Up-regulation of miR-21 by HER2/neu Signaling Promotes Cell Invasion
2009; Elsevier BV; Volume: 284; Issue: 27 Linguagem: Inglês
10.1074/jbc.m109.006676
ISSN1083-351X
AutoresTzu Hsuan Huang, Fangting Wu, Gabriel B. Loeb, Ruby Hsu, Amy Heidersbach, Allison Brincat, Dai Horiuchi, Robert Jan Lebbink, Yin Yuan Mo, Andrei Goga, Michael T. McManus,
Tópico(s)Circular RNAs in diseases
ResumoThe cell surface receptor tyrosine kinase HER2/neu enhances tumor metastasis. Recent studies suggest that deregulated microRNA (miRNA) expression promotes invasion and metastasis of cancer cells; we therefore explored the possibility that HER2/neu signaling induces the expression of specific miRNAs involved in this process. We identified a putative oncogenic miRNA, miR-21, whose expression is correlated with HER2/neu up-regulation and is functionally involved in HER2/neu-induced cell invasion. We show that miR-21 is up-regulated via the MAPK (ERK1/2) pathway upon stimulation of HER2/neu signaling in breast cancer cells, and overexpression of other ERK1/2 activators such as RASV12 or ID-1 is sufficient to induce miR-21 up-regulation in HER2/neu-negative breast cancer cells. Furthermore, the metastasis suppressor protein PDCD4 (programmedcell death 4) is down-regulated by miR-21 in breast cancer cells expressing HER2/neu. Our data reveal a mechanism for HER2/neu-induced cancer cell invasion via miRNA deregulation. In addition, our results identify miR-21 as a potential therapeutic target for the prevention of breast cancer invasion and metastasis. The cell surface receptor tyrosine kinase HER2/neu enhances tumor metastasis. Recent studies suggest that deregulated microRNA (miRNA) expression promotes invasion and metastasis of cancer cells; we therefore explored the possibility that HER2/neu signaling induces the expression of specific miRNAs involved in this process. We identified a putative oncogenic miRNA, miR-21, whose expression is correlated with HER2/neu up-regulation and is functionally involved in HER2/neu-induced cell invasion. We show that miR-21 is up-regulated via the MAPK (ERK1/2) pathway upon stimulation of HER2/neu signaling in breast cancer cells, and overexpression of other ERK1/2 activators such as RASV12 or ID-1 is sufficient to induce miR-21 up-regulation in HER2/neu-negative breast cancer cells. Furthermore, the metastasis suppressor protein PDCD4 (programmedcell death 4) is down-regulated by miR-21 in breast cancer cells expressing HER2/neu. Our data reveal a mechanism for HER2/neu-induced cancer cell invasion via miRNA deregulation. In addition, our results identify miR-21 as a potential therapeutic target for the prevention of breast cancer invasion and metastasis. The HER2/neu (c-erbB-2) proto-oncogene encodes a transmembrane protein-tyrosine kinase growth factor receptor, p185HER2, which is a member of the human epidermal growth factor receptor family. HER2/neu overexpression is found in about 30% of human breast cancers and several other cancer types. HER2/neu overexpression is associated with a poor clinical outcome, including a positive correlation with metastasis (1.Slamon D.J. Godolphin W. Jones L.A. Holt J.A. Wong S.G. Keith D.E. Levin W.J. Stuart S.G. Udove J. Ullrich A. Science. 1989; 244: 707-712Crossref PubMed Scopus (6289) Google Scholar, 2.Yu D. Hung M.C. Oncogene. 2000; 19: 6115-6121Crossref PubMed Scopus (353) Google Scholar). The involvement of HER2/neu in metastasis is supported by studies demonstrating that HER2/neu increases the metastatic potential of human and murine cancer cell lines (3.Tan M. Yao J. Yu D. Cancer Res. 1997; 57: 1199-1205PubMed Google Scholar) and induces lung metastasis in transgenic animal models (4.Guy C.T. Webster M.A. Schaller M. Parsons T.J. Cardiff R.D. Muller W.J. