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Ets-1 upregulates HER2-induced MMP-1 expression in breast cancer cells
Biochemical and Biophysical Research Communications 377 (2008) 389–394
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Biochemical and Biophysical Research Communications j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y b b r c
Ets-1 upregulates HER2-induced MMP-1 expression in breast cancer cells Yeon Hee Park, Hae Hyun Jung, Jin Seok Ahn, Young-Hyuck Im * Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-Dong, Gangnam-Gu, Seoul, Republic of Korea Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-Dong, Gangnam-Gu, Seoul, Republic of Korea
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Article history: Received 25 September 2008 Available online 11 October 2008 Keywords: Ets-1 HER2 MMP-1 Breast cancer
a b s t r a c t The human epidermal growth factor receptor-2 (HER2) plays an important role in breast cancer. Enhanced Ets-1 activity has recently been shown to be associated with breast cancer pathog enesis. To test the role of Ets-1 in breast cancer cells in relation to the expression of HER2 and MMP-1, we transiently overex pressed Ets-1 and/or HER2 in MCF-7 breast cancer cells and comprehensively searched for genes related to HER2 and Ets-1 using cDNA microarray analysis. The expression of matrix metalloproteinase (MMP) genes was enhanced by the overexpression of HER2/Ets-1. We analyzed the relationship between HER2induced MMP-1 expression and the transcription factor Ets-1, which has significant activity in breast can cer pathog enes is. Our results demonstrate that HER2-induced MMP-1 expression is positively regulated by Ets-1 in breast cancer cells. This study confirms that Ets-1 is a downstream effector of oncogenic HER2, associated with MMP-1. © 2008 Elsevier Inc. All rights reserved.
The Ets proteins are expressed in both primary human breast cancers and breast cancer cell lines, and their expression has been associated with disease progression and metastasis [1,2]. Ets-1 is an independent prognostic marker for relapse-free survival in breast cancer, and has not been linked to other prognostic factors, such as nodal status, tumor size, histological grade, or estrogen receptor status [1]. The degree of Ets-1 expression correlates with the extent of breast carcinoma invasion, and the atypism of the carcinoma correlates significantly with Ets-1 expression [3]. Ets-1 has also been shown to be correlated positively with HER2 expres sion, which is a representative prognostic and predictive marker of breast cancer [4]. Several other functional roles of Ets-1 in tumor igenesis of breast cancer have also been postulated. Ets-1 regu lates the expression of genes encoding enzymes involved in the degradation of the extracellular matrix, such as MMP-1, MMP-3, MMP-7, and MMP-9 [5]. MMP-1 and MMP-9 are involved in the 70 genes that identify the “gene-expression signature” which predicts distant metastasis in lymph-node-negat ive breast cancer patients [6,7]. MMPs have been implicated in normal matrix remodeling events and pathological conditions, including tumor invasion and metastasis [7]. Abnormalities in growth factor signaling path ways play an intrinsic role in disease progression. In humans, the growth factor receptor HER2 is overexpressed in 20–30% of breast cancers and overexpression of HER2 is associated with enhanced tumorigenicity and propensity to metastasize and resistance to * Corresponding author. Address: Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medi cine, 50 Irwon-Dong, Gangnam-Gu, Seoul, Republic of Korea. Fax: +82 2 3410 1757. E-mail address: [email protected] (Y.-H. Im). 0006-291X/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.09.135
endocrine therapy [8,9]. The targeting of HER2 as a therapeutic strategy for patients with HER2 overexpressing breast cancer has already shown survival advantage when combined with many che motherapeutic agents [10]. Assuming that the action of HER2 is related to Ets-1, we transiently overexpressed Ets-1 and/or HER2 in MCF-7 breast cancer cells and comprehensively searched for genes related to HER2 and Ets-1 using cDNA microarray analysis. The expression of metalloproteinase (MMP) genes was enhanced by the overexpression of HER2/Ets-1. Our cDNA microarray analy sis showed that MMP genes are associated with the action of HER2 and Ets-1. We wondered whether Ets-1 could thus potentially function as a downstream effector of oncogenic HER2 in associa tion with MMP-1. Based on this knowledge, we were interested in the relationships between MMP-1, HER2, and Ets-1 in breast can cer. We hypothes ized that HER2 induces MMP-1 expression and Ets-1 cooperates with this function. Here, we performed reverse transcription-polymerase chain reaction (RT-PCR), western blot analysis, an MMP-1 enzyme-linked immunosorbent assay (ELISA), and an MMP-1 promoter assay after transfecting the MCF-7 human breast cancer cell line with HER2. We then investigated how Ets-1 influences the MMP-1 expression induced by HER2. Materials and methods Antibodies and reagents. Anti-Ets-1 and anti-HER2 were pur chased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-bactin was purchased from Sigma (Salt Lake City, UT). Anti-MMP-1 was purchased from Calbiochem (Cambridge, MA). Trastuzumab (Herceptin®) and Erlotinib (Tarceva®) were provided by Roche
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Korea. MMP ELISA kits were obtained from R&D Systems. [c-32P] ATP was purchased from NEN Life Science Products (Boston, MA). Tumor cell lines and culture. The human breast cancer cell line MCF-7 was cultured and maintained in RPMI 1640 medium (Bio Whittaker, Walkersville, MD) supplemented with 10% fetal bovine serum, penicillin, and streptomycin. Transient and stable transfection of MCF-7 cells with HER2 and Ets-1. An expression vector for FLAG-tagged Ets-1 constructs was generated by subcloning PCR-amplified cDNA into the G418-resis tance plasmid pcDNA3.1. HER2/pCMV6–XL4 constructs were pur chased from Origene. The HER2/pCMV6–XL4 plasmid was digested with the restriction enzyme Not1 to release the inserted fragment. The fragment was re-subcloned into the G418-resistance plasmid pcDNA3.1 (Invitrogen) at the Not1 site. MCF-7 cells were tran siently transfected using Effectene Transfection Reagent (Qiagen Inc., Valencia, CA). MCF-7 cells were stably transfected with either the HER2/pcDNA3.1 or pcDNA3.1 vector in the presence of Effec tene Transfection Reagent for 48 h and were treated with 500 lg/ mL G418. RNA isolation and microarray analysis. The enriched plasma cells were stored in RNAlater (Ambion) for RNA extraction and gene chip experiments. RNA was isolated with an RNeasy Mini Kit (Qiagen, Valencia, CA) and the RNeasy Mini Kit (Qiagen, Valencia, CA). After cleanup with a Sample Cleanup Module (Affymetrix), the doublestranded cDNA was used for in vitro transcription. After cleanup with a Sample Cleanup Module (Affymetrix), 10–15 lg of labeled cRNA was fragmented into 35–200 bp lengths in fragmentation buffer (Affymetrix). The fragmented cRNA was hybridized to the Human Genome U133A 2.0 gene chip (Affymetrix) at 45 °C for 16 h. After hybridization, the arrays were washed in a GeneChip Fluid ics Station 450 with a nonstringent wash buffer at 25 °C, followed
