Therapeutic targeting of erbB3 with MM-121/SAR256212 enhances antitumor activity of paclitaxel against erbB2-overexpressing breast cancer

Introduction Elevated expression of erbB3 rendered erbB2-overexpressing breast cancer cells resistant to paclitaxel via PI-3 K/Akt-dependent upregulation of Survivin. It is unclear whether an erbB3-targeted therapy may abrogate erbB2-mediated paclitaxel resistance in breast cancer. Here, we study the antitumor activity of an anti-erbB3 antibody MM-121/SAR256212 in combination with paclitaxel against erbB2-overexpressing breast cancer. Methods Cell growth assays were used to determine cell viability. Cells undergoing apoptosis were quantified by a specific apoptotic ELISA. Western blot analyses were performed to assess the protein expression and activation. Lentiviral vector containing shRNA was used to specifically knockdown Survivin. Tumor xenografts were established by inoculation of BT474-HR20 cells into nude mice. The tumor-bearing mice were treated with paclitaxel and/or MM-121/SAR256212 to determine whether the antibody (Ab) enhances paclitaxel’s antitumor activity. Immunohistochemistry was carried out to study the combinatorial effects on tumor cell proliferation and induction of apoptosis in vivo. Results MM-121 significantly facilitated paclitaxel-mediated anti-proliferative/anti-survival effects on SKBR3 cells transfected with a control vector or erbB3 cDNA. It specifically downregulated Survivin associated with inactivation of erbB2, erbB3, and Akt. MM-121 enhances paclitaxel-induced poly(ADP-ribose) polymerase (PARP) cleavage, activation of caspase-8 and -3, and apoptosis in both paclitaxel-sensitive and -resistant cells. Specific knockdown of Survivin in the trastuzumab-resistant BT474-HR20 cells dramatically enhanced paclitaxel-induced apoptosis, suggesting that increased Survivin caused a cross-resistance to paclitaxel. Furthermore, the studies using a tumor xenograft model-established from BT474-HR20 cells revealed that either MM-121 (10 mg/kg) or low-dose (7.5 mg/kg) paclitaxel had no effect on tumor growth, their combinations significantly inhibited tumor growth in vivo. Immunohistochemical analysis showed that the combinations of MM-121 and paclitaxel significantly reduced the cells with positive staining for Ki-67 and Survivin, and increased the cells with cleaved caspase-3. Conclusions The combinations of MM-121 and paclitaxel not only inhibit tumor cell proliferation, but also promote erbB2-overexpressing breast cancer cells to undergo apoptosis via downregulation of Survivin in vitro and in vivo, suggesting that inactivation of erbB3 with MM-121 enhances paclitaxel-mediated antitumor activity against erbB2-overexpressing breast cancers. Our data supports further exploration of the combinatorial regimens consisting of MM-121 and paclitaxel in breast cancer patients with erbB2-overexpressing tumors, particularly those resistant to paclitaxel.


