Downregulation of erbB3 abrogates erbB2-mediated tamoxifen resistance in breast cancer cells

Authors

  • Bolin Liu,

    Corresponding author
    1. Department of Pathology and College of Medicine, University of Colorado at Denver and Health Sciences Center (UCDHSC), Aurora, CO
    • Department of Pathology, Mail Stop 8104, UCDHSC at Fitzsimons, P.O. Box 6511, 12801 E. 17th Ave., Aurora, CO 80045
    Search for more papers by this author
    • Fax: +303-724-3712.

  • Dalia Ordonez-Ercan,

    1. Department of Pathology and College of Medicine, Oklahoma University Health Sciences Center (OUHSC), Oklahoma, OK
    Search for more papers by this author
  • Zeying Fan,

    1. Department of Pathology and College of Medicine, University of Colorado at Denver and Health Sciences Center (UCDHSC), Aurora, CO
    Search for more papers by this author
  • Susan M. Edgerton,

    1. Department of Pathology and College of Medicine, University of Colorado at Denver and Health Sciences Center (UCDHSC), Aurora, CO
    Search for more papers by this author
  • XiaoHe Yang,

    1. Department of Pathology and College of Medicine, Oklahoma University Health Sciences Center (OUHSC), Oklahoma, OK
    Search for more papers by this author
  • Ann D. Thor

    1. Department of Pathology and College of Medicine, University of Colorado at Denver and Health Sciences Center (UCDHSC), Aurora, CO
    Search for more papers by this author

Abstract

Receptor tyrosine kinase activity is essential for erbB2 (HER2/neu) promotion of breast carcinogenesis, metastasis and therapeutic resistance. erbB2 kinase can be activated by dimerization with another erbB receptor, most of which bind ligands. Of these, the erbB2/erbB3 heterodimer is the most potent oncogenic complex. erbB2 reportedly requires erbB3 to promote cellular proliferation, although this may occur without changes in erbB2 tyrosine kinase activity in some model systems. Our investigations focus on the role(s) of erbB3 in erbB2-associated kinase activity and tamoxifen resistance. Using tumor-derived cell lines from wild type rat c-neu transgenic mice and human breast cancers, we demonstrate that erbB3 plays a critical role in the activation of erbB2 tyrosine kinase activity and erbB2-associated tumorigenesis. Mechanistically, downregulation of erbB3 by specific siRNA reduces erbB2 tyrosine phosphorylation, decreases the PI-3K/Akt signaling, and inhibits mammary/breast cancer cell proliferation and colony formation. Specific erbB3 siRNA sensitizes erbB2 transfected MCF-7 cells (MCF-7/erbB2) to tamoxifen-associated inhibition of both cell growth and colony formation and enhances tamoxifen-induced apoptosis, in contrast to control siRNA transfected MCF-7/erbB2 cells which are tamoxifen-resistant. Our data indicates that erbB2/erbB3 heterodimerization is a prerequisite for erbB2 tyrosine kinase activation in mammary/breast cancer cells and that downregulation of erbB3 inhibits erbB2-associated procarcinogenic activity via inactivation of the PI-3K/Akt pathway. Furthermore, erbB3 also contributes to erbB2-mediated tamoxifen resistance and therefore may be a clinically relevant therapeutic target in addition to erbB2. © 2007 Wiley-Liss, Inc.

Aberrant activation of erbB receptor tyrosine kinase (RTK) has been widely implicated in human carcinogenesis.1 In general, erbB receptors may be activated via both ligand-dependent and -independent mechanisms. Once activated, these receptors may induce signaling via tyrosine kinase activity that add phosphorylated tyrosine residues to downstream signaling molecules, [e.g. the p85 subunit of phosphatidylinositol 3-kinase (PI-3K); Shc and/or Grb2 of the mitogen-activated protein kinase (MAPK) pathway]. Although these 2 pathways are the predominant downstream effectors of RTK activation, they may be significantly influenced by other molecular factors and signaling pathways. These complex interconnected pathways provide mechanisms for fine tuning critical intracellular processes, such as proliferation, maturation, survival, apoptosis and angiogenesis.2, 3, 4, 5

erbB2 (HER2/neu) is believed to play an important role in normal mammary gland (breast) development.6 Like the related epidermal growth factor receptor (EGFR) and erbB4, erbB2 has intrinsic tyrosine kinase activity.7, 8 Alterations of erbB2 are frequently identified in human cancers. This typically involves gene amplification with overexpression of erbB2-encoded p185, which occurs in ∼30% of invasive breast cancers.9, 10erbB2 alterations are significantly associated with a worse disease free and overall survival (i.e. prognosis), particularly for node positive breast cancer patients.9, 11 We have shown enhanced prognostic significance when multiple erbB family members are altered, which result in enhanced activation of erbB2 and modification of downstream signaling molecules.12, 13, 14, 15