by a stringent wash buffer at 50 °C. After washing, the arrays were stained with a streptavidin–phycoeryt hrin complex. After staining, the intensities were determined with a GeneChip Scanner 3000 (Affymetrix). Data analysis. Expression profiles were analyzed using the GeneChip Operating Software (Affymetrix). Transfection of Stealth RNAi. Ets-1 Validated Stealth™ RNAi DuoPak and ERBB2 Stealth™ Select 3 RNAi were purchased from Invitrogen. MCF-7 cells were transiently transfected using Lipo fectamine™ RNAiMAX (Invitrogen). Preparation of promoter–reporter constructs. A promoter frag ment of the human MMP-1 gene, from ¡4334 to +5218 relative to the transcription start point, was amplified from human DNA by PCR, using a pair of primers (59-agatgtaagagctgggaaaggacgg-39/59tcagtgcaaggtaagtgatggcttc-39) and Accuprime Pfx DNA polymerase (Invitrogen). The fragment was subcloned into the pCR–XL–TOPO vector (Invitrogen) for propagation in bacteria. The isolated plas mid was digested with the restriction enzymes MluI and XhoI to release the inserted fragment. The fragment was re-subcloned in the sense orientation into the promoter-free pGL3-Basic vector (Promega) at the MluI and XhoI sites in the 59 flanking region of the luciferase sequence. Isolation of RNA and RT-PCR. Total cellular RNA was isolated using TRIzol™ Reagent (Gibco BRL, Carlsbad, CA). For RT-PCR, 2 lg of RNA was treated with RNase-free DNase, and cDNA was obtained using the SuperScript™ III First-Strand Synthesis System for RT-PCR. The cDNA was amplified by PCR. The primers used in this analysis are shown in Supplemental Table 1. Western blot analysis. Cells were extracted with RIPA buffer. The samples were placed on ice for 20 min with occasional vortexing. Protein concentrations were determined using the BCA Protein
Fig. 1. HER2 overexpression increases MMP-1 expression in MCF-7 cells. (A) MCF-7 cells were transfected with pcDNA3.1(+) or HER2/pcDNA3.1(+) for 24 h. The mRNA expres sion of HER2 and MMP-1 analyzed by RT-PCR. (B) At 48 h post-transfection, the conditioned medium was subjected to ELISA to quantify secreted MMP-1. Data were shown as means ± SD from three separate experiments. *P < 0.05 compared with the Vector group. (C) MCF-7 cells were co-transfected with MMP-1 promoter-reporter construct together with empty expression vector or with expression plasmid for HER2. The cellular luciferase activity was measured. Data were shown as means ± SD from three sepa rate experim ents. ***P < 0.0005 compared with the control group. (D) MCF-7 cells were stably transfected with HER2 expression vector. The expression of HER2 was analyzed by immunoblotting. b-Actin was used as a loading control. (E) MMP-1 mRNA expression was increased in MCF-7/HER2 clones. †The molecular sizes of different bands: b-actin 331 bp; HER2 281 bp; MMP-1 444 bp.
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Assay Kit (Pierce). Equal amounts of sample were subjected to SDS–PAGE. Membranes were incubated for 2 h then, incubated with primary antibody for 2 h. After several washes in PBS and incubation with horseradish-peroxidase-conjugated secondary antibodies (Zymed), bound primary antibodies were detected with ECL chemiluminescent reagents (Amersham). ELISA of secreted MMP-1. MMP-1 secreted from MCF-7 cells was quantified using the Human Pro-MMP-1 ELISA kit (R&D Systems). The absorbance at 450 nm was measured with a spectrophotomet ric plate reader. Luciferase assays. MCF-7 cells were transiently transfected with the reporter constructs using Effectene Transfection Reagent. After transfection, whole-cell lysates were prepared and luciferase activ ity was measured using a luciferase activity assay kit (Promega). Preparation of nuclear protein extracts. The washed cells were scraped from the culture plates and collected by centrifugation. The pellets were resuspended in 0.4 mL of hypotonic lysis buffer. After 15 min on ice, Nonidet P-40 was added to a final concentra tion of 0.5%, and the cells were vortexed for 10 s and centrifuged (30 s, 4 °C, 12,000g). The supernatant was frozen immediately and the nuclear pellets were resuspended in 130 lL of buffer and vor texed for 30 min at 4 °C. After centrifugation (5 min, 4 °C, 12,000g), the supernatant was frozen at ¡70 °C. Immunoprecipit ation assays. At 24 h after incubation on 60-mm Petri dishes, cells were extracted with lysis buffer. After the lysates had been precleared with Protein-A/G PLUS-Agarose, 2 lg HER2 monoclonal antibodies (Calbiochem) or EGFR (biosource) and Protein-A/G PLUS-Agarose were added and incubated overnight at 4 °C on a rotating wheel. Immunocomplexes were collected by centrifugation, washed five times with lysis buffer, separated by SDS–PAGE.
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Statistical analysis. Student’s T-test was done to determine sta tistical significance for ELISA and luciferase assay. Results cDNA microarray revealed that MMP genes are related to the action of HER2 and Ets-1 To identify genes biologically related to HER2 and Ets-1 activity in breast cancer cells, we analyzed the expression profiles of cells after transfection with HER2 and/or Ets-1 in MCF breast cancer cell using the cDNA microarray method. The expression profiles showed high expression of MMP’s and collagenase genes in the cell transfected with HER2 and Ets-1, suggesting that these genes were closely associated with the interaction of HER2 and Ets-1 (Supple mental Table 2). Induction by HER2 of MMP-1 expression and promoter activity To determine whether HER2 is involved in the regulation of MMP-1 expression and promoter activity, HER2-transfected MCF-7 breast cancer cells were incubated, and RT-PCR with MMP-1 spe cific primers and Western immunoblotting were performed (Fig. 1A). Transient overexpression of HER2 gene led to an enhancement of MMP-1 mRNA expression levels. We also quantified the MMP-1 secreted from MCF-7 cells using an ELISA, and the MMP-1 pro moter activity was analyzed by measuring the luciferase activity (Fig. 1B and C). Fig. 1A shows that the expression of MMP-1 mRNA was upregulated after transfection of cells with HER2. We used western immunoblotting analysis to determine if the observed increase in MMP-1 mRNA transcription correlated with MMP-1
Fig. 2. Down-regulation of HER2 inhibits HER2-induced MMP-1 expression. (A) To knock down HER2 expression in MCF-7/HER2-14 cells, the cells were transfected with control siRNA oligo or three different siRNA oligos against HER2. RNA was collected 48 h after transfection. The mRNA expression of HER2 and MMP-1 were analyzed by RTPCR. (B) ELISA was performed to assess the effects of different kinds of HER2 siRNA on MMP-1 expression. (1 lane, MCF-7/Vector; 2–9 lane, MCF-7/HER2-14; NC, negative control siRNA). *P < 0.05 compared with the MCF-7HER2-14 group; **P < 0.01 compared with the MCF-7HER2-14 group. (C) MCF-7/HER2-14 cells were incubated in medium supplemented with Trastuzumab at 10 lg/ml for 48 h. Cell lysates were prepared and immunoblotted with HER2 and b-actin. b-Actin was used as a loading control. MMP-1 expression was analyzed by RT-PCR. (D) The conditioned medium was subjected to ELISA to quantify secreted MMP-1. §P < 0.005 compared with the MCF-7/HER2-14 control group. The experiment was replicated three times.
