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• W3044149721 abstract "Transcriptional repressor zinc finger and BTB domain containing 1 (ZBTB1) is required for DNA repair. Because DNA repair defects often underlie genome instability and tumorigenesis, we determined to study the role of ZBTB1 in cancer. In this study, we found that ZBTB1 is down-regulated in breast cancer and this down-regulation is associated with poor outcome of breast cancer patients. ZBTB1 suppresses breast cancer cell proliferation and tumor growth. The majority of breast cancers are estrogen receptor (ER) positive and selective estrogen receptor modulators such as tamoxifen have been widely used in the treatment of these patients. Unfortunately, many patients develop resistance to endocrine therapy. Tamoxifen-resistant cancer cells often exhibit higher HER2 expression and an increase of glycolysis. Our data revealed that ZBTB1 plays a critical role in tamoxifen resistance in vitro and in vivo. To see if ZBTB1 regulates HER2 expression, we tested the recruitments of ZBTB1 on HER2 regulatory sequences. We observed that over-expressed ZBTB1 occupies the estrogen receptor α (ERα)-binding site of the HER2 intron in tamoxifen-resistant cells, suppressing tamoxifen-induced transcription. In an effort to identify potential microRNAs (miRNAs) regulating ZBTB1, we found that miR-23b-3p directly targets ZBTB1. MiR-23b-3p regulates HER2 expression and tamoxifen resistance via targeting ZBTB1. Finally, we found that miR-23b-3p/ZBTB1 regulates aerobic glycolysis in tamoxifen-resistant cells. Together, our data demonstrate that ZBTB1 is a tumor suppressor in breast cancer cells and that targeting the miR-23b-3p/ZBTB1 may serve as a potential therapeutic approach for the treatment of tamoxifen resistant breast cancer. Transcriptional repressor zinc finger and BTB domain containing 1 (ZBTB1) is required for DNA repair. Because DNA repair defects often underlie genome instability and tumorigenesis, we determined to study the role of ZBTB1 in cancer. In this study, we found that ZBTB1 is down-regulated in breast cancer and this down-regulation is associated with poor outcome of breast cancer patients. ZBTB1 suppresses breast cancer cell proliferation and tumor growth. The majority of breast cancers are estrogen receptor (ER) positive and selective estrogen receptor modulators such as tamoxifen have been widely used in the treatment of these patients. Unfortunately, many patients develop resistance to endocrine therapy. Tamoxifen-resistant cancer cells often exhibit higher HER2 expression and an increase of glycolysis. Our data revealed that ZBTB1 plays a critical role in tamoxifen resistance in vitro and in vivo. To see if ZBTB1 regulates HER2 expression, we tested the recruitments of ZBTB1 on HER2 regulatory sequences. We observed that over-expressed ZBTB1 occupies the estrogen receptor α (ERα)-binding site of the HER2 intron in tamoxifen-resistant cells, suppressing tamoxifen-induced transcription. In an effort to identify potential microRNAs (miRNAs) regulating ZBTB1, we found that miR-23b-3p directly targets ZBTB1. MiR-23b-3p regulates HER2 expression and tamoxifen resistance via targeting ZBTB1. Finally, we found that miR-23b-3p/ZBTB1 regulates aerobic glycolysis in tamoxifen-resistant cells. Together, our data demonstrate that ZBTB1 is a tumor suppressor in breast cancer cells and that targeting the miR-23b-3p/ZBTB1 may serve as a potential therapeutic approach for the treatment of