Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin

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Abstract

The coexpression of erbB3 and erbB2 is frequently observed in breast cancer; and erbB3 has a critical role in erbB2 promotion of breast cancer progression and anti-estrogen resistance. In this study, we determine the role of erbB3 in erbB2-mediated paclitaxel resistance in breast cancer cells. The overexpression of exogenous erbB3 via either stable or transient transfection in erbB2-overexpressing, but not epidermal growth factor receptor (EGFR)-expressing, breast cancer cells significantly decreases paclitaxel-induced growth inhibition and apoptosis. Consistently, knockdown of erbB3 expression with a specific short hairpin RNA (shRNA) in breast cancer cells with coexpression of both erbB2 and erbB3 enhances paclitaxel-induced apoptosis evidenced by increased DNA fragmentation, poly (ADP-ribose) polymerase (PARP) cleavage and activation of caspase-3 and -8. Furthermore, while forced overexpression of erbB3 increases, specific knockdown of erbB3 decreases the expression levels of Survivin only in the erbB2-overexpressing breast cancer cells. Targeting Survivin with specific shRNA overcomes paclitaxel resistance without effect on the expression levels of either erbB2 or erbB3. Mechanistic studies indicate that the specific phosphoinositide 3-kinase (PI-3K), Akt and mammalian target of rapamycin (mTOR) inhibitors, but not the mitogen-activated protein kinase kinase (MEK) inhibitor, not only abrogate erbB3-mediated upregulation of Survivin, but also reinforce the erbB2/erbB3-coexpressing breast cancer cells to paclitaxel-induced growth inhibition. These data demonstrate that heterodimerization of erbB2/erbB3 is a prerequisite for erbB2 tyrosine kinase activation; and elevated expression of erbB3 is required for erbB2-mediated paclitaxel resistance in breast cancer cells via PI-3K/Akt/mTOR signaling pathway-dependent upregulation of Survivin. Our studies suggest that new strategies targeting erbB3 or Survivin may enhance the efficacy of chemotherapeutic agents against erbB2-overexpressing breast cancer.

Introduction

The erbB receptor tyrosine kinase family is arguably the most important receptor family in the context of development and tumorigenesis. In addition to their normal physiological role in cell growth and differentiation, erbB receptors have a key role in transformation and tumor initiation and progression. One of the most well-characterized oncogenes involved in breast carcinogenesis is erbB2 (HER2 or neu). ErbB2 is a 185-kDa transmembrane glycoprotein that shares extensive sequence homology with family members, including epidermal growth factor receptor (EGFR), erbB3 and erbB4 (Bargmann et al., 1986; Yamamoto et al., 1986). The amplification and/or overexpression of ErbB2 are significantly and independently associated with a worse prognosis for breast cancer patients (Slamon et al., 1987; Thor et al., 1989). Studies on the underlying mechanisms suggest two major biological properties associated with erbB2-mediated poor outcome of breast cancers. First, erbB2 alterations enhance the ability of a cancer cell to invade and metastasize. Second, overexpression of erbB2 is associated with an increase in therapeutic resistance to tamoxifen and some chemotherapeutic agents, such as methotrexate and paclitaxel (Yu and Hung, 2000). The erbB2 receptor is therefore an ideal target for breast cancer therapy.

Unlike other erbB family members, erbB3 lacks intrinsic kinase activity, but has most tyrosine residues in its intracellular domain ready to be phosphorylated. It must interact with other receptor tyrosine kinases to exhibit its biological functions (Schulze et al., 2005). Among many interactive partners of erbB3, erbB2 is the most important one. In association with erbB2 receptors, erbB3 not only benefits from their strong kinase activity for phosphorylation on its intracellular tyrosine residues, it also serves like a sensitive ‘antenna’ through specific binding with its ligand, heregulin, to capture extracellular stimuli, and subsequently transduce the outside signals into downstream pathway through its phosphorylated tyrosine residues by erbB2, which has no known ligand to respond to the environmental stimulation. Together, the ‘dumb’ erbB3 (no kinase activity, but has ligand) and the ‘deaf’ erbB2 (no known ligand, but has kinase activity) make a perfect sense to form a potent erbB2/erbB3 heterodimer leading to the activation of the downstream signaling pathways, such as phosphoinositide 3-kinase (PI-3K)/Akt and mitogen-activated protein kinase kinase/ mitogen-activated protein kinase (MAPK), which promote cancer cell proliferation and survival (Citri et al., 2003). In fact, the erbB2/erbB3 heterodimer complex is now believed to be the most biologically active and pro-tumorigenic form of all erbB receptor complexes (Alimandi et al., 1995; Wallasch et al., 1995).

