbB3 and P-Akt as compared to trastuzumab alone in SKBR3 cells and to a less extend in BT474 cells (Figure 1A). Neither MM-121 or trastuzumab alone, nor their combinations had significant effects on erbB2 kinase activity, MAPK signaling, and the expression of erbB2/erbB3 receptors (Figure 1A). Cell growth assays revealed that trastuzumab inhibited proliferation of SKBR3 and BT474 cell lines in a dose-dependent manner, consistent with our previous findings [26]. The addition of MM-121 significantly enhanced trastuzumab-mediated growth inhibition in both SKBR3 and BT474 cell lines (Figure 1B). Since activation of the erbB3 signaling plays an important role in the development of trastuzumab resistance [26], we next studied whether MM-121 might overcome the resistance and enhance trastuzumab-mediated growth inhibition in two otherwise resistant breast cancer cell lines. SKBR3-pool2 and BT474-HR20 are trastuzumab-resistant sublines-derived from SKBR3 and BT474 cell lines, respectively [26, 28]. While SKBR3-pool2 cells were kindly provided by Dr. Francisco Esteva at MD Anderson Cancer Center [28, 29], the BT474-HR20 subline was developed by our laboratory through continuously exposing BT474 cells to trastuzumab in culture for 4 months [26, 30]. Indeed, both SKBR3-pool2 and BT474-HR20 cells maintained their resistant phenotype to trastuzumab treatment as compared to their sensitive counterparts (Figure 2A vs Figure 1B). However, the presence of MM-121 significantly enhanced trastuzumab-mediated growth inhibition in both SKBR3-pool2 and BT474-HR20 cell lines (Figure 2A). Further studies on erbB3 activation and the downstream signaling showed that while either MM-121 or trastuzumab alone induced a clear reduction of P-erbB3 and P-Akt and had no significant effects on P-erbB2 and P-MAPK, the combinations of MM-121 and trastuzumab dramatically reduced P-erbB3 and P-Akt in both SKBR3-pool2 and BT474-HR20 cell lines (Figure 2B). Taken together, our data indicate that the erbB3 blocking Ab MM-121 significantly enhances trastuzumab-induced growth inhibition in two erbB2+ breast cancer cell lines and exhibits potential to overcome trastuzumab resistance mainly through inactivation of the erbB3/PI-3K/Akt signaling.

To study the molecular mechanism by which MM-121 overcomes trastuzumab resistance and enhances trastuzumab’s efficacy on inhibition of cell proliferation and/or survival in the studied cell lines, we considered the mechanism of action of trastuzumab inducing cell cycle G1 arrest [4, 31, 32] and thus investigated the combinatorial effects of MM-121 and trastuzumab on the expression levels of several critical molecules participating in G1-S transition and cell cycle progression in erbB2+ breast cancer cell lines. In the trastuzumab-sensitive cells, trastuzumab alone induced a minor reduction of E2F-1 and a slight increase of p27^kip1 in SKBR3 cells, and it only upregulated p27^kip1 in BT474 cells (Figure 3A). MM-121 alone did not alter the expression levels of E2F-1 and p27^kip1 in either cell lines. However, the combinations of trastuzumab and MM-121 clearly increased the levels of p27^kip1 in both cell lines and led to a minor reduction of E2F-1 in SKBR3 cells (Figure 3A). The expression levels of cyclin D1 were not significantly changed upon treatment with trastuzumab and/or MM-121. Flow cytometry analysis of cell cycle distribution showed that trastuzumab alone increased the cells at G1 phase in both cell lines, whereas MM-121 enhanced G1 population only in SKBR3 cells. Importantly, MM-121 in combination with trastuzumab more dramatically increased the percentage cells at G1 phase and decreased the cells at S phase in both cell lines (Figure 3B), suggesting a further induction of G1 arrest. In the trastuzumab-resistant cells, the expression levels of p27^kip1 were slightly increased upon treatment with either trastuzumab or MM-121 alone, whereas the combinations of MM-121 and trastuzumab not only upregulated p27^kip1 in both SKBR3-pool2 and BT474-HR20 cell lines, but also decreased E2F-1 in SKBR3-pool2 cells (Figure 4A). Furthermore, cell cycle analysis confirmed that the combinations of MM-121 and trastuzumab exhibited more potent activity than either agent alone to increase the G1 population and decrease the cells at S phase (Figure 4B). MM-121 and/or trastuzumab had no significant effect on G2-M transition in both trastuzumab-sensitive and -resistant cells (Figures 3B & 4B). While Figures 3B and 4B show the representative data, statistical analyses of G1 population from multiple cell cycle assays were also performed, and we found that the combinations of MM-121 and trastuzumab as compared to trastuzumab alone significantly increased G1 population in SKBR3, SKBR3-pool2, and BT474-HR20 cells (Additional file 1: Figure S1). Additional studies on induction of apoptosis showed that MM-121 and/or trastuzumab did not induce apoptosis in our cell culture condition (data not shown). Collectively, our studies suggest that the combinations of MM-121 and trastuzumab inhibited proliferation of both trastuzumab-sensitive and trastuzumab-resistant breast cancer cells mainly through cell cycle G1 arrest, which was correlated with the upregulation of p27^kip1 and sometimes a concomitant downregulation of E2F-1.

