Induction of miR-21 by Retinoic Acid in Estrogen Receptor-positive Breast Carcinoma Cells

Retinoids are promising agents for the treatment/prevention of breast carcinoma. We examined the role of microRNAs in mediating the effects of all-trans-retinoic acid (ATRA), which suppresses the proliferation of estrogen receptor-positive (ERα+) breast carcinoma cells, such as MCF-7, but not estrogen receptor-negative cells, such as MDA-MB-231. We found that pro-oncogenic miR-21 is selectively induced by ATRA in ERα+ cells. Induction of miR-21 counteracts the anti-proliferative action of ATRA but has the potentially beneficial effect of reducing cell motility. In ERα+ cells, retinoid-dependent induction of miR-21 is due to increased transcription of the MIR21 gene via ligand-dependent activation of the nuclear retinoid receptor, RARα. RARα is part of the transcription complex present in the 5′-flanking region of the MIR21 gene. The receptor binds to two functional retinoic acid-responsive elements mapping upstream of the transcription initiation site. Silencing of miR-21 enhances ATRA-dependent growth inhibition and senescence while reverting suppression of cell motility afforded by the retinoid. Up-regulation of miR-21 results in retinoid-dependent inhibition of the established target, maspin. Knockdown and overexpression of maspin in MCF-7 cells indicates that the protein is involved in ATRA-induced growth inhibition and contributes to the ATRA-dependent anti-motility responses. Integration between whole genome analysis of genes differentially regulated by ATRA in MCF-7 and MDA-MB-231 cells, prediction of miR-21 regulated genes, and functional studies led to the identification of three novel direct miR-21 targets: the pro-inflammatory cytokine IL1B, the adhesion molecule ICAM-1 and PLAT, the tissue-type plasminogen activator. Evidence for ICAM-1 involvement in retinoid-dependent inhibition of MCF-7 cell motility is provided. Retinoids are promising agents for the treatment/prevention of breast carcinoma. We examined the role of microRNAs in mediating the effects of all-trans-retinoic acid (ATRA), which suppresses the proliferation of estrogen receptor-positive (ERα+) breast carcinoma cells, such as MCF-7, but not estrogen receptor-negative cells, such as MDA-MB-231. We found that pro-oncogenic miR-21 is selectively induced by ATRA in ERα+ cells. Induction of miR-21 counteracts the anti-proliferative action of ATRA but has the potentially beneficial effect of reducing cell motility. In ERα+ cells, retinoid-dependent induction of miR-21 is due to increased transcription of the MIR21 gene via ligand-dependent activation of the nuclear retinoid receptor, RARα. RARα is part of the transcription complex present in the 5′-flanking region of the MIR21 gene. The receptor binds to two functional retinoic acid-responsive elements mapping upstream of the transcription initiation site. Silencing of miR-21 enhances ATRA-dependent growth inhibition and senescence while reverting suppression of cell motility afforded by the retinoid. Up-regulation of miR-21 results in retinoid-dependent inhibition of the established target, maspin. Knockdown and overexpression of maspin in MCF-7 cells indicates that the protein is involved in ATRA-induced growth inhibition and contributes to the ATRA-dependent anti-motility responses. Integration between whole genome analysis of genes differentially regulated by ATRA in MCF-7 and MDA-MB-231 cells, prediction of miR-21 regulated genes, and functional studies led to the identification of three novel direct miR-21 