Studies of the molecular and cellular mechanisms underlying erbB2-associated breast cancer aggression suggest 2 major biologic features of this phenomenon. First, overexpression/amplification of erbB2 increases the ability of a cancer cell to metastasize via enhanced invasion, angiogenesis and survival signals. A recent report indicates that CXCR4 (CXC chemokine receptor 4) is an essential element of erbB2-mediated tumor metastasis. In brief, erbB2 activation inhibits CXCR4 ubiquitination resulting in upregulation, enhanced metastatic potential and tumor homing.16 erbB2 activation has also been associated with variable chemotherapeutic or hormonal sensitivity.17 For example, while the majority of estrogen receptor (ER) positive breast cancer patients benefit from tamoxifen, some cancers demonstrate primary or secondary tamoxifen resistance,18, 19 which has been linked to alterations of erbB2 (and in some cases EGFR).20, 21, 22, 23In vitro studies also support a link between tamoxifen resistance, EGFR24, 25 and erbB2 alterations.25, 26, 27 This deleterious interaction may be reversed or delayed by the concordant administration of hormonal and RTK inhibiting agents.28 In aggregate, these in vivo and in vitro data substantiate cross talk between the ER and RTK (erbB2 and EGFR) signaling pathways. While others have not yet linked tamoxifen resistance to alterations of the third member of the RTK family, namely erbB3, data presented herein suggests that erbB3 may also participate in this phenomenon.

erbB3 expression is frequently identified in erbB2 altered breast, ovarian and bladder cancers,29, 30, 31, 32 The etiologic, prognostic or predictive roles of erbB3 in breast carcinogenesis, however, are not yet well defined. Structural and functional studies of erbB3 show that it lacks a functional tyrosine kinase activity. Notably associated with other family members, therefore, erbB3 cannot initiate cell signaling in the absence of a heterodimer partner bearing tyrosine kinase activity.1, 33 Of the many possible RTK heterodimer complexes, erbB2/erbB3 is believed to be the most biologically active and protumorigenic.34, 35 Coexpression of erbB2 and erbB3 has also been associated with upregulation of erbB3 ligands, suggesting critical feedback mechanisms.36 While it is well recognized that ligand-induced heterodimerization [e.g. Heregulin (HRG) bound erbB3/erbB2] occurs and may lead to rapid receptor activation, it has also been proposed that high expression of erbB2 may recruit autoinhibited erbB3 (without bound ligand) into heterodimers. These later heterodimers may also assume the ligand-induced conformation, resulting in weak, but sustained (and hence profound) activation of signaling complexes.37 We have recently demonstrated physical and functional interactions between the wild type (wt) rat neu/erbB2 transgene and upregulated mouse erbB3, in the presence or absence of HRG in mammary tumor cell lines-derived from the wt rat c-neu/erbB2 transgenic mice.38 Our results are consistent with both mechanistic hypotheses.

Others have been shown that inactivation of erbB3 blocks proliferation of breast cancer cells as efficiently as impeding erbB2 signaling without an effect on erbB2 tyrosine kinase activity.2 These data suggest complex interactions between erbB2 and erbB3 possibly involving coregulatory or feedback processes. In this report, we further investigate the role(s) of erbB3 in erbB2 RTK activity and erbB2-mediated tumorigenesis and tamoxifen resistance.

Abbreviations:

dUTP, deoxyuridine triphosphate-digoxigenin; EGFR, epidermal growth factor receptor; ELISA, enzyme-linked immunosorbent assay; ER, estrogen receptor; HRG, heregulin; IGF-1R, insulin-like growth factor-1 receptor; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; PAGE, polyacrylamide gel electrophoresis; PARP, poly(ADP-ribose) polymerase; PI-3K, phosphoinositide 3-kinase; RTK, receptor tyrosine kinase; siRNA, small interfering RNA; TdT, terminal deoxynucleotidyl transferase; TUNEL, TdT-mediated dUTP nick end labeling.

Material and methods

Reagents

Both human and mouse erbB3 siRNA and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 4-Hydroxytamoxifen (for convenience, described as tamoxifen herein) was obtained from Sigma Chemical (St. Louis, MO).

Antibodies used for Western blot analysis were from the following sources: erbB2 (c-neu Ab-3, Oncogene Research Products, San Diego, CA); phospho-erbB2 (P-erbB2, clone PN2A, NeoMarkers, Fremont, CA); erbB3 (Ab-7, NeoMarkers), phospho-erbB3 (P-erbB3, clone 21D3, Cell Signaling Technology, Beverly, MA); phospho-Akt (Ser473) and Akt (Cell Signaling Technology); total ERK2 (polyclonal antibody C-14, Santa Cruz Biotechnology); phospho-MAPK (P-MAPK, E10 mAb, Cell Signaling Technology); phospho-ERα (Ser118) polyclonal antibody, phospho-ERα (Ser167) polyclonal antibody, and ERα 62A3 monoclonal antibody (Cell Signaling Technology); poly (ADP-ribose) polymerase (PARP) monoclonal antibody (C-2-10, BIOMOL Research Laboratories Plymouth Meeting, PA); β-actin (clone AC-74, mAb, Sigma Chemical). All other reagents were purchased from Sigma Chemical unless otherwise specified.

Cells and cell culture

Murine mammary tumor cell lines 78717, 85815 and 85819 were established from mammary tumors derived from wt rat c-neu transgenic mice as previously described.38, 39 The human breast cancer cell lines MCF-7, MDA-MB-453, BT-474, and SKBR-3 were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and maintained in Dulbecco's Modified Eagle's Medium: Nutrient Mix F-12 (D-MEM/F-12 1:1) (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Invitrogen). erbB2 transfected MCF-7 cells (MCF-7/erbB2) were obtained from Dr. Christopher C. Benz, Buck Institute for Age Research, Novato, CA. All the cell lines were cultured in a 37°C humidified atmosphere containing 95% air and 5% CO2 and were split twice a week.