Increased resistance to therapeutic agents is one of the mechanisms by which erbB2 contributes to breast tumorigenesis, because of its pivotal role both within the erbB receptor family and in major downstream signaling pathways. Nonetheless, erbB2 receptor does not act in isolation. Although simultaneous suppression of EGFR and erbB2 results in a less malignant phenotype in ovarian cancer cells (Pack et al., 2004), only a minority of erbB2 altered breast cancers have co-existent overexpression of EGFR. Most metastatic breast cancers show expression for either EGFR or erbB2, and less often for both (Grupka et al., 2004). In contrast, coexpression of erbB3 and erbB2 is frequently observed in breast cancers (Bieche et al., 2003) and cell lines (deFazio et al., 2000). It has been reported that erbB2 requires erbB3 to promote breast cancer cell proliferation (Holbro et al., 2003); and erbB3 has a critical role in erbB2 altered breast cancers (Lee-Hoeflich et al., 2008). These data suggest that the erbB3 receptor, but not EGFR, probably has an important role in erbB2-mediated therapeutic resistance in breast cancer. Our recent studies strongly support this hypothesis showing that estrogen promotion of erbB2 tyrosine kinase activation in mammary tumor cells requires erbB3 signaling (Liu et al., 2009), and elevated expression of erbB3 contributes to erbB2-mediated tamoxifen resistance (Liu et al., 2007).

We have also reported physical and functional interactions between rat c-neu/erbB2 and endogenous mouse erbB3, which led to the activation of PI-3K/Akt signaling, in mammary tumor cells derived from MMTV-neu transgenic mice (Kim et al., 2005); and activation of PI-3K/Akt signaling is a key pathway that induces resistance to chemotherapeutic agents in the breast cancer cells (Knuefermann et al., 2003). Although the taxanes, paclitaxel and docetaxel, have emerged as critical drugs in the treatment of breast cancer patients, acquired and intrinsic resistance to taxanes currently represents a significant clinical problem (Saloustros et al., 2008; Morris and Fornier, 2009). Although several molecular mechanisms contributing to paclitaxel resistance have been proposed (Orr et al., 2003; Ferlini et al., 2007), a series of studies demonstrates that overexpression of erbB2 inhibits paclitaxel-induced apoptosis in breast cancer cells (Yu et al., 1996, 1998; Tan et al., 2002). However, the role of erbB3 in paclitaxel resistance remains unclear. We have focused our studies on erbB3 receptor, its interactions with erbB2 and subsequent activation of downstream signaling pathways. Our data indicate that expression of erbB3 is required for erbB2-mediated paclitaxel resistance via PI-3K/Akt-dependent upregulation of Survivin.

Results

Elevated expression of erbB3 inhibits paclitaxel-induced apoptosis in erbB2-overexpressing breast cancer cells

To investigate the potential role of erbB3 in resistance to chemotherapeutic agents, we wondered whether erbB3 receptor might cooperate with EGFR or erbB2 to induce paclitaxel resistance in breast cancer. First, we generated stable erbB3-transfectant clones from MDA-MB-231 cells, which have express EGFR but not erbB2, and SKBR3 cells, which overexpress erbB2 but not EGFR. The erbB3-transfectant single and pooled clones expressed much higher levels of erbB3 in both MDA-MB-231 and SKBR3 cells, as compared with their corresponding parental cell lines and the empty vector-transfected neo controls (Figure 1a). The overexpression of erbB3 did not alter the expression of either EGFR or erbB2 in both cell lines. Although the downstream signaling, such as P-Akt and P-MAPK pathways, remained unchanged in MDA-MB-231 cells, elevated expression of erbB3 significantly increased the levels of P-Akt, but not P-MAPK, in SKBR3 cells (Figure 1a). Cell proliferation assays showed that forced expression of erbB3 in MDA-MB-231 cells did not affect their response to paclitaxel-induced cytotoxicity (Figure 1b); however, it did significantly increase cell viability on paclitaxel treatment in SKBR3 cells (Figure 1c). These data suggest that expression of erbB3 may reduce paclitaxel-mediated cell killing only in erbB2-overexpressing, but not EGFR-expressing, breast cancer cells. To confirm these results, we next performed transient transfection to avoid the potential problem of long-term selection and clonal variation. MDA-MB-231 and SKBR3 cells were infected with a lentivirus containing either control vector or the same vector with human erbB3 complementary DNA, as described in the Materials and methods section. After 48-h infection, the expression levels of erbB3 were clearly enhanced in both cells (Figure 2a). This transient upregulation of erbB3 significantly increased P-Akt levels and attenuated paclitaxel-induced cytotoxicity only in SKBR3 cells (Figure 2b). These data were consistent with the observations of stable erbB3-transfectant clones (Figure 1). Furthermore, western blots on PARP and caspases and apoptotic enzyme-linked immunosorbent assay revealed that transient induction of erbB3 expression reduced paclitaxel-induced PARP cleavage, caspase-3 activation and apoptosis in SKBR3 cells, whereas MDA-MB-231 cells, regardless their erbB3 expression levels, retained same sensitivity to paclitaxel-induced apoptosis (Figures 2c and d). Collectively, these data indicate that elevated expression of erbB3 inhibits paclitaxel-induced apoptosis in erbB2-overexpressing, but not EGFR-expressing, breast cancer cells.