targets: the pro-inflammatory cytokine IL1B, the adhesion molecule ICAM-1 and PLAT, the tissue-type plasminogen activator. Evidence for ICAM-1 involvement in retinoid-dependent inhibition of MCF-7 cell motility is provided. IntroductionAll-trans-retinoic acid (ATRA) 2The abbreviations used are: ATRA, all-trans-retinoic acid; ERα, estrogen receptor type α; miRNA, microRNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ICAM-1, intercellular adhesion molecule 1. and derivatives (retinoids) are promising agents in the treatment/chemoprevention of hematologic and other malignancies (1Garattini E. Gianni M. Terao M. Curr. Pharm. Des. 2007; 13: 1375-1400Crossref PubMed Scopus (65) Google Scholar, 2Garattini E. Gianni M. Terao M. Curr. Pharm. Des. 2004; 10: 433-448Crossref PubMed Scopus (58) Google Scholar), including breast carcinoma (3Uray I.P. Brown P.H. Expert Opin. Investig. Drugs. 2006; 15: 1583-1600Crossref PubMed Scopus (21) Google Scholar). Breast cancer is an heterogeneous group of tumors with variable response to therapeutic agents, including retinoids. Generally, breast carcinoma cells expressing estrogen receptor type α (ERα+) are sensitive to, whereas the ERα-negative (ERα−) counterparts are refractory to, the anti-proliferative activity of retinoids (1Garattini E. Gianni M. Terao M. Curr. Pharm. Des. 2007; 13: 1375-1400Crossref PubMed Scopus (65) Google Scholar).MicroRNAs (miRNAs) are short RNAs controlling the stability of target mRNAs or their translation into protein products (4Brodersen P. Voinnet O. Nat. Rev. Mol. Cell Biol. 2009; 10: 141-148Crossref PubMed Scopus (538) Google Scholar). They influence cell homeostasis and response to drugs (5Gregory P.A. Bert A.G. Paterson E.L. Barry S.C. Tsykin A. Farshid G. Vadas M.A. Khew-Goodall Y. Goodall G.J. Nat. Cell Biol. 2008; 10: 593-601Crossref PubMed Scopus (3115) Google Scholar, 6Bhat-Nakshatri P. Wang G. Collins N.R. Thomson M.J. Geistlinger T.R. Carroll J.S. Brown M. Hammond S. Srour E.F. Liu Y. Nakshatri H. Nucleic Acids Res. 2009; 37: 4850-4861Crossref PubMed Scopus (276) Google Scholar), modulating the activity of numerous target transcripts simultaneously, via binding to the 3′-untranslated region. Little is known about the effects of retinoids on miRNAs in breast carcinoma and/or other neoplasias (7Fazi F. Rosa A. Fatica A. Gelmetti V. De Marchis M.L. Nervi C. Bozzoni I. Cell. 2005; 123: 819-831Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar).MCF-7 breast carcinoma cells are ERα+, whereas the MDA-MB-231 counterparts are ERα− (8Mandal S. Davie J.R. BMC Cancer. 2007; 7: 181Crossref PubMed Scopus (10) Google Scholar) (9Jönsson G. Staaf J. Olsson E. Heidenblad M. Vallon-Christersson J. Osoegawa K. de Jong P. Oredsson S. Ringnér M. Höglund M. Borg A. Genes Chromosomes Cancer. 2007; 46: 543-558Crossref PubMed Scopus (162) Google Scholar). MCF-7 are sensitive, whereas MDA-MB-231 cells are refractory to the transcriptional and proliferative effects of E2. The pair of cell lines is a model (10Patel J.B. Mehta J. Belosay A. Sabnis G. Khandelwal A. Brodie A.M. Soprano D.R. Njar V.C. Br. J. Cancer. 2007; 96: 1204-1215Crossref PubMed Scopus (35) Google Scholar, 11del Rincón S.V. Guo Q. Morelli C. Shiu H.Y. Surmacz E. Miller W.H. Oncogene. 2004; 23: 9269-9279Crossref PubMed Scopus (52) Google Scholar, 12Kogai T. Schultz J.J. Johnson L.S. Huang M. Brent G.A. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 8519-8524Crossref PubMed Scopus (131) Google Scholar, 13Chen Y. Dokmanovic M. Stein W.D. Ardecky R.J. Roninson I.B. Cancer Res. 2006; 66: 8749-8761Crossref PubMed Scopus (36) Google Scholar, 14Fu H. Yang G. Lu F. Wang R. Yao L. Lu Z. Biochem. Biophys. Res. Commun. 