Introduction
The erbB receptor tyrosine kinase (RTK) family, including the epidermal growth factor receptor (EGFR), erbB2 (or Her2/neu), erbB3, and erbB4, is arguably the most important receptor family in the context of development and tumorigenesis [1,2]. Amplification and/or overexpression of erbB2 occur in approximately 25 to 30% of invasive breast cancers and are significantly associated with a worse prognosis in breast cancer patients [3,4]. Numerous studies indicate that increased treatment resistance and enhanced metastatic potential are two of the major mechanisms by which erbB2 contributes to breast carcinogenesis [5,6]. Most metastatic breast cancers show expression for either EGFR or erbB2, and less often for both [7]. In contrast, co-expression of erbB2 and erbB3 frequently occurs in breast cancers [8] and breast cancer cell lines [9]. The erbB3 receptor is unique among the four erbB family members. Unlike EGFR, erbB2, and erbB4, it lacks kinase activity [10,11] or possesses weak kinase activity [12]. However, erbB3 has been shown to serve as a critical co-receptor of erbB2, and its expression is a rate-limiting factor for erbB2mediated breast cancer cell survival and proliferation [13,14]. We and others have also observed an elevated expression of the endogenous mouse erbB3 in the mammary tumors derived from erbB2/neu-transgenic mice [15,16]; and the increased erbB3 forms physical and functional interactions with the transgene-encoded erbB2 to promote mammary tumorigenesis [15]. In fact, the erbB2/ erbB3 heterodimer has been identified as the most potent form of all erbB receptor complexes to activate the oncogenic signaling, such as PI-3 K/protein kinase B (Akt), mitogen-activated protein kinase kinase (MEK)/mitogenactivated protein kinase (MAPK), and/or janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathways, and/or Src kinase, in breast cancers [17][18][19].
Mechanistic studies implicate the function of erbB3 as a major cause of treatment failure in human cancers [20]. Therapeutic targeting of erbB3 is being investigated. Currently, no erbB3-targeted therapy has been approved for cancer treatment. Several erbB3 blocking antibodies (Abs) that prevent ligand-induced activation of erbB3, such as MM-121/SAR256212 (which is being codeveloped by Merrimack Pharmaceuticals, Cambridge, MA, USA and Sanofi, Cambridge, MA, USA), MM-111 (Merrimack Pharmaceuticals) and U3-1287/AMG 888 (Amgen Inc., Thousand Oaks, CA, USA) are actively under preclinical and clinical studies [21] and show significant antitumor activity in preclinical studies [22][23][24][25]. MM-121 is a fully humanized anti-erbB3 monoclonal IgG2 Ab. It inhibits ligand-induced activation of erbB3, and exerts antitumor activity in human cancer models [23,24]. Nonetheless, MM-121's therapeutic potential against erbB2-overexpressing breast cancers that are resistant to chemotherapy and/or the erbB2-targeted therapy has not been explored.
We have reported that activation of erbB3, mainly through PI-3 K/Akt signaling, plays a critical role in erbB2-mediated therapeutic resistance to tamoxifen [26] and paclitaxel [27]. The erbB3 receptor also interacts with both erbB2 and the insulin-like growth factor-1 receptor (IGF-1R) to form a heterotrimeric complex, which activates the PI-3 K/Akt signaling and Src kinase and subsequently results in resistance to erbB2-targeted therapy, trastuzumab (Herceptin) [28]. Since activation of the PI-3 K/Akt signaling is the major determinant of treatment resistance [29], we investigated, in vitro and in vivo, whether the anti-erbB3 Ab MM-121 would be able to overcome resistance and enhance the efficacy of chemotherapy or trastuzumab against erbB2-overexpressing breast cancer models via inhibition of the erbB3/PI-3 K/Akt signaling. In the current report, we sought to determine the antitumor activity of MM-121 in combination with paclitaxel against erbB2-overexpressing breast cancer using both in vitro and in vivo models. Our previous studies indicated that elevated expression of erbB3 led to paclitaxel resistance in erbB2-overexpressing breast cancer cells via PI-3 K/Akt signaling-dependent upregulation of Survivin [27]. Thus, we have focused on studying whether inactivation of erbB3 signaling with MM-121 may specifically downregulate Survivin, and subsequently re-sensitize the otherwise resistant breast cancer cells to paclitaxel-mediated anti-proliferative/ anti-survival effects and apoptosis.

Cell proliferation assay
The CellTiter96 AQ nonradioactive cell proliferation kit (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to determine cell viability as previously described [27,28,30]. 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 paclitaxel 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 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS).

Specific knockdown of Survivin expression with a lentiviral system
Lentiviral production and specific knockdown of Survivin expression with an shRNA strategy were carried out as described previously [27]. In brief, the lentivirus-containing either control shRNA or Survivin specific shRNA (Survivin-S3 or Survivin-S5) were produced in 293 T cells following the standard procedure. The virus in conditioned medium were harvested, aliquoted, and stored in a −80°C freezer. Prior to infection, the lentivirus-containing media were thawed completely at room temperature, and mixed with the same amount of fresh medium containing polybrene (8 μg/ml). The culture media of the candidate breast cancer cells were then replaced with the lentivirus-containing media. After 24 h, the virus-infected cells were selected with puromycin (1 μg/ml) for 48 h, and then subjected to required experiments.

Quantification of apoptosis
An apoptotic ELISA kit (Roche Diagnostics Corp., Indianapolis, IN, USA) was used to quantitatively measure cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes) as previously reported [27,28,30]. This enzyme immunoassay was performed according to the manufacturer's instructions.