tamoxifen resistant breast cancer. Estrogen receptor (ER) α is the key mediator of estrogen functions in the breast and plays prominent roles in breast cancer (1Clemons M. Goss P. Estrogen and the risk of breast cancer.N. Engl. J. Med. 2001; 344 (11172156): 276-28510.1056/NEJM200101253440407Crossref PubMed Scopus (801) Google Scholar, 2Germain D. Estrogen carcinogenesis in breast cancer.Endocrinol. Metab. Clinics N. Am. 2011; 40 (vii) (21889715): 473-48410.1016/j.ecl.2011.05.009Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 3Cuesta R. Berman A.Y. Alayev A. Holz M.K. Estrogen receptor alpha promotes protein synthesis by fine-tuning the expression of the eukaryotic translation initiation factor 3 subunit f (eIF3f).J. Biol. Chem. 2019; 294 (30573685): 2267-227810.1074/jbc.RA118.004383Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar). In common with other nuclear receptors, ERα regulates target genes by recruiting transcriptional co-regulators and components of the basal transcription machinery (4Thomas C. Gustafsson J.A. The different roles of ER subtypes in cancer biology and therapy.Nat. Rev. Cancer. 2011; 11 (21779010): 597-60810.1038/nrc3093Crossref PubMed Scopus (441) Google Scholar, 5Manavathi B. Dey O. Gajulapalli V.N. Bhatia R.S. Bugide S. Kumar R. Derailed estrogen signaling and breast cancer: an authentic couple.Endocr. Rev. 2013; 34 (22947396): 1-3210.1210/er.2011-1057Crossref PubMed Scopus (71) Google Scholar, 6Shang Y. Hu X. DiRenzo J. Lazar M.A. Brown M. Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription.Cell. 2000; 103 (11136970): 843-85210.1016/S0092-8674(00)00188-4Abstract Full Text Full Text PDF PubMed Scopus (1394) Google Scholar). The ligand-bound ERα, depending on the nature of the ligand, recruits and interacts with coregulatory proteins that can either enhance (co-activators) or repress (co-repressors) its transcriptional activity. Ligand-activated ERα binds to estrogen-response elements (ERE) of target genes such as trefoil factor 1 (TFF1) and c-Myc (7Klinge C.M. Estrogen receptor interaction with estrogen response elements.Nucleic Acids Res. 2001; 29 (11452016): 2905-291910.1093/nar/29.14.2905Crossref PubMed Scopus (734) Google Scholar, 8Klinge C.M. Studinski-Jones A.L. Kulakosky P.C. Bambara R.A. Hilf R. Comparison of tamoxifen ligands on estrogen receptor interaction with estrogen response elements.Mol. Cell. Endocrinol. 1998; 143 (9806352): 79-9010.1016/S0303-7207(98)00130-0Crossref PubMed Scopus (22) Google Scholar, 9Green K.A. Carroll J.S. Estrogen-receptor-mediated transcription and the influence of co-factors and chromatin state.Nat. Rev. Cancer. 2007; 7 (17721435): 713-72210.1038/nrc2211Crossref PubMed Scopus (159) Google Scholar). Approximately 70% of all breast cancers are ERα positive at the time of diagnosis, and anti-estrogen therapies, such as tamoxifen, are very important in premenopausal women breast cancer management (10Riggs B.L. Hartmann L.C. Selective estrogen-receptor modulators – mechanisms of action and application to clinical practice.N. Engl. J. Med. 2003; 348 (12584371): 618-62910.1056/NEJMra022219Crossref PubMed Scopus (758) Google Scholar, 11Nilsson S. Koehler K.F. Gustafsson J.A. Development of subtype-selective estrogen receptor-based therapeutics.Nat. Rev. Drug Discov. 2011; 10 (21921919): 778-79210.1038/nrd3551Crossref PubMed Scopus (193) Google Scholar). The selective estrogen receptor modulator, tamoxifen, can bind to the ER and block the interaction between estrogen and the ER (12Shou J. Massarweh S. Osborne C.K. Wakeling A.E. Ali S. Weiss H. Schiff R. Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer.J. Natl. Cancer Inst. 2004; 96 (15199112): 926-93510.1093/jnci/djh166Crossref PubMed Scopus (926) Google Scholar, 13Zheng D. Williams C. Vold J.A. Nguyen J.H. Harnois D.M. Bagaria S.P. McLaughlin S.A. Li Z. Regulation of sex hormone receptors in sexual dimorphism of human cancers.Cancer Lett. 2018; 438 (30223066): 24-3110.1016/j.canlet.2018.09.001Crossref PubMed Scopus (4) Google Scholar). Tamoxifen is the most prolific therapeutic drug for the treatment of ER-positive breast cancer, showing effective tumor growth inhibition and prevention of disease recurrence (14Early Breast Cancer T. Collaborative G. Effects of chemotherapy and hormonal therapy for early breast cancer on recurrence and 15-year survival: an overview of the randomised trials.Lancet. 2005; 365: 1687-171710.1016/S0140-6736(05)66544-0Abstract Full Text Full Text PDF PubMed Scopus (5876) Google Scholar, 15Smith C.L. Nawaz Z. O'Malley B.W. Coactivator and corepressor regulation of the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen.Mol. Endocrinol. 1997; 11 (9171229): 657-66610.1210/mend.11.6.0009Crossref PubMed Scopus (0) Google Scholar). Unfortunately, not all patients with ER-positive breast cancer respond to tamoxifen. Moreover, many patients that receive tamoxifen as adjuvant therapy eventually acquire tamoxifen resistance. Therefore, studies on the mechanism of tamoxifen resistance are very important to improve the prognosis of breast cancer patients (16Ali S. Coombes R.C. Endocrine-responsive breast cancer and strategies for combating resistance.Nat. Rev. Cancer. 2002; 2 (12635173): 101-11210.1038/nrc721Crossref PubMed Scopus (675) Google Scholar, 17Osborne C.K. Schiff R. Mechanisms of endocrine resistance in breast cancer.Annu. Rev. Med. 2011; 62 (20887199): 233-24710.1146/annurev-med-070909-182917Crossref PubMed Scopus (715) Google Scholar, 18Santiago-Gómez A. Kedward T. Simoes B.M. Dragoni I. NicAmhlaoibh R. Trivier E. Sabin V. Gee J.M. Sims A.H. Howell S.J. Clarke R.B. PAK4 regulates stemness and progression in endocrine resistant ER-positive metastatic breast cancer.Cancer Lett. 2019; 458 (31121213): 66-7510.1016/j.canlet.2019.05.014Crossref PubMed Scopus (13) Google Scholar, 19Gu J. Wang Y. Wang X. Zhou D. Shao C. Zhou M. He Z. Downregulation of lncRNA GAS5 confers tamoxifen resistance by activating miR-222 in breast cancer.Cancer Lett. 2018; 434 (29969658): 1-1010.1016/j.canlet.2018.06.039Crossref PubMed Scopus (90) Google Scholar). It has been recognized that activation of the tyrosine kinase HER2 (human epidermal growth factor receptor-2) is one of the major mechanisms contributing to the tamoxifen resistance (20Osborne C.K. Bardou V. Hopp T.A. Chamness G.C. Hilsenbeck S.G. Fuqua S.A. Wong J. Allred D.C. Clark G.M. Schiff R. Role of the estrogen receptor coactivator AIB1 (SRC-3) and HER-2/neu in tamoxifen resistance in breast cancer.J. Natl. Cancer Inst. 2003; 95 (12618500): 353-36110.1093/jnci/95.5.353Crossref PubMed Scopus (665) Google Scholar). HER2 is amplified and overexpressed in 20 to 30% of invasive breast cancers and has been implicated as a major player in tamoxifen resistance (12Shou J. Massarweh S. Osborne C.K. Wakeling A.E. Ali S. Weiss H. Schiff R. Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer.J. Natl. Cancer Inst. 2004; 96 (15199112): 926-93510.1093/jnci/djh166Crossref PubMed Scopus (926) Google Scholar, 21Shang Y. Brown M. Molecular determinants for the tissue specificity of SERMs.Science. 