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protein expression. Fig. 1B shows the strongly augmented expres sion of MMP-1 protein relative to that before the transfection of cells with HER2. Fig. 1C shows enhanced MMP-1 promoter activity by HER2. Fig. 1D shows the expression of HER2 by Western blot ting. MMP-2 mRNA expression was increased in several MCF-7/ HER2 clones (Fig. 1E).
Actin expression was unaffected by either control or HER2 siRNA treatment, indicating that non-specific down-regulation did not occur. This result was quantified by ELISA (Fig. 2B). Trastuzumab, HER2-targeting agent, inhibits HER2-induced MMP-1 expression (Fig. 2C and D). This finding confirmed MMP-1 upregulation by HER2.
Stable transfection of HER2 into the MCF-7 breast cancer cell line
EGFR tyrosine kinase inhibit or inhibits (TKI) ligand-induced phosphorylation of EGFR, HER2 and HER2/EGFR and MMP-1
To test the efficiency of HER2 transfection into MCF-7 cells, we compared the HER2 expression profile with that of MCF-7 cells transfected with the G418-resistance plasmid pcDNA3.1. We also compared it with that of cells transfected with HER2 Stealth RNAi (50 mg or 100 mg). HER2 mRNA expression was markedly increased in MCF-7 cells after transfection with HER2 when analyzed by RTPCR. The ability of small interfering RNA (siRNA) of HER2 to sup press HER2 expression was confirmed by RT-PCR. We determined the effect of HER2 siRNA at different doses on HER2 expression in MCF-7 cells, which indicated that HER2 expression was blocked by HER2 siRNA treatment (Fig. 2A). Down-regulation of HER2 inhibits HER2-induced MMP-1 expression siRNA to HER2 provide further evidence for the positive regu latory role of HER2 in the regulation of MMP-1 (Fig. 2A). The abil ity of HER2 siRNA to suppress MMP-1 expression was performed by RT-PCR. RNA was collected 48 h after transfection. The mRNA expression of HER2 and MMP-1 were down-regulated in accor dance with increasing HER2-specific siRNA by three kinds of siRNA.
We investigated whether erlotinib, EGFR TKI, inhibits HER2induced MMP-1 overexpression. The phosphorylation of EGFR and HER2 in MCF-7/HER2 was analyzed by immunoblotting (Fig. 3A). Immunoprecipitation in Fig. 3B indicated that HER2 has increased associated with EGFR in MCF-7/HER2-14. To examine the inhibitory effect of erlotinib on phosphorylation of EGFR and HER2 in MCF-7/ HER2 cells, MCF-7/HER2-14 cells were treated with erlotinib with indicated concentrations for 24 h. followed by immunoblotting for the indicated proteins. As shown in Fig. 3C, HER2 expression was decreased in a concentration-dependent manner. The mRNA expression of MMP-1 was decreased after erlotinib treatment in concentration-dependent manner by RT-PCR (Fig. 3D). Ets-1 enhances HER2-induced MMP-1 expression We investigated whether MMP-1, which was activated with HER2, is affected by Ets-1 treatment in MCF-7 cells. After treat ment with Ets-1, MMP-1 mRNA expression was enhanced in estab lished a stable MCF-7 cell line transfected with either Ets-1 or
Fig. 3. Erlotinib inhibits HER2-induced MMP-1 expression. (A) Expression levels of EGFR and HER2 phosphorylation in MCF-7/HER2 clones. Cell lysates were prepared and analyzed by immunoblotting using the indicated antibodies. b-Actin was used as a loading control. (B) HER2 has increased association with EGFR in MCF-7/HER2-14. MCF-7/Vector and MCF-7/HER2-14 cells were lysed in RIPA buffer. HER2 or EGFR in 0.5 mg cell lysate was immunoprecipitated. The immunoprecipitates were fractionated on SDS–PAGE followed by immunoblotting for the indicated proteins. (C) Erlotinib inhibits EGFR and HER2 phosphorylation in MCF-7/HER2-14. MCF-7/HER2-14 cells were treated with erlotinib for 24 h. Cell lysates were analyzed by immunoblotting. b-Actin was used as a loading control. (D) The mRNA expression of MMP-1 and HER2 analyzed by RT-PCR. The experiment was replicated three times.
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Fig. 4. The effect of Ets-1 on HER2-induced MMP-1 expression. (A) HER2 and Ets-1 enhance expression of the MMP-1. MCF-7 cells were transiently transfected with expres sion plasmids for HER2 (2 lg) or Ets-1 (2 lg). (B) MCF-7 cells were co-transfected with MMP-1 promoter-reporter construct together with expression plasmid for HER2 or with expression plasmid for Ets-1. The cellular luciferase activity was measured. Data were shown as means ± SD from three separate experiments. *P < 0.05 compared with the pcDNA group; ***P < 0.005 compared with the pcDNA group. (C) HER2-induced MMP-1 expression was inhibited by Ets-1 siRNA in MDA-MB-231 cells. To knock down Ets-1 expression in MDA-MB-231 cells, the cells were transfected with control siRNA oligo or two different siRNA oligos against Ets-1. The expression of Ets-1 was analyzed by immunoblotting. b-Actin was used as a loading control. RNA was b-actin was used as a loading control. RNA was collected 48 h after transfection. The mRNA expression of Ets-1 and MMP-1 analyzed by RT-PCR.