Figure 1
figure1

Elevated expression of erbB3 via stable transfection induces Akt activation and attenuates paclitaxel cyototoxicity in erbB2-overexpressing breast cancer cells. (a) MDA-MB-231 and SKBR3 cells were transfected with either pDsRed-Monimer-N1 or pDsRed-erbB3. After a 2-month selection with Geneticin, the stable clones of vector control (neo.1) or human erbB3 complimentary DNA (cDNA; B3.pool, B3.1, B3.2) were established, and the total cell lysates were subjected to western blot analyses with specific antibodies directed against erbB3, erbB2, epidermal growth factor receptor (EGFR), P-Akt, Akt, P- mitogen-activated protein kinase (P-MAPK), MAPK or β-actin. (b, c) The series of stable transfectants derived from MDA-MB-231 or SKBR3 cells were plated onto 96-well plates with complete culture medium (Dulbecco's modified Eagle medium (DMEM)/F12, 10% fetal bovine serum (FBS)). After 24 h, the medium was replaced with control medium (fresh DMEM/F12, 0.5% FBS) or same medium containing indicated concentrations of paclitaxel for another 72-h incubation. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt)). Data shows the representative of at least three independent experiments. Bars represent s.d. values.

Figure 2
figure2

Transient induction of erbB3 expression activates Akt and inhibits paclitaxel-induced apoptosis in erbB2-overexpressing breast cancer cells. MDA-MB-231 (MDA-231) and SKBR3 cells were infected with lentivirus containing either control vector or pLEX-erbB3 for 24 h. After selection with puromycin (1 μg/ml) for 48 h, the cells were subjected to the following experiments. (a) Western blot analyses of erbB3, P-erbB3, P-Akt, Akt, P-mitogen-activated protein kinase (P-MAPK), MAPK or β-actin. (b) MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt)) assays to determine the percentages of surviving cells upon paclitaxel treatment from each cell line relative to controls, defined as 100% survival. Data shows the representative of at least three independent experiments. Bars represent s.d. values. (c, d) The cells untreated or treated with paclitaxel (5 nM for SKBR3 cells, 20 nM for MDA-MB-231 cells), for additional 24 h, were then collected and subjected to western blot analyses of poly (ADP-ribose) polymerase (PARP; F-PARP, full length PARP; C-PARP, cleaved PARP), caspase-3 (F-Casp3, full-length caspase-3; C-Casp3, cleaved caspase-3) or β-actin (c) and apoptosis enzyme-linked immunosorbent assay (ELISA) (d).

Specific knockdown of erbB3 expression enhances paclitaxel-induced apoptosis and abrogates erbB2-mediated paclitaxel resistance in breast cancer cells

To explore whether erbB3 expression is required for erbB2-mediated paclitaxel resistance in breast cancer cells, we studied whether specific knockdown of erbB3 expression might potentiate paclitaxel-induced apoptosis in breast cancer cells with the coexpression of both erbB2 and erbB3. The expression levels of erbB3 in both SKBR3 and MDA-MB-453 cells were downregulated with a lentiviral system containing human erbB3 shRNA, as we proved this erbB3 shRNA was specific and efficient (Huang et al., 2009). Specific knockdown of erbB3 not only dramatically decreased the P-Akt levels (Figure 3a), but also significantly enhanced paclitaxel-mediated cell killing (Figure 3b). The assessment of apoptotic cells confirmed that erbB3 knockdown using specific shRNA in both SKBR3 and MDA-MB-453 cells potentiated paclitaxel-induced apoptosis evidenced by increased PARP cleavage, activation of caspase-8 and -3 (Figure 3c) and DNA fragmentation measured by enzyme-linked immunosorbent assay (Figure 3d).

Figure 3
figure3

Specific knock down of erbB3 expression reduces P-Akt levels and significantly enhances paclitaxel-induced apoptosis. SKBR3 and MDA-MB-453 (MDA-453) cells were infected with lentivirus containing either ConshRNA or erbB3shRNA for 24 h. After selection with puromycin (1 μg/ml) for 48 h, the cells were subjected to the following experiments. (a) Western blot analyses of erbB3, P-erbB3, erbB2, P-erbB2, P-Akt, Akt, P-mitogen-activated protein kinase (P-MAPK), MAPK or β-actin. (b) MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt)) assays to determine the percentages of surviving cells upon paclitaxel treatment from each cell line relative to controls, defined as 100% survival. Data shows the representative of at least three independent experiments. Bars represent s.d. values. (c, d) The cells untreated or treated with paclitaxel (5 nM) for additional 24 h were collected and subjected to western blot analyses of poly (ADP-ribose) polymerase (PARP), caspase-8 (F-Casp8, full-length caspase-8; C-Casp8, cleaved caspase-8), caspase-3 or β-actin (c) and apoptosis enzyme-linked immunosorbent assay (ELISA) (d).