2002; 295 (11923541): 2465-246810.1126/science.1068537Crossref PubMed Scopus (967) Google Scholar, 22Cui J. Germer K. Wu T. Wang J. Luo J. Wang S.C. Wang Q. Zhang X. Cross-talk between HER2 and MED1 regulates tamoxifen resistance of human breast cancer cells.Cancer Res. 2012; 72 (22964581): 5625-563410.1158/0008-5472.CAN-12-1305Crossref PubMed Scopus (67) Google Scholar, 23Wang M. Hu Y. Yu T. Ma X. Wei X. Wei Y. Pan-HER-targeted approach for cancer therapy: mechanisms, recent advances and clinical prospect.Cancer Lett. 2018; 439 (30218688): 113-13010.1016/j.canlet.2018.07.014Crossref PubMed Scopus (6) Google Scholar). Some breast tumors that exhibit tamoxifen resistance are characterized by elevated HER2 levels, and acquired tamoxifen-resistant cell lines exhibit HER2 overexpression. Some ERα-positive tumors with the poor outcome tend to have high HER2 levels (24Knowlden J.M. Hutcheson I.R. Jones H.E. Madden T. Gee J.M. Harper M.E. Barrow D. Wakeling A.E. Nicholson R.I. Elevated levels of epidermal growth factor receptor/c-erbB2 heterodimers mediate an autocrine growth regulatory pathway in tamoxifen-resistant MCF-7 cells.Endocrinology. 2003; 144 (12586780): 1032-104410.1210/en.2002-220620Crossref PubMed Scopus (449) Google Scholar, 25Mills J.N. Rutkovsky A.C. Giordano A. Mechanisms of resistance in estrogen receptor positive breast cancer: overcoming resistance to tamoxifen/aromatase inhibitors.Curr. Opin. Pharmacol. 2018; 41 (29719270): 59-6510.1016/j.coph.2018.04.009Crossref PubMed Scopus (48) Google Scholar, 26Kirkegaard T. McGlynn L.M. Campbell F.M. Muller S. Tovey S.M. Dunne B. Nielsen K.V. Cooke T.G. Bartlett J.M. Amplified in breast cancer 1 in human epidermal growth factor receptor: positive tumors of tamoxifen-treated breast cancer patients.Clin. Cancer Res. 2007; 13 (17332282): 1405-141110.1158/1078-0432.CCR-06-1933Crossref PubMed Scopus (96) Google Scholar). Although HER2 overexpression is not the universal mechanism for all tamoxifen-resistant tumors, it is an important strategy to repress HER2 expression for overcoming tamoxifen resistance in some breast cancer patients. Paired box gene 2 has been shown to play a critical role in HER2 expression in tamoxifen-resistant cells and paired box gene 2 predicts clinical outcome of tamoxifen therapy in breast cancer patients (12Shou J. Massarweh S. Osborne C.K. Wakeling A.E. Ali S. Weiss H. Schiff R. Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer.J. Natl. Cancer Inst. 2004; 96 (15199112): 926-93510.1093/jnci/djh166Crossref PubMed Scopus (926) Google Scholar, 27Hurtado A. Holmes K.A. Geistlinger T.R. Hutcheson I.R. Nicholson R.I. Brown M. Jiang J. Howat W.J. Ali S. Carroll J.S. Regulation of ERBB2 by estrogen receptor-PAX2 determines response to tamoxifen.Nature. 2008; 456 (19005469): 663-66610.1038/nature07483Crossref PubMed Scopus (246) Google Scholar). The cross-talk between HER2 and ERα has been well-established. There is an ERα-binding site on the HER2 intron and ERα is responsible for HER2 regulation (27Hurtado A. Holmes K.A. Geistlinger T.R. Hutcheson I.R. Nicholson R.I. Brown M. Jiang J. Howat W.J. Ali S. Carroll J.S. Regulation of ERBB2 by estrogen receptor-PAX2 determines response to tamoxifen.Nature. 