HER2, as detected by RT–PCR (Fig. 4A). MMP-1 levels increased relative to those before the addition of Ets-1, as determined by ELISA (Fig. 4B). The effect of Ets-1 siRNA in suppressing MMP-1 expression was confirmed by RT-PCR and Western blotting in MDA-MB-231 cells, which overexpress HER2. When MDA-MB-231 cells were treated with Ets-1 siRNA, Ets-1 expression was blocked and MMP-1 expression was inhibited (Fig. 4C). The expression of MMP-1 was down-regulated by the addition of Ets-1 siRNA to the MDA-MB-231 cell line. Discussion In this study, we tested the role of Ets-1 in breast cancer cells in relation to the expression of HER2 and MMP-1. HER2 overexpres sion in breast cancer cells are known to be poor prognostic markers and are strong predictors of benefit from treatment with HER2targeting agents [11]. Furthermore, several recent clinical studies have reported that the status of HER2 in the tumor can predict the results of other breast cancer treatments, including cytotoxic che motherap eutic agents and endocrine treatments [12,13]. After the discovery of the HER2-directed therapeutic strategy, its availabil ity strongly affected the treatment and clinical research of breast cancer [14,15]. Despite of the failure of MMP inhibition strategies in many clinical trials [16,17], MMPs are important contributors to tumor progression and provided the rationale for develop ing new cancer drugs that targeted MMP activity. Unfortunately, there is still lack of the knowledge in the contribution of MMPs to the progression of specific cancer types, stages and appropri ate tools for evaluating MMP inhibit ors. Our results showed that MMP-1 was overexpressed in HER2 transfected breast cancer cells
(Figs. 1 and 2). It is postulated that MMP-1 could function in breast cancer in relation to HER2, which is already known as target against breast cancer. Interestingly, erlotinib (EGFR tyrosine kinase inhibi tor) treatment as well as trastuzumab (HER2 humanized monoclo nal antibody) resulted in inhibition of MMP-1, HER2 and ligandinduced phosphorylation of EGFR, HER2 and HER2/EGFR (Fig. 3). It strongly suggests that MMP-1 can be a rational therapeutic target for breast cancer with new insight for EGFR in relation to HER2. In addition, we tested the role of Ets-1 using HER2-induced MMP-1 expression. Ets proteins are a family of mitogen-activated protein kinase (MAPK)-dependent transcription factors that have been implicated as downstream effectors of HER2 signaling [18]. Con sidering the association between Ets-1 and the invasiveness and metastasis of breast cancer [1–3], it also was postulated that Ets-1 could affect HER2 activity in breast cancer. However, this remained uncertain. Ets-1 is also required for the activation of several genes involved in angiogenes is and extracellular matrix remodeling in breast cancer [19]. Furthermore, Ets-1 is involved in the upregu lation of MMP-1 gene expression as well [2]. Based on our cDNA microarray analysis, we further clarified the relationship between MMPs and the actions of HER2 and Ets-1. The purpose of this study was to identify the role of Ets-1 in breast cancer as it relates to HER2 and MMP-1. First, we tested whether HER2 stimulates MMP-1 expression in MCF-7 breast cancer cells. MMP-1 expression showed a linear correlation with HER2 transfection in a time- and concentration-dependent man ner, which may have resulted from the collaborative activation of HER2 and MMP-1 protein expression. Trastuzumab and erl otinib abolished the MMP-1 expression stimulated by HER2. We have clearly demonstrated that Ets-1 enhances the HER2-induced
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expression of MMP-1 (Fig. 4). Therefore, Ets-1 may have clinical utility as a new target in breast cancer therapy, in cooperation with HER2 and MMP-1. It suggests that MMP-1 has its role for breast cancer in relation with HER2 and Ets-1. We found no correlation with other MMPs. Recently, other HER2-targeting agents apart from Trastuzumab have shown activity against breast cancer. Our results indicate that MMP-1 expression can exaggerated by HER2 in MCF-7 breast cancer cells, and Ets-1 could affect HER2-induced MMP-1 expression. There might be an association and interaction between Ets-1 and HER2 through MMP-1. These relations require further investigation, especially to a detailed analysis for its action mechanism and clinical relevance to breast cancer. Acknowledgment This work was supported by the Samsung Biomedic al Research Institute Grant #SBRI C-A6-423-1. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2008.09.135. References [1] P.N. Span, P. Manders, J.J. Heuvel, C.M. Thomas, R.R. Bosch, L.V. Beex, C.G. Sweep, Expression of the transcription factor Ets-1 is an independent prog nostic marker for relapse-free survival in breast cancer, Oncogene 21 (2002) 8506–8509. [2] T. Shepherd, J.A. Hassell, Role of Ets transcription factors in mammary gland development and oncogenes is, J. Mammary Gland Biol. Neoplasia 6 (2001) 129–140. [3] E. Myers, A.D.K. Hill, G. Kelly, E.W. McDermott, N.J. O’Higgins, Y. Buggy, L.S. Young, Associations and interactions between Ets-1 and Ets-2 and coregu latory proteins, SRC-1, AIB1, and NCoR in breast cancer, Clin. Cancer Res. 11 (2005) 2111–2122. [4] S. Katayama, T. Nakayama, M. Ito, S. Naito, I. Seline, Expression of the ets-1 proto-oncogene in human breast carcinoma: differential expression with his tological grading and growth pattern, Histol. Histopathol. 20 (2005) 119–126. [5] V.I. Sementchenko, D.K. Watson, Ets target genes: past, present and future, Oncogene 19 (2000) 6533–6548. [6] L.J. van’t Veer, H.Y. Dai, M.J. van de Vijver, Y.D. He, A.A. Hart, M. Mao, H.L. Peterse, K. van der Kooy, M.J. Marton, A.T. Witteveen, G.J. Schreib er, R.M. Ker
khoven, C. Roberts, P.S. Linsley, R. Bernards, S.H. Friend, Gene expression pro filing predicts clinical outcome of breast cancer, Nature 415 (2002) 530–536. [7] V. Chabottaux, A. Noel, Breast cancer progression: insights into multifaceted matrix metalloproteinases, Clin. Exp. Metastasis 24 (2007) 647–656. [8] D.J. Slamon, G.M. Clark, S.G. Wong, W.J. Levin, A. Ullric, W.L. McGuire, Human breast cancer: correlation of relapse and survival with amplification of the HER2/neu oncogene, Science 244 (1987) 707–712. [9] C. Wright, S. Nicholson, B. Angus, J.R. Sainsbury, J. Farndon, J. Cairns, A.L. Har ris, C.H. Horne, Relationship between c-erbB-2 protein product expression and response to endocrine therapy in advanced breast cancer, Br. J. Cancer 65 (1992) 118–121. [10] M. De Laurentiis, G. Cancello, L. Zinno, E. Montagna, L. Malorni, A. Esposito, R. Pennacchio, L. Silvestro, M. Giuliano, A. Giordano, F. Caputo, A. Accurso, S. De Placido, Targeting HER2 as a therapeutic strategy for breast cancer: a par adigmatic shift of drug development in oncology, Ann. Oncol. Suppl. 