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Molecular Oncology, Markers, Clinical Correlates |
in Breast Cancer Cells
Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas
ABSTRACT
Purpose: Approximately two-thirds of breast cancer patients respond to endocrine therapy, and this population of patients is estrogen receptor (ER) positive. However, a significant proportion of patients do not respond to hormone therapy. ER hormone responsiveness is widely believed to be influenced by enhanced cross-talk of ER with overexpressed human epidermal growth factor receptor 2 (HER2), and a subgroup of ER-positive tumors coexpress high HER2.
Experimental Design: Breast cancer cells with or without HER2 overexpression were analyzed for ER status, subcellular localization, and interactions with HER2 signaling components by biochemical and immunological methods. Experiments explored the regulatory interactions between the HER2 and ER pathways and the sensitivity of breast cancer cells to tamoxifen.
Results: Stable or transient or natural HER2 overexpression in ER-positive breast cancer cells promoted the nucleus-to-cytoplasm relocalization of ER, enhanced interactions of ER with HER2, inhibited ER transactivation function, and induced resistance to tamoxifen-mediated growth inhibition of breast cancer cells. In addition, HER2 up-regulation resulted in ER interaction with Sos, a component of Ras signaling, and hyperstimulation of the mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 (ERK1/2). Conversely, down-regulation of HER2 by the anti-HER2 monoclonal antibody Herceptin led to suppression of ERK1/2 stimulation, restoration of ER to the nucleus, and potentiation of the growth-inhibitory action of tamoxifen.
Conclusion: The results presented here show for the first time that ER redistribution to the cytoplasm and its interaction with HER2 are important downstream effects of HER2 overexpression, that ERK1/2 is important for ER cytoplasmic localization, and that subcellular localization of ER may play a mechanistic role in determining the responsiveness of breast cancer cells to tamoxifen.
INTRODUCTION
Breast cancer is one of the most common malignancies in the United States, affecting one in nine women during their lifetime. Localized breast cancer, before metastasis, can be cured by surgery. The high mortality rate associated with breast cancer, however, is due to a propensity for the tumor to metastasize while the primary tumor is small and undetected. The process of breast cancer metastasis requires, among other steps, changes in cell signaling pathways, enhanced cell survival, and development of a hormone-independent state. The development of human breast cancer is promoted by estrogen stimulation of mammary epithelial cell growth. The biological effects of estrogen are mediated by its binding to the structurally and functionally distinct estrogen receptors (ER
and ERß). ER (ER) is the major estrogen receptor in the human mammary epithelium (1)
, and targeted disruption of estrogen signaling inhibits the mammary gland development (2
, 3)
. The principal target of estrogen, ER is found in 60–70% of breast tumors at presentation, together with a profile of ER-regulated genes. These tumors are generally responsive to antiestrogen therapy (4)
. Several studies have demonstrated that approximately two-thirds of breast cancer patients respond to antihormone therapy and that tumors in these patients are ER positive; i.e., ER is localized in the cell nucleus. Regardless of the presence of ER, a significant proportion of patients do not respond to antihormone therapy, and most who do initially respond will eventually develop acquired hormone-independent disease (5
, 6)
. At present, it is also not known whether a correlation exists between the between the ER location and tamoxifen sensitivity.
The molecular mechanisms leading to breast cancer progression and advanced malignancy are not completely understood at present but are widely believed to involve intracellular growth-factor-triggered signaling cascades. Studies have established that the human epidermal growth factor receptor (HER) family of receptor tyrosine kinases and their ligands have an essential role in the regulation of proliferation of mammary epithelial cells and in the pathogenesis of breast cancer (7 , 8) . For example, overexpression of the HER2 receptor gene product is frequently associated with a decrease in disease-free survival, poor prognosis, increased metastasis, and an aggressive clinical course of human breast cancer (9, 10, 11, 12, 13) . Among the various factors that could participate in the development of anti-hormone-resistant breast tumors, deregulation of HER2 expression/signaling has emerged as one of the most recognizable molecular dysfunctions in breast tumors. A variety of in vitro and in vivo models have shown that suppression of HER2 enhances the antiproliferative effects of tamoxifen (14, 15, 16, 17, 18) , thus demonstrating a causal association between HER2 overexpression and acquisition of resistance to tamoxifen. Although a subgroup of ER-positive tumors coexpress high HER2 levels, the mechanism by which HER2 influences the ER pathway in breast cancer cells remains poorly understood.