As we have reported that the 435.eB1 cells (erbB2 transfectant of MDA-MB-435 cell line) are more resistant to paclitaxel-induced apoptosis (Yu et al., 1996, 1998); and the MDA-MB-435 cells express high levels of erbB3 (Knuefermann et al., 2003), we then performed additional experiments to further address the hypothesis that overexpression of erbB2 in 435.eB1 cells may require the existence of erbB3 to induce paclitaxel resistance. Specific human erbB3 shRNA, not the control shRNA, reduced the levels of erbB3, P-erbB3, P-erbB2, P-Akt and P-MAPK in 435.eB1 cells (Supplementary Figure S1A), suggesting that the expression of erbB3 was essential for erbB2 tyrosine kinase activation and the downstream signaling. More importantly, specific knock down of erbB3 expression resensitized 435.eB1 cells to paclitaxel-induced PARP cleavage, caspase-8 and -3 activation and apoptosis (Supplementary Figures S1B and C). Collectively, our studies strongly support an important role for the erbB3 receptor in erbB2-mediated paclitaxel resistance.

The levels of erbB3 modulate Survivin expression in erbB2-overexpressing breast cancer cells via PI-3K/Akt/mTOR signaling-dependent mechanism

To determine the molecular mechanism how erbB3 expression contributes to erbB2-mediated paclitaxel resistance, we considered the downstream signaling pathways, class III β-tubulin (TUBB3) and several regulators of apoptosis. Although several studies indicate that elevated expression of TUBB3 may result in therapeutic resistance to microtubule-targeting agents in many cancers (Ferlini et al., 2007; Seve and Dumontet, 2008), we saw no change in TUBB3 levels under our experimental conditions (data not shown). In addition, overexpression of erbB3 with either stable clones or transient transfection did not alter the expression levels of C-X-C chemokine receptor type 4, Mcl-1 and Bcl-xL. However, a 1.5–2.0-fold induction of Survivin was observed only in erbB3-transfected SKBR3, but not MDA-MB-231 cells (Figure 4a). Interestingly, the upregulation of Survivin was also observed in 435.eB1 cells as compared with the parental cells (Lu et al., 2009). In contrast, specific knock down of erbB3 expression in the erbB2/erbB3-coexpressing breast cancer cells significantly decreased Survivin, but had no effect on C-X-C chemokine receptor type 4, Mcl-1 and Bcl-xL (Figure 4b). These data suggest that the erbB3 levels specifically modulate Survivin expression in erbB2-overexpressing breast cancer cells. As elevated expression of erbB3 mainly activated Akt in SKBR3 cells (Figures 1a and 2a), we studied whether there was a causal relationship between the activation of PI-3K/Akt signaling and erbB3-mediated upregulation of Survivin. Although mitogen-activated protein kinase kinase inhibitor, PD98059, efficiently reduced P-MAPK levels in erbB3-tarnsfectant clone 1 and 2 (SKBR3.B3.1 and SKBR3.B3.2), it had no significant effect on Survivin. However, the PI-3K inhibitor, LY294002, simultaneously reduced the levels of both P-Akt and Survivin (Figure 4c). Moreover, the PI-3K inhibitor, LY294002, but not the mitogen-activated protein kinase kinase inhibitor, PD98059, was able to significantly sensitize the SKBR3.B3.1 and SKBR3.B3.2 cells to paclitaxel-mediated cytotoxicity (Figure 4d). These results were also confirmed with the same assays performed on the pooled erbB3-tarnsfectant clones of SKBR3 cells, SKBR3.B3.pool (Supplementary Figures S2A and B). Furthermore, additional experiments using specific inhibitor of Akt or mTOR revealed that inhibition of Akt or mTOR not only decreased the expression levels of Survivin in SKBR3.B3.pool, SKBR3.B3.1 and SKBR3.B3.2 cells (Supplementary Figures S2C and D), but also resensitized these cells to paclitaxel-mediated cytotoxicity (Supplementary Figure S2E). Taken together, our data demonstrate that the overexpression of erbB3 upregulates Survivin through a PI-3K/Akt/mTOR signaling-dependent mechanism, and activation of PI-3K/Akt signaling is critical for erbB3 promotion of erbB2-induced paclitaxel resistance in breast cancer cells.

Figure 4
figure4

The expression levels of erbB3 modulate Survivin expression in erbB2-overexpressing breast cancer cells through a PI-3K/Akt-dependent mechanism. (a) MDA-MB-231, SKBR3 cells, their stable transfectants, and the MDA-MB-231 and SKBR3 cells infected with lentivirus containing either control vector or pLEX-erbB3, as performed in Figure 2, were collected and subjected to western blot analyses of Survivin, C-X-C chemokine receptor type 4 (CXCR4), Mcl-1, Bcl-xL or β-actin. (b) SKBR3, MDA-MB-453 and 435.eB1 cells infected with lentivirus containing either ConshRNA or erbB3shRNA, as performed in Figure 3, were collected and subjected to western blot analyses of Survivin, CXCR4, Mcl-1, Bcl-xL or β-actin. (c and d) SKBR3.B3.1 and SKBR3.B3.2 cells, untreated or treated with PD98059 (PD, 20 μM) or LY294002 (LY, 10 μM) for 6 h, were then collected and subjected to (c) western blot analyses of Survivin, P-Akt, Akt, P-mitogen-activated protein kinase (P-MAPK), MAPK or β-actin and (d) MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt)) assays to determine the percentages of surviving cells on paclitaxel treatment from each cell line relative to controls, defined as 100% survival. Data shows the representative of at least three independent experiments. Bars represent s.d. values. The densitometry analyses of Survivin signals were shown underneath, and the arbitrary numbers indicate the intensities of each cell line relative to controls, defined as 1.0.