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 10578-10582Crossref PubMed Scopus (1025) Google Scholar). Additionally, HER2/neu signaling up-regulates genes that play important roles in cell invasion and metastasis, such as cyclooxygenase-2, CXCR4, and matrix metalloproteinases (5.Subbaramaiah K. Norton L. Gerald W. Dannenberg A.J. J. Biol. Chem. 2002; 277: 18649-18657Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 6.Li Y.M. Pan Y. Wei Y. Cheng X. Zhou B.P. Tan M. Zhou X. Xia W. Hortobagyi G.N. Yu D. Hung M.C. Cancer Cell. 2004; 6: 459-469Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar, 7.Bosc D.G. Goueli B.S. Janknecht R. Oncogene. 2001; 20: 6215-6224Crossref PubMed Scopus (97) Google Scholar). Given the complex signaling network initiated by HER2/neu overexpression in cancer cells, it is likely that HER2/neu regulates additional unidentified players involved in these processes. miRNAs 4The abbreviations used are: miRNAmicroRNAMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinaseMEKMAPK/ERK kinaseGFPgreen fluorescent proteinqRTquantitative real timeScFVsingle chain FvTNFtumor necrosis factorRTKreceptor tyrosine kinase. constitute a class of 21 or 22 nucleotides noncoding RNAs that play an important role in development and cellular processes. Aberrant expression of miRNAs is associated with cancer (8.He L. Thomson J.M. Hemann M.T. Hernando-Monge E. Mu D. Goodson S. Powers S. Cordon-Cardo C. Lowe S.W. Hannon G.J. Hammond S.M. Nature. 2005; 435: 828-833Crossref PubMed Scopus (3155) Google Scholar), suggesting that some miRNAs can function as tumor suppressor genes or oncogenes. miRNAs may also cooperate with the loss of tumor suppressors or overexpression oncogenes in cancer cells to contribute to a fully malignant phenotype. Up-regulation of several miRNAs in breast cancer cells, such as miR-21 and miR-10b, can increase cell invasion and metastasis (9.Zhu S. Wu H. Wu F. Nie D. Sheng S. Mo Y.Y. Cell Res. 2008; 18: 350-359Crossref PubMed Scopus (990) Google Scholar, 10.Ma L. Teruya-Feldstein J. Weinberg R.A. Nature. 2007; 449: 682-688Crossref PubMed Scopus (2236) Google Scholar). HER2/neu signaling activates a variety of transcription factors, such as AP-1, Myc, and NF-κB that alter miR-21 and other miRNA transcription (8.He L. Thomson J.M. Hemann M.T. Hernando-Monge E. Mu D. Goodson S. Powers S. Cordon-Cardo C. Lowe S.W. Hannon G.J. Hammond S.M. Nature. 2005; 435: 828-833Crossref PubMed Scopus (3155) Google Scholar, 11.Fujita S. Ito T. Mizutani T. Minoguchi S. Yamamichi N. Sakurai K. Iba H. J. Mol. Biol. 2008; 378: 492-504Crossref PubMed Scopus (367) Google Scholar, 12.Taganov K.D. Boldin M.P. Chang K.J. Baltimore D. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 12481-12486Crossref PubMed Scopus (3569) Google Scholar). We therefore hypothesize that HER2/neu signaling may induce the expression of specific miRNAs, which contribute to the increased metastatic potential of HER2/neu-overexpressing cancer cells. microRNA mitogen-activated protein kinase extracellular signal-regulated kinase MAPK/ERK kinase green fluorescent protein quantitative real time single chain Fv tumor necrosis factor receptor tyrosine kinase. Here we describe a putative oncogenic miRNA, miR-21, whose expression is correlated with HER2/neu up-regulation. We found that HER2/neu signaling up-regulates miR-21 via the MAPK (ERK1/2) pathway and that its increased expression promotes cell invasion. Furthermore, miR-21 suppressed expression of the metastasis suppressor PDCD4 in HER2/neu-expressing breast cancer cells. Our data show a new mechanism by which HER2/neu induces cell invasion via miR-21 deregulation in breast cancer cells. This raises the possibility that anti-miR-21 therapies might exhibit a synergistic effect with other anti-HER2/neu therapeutics, for