Transfection of siRNA

Specific erbB3 siRNA or control siRNA was transfected into cultured cells with Lipofectamine™ 2000 reagent (Invitrogen) exactly following the procedures provided by Santa Cruz Biotechnology.

Cell proliferation assay

A CellTiter96™ AQ nonradioactive cell proliferation kit (Promega, Madison, WI) was used to determine cell viability as previously described.38, 39 In brief, 24 hr after siRNA transfections, cells were plated onto 96-well plates with complete medium for an additional 60 hr incubation at 37°C with 5% CO2. For tamoxifen treatment experiments, 24 hr after plating onto 96-well plates, cells were grown in either 0.1 ml phenol-red free D-MEM/F-12 medium (Invitrogen) with 1% charcoal-stripped FBS (Cocalico Biologicals, Reamstown, PA) as control, or 0.1 ml of the same medium containing 2.5 μM tamoxifen and incubated at 37°C with 5% CO2 for another 60 hr. After reading all wells at 490 nM with a microplate reader, the percentages of surviving cells from each group relative to controls, defined as 100% survival, were determined by reduction of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt).

Clonogenic assay

Clonogenic assays were performed as described previously.40 In brief, following siRNA transfections, cells were seeded into 6-well plates in triplicates at a density of 500 cells/well in 2 ml of medium containing 10% FBS and cultured for 14 days in a 37°C humidified atmosphere containing 95% air and 5% CO2. The cell clones were stained for 15 min with a solution containing 0.5% crystal violet and 25% methanol, followed by 3 rinses with tap water to remove excess dye. The colony numbers were counted by gel documentation system EAGLE EYE™ II (Stratagene, La Jolla, CA).

TUNEL assay

Following siRNA transfections, untreated or tamoxifen-treated cells were cytocentrifuged onto glass slides as described previously.41 Cells were fixed with 4% paraformaldehyde for 1 hr at room temperature and then incubated with permeabilisation solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min on ice. Apoptotic cells with DNA strand breaks were determined by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling) using an in situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions. The cells were evaluated using a Leica TCS NT microscope (Leica Microsystems, Heildelburg, Germany).

Quantification of apoptosis

An apoptosis ELISA kit (Roche Diagnostics) was used to quantitatively measure cytoplasmic histone-associated DNA fragments (mononucleosomes and oligonucleosomes) as previously reported.42 This photometric enzyme immunoassay was performed according to the manufacturer's instructions.

Western blotting analysis

Protein expression levels were determined by western blot analysis as previously described.39, 42 Briefly, cells were lysed in a buffer containing 50 mM Tris, pH 7.4, 50 mM NaCl, 0.5% NP-40, 50 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, 25 μg/ml leupeptin and 25 μg/ml aprotinin. The lysates were centrifuged at full speed in a microcentrifuge for 20 min and the supernatants were collected for protein concentration determination by the Coomassie Plus protein assay reagent (Pierce Chemical, Rockford, IL). Equal amounts of cell lysates were boiled in Laemmli SDS-sample buffer, resolved by SDS-PAGE, and western blot analysis with specific antibodies as described in the figure legends.

Statistical analysis

Statistical analyses of experimental data were performed using a 2-sided Student's t-test. Significance was set at p < 0.05.

Results

Downregulation of erbB3 expression by specific siRNA reduces erbB2 tyrosine phosphorylation and decreases activity of Akt, but not MAPK, in mammary/breast cancer cells

Frequent upregulation of erbB3 has been observed in mammary tumors of transgenic mice bearing either wt or activated rat c-neu/erbB2.38, 43 These data suggest that concomitant upregulation of erbB3 and erbB2, facilitating ligand-associated RTK signaling may be a critical step in erbB2-induced mammary tumorigenesis. We have previously demonstrated that endogenous mouse erbB3 interacts with the transgene (wt rat neu/erbB2)-encoded protein, both in the presence and absence of HRG.38 In the current studies, we used siRNA to specifically knockdown erbB3, allowing us to determine the role(s) of erbB3 in erbB2 tyrosine kinase activity and erbB2-mediated signal transduction. We first examined the efficiency and specificity of the erbB3 siRNAs on our novel mammary tumor cell lines, established from the mammary tumors which arose from the wt rat c-neu transgenic mice.38, 39 We then performed similar knockdown experiments on human breast cancer cell lines. As shown in Figure 1a, the erbB3 siRNA, but not control siRNA, dramatically reduced erbB3 expression levels in both mouse and human mammary/breast cancer cells. In contrast, erbB2 levels remained unchanged in all cells. These data indicate that the siRNAs were specific and efficiently downregulated erbB3 expression in both murine and human cell line models. Downregulation of erbB3 in cells that coexpressed erbB2 and erbB3 (85815, 85819 and MDA-MB-453) significantly decreased the levels of phosphorylated Akt (P-Akt). Downregulation of erbB3 had no appreciable effect on the levels of phosphorylated MAPK (P-MAPK) and ERK2 (Fig. 1a). These findings are consistent with the literature,1, 44 including a recent report indicating that loss of erbB3 by an artificial transcription factor (E3) had no major effect on P-MAPK but resulted in a significant decrease in P-Akt in SKBR-3 cells.2

Figure 1.