2006; 343: 1009-1016Crossref PubMed Scopus (8) Google Scholar, 15Lu M. Mira-y-Lopez R. Nakajo S. Nakaya K. Jing Y. Oncogene. 2005; 24: 4362-4369Crossref PubMed Scopus (48) Google Scholar, 16Zhu W.Y. Jones C.S. Amin S. Matsukuma K. Haque M. Vuligonda V. Chandraratna R.A. De Luca L.M. Cancer Res. 1999; 59: 85-90PubMed Google Scholar) for the association between ERα positivity and response to the anti-proliferative effects of retinoids.We used predominantly the MCF-7 and MDA-MB-231 cell lines to study the effects of ATRA and derivatives on miRNA expression. miR-21 was the only miRNA whose expression was perturbed by the retinoid. Retinoid-dependent induction of the miRNA was observed in MCF-7 and other ERα+ cell lines. The consequences of miR-21 induction were evaluated in terms of retinoid-dependent functional responses and gene expression.DISCUSSIONWe demonstrated that treatment of retinoid-sensitive ERα+ breast carcinoma cells with ATRA resulted in the induction of miR-21. Up-regulation of miR-21 was the consequence of increased transcription of the corresponding gene via selective activation of RARα. This was due to a direct effect of the ligand-activated receptor on two functional RAREs, mapping to the 5′-flanking region of MIR21.Induction of the oncogenic miR-21 by an anti-proliferative agent like ATRA in ERα+ breast carcinoma cells was unexpected. For this reason, we deemed it important to establish whether miR-21 induction was involved in some of the cellular responses potentially underlying the therapeutic activity of ATRA. In the MCF-7 context, miR-21 counteracted the anti-proliferative and pro-senescence effects of ATRA. This is consistent with the reported role of miR-21 on the growth and progression of breast carcinoma, suggesting that induction by ATRA is part of an uncharacterized negative feedback loop similar to the one activated by E2 in ERα+ breast cancer cells (6Bhat-Nakshatri P. Wang G. Collins N.R. Thomson M.J. Geistlinger T.R. Carroll J.S. Brown M. Hammond S. Srour E.F. Liu Y. Nakshatri H. Nucleic Acids Res. 2009; 37: 4850-4861Crossref PubMed Scopus (276) Google Scholar, 58Castellano L. Giamas G. Jacob J. Coombes R.C. Lucchesi W. Thiruchelvam P. Barton G. Jiao L.R. Wait R. Waxman J. Hannon G.J. Stebbing J. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 15732-15737Crossref PubMed Scopus (289) Google Scholar) or interferon in colon cancer cells (59Yang C.H. Yue J. Fan M. Pfeffer L.M. Cancer Res. 2010; 70: 8108-8116Crossref PubMed Scopus (143) Google Scholar). On the other hand, the results of the studies on the motility of MCF-7 and T47D cells performed with anti-miR-21 imply a role for the miRNA in mediating inhibition of this process by ATRA. The anti-motility action of miR-21 in ATRA-treated ERα+ cells is apparently against the idea that the miRNA is pro-invasive for breast cancer cells, when three-dimensional in vitro assays are used (54Zhu S. Wu H. Wu F. Nie D. Sheng S. Mo Y.Y. Cell Res. 2008; 18: 350-359Crossref PubMed Scopus (970) Google Scholar). This discrepancy probably reflects the absence of localized stimuli guiding the direction of the cell movement in the motility assay used in this study. Another point to be considered stems from the results obtained after calculation of persistence (ratio of the direct distance from start point to end point divided by the total track distance) in ATRA-treated MCF-7 and T47D cells transfected with anti-miR-21 or anti-NC. In these conditions, silencing