Western blot analysis
Protein expression levels were determined by western blot analysis as described previously [27,28,30,31]. 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, USA), 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, USA), the signals were detected using the enhanced chemiluminescence reagents (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA).
Survivin was optimized under ambient conditions with the Rabbit ImmPress polymer detection system (Vector Laboratories, Burlingame, CA, USA; cat# MP-7401). ErbB2 was incubated for 32 minutes and detected with the standard I-VIEW detection. ErbB3 was incubated for 32 minutes and detected with a modified I-VIEW DAB kit in which the secondary antibody was replaced with Rabbit ImmPress (Vector Laboratories; 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 minutes in 3% hydrogen peroxide (v/v) and 30 minutes in Rodent Block M (Biocare; cat# RBM961), followed by primary antibody incubation for 30 minutes and 30 minutes 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 minutes, nuclei blued in 1% ammonium hydroxide (v/v), dehydrated in graded alcohols, cleared in xylene and coverglassmounted using synthetic resin.

Tumor xenograft model
Athymic nu/nu mice (Harlan Laboratories, Inc., Indianapolis, IN, USA) were maintained in accordance with the Institutional Animal Care and Use Committee (IACUC) procedures and guidelines approved by University of Colorado Health Sciences Center Animal Care and Use Committee. We suspended 8 × 10 6 BT474-HR20 cells in 100 μL of PBS mixed with 50% Matrigel (BD Biosciences) and injected these subcutaneously into the flanks of 5week-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 shown below, where length was the longest axis and width the measurement at a right angle to the length: This was followed by statistical analysis as we described previously [32]. When tumors reached approximately 150 mm 3 or 100 mm 3 , mice were randomly assigned into four groups (n = 5): 1) control-group mice received intraperitoneal (i.p.) injection of 100 μl PBS; 2) mice received i.p. injection of paclitaxel (15 mg/kg or 7.5 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, or 4) mice received i.p. injection of paclitaxel (15 mg/kg or 7.5 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 ability while in their cages. At the end of study, mice were euthanized according to the approved IACUC protocol. Tumors from all animals were excised and embedded in paraffin for IHC analyses.

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

MM-121 overcomes paclitaxel resistance induced by coexpression of erbB2 and erbB3 in breast cancer cells and significantly enhances inhibitory activity of paclitaxel
To study whether inactivation of erbB3 signaling with MM-121 may overcome paclitaxel resistance and facilitate paclitaxel-mediated inhibitory activity against erbB2overexpressing breast cancer, the SKBR3.B3.1 and SKBR3. B3.2 cells that show resistance to paclitaxel due to ectopic expression of erbB3 in SKBR3 cells [27] were used to investigate inhibitory effects of MM-121 on erbB3 signaling and enhancement of paclitaxel-mediated antiproliferative/anti-survival effects. Although SKBR3.B3.1 and SKBR3.B3.2 cell lines were less sensitive to paclitaxel than the parental SKBR3 and vector control SKBR3.neo1 cell lines [27], (this lower responsiveness was further confirmed in the current studies shown in Additional file 2), paclitaxel was still able to inhibit proliferative/survival of SKBR3.neo1, SKBR3.B3.1, and SKBR3.B3.2 cells in a dose-dependent manner ( Figure 1A). Importantly, the presence of MM-121 significantly enhanced paclitaxel-mediated inhibitory activity in all three cell lines ( Figure 1A). Western blot analyses showed that treatment of SKBR3.neo1, SKBR3.B3.1, and SKBR3.B3.2 cell lines with MM-121 reduced the levels of phosphorylated erbB3 (P-erbB3) and phosphorylated erbB2 (P-erbB2) in a time-dependent manner ( Figure 1B). These data suggest that MM-121 mainly inhibits activation of erbB3, which is in agreement with other reports [23,24]. Since erbB3 interacted with the erbB2 receptor to activate erbB2, it is understandable that inactivation of erbB3 with MM-121 might disrupt the heterodimerization of erbB2/erbB3 receptors, and thus decrease the kinase activity of erbB2 (phosphorylation). Furthermore, treatment with MM-121 gradually reduced the levels of phosphorylated Akt (P-Akt), but had no significant effects on phosphorylated MAPK (P-MAPK) ( Figure 1B). These data are consistent with our previous findings showing that the PI-3 K/Akt pathway is the major downstream signaling mechanism for erbB2/erbB3 interactions in breast cancer cell lines [26,27]. Taken together, our data indicate that the anti-erbB3 Ab MM-121 exhibits the potential to overcome paclitaxel resistance and significantly enhances paclitaxelmediated anti-proliferative/anti-survival effects in erbB2-overexpressing breast cancer cell lines, with medium and high erbB3 expression, associated with its inhibition of the erbB3/PI-3 K/Akt signaling.