To further explore whether MM-121 holds potential to overcome trastuzumab resistance in an in vivo model for breast cancer treatment, we took advantage of the tumor xenografts model established from the trastuzumab-resistant breast cancer cell line BT474-HR20. There is a general concern that erbB2+ breast cancer cell lines are difficult to form spontaneous xenografts in athymic nu/nu mice [33], and it is not known whether the BT474-HR20 cells would maintain their trastuzumab-resistant phenotype in vivo. We first compared the ability of trastuzumab-sensitive and -resistant cells to form tumors in nude mice. We found that the BT474-HR20 cells formed tumors with a shorter latency than BT474 cells, and the tumors established from the resistant cells grew significantly faster than those from the parental cells (Additional file 2: Figure S2A), suggesting the aggressive phenotypes of BT474-HR20 cells. Importantly, the tumors-derived from BT474-HR20 cells were still growing under the treatment of trastuzumab (Additional file 2: Figure S2B), whereas the tumors-derived from BT474 cells were eliminated after three doses of trastuzumab (Additional file 2: Figure S2C). These data suggest that although BT474-HR20 cells were obtained in in vitro cell culture condition, they still maintained the trastuzumab-resistant phenotype in vivo. We next performed the following in vivo experiments with Ab treatment. When BT474-HR20 tumor volumes reached ~65 mm³, the nude mice were treated with either PBS (control), or MM-121 or trastuzumab alone, or the combinations of MM-121 and trastuzumab. Treatment with trastuzumab alone resulted in a minor and statistically insignificant inhibition (Figure 5A). It appeared that MM-121 alone had a stimulatory effect on the growth of BT474-HR20 tumor xenograft, although the differences were statistically insignificant. However, this phenomenon was not observed consistently. In our recent publication, MM-121 alone had neither positive nor negative effect on in vivo tumor growth of BT474-HR20 cells . More importantly, the combinations of MM-121 and trastuzumab significantly inhibited tumor growth of BT474-HR20 cells (Figure 5A). After 6-time treatments, the remaining tumors from the combinatorial treatment were very small. We did observe tumor regression in the time frame of our experiments. Histology and immunohistochemistry (IHC) assays revealed that treatment with MM-121 or trastuzumab alone did not alter tumor cell morphology and the expression of erbB2/erbB3 receptors (Figure 5B). In contrast, the combinatorial treatment resulted in much less tumor cells remaining, lost tumor architecture, and increased fibroblast cells in the tissues. Nonetheless, the remaining tumor cells maintained a similar expression levels of both erbB2 and erbB3 receptors (Figure 5B), which was consistent with the results of our cell culture studies (Figure 2B).