Downregulation of erbB3 expression by specific siRNA in mammary/breast cancer cells reduces erbB2 tyrosine phosphorylation and results in inactivation of Akt, but not MAPK. (a) Mouse mammary tumor cell lines (85815 and 85819) and human breast cancer cell lines (MCF-7 and MDA-MB-453) were transiently transfected with either control siRNA or erbB3 specific siRNA. After 24 hr, cells were collected, lysed and subjected to western blots analyses with specific antibodies directed against erbB3, erbB2, P-Akt, Akt, P-MAPK, ERK2 and β-actin. (b) and (c) Mouse mammary tumor cell lines (78717 and 85819) or human breast cancer cell lines (BT474 and SKBR-3) were transiently transfected with either control siRNA or specific erbB3 siRNA. After 24 hr, cells were collected, lysed and subjected to western blots analyses with specific antibodies directed against erbB3, P-erbB3, erbB2, P-erbB2, P-Akt, Akt and β-actin. The bar graphs underneath were obtained by calculating the percentages of P-Akt signal intensities in erbB3 siRNA-transfected cells comparing to their control siRNA-transfected counterparts (defined as 100%). The relative signal intensities of P-Akt were measured by densitometry analysis with EAGLE EYE™ II (Stratagene, La Jolla, CA).

To investigate if expression of erbB3 was required for the maintainence of erbB2 tyrosine kinase activity, we chose a pair of mammary cancer cell lines and a pair of human breast cancer cell lines for further study. Both 78717 and 85819 mouse mammary tumor cell lines have high mouse erbB3 expression levels. 78717 cells express very low levels of the transgene erbB2, whereas 85819 cells coexpress both the transgene erbB2 protein and endogenous erbB3 at high levels.38 We then used 2 human breast cancer cell lines with high erbB2 expression. BT474 cells have high erbB3, whereas SKBR-3 cells have less erbB3 expression (Fig. 1c). Using cells harvested post siRNA transfection, we determined erbB2 and erbB3 levels as well as their tyrosine phosphorylation, and downstream signaling by western blot analyses (Figs. 1b and 1c). erbB3 siRNA specifically decreased erbB3 protein but had no effect on erbB2 expression levels in all cell lines. Consistent with the data described earlier (Fig. 1a), the downregulation of erbB3 resulted in reduced levels of P-Akt in the 85819, BT474 and SKBR-3 cells with coexpression of both erbB2 and erbB3, whereas the erbB3 siRNA had no effect on P-Akt in the 78717 cells (Fig. 1b) or on P-MAPK levels in all cell lines (data not shown). Densitometry analysis of the P-Akt signals showed that following erbB3 siRNA transfection, the P-Akt intensity of 85819, BT474 and SKBR-3 cells were reduced to 71%, 74% and 90% of their corresponding controls, respectively. (Figs. 1b and 1c, bargraph). These reductions of P-Akt levels appeared to be correlated with their expression levels of erbB3, i.e. the higher the cells exhibited erbB3 levels, the more reduction of P-Akt the cells had (Figs. 1b and 1c). Because activation of erbB2 kinase leads to tyrosine phosphorylation of itself (autophosphorylation) as well as its dimerization partner (erbB3), the phosphorylation levels of erbB2 and erbB3 are frequently used as indicators of erbB2 tyrosine kinase activity. In 78717 cells with low levels of erbB2 protein expression, basal levels of phosphorylated erbB3 (P-erbB3) were undetectable although these cells expressed similar levels of erbB3 protein as the 85819 cells. In contrast, P-erbB3 was clearly detected in the 85819 cells which expressed both erbB2 and erbB3 (Fig. 1b, lane 1 vs. 3). These data suggest that the presence of erbB2 may be neccesary for erbB3 phosphorylation. More importantly, while downregulation of erbB3 had no effect on erbB2 protein levels, the levels of both P-erbB2 and P-erbB3 were dramatically reduced following specific erbB3 siRNA transfections in both the 85819 and BT474, and to a lesser extent in the SKBR-3 cells. All of these cell lines coexpressed both erbB2 and erbB3 (Fig. 1b, lane 3 vs. 4 and Fig. 1c, lane 1 vs. 2, 3 vs. 4). Taken together, our results suggest that the presence of erbB3 may be critical for maintenance of erbB2 tyrosine kinase activity, most likely via heterodimerization complex formation between these two receptors.

Downregulation of erbB3 expression by specific siRNA inhibits breast tumor cell proliferation and colony formation

To investigate the role of erbB3 in erbB2-associated mammary/breast cancer cell proliferation, transformation and tamoxifen resistance, both cell proliferation analyses and colony formation assays were carried out in control or erbB3 siRNA transfected mammary/breast cancer cell lines. The siRNA directed against erbB3 significantly inhibited cellular proliferation as measured by a MTS assay, 3 days post siRNA transfections in 78717, 85815 and 85819 cells (Fig. 2a, p < 0.001 for each). Since synthetic siRNA-mediated RNA interference in mammalian cells is transitory (with cells recovering in 4–6 days45, 46), we next performed clonogenic assays to study whether short-term knockdown of erbB3 would have long-term effects on tumor cell growth. As shown in Figure 2b, anchorage-dependent cell clones formed 2 weeks post siRNA transfection, with erbB3 siRNA treated cells showing attenuation of colony formation in all three (78717, 85815 and 85819) cell lines. The reduction of colony numbers was statistically significant. To verify these results in human breast cancer cells, we performed similar studies using BT474 and SKBR-3 cells. The siRNA we used for human erbB3 was specific and efficient, since the erbB3 siRNA significantly knocked down erbB3 (but not erbB2) levels and reduced P-Akt levels as compared to control siRNA (Fig. 1c). In these human breast cancer cell lines, downregulation of erbB3 expression significantly inhibited cell proliferation (Fig. 3a) and had a long-term effect on anchorage-dependent colony formation (Fig. 3b).