MM-121 specifically downregulates Survivin in paclitaxel-resistant breast cancer cell lines and significantly enhances paclitaxel-induced apoptosis in both sensitive and resistant cells
To study the molecular mechanism by which MM-121 overcomes paclitaxel resistance and increases paclitaxelmediated anti-proliferative/anti-survival effects in the studied erbB2-overexpressing breast cancer cell lines, we investigated whether MM-121 might enhance paclitaxelinduced apoptosis. Because activation of erbB2/erbB3 signaling resulted in paclitaxel resistance via PI-3 K/Aktdependent upregulation of Survivin [27] and we showed that MM-121 mainly inactivated Akt in all three SKBR3 sublines ( Figure 1B), we hypothesized that MM-121 might inhibit Survivin expression in both paclitaxelsensitive and -resistant breast cancer cells. Indeed, western blot analyses revealed that treatment of SKBR3.neo1, SKBR3.B3.1, and SKBR3.B3.2 cell lines with MM-121 led to decreased Survivin, but had no effects on the functionally related proteins, such as Bcl-xL and Mcl-1, in a time-dependent manner ( Figure 2A). These data are consistent with our previous studies indicating that the erbB3 signaling specifically regulates Survivin expression in erbB2-overexpressing breast cancer cells [27]. Moreover, although SKBR3.B3.1 and SKBR3.B3.2 cells were less responsive than the parental SKBR3 and vector control SKBR3.neo1 cells to paclitaxel-induced apoptosis, the addition of MM-121 significantly enhanced paclitaxel-induced apoptosis in both paclitaxel-sensitive and -resistant breast cancer cells ( Figure 2B and C). We showed evidence that the combinations of MM-121 and paclitaxel, as compared to either agent alone, gave rise to a profound induction of cleaved PARP and activation of caspase-8 and −3 ( Figure 2B), as well as histone-associated DNA fragments ( Figure 2C). Thus, our studies demonstrate that MM-121 overcomes paclitaxel resistance and enhances paclitaxel-induced apoptosis in the studied erbB2-overexpressing breast cancer cell lines via specific downregulation of Survivin.
Elevated expression of Survivin is observed in one trastuzumab-resistant breast cancer cell line and shows cross-resistance to paclitaxel that can be abrogated by MM-121 We previously reported that the three RTKs, erbB2, erbB3, and IGF-1R interacted with each other to form a heterotrimeric complex, which activates the downstream signaling, such as PI-3 K/Akt or MEK/MAPK pathways and Src kinase in trastuzumab-resistant breast cancer cells [28]. We explored whether the trastuzumab-resistant sublines BT474-HR20 and SKBR3-pool2 might also have increased expression of Survivin due to the activation of PI-3 K/Akt signaling, and subsequently exhibit resistance to paclitaxel-induced apoptosis. Compared to the parental BT474 cells, the trastuzumab-resistant BT474-HR20 cells expressed much higher levels of Survivin and had a minor increase in Mcl-1. The expression levels of Bcl-xL showed no difference between BT474 and BT474-HR20 cells (Additional file 3: Figure S2A). Interestingly, BT474-HR20 cells were significantly more resistant to paclitaxel-mediated anti-proliferative/anti-survival effects than BT474 cells (Additional file 3: Figure S2B). However, this phenomenon was not observed in another pair of trastuzumab-sensitive SKBR3 and -resistant SKBR3-pool2 cells, as we did not find a significant induction of Survivin, Mcl-1, or Bcl-xL in SKBR3-pool2 cells (data not shown). The difference might be due to the fact that BT474-HR20 cells exhibited a dramatic activation of Akt as compared to BT474 cells, whereas significant activation of MAPK, but not Akt was discovered in SKBR3-pool2 cells [28]. To study whether the enhanced expression of Survivin in BT474-HR20 cells causally induced paclitaxel resistance, two shRNA sequences (Survivin-S3 and -S5) were used to specifically knock down Survivin ( Figure 3A). Compared to control shRNA, both Survivin-S3 and Survivin-S5 significantly enhanced paclitaxel-induced apoptosis evidenced by increased PARP cleavage, caspase-3 activation ( Figure 3B), and an apoptotic ELISA ( Figure 3C). These data strongly suggest that the increased Survivin in the studied trastuzumab-resistant cell line causally elicited cross-resistance to paclitaxel. Because activation of the erbB3 signaling played an important role in the development of BT474-HR20 cells, we next studied whether MM-121 might also reduce Survivin in BT474-HR20 cells and therefore re-sensitize the cells to paclitaxel-mediated inhibitory activity and apoptosis. We discovered that MM-121 not only reduced the levels of P-erbB3, but also specifically downregulated Survivin (not Bcl-xL and Mcl-1) in BT474-HR20 cells ( Figure 4A), consistent with our findings in the SKBR3 sublines ( Figures 1B and 2A). More importantly, MM-121 significantly enhanced both paclitaxel-mediated anti-proliferative/anti-survival effects ( Figure 4B) and apoptosis ( Figure 4D), and facilitated paclitaxel-induced PARP cleavage and activation of caspase-8 and −3 ( Figure 4C) in BT474-HR20 cells.
Collectively, our data demonstrate that MM-121 was also able to abrogate paclitaxel resistance-induced by elevated expression of Survivin in the trastuzumabresistant breast cancer cells.  Figure 5A). These data suggest that MM-121 enhances low-dose paclitaxel-mediated antitumor activity against erbB2-overexpressing breast cancer in this in vivo mouse model. After 3-week (twice/week) treatments, the remaining tumors obtained from the second study were subjected to histology and IHC analyses. Our data revealed that treatment with either MM-121 or paclitaxel (7.5 mg/kg) had no significant effects on tumor cell morphology, tumor mass architecture, and the expression of erbB2/erbB3 receptors ( Figure 5B). In contrast, smaller tumor mass and bigger empty spaces among tumor cells were found with the combinatorial treatment. Nonetheless, the remaining tumor cells expressed similar levels of erbB2 and erbB3 ( Figure 5B).

