Oncogene (2010) 29, 4225–4236 & 2010 Macmillan Publishers Limited All rights reserved 0950-9232/10 www.nature.com/onc ORIGINAL ARTICLE Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cells via upregulation of Survivin

S Wang1, X Huang1, C-K Lee2 and B Liu1

1Department of Pathology, University of Colorado Denver School of Medicine, Aurora, CO, USA and 2The Myeloma and Amyloidosis Program, Department of Medicine, University of Colorado Denver School of Medicine, Aurora, CO, USA

The coexpression of erbB3 and erbB2 is frequently observed Introduction in ; and erbB3 has a critical role in erbB2 promotion of breast cancer progression and anti-estrogen The erbB family is arguably resistance. In this study, we determine the role of erbB3 in the most important receptor family in the context of erbB2-mediated paclitaxel resistance in breast cancer cells. development and tumorigenesis. In addition to their The overexpression of exogenous erbB3 via either stable normal physiological role in and differentia- or transient transfection in erbB2-overexpressing, but not tion, erbB receptors have a key role in transformation epidermal receptor (EGFR)-expressing, and tumor initiation and progression. One of the most breast cancer cells significantly decreases paclitaxel-induced well-characterized oncogenes involved in breast carci- growth inhibition and . Consistently, knockdown nogenesis is erbB2 (HER2 or neu). ErbB2 is a 185-kDa of erbB3 expression with a specific short hairpin RNA transmembrane glycoprotein that shares extensive (shRNA) in breast cancer cells with coexpression of sequence homology with family members, including both erbB2 and erbB3 enhances paclitaxel-induced apop- epidermal growth factor receptor (EGFR), erbB3 and tosis evidenced by increased DNA fragmentation, poly erbB4 (Bargmann et al., 1986; Yamamoto et al., 1986). (ADP-ribose) polymerase (PARP) cleavage and activation The amplification and/or overexpression of ErbB2 are of caspase-3 and -8. Furthermore, while forced over- significantly and independently associated with a worse expression of erbB3 increases, specific knockdown of erbB3 prognosis for breast cancer patients (Slamon et al., 1987; decreases the expression levels of Survivin only in the Thor et al., 1989). Studies on the underlying mechan- erbB2-overexpressing breast cancer cells. Targeting Survi- isms suggest two major biological properties associated vin with specific shRNA overcomes paclitaxel resistance with erbB2-mediated poor outcome of breast . without effect on the expression levels of either erbB2 First, erbB2 alterations enhance the ability of a cancer or erbB3. Mechanistic studies indicate that the specific cell to invade and metastasize. Second, overexpression phosphoinositide 3-kinase (PI-3K), Akt and mammalian of erbB2 is associated with an increase in therapeutic target of rapamycin (mTOR) inhibitors, but not the resistance to tamoxifen and some chemotherapeutic -activated kinase kinase (MEK) inhibitor, agents, such as methotrexate and paclitaxel (Yu and not only abrogate erbB3-mediated upregulation of Survivin, Hung, 2000). The erbB2 receptor is therefore an ideal but also reinforce the erbB2/erbB3-coexpressing breast target for breast cancer therapy. cancer cells to paclitaxel-induced growth inhibition. These Unlike other erbB family members, erbB3 lacks data demonstrate that heterodimerization of erbB2/erbB3 intrinsic kinase activity, but has most tyrosine residues is a prerequisite for erbB2 tyrosine kinase activation; and in its intracellular domain ready to be phosphorylated. elevated expression of erbB3 is required for erbB2-mediated It must interact with other receptor tyrosine kinases paclitaxel resistance in breast cancer cells via PI-3K/Akt/ to exhibit its biological functions (Schulze et al., 2005). mTOR signaling pathway-dependent