example Herceptin, in treating HER2/neu-associated breast cancers. Human breast cancer cell line SKBR3 (ATCC) was cultured in McCoy's 5A medium with 10% fetal bovine serum. BT-474 (ATCC) was cultured in RPMI 1640 with 10% fetal bovine serum. The HeLa (ATCC) cell line and its HER2/neu transfectant were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum. The human breast cancer cell line MDA-MB-435 transfected with a neo vector or a HER2-expressing vector were gifts from Dr. Mien-Chie Hung (M. D. Anderson Cancer Center, Houston, TX) and have been characterized and described previously (6.Li Y.M. Pan Y. Wei Y. Cheng X. Zhou B.P. Tan M. Zhou X. Xia W. Hortobagyi G.N. Yu D. Hung M.C. Cancer Cell. 2004; 6: 459-469Abstract Full Text Full Text PDF PubMed Scopus (457) Google Scholar). These cells were maintained in Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal bovine serum and 400 μg/ml geneticin (Invitrogen). pEBS7 and pEBS7-HER2/neu vectors were gifts from Dr. Mikala Egeblad (13.Egeblad M. Jäättelä M. Int. J. Cancer. 2000; 86: 617-625Crossref PubMed Scopus (28) Google Scholar). Ets-1 small hairpin RNA-expressing vector was obtained from SuperArray Bioscience Corp. (Frederick, MD). Replication-defective mouse stem cell (MSCV-IRES-GFP) retrovirus encoding control vector, activated H-Ras (G12V), activated myristoylated AKT, Myc, or ID-1 were prepared as previously described (14.Goga A. Yang D. Tward A.D. Morgan D.O. Bishop J.M. Nat. Med. 2007; 13: 820-827Crossref PubMed Scopus (248) Google Scholar). Nontransformed human MCF10A breast epithelial cells were infected with each of the recombinant retrovirus to generate stable cell populations. Greater than 80% of MCF10A stable cell populations expressed cis-linked GFP fluorescence marker as judged by fluorescence microscopy. The wild type ERK2 and dominant negative ERK2 were gift from Dr. Mien-Chie Hung (M. D. Anderson Cancer Center) and have been characterized and described previously (15.Yang J.Y. Zong C.S. Xia W. Yamaguchi H. Ding Q. Xie X. Lang J.Y. Lai C.C. Chang C.J. Huang W.C. Huang H. Kuo H.P. Lee D.F. Li L.Y. Lien H.C. Cheng X. Chang K.J. Hsiao C.D. Tsai F.J. Tsai C.H. Sahin A.A. Muller W.J. Mills G.B. Yu D. Hortobagyi G.N. Hung M.C. Nat. Cell Biol. 2008; 10: 138-148Crossref PubMed Scopus (538) Google Scholar). To construct the mCherry miR-21 and mCherry miR-155 sensor vectors, the GFP gene in the pSicoR vector (16.Ventura A. Meissner A. Dillon C.P. McManus M. Sharp P.A. Van Parijs L. Jaenisch R. Jacks T. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10380-10385Crossref PubMed Scopus (514) Google Scholar) was replaced with the mCherry gene using the Age1 and BsrG1 sites. The oligonucleotides with either two complementary miR-21-binding sites (5′-GTACATCAACATCAGTCTGATAAGCTACCCGGGTCAACATCAGTCTGATAAGCTAG-3′) or miR-155-binding sites (5′-GTACACCCCTATCACGATTAGCATTAACCCGGGCCCCTATCACGATTAGCATTAAG-3′) were cloned downstream the mCherry gene using the BsrGI and EcoRI sites to produce the mCherry miR-21 sensor vector and the mCherry miR-155 sensor vector, respectively. To construct the miR-21 expression vector, 550 bp including the miR-21 pri-cursor was amplified by PCR from human genomic DNA with the forward primer 5′-AGTTTTCTTGCCGTTCTGTAAGT-3′ and the reverse primer 5′-GTCATGAAGACTATCCCCATTTC-3′. The final PCR product was ligated into a PCR2.1 TOPO TA cloning vector (Invitrogen) according to the manufacturer's instructions. The resulting vector was sequence verified and subsequently digested with BamHI and XhoI to release the DNA fragment, which was inserted into a modified pSicoR vector downstream from the GFP gene. 1,544 modified oligonucleotides with C6 5′ amino linkers (IDT) were printed in triplicate using a microarrayer on house-made poly-l-lysine slides (17.DeRisi J.L. Iyer V.R. Brown P.O. Science. 