Figure 2.

Downregulation of erbB3 expression by specific siRNA inhibits mouse mammary tumor cell proliferation and colony formation. Mouse mammary tumor cells (78717, 85815 and 85819) were transiently transfected with either control siRNA or specific erbB3 siRNA for 24 hr. (a) Transfected cells were trypsinized and 5 × 103 cells in 0.1 ml culture medium with 10% FBS were plated onto 96-well plates and incubated at 37°C with 5% CO2 for an additional 60 hr. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of MTS. Data shows the representative of 3 independent experiments. Bars, SD. *, p < 0.001 versus control siRNA transfected cells. (b) Cells from the same batch of transfected cells were also plated onto 6-well plates (500 cell/well) in triplicates with fresh medium containing 10% FBS and the culture medium was changed once every 3 days for 2 weeks. The pictures of 6-well plates with colonies were taken by a digital camera and the bar graph underneath was obtained by calculating the percentages of colony numbers from each cell line relative to controls, defined as 100%, measured by EAGLE EYE™ II. Data shows the representative of 3 independent experiments. Bars, SD.

Figure 3.

Downregulation of erbB3 expression by specific siRNA inhibits proliferation and colony formation in human breast cancer cells. Human breast cancer cells (BT474 and SKBR-3) were transiently transfected with either control siRNA or specific erbB3 siRNA for 24 hr. (a) Transfected cells were trypsinized and 5 × 103 cells in 0.1 ml culture medium with 10% FBS were plated onto 96-well plates and incubated at 37°C with 5% CO2 for an additional 60 hr. The percentages of surviving cells from each cell line relative to controls, defined as 100% survival, were determined by reduction of MTS. Data shows the representative of 3 independent experiments. Bars, SD. *, p < 0.001 versus control siRNA transfected cells. (b) Cells from the same batch of transfected cells were also plated onto 6-well plates (500 cell/well) in triplicates with fresh medium containing 10% FBS and the culture medium was changed once every 3 days for 2 weeks. The pictures of 6-well plates with colonies were taken by a digital camera and the bar graph underneath was obtained by calculating the percentages of colony numbers from each cell line relative to controls, defined as 100%, measured by EAGLE EYE™ II. Data shows the representative of 3 independent experiments. Bars, SD.

These data suggest that the specific antiproliferative effects of erbB3 siRNA may be through inactivation of PI-3K/Akt signaling in erbB2-overexpressing mammary/breast cancer cells. Interestingly, downregulation of erbB3 was also able to significantly inhibit cell growth of the 78717 cells which have low erbB2 expression as shown by both MTS and clonogenic assays (Fig. 2). In these cells, however, the activity of Akt was not affected by erbB3 siRNA (Fig. 1b), suggesting that in erbB3-high and erbB2-low expressing mammary/breast cancer cells, erbB3 may promote cell growth through a PI-3K/Akt signaling-independent mechanism.

Downregulation of erbB3 sensitizes erbB2-overexpressing breast cancer cells to tamoxifen-mediated growth inhibition

MCF-7 is a well-known ER α positive (ERα+), tamoxifen sensitive human breast cancer cell line. Transfection of MCF-7 cells with erbB2 to generate a subline MCF-7/erbB2 induces tamoxifen resistance.26 To study whether erbB3 plays a role in erbB2-mediated tamoxifen resistance, both MCF-7 and MCF-7/erbB2 cells were transfected with erbB3 siRNA prior to tamoxifen treatment. erbB3 siRNA specifically downregulated erbB3 expression and had no apparent effect on erbB2 expression (Fig. 4a, consistent with our results shown in Fig. 1). Both the expression levels and activity of ERα can modify tamoxifen sensitivity of breast cancer cells. The phosphorylation of ERα provides an important mechanism to regulate ERα activity47, 48, 49 and it is well known that ERα can be phosphorylated on multiple sites.50 Both MAPK and Akt, the 2 major downstream signaling kinases of erbB2/erbB3, are capable of activating ERα via a ligand-independent mechanism by phosphorylating ERα at serine 118 and serine 167, respectively.47, 48 To better define the role of erbB3 in this process, we investigated whether downregulation of erbB3 would affect the expression and phosphorylation status of ERα. For both MCF-7 and MCF-7/erbB2 cells, downregulation of erbB3 with siRNA transfection had no effect on ERα expression. In contrast, tamoxifen dramatically increased the levels of ERα (Fig. 4a), which is consistent with the literature.51 The phosphorylation of ERα at serine 118 (P118-ERα) was not affected by downregulation of erbB3 in either cell line, although the level of P118-ERα was much lower in MCF-7/erbB2 cells as compared to parental MCF-7 cells (P118-ERα, Fig. 4a). P118-ERα was reduced post tamoxifen treatment in MCF-7 cells transfected with either control or erbB3 specific siRNA. Interestingly, tamoxifen increased the level of P118-ERα in control siRNA-, but not erbB3 siRNA-, transfected MCF-7/erbB2 cells (P118-ERα, Fig. 4a). The phosphorylation of ERα at serine 167 (P167-ERα) was also studied using a specific antiphospho-Ser167 antibody from Cell Signaling Technology, although we were unable to detect P167-ERα signals using any of our experimental conditions (data not shown).