of miR-21 was associated with decreased persistence in both cell lines (MCF-7: anti-miR-21 = 0.17 ± 0.01, anti-NC = 0.24 ± 0.02; mean ± S.E., n = 30; and T47D: anti-miR-21 = 0.15 ± 0.01, anti-NC = 0.27 ± 0.02; mean ± S.E., n = 30). Altogether, our data suggest that miR-21 enhances instantaneous velocity. However, this phenomenon does not translate into efficient migration because of the observed decrease in persistence. Clearly the effect of miR-21 on motility is complex and further complicated by the concomitant cellular responses activated by ATRA.Apparently, our results provide contrasting evidence regarding the role of miR-21 induction in terms of the overall anti-cancer activity of retinoids. In fact, induction of miR-21 seems to be detrimental for the growth inhibitory effect of ATRA. As such, strategies aimed at suppressing this effect should enhance the anti-proliferative action of the retinoid. On the other hand, the observations on cell motility suggest a potentially beneficial role of miR-21 induction for the anti-metastatic activity of retinoids. However, caution should be exercised in drawing any conclusion on this point. Indeed, it must be stressed that motility is clearly only one component of the complex process of cancer cell dissemination, which is modulated by many other factors that are not considered with the random motility assay employed in this study.Among the few validated miR-21 targets, maspin is the only one modulated by ATRA via induction of the miRNA in MCF-7 cells. In this context, it is of particular relevance that expression of two established miR-21 target genes, PTEN and PDCD4, inhibiting growth and survival of cancer cells (37Frankel L.B. Christoffersen N.R. Jacobsen A. Lindow M. Krogh A. Lund A.H. J. Biol. Chem. 2008; 283: 1026-1033Abstract Full Text Full Text PDF PubMed Scopus (983) Google Scholar, 60Knobbe C.B. Lapin V. Suzuki A. Mak T.W. Oncogene. 2008; 27: 5398-5415Crossref PubMed Scopus (99) Google Scholar) was left unaltered (PTEN) or was increased (PDCD4) in ATRA-treated MCF-7 cells. This indicates that the two proteins do not play a significant role in miR-21-dependent suppression of retinoid-triggered growth inhibition. In contrast, maspin, which is also endowed with onco-suppressor properties, is one of the miR-21 targets contributing to ATRA-dependent growth inhibition of MCF-7 cells, as suggested by our silencing and overexpression experiments. In line with down-regulation of maspin by miR-21, our data support a role for the protein in mediating ATRA- and miR-21-dependent inhibition of cell motility as well. The relevance of our observations for the reported role of certain forms of maspin in suppressing the metastatic potential of cancer cells (47Latha K. Zhang W. Cella N. Shi H.Y. Zhang M. Mol. Cell Biol. 2005; 25: 1737-1748Crossref PubMed Scopus (73) Google Scholar, 61Joensuu K.M. Leidenius M.H. Andersson L.C. Heikkila P.S. Hum. Pathol. 2009; 40: 1143-1151Crossref PubMed Scopus (19) Google Scholar) remains to be established.One of the most important outcomes of our study is the identification of three novel and functionally validated miR-21 targets: ICAM-1, PLAT, and IL1B. These three genes are regulated by miR-21 primarily at the mRNA level.ICAM-1 is an adhesion molecule, and its down-regulation by miR-21 is not involved in cell growth. In contrast, silencing of ICAM-1 resulted in reduction of MCF-7 random cell motility, whereas overexpression of the protein exerted an opposite effect. These data indicate that inhibition of ICAM-1 by ATRA-dependent induction of miR-21 mediates at least part of the anti-motility effects exerted by the miRNA in retinoid-treated cells, consistent with the described pro-motility action of the protein in breast carcinoma cells (62Rosette C. Roth R.B. Oeth P. Braun A. Kammerer S. Ekblom J. Denissenko M.F. Carcinogenesis. 