2008; 456 (19005469): 663-66610.1038/nature07483Crossref PubMed Scopus (246) Google Scholar). In tamoxifen-sensitive cell line MCF-7, tamoxifen represses HER2 expression through co-repressor recruitments by ERα. However, tamoxifen does not repress HER2 expression in tamoxifen-resistant cells for ERα recruits co-activators instead of co-repressors onto HER2 intron (27Hurtado A. Holmes K.A. Geistlinger T.R. Hutcheson I.R. Nicholson R.I. Brown M. Jiang J. Howat W.J. Ali S. Carroll J.S. Regulation of ERBB2 by estrogen receptor-PAX2 determines response to tamoxifen.Nature. 2008; 456 (19005469): 663-66610.1038/nature07483Crossref PubMed Scopus (246) Google Scholar). These findings suggest a new mechanism for HER2 regulation in ERα positive breast cancer (22Cui J. Germer K. Wu T. Wang J. Luo J. Wang S.C. Wang Q. Zhang X. Cross-talk between HER2 and MED1 regulates tamoxifen resistance of human breast cancer cells.Cancer Res. 2012; 72 (22964581): 5625-563410.1158/0008-5472.CAN-12-1305Crossref PubMed Scopus (67) Google Scholar, 27Hurtado A. Holmes K.A. Geistlinger T.R. Hutcheson I.R. Nicholson R.I. Brown M. Jiang J. Howat W.J. Ali S. Carroll J.S. Regulation of ERBB2 by estrogen receptor-PAX2 determines response to tamoxifen.Nature. 2008; 456 (19005469): 663-66610.1038/nature07483Crossref PubMed Scopus (246) Google Scholar, 28Mansouri S. Naghavi-Al-Hosseini F. Farahmand L. Majidzadeh-A K. MED1 may explain the interaction between receptor tyrosine kinases and ERα66 in the complicated network of tamoxifen resistance.Eur. J. Pharmacol. 2017; 804 (28322840): 78-8110.1016/j.ejphar.2017.03.026Crossref PubMed Scopus (9) Google Scholar). Transcriptional repressor ZBTB1 is required for DNA repair and lymphoid development. However, the role of ZBTB1 in cancer remains to be investigated (29Kim H. Dejsuphong D. Adelmant G. Ceccaldi R. Yang K. Marto J.A. D'Andrea A.D. Transcriptional repressor ZBTB1 promotes chromatin remodeling and translesion DNA synthesis.Mol. Cell. 2014; 54 (24657165): 107-11810.1016/j.molcel.2014.02.017Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 30Siggs O.M. Li X. Xia Y. Beutler B. ZBTB1 is a determinant of lymphoid development.J. Exp. Med. 2012; 209 (22201126): 19-2710.1084/jem.20112084Crossref PubMed Scopus (27) Google Scholar, 31Cao X. Ma X.X. Du J.L. Zeng Y. Zhang X.Y. Lu Y. Xue Y.J. Ma P. Chang Q.Y. Li L.J. Zhou X.Y. Cai K.Z. Kovalovsky D. Ma Z.R. Reciprocal suppression between Zbtb1 expression and IL-7Rα signaling during T-cell development.J. Cell. Mol. Med. 2018; 22: 4012-401510.1111/jcmm.13663Crossref Scopus (3) Google Scholar). Here, we found that ZBTB1 is down-regulated in breast cancer and the low expression of ZBTB1 indicates poor outcome in breast cancer treatments. ZBTB1 sensitized resistant cells to tamoxifen through HER2 regulation. Moreover, we found that miR-23b-3p directly target ZBTB1 and then regulate tamoxifen resistance and aerobic glycolysis. POK/ZBTB is an emerging family of transcriptional factors that regulates cancer initiation and progression. In search of novel POK/ZBTB proteins that may play critical roles in breast cancer, we interrogated the Cancer Genome Atlas (TCGA) data using UALCAN (32Chandrashekar D.S. Bashel B. Balasubramanya S.A.H. Creighton C.J. Ponce-Rodriguez I. Chakravarthi B. Varambally S. UALCAN: a portal for facilitating tumor subgroup gene expression and survival analyses.Neoplasia. 