upregulation of Survi- Among many interactive partners of erbB3, erbB2 vin. Our studies suggest that new strategies targeting erbB3 is the most important one. In association with erbB2 or Survivin may enhance the efficacy of chemotherapeutic receptors, erbB3 not only benefits from their strong agents against erbB2-overexpressing breast cancer. kinase activity for phosphorylation on its intra- Oncogene (2010) 29, 4225–4236; doi:10.1038/onc.2010.180; cellular tyrosine residues, it also serves like a sensitive published online 24 May 2010 ‘antenna’ through specific binding with its , heregulin, to capture extracellular stimuli, and subse- Keywords: erbB3; erbB2; Survivin; paclitaxel resistance; quently transduce the outside signals into down- breast cancer stream pathway through its phosphorylated tyrosine residues by erbB2, which has no known ligand to respond to the environmental stimulation. Together, the Correspondence: Dr B Liu, Department of Pathology, University of ‘dumb’ erbB3 (no kinase activity, but has ligand) and Colorado Denver School of Medicine, MS-8104, PO Box 6511, 12801 the ‘deaf’ erbB2 (no known ligand, but has kinase E. 17th Ave., Aurora, CO 80045, USA. E-mail: [email protected] activity) make a perfect sense to form a potent erbB2/ Received 19 January 2010; revised 10 April 2010; accepted 17 April 2010; erbB3 heterodimer leading to the activation of the published online 24 May 2010 downstream signaling pathways, such as phosphoinositide ErbB3 induces Survivin expression S Wang et al 4226 3-kinase (PI-3K)/Akt and mitogen-activated protein Results kinase kinase/ mitogen-activated protein kinase (MAPK), which promote proliferation and Elevated expression of erbB3 inhibits paclitaxel-induced survival (Citri et al., 2003). In fact, the erbB2/erbB3 apoptosis in erbB2-overexpressing breast cancer cells heterodimer complex is now believed to be the most To investigate the potential role of erbB3 in resistance to biologically active and pro-tumorigenic form of all erbB chemotherapeutic agents, we wondered whether erbB3 receptor complexes (Alimandi et al., 1995; Wallasch receptor might cooperate with EGFR or erbB2 to et al., 1995). induce paclitaxel resistance in breast cancer. First, we Increased resistance to therapeutic agents is one generated stable erbB3-transfectant clones from MDA- of the mechanisms by which erbB2 contributes to breast MB-231 cells, which have express EGFR but not erbB2, tumorigenesis, because of its pivotal role both within and SKBR3 cells, which overexpress erbB2 but not the erbB receptor family and in major downstream EGFR. The erbB3-transfectant single and pooled clones signaling pathways. Nonetheless, erbB2 receptor does expressed much higher levels of erbB3 in both MDA- not act in isolation. Although simultaneous suppres- MB-231 and SKBR3 cells, as compared with their sion of EGFR and erbB2 results in a less malignant corresponding parental cell lines and the empty vector- phenotype in ovarian cancer cells (Pack et al., 2004), transfected neo controls (Figure 1a). The overexpression only a minority of erbB2 altered breast cancers have of erbB3 did not alter the expression of either EGFR co-existent overexpression of EGFR. Most metastatic or erbB2 in both cell lines. Although the down- breast cancers show expression for either EGFR or stream signaling, such as P-Akt and P-MAPK path- erbB2, and less often for both (Grupka et al., 2004). ways, remained unchanged in MDA-MB-231 cells, In contrast, coexpression of erbB3 and erbB2 is elevated expression of erbB3 significantly increased the frequently observed in breast cancers (Bieche et al., levels of P-Akt, but not P-MAPK, in SKBR3 cells 2003) and cell lines (deFazio et al., 2000). It has (Figure 1a). Cell proliferation assays showed that forced been reported that erbB2 requires erbB3 