Upregulation of Survivin is required for erbB3-induced paclitaxel resistance in erbB2-overexpressing breast cancer cells

We next evaluated whether the upregulation of Survivin is required for erbB3-mediated antiapoptotic cell death-induced by paclitaxel. Similar strategy was used to specifically knock down Survivin expression with a lentiviral system containing Survivin shRNA. Three specific shRNA sequences as compared with control shRNA all efficiently downregulated Survivin expression in both SKBR3.B3.1 and SKBR3.B3.2 cells, but did not alter the levels of erbB2, erbB3, P-Akt and P-MAPK (Figure 5a), suggesting Survivin acted downstream of erbB3/PI-3K/Akt signaling pathway. Importantly, all three Survivin shRNA: S3, S4 and S5 were able to significantly enhance paclitaxel-induced cytotoxicity (Figure 5b), PARP cleavage and activation of caspase-8 and -3 (Figure 5c) and apoptosis (Figure 5d). Similar results were obtained with SKBR3.B3.pool cells (Supplementary Figure S3).

Figure 5
figure5

Specific knockdown of Survivin expression abrogates erbB3-mediated paclitaxel resistance in erbB2-overexpressing breast cancer cells. (a) SKBR3.B3.1 and SKBR3.B3.2 cells infected with lentivirus containing either ConshRNA or SurshRNA (S3, S4 and S5) were subjected to the following experiments. (a) Western blot analyses of Survivin, erbB2, erbB3, P-Akt, Akt, P-mitogen-activated protein kinase (P-MAPK), MAPK or β-actin. (b) MTS ((3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt)) assays to determine the percentages of surviving cells on paclitaxel treatment from each cell line relative to controls, defined as 100% survival. Data shows the representative of at least three independent experiments. Bars represent s.d. values. (c, d) The cells untreated or treated with paclitaxel (5 nM) for additional 24 h were collected and subjected to (c) western blot analyses of poly (ADP-ribose) polymerase (PARP), caspase-8, caspase-3 or β-actin and (d) apoptosis enzyme-linked immunosorbent assay (ELISA). *P<0.005, **P=0.0133, ***P=0.0064 vs ConshRNA+paclitaxel.

As dimerization of erbB2/erbB3 is essential for erbB2 tyrosine kinase activation (Liu et al., 2007), and erbB3 expression closely correlated to the P-Akt levels only in erbB2-overexpressing, but not EGFR-expressing, breast cancer cells (Figures 1a, 2a and 3a), we believed that coexpression of erbB3 may preferably form a potent heterodimer complex with erbB2, but not EGFR, to activate the erbB receptors. To test this hypothesis, co-immunoprecipitation assays with an anti-erbB3 antibody revealed that erbB3 indeed interacted with erbB2 in those breast cancer cells with coexpression of both erbB2 and erbB3, and elevated expression of erbB3 further increased their associations, however, the similarly elevated expression of erbB3 never formed dimerization with EGFR in MDA-MB-231 cells (Figure 6a). Consistent with these data, increased associations of erbB2/erbB3 resulted in higher levels of both P-erbB2 and P-erbB3 in SKBR3 cells, in contrast, P-erbB3 was undetectable and P-EGFR levels remained unchanged in MDA-MB-231 cells, although their protein levels seemed to be as high as erbB3 and erbB2 levels in SKBR3 cells (Figure 6b). Thus, our studies confirm that heterodimerization of erbB2/erbB3 is a prerequisite for erbB2 tyrosine kinase activation; and PI-3K/Akt signaling-dependent upregulation of Survivin is required for erbB3 potentiating erbB2-mediated paclitaxel resistance in breast cancer cells (Figure 6c).

Figure 6
figure6

Expression of erbB3 forms dimerization with erbB2, but not epidermal growth factor receptor (EGFR), and enhances erbB2 tyrosine kinase activation in breast cancer cells. (a) SKBR3 and MDA-231 cells infected with lentivirus containing either control vector or pLEX-erbB3 along with 435.eB1 and MDA-453 cells were collected and subjected to immunoprecipitation with anti-erbB3 mAb (mouse IgG was used as negative control with the same amount of total lysates of 435.eB1 and MDA-453 cells), and then followed by western blot analyses of erbB2, erbB3 or EGFR. (b) SKBR3 and MDA-231 cells infected with lentivirus containing either control vector or pLEX-erbB3 along with 435.eB1 and MDA-453 cells were collected and subjected to western blot analyses of erbB2, P-erbB2, erbB3, P-erbB3, EGFR, P-EGFR or β-actin. (c) Diagram of proposed model underlying the mechanism of erbB3-mediated paclitaxel resistance in erbB2-overexpressing breast cancer cells.