It is generally accepted that estrogen activates components of growth factor signaling pathways, i.e., extracellular signal-regulated kinase 1/2 (ERK1/2) and Shc/Grb2/Sos, in ER-positive breast cancer cells (19, 20, 21, 22) . These findings are significant because ERK1/2 activity has been shown to be frequently up-regulated in human breast tumors. Furthermore, the ERK1/2 pathway has been shown to regulate the expression of genes with roles in the invasiveness of breast cancer cells (23, 24, 25, 26) . In this context, recent studies have demonstrated the presence of hyperstimulation of ERK1/2 in HER2-overexpressing, tamoxifen-resistant MCF-7 breast cancer cells and that inhibition of ERK1/2 enhanced tamoxifen-mediated growth inhibition (18) . ER signaling has also been shown to regulate cell growth, presumably as a result of cross-talk between growth factors and ER. For example, HER2 promotes the development of the hormone-independent state and a more aggressive phenotype in breast cancer cells (14, 15, 16, 17, 18) . Estrogen stimulation of growth by ERK1/2 can also occur via a nongenomic action of ER because estrogen-induced ERK1/2 activation and DNA synthesis can be effectively blocked by the mitogen-activated protein kinase kinase (MEK1) inhibitor PD 98059 (19 , 22) . In brief, enhanced cross-talk of ER with growth factor signaling components may be involved in the development of a hormone-independent phenotypes in breast cancer cells. This study was undertaken to further explore the regulatory interactions between the HER2 and ER pathways in breast cancer cells.
MATERIALS AND METHODS
Cell Cultures and Reagents.
MCF-7, MCF-7/HER2 (14
, 27
, 28)
, and SKBR3 (28)
human breast cancer cells (23)
were maintained in DMEM-F12 (1:1) supplemented with 10% FCS. The following antibodies were used: (a) anti-ER and anti-Grb2 (Upstate Biotechnology, Lake Placid, NY); (b) anti-HER2, anti-Sos, and anti-ERK1/2 antibodies (Neomarkers, Fremont, CA); (c) anti-phospho-ERK1/2 (Thr202/Tyr204, P-ERK1/2) and anti-phospho-ER (Ser118; Transduction Laboratories, Franklin Lakes, NJ); (d) antiactin and antivinculin (Sigma Chemical Co., St. Louis, MO); and (e) antimouse and antirabbit horseradish peroxidase-conjugated secondary antibodies (Amersham, Piscataway, NJ). The following reagents were also used: (a) anti-HER2 monoclonal antibody Herceptin (Genentech, San Francisco, CA); (b) the pure antiestrogen ICI 182780 (Tocris, Ellisville, MO); (c) the steroid hormone 17ß-estradiol (E2) and charcoal-stripped serum (DCC serum; Sigma).
Reverse Transcription-PCR.
Isolated RNA was reverse-transcribed and amplified with the ONE-STEP RT-PCR System (Promega). The primer sequences used were as follows: PS2 forward (5'-ATA CCA TCG ACG TCC CTC CA-3') and PS2 reverse (5'-CAC CTC AGA CAC GCT T-3'), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward (5'-CCA TCT TCC AGG AGC GAG ATC-3') and GAPDH reverse (5'-CGT TCA GCT CAG GGA TGA CC-3'). Amplification was conducted in 20-µl reactions, each containing 50 ng of total RNA, 0.2 mM deoxynucleotide triphosphates, 1 mM MgCl2, 1 µM each primer, 0.5 units of AMV, 0.5 units of Tfl DNA polymerase, and reaction buffer. The cycling conditions included an initial incubation at 48°C for 45 min and a second incubation at 94°C for 2 min, followed by 35 cycles of 30 s at 94°C, 30 s at 60°C, and 1 min at 68°C. The final extension was for 7 min at 68°C. The reverse transcription-PCR products were 156 bp for PS2 and 451 bp for GAPDH. Reverse transcription-PCR products were separated on 2% agarose gels.
Metabolic Labeling of Cells.
Cells (3 x 105) were plated in F-12/DMEM in each well of a 6-well dish. After 24 h, cultures were washed with phosphate-free medium and incubated for up to 15 h in phosphate-free F-12/DMEM containing 0.4 mCi/ml 32P in the presence or absence of 100 nM Herceptin. After 16 h, cells were washed three times with PBS and then lysed in 400 µl of RIPA buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, 0.1% SDS, 0.1% sodium deoxycholate, 1x protease inhibitor cocktail (Roche Biochemical), and 1 mM sodium vanadate] for 15 min on ice. The lysates were centrifuged at 12,000 rpm in an Eppendorf microfuge for 10 min. For labeling with [35S]methionine, the cells were washed with methionine-free medium and incubated for up to 16 h with methionine-free F-12/DMEM containing 0.15 mCi/ml 35S with or without 100 µM Herceptin. Lysates containing equal amounts of perceptible trichloroacetic acid counts were immunoprecipitated with the anti-HER2 or anti-ER monoclonal antibodies, resolved on 10% SDS-polyacrylamide gels, and analyzed by autoradiography.