to promote expression of erbB3 in MDA-MB-231 cells did not breast cancer cell proliferation (Holbro et al., 2003); affect their response to paclitaxel-induced cytotoxicity and erbB3 has a critical role in erbB2 altered breast (Figure 1b); however, it did significantly increase cell cancers (Lee-Hoeflich et al., 2008). These data suggest viability on paclitaxel treatment in SKBR3 cells that the erbB3 receptor, but not EGFR, probably (Figure 1c). These data suggest that expression of erbB3 has an important role in erbB2-mediated therap- may reduce paclitaxel-mediated cell killing only in eutic resistance in breast cancer. Our recent studies erbB2-overexpressing, but not EGFR-expressing, breast strongly support this hypothesis showing that estrogen cancer cells. To confirm these results, we next performed promotion of erbB2 tyrosine kinase activation in transient transfection to avoid the potential problem of mammary tumor cells requires erbB3 signaling (Liu long-term selection and clonal variation. MDA-MB-231 et al., 2009), and elevated expression of erbB3 con- and SKBR3 cells were infected with a lentivirus tributes to erbB2-mediated tamoxifen resistance (Liu containing either control vector or the same vector with et al., 2007). human erbB3 complementary DNA, as described in the We have also reported physical and functional interac- Materials and methods section. After 48-h infection, the tions between rat c-neu/erbB2 and endogenous mouse expression levels of erbB3 were clearly enhanced in both erbB3, which led to the activation of PI-3K/Akt signaling, cells (Figure 2a). This transient upregulation of erbB3 in mammary tumor cells derived from MMTV-neu significantly increased P-Akt levels and attenuated transgenic mice (Kim et al., 2005); and activation of PI- paclitaxel-induced cytotoxicity only in SKBR3 cells 3K/Akt signaling is a key pathway that induces resistance (Figure 2b). These data were consistent with the to chemotherapeutic agents in the breast cancer cells observations of stable erbB3-transfectant clones (Fig- (Knuefermann et al., 2003). Although the taxanes, ure 1). Furthermore, western blots on PARP and paclitaxel and docetaxel, have emerged as critical drugs caspases and apoptotic -linked immunosorbent in the treatment of breast cancer patients, acquired and assay revealed that transient induction of erbB3 expres- intrinsic resistance to taxanes currently represents a sion reduced paclitaxel-induced PARP cleavage, cas- significant clinical problem (Saloustros et al., 2008; Morris pase-3 activation and apoptosis in SKBR3 cells, whereas and Fornier, 2009). Although several molecular mechan- MDA-MB-231 cells, regardless their erbB3 expression isms contributing to paclitaxelresistancehavebeen levels, retained same sensitivity to paclitaxel-induced proposed (Orr et al., 2003; Ferlini et al., 2007), a series apoptosis (Figures 2c and d). Collectively, these data of studies demonstrates that overexpression of erbB2 indicate that elevated expression of erbB3 inhibits inhibits paclitaxel-induced apoptosis in breast cancer paclitaxel-induced apoptosis in erbB2-overexpressing, cells (Yu et al., 1996, 1998; Tan et al., 2002). How- but not EGFR-expressing, breast cancer cells. ever, the role of erbB3 in paclitaxel resistance remains unclear. We have focused our studies on erbB3 receptor, its interactions with erbB2 and subsequent Specific knockdown of erbB3 expression enhances activation of downstream signaling pathways. Our data paclitaxel-induced apoptosis and abrogates erbB2- indicate that expression of erbB3 is required for erbB2- mediated paclitaxel resistance in breast cancer cells mediated paclitaxel resistance via PI-3K/Akt-dependent To explore whether erbB3 expression is required for upregulation of Survivin. erbB2-mediated paclitaxel resistance in breast cancer