Figure 4.

Downregulation of erbB3 expression abrogates resistance to tamoxifen-mediated growth inhibition in erbB2-overexpressing breast cancer cells. (a) MCF-7 and erbB2-overexpressing MCF-7 (MCF-7/erbB2) cells were transfected with either control siRNA or specific erbB3 siRNA for 24 hr. The cells were then incubated in phenol-red free medium with 1% charcoal-stripped FBS in the presence or absence of 2.5 μM tamoxifen for another 24 hr. The cells were collected and equal amounts of total cell lysates were used for western blot analyses with antibodies directed against erbB3, erbB2, phospho-ERα at serine 118 (P118-ERα), ERα or β-actin. (b) MCF-7 and MCF-7/erbB2 cells were transfected with either control siRNA or erbB3 siRNA for 24 hr. The cells were then trypsinized and 5 × 103 cells in 0.1 ml culture media were plated onto 96-well plates. After another 24 hr incubation, cells were grown in either 0.1 ml phenol-red free medium with 1% charcoal-stripped FBS as control, or 0.1 ml same medium containing 2.5 μM tamoxifen. Cells were incubated at 37°C with 5% CO2 for another 60 hr, and the percentages of surviving cells from each group relative to controls, defined as 100% survival, were determined by reduction of MTS. Data shows the representative of 3 independent experiments. Bars, SD. *, p < 0.05 versus control siRNA transfected and tomoxifen-treated MCF-7 cells. **, p < 0.005 versus control siRNA transfected and tomoxifen-treated MCF-7/erbB2 cells. ***, p < 0.05 versus specific erbB3 siRNA transfected (without tamoxifen treatmen) MCF-7/erbB2 cells. (c) Cells from the same batch of transfected cells were plated onto 6-well plates (500 cell/well) in triplicates with fresh medium containing 10% FBS for 24 hr. Cell culture media were then changed with fresh complete medium (10% FBS) or the same medium containing 2.5 μM tamoxifen once every 3 days for 2 weeks. The pictures of 6-well plates with colonies were taken by a digital camera and the bar graph underneath was obtained by calculating the percentages of colony numbers from each cell line relative to controls, defined as 100%, measured by EAGLE EYE™ II. Data shows the representative of 3 independent experiments. Bars, SD. *, p < 0.05 versus cells with control siRNA transfection and no tomoxifen treatment. UT, untreated; TAM, tamoxifen.

To further study whether downregulation of erbB3 could sensitize MCF-7 and MCF-7/erbB2 cells to tamoxifen-mediated growth inhibition, both cell proliferation and clonogenic assays were carried out. erbB3 siRNA transfection alone slightly decreased MCF-7/erbB2 cell proliferation but had little effect on the proliferation of MCF-7 cells (Fig. 4b, column 7 vs. 5, 3 vs. 1). In control siRNA transfected MCF-7 cells, tamoxifen clearly inhibited cell proliferation but had no effect on similarly treated MCF-7/erbB2 cells (Fig. 4b, column 2 vs. 1, 6 vs. 5). Importantly, specific erbB3 siRNA transfections significantly enhanced tamoxifen-associated growth inhibition not only in MCF-7, but also in MCF-7/erbB2 cells (Fig. 4b, column 4 vs. 2, 8 vs. 6). These data indicate that erbB2-associated tamoxifen resistance may be overcome by downregulation of erbB3 expression via siRNA transfection. Clonogenic assays showed that erbB3 siRNA transfection alone significantly reduced the colony numbers in both MCF-7 and MCF-7/erbB2 cells (p < 0.05), and the combination of erbB3 siRNA transfection and tamoxifen treatment resulted in remarkable inhibition of colony formation in MCF-7/erbB2 cells (Fig. 4c, p = 0.0038). Downregulation of erbB3 may therefore abrogate erbB2-induced tamoxifen resistance in breast cancer cells.

Specific erbB3 siRNA enhances tamoxifen-induced apoptosis in erbB2-overexpressing breast cancer cells

To explore the molecular mechanisms by which downregulation of erbB3 might sensitize erbB2-overexpressing breast cancer cells to tamoxifen-associated growth inhibition, we investigated whether erbB3 siRNA transfection could enhance tamoxifen-induced apoptosis. Using PARP cleavage as an indicator of apoptosis, treatment with erbB3 siRNA and tamoxifen enhanced apoptosis in both MCF-7 and MCF-7/erbB2 cells as compared to cells transfected with erbB3 siRNA in the absence of tamoxifen (Fig. 5a, lane 4 vs. 3 and 8 vs. 7). We also observed cleaved PARP in control siRNA transfected, tamoxifen-treated MCF-7/erbB2 cells. However, the levels of full-length PARP in erbB3 siRNA transfected, tamoxifen-treated MCF-7/erbB2 cells were much less than control or other groups of MCF-7/erbB2 cells (Fig. 5a, lane 8 vs. 5–7). These data suggest that a major mechanism by which downregulation of erbB3 restores tamoxifen sensitivity involves modulation of apoptosis.

Figure 5.