1997; 278: 680-686Crossref PubMed Scopus (3707) Google Scholar). The probes were reverse complements of annotated (18.Griffiths-Jones S. Grocock R.J. van Dongen S. Bateman A. Enright A.J. Nucleic Acids Res. 2006; 34: D140-144Crossref PubMed Scopus (3688) Google Scholar) and predicted miRNAs (19.Berezikov E. Guryev V. van de Belt J. Wienholds E. Plasterk R.H. Cuppen E. Cell. 2005; 120: 21-24Abstract Full Text Full Text PDF PubMed Scopus (1070) Google Scholar), with some probes shortened to standardize melting temperature. Probe sequences are listed in supplemental Table S1. Before hybridization, the arrays were prehybridized, and probes were UV cross-linked. miRNA microarrays were performed essentially as described (20.Thomson J.M. Parker J. Perou C.M. Hammond S.M. Nat. Methods. 2004; 1: 47-53Crossref PubMed Scopus (677) Google Scholar) with the following modifications. RNA was extracted with TRIzol (Invitrogen) according to the manufacturer's protocol. 5 μg of total RNA was added to a ligation reaction containing either 250 ng of 5′-phospate-citidyl-uridyl-Cy3-3′ or 1 μg of 5′- phosphate-citidyl-citidyl-uridyl-Dy647-3′ (Dharmacon), and labeling was allowed to proceed overnight on ice. Two samples labeled with different dyes were then combined and precipitated with ethanol. Precipitated RNA was resuspended in 120 μl of hybridization buffer and hybridized for 2 h on arrays using a 1.7 × 2.8-cm Gene Frame (ABgene, Rochester, NY). The arrays were washed and then immediately scanned using a GenePix 4000B scanner (Axon Instruments, Sunnyvale, CA). The data were extracted, and local background was subtracted using Genepix Pro-4 software (Axon Instruments). The data were processed in Excel to calculate the median intensity for each set of triplicate probes in each channel. The displayed data (see Fig. 1B) summarizes the results from three arrays in which labeling of each sample was done with a different fluorophore on each array, known as dye swapping, to eliminate dye bias. The mean fluorescence for each probe from the two arrays was normalized to the maximum mean fluorescence for each sample. Only the 330 probes on the array corresponding to annotated human miRNAs are shown. Fluorescein isothiocyanate-conjugated anti-human HER2/neu antibody was obtained from Abcam Inc. (Cambridge, MA). AKT, phospho-AKT (Ser473), and hemagglutinin tag (clone 6E2) are from Cell Signaling (Danvers, MA). Ras (Ab-1) is from Thermo Scientific (Waltham, MA). Myc (clone Y69) is from Epitomics (Burlingame, CA). β-Actin (clone AC-15) is from Sigma. HER2/neu antibody for Western blot is from Calbiochem (Gibbstown, NJ). The HER2/neu agonist antibody anti-HER2/neu ScFv-TNF-α (S147Y) was a gift from Dr. Sherie Morrison (UCLA, Los Angeles) and has been characterized and described previously (21.Huang T.H. Morrison S.L. J. Pharmacol. Exp. Ther. 2006; 316: 983-991Crossref PubMed Scopus (27) Google Scholar). The Akt inhibitor LY294002 and ERK1/2 inhibitor U0126 were obtained from Cell Signaling. For real time PCR of primary miR-21 transcripts (pri-miR-21) and Ets-1, total RNA was prepared using the mirVana miRNA isolation kit (Ambion, Austin, TX) and quantified by Nanodrop (Wilmington, DE). Reverse transcription was performed with a Superscript III first strand synthesis system for real time PCR (Invitrogen). Real time PCR was performed using 7900HT Fast real time PCR system (Applied Biosystems, Foster City, CA). The pri-miR-21 was amplified using the forward primer 5′-CATTGTGGGTTTTGAAAAGGTTA-3′ and the reverse primer 5′-CCACGACTAGAGGCTGACTTAGA-3′, and the specificity of the pri-miR-21 amplification was validated previously (22.Löffler D. Brocke-Heidrich K. Pfeifer G. Stocsits C. Hackermller J. Kretzschmar A.K. Burger R. Gramatzki M. Blumert C. Bauer K. Cvijic H. Ullmann A.K. Stadler P.F. Horn F. Blood. 