Discussion

Recent advances in our understanding of breast cancer biology have led to improved patient survival with target therapies, for example, anti-estrogen agents (tamoxifen) in the treatment of estrogen receptor-positive breast cancers (Jensen and Jordan, 2003); anti-erbB2 antibody (trastuzumab, also known as Herceptin, Genentech, South San Francisco, CA, USA) for patients with erbB2 amplification and/or overexpression (Slamon et al., 2001). Although Herceptin has been successfully used in early-stage and metastatic breast cancer patients with erbB2-overexpressing tumors as monotherapy (Cobleigh et al., 1999; Vogel et al., 2002) and in combination with other agents (Slamon et al., 2001; Hudis, 2007), both primary (de novo) and acquired resistances to Herceptin frequently occur (Nahta et al., 2006; Nahta and Esteva, 2007); and it is in urgent need to explore additional molecular markers accurately predicting erbB2-targeted therapies, and to identify new strategies enhancing the efficacy of therapeutic agents against erbB2-overexpressing breast cancers. With a focus on studying the unique biological features of erbB3 receptor in breast cancer, we have recently reported that erbB3 not only contributes to erbB2-mediated tamoxifen resistance (Liu et al., 2007), but also has a vital role in Herceptin resistance through a heterotrimeric complex consisting of erbB3/erbB2/IGF-1R (Huang et al., 2010). The data presented in this study strongly support that elevated expression of erbB3 is required for erbB2-mediated paclitaxel resistance. Thus, a series of articles published by our laboratory have provided compelling evidence, suggesting that erbB3 should be developed as another important therapeutic target and the new therapeutics targeting erbB3 may significantly enhance the therapeutic efficacy of tamoxifen, paclitaxel and erbB2-targeted therapies, such as Herceptin and lapatinib, for erbB2-overexpressing breast cancer.

Survivin, the smallest member of IAP (inhibitor of apoptosis) family, is a dual functional protein acting as an important apoptosis inhibitor and key cell cycle regulator (Liu et al., 2008; Mita et al., 2008). Survivin is usually expressed in embryonic tissues during development, but undetectable in most normal adult tissues. Numerous studies show that Survivin is selectively expressed in almost all types of human malignancies and its overexpression positively correlates with poor prognosis, tumor recurrence and therapeutic resistance (Liu et al., 2008; Mita et al., 2008). This differential expression of Survivin in tumors and normal tissues draws a great interest to identify strategies targeting Survivin for cancer treatment (Altieri, 2008; Mita et al., 2008). However, the molecular mechanisms controlling Survivin expression in human tumors have not been fully elucidated. Although expression of both EGFR and erbB2 have been shown to upregulate Survivin (Asanuma et al., 2005; Xia et al., 2006), to the best of our knowledge, this is the first report indicating that elevated expression of erbB3 is also capable of inducing Survivin expression through a PI-3K/Akt-dependent mechanism in erbB2-overexpressing, but not EGFR-expressing, breast cancer cells. Our data is consistent with previous studies supporting a critical role of PI-3K/Akt signaling pathway in the induction of Survivin expression; however, the detailed mechanism of how PI-3K/Akt activation results in the upregulation of Survivin remains less understood. Recent studies suggest that activation of insulin-like growth factor 1/ insulin-like growth factor 1 receptor signaling increases Survivin levels independent of gene transcription, rather through an enhanced protein translation-induced by Akt/mTOR/p70S6K1 signaling pathway (Vaira et al., 2007; Oh et al., 2008). Although regulating protein translation of target gene is one of the major mechanisms of miRNA to exert its biological functions, it has been reported that different isoforms of Akt regulate epithelial–mesenchymal transition by controlling expression of miR-200 family in cancer cells (Iliopoulos et al., 2009). Noticeably, miR-200c is recently found to modulate paclitaxel sensitivity by targeting TUBB3 (Cochrane et al., 2009). Thus, it will be very interesting to explore whether elevated expression of erbB3 may modulate certain miRNAs that specifically target Survivin mRNA in erbB2-overexpressing breast cancer cells. As the interactions of EGFR and IGF-1R induce Survivin expression and counteract erlotinib's antitumor activity in non-small cell lung carcinoma (Morgillo et al., 2006), we are currently testing whether the heterotrimerization of erbB3/erbB2/IGF-1R, which we have observed in Herceptin-resistant breast cancer cells (Huang et al., 2010), may also regulate Survivin expression. Such studies may shed new lights on identifying Survivin as a molecular linker of cross-resistance to paclitaxel and Herceptin treatment of breast cancer patients whose tumors overexpress erbB2.