4 (2005) iv7–iv13. [11] R.D. Mass, M.F. Press, S. Anderson, M.A. Cobleigh, C.L. Vogel, N. Dybdal, G. Leiberman, D.J. Slamon, Evaluation of clinical outcomes according to HER2 detection by fluorescence in situ hybridization in women with metastatic breast cancer treated with Trastuzumab, Clin. Breast Cancer 6 (2005) 240– 246. [12] D.F. Hayes, A.D. Thor, L.G. Dressler, D. Weaver, S. Edgerton, D. Cowan, G. Broad water, L.J. Goldstein, S. Martino, J.N. Ingle, I.C. Henderson, L. Norton, E.P. Winer, C.A. Hudis, M.J. Ellis, D.A. Berry, Cancer and leukemia group B (CALGB) inves tigators: HER2 and response to paclitaxel in node-positive breast cancer, N. Engl. J. Med. 357 (2007) 1496–1506. [13] E. Azambuja, V. Durbecq, D.D. Rosa, M. Colozza, D. Larsimont, M. Piccart-Geb hart, F. Cardoso, HER2 overexpression/amplification and its interaction with taxane-based therapy in breast cancer, Ann. Oncol. 19 (2008) 223–232. [14] M.A. Cobleigh, C.L. Vogel, D. Tripathy, N.J. Robert, S. Scholl, L. Fehrenbacher, J.M. Wolter, V. Paton, S. Shak, G. Lieberman, D.J. Slamon, Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has pro gressed after chemotherapy for metastatic disease, J. Clin. Oncol. 17 (1999) 2639–2648. [15] D.J. Slamon, B. Leyland-Jones, S. Shak, H. Fuchs, V. Paton, A. Bajamonde, T. Fleming, W. Eiermann, J. Wolter, M. Pegram, J. Baselga, L. Norton, Use of che motherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2, N. Engl. J. Med. 344 (2001) 783–792. [16] L.M. Coussens, B. Fingleton, L.M. Matrisian, Matrix metalloproteinase inhibi tors and cancer: trials and tributions, Science 295 (2002) 2387–2392. [17] C.M. Overall, C. Lopez-Otin, Strateg ies for MMP inhibition in cancer: innova tions for the post-trial era, Nat. Rev. Cancer 2 (2002) 657–672. [18] C.K. Galang, J.J. Garcia-Ramirez, P.A. Solski, J.K. Westwick, C.J. Der, N.N. Nezna nov, R.G. Oshima, C.A. Hauser, Oncogenic Neu/ErbB-2 increases Ets, AP-1 and NF-jB-dependent gene expression and inhibiting Ets activation blocks neumediated cellular transformation, J. Biol. Chem. 271 (1996) 7992–7998. [19] J. Westermarck, A. Seth, V.M. Kähäri, Differential regulation of interstitial col lagenase (MMP-1) gene expression by ETS transcription factors, Oncogene 14 (1997) 2651–2660.
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Ets-1 upregulates HER2-induced MMP-1 expression in breast cancer cells Yeon Hee Park, Hae Hyun Jung, Jin Seok Ahn, Young-Hyuck Im * Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-Dong, Gangnam-Gu, Seoul, Republic of Korea Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine, 50 Irwon-Dong, Gangnam-Gu, Seoul, Republic of Korea
a r t i c l e
i n f o
Article history: Received 25 September 2008 Available online 11 October 2008 Keywords: Ets-1 HER2 MMP-1 Breast cancer
a b s t r a c t The human epidermal growth factor receptor-2 (HER2) plays an important role in breast cancer. Enhanced Ets-1 activity has recently been shown to be associated with breast cancer pathog enesis. To test the role of Ets-1 in breast cancer cells in relation to the expression of HER2 and MMP-1, we transiently overex pressed Ets-1 and/or HER2 in MCF-7 breast cancer cells and comprehensively searched for genes related to HER2 and Ets-1 using cDNA microarray analysis. The expression of matrix metalloproteinase (MMP) genes was enhanced by the overexpression of HER2/Ets-1. We analyzed the relationship between HER2induced MMP-1 expression and the transcription factor Ets-1, which has significant activity in breast can cer pathog enes is. Our results demonstrate that HER2-induced MMP-1 expression is positively regulated by Ets-1 in breast cancer cells. This study confirms that Ets-1 is a downstream effector of oncogenic HER2, associated with MMP-1. © 2008 Elsevier Inc. All rights reserved.
The Ets proteins are expressed in both primary human breast cancers and breast cancer cell lines, and their expression has been associated with disease progression and metastasis [1,2]. Ets-1 is an independent prognostic marker for relapse-free survival in breast cancer, and has not been linked to other prognostic factors, such as nodal status, tumor size, histological grade, or estrogen receptor status [1]. The degree of Ets-1 expression correlates with the extent of breast carcinoma invasion, and the atypism of the carcinoma correlates significantly with Ets-1 expression [3]. Ets-1 has also been shown to be correlated positively with HER2 expres sion, which is a representative prognostic and predictive marker of breast cancer [4]. Several other functional roles of Ets-1 in tumor igenesis of breast cancer have also been postulated. Ets-1 regu lates the expression of genes encoding enzymes involved in the degradation of the extracellular matrix, such as MMP-1, MMP-3, MMP-7, and MMP-9 [5]. MMP-1 and MMP-9 are involved in the 70 genes that identify the “gene-expression signature” which predicts distant metastasis in lymph-node-negat ive breast cancer patients [6,7]. MMPs have been implicated in normal matrix remodeling events and pathological conditions, including tumor invasion and metastasis [7]. Abnormalities in growth factor signaling path ways play an intrinsic role in disease progression. In humans, the growth factor receptor HER2 is overexpressed in 20–30% of breast cancers and overexpression of HER2 is associated with enhanced tumorigenicity and propensity to metastasize and resistance to * Corresponding author. Address: Division of Hematology-Oncology, Department of Medicine, Samsung Medical Center, Sungkyunkwan University School of Medi cine, 50 Irwon-Dong, Gangnam-Gu, Seoul, Republic of Korea. Fax: +82 2 3410 1757. E-mail address: [email protected] (Y.-H. Im). 0006-291X/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.09.135
endocrine therapy [8,9]. The targeting of HER2 as a therapeutic strategy for patients with HER2 overexpressing breast cancer has already shown survival advantage when combined with many che motherapeutic agents [10]. Assuming that the action of HER2 is related to Ets-1, we transiently overexpressed Ets-1 and/or HER2 in MCF-7 breast cancer cells and comprehensively searched for genes related to HER2 and Ets-1 using cDNA microarray analysis. The expression of metalloproteinase (MMP) genes was enhanced by the overexpression of HER2/Ets-1. Our cDNA microarray analy sis showed that MMP genes are associated with the action of HER2 and Ets-1. We wondered whether Ets-1 could thus potentially function as a downstream effector of oncogenic HER2 in associa tion with MMP-1. Based on this knowledge, we were interested in the relationships between MMP-1, HER2, and Ets-1 in breast can cer. We hypothes ized that HER2 induces MMP-1 expression and Ets-1 cooperates with this function. Here, we performed reverse transcription-polymerase chain reaction (RT-PCR), western blot analysis, an MMP-1 enzyme-linked immunosorbent assay (ELISA), and an MMP-1 promoter assay after transfecting the MCF-7 human breast cancer cell line with HER2. We then investigated how Ets-1 influences the MMP-1 expression induced by HER2. Materials and methods Antibodies and reagents. Anti-Ets-1 and anti-HER2 were pur chased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-bactin was purchased from Sigma (Salt Lake City, UT). Anti-MMP-1 was purchased from Calbiochem (Cambridge, MA). Trastuzumab (Herceptin®) and Erlotinib (Tarceva®) were provided by Roche