Cell Extracts, Immunoblotting, and Immunoprecipitation.
To prepare cell extracts, cells were washed three times with PBS and then lysed in RIPA buffer for 15 min on ice. The lysates were centrifuged in an Eppendorf centrifuge at 4°C for 15 min. Cell lysates containing 200 µg of protein were resolved on SDS-polyacrylamide gels (8% acrylamide), transferred to nitrocellulose membranes, probed with the appropriate antibodies, and developed by either the enhanced chemiluminescence method or the alkaline phosphatase-based color reaction method. Immunoprecipitation was performed for 2 h at 4°C with 1 µg antibody/mg of protein (28)
.
Reporter Gene Assays.
For reporter gene transient transfections, MCF-7 and MCF-7/HER2 cells were cultured in 6-well plates (105 cells/well) in MEM, without phenol red, containing 10% DCC serum for 48 h and then transfected with 100 ng of estrogen response element-luciferase reporter plasmid per well using Fugene-6 reagent according with the manufacturer’s instructions (Roche Molecular Biochemicals). After 24 h, cells were treated with or without 10–9 M estrogen for 16 h. Cells were then lysed with passive lysis buffer, and the luciferase assay was performed with a luciferase reporter assay kit (Promega, Madison, WI); luciferase activity was measured with a Lumant Luminometer. The activity of ß-galactosidase reporter was used to correct the transfection efficiencies. Each transfection was performed in triplicate wells (28)
. Each experiment was repeated three to six times, and transfection efficiency varied between 30 and 50%.
Northern Blot Hybridization.
Total cytoplasmic RNA was isolated with use of Trizol reagent, and 20 µg of RNA was analyzed by Northern hybridization using a 496-nucleotide fragment of the PS2 full-length cDNA. rRNA (28S and 18S) was used to assess the integrity of the RNA, and the blot was routinely reprobed with human GAPDH cDNA for RNA loading and transfer control as described previously (28)
.
Transient Transfection with Human HER2.
The breast cancer cell lines MCF-7 and ZR-75 growing on glass coverslips in DMEM-F12 (1:1) supplemented with 10% FCS were transfected with human HER2 plasmid (kindly provided by Dr. Mien-Chie Hung; University of Texas M. D. Anderson Cancer Center, Houston, TX) using Fugene-6 reagent according with the manufacturer’s instructions (Roche Molecular Biochemicals). Thirty-six h after transfection, cells were fixed with methanol at –20°C for 6 min and then prepared for confocal study as described below.
Immunofluorescence and Confocal Studies.
Cellular localization of different proteins was determined by indirect immunofluorescence as described previously (29
, 30)
. Briefly, cells grown on glass coverslips were fixed in either ice-cold methanol for 6 min or 4% paraformaldehyde in PBS at room temperature for 15 min. Cells were incubated with the respective primary antibodies for 2 h at room temperature, washed three times in PBS, and then incubated with 546-Alexa- (red) or 488-Alexa-labeled (green) secondary antibodies (Molecular Probes, Eugene, OR). We used 488-Alexa-phalloidin to localize F-actin. The DNA dye Topro-3 (Molecular Probes), used to costain the DNA, gives an emission in the far-red segment of the light spectrum and was color-coded in blue. For controls, cells were treated only with the secondary antibodies or pretreated with the peptide against which the antibodies had been raised. Confocal analysis was performed with a Zeiss laser-scanning confocal microscope with established methods (29)
. Each image represents Z-sections at the same cellular level and magnification. Colocalization of two proteins as a result of red and green overlapping pixels is indicated by a yellow color.
Cell Growth Assay.