In addition to the upregulation of Survivin, several other factors contributing to paclitaxel resistance in breast cancer treatment have been described. Paclitaxel's therapeutic efficacy may be compromised by p170mdr−1 and multi-drug resistance protein-mediated drug efflux (Saloustros et al., 2008; Morris and Fornier, 2009). Changes in β-tubulin isotype expression with a switch from class I to III and overexpression of TXR1 (taxol-resistance gene 1) lead to paclitaxel resistance (van Amerongen and Berns, 2006; Ferlini et al., 2007). Although we did not observe any changes in TUBB3 levels upon manipulation of erbB3 expression in our experimental conditions, there is no report showing whether erbB3 expression may regulate tyrosinase-related protein 1 expression. As overexpression of erbB2 confers paclitaxel resistance via mdr-1-independent mechanism (Yu et al., 1996), and our current data show that erbB3 expression induces paclitaxel resistance only in erbB2-overexpressing breast cancer cells, suggesting that erbB3-mediated paclitaxel resistance might also be independent of mdr-1. Nonetheless, there is clear difference between erbB2- and erbB3-associated downstream effectors, resulting in paclitaxel resistance of breast cancer cells. The overexpression of erbB2 not only increases Survivin levels via enhanced protein translation (Lu et al., 2009), it also induces Src/signal transducer and activator of transcription 3 activation-mediated p21waf1 transcriptional upregulation (Hawthorne et al., 2009), and both Survivin and p21waf1 contribute to erbB2-mediated paclitaxel resistance. Our studies found no change in p21waf1 expression followed by either transfection or knock down of erbB3 (data not shown). It seems that elevated expression of erbB3 specifically upregulates Survivin in erbB2-overexpressing breast cancer cells. One potential explanation might be that overexpression of erbB3 can only form heterodimerization with endogenous erbB2, but not EGFR (Figure 6a). In contrast, the overexpression of erbB2 not only interacts with endogenous erbB3, it may also dimerize with other receptor tyrosine kinases, such as EGFR, and subsequently activates Src/ signal transducer and activator of transcription 3 signaling to induce p21waf1 transcriptional upregulation. Further studies using animal model and human breast cancer tissues are warranted to elucidate this hypothesis.

In summary, we have demonstrated that erbB3 expression in erbB2-overexpressing breast cancer cells confers paclitaxel resistance via PI-3K/Akt-dependent upregulation of Survivin. These findings, together with our previous reports on the role of erbB3 receptor in tamoxifen and Herceptin resistance, provide a strong rationale to develop erbB3-targeted therapies. Such strategies should significantly enhance the efficacy of tamoxifen-, paclitaxel- and erbB2-targeted therapies, such as Herceptin and lapatinib, for the treatment of erbB2-overexpressing breast cancer.

Materials and methods

Reagents and antibodies

Paclitaxel (Ben Venue Labs, Bedford, OH, USA) was obtained from University of Colorado Hospital Pharmacy. MISSION Non-target shRNA, which does not target human and mouse genes, control vector (pLKO.1-ConshRNA), pLKO.1 containing human Survivin shRNA (pLKO.1-SurshRNA) were purchased from Sigma (St Louis, MO, USA). The packaging plasmids psPAX2 and pMD2.G for lentiviral expression vector were from Addgene (Cambridge, MA, USA).

Antibodies were obtained as follows: erbB2 (Ab3) (from EMD Chemicals, Gibbstown, NJ, USA); erbB3 (Ab7) and P-erbB2 (PN2A) (from LabVision, Fremont, CA, USA); P-erbB3 (21D3), caspase-8 (1C12) and caspase-3 (8G10), P-MAPK (E10), MAPK, P-Akt (Ser473), Akt, Survivin (6E4), Bcl-xL (from Cell Signaling Technology, Beverly, MA, USA); C-X-C chemokine receptor type 4 (from IMGENEX, San Diego, CA, USA), Mcl-1 (clone 22) (from BD Biosciences, San Jose, CA, USA); PARP mAb (C-2-10) (from BIOMOL Research Laboratories, Plymouth Meeting, PA, USA); EGFR (F4), P-EGFR (Tyr1068) and β-actin (AC-75) (from Sigma). All other reagents were purchased from Sigma unless otherwise specified.

Cells and cell culture

Human breast cancer cell lines SKBR3, MDA-MB-231, MDA-MB-453 and MDA-MB-435 were obtained from American Type Culture Collection (Manassas, VA, USA). The erbB2-transfectant 435.eB1 cell line (Yu et al., 1996) was kindly provided by Dr Dihua Yu (MD Anderson Cancer Center). The HEK293T human embryonic kidney cells were kindly provided by Dr Haihua Gu (University of Colorado Denver). All cells were maintained in Dulbecco's modified Eagle's medium: Nutrient Mix F-12 (D-MEM/F-12 1:1) supplemented with 10% fetal bovine serum. All cell lines were cultured in a 37 °C humidified atmosphere containing 95% air and 5% CO2 and were split twice a week.

Establishment of stable erbB3-tranfectant clones of breast cancer cells

The MDA-MB-231 or SKBR3 cells were transfected with either control vector pDsRed-Monimer-N1 (Clontech Laboratories, Palo Alto, CA, USA) or human erbB3-expressing vector pDsRed-erbB3 (Huang et al., 2009) using FuGENE-6 transfection kit (Roche Diagnostics, Indianapolis, IN, USA). The cells were selected with Geneticin (Invitrogen, Carlsbad, CA, USA) for 2 months. The resulting single clone was picked, amplified and grown into stable cell line. The remaining clones were cultured together and grown into pooled stable transfectants.