Oncogene ErbB3 induces Survivin expression S Wang et al 4227 cells, we studied whether specific knockdown of erbB3 potentiated paclitaxel-induced apoptosis evidenced by expression might potentiate paclitaxel-induced apopto- increased PARP cleavage, activation of caspase-8 and -3 sis in breast cancer cells with the coexpression of both (Figure 3c) and DNA fragmentation measured by erbB2 and erbB3. The expression levels of erbB3 in both enzyme-linked immunosorbent assay (Figure 3d). SKBR3 and MDA-MB-453 cells were downregulated As we have reported that the 435.eB1 cells (erbB2 with a lentiviral system containing human erbB3 transfectant of MDA-MB-435 cell line) are more shRNA, as we proved this erbB3 shRNA was specific resistant to paclitaxel-induced apoptosis (Yu et al., and efficient (Huang et al., 2009). Specific knockdown of 1996, 1998); and the MDA-MB-435 cells express high erbB3 not only dramatically decreased the P-Akt levels levels of erbB3 (Knuefermann et al., 2003), we then (Figure 3a), but also significantly enhanced paclitaxel- performed additional experiments to further address the mediated cell killing (Figure 3b). The assessment of hypothesis that overexpression of erbB2 in 435.eB1 cells apoptotic cells confirmed that erbB3 knockdown using may require the existence of erbB3 to induce paclitaxel specific shRNA in both SKBR3 and MDA-MB-453 cells 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 (Supple- mentary Figure S1A), suggesting that the expression of erbB3 was essential for erbB2 tyrosine kinase activation and the downstream signaling. More importantly, spec- ific knock down of erbB3 expression resensitized 435.eB1 cells to paclitaxel-induced PARP cleavage, caspase-8 and -3 activation and apoptosis (Supplemen- tary 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 resis- tance, we considered the downstream signaling pathways, class III b-tubulin (TUBB3) and several regulators of apoptosis. Although several studies indicate that elevated expression of TUBB3 may result in therapeutic resis- tance 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 condi- tions (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