2005; 26: 943-950Crossref PubMed Scopus (205) Google Scholar, 63Strell C. Lang K. Niggemann B. Zaenker K.S. Entschladen F. Exp. Cell Res. 2010; 316: 138-148Crossref PubMed Scopus (66) Google Scholar).PLAT codes for a fibrinolytic factor whose induction in cells lacking miR-21 up-regulation may be part of a stereotyped response to ATRA, which is anti-thrombotic (64Marchetti M. Vignoli A. Bani M.R. Balducci D. Barbui T. Falanga A. Haematologica. 2003; 88: 895-905PubMed Google Scholar). Inhibition of PLAT expression by miR-21, as observed in MCF-7 cells, does not seem to play a role in the anti-proliferative effects exerted by ATRA. Nevertheless, PLAT down-regulation may be therapeutically desirable, because stimulation of a thrombotic response in cancer cells is associated with tumor growth and progression in vivo (65Rickles F.R. Thromb. Res. 2009; 123: S16-S20Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Although PLAT activates the motility of neural crest cells (66Erickson C.A. Isseroff R.R. J. Exp. Zool. 1989; 251: 123-133Crossref PubMed Scopus (44) Google Scholar), involvement of the protein in the miR-21-dependent anti-motility effects observed in ATRA-treated ERα+ cells is once again ruled out by silencing and overexpression studies.Finally, IL1B is an inflammatory protein, and our microarray data indicate that ATRA up-regulates inflammation-related genes in ERα− MDA-MB-231 cells but not in ERα+ MCF-7 cells. Based on the presence of several potential miR-21 targets in this group of genes, including IL1B, we propose that retinoid-dependent miR-21 induction inhibits, either directly or indirectly, certain aspects of the inflammatory responses otherwise activated by retinoids in breast cancer cells. Because inflammation is known to be involved in tumor growth in vivo (67Mantovani A. Allavena P. Sica A. Balkwill F. Nature. 2008; 454: 436-444Crossref PubMed Scopus (7612) Google Scholar), the proposed anti-inflammatory action of miR-21 may play a positive role in the overall therapeutic responses to retinoids.In conclusion, this is the first demonstration that MIR21 is a direct retinoid target gene only in retinoid-sensitive ERα+ breast carcinoma cells. The mechanisms underlying cell context specificity are unknown, although simple anti-estrogenic effects are unlikely. The role of miR-21 and the three novel targets: ICAM-1, PLAT, and IL1B, in retinoid-induced anti-proliferative, pro-senescence, and anti-motility effects requires further study, because it has implications for the therapeutic use of these agents. IntroductionAll-trans-retinoic acid (ATRA) 2The abbreviations used are: ATRA, all-trans-retinoic acid; ERα, estrogen receptor type α; miRNA, microRNA; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ICAM-1, intercellular adhesion molecule 1. and derivatives (retinoids) are promising agents in the treatment/chemoprevention of hematologic and other malignancies (1Garattini E. Gianni M. Terao M. Curr. Pharm. Des. 2007; 13: 1375-1400Crossref PubMed Scopus (65) Google Scholar, 2Garattini E. Gianni M. Terao M. Curr. Pharm. Des. 2004; 10: 433-448Crossref PubMed Scopus (58) Google Scholar), including breast carcinoma (3Uray I.P. Brown P.H. Expert Opin. Investig. Drugs. 