2017; 19 (28732212): 649-65810.1016/j.neo.2017.05.002Crossref PubMed Scopus (1381) Google Scholar) and found that ZBTB1, a member of POK/ZBTB family was significantly down-regulated at mRNA level in breast tumors compared with normal tissues. Although each major subtype of breast cancer exhibits lower level of ZBTB1, a significant lower level of ZBTB1 was observed in HER2-positive and triple-negative than luminal breast tumors (Fig. 1A). To study the alteration of ZBTB1 protein expression, we performed immunohistochemistry (IHC) using patient breast tissues and found that ZBTB1 protein exhibited a lower level in breast tumors than normal tissues (Fig. 1, B and C). Breast cancer with low ZBTB1 expressions had shorter overall survival (Fig. 1D). These results indicated that ZBTB1 may be a novel suppressor for breast cancer. Because ZBTB1 is down-regulated in breast cancer, we decided to carry out a series of experiments to examine the effect of ZBTB1 on the growth of breast cancer cells. Cell proliferation assays revealed that overexpression of ZBTB1 reduced the proliferation of MCF-7 and T47D cells (Fig. 2, A and B), whereas ZBTB1 shRNA treatment enhanced the proliferation of these two cell lines (Fig. 2, C and D). To exclude the off-target effect of ZBTB1 shRNA, we reintroduced shRNA-resistant ZBTB1 into knockdown MCF-7 cells and found that this construct of ZBTB1 restored cell proliferation (Fig. S1). Soft agar assay showed that ZBTB1 inhibited anchorage-independent MCF-7 and T47D cell growth (Fig. 2, E and F). To examine the effect of ZBTB1 on tumor growth in vivo, ZBTB1 stably expressing cells were injected into nude mice. As shown in Fig. 2, G and H, the decreases in the sizes and weights of tumors excised from animals of the ZBTB1-overexpressing group were observed as compared with those of the control group. Taken together, these data indicate that ZBTB1 plays an important role in the growth of breast cancer cells both in vitro and in vivo. ∼70% of breast cancers are ER-positive and thus disruption of ER function using tamoxifen is the main therapeutic strategy employed in targeting the disease. However, tamoxifen resistance is a major challenge in breast cancer treatment (12Shou J. Massarweh S. Osborne C.K. Wakeling A.E. Ali S. Weiss H. Schiff R. Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer.J. Natl. Cancer Inst. 2004; 96 (15199112): 926-93510.1093/jnci/djh166Crossref PubMed Scopus (926) Google Scholar, 22Cui J. Germer K. Wu T. Wang J. Luo J. Wang S.C. Wang Q. Zhang X. Cross-talk between HER2 and MED1 regulates tamoxifen resistance of human breast cancer cells.Cancer Res. 2012; 72 (22964581): 5625-563410.1158/0008-5472.CAN-12-1305Crossref PubMed Scopus (67) Google Scholar). To investigate the role of ZBTB1 in tamoxifen resistance in breast cancer, we generated the tamoxifen-resistant cell line MCF-7/TamR. We then measured ZBTB1 expression using Western blotting in cells. As shown in Fig. 3A, ZBTB1 presented at a scarcely detectable level in MCF-7/TamR cells. We next investigated if ZBTB1 could be involved in tamoxifen resistance in MCF-7/TamR cells. We found that re-expression of ZBTB1 was capable of sensitizing MCF-7/TamR cells to tamoxifen (Fig. 3B). To confirm this result, we carried out these experiments in another HER2 overexpression and tamoxifen-resistant cell line, BT474, and we obtained similar result (Fig. S2, A and B). In addition, we found that knockdown of ZBTB1 conferred tamoxifen resistance in MCF-7 cells (Fig. S3, A and B). An increasing number of studies have shown that tamoxifen plays an agonist role in ERα-mediated transcription in tamoxifen-resistant cells (12Shou J. Massarweh S. Osborne C.K. Wakeling A.E. Ali S. Weiss H. Schiff R. Mechanisms of tamoxifen resistance: increased estrogen receptor-HER2/neu cross-talk in ER/HER2-positive