Figure 1 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 compli- mentary 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 b-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-carboxy- methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt)). Data shows the representative of at least three independent experiments. Bars represent s.d. values.

Oncogene ErbB3 induces Survivin expression S Wang et al 4228 receptor type 4, Mcl-1 and Bcl-xL. However, a 1.5–2.0- Similar results were obtained with SKBR3.B3.pool cells fold induction of Survivin was observed only in erbB3- (Supplementary Figure S3). transfected SKBR3, but not MDA-MB-231 cells As dimerization of erbB2/erbB3 is essential for erbB2 (Figure 4a). Interestingly, the upregulation of Survivin tyrosine kinase activation (Liu et al., 2007), and erbB3 was also observed in 435.eB1 cells as compared with the expression closely correlated to the P-Akt levels only in parental cells (Lu et al., 2009). In contrast, specific knock erbB2-overexpressing, but not EGFR-expressing, breast down of erbB3 expression in the erbB2/erbB3-coexpressing cancer cells (Figures 1a, 2a and 3a), we believed that breast cancer cells significantly decreased Survivin, but had coexpression of erbB3 may preferably form a potent no effect on C-X-C chemokine receptor type 4, Mcl-1 and heterodimer complex with erbB2, but not EGFR, to Bcl-xL (Figure 4b). These data suggest that the erbB3 activate the erbB receptors. To test this hypothesis, co- levels specifically modulate Survivin expression in erbB2- immunoprecipitation assays with an anti-erbB3 anti- overexpressing breast cancer cells. As elevated expression body revealed that erbB3 indeed interacted with erbB2 of erbB3 mainly activated Akt in SKBR3 cells (Figures 1a in those breast cancer cells with coexpression of both and 2a), we studied whether there was a causal relationship erbB2 and erbB3, and elevated expression of erbB3 between the activation of PI-3K/Akt signaling and erbB3- further increased their associations, however, the simi- mediated upregulation of Survivin. Although mitogen- larly elevated expression of erbB3 never formed dimeri- activated protein kinase kinase inhibitor, PD98059, zation with EGFR in MDA-MB-231 cells (Figure 6a). efficiently reduced P-MAPK levels in erbB3-tarnsfectant Consistent with these data, increased associations of clone 1 and 2 (SKBR3.B3.1 and SKBR3.B3.2), it had no erbB2/erbB3 resulted in higher levels of both P-erbB2 significant effect on Survivin. However, the PI-3K and P-erbB3 in SKBR3 cells, in contrast, P-erbB3 was inhibitor, LY294002, simultaneously reduced the levels undetectable and P-EGFR levels remained unchanged of both P-Akt and Survivin (Figure 4c). Moreover, the PI- in MDA-MB-231 cells, although their protein levels 3K inhibitor, LY294002, but not the mitogen-activated seemed to be as high as erbB3 and erbB2 levels in protein kinase kinase inhibitor, PD98059, was able to SKBR3 cells (Figure 6b). Thus, our studies confirm that significantly sensitize the SKBR3.B3.1 and SKBR3.B3.2 heterodimerization of erbB2/erbB3 is a prerequisite for cells to paclitaxel-mediated cytotoxicity (Figure 4d). These erbB2 tyrosine kinase activation; and PI-3K/Akt signal- resultswerealsoconfirmedwiththesameassaysper- ing-dependent upregulation of Survivin is required for formed on the pooled erbB3-tarnsfectant clones of SKBR3 erbB3 potentiating erbB2-mediated paclitaxel resistance cells, SKBR3.B3.pool (Supplementary Figures S2A and in breast cancer cells (Figure 6c). 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 Sur- vivin in SKBR3.B3.pool, SKBR3.B3.1 and SKBR3.B3.2 Discussion cells (Supplementary Figures S2C and D), but also resensitized these cells to paclitaxel-mediated cytotoxicity Recent advances in our understanding of breast cancer (Supplementary Figure S2E). Taken together, our data biology have led to improved patient survival with demonstrate that the overexpression of erbB3 upregulates target therapies, for example, anti-estrogen agents Survivin through a PI-3K/Akt/mTOR signaling-depen- (tamoxifen) in the treatment of estrogen receptor- dent mechanism, and activation of PI-3K/Akt signaling is positive breast cancers (Jensen and Jordan, 2003); anti- critical for erbB3 promotion of erbB2-induced paclitaxel erbB2 antibody (trastuzumab, also known as Herceptin, resistance in breast cancer cells. Genentech, South San Francisco, CA, USA) for patients with erbB2 amplification and/or overexpression (Slamon et al., 2001). Although Herceptin has been successfully Upregulation of Survivin is required for erbB3-induced used in early-stage and metastatic breast cancer patients paclitaxel resistance in erbB2-overexpressing breast with erbB2-overexpressing tumors as monotherapy cancer cells (Cobleigh et al., 1999; Vogel et al., 2002) and in We next evaluated whether the upregulation of Survivin combination with other agents (Slamon et al., 2001; is required for erbB3-mediated antiapoptotic cell death- Hudis, 2007), both primary (de novo) and acquired induced by paclitaxel. Similar strategy was used to resistances to Herceptin frequently occur (Nahta et al., specifically knock down Survivin expression with a 2006; Nahta and Esteva, 2007); and it is in urgent need lentiviral system containing Survivin shRNA. Three to explore additional molecular markers accurately specific shRNA sequences as compared with control predicting erbB2-targeted therapies, and to identify shRNA all efficiently downregulated Survivin expres- new strategies enhancing the efficacy of therapeutic sion in both SKBR3.B3.1 and SKBR3.B3.2 cells, agents against erbB2-overexpressing breast cancers. but did not alter the levels of erbB2, erbB3, P-Akt and With a focus on studying the unique biological features P-MAPK (Figure 5a), suggesting Survivin acted down- of erbB3 receptor in breast cancer, we have recently stream of erbB3/PI-3K/Akt signaling pathway. Impor- reported that erbB3 not only contributes to erbB2- tantly, all three Survivin shRNA: S3, S4 and S5 were mediated tamoxifen resistance (Liu et al., 2007), but also able to significantly enhance paclitaxel-induced cyto- has a vital role in Herceptin resistance through a toxicity (Figure 5b), PARP cleavage and activation of heterotrimeric complex consisting of erbB3/erbB2/ caspase-8 and -3 (Figure 5c) and apoptosis (Figure 5d). IGF-1R (Huang et al., 2010). The data presented in

Oncogene ErbB3 induces Survivin expression S Wang et al 4229

Figure 2 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 mg/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 b-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 b-actin (c) and apoptosis enzyme-linked immunosorbent assay (ELISA) (d).

Oncogene ErbB3 induces Survivin expression S Wang et al 4230

Figure 3 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 mg/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 b-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 b-actin (c) and apoptosis enzyme-linked immunosorbent assay (ELISA) (d).