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Korea. MMP ELISA kits were obtained from R&D Systems. [c-32P] ATP was purchased from NEN Life Science Products (Boston, MA). Tumor cell lines and culture. The human breast cancer cell line MCF-7 was cultured and maintained in RPMI 1640 medium (Bio Whittaker, Walkersville, MD) supplemented with 10% fetal bovine serum, penicillin, and streptomycin. Transient and stable transfection of MCF-7 cells with HER2 and Ets-1. An expression vector for FLAG-tagged Ets-1 constructs was generated by subcloning PCR-amplified cDNA into the G418-resis tance plasmid pcDNA3.1. HER2/pCMV6–XL4 constructs were pur chased from Origene. The HER2/pCMV6–XL4 plasmid was digested with the restriction enzyme Not1 to release the inserted fragment. The fragment was re-subcloned into the G418-resistance plasmid pcDNA3.1 (Invitrogen) at the Not1 site. MCF-7 cells were tran siently transfected using Effectene Transfection Reagent (Qiagen Inc., Valencia, CA). MCF-7 cells were stably transfected with either the HER2/pcDNA3.1 or pcDNA3.1 vector in the presence of Effec tene Transfection Reagent for 48 h and were treated with 500 lg/ mL G418. RNA isolation and microarray analysis. The enriched plasma cells were stored in RNAlater (Ambion) for RNA extraction and gene chip experiments. RNA was isolated with an RNeasy Mini Kit (Qiagen, Valencia, CA) and the RNeasy Mini Kit (Qiagen, Valencia, CA). After cleanup with a Sample Cleanup Module (Affymetrix), the doublestranded cDNA was used for in vitro transcription. After cleanup with a Sample Cleanup Module (Affymetrix), 10–15 lg of labeled cRNA was fragmented into 35–200 bp lengths in fragmentation buffer (Affymetrix). The fragmented cRNA was hybridized to the Human Genome U133A 2.0 gene chip (Affymetrix) at 45 °C for 16 h. After hybridization, the arrays were washed in a GeneChip Fluid ics Station 450 with a nonstringent wash buffer at 25 °C, followed
by a stringent wash buffer at 50 °C. After washing, the arrays were stained with a streptavidin–phycoeryt hrin complex. After staining, the intensities were determined with a GeneChip Scanner 3000 (Affymetrix). Data analysis. Expression profiles were analyzed using the GeneChip Operating Software (Affymetrix). Transfection of Stealth RNAi. Ets-1 Validated Stealth™ RNAi DuoPak and ERBB2 Stealth™ Select 3 RNAi were purchased from Invitrogen. MCF-7 cells were transiently transfected using Lipo fectamine™ RNAiMAX (Invitrogen). Preparation of promoter–reporter constructs. A promoter frag ment of the human MMP-1 gene, from ¡4334 to +5218 relative to the transcription start point, was amplified from human DNA by PCR, using a pair of primers (59-agatgtaagagctgggaaaggacgg-39/59tcagtgcaaggtaagtgatggcttc-39) and Accuprime Pfx DNA polymerase (Invitrogen). The fragment was subcloned into the pCR–XL–TOPO vector (Invitrogen) for propagation in bacteria. The isolated plas mid was digested with the restriction enzymes MluI and XhoI to release the inserted fragment. The fragment was re-subcloned in the sense orientation into the promoter-free pGL3-Basic vector (Promega) at the MluI and XhoI sites in the 59 flanking region of the luciferase sequence. Isolation of RNA and RT-PCR. Total cellular RNA was isolated using TRIzol™ Reagent (Gibco BRL, Carlsbad, CA). For RT-PCR, 2 lg of RNA was treated with RNase-free DNase, and cDNA was obtained using the SuperScript™ III First-Strand Synthesis System for RT-PCR. The cDNA was amplified by PCR. The primers used in this analysis are shown in Supplemental Table 1. Western blot analysis. Cells were extracted with RIPA buffer. The samples were placed on ice for 20 min with occasional vortexing. Protein concentrations were determined using the BCA Protein
Fig. 1. HER2 overexpression increases MMP-1 expression in MCF-7 cells. (A) MCF-7 cells were transfected with pcDNA3.1(+) or HER2/pcDNA3.1(+) for 24 h. The mRNA expres sion of HER2 and MMP-1 analyzed by RT-PCR. (B) At 48 h post-transfection, the conditioned medium was subjected to ELISA to quantify secreted MMP-1. Data were shown as means ± SD from three separate experiments. *P < 0.05 compared with the Vector group. (C) MCF-7 cells were co-transfected with MMP-1 promoter-reporter construct together with empty expression vector or with expression plasmid for HER2. The cellular luciferase activity was measured. Data were shown as means ± SD from three sepa rate experim ents. ***P < 0.0005 compared with the control group. (D) MCF-7 cells were stably transfected with HER2 expression vector. The expression of HER2 was analyzed by immunoblotting. b-Actin was used as a loading control. (E) MMP-1 mRNA expression was increased in MCF-7/HER2 clones. †The molecular sizes of different bands: b-actin 331 bp; HER2 281 bp; MMP-1 444 bp.
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Assay Kit (Pierce). Equal amounts of sample were subjected to SDS–PAGE. Membranes were incubated for 2 h then, incubated with primary antibody for 2 h. After several washes in PBS and incubation with horseradish-peroxidase-conjugated secondary antibodies (Zymed), bound primary antibodies were detected with ECL chemiluminescent reagents (Amersham). ELISA of secreted MMP-1. MMP-1 secreted from MCF-7 cells was quantified using the Human Pro-MMP-1 ELISA kit (R&D Systems). The absorbance at 450 nm was measured with a spectrophotomet ric plate reader. Luciferase assays. MCF-7 cells were transiently transfected with the reporter constructs using Effectene Transfection Reagent. After transfection, whole-cell lysates were prepared and luciferase activ ity was measured using a luciferase activity assay kit (Promega). Preparation of nuclear protein extracts. The washed cells were scraped from the culture plates and collected by centrifugation. The pellets were resuspended in 0.4 mL of hypotonic lysis buffer. After 15 min on ice, Nonidet P-40 was added to a final concentra tion of 0.5%, and the cells were vortexed for 10 s and centrifuged (30 s, 4 °C, 12,000g). The supernatant was frozen immediately and the nuclear pellets were resuspended in 130 lL of buffer and vor texed for 30 min at 4 °C. After centrifugation (5 min, 4 °C, 12,000g), the supernatant was frozen at ¡70 °C. Immunoprecipit ation assays. At 24 h after incubation on 60-mm Petri dishes, cells were extracted with lysis buffer. After the lysates had been precleared with Protein-A/G PLUS-Agarose, 2 lg HER2 monoclonal antibodies (Calbiochem) or EGFR (biosource) and Protein-A/G PLUS-Agarose were added and incubated overnight at 4 °C on a rotating wheel. Immunocomplexes were collected by centrifugation, washed five times with lysis buffer, separated by SDS–PAGE.