breast cancer.J. Natl. Cancer Inst. 2004; 96 (15199112): 926-93510.1093/jnci/djh166Crossref PubMed Scopus (926) Google Scholar, 22Cui J. Germer K. Wu T. Wang J. Luo J. Wang S.C. Wang Q. Zhang X. Cross-talk between HER2 and MED1 regulates tamoxifen resistance of human breast cancer cells.Cancer Res. 2012; 72 (22964581): 5625-563410.1158/0008-5472.CAN-12-1305Crossref PubMed Scopus (67) Google Scholar). In this study, we found that re-expression of ZBTB1 could inhibit ERα transcriptional activity by tamoxifen treatment in MCF-7/TamR cells (Fig. 3C). However, knockdown of ZBTB1 induced ERα transcriptional activity by tamoxifen treatment in MCF-7 cells (Fig. S3C). To determine the role of ZBTB1 on tamoxifen resistance in vivo, ZBTB1 stably expressing cells were injected into nude mice and then mice were treated with tamoxifen. As expected, tamoxifen treatment alone did not significantly alter tumor growth compared with the vehicle control group in mice injected with MCF-7/TamR cells. However, tamoxifen treatment could further potentiate tumor growth inhibition by ZBTB1 (Fig. 3, D and E). These results suggest that ZBTB1 sensitizes breast cancer cells to tamoxifen in vivo. HER2 has been shown to play a critical role in tamoxifen resistance. Our data above showed that ZBTB1 exhibits a much lower level in HER2 overexpressing than luminal breast cancer as well normal tissues. This result indicates a reverse association between HER2 and ZBTB1 expressions. To investigate whether ZBTB1 could regulate HER2 expression in tamoxifen-resistant cells, we first compared HER2 expression in MCF-7/TamR with MCF-7 cells and found that HER2 was present at significantly higher levels in MCF-7/TamR cells (Fig. 4A). We then investigated the effect of ZBTB1 on HER2 expression and found that re-expression of ZBTB1 inhibited HER2 expression at both mRNA and protein levels in the presence of tamoxifen (Fig. 4, B and C, Fig. S4, A and B). Our and other groups' studies show that ERα and its co-activators, such as amplified in breast cancer 1 (AIB1) and mediator subunit 1 (MED1) bind HER2 intron to regulate HER2 expression in tamoxifen-resistant cells (22Cui J. Germer K. Wu T. Wang J. Luo J. Wang S.C. Wang Q. Zhang X. Cross-talk between HER2 and MED1 regulates tamoxifen resistance of human breast cancer cells.Cancer Res. 2012; 72 (22964581): 5625-563410.1158/0008-5472.CAN-12-1305Crossref PubMed Scopus (67) Google Scholar, 27Hurtado A. Holmes K.A. Geistlinger T.R. Hutcheson I.R. Nicholson R.I. Brown M. Jiang J. Howat W.J. Ali S. Carroll J.S. Regulation of ERBB2 by estrogen receptor-PAX2 determines response to tamoxifen.Nature. 2008; 456 (19005469): 663-66610.1038/nature07483Crossref PubMed Scopus (246) Google Scholar). To study the molecular mechanisms under HER2 regulation by ZBTB1, we performed chromatin immunoprecipitation (ChIP) assays to detect the ERα, AIB1, and MED1 recruitments on ERα-binding region of HER2 intron in MCF-7/TamR and BT474 cells. As a result, we found that re-expression of ZBTB1 diminished MED1 and AIB1 but not ERα recruitments induced by tamoxifen (Fig. 4D, Fig. S4C). However, re-expression of ZBTB1 restored co-repressors such as N-CoR (nuclear receptor co-repressor 1) and HDAC1 (histone deacetylase 1) recruitments by ERα onto HER2 intron (Fig. 4E, Fig. S4D). In addition, we found that knockdown of ZBTB1 induced co-activator recruitments but not co-repressor under tamoxifen treatment in MCF-7 cells (Fig. S3, D and E). The above data suggest that ZBTB1 alter