MCF-7/HER2 cells were plated in 24-well plates maintained in DMEM-F12 (1:1) supplemented with 10% FCS. After an overnight incubation, cells were treated with various concentrations of the antiestrogen Tamoxifen (Sigma), the anti-HER2 antibody Herceptin, or both agents. Cells were allowed to grow at 37°C in an atmosphere of 5% CO2. After 4 days, the medium was removed, and 1 ml of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Thiazolyl blue; Sigma) was added. After a 1-h incubation at 37°C in a 5% CO2 atmosphere, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was removed, and isopropanol-1 N HCl (96:4 v/v) was added. The absorbance at 570 nm was measured on a Bio-tek Instruments (Winooski, VT) automated microtiter plate reader. Identical concentrations and combinations were tested in four separate wells/assay, and the assay was performed four times (16)
.
RESULTS AND DISCUSSION
Evidence of Cytoplasmic Localization of ER in HER2-Overexpressing Cells.
It is increasingly accepted that a causal association exists between HER2 overexpression and acquisition of resistance to tamoxifen, both in human breast cancer cell lines (14
, 16
, 18) and in clinical studies of patients with ER-positive, hormone-dependent tumors (9, 10, 11, 12, 13)
. However, there are few molecular explanations for this well-recognized cellular phenomenon in HER2-overexpressing ER-positive breast cancer cells. To explore the potential effect of HER2 deregulation on the ER pathway in breast cancer cells, we used MCF-7 and a well-characterized clone of MCF-7/HER2 breast cancer cells with aggressive phenotypes (14
, 18
, 27
, 28
, 30)
. HER2 overexpression in MCF-7 cells (Fig. 1A)
was accompanied by a significant inhibition of both baseline and E2-induced stimulation of ER transactivation function as assessed in an estrogen response element-luciferase reporter system (Fig. 1B)
and by suppression of ER target gene PS2 mRNA (Fig. 1C)
, These results suggested that HER2 overexpression antagonizes genomic responses to ER transactivation in breast cancer cells. Consistent with these results, we also found that estrogen induces expression of endogenous ER target PS2 mRNA in a tamoxifen-sensitive manner in MCF-7 but not in MCF-7/HER2 cells (Fig. 1D)
. In addition, tamoxifen was able to inhibit the basal levels of PS2 in exponentially growing MCF-7 cells (Fig. 1E)
. Because subcellular localization of several other proteins (i.e., FKHR and 14-1-1) has been shown to affect their nuclear functions and because there was no effect of HER2 on the steady-state level of ER, we next explored whether HER2 overexpression alters ER subcellular localization. Results from confocal scanning microscopy indicated that HER2 deregulation was accompanied by the presence of ER in the cytoplasmic compartment with a concurrent reduction in the level of nuclear ER (Fig. 1F)
, suggesting that exclusion of ER from the nucleus may well act to deprive the antiestrogenic agents of their target in the proper cellular compartment. Estrogen treatment was able to translocate ER to the nucleus in MCF-7 but not in MCF-7/HER2 cells (Fig. 1F)
. This potential mechanism may be related to the reduced tamoxifen response in HER2-overexpressing breast tumors. To evaluate whether the observed phenomenon is restricted to the MCF-7/HER2 clone or whether it could be demonstrated in other ER-positive breast cancer cells, we transiently transfected HER2 into ER-positive MCF-7 and ZR-75 breast cancer cells. Our results demonstrated that HER2 overexpression did indeed promote ER translocation and colocalization with HER2 in the cytoplasm (Fig. 1G)
. Because MCF-7/HER2 cells have the same expression of ER as MCF-7 cells (Fig. 1A)
, there was no apparent effect of the altered ER localization on its steady-state level. A recent study showed that peroxisome proliferator-activated receptor-
agonists induce proteasome-dependent degradation of ER (31)
. However, HER2 overexpression antagonizes the sensitivity of peroxisome proliferator-activated receptor- to its ligand (32)
, which may explain, at least in part, why cytoplasmic ER in MCF-7/HER2 cells is not subject to degradation.
|
or by confocal microscopy (Fig. 2B)
. The ERK1/2 pathway was rapidly stimulated in MCF-7 cells by 10–9 M E2, and maximum activation was attained by 5 min after E2 stimulation (Fig. 2C)
. A comparative analysis of E2 stimulation for 5 min indicated that ERK1/2 phosphorylation was further induced over already elevated baseline ERK1/2 levels in MCF-7/HER2 cells (Fig. 2D)
. The specificity of E2 stimulation of ERK1/2 was confirmed by pretreating the cells with 10–8 M of the pure antiestrogen ICI 182780 for 30 min (Fig. 2D)
. ICI 182780 destroys the ER and decreases ERK1/2. Of interest, the ERK1/2 inhibitor PD 98059 was able to partially restore nuclear ER in HER2-overexpressing MCF-7 cells, implying a functional role for ERK1/2 signaling in ER cytoplasmic localization (Fig. 2E)
. Overall, these findings support the notion of a close relationship between ERK1/2 hyperstimulation and HER2 overexpression in breast cancer cells.