Specific erbB3 siRNA sensitizes erbB2-overexpressing breast cancer cells to tamoxifen-induced apoptosis. (a) MCF-7 and MCF-7/erbB2 cells were transfected with either control siRNA or specific erbB3 siRNA for 24 hr. The cells were then incubated in phenol-red free medium with 1% charcoal-stripped FBS in the presence or absence of 2.5 μM tamoxifen for another 24 hr. An equal amount of protein for each sample was resolved by 8% SDS-PAGE, followed by western blot analysis with specific antibodies directed against PARP or β-actin. (b) TUNEL assay. Following siRNA transfections, untreated (UT) or tamoxifen-treated (TAM) cells cytospun onto glass slides were subjected to TUNEL reaction as described in the Material and methods. The images of fluorescence (TUNEL) and differential interference contrast (DIC) of identical areas were captured by a confocal microscope. TUNEL-positive indicates the apoptotic cells with DNA strand breaks. Arrows identify identical nuclei in fluorescence (TUNEL) and DIC images. Magnification ×40 in all images. (c) Cells from the same batch of cell lysates as prepared in (a) were subjected to analysis of apoptosis determined using an ELISA that quantified cytoplasmic DNA-histone complexes.

These findings were also confirmed by DNA fragmentation analyses. erbB3 siRNA transfected, tamoxifen-treated MCF-7/erbB2 cells showed significant DNA fragmentation, as measured by a TUNEL assay (Fig. 5b). erbB3 siRNA transfection in the absence of tamoxifen resulted in minimal apoptotic activity in both MCF-7 and MCF-7/erbB2 cells. Likewise, tamoxifen treated, control siRNA transfected MCF-7 cells also showed low level DNA fragmentation that was not seen in similarly treated MCF-7/erbB2 cells (Fig. 5b). These data were further verified using a quantitative apoptotic ELISA assay. Tamoxifen induced the most cell death in MCF-7/erbB2 cells transfected with erbB3 siRNA (Fig. 5c). erbB3 knockdown therefore sensitized the MCF-7/erbB2 cells to tamoxifen-induced apoptosis. In aggregate, these data suggest that erbB3 contributes to erbB2-mediated tamoxifen resistance via effects on cellular proliferation, clonogenecity and apoptosis.

Discussion

Given the complexity of erbB receptor biology, it is possible that each erbB family member makes a unique contribution to cell transformation and tumorigenesis.52, 53 While numerous studies have established an association between the amplification/overexpression of erbB2, breast cancer prognosis and therapeutic response, the role of erbB3 in this process is less well defined. Using both mouse mammary tumor cell lines and human breast cancer cell lines with significant erbB2 expression, we have shown that erbB3 modulates erbB2-mediated proliferation, cloning efficiency and resistance to tamoxifen. Although erbB3 has no intrinsic kinase activity to initiate signaling process,1, 33 ligand bound or even ligand-independent erbB3 may form heterodimers with erbB2 that are potent signaling complexes.34, 35, 37, 38

Our data provide several lines of evidence indicating that the combined effects of erbB2 and erbB3 are dependent upon the presence of both receptors. Mechanistically, their interactions most likely occur via heterodimerization, which may stabilize and lead to activation of erbB2 tyrosine kinase. The kinase activity of erbB2 phosphorylates erbB3 and subsequent activation of downstream signaling (including PI-3K/Akt). The role of erbB2 in phosphorylating erbB3 is indicated by the absence of P-erbB3 in 78717 cells, whose protein levels of erbB2 were undetectable, compared to the levels of P-erbB3 in 85819 cells which express erbB2 and comparable levels of erbB3 (Fig. 1b). This suggests that erbB2 may be the major kinase responsible for erbB3 phosphorylation. We have previously shown that detectable P-erbB3 (as well as P-erbB2) can only be observed in mammary tumor cell lines that coexpress both erbB2 and erbB3 proteins.38 Specific downregulation of erbB3 by siRNA significantly decreased the levels of P-erbB2 in multiple mammary/breast cancer cell lines (Figs. 1b and 1c), suggesting the activation of erbB2 tyrosine kinase may require the presence of erbB3 protein, or alternatively is stabilized by erbB3. Finally, downregulation of erbB3 by specific siRNA disrupts the erbB2/erbB3 heterodimer formation and results in inactivation of PI-3K/Akt downstream signaling. This only occurs in cells which coexpress both erbB2 and erbB3 receptors (Fig. 1). These data were in agreement with a recent report showing that loss of erbB3 had no major effects on P-MAPK but led to a strong reduction in P-Akt in SKBR-3 cells.2 Our data indicating that levels of P-erbB2 are modulated by the levels of erbB3 (Fig. 1) differ from previous studies that showed that blockade of erbB3 expression did not reduce the levels of P-erbB2 in SKBR-3 and MDA-MB-361 cells.2 These differences might be due to the differences in methodologies used to reduce erbB3 expression, since we used an erbB3-specific siRNA while other investigators used an artificial transcription factor (E3).2