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Statistical analysis. Student’s T-test was done to determine sta tistical significance for ELISA and luciferase assay. Results cDNA microarray revealed that MMP genes are related to the action of HER2 and Ets-1 To identify genes biologically related to HER2 and Ets-1 activity in breast cancer cells, we analyzed the expression profiles of cells after transfection with HER2 and/or Ets-1 in MCF breast cancer cell using the cDNA microarray method. The expression profiles showed high expression of MMP’s and collagenase genes in the cell transfected with HER2 and Ets-1, suggesting that these genes were closely associated with the interaction of HER2 and Ets-1 (Supple mental Table 2). Induction by HER2 of MMP-1 expression and promoter activity To determine whether HER2 is involved in the regulation of MMP-1 expression and promoter activity, HER2-transfected MCF-7 breast cancer cells were incubated, and RT-PCR with MMP-1 spe cific primers and Western immunoblotting were performed (Fig. 1A). Transient overexpression of HER2 gene led to an enhancement of MMP-1 mRNA expression levels. We also quantified the MMP-1 secreted from MCF-7 cells using an ELISA, and the MMP-1 pro moter activity was analyzed by measuring the luciferase activity (Fig. 1B and C). Fig. 1A shows that the expression of MMP-1 mRNA was upregulated after transfection of cells with HER2. We used western immunoblotting analysis to determine if the observed increase in MMP-1 mRNA transcription correlated with MMP-1
Fig. 2. Down-regulation of HER2 inhibits HER2-induced MMP-1 expression. (A) To knock down HER2 expression in MCF-7/HER2-14 cells, the cells were transfected with control siRNA oligo or three different siRNA oligos against HER2. RNA was collected 48 h after transfection. The mRNA expression of HER2 and MMP-1 were analyzed by RTPCR. (B) ELISA was performed to assess the effects of different kinds of HER2 siRNA on MMP-1 expression. (1 lane, MCF-7/Vector; 2–9 lane, MCF-7/HER2-14; NC, negative control siRNA). *P < 0.05 compared with the MCF-7HER2-14 group; **P < 0.01 compared with the MCF-7HER2-14 group. (C) MCF-7/HER2-14 cells were incubated in medium supplemented with Trastuzumab at 10 lg/ml for 48 h. Cell lysates were prepared and immunoblotted with HER2 and b-actin. b-Actin was used as a loading control. MMP-1 expression was analyzed by RT-PCR. (D) The conditioned medium was subjected to ELISA to quantify secreted MMP-1. §P < 0.005 compared with the MCF-7/HER2-14 control group. The experiment was replicated three times.
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protein expression. Fig. 1B shows the strongly augmented expres sion of MMP-1 protein relative to that before the transfection of cells with HER2. Fig. 1C shows enhanced MMP-1 promoter activity by HER2. Fig. 1D shows the expression of HER2 by Western blot ting. MMP-2 mRNA expression was increased in several MCF-7/ HER2 clones (Fig. 1E).
Actin expression was unaffected by either control or HER2 siRNA treatment, indicating that non-specific down-regulation did not occur. This result was quantified by ELISA (Fig. 2B). Trastuzumab, HER2-targeting agent, inhibits HER2-induced MMP-1 expression (Fig. 2C and D). This finding confirmed MMP-1 upregulation by HER2.
Stable transfection of HER2 into the MCF-7 breast cancer cell line
EGFR tyrosine kinase inhibit or inhibits (TKI) ligand-induced phosphorylation of EGFR, HER2 and HER2/EGFR and MMP-1
To test the efficiency of HER2 transfection into MCF-7 cells, we compared the HER2 expression profile with that of MCF-7 cells transfected with the G418-resistance plasmid pcDNA3.1. We also compared it with that of cells transfected with HER2 Stealth RNAi (50 mg or 100 mg). HER2 mRNA expression was markedly increased in MCF-7 cells after transfection with HER2 when analyzed by RTPCR. The ability of small interfering RNA (siRNA) of HER2 to sup press HER2 expression was confirmed by RT-PCR. We determined the effect of HER2 siRNA at different doses on HER2 expression in MCF-7 cells, which indicated that HER2 expression was blocked by HER2 siRNA treatment (Fig. 2A). Down-regulation of HER2 inhibits HER2-induced MMP-1 expression siRNA to HER2 provide further evidence for the positive regu latory role of HER2 in the regulation of MMP-1 (Fig. 2A). The abil ity of HER2 siRNA to suppress MMP-1 expression was performed by RT-PCR. RNA was collected 48 h after transfection. The mRNA expression of HER2 and MMP-1 were down-regulated in accor dance with increasing HER2-specific siRNA by three kinds of siRNA.
We investigated whether erlotinib, EGFR TKI, inhibits HER2induced MMP-1 overexpression. The phosphorylation of EGFR and HER2 in MCF-7/HER2 was analyzed by immunoblotting (Fig. 3A). Immunoprecipitation in Fig. 3B indicated that HER2 has increased associated with EGFR in MCF-7/HER2-14. To examine the inhibitory effect of erlotinib on phosphorylation of EGFR and HER2 in MCF-7/ HER2 cells, MCF-7/HER2-14 cells were treated with erlotinib with indicated concentrations for 24 h. followed by immunoblotting for the indicated proteins. As shown in Fig. 3C, HER2 expression was decreased in a concentration-dependent manner. The mRNA expression of MMP-1 was decreased after erlotinib treatment in concentration-dependent manner by RT-PCR (Fig. 3D). Ets-1 enhances HER2-induced MMP-1 expression We investigated whether MMP-1, which was activated with HER2, is affected by Ets-1 treatment in MCF-7 cells. After treat ment with Ets-1, MMP-1 mRNA expression was enhanced in estab lished a stable MCF-7 cell line transfected with either Ets-1 or
Fig. 3. Erlotinib inhibits HER2-induced MMP-1 expression. (A) Expression levels of EGFR and HER2 phosphorylation in MCF-7/HER2 clones. Cell lysates were prepared and analyzed by immunoblotting using the indicated antibodies. b-Actin was used as a loading control. (B) HER2 has increased association with EGFR in MCF-7/HER2-14. MCF-7/Vector and MCF-7/HER2-14 cells were lysed in RIPA buffer. HER2 or EGFR in 0.5 mg cell lysate was immunoprecipitated. The immunoprecipitates were fractionated on SDS–PAGE followed by immunoblotting for the indicated proteins. (C) Erlotinib inhibits EGFR and HER2 phosphorylation in MCF-7/HER2-14. MCF-7/HER2-14 cells were treated with erlotinib for 24 h. Cell lysates were analyzed by immunoblotting. b-Actin was used as a loading control. (D) The mRNA expression of MMP-1 and HER2 analyzed by RT-PCR. The experiment was replicated three times.