|
. Of interest, HER2 and Sos proteins were selectively associated with ER in MCF-7/HER2, whereas there was no change in the level of ER-associated Grb2 (Fig. 3A
, upper panel). We further validated the observed ER-HER2 interaction in MCF-7/HER2 cells by confocal scanning microscopy (Fig. 3B)
. We next extended these observations to ER-positive BT-474 breast cancer cells, which naturally express high HER2 levels. Our results showed that ER was also localized predominantly in the cytoplasm and colocalized with HER2 on the cellular membrane in BT474 cells (Fig. 3C)
and raised the possibility of a causal role of HER2 signaling in ER translocation.
|
. E2-enhanced ER-HER2 interaction was sensitive to the pure antiestrogen ICI 182780, which effectively blocked ER phosphorylation on Ser118 (Fig. 3D
, lower panel). Because ICI 182780 effectively inhibited E2-mediated stimulation of mitogen-activated protein kinase (Fig. 2D)
, ER activation, and ER-HER2 interactions (Fig. 3D)
, these findings suggested an apparent role for mitogen-activated protein kinase stimulation in the observed ER-HER2 interactions in MCF-7/HER2 cells.
HER2 Down-Regulation Promotes Nuclear Localization of ER.
Together, the results illustrated in Figs. 1
2
3
suggested that HER2 overexpression leads to cytoplasmic localization of ER and its interaction with HER2. To further establish a role for HER2 in the observed cytoplasmic localization of ER in MCF-7/HER2 cells, we next examined the effect of HER2 down-regulation by the anti-HER2 monoclonal antibody Herceptin on the subcellular localization of ER in MCF-7/HER2 cells. As expected, treatment of cells with 100 nM Herceptin for 16 h down-regulated the amount of phosphorylated HER2 bound by ER in MCF-7/HER2 cells metabolically labeled with 32P (Fig. 4A)
and, hence, down-regulated the level of [35S]methionine-labeled HER2 in MCF-7/HER2 cells (Fig. 4B)
. Herceptin treatment also reduced the level of activated ERK1/2 in MCF-7/HER2 cells (Fig. 4C)
. Importantly, Herceptin treatment restored the primary localization of nuclear ER in a significant proportion of MCF-7/HER2 cells (93% of Herceptin-treated cells compared with 12% in untreated cells; Fig. 4D
). These results raised the possibility that Herceptin treatment and restoration of nuclear ER may promote the growth-inhibitory action of tamoxifen in MCF-7/HER2 cells, which were previously reported to be tamoxifen resistant (14
, 16
, 18)
. Indeed, the combination of tamoxifen with Herceptin provided a beneficial growth-inhibitory action compared with treatment with individual agents alone (Fig. 4E)
. Because inhibition of ERK1/2 has also been shown to augment the action of tamoxifen (18)
and because Herceptin treatment was accompanied by inhibition of ERK1/2 as well as nuclear localization of ER (this study), these findings suggested that Herceptin-mediated down-regulation of ERK1/2 may be associated with increased sensitivity to tamoxifen, presumably because of nuclear localization of ER. These observations may provide a potential rationale for ongoing and planned clinical studies combining Herceptin and hormone-based therapies (34)
. In brief, these findings suggest that HER2 down-regulation by Herceptin might promote the action of tamoxifen by inhibiting the ER-HER2 interaction and/or ERK1/2 activation. These findings are consistent with a mechanistic role for the subcellular localization of ER in the determination of the responsiveness of breast cancer cells to tamoxifen.
|
ACKNOWLEDGMENTS
We thank Mahitosh Mandal for the PS2 Northern blot and assisting in the initial part of this study.
FOOTNOTES
Grant support: NIH Grant CA65746 and CA098823.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Rakesh Kumar, Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center-108, 1515 Holcombe Boulevard, Houston, TX 77030. Fax: (713) 745-3792; E-mail: rkumar{at}mdanderson.org
Received 7/23/03; revised 2/25/04; accepted 3/ 1/04.
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), Herceptin alone (
), or the combination of the two agents (•) on the proliferation of MCF-7/HER2 cells as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay at 570 nm. Identical concentrations and combinations were tested in four separate wells per assay in three independent experiments. Representative results are shown. Bars, SE.