Oncogene ErbB3 induces Survivin expression S Wang et al 4231

Figure 4 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 b-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 b-actin. (c and d) SKBR3.B3.1 and SKBR3.B3.2 cells, untreated or treated with PD98059 (PD, 20 mM) or LY294002 (LY, 10 mM) 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 b-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. this study strongly support that elevated expression of laboratory have provided compelling evidence, suggest- erbB3 is required for erbB2-mediated paclitaxel resis- ing that erbB3 should be developed as another tance. Thus, a series of articles published by our important therapeutic target and the new therapeutics

Oncogene ErbB3 induces Survivin expression S Wang et al 4232

Figure 5 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 b-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 b-actin and (d) apoptosis enzyme-linked immunosorbent assay (ELISA). *Po0.005, **P ¼ 0.0133, ***P ¼ 0.0064 vs ConshRNA þ paclitaxel.

targeting erbB3 may significantly enhance the therapeu- an important apoptosis inhibitor and key tic efficacy of tamoxifen, paclitaxel and erbB2-targeted regulator (Liu et al., 2008; Mita et al., 2008). Survivin is therapies, such as Herceptin and , for erbB2- usually expressed in embryonic tissues during develop- overexpressing breast cancer. ment, but undetectable in most normal adult tissues. Survivin, the smallest member of IAP (inhibitor of Numerous studies show that Survivin is selectively apoptosis) family, is a dual functional protein acting as expressed in almost all types of human malignancies

Oncogene ErbB3 induces Survivin expression S Wang et al 4233 and its overexpression positively correlates with poor Survivin expression in human tumors have not been prognosis, tumor recurrence and therapeutic resistance fully elucidated. Although expression of both EGFR (Liu et al., 2008; Mita et al., 2008). This differential and erbB2 have been shown to upregulate Survivin expression of Survivin in tumors and normal tissues (Asanuma et al., 2005; Xia et al., 2006), to the best of draws a great interest to identify strategies targeting our knowledge, this is the first report indicating that Survivin for cancer treatment (Altieri, 2008; Mita et al., elevated expression of erbB3 is also capable of inducing 2008). However, the molecular mechanisms controlling 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 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 trans- lation 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 expres- sion 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 (Morgillo et al., 2006), we are currently testing whether the hetero- trimerization of erbB3/erbB2/IGF-1R, which we have observed in Herceptin-resistant breast cancer cells (Huang et al., 2010), may also regulate Survivin ex- pression. Such studies may shed new lights on identi- fying 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 p170mdrÀ1

Figure 6 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 b-actin. (c) Diagram of proposed model underlying the mechanism of erbB3-mediated paclitaxel resistance in erbB2-overexpressing breast cancer cells.