De novo (primary) and acquired (secondary) resistance to tamoxifen is a significant problem in the treatment of ER positive breast cancer patients. Some of the mechanisms proposed to explain tamoxifen resistance include direct or indirect links to the increased expression of EGFR and/or erbB2. Studies have also shown that tamoxifen resistance is associated with activation of PI-3K/Akt and/or MEK/MAPK signaling.27, 54, 55, 56 In addition, cross talk between ER, the ER coactivator AIB1, erbB2, p38 (MAPK) and ERK could contribute to tamoxifen resistance.22, 57, 58 Other reported mechanisms that may influence erbB2-associated tamoxifen resistance include mutations of ERα or altered subcellular localization of ERα.59, 60 We attempted to study whether downregulation of erbB3 could change the expression or activity of ERα, and therefore modify tamoxifen sensitivity in breast cancer cells. Our data indicate that downregulation of erbB3 has no apparent effects on the levels or serine 118 phosphorylation (activation) of ERα (Fig. 4a). We believe that the lower levels of P118-ERα observed in MCF-7/erbB2 cells might be due to the lower MAPK activity (unexpectedly) in these erbB2-transfected MCF-7 cells as compared to the parental MCF-7 cells (Yang and Thor, unpublished data). In the tamoxifen-sensitive cell line, P118-ERα level was reduced post tamoxifen treatment in either control siRNA- or erbB3 siRNA-transfected MCF-7 cells. In the tamoxifen-resistant MCF-7/erbB2 cell line, however, tamoxifen treatment increased P118-ERα level only in control siRNA-transfected cells (Fig. 4a). This latter result was in agreement with a recent report showing that tamoxifen induced serine 118 phosphorylation of ERα in BT474 breast cancer cells,51 which are also ERα positive and erbB2-overexpressing cells. Interestingly, knockdown of erbB3 expression abrogated tamoxifen-induced P118-ERα in MCF-7/erbB2 cells (Fig. 4a). We believe that more experiments will be needed to elucidate if this is the molecular mechanism by which downregulation of erbB3 resensitizes MCF-7/erbB2 cells to tamoxifen-mediated growth inhibition/cell death. We also examined the ER coactivator AIB1 in our experiments. Knockdown expression of erbB3 with siRNA transfection had no effect on the levels of AIB1 in both MCF-7 and MCF-7/erbB2 cells (data not shown). Since there is no commercially available antibody against phosphorylated AIB1, we don't know if downregulation of erbB3 affects the phosphorylation status of AIB1. In aggregate, we believe that erbB3 and its cognate ligands may have a profound influence on tamoxifen resistance. If verified, the erbB3 receptor may be a useful molecular target for modulating tamoxifen resistance in ER positive, erbB2 abnormal breast cancer patients.

It is worth noting that in the 78717 cells, which express low levels of erbB2 and thus erbB2 may not drive proliferation of the tumor cells (Fig. 1b), siRNA-mediated downregulation of erbB3 was still able to significantly inhibit cell proliferation and colony formation (Fig. 2). Therefore, it is possible to hypothesize that in the absence of erbB2, erbB3 may interact with other RTKs, such as EGFR, erbB4 or IGF-1R, to promote cell growth and survival through PI-3K/Akt-independent pathways. Nevertheless, the majority of mammary tumors and tumor-derived cell lines from either wt or mutant rat c-neu transgenic mice coexpress both erbB2 and erbB3.38, 43 We and others have observed elevated expression of erbB3 in the mammary tumor cells derived from wt and mutated c-neu/erbB2 transgenic mice,38, 43 suggesting that erbB2 modulates the protein or mRNA levels of erbB3. It has been reported that erbB3 expression can be transcriptionally upregulated by the transcription factor AP-2γ.61 However, in mammary tumors derived from active mutated c-neu transgenic mice, Siegel et al.43 observed only a modest elevation (<3-fold) in erbB3 mRNA in cells that had a 10- to 15-fold increase in erbB3 protein. Our western blots showed increased expression levels of the erbB3 protein in MCF-7/erbB2 cells as compared to parental MCF-7 cells (Fig. 4a), however, semiquantitative RT-PCR analysis revealed that both cells expressed similar erbB3 mRNA levels (data not shown). Thus, we hypothesize that overexpression of erbB2 results in elevated levels of erbB3 protein, either through enhanced protein translation or by stabilizing erbB3 protein. The precise molecular mechanisms that contribute to increased levels of erbB3 protein in erbB2-induced tumor cells require further investigation.

SiRNA only results in a temporary reduction of gene expression,45, 46 however, it is a useful tool to study the biologic effects of target gene knockdown. In our studies using specific erbB3 siRNA, we observed long term effects on growth of tumor cells as measured by clonogenic assays (Figs. 2b, 3b and 4b). There are 2 possible explanations for this effect: first, cells with decreased erbB3 expression may have died, leaving cells with normal erbB3 expression. Alternatively, cells without erbB3 may have a reduced plating efficiency and/or reduced growth potential. Because our data shows apoptotic cells in erbB3 siRNA transfected cells in the absence of tamoxifen treatment (Fig. 5), the first explanation may be most likely. Recent studies of human lung adenocarcinoma cells support this hypothesis, since downregulation of erbB3 using siRNA promoted apoptosis.62

In summary, we have demonstrated that erbB3 plays an important role in the maintenance of erbB2 tyrosine kinase activity and erbB2-associated tumorigenesis in both mouse and human derived mammary/breast cancer cells. Most significantly, erbB3 also contributes to erbB2-associated tamoxifen resistance and reduction of erbB3 levels restores tamoxifen sensitivity. Therefore, we suggest that erbB3 might be a useful molecular target for anti-cancer treatments, particularly in restoring sensitivity to anti-hormone therapy. The addition of anti-erbB3 agents may also be a useful strategy to reduce or overcome secondary tamoxifen resistance in breast cancers.

Acknowledgements

The authors are grateful to Dr. Steve Anderson for his insightful comments and thoughtful editing of this manuscript, and to Ms. Lisa Litzenberger for her excellent assistance in arts preparation.

Ancillary