Discussion
The erbB3 receptor has long been considered as an inactive pseudokinase [10,11], although a recent study suggests that erbB3 has weak kinase activity that can trans-autophosphorylate its intracellular region [12]. In order to fully transduce cell signaling, however, erbB3 has to be phosphorylated by its interactive partners; of these, erbB2 is the most important one [33]. It has been well-documented that activation of the erbB3 signaling plays a pivotal role in the development of erbB2overexpressing breast cancer [13,14]. Although a number of strategies targeting erbB2 are being used in the clinic [34], no erbB3-targeted therapy has been approved for cancer treatment. MM-121 is an erbB3-blocking Ab that is being actively investigated in clinical trials of cancer patients with solid tumors, such as advanced nonsmall cell lung cancer, colorectal cancer, squamous cell head and neck cancer, and platinum resistant/refractory ovarian cancer [35]. In breast cancer, the therapeutic potential of MM-121 is being tested in patients with estrogen receptor-and/or progesterone receptor-positive and erbB2-negative breast cancers in combination with the aromatase inhibitor exemestane, and in patients with triple-negative or erbB2-negative breast cancers in combination with paclitaxel. To date, no study has been initiated to test the activity of MM-121 in breast cancer patients with erbB2-overexpressing tumors, including those resistant to paclitaxel. Here we provide experimental evidence indicating that MM-121 inactivates erbB3 ( Figures 1B and 4A), significantly enhances paclitaxelinduced anti-proliferative/anti-survival effects ( Figures 1A,  4B, 5A, and 6), and apoptosis ( Figures 2B, 2C, 4C, 4D, and 6) in erbB2-overexpressing breast cancer cells with either medium (SKBR3, SKBR3.neo1, and BT474-HR20) or high erbB3 (SKBR3.B3.1 and SKBR3.B3.2) expression. Our data suggest that MM-121 is efficacious in all erbB2overexpressing breast cancer cell lines tested. We believe that MM-121 will exhibit therapeutic potential in all erbB2-positive breast cancer patients (not only those with strong erbB3-expressing tumors) as long as the tumors show active erbB3 signaling. The concentrations of paclitaxel used in the in vitro studies (0 to 16 nmol/L or 0 to 32 nmol/L) are much lower than the average steady-state plasma concentrations of paclitaxel (300 to 800 nmol/L) found in patients [36,37]. This difference may be explained by the two very distinct systems. In the 2-dimensional cell culture condition, paclitaxel should easily get into the tumor cells which grow as a monolayer. However, due to the complexity of the human body, some substances in the circulation may attenuate the efficacy of paclitaxel. The presence of fibroblast, microphage, immune cells and others in the tumor mass may block or decrease the drug's entry into the tumor cells. In addition, because of the heterogeneicity of tumors and their 3-dimensional architecture, drug uptake varies in tumor cells. Thus, the steady-state plasma concentration of paclitaxel may not reflect the drug concentration inside the tumor cells. The data obtained from our animal studies provide further support of this point. It has been reported that administration of 10 mg/kg paclitaxel to mice gives rise to a peak plasma concentration of approximately 3 μmol/L, which gradually and near-linearly declines to 0 at 16 h [38]. This peak concentration of paclitaxel is much higher than that we used in our cell culture studies. However, in the in vivo studies, although administration of 15 mg/kg paclitaxel to mice had significant inhibitory effects on tumor growth (Additional file 4), we found no such effect when paclitaxel was used at 7.5 mg/kg ( Figure 5A). Thus, both our in vitro and in vivo data seem to indicate that the ability of MM-121 to enhance the therapeutic efficacy of paclitaxel against erbB2overexpressing breast cancer may be restricted to the ineffective doses of paclitaxel. Although a recent report shows that as a single agent, neoadjuvant treatment with paclitaxel induces tumor response in 92% of patients with erbB2-overexpressing breast cancer [39], our findings suggest that the presence of MM-121 may convert the tumors from non-responsive to responsive to the lower doses of paclitaxel with less side effects, and therefore merit translational implications in the clinic.