Oncogene ErbB3 induces Survivin expression S Wang et al 4234 and multi-drug resistance protein-mediated drug efflux containing human Survivin shRNA (pLKO.1-SurshRNA) were (Saloustros et al., 2008; Morris and Fornier, 2009). purchased from Sigma (St Louis, MO, USA). The packaging Changes in b-tubulin isotype expression with a switch psPAX2 and pMD2.G for lentiviral expression vector from class I to III and overexpression of TXR1 (taxol- were from Addgene (Cambridge, MA, USA). resistance gene 1) lead to paclitaxel resistance (van Antibodies were obtained as follows: erbB2 (Ab3) (from Amerongen and Berns, 2006; Ferlini et al., 2007). EMD Chemicals, Gibbstown, NJ, USA); erbB3 (Ab7) and P-erbB2 (PN2A) (from LabVision, Fremont, CA, USA); Although we did not observe any changes in TUBB3 P-erbB3 (21D3), caspase-8 (1C12) and caspase-3 (8G10), levels upon manipulation of erbB3 expression in our P-MAPK (E10), MAPK, P-Akt (Ser473), Akt, Survivin experimental conditions, there is no report showing (6E4), Bcl-xL (from Cell Signaling Technology, Beverly, whether erbB3 expression may regulate tyrosinase- MA, USA); C-X-C chemokine receptor type 4 (from related protein 1 expression. As overexpression of erbB2 IMGENEX, San Diego, CA, USA), Mcl-1 (clone 22) (from confers paclitaxel resistance via mdr-1-independent BD Biosciences, San Jose, CA, USA); PARP mAb (C-2-10) mechanism (Yu et al., 1996), and our current data show (from BIOMOL Research Laboratories, Plymouth Meeting, that erbB3 expression induces paclitaxel resistance only PA, USA); EGFR (F4), P-EGFR (Tyr1068) and b-actin (AC- in erbB2-overexpressing breast cancer cells, suggesting 75) (from Sigma). All other reagents were purchased from that erbB3-mediated paclitaxel resistance might also be Sigma unless otherwise specified. independent of mdr-1. Nonetheless, there is clear difference between erbB2- and erbB3-associated down- Cells and cell culture stream effectors, resulting in paclitaxel resistance of Human breast cancer cell lines SKBR3, MDA-MB-231, breast cancer cells. The overexpression of erbB2 not MDA-MB-453 and MDA-MB-435 were obtained from only increases Survivin levels via enhanced protein American Type Culture Collection (Manassas, VA, USA). The erbB2-transfectant 435.eB1 cell line (Yu et al., 1996) was translation (Lu et al., 2009), it also induces Src/signal kindly provided by Dr Dihua Yu (MD Anderson Cancer transducer and activator of transcription 3 activation- Center). The HEK293T human embryonic kidney cells were mediated p21waf1 transcriptional upregulation (Hawthorne kindly provided by Dr Haihua Gu (University of Colorado et al., 2009), and both Survivin and p21waf1 contribute to Denver). All cells were maintained in Dulbecco’s modified erbB2-mediated paclitaxel resistance. Our studies found Eagle’s medium: Nutrient Mix F-12 (D-MEM/F-12 1:1) no change in p21waf1 expression followed by either supplemented with 10% fetal bovine serum. All cell lines were transfection or knock down of erbB3 (data not shown). cultured in a 37 1C humidified atmosphere containing 95% air It seems that elevated expression of erbB3 specifically and 5% CO2 and were split twice a week. upregulates Survivin in erbB2-overexpressing breast cancer cells. One potential explanation might be that Establishment of stable erbB3-tranfectant clones of breast overexpression of erbB3 can only form heterodimerization cancer cells with endogenous erbB2, but not EGFR (Figure 6a). In The MDA-MB-231 or SKBR3 cells were transfected with contrast, the overexpression of erbB2 not only interacts either control vector pDsRed-Monimer-N1 (Clontech Labora- with endogenous erbB3, it may also dimerize with other tories, Palo Alto, CA, USA) or human erbB3-expressing vector receptor tyrosine kinases, such as EGFR, and subse- pDsRed-erbB3 (Huang et al., 2009) using FuGENE-6 trans- fection (Roche Diagnostics, Indianapolis, IN, USA). The quently activates Src/ signal transducer and activator of waf1 cells were selected with Geneticin (Invitrogen, Carlsbad, CA, transcription 3 signaling to induce transcriptional USA) for 2 months. The resulting single clone was picked, upregulation. Further studies using animal model and amplified and grown into stable cell line. The remaining human breast cancer tissues are warranted to elucidate clones were cultured together and grown into pooled stable this hypothesis. transfectants. In summary, we have demonstrated that erbB3 expression in erbB2-overexpressing breast cancer cells Construction of lentiviral expression vector pLEX-erbB3 confers paclitaxel resistance via PI-3K/Akt-dependent The entire coding sequence of human erbB3 was amplified upregulation of Survivin. These findings, together with from pDsRed-erbB3 by PCR using Phusion High-Fidelity our previous reports on the role of erbB3 receptor in DNA Polymerase (New England BioLabs, Ipswich, MA, tamoxifen and Herceptin resistance, provide a strong USA) with the primer set: forward primer 50-ATAGCGGCCG rationale to develop erbB3-targeted therapies. Such CATGAGGGCGAACGACGCTC-30; reverse primer 50-GCG strategies should significantly enhance the efficacy of ACGCGTTTACGTTCTCTGGGCATTAGC-30. The amplified tamoxifen-, paclitaxel- and erbB2-targeted therapies, fragments were purified and digested with NotIandMluI such as Herceptin and lapatinib, for the treatment of (New England BioLabs), and inserted into the lentiviral expression vector LentiORF pLEX-MCS (Open Biosystem, erbB2-overexpressing breast cancer. Huntsville, AL, USA) using Quick Ligation Kit (New England BioLabs). After verification by DNA sequencing, the recombi- nant with accurate sequence of human erbB3 complementary Materials and methods DNA was nominated as pLEX-erbB3.