2007; 110: 1330-1333Crossref PubMed Scopus (562) Google Scholar). The human Ets-1 gene was amplified using the forward primer 5′-AGGAGATGGGGAAAGAGGAA-3′ and the reverse primer 5′-AGCGGTACACGTAGCGTTTC-3′. The HPRT1 gene was amplified using the forward primer 5′-TGACACTGGCAAAACAATGCA-3′ and the reverse primer 5′-GGTCCTTTTCACCAGCAAGCT-3′, which was validated previously (23.Vandesompele J. De Preter K. Pattyn F. Poppe B. Van Roy N. De Paepe A. Speleman F. Genome Biol. 2002; 3 (RESEARCH0034/1)Crossref PubMed Google Scholar) and served as an internal control during the pri-miR-21 and Ets-1 mRNA amplification. The PCR was established with the Power SYBR Green PCR Master Mix (Applied Biosystems), and only one PCR product was generated in these reactions. PCR-based detection of mature miR-21 and other microRNAs was performed by the TaqMan miRNA assays (Applied Biosystems) as described previously (24.Chen C. Ridzon D.A. Broomer A.J. Zhou Z. Lee D.H. Nguyen J.T. Barbisin M. Xu N.L. Mahuvakar V.R. Andersen M.R. Lao K.Q. Livak K.J. Guegler K.J. Nucleic Acids Res. 2005; 33: e179Crossref PubMed Scopus (4083) Google Scholar). The PCR results were normalized with U6 RNA as an internal control and then expressed as relative expression compared with untreated samples. For detection of HER2/neu expression, the cells were treated with fluorescein isothiocyanate-conjugated anti-ErbB2 antibody (Abcam) at 4 °C for 30 min and analyzed with a LSRII flow cytometer (BD, Franklin Lakes, NJ). For detection of mCherry expression, the cells were directly analyzed with the LSRII flow cytometer. For detection of miR-21 by Northern blotting, RNA was extracted by using the TRIzol reagent according to the manufacturer's protocol (Invitrogen). 10 μg of total RNA was electrophoretically separated on a 10% polyacrylamide denaturing gel. Subsequently RNA was transferred to a Hybond-N+ membrane (Amersham Biosciences) by using a semi-dry Transblot electrophoresis apparatus (Bio-Rad). The RNA was cross-linked to the membrane by using UV radiation. Hybridization was carried out by using ULTRAHybOligo solution according to the manufacturer's instructions (Ambion). The probe sequence was complementary to the mature form of miR-21 and was labeled with γ-32P. After washing, the membranes were imaged using phosphorimaging. Western blot analysis of PDCD4 expression was performed as previously described (25.Zhu S. Si M.L. Wu H. Mo Y.Y. J. Biol. Chem. 2007; 282: 14328-14336Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar). In brief, the protein concentration was determined by protein assay kit (Bio-Rad), and the samples were separated in 9% SDS-polyacrylamide gels. After probing with a primary antibody, the membrane was incubated with secondary antibodies labeled with IRDye 800CW. Finally; signal intensity was determined with the Odyssey infrared imaging system and associated software (LI-COR Biosciences, Lincoln, NE). Relative expression of PDCD4 was normalized against the internal control β-actin. Transfection of MDA-MB-435 and BT-474 cells with anti-miR-21 or a control inhibitor (Dharmacon, Chicago, IL) was performed using the DharmaFECT small interfering RNA transfection reagents (Dharmacon) according to the manufacturer's protocol. Transfection of HeLa cells and BT-474 cells with DNA plasmids was performed using Lipofectamine 2000 according to the manufacturer's protocol (Invitrogen). Lentiviruses were generated essentially as previously described (16.Ventura A. Meissner A. Dillon C.P. McManus M. Sharp P.A. Van Parijs L. Jaenisch R. Jacks T. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 10380-10385Crossref PubMed Scopus (514) Google Scholar). Briefly, 4 μg of lentiviral vector and 4 μg of a mixture of packaging vectors were cotransfected in 293T cells using FuGENE 6 (Roche Applied Science). The supernatants were collected 48–96 h after transfection, filtered through a 0.45-μm filter, and used directly to infect cancer cells. The invasive potential of cells was measured in 24-well Matrigel-coated invasion chambers following the manufacturer's instructions (Millipore, Billerica, MA). After the cells were incubated for 24–48 h at 37 °C in a humidified incubator with 5% CO2, the noninvading cells that remained on the upper surface of the membrane were removed by scraping. Invading cells were stained using 4′,6-diamidino-2-phenylindole and counted in three randomly selected fields in the center of each membrane under fluorescence microscopy at medium power fields (100× or 200×). To detect the effect of HER2/neu on the direct miR-21 target PDCD4 expression at the cellular level, both MDA-MB-435/HER2 MDA-MB-435/Neo cells were directly immunostained with anti-PDCD4 antibody. Alternatively, MDA-MB-435/HER2 cells were transfected with anti-miR-21, or MDA-MB-435/Neo cells were transfected with miR-21 expression vector, followed by immunostaining with anti-PDCD4 antibody. The transfected cells were seeded on cover slides 24 h after transfection and fixed with 3% paraformaldehyde as described previously (26.Wu F. Chiocca S. Beck W.T. Mo Y.Y. Mol. Cancer Ther. 2007; 6: 1823-1830Crossref PubMed Scopus (26) Google Scholar). After incubation with primary PDCD4 antibody (Rockland, Gilbertsville, PA), second antibody conjugated with Alexa Fluor 560 nm (Invitrogen) was used to reveal PDCD4 signals. The images were taken under a fluorescent microscope (Olympus, Center Valley, PA). Overexpression of HER2/neu enhances local tumor invasion and lung metastasis of breast cancer cells (4.Guy C.T. Webster M.A. Schaller M. Parsons T.J. Cardiff R.D. Muller W.J. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 10578-10582Crossref PubMed Scopus (1025) Google Scholar). Previously, miR-21 and other MicroRNAs have been shown to increase the metastatic potential of breast cancers (9.Zhu S. Wu H. Wu F. Nie D. Sheng S. Mo Y.Y. Cell Res. 2008; 18: 350-359Crossref PubMed Scopus (990) Google Scholar, 10.Ma L. Teruya-Feldstein J. Weinberg R.A. Nature. 2007; 449: 682-688Crossref PubMed Scopus (2236) Google Scholar), and HER2/neu signaling activates several transcription factors that have been shown to regulate miR-21 expression in various cancer cell lines (11.Fujita S. Ito T. Mizutani T. Minoguchi S. Yamamichi N. Sakurai K. Iba H. J. Mol. Biol. 2008; 378: 492-504Crossref PubMed Scopus (367) Google Scholar, 22.Löffler D. Brocke-Heidrich K. Pfeifer G. Stocsits C. Hackermller J. Kretzschmar A.K. Burger R. Gramatzki M. Blumert C. Bauer K. Cvijic H. Ullmann A.K. Stadler P.F. Horn F. Blood. 2007; 110: 1330-1333Crossref PubMed Scopus (562) Google Scholar). We therefore explored the possibility that HER2/neu signaling induces the expression of miR-21 and/or other microRNAs, which could in turn contribute to the increased metastatic potential of these cancer cells. We first examined whether miR-21 level is up-regulated in the HER2/neu-transfected MDA-MB-435 human breast cancer cell line, because overexpression of HER2/neu significantly enhances invasion and metastasis in this cell line without altering its cell growth rates, anchorage-independent growth, and other indices of tumorigenic potential (3.Tan M. Yao J. Yu D. Cancer Res. 1997; 57: 1199-1205PubMed Google Scholar). We observed that HER2/neu expression was sufficient to up-regulate mature miR-21 levels 2.9 ± 0.3-fold in MDA-MB-435 cells (Fig. 1A). To verify that HER2/neu is sufficient to induce miR-21 up-regulation in other cancer cells and identify other HER2/neu-dependent microRNAs, we assessed the changes in miRNA expression in the HeLa/HER2 cells compared with vector control cells by using miRNA microarrays. We chose HeLa cells because of the fact that some cervical carcinoma tumors overexpress HER2/neu (27.Chavez-Blanco A. Perez-Sanchez V. Gonzalez-Fierro A. Vela-Chavez T. Candelaria M. Cetina L. Vidal S. Dueñas-Gonzalez A. BMC Cancer. 