While our cell culture studies discovered that MM-121 in combination with trastuzumab inhibited proliferation of SKBR3-pool2 and BT474-HR20 cell lines (Figure 2A) without induction of apoptosis (data not shown), we wondered whether the combinations of MM-121 and trastuzumab would have similar effects on the trastuzumab-resistant cells in vivo. By utilizing the tumor tissues obtained from the BT474-HR20 xenograft animal studies described above, we then performed IHC studies on the classic cell proliferative marker Ki67 and cleaved caspase-3, an indicative of cells undergoing apoptosis. The mice treated with MM-121 or trastuzumab exhibited a minor reduction in the number of tumor cells with positive staining of Ki67 as compared to the control mice (Figure 6A). Neither MM-121 nor trastuzumab induced caspase-3 cleavage in the tumor tissues. However, the mice treated with both MM-121 and trastuzumab showed a dramatic reduction in the number of tumor cells with positive staining of Ki67 and a significant increase of the tumor cells with cleaved caspase-3 (Figure 6 and Additional file 3: Figure S3). These data indicate that MM-121 enhances trastuzumab’s antitumor activity against the otherwise resistant breast cancer cells via induction of both cell growth inhibition and apoptosis in this in vivo model.

MM-121 was kindly provided by Merrimack Pharmaceuticals, Inc. (Cambridge, MA). Trastuzumab (Herceptin®, Genentech, South San Francisco, CA) was obtained from University of Colorado Hospital pharmacy. Antibodies used for western blots were as follows: erbB2 (EMD Chemicals, Inc., Gibbstown, NJ); erbB3 and P-erbB2 (Tyr1248) (LabVision Corp., Fremont, CA); P-erbB3 (Tyr1289), P-MAPK (Thr202/Tyr204), MAPK, P-Akt (Ser473), and Akt (Cell Signaling Technology, Inc., Beverly, MA); Cyclin D1 (M-20), E2F1 (KH95), and p27^kip1 (F-8) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); and β-actin (Sigma Co., St. Louis, MO). All other reagents were purchased from Sigma unless otherwise specified.

Human breast cancer cell lines SKBR3 and BT474 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The trastuzumab-resistant sublines SKBR3-pool2 and BT474-HR20, derived from SKBR3 and BT474, respectively, were described previously [26]. All cell lines were maintained in DMEM/F-12 (1:1) medium (Sigma) containing 10% fetal bovine serum (Sigma), and cultured in a 37°C humidified atmosphere containing 95% air and 5% CO2 and split twice a week.

The CellTiter96 AQ nonradioactive cell proliferation kit (Thermo Fisher Scientific Inc., Waltham, MA) was used to determine cell viability as previously described [25, 26]. Briefly, cells were plated onto 96-well plates for 24 h, and then grown in either DMEM/F12 medium with 0.5% FBS as control, or the same medium containing different concentrations of trastuzumab in the presence or absence of MM-121, and then incubated for another 72 h. After reading all wells at 490 nm with a microplate reader, the percentages of surviving cells from each group relative to controls, defined as 100% survival, were determined by reduction of MTS.

Flow cytometric assays were performed as described previously [52] to define the cell cycle distribution. In brief, cells grown in culture dishes were harvested by trypsinization and fixed with 70% ethanol. Cells were stained for total DNA content with a solution containing 50 μg/ml propidium iodide and 100 μg/ml RNase I in PBS for 30 min at 37°C. Cell cycle distribution was analyzed at the Flow Cytometry Core Facility of University of Colorado Cancer Center with a FACScan flow cytometer (BD Biosciences, San Jose, CA).

Protein expression levels were determined by western blot analysis as described previously [26, 30, 52]. Equal amounts of total cell lysates were boiled in Laemmli SDS-sample buffer, resolved by SDS-PAGE, transferred to nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA), and probed with the primary antibodies described in the figure legends. After the blots were incubated with horseradish peroxidase-labeled secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA), the signals were detected using the enhanced chemiluminescence reagents (GE Healthcare Bio-Sciences Corp., Piscataway, NJ).

Five micron thick paraffin sections were deparaffinized, antigens unmasked and immunohistochemically stained for Ki67 (Thermo Fisher Scientific; rabbit monoclonal SP6; cat# RM-9106-SO; dilution 1:500 in TBST + 1% BSA w/v), cleaved Caspase-3 (Cell Signaling Technology; rabbit polyclonal; cat#: 9661, 1:1000 in TBST + 1% BSA w/v), erbB2 (EMD Chemicals; mouse monoclonal 96G; cat#OP14T; dilution 1:500 in TBST + 1% BSA w/v), and erbB3 (Spring Bioscience, Pleasanton, CA; rabbit monoclonal SP71; cat# M3710; dilution 1:200 in TBST + 1% BSA w/v). The specificity of all antibodies has been confirmed by both positive and negative controls. For erbB2 and erbB3, SKBR3 cells were used as a positive control. For Ki67 and cleaved caspase-3, the human tonsil tissues were used a positive control. All the negative controls were performed with the same cells/tissues without addition of the primary antibodies.