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Fig. 4. The effect of Ets-1 on HER2-induced MMP-1 expression. (A) HER2 and Ets-1 enhance expression of the MMP-1. MCF-7 cells were transiently transfected with expres sion plasmids for HER2 (2 lg) or Ets-1 (2 lg). (B) MCF-7 cells were co-transfected with MMP-1 promoter-reporter construct together with expression plasmid for HER2 or with expression plasmid for Ets-1. The cellular luciferase activity was measured. Data were shown as means ± SD from three separate experiments. *P < 0.05 compared with the pcDNA group; ***P < 0.005 compared with the pcDNA group. (C) HER2-induced MMP-1 expression was inhibited by Ets-1 siRNA in MDA-MB-231 cells. To knock down Ets-1 expression in MDA-MB-231 cells, the cells were transfected with control siRNA oligo or two different siRNA oligos against Ets-1. The expression of Ets-1 was analyzed by immunoblotting. b-Actin was used as a loading control. RNA was b-actin was used as a loading control. RNA was collected 48 h after transfection. The mRNA expression of Ets-1 and MMP-1 analyzed by RT-PCR.
HER2, as detected by RT–PCR (Fig. 4A). MMP-1 levels increased relative to those before the addition of Ets-1, as determined by ELISA (Fig. 4B). The effect of Ets-1 siRNA in suppressing MMP-1 expression was confirmed by RT-PCR and Western blotting in MDA-MB-231 cells, which overexpress HER2. When MDA-MB-231 cells were treated with Ets-1 siRNA, Ets-1 expression was blocked and MMP-1 expression was inhibited (Fig. 4C). The expression of MMP-1 was down-regulated by the addition of Ets-1 siRNA to the MDA-MB-231 cell line. Discussion In this study, we tested the role of Ets-1 in breast cancer cells in relation to the expression of HER2 and MMP-1. HER2 overexpres sion in breast cancer cells are known to be poor prognostic markers and are strong predictors of benefit from treatment with HER2targeting agents [11]. Furthermore, several recent clinical studies have reported that the status of HER2 in the tumor can predict the results of other breast cancer treatments, including cytotoxic che motherap eutic agents and endocrine treatments [12,13]. After the discovery of the HER2-directed therapeutic strategy, its availabil ity strongly affected the treatment and clinical research of breast cancer [14,15]. Despite of the failure of MMP inhibition strategies in many clinical trials [16,17], MMPs are important contributors to tumor progression and provided the rationale for develop ing new cancer drugs that targeted MMP activity. Unfortunately, there is still lack of the knowledge in the contribution of MMPs to the progression of specific cancer types, stages and appropri ate tools for evaluating MMP inhibit ors. Our results showed that MMP-1 was overexpressed in HER2 transfected breast cancer cells
(Figs. 1 and 2). It is postulated that MMP-1 could function in breast cancer in relation to HER2, which is already known as target against breast cancer. Interestingly, erlotinib (EGFR tyrosine kinase inhibi tor) treatment as well as trastuzumab (HER2 humanized monoclo nal antibody) resulted in inhibition of MMP-1, HER2 and ligandinduced phosphorylation of EGFR, HER2 and HER2/EGFR (Fig. 3). It strongly suggests that MMP-1 can be a rational therapeutic target for breast cancer with new insight for EGFR in relation to HER2. In addition, we tested the role of Ets-1 using HER2-induced MMP-1 expression. Ets proteins are a family of mitogen-activated protein kinase (MAPK)-dependent transcription factors that have been implicated as downstream effectors of HER2 signaling [18]. Con sidering the association between Ets-1 and the invasiveness and metastasis of breast cancer [1–3], it also was postulated that Ets-1 could affect HER2 activity in breast cancer. However, this remained uncertain. Ets-1 is also required for the activation of several genes involved in angiogenes is and extracellular matrix remodeling in breast cancer [19]. Furthermore, Ets-1 is involved in the upregu lation of MMP-1 gene expression as well [2]. Based on our cDNA microarray analysis, we further clarified the relationship between MMPs and the actions of HER2 and Ets-1. The purpose of this study was to identify the role of Ets-1 in breast cancer as it relates to HER2 and MMP-1. First, we tested whether HER2 stimulates MMP-1 expression in MCF-7 breast cancer cells. MMP-1 expression showed a linear correlation with HER2 transfection in a time- and concentration-dependent man ner, which may have resulted from the collaborative activation of HER2 and MMP-1 protein expression. Trastuzumab and erl otinib abolished the MMP-1 expression stimulated by HER2. We have clearly demonstrated that Ets-1 enhances the HER2-induced
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expression of MMP-1 (Fig. 4). Therefore, Ets-1 may have clinical utility as a new target in breast cancer therapy, in cooperation with HER2 and MMP-1. It suggests that MMP-1 has its role for breast cancer in relation with HER2 and Ets-1. We found no correlation with other MMPs. Recently, other HER2-targeting agents apart from Trastuzumab have shown activity against breast cancer. Our results indicate that MMP-1 expression can exaggerated by HER2 in MCF-7 breast cancer cells, and Ets-1 could affect HER2-induced MMP-1 expression. There might be an association and interaction between Ets-1 and HER2 through MMP-1. These relations require further investigation, especially to a detailed analysis for its action mechanism and clinical relevance to breast cancer. Acknowledgment This work was supported by the Samsung Biomedic al Research Institute Grant #SBRI C-A6-423-1. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2008.09.135. References [1] P.N. Span, P. Manders, J.J. Heuvel, C.M. Thomas, R.R. Bosch, L.V. Beex, C.G. Sweep, Expression of the transcription factor Ets-1 is an independent prog nostic marker for relapse-free survival in breast cancer, Oncogene 21 (2002) 8506–8509. [2] T. Shepherd, J.A. Hassell, Role of Ets transcription factors in mammary gland development and oncogenes is, J. Mammary Gland Biol. Neoplasia 6 (2001) 129–140. [3] E. Myers, A.D.K. Hill, G. Kelly, E.W. McDermott, N.J. O’Higgins, Y. Buggy, L.S. Young, Associations and interactions between Ets-1 and Ets-2 and coregu latory proteins, SRC-1, AIB1, and NCoR in breast cancer, Clin. Cancer Res. 11 (2005) 2111–2122. [4] S. Katayama, T. Nakayama, M. Ito, S. Naito, I. Seline, Expression of the ets-1 proto-oncogene in human breast carcinoma: differential expression with his tological grading and growth pattern, Histol. Histopathol. 20 (2005) 119–126. [5] V.I. Sementchenko, D.K. Watson, Ets target genes: past, present and future, Oncogene 19 (2000) 6533–6548. [6] L.J. van’t Veer, H.Y. Dai, M.J. van de Vijver, Y.D. He, A.A. Hart, M. Mao, H.L. Peterse, K. van der Kooy, M.J. Marton, A.T. Witteveen, G.J. Schreib er, R.M. Ker
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