We have reported that the underlying mechanism that elevated expression of erbB3 results in paclitaxel resistance in erbB2-overexpressing breast cancer cells is attributed to PI-3 K/Akt-dependent upregulation of Survivin [27]. Numerous studies indicate that Survivin functions as a critical inhibitor of apoptosis to promote cell survival and proliferation, and regulates mitosis during cell cycle progression [40]. Survivin is selectively expressed in a variety of human malignancies and its overexpression positively correlates with poor prognosis, tumor recurrence and therapeutic resistance [40]. A number of different strategies targeting Survivin, including antisense oligonucleotide and pharmacological inhibitors have been developed and are currently under clinical trials for cancer treatment [40][41][42]. Our data showed that inactivation of erbB3 signaling with MM-121 specifically downregulated Survivin in our in vitro models, and significantly enhanced paclitaxel-induced cytotoxicity and apoptosis in the otherwise resistant breast cancer cells (Figures 1 and 2). In our mouse model, although treatment with MM-121 alone had no significant effects on Survivin expression, MM-121 did dramatically downregulate Survivin when combined with paclitaxel ( Figure 6). It is possible that MM-121 at the dose we used only partially inhibits the PI-3 K/Akt signaling in vivo, and the inactivation of Akt to such an extent alone could not significantly alter the expression of Survivin. Alternatively, other signaling pathways may also be critical to control Survivin expression in the in vivo circumstance. A marked reduction of Survivin was only observed with the combinatorial treatment. In addition, we have found that hMP-RM-1, a humanized version of the anti-erbB3 Ab MP-RM-1 [43], exhibited similar in vitro activity to specifically downregulate Survivin in erbB2overexpressing breast cancer cells (data not shown). Thus, inhibition of erbB3 with blocking Ab may be a novel strategy to target Survivin for cancer treatment. As MP-RM-1 inhibits both ligand-dependent and -independent activation of erbB3 [43], we speculate that hMP-RM-1 might exert more potent antitumor activity than MM-121 against erbB2-overexpressing breast cancer. More detailed studies are needed to confirm our hypothesis. Elevated expression of Survivin was also observed in the trastuzumab-resistant subline BT474-HR20 and causally led the cells resistant to paclitaxel-induced apoptosis (Additional files 3 and 4). However, we did not find Survivin upregulation in another trastuzumab-resistant subline SKBR3-pool2. These data suggest that elevated expression of Survivin may cause cross-resistance to paclitaxel treatment in some trastuzumab-resistant breast cancers. We believe that profound activation of PI-3 K/Akt signaling, which we observed in BT474-HR20, but not in SKBR3-pool2 cells [28], is the major mechanism contributing to the upregulation of Survivin. At this moment, it remains unclear whether activation of the PI-3 K/Akt signaling by any means, such as PIK3CA mutation or phosphatase and tensin homolog (PTEN) deletion in addition to erbB2/ erbB3 receptors, would also enhance expression of Survivin in breast cancer cells. The interesting phenomenon of Survivin-mediated cross-resistance to paclitaxel and trastuzumab warrants further investigation.
Activation of the PI-3 K/Akt signaling has been identified as the major determinant of trastuzumab resistance [29]. In addition to inducing paclitaxel resistance, the erbB2/erbB3/PI-3 K/Akt signaling also results in resistance to hormonal therapy [44] and other chemotherapy [45] in breast cancer treatment. Because MM-121 mainly inhibits activation of erbB3 and Akt in erbB2overexpressing breast cancer cells, it is conceivable to hypothesize that MM-121 may abrogate erbB3 signalingmediated therapeutic resistance to tamoxifen, trastuzumab, and other chemotherapeutic agents, such as doxorubicin.
By taking advantage of the trastuzumab-resistant breast cancer model (BT474-HR20 and SKBR3-pool2), we have discovered that MM-121 is able to overcome trastuzumab resistance and significantly enhance trastuzumab-induced growth inhibition and/or apoptosis in vitro and in vivo (submitted separately).