2004; 4: 59Crossref PubMed Scopus (58) Google Scholar), and the transfection of HER2/neu expression vector is more feasible and efficient in this cell line. A HeLa cell line was generated that stably expresses HER2/neu (Fig. 1B), and surprisingly, only a single signature from the array was significantly elevated in the HeLa/HER2 cells: the highly conserved miR-21 miRNA (Fig. 1C). This observation was validated using qRT-PCR, which showed that mature miR-21 expression in HeLa/HER2 cells was increased 2.5 ± 0.5-fold compared with control cells (Fig. 1D). Northern blot analysis further confirmed these analyses, showing a moderate but significant up-regulation of miR-21 expression (Fig. 1E). Our results demonstrate that HER2/neu overexpression up-regulated miR-21 significantly in both MDA-MB-435 and HeLa cells. HER2/neu overexpression may stabilize the miR-21 level in breast cancer cells, thus resulting in higher miR-21 level compared with control cells. To examine whether HER2/neu signaling is able to induce de novo miR-21 expression, we followed the kinetics of miR-21 induction by assessing the relative abundance of its primary transcript and mature sequence upon HER2/neu signaling in different cancer cells. A HER2/neu agonist antibody (anti-HER2/neu ScFv-TNF-α (S147Y)) was used to induce HER2/neu signaling in various HER2/neu-expressing cells lines (21.Huang T.H. Morrison S.L. J. Pharmacol. Exp. Ther. 2006; 316: 983-991Crossref PubMed Scopus (27) Google Scholar). Anti-HER2/neu ScFv-TNF-α (S147Y) (HER2/neu agonist) is a ScFv antibody specific for human HER2/neu fused to a mutant TNF-α with abrogated biological activity. This HER2/neu agonist has previously been shown to form a homotrimeric structure via the noncovalent binding of the mutant TNF-α and is therefore able to cross-link multiple HER2/neu receptors, thereby potently stimulating the tyrosine phosphorylation of HER2/neu receptor and the downstream AKT and MAPK signaling (21.Huang T.H. Morrison S.L. J. Pharmacol. Exp. Ther. 2006; 316: 983-991Crossref PubMed Scopus (27) Google Scholar). HER2/neu agonist treatment induced a rapid and substantial pri-miR-21 up-regulation in HeLa/HER2 (Fig. 2A), SKBR3 (Fig. 2B), and BT-474 (Fig. 2C) cells after 4 h, and the level of pri-miR-21 slightly decreased after 8 h of stimulation. In contrast, the kinetics of mature miR-21 processing in these cells is highly distinct. HER2/neu agonist treatment of HeLa/HER2 cells for 10 h caused a 3.4 ± 0.4-fold up-regulation of mature miR-21 expression as compared with untreated HeLa/HER2 cells. In contrast, no significant miR-21 up-regulation was observed in vector control cells treated with the HER2/neu agonist as compared with untreated cells (Fig. 2A). In contrast to HeLa/HER2 cells, up-regulation of mature miR-21 was delayed in the HER2/neu-overexpressing breast cancer cell lines SKBR3 (Fig. 2B) and BT-474 (Fig. 2C). Mature miR-21 was increased 3.2 ± 0.2- and 2.6 ± 0.2-fold in SKBR3 and BT-474 cells, respectively, stimulated with HER2/neu agonist for 4 days. These results suggest that HER2/neu signaling induces rapid transcription of pri-miR-21; however, processing to mature miR-21 is less efficient in SKBR3 and BT-474 breast cancer cells. Recently it has been shown that the expression of DICER1 is low in several HER2/neu-positive tumors (28.Blenkiron C. Goldstein L.D. Thorne N.P. Spiteri I. Chin S.F. Dunning M.J. Barbosa-Morais N.L. Teschendorff A.E. Green A.R. Ellis