Construction of lentiviral expression vector pLEX-erbB3

The entire coding sequence of human erbB3 was amplified from pDsRed-erbB3 by PCR using Phusion High-Fidelity DNA Polymerase (New England BioLabs, Ipswich, MA, USA) with the primer set: forward primer 5′-IndexTermATAGCGGCCGCATGAGGGCGAACGACGCTC-3′; reverse primer 5′-IndexTermGCGACGCGTTTACGTTCTCTGGGCATTAGC-3′. The amplified fragments were purified and digested with NotI and MluI (New England BioLabs), and inserted into the lentiviral expression vector LentiORF pLEX-MCS (Open Biosystem, Huntsville, AL, USA) using Quick Ligation Kit (New England BioLabs). After verification by DNA sequencing, the recombinant with accurate sequence of human erbB3 complementary DNA was nominated as pLEX-erbB3.

Production of lentivirus

Lentiviral production was carried out as described previously (Huang et al., 2009, 2010). In brief, the lentiviral expression vector pLKO.1-ConshRNA or pLKO.1-SurshRNA and lentivirus packaging plasmids pCMV-VSVG and pCMV-ΔA.9 were co-transfected into virus packaging cell line 293T using FuGENE-6 following the standard procedure. Similar transfections were also performed with the control vector pLEX-MCS or pLEX-erbB3 and the packaging plasmids psPAX2 and pMD2.G. After 24 h, the culture media were changed with fresh DMEM/F12 medium (10% fetal bovine serum). The virus in conditioned medium were collected in the following 3 consecutive days, and then filtered with low protein-binding filters (Millex-hV 0.45-mm polyvinylidene fluoride, Millipore, Burlington, MA, USA) before they were aliquoted and stored in −80 °C freezer.

ErbB3 overexpression or specific knockdown of erbB3 expression with a lentiviral system

Before infection, the lentivirus-containing media (5 ml in each tube) were thawed completely at room temperature. Another 5 ml fresh medium and polybrene (8 μg/ml) were added into the virus-containing media. Then the culture media of the candidate breast cancer cells were 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 collected and subjected to required experiments.

Cell proliferation assay

The CellTiter96 AQ non-radioactive cell proliferation kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine cell viability (Huang et al., 2009, 2010). In brief, cells were plated onto 96-well plates with complete medium for 24 h. Cells were then grown in either control medium or the same medium containing a series of doses of paclitaxel for another 72 h. After reading all wells at 490 nm with a micro-plate reader, the percentages of surviving cells from each group relative to controls, defined as 100%, were determined by reduction of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt).

Quantification of apoptosis

An apoptosis enzyme-linked immunosorbent assay kit (Roche) was used to quantitatively measure cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes) as previously reported (Liu et al., 2007; Huang et al., 2009). This enzyme immunoassay was performed according to the manufacturer's instructions.

Immunoprecipitation and western blot analysis

Immunoprecipitation and western blot assays were performed as described (Huang et al., 2010). Briefly, equal amounts of cell lysates were incubated with specific antibody for 2 h at 4 °C, followed by incubation with protein A–agarose or protein G–agarose (Roche) at 4 °C overnight. The immunoprecipitates or equal amounts of cell lysates were boiled in sodium dodecyl sulfate sample buffer, resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotted with specific antibodies, as described in the figure legends.

Statistical analysis

Statistical analyses of the experimental data were performed using a two sided Student's t-test. Significance was set at P<0.05.

Abbreviations

ER:

estrogen receptor

EGFR:

epidermal growth factor receptor

MAPK:

mitogen-activated protein kinase

MEK:

MAPK kinase

PI-3K:

phosphoinositide 3-kinase

mAb:

monoclonal antibody

mTOR:

mammalian target of rapamycin

PARP:

poly(ADP-ribose) polymerase

TUBB3:

class III β-tubulin

CXCR4:

C-X-C chemokine receptor type 4

MRP1:

multi-drug resistance protein 1

TXR1:

taxol-resistance gene 1

NSCLC:

non-small cell lung cancer

shRNA:

short-hairpin RNA

ELISA:

enzyme-linked immunosorbent assay

PAGE:

polyacrylamide gel electrophoresis

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Acknowledgements

We thank Dr Haihua Gu for providing the pLKO.1-ErbB3shRNA expression vector and lentivirus packaging plasmids pCMV-VSVG and pCMV-ΔA.9. We also thank Ms Lisa Litzenberger for her excellent art preparation. This work was supported in part by a research Grant from Susan G Komen for the Cure (to BL).

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Correspondence to B Liu.

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Supplementary Information accompanies the paper on the Oncogene website

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Wang, S., Huang, X., Lee, C. et al. Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin. Oncogene 29, 4225–4236 (2010) doi:10.1038/onc.2010.180

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Keywords

  • erbB3
  • erbB2
  • Survivin
  • paclitaxel resistance
  • breast cancer

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