Conclusions
We demonstrate that targeting of erbB3 with the blocking Ab MM-121 significantly enhances paclitaxel antitumor activity against erbB2-overexpressing breast cancer cells in our in vitro and in vivo models. In those models, MM-121 is active to overcome the resistance to paclitaxel, and such a capability of MM-121 may be restricted to the ineffective doses of paclitaxel. Mechanistically, MM-121 inhibits the PI-3 K/Akt signaling, downregulates Survivin, and subsequently enhances paclitaxel-mediated cytotoxicity and apoptosis in vitro. The combinations of MM-121 and paclitaxel significantly inhibit tumor cell proliferation, reduce expression of Survivin, and induce apoptosis in vivo. Our data support further studies to explore the therapeutic potential of MM-121 in combination with paclitaxel in breast cancer patients with erbB2-overexpressing tumors.

Additional files
Additional file 1: Figure S4. Evaluation of specificity of the antibodies used in the immunohistochemistry (IHC) studies. (A) The anti-Ki67 antibody was tested using the tumor xenografts established by the human breast cancer cell line MDA-MB-231 in a nude mouse. The picture was taken at ×100 magnification by an Olympus BX40 microscope. Both positive-staining MDA-MB-231 tumor cells and negative staining of the adjacent mouse lymphocytes are indicated as (+) and (-), respectively. (B) The human breast cancer cell line MCF-7, which has no erbB2 expression, and SKBR3, which is a well-known erbB2-overexpressing breast cancer cell line, were used to assess the anti-erbB2 antibody. Both cell lines under normal culture condition were collected, paraffin-embedded, and then followed by the standard procedure of IHC analysis. The pictures were taken at ×200 magnification. (C) Tumor sections obtained from the BT474-HR20 tumor xenografts described in the current studies (in the animals without treatment) were used to evaluate the antibodies against erbB3, Survivin, and cleaved caspase-3. All pictures were taken at ×200 magnification. Wheareas the BT474-HR20 tumor cells showed positive staining of erbB3 (+), Survivin (+), and rare cleaved caspase-3 (+), the fibroblast cells (likely from the host) stained negative for erbB3 (-) and the adjacent mouse lymphocytes stained negative for Survivin (-). The majority of the BT474-HR20 tumor cells were negative-stained for cleaved caspase-3 (-), because the animals received no treatment.