I.O. Tavaré S. Caldas C. Miska E.A. Genome Biol. 2007; 8: R214Crossref PubMed Scopus (810) Google Scholar), which may explain the delay in achieving increased mature miR-21 expression in SKBR3 and BT-474 cells. Given the significant but moderate level of miR-21 up-regulation, we wanted to determine whether a ∼3-fold change in miR-21 expression would mitigate a change in target mRNA expression. To study the potential functional consequence of miR-21 up-regulation in response to HER2/neu signaling, we took advantage of a lentivirus construct encoding a fluorescent reporter gene (mCherry) containing two exact miR-21-binding sites in its 3′-untranslated region (mCherry miR-21 sensor). A similar construct with miR-155-binding sites (mCherry miR-155 sensor) was used as a control vector because miR-155 levels do not change significantly in response to HER2/neu expression (data not shown). SKBR3 cells infected with the mCherry miR-155 sensor exhibited strong reporter expression, indicating that the miR-155 level is very low or undetectable in HER2/neu-overexpressing breast cancer cells (Fig. 2D, left panel). Upon treatment with the HER2/neu agonist, the mCherry miR-155 sensor exhibited a slight increase of the reporter signal (Fig. 2D, left panel), probably because of the widespread activation of transcription factors induced by HER2/neu signaling. In contrast, SKBR3 cells infected with the mCherry miR-21 sensor exhibited a moderate reporter signal, indicating that the mCherry mRNA sequence is silenced by endogenous miR-21 expression in these cells (Fig. 2D, right panel). After treatment with HER2/neu agonist, the miR-21 sensor exhibited an even greater repression (50%) of the reporter signal (Fig. 2D, right panel), indicating that the ∼3-fold miR-21 expression level change induced by the HER2/neu signaling is sufficient to repress target gene expression and may therefore have a functional consequence in cancer cells by repressing endogenous target mRNAs. The RAS-MEK1/2-ERK1/2 and phosphatidylinositol 3-kinase-AKT pathways are two major signaling cascades downstream of activated HER2/neu. To test which pathway is responsible for the miR-21 up-regulation, we treated HER2/neu-overexpressing cells BT-474 with an inhibitor of ERK1/2 (U0126) or AKT (LY294002) and subsequently stimulated these cells for 4 days with HER2/neu agonist. Vehicle-treated cells up-regulated miR-21 levels by 3.2 ± 0.2-fold after stimulation, whereas the ERK1/2 inhibitor completely blocked miR-21 induction by the HER2/neu agonist (Fig. 3A) in a dose-dependent manner (Fig. 3B). In contrast, the AKT inhibitor (LY294002) did not reduce HER2/neu-dependent miR-21 up-regulation (Fig. 3A). We also observed that treatment with the ERK1/2 inhibitor repressed the miR-21 induction by HER2/neu agonist in SKBR3 cells and HER2/neu transfected HeLa cells (Fig. 3C). Finally, miR-21 level increased 100% in 293T cells transfected with a wild type ERK2 but decreased 30% in 293T cells transfected with a dominant negative ERK2 (Fig. 3D). These findings indicate that ERK1/2 is a key mediator for the HER2/neu-dependent miR-21 up-regulation and that this regulatory mechanism is conserved among several different cell types. We next examined which transcription factors activate miR-21 transcription. The miR-21 promoter contains a highly conserved region spanning 300 bp in which two STAT3-binding sites are present, and interleukin-6 treatment can induce miR-21 expression via STAT3 in myeloma cells (22.Löffler D. Brocke-Heidrich K. Pfeifer G. Stocsits C. Hackermller J. Kretzschmar A.K. Burger R. Gramatzki M. Blumert C. Bauer K. Cvijic H. Ullmann A.K. Stadler P.F. Horn F. Blood. 2007; 110: 1330-1333Crossref PubMed Scopus (562) Google Scholar). Three
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