Ki67 and cleaved caspase-3 antigens were revealed in pH 9.5 BORG solution (Biocare Medical, Concord, CA) for 5 min at 125°C (22 psi; Decloaking chamber, Biocare). ErbB2 required modest retrieval in 10 mmol/L sodium citrate for 5 min at 125°C in the Decloaking chamber. ErbB3 required retrieval in Cell Conditioner 1 (standard retrieval time, Ventana). Immunodetection of Ki67, cleaved Caspase-3 and erbB2 was performed on the NexES stainer (Ventana Medical Systems, Tucson, AZ) at an operating temperature of 37°C. Ki67 and cleaved caspase-3 antibodies were incubated for 32 min and detected with a modified I-VIEW DAB (Ventana) detection kit. The I-VIEW secondary antibody and enzyme were replaced with a species specific secondary antibody (biotinylated goat anti-rabbit; 1:75; cat# 111-065-144; Jackson ImmunoResearch; 8 min) and streptavidin-horseradish (SA-HRP; 1:50; cat# SA-5004; DAKO Cytomation, Carpinteria, CA; 8 min). ErbB2 was incubated for 32 min and detected with the standard I-VIEW detection. ErbB3 was incubated for 32 min and detected with a modified I-VIEW DAB kit in which the secondary antibody was replaced with Rabbit ImmPress (Vector Labs; Burlingame, CA; cat# MP-7401; 8 minutes at 37°C) and enzyme was replaced with Rabbit ImmPress (Diluted 1:1 in PBS pH 7.6; 8 minutes at 37°C). Sections were sequentially blocked for 10 min in 3% hydrogen peroxide (v/v) and 30 min in Rodent Block M (Biocare, cat# RBM961), followed by primary antibody incubation for 30 min and 30 min in polymer. Antibody complexes were visualized with IP Flex DAB (Biocare; cat# IPK5010 G80; 4.5%). All sections were counterstained in Mayer’s hematoxylin for 2 min, nuclei blued in 1% ammonium hydroxide (v/v), dehydrated in graded alcohols, cleared in xylene and coverglass mounted using synthetic resin.

Athymic nu/nu mice (Harlan Laboratories, Inc., Indianapolis, IN) were maintained in accordance with the Institutional Animal Care and Use Committee (IACUC) procedures and guidelines. Eight ×10⁶ BT474-HR20 cells were suspended in 100 μL of PBS, mixed with 50% Matrigel (BD Biosciences) and injected subcutaneously (S.C.) into the flanks of 5-week-old female mice. Tumor formation was assessed by palpation and measured with fine calipers three times a week. Tumor volume was calculated by the formula: volume = (length × width²)/2, where length was the longest axis and width the measurement at a right angle to the length, and followed by statistical analysis as we described previously [52]. When tumors reach ~65 mm³, mice were randomly assigned to four groups (n = 5): 1) control group-mice received intraperitoneally (i.p) injection of 100 μl PBS only; 2) mice received i.p. injection of trastuzumab (10 mg/kg) in 100 μl PBS twice a week; 3) mice received i.p. injection of MM-121 (10 mg/kg) in 100 μl PBS twice a week; 4) mice received i.p. injection of trastuzumab (10 mg/kg) and MM-121 (10 mg/kg) in 100 μl PBS twice a week. The animals’ health status was monitored daily for weight loss or for signs of altered motor while in their cages. At the end of study, mice were euthanized according to approved IACUC protocol. Tumors from all animals were excised and embedded in paraffin for immunohistochemical analyses.

Statistical analyses of the experimental data were performed using either a two-sided t test or ANOVA for each time point followed by post-hoc testing between groups. Significance was set at a P value of <0.05. All statistical analyses were conducted with the software StatView v5.1 from SAS Institute Inc., Cary, NC.