Reagents and antibodies Production of lentivirus Paclitaxel (Ben Venue Labs, Bedford, OH, USA) was obtained Lentiviral production was carried out as described previously from University of Colorado Hospital Pharmacy. MISSION (Huang et al., 2009, 2010). In brief, the lentiviral expression Non-target shRNA, which does not target human and vector pLKO.1-ConshRNA or pLKO.1-SurshRNA and lenti- mouse , control vector (pLKO.1-ConshRNA), pLKO.1 virus packaging plasmids pCMV-VSVG and pCMV-DA.9

Oncogene ErbB3 induces Survivin expression S Wang et al 4235 were co-transfected into virus packaging cell line 293T using Immunoprecipitation and western blot analysis FuGENE-6 following the standard procedure. Similar trans- Immunoprecipitation and western blot assays were performed as fections were also performed with the control vector pLEX- described (Huang et al., 2010). Briefly, equal amounts of MCS or pLEX-erbB3 and the packaging plasmids psPAX2 cell lysates were incubated with specific antibody for 2 h at and pMD2.G. After 24 h, the culture media were changed with 4 1C, followed by incubation with protein A–agarose or protein fresh DMEM/F12 medium (10% fetal bovine serum). The G–agarose (Roche) at 4 1C overnight. The immunoprecipitates or virus in conditioned medium were collected in the following 3 equal amounts of cell lysates were boiled in sodium dodecyl consecutive days, and then filtered with low protein-binding sulfate sample buffer, resolved by sodium dodecyl sulfate filters (Millex-hV 0.45-mm polyvinylidene fluoride, Millipore, polyacrylamide gel electrophoresis and western blotted with Burlington, MA, USA) before they were aliquoted and stored specific antibodies, as described in the figure legends. in À80 1C freezer. Statistical analysis Statistical analyses of the experimental data were performed ErbB3 overexpression or specific knockdown of erbB3 using a two sided Student’s t-test. Significance was set at P 0.05. expression with a lentiviral system o 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 mg/ml) were added into Abbreviations the virus-containing media. Then the culture media of the candidate breast cancer cells were replaced with the lentivirus- ER, estrogen receptor; EGFR, epidermal growth factor containing media. After 24 h, the virus-infected cells were receptor; MAPK, mitogen-activated protein kinase; MEK, selected with puromycin (1 mg/ml) for 48 h, and then collected MAPK kinase; PI-3K, phosphoinositide 3-kinase; mAb, and subjected to required experiments. monoclonal antibody; mTOR, mammalian target of rapamy- cin; PARP, poly(ADP-ribose) polymerase; TUBB3, class III b-tubulin; CXCR4, C-X-C chemokine receptor type 4; MRP1, Cell proliferation assay multi-drug resistance protein 1; TXR1, taxol-resistance gene 1; The CellTiter96 AQ non-radioactive cell proliferation kit NSCLC, non-small cell ; shRNA, short-hairpin (Thermo Fisher Scientific, Waltham, MA, USA) was used to RNA; ELISA, enzyme-linked immunosorbent assay; PAGE, determine cell viability (Huang et al., 2009, 2010). In brief, cells polyacrylamide gel electrophoresis. 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 Conflict of interest reader, the percentages of surviving cells from each group relative to controls, defined as 100%, were determined by The authors declare no conflict of interest. reduction of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy- methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt). Acknowledgements Quantification of apoptosis An apoptosis enzyme-linked immunosorbent assay kit (Roche) We thank Dr Haihua Gu for providing the pLKO.1- was used to quantitatively measure cytoplasmic histone- ErbB3shRNA expression vector and lentivirus packaging associated DNA fragments (mononucleosomes and oligonu- plasmids pCMV-VSVG and pCMV-DA.9. We also thank Ms cleosomes) as previously reported (Liu et al., 2007; Huang Lisa Litzenberger for her excellent art preparation. This work et al., 2009). This enzyme immunoassay was performed was supported in part by a research Grant from Susan G according to the manufacturer’s instructions. Komen for the Cure (to BL).

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

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