2006; 15: 1583-1600Crossref PubMed Scopus (21) Google Scholar). Breast cancer is an heterogeneous group of tumors with variable response to therapeutic agents, including retinoids. Generally, breast carcinoma cells expressing estrogen receptor type α (ERα+) are sensitive to, whereas the ERα-negative (ERα−) counterparts are refractory to, the anti-proliferative activity of retinoids (1Garattini E. Gianni M. Terao M. Curr. Pharm. Des. 2007; 13: 1375-1400Crossref PubMed Scopus (65) Google Scholar).MicroRNAs (miRNAs) are short RNAs controlling the stability of target mRNAs or their translation into protein products (4Brodersen P. Voinnet O. Nat. Rev. Mol. Cell Biol. 2009; 10: 141-148Crossref PubMed Scopus (538) Google Scholar). They influence cell homeostasis and response to drugs (5Gregory P.A. Bert A.G. Paterson E.L. Barry S.C. Tsykin A. Farshid G. Vadas M.A. Khew-Goodall Y. Goodall G.J. Nat. Cell Biol. 2008; 10: 593-601Crossref PubMed Scopus (3115) Google Scholar, 6Bhat-Nakshatri P. Wang G. Collins N.R. Thomson M.J. Geistlinger T.R. Carroll J.S. Brown M. Hammond S. Srour E.F. Liu Y. Nakshatri H. Nucleic Acids Res. 2009; 37: 4850-4861Crossref PubMed Scopus (276) Google Scholar), modulating the activity of numerous target transcripts simultaneously, via binding to the 3′-untranslated region. Little is known about the effects of retinoids on miRNAs in breast carcinoma and/or other neoplasias (7Fazi F. Rosa A. Fatica A. Gelmetti V. De Marchis M.L. Nervi C. Bozzoni I. Cell. 2005; 123: 819-831Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar).MCF-7 breast carcinoma cells are ERα+, whereas the MDA-MB-231 counterparts are ERα− (8Mandal S. Davie J.R. BMC Cancer. 2007; 7: 181Crossref PubMed Scopus (10) Google Scholar) (9Jönsson G. Staaf J. Olsson E. Heidenblad M. Vallon-Christersson J. Osoegawa K. de Jong P. Oredsson S. Ringnér M. Höglund M. Borg A. Genes Chromosomes Cancer. 2007; 46: 543-558Crossref PubMed Scopus (162) Google Scholar). MCF-7 are sensitive, whereas MDA-MB-231 cells are refractory to the transcriptional and proliferative effects of E2. The pair of cell lines is a model (10Patel J.B. Mehta J. Belosay A. Sabnis G. Khandelwal A. Brodie A.M. Soprano D.R. Njar V.C. Br. J. Cancer. 2007; 96: 1204-1215Crossref PubMed Scopus (35) Google Scholar, 11del Rincón S.V. Guo Q. Morelli C. Shiu H.Y. Surmacz E. Miller W.H. Oncogene. 2004; 23: 9269-9279Crossref PubMed Scopus (52) Google Scholar, 12Kogai T. Schultz J.J. Johnson L.S. Huang M. Brent G.A. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 8519-8524Crossref PubMed Scopus (131) Google Scholar, 13Chen Y. Dokmanovic M. Stein W.D. Ardecky R.J. Roninson I.B. Cancer Res. 2006; 66: 8749-8761Crossref PubMed Scopus (36) Google Scholar, 14Fu H. Yang G. Lu F. Wang R. Yao L. Lu Z. Biochem. Biophys. Res. Commun. 2006; 343: 1009-1016Crossref PubMed Scopus (8) Google Scholar, 15Lu M. Mira-y-Lopez R. Nakajo S. Nakaya K. Jing Y. Oncogene. 2005; 24: 4362-4369Crossref PubMed Scopus (48) Google Scholar, 16Zhu W.Y. Jones C.S. Amin S. Matsukuma K. Haque M. Vuligonda V. Chandraratna R.A. De Luca L.M. Cancer Res. 1999; 59: 85-90PubMed Google Scholar) for the association between ERα positivity and response to the anti-proliferative effects of retinoids.We used predominantly the MCF-7 and MDA-MB-231 cell lines to study the effects of ATRA and derivatives on miRNA expression. miR-21 was the only miRNA whose expression was perturbed by the retinoid. Retinoid-dependent induction of the miRNA was observed in MCF-7 and other ERα+ cell lines. The consequences of miR-21 induction were evaluated in terms of retinoid-dependent functional responses and gene expression.