ERα co-regulator recruitments onto HER2 intron. Then, we examined the possibility of interaction between ZBTB1 and ERα. The endogenous ZBTB1 protein from MCF-7 cells was immunoprecipitated with an anti-ZBTB1 antibody. Subsequent immunoblotting with anti-ERα antibody indicated that the endogenous ERα was co-precipitated with ZBTB1 in the presence of tamoxifen (Fig. 4F). ChIP–re-ChIP assay confirmed that ZBTB1 interacted with ERα onto the ERα-binding site of HER2 intron (Fig. S5). Finally, we investigate ZBTB1 recruitments by tamoxifen in MCF-7 and MCF-7/TamR cells. As shown in Fig. 4G, ZBTB1 was present on the ERα-binding site of HER2 intron in MCF-7 but not MCF-7/TamR cells. However, re-expression of ZBTB1 restored its occupy on HER2 intron in MCF-7/TamR cells. To further investigate the role of ZBTB1 in tamoxifen resistance, we used 2 target prediction programs, TargetScan and miRanda, to predict miRNAs that target ZBTB1. Our analysis predicted 6 potential ZBTB1-targeting miRNAs. First, we investigate microRNAs, which are dysregulated in tamoxifen-resistant cells and found only miR-23b-3p exhibited a higher level in MCF-7/TamR than MCF-7 cells (Fig. S6). Then, we performed Western blotting and found miR-23b-3p overexpression decreased ZBTB1 expression in MCF-7 and T47D cells, however, miR-23b-3p inhibition increased ZBTB1 expression in MCF-7/TamR and BT474 cells (Fig. 5, A–D, Fig. S7, A–D). Thus, we decided to study the role of miR-23b-3p in tamoxifen resistance. We generated WT and mutant ZBTB1 3′-UTR expression plasmids that were fused to a luciferase reporter according to the matched sequence between ZBTB1 3′-UTR and miR-23b-3p (Fig. 5E). We found that miR-23b-3p overexpression significantly inhibited luciferase activity of WT but not mutant reporter genes in MCF-7 and T47D cells. However, miR-23b-3p silencing specifically enhanced the luciferase activity of WT but not mutant ZBTB1 3′-UTR in MCF-7/TamR and BT474 cells (Fig. 5, F and G, Fig. S7, E and F). To investigate the role of miR-23b-3p in tamoxifen resistance, we transfected MCF-7/TamR and BT474 cells with anti-miR-23b-3p oligo and performed MTT (3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Our results showed that miR-23b-3p inhibition significantly sensitized resistant cells to tamoxifen treatment. In addition, knockdown of ZBTB1 diminished the effect of miR-23b-3p inhibition on tamoxifen response in MCF-7/TamR and BT474 cells (Fig. 6A, Fig. S8A). Our data also indicated that miR-23b-3p inhibition suppressed tamoxifen-induced HER2 expression at mRNA and protein levels via targeting ZBTB1 (Fig. 6, B and C, Fig. S8, B and C). We then performed ChIP assays to detect the effect of miR-23b-3P on ERα and its coregulator recruitments on the ERα-binding region of HER2 intron in MCF-7/TamR and BT474 cells. As a result, we found that miR-23b-3p knockdown diminished MED1 and AIB1 but not ERα recruitments induced by tamoxifen (Fig. 6D, Fig. S8D). However, miR-23b-3p knockdown restored co-repressors such as N-CoR and HDAC1 recruitments by ERα onto HER2 intron (Fig. 6E, Fig. S8E). Recent studies established the association of aerobic glycolysis and tamoxifen resistance. Inhibition of aerobic glycolysis can reverse tamoxifen resistance (33He M. Jin Q. Chen C. Liu Y. Ye X. Jiang Y. Ji F. Qian H. Gan D. Yue S. Zhu W. Chen T. The miR-186-3p/EREG axis orchestrates tamoxifen resistance and aerobic glycolysis in breast cancer cells.Oncogene. 2019; 38: 5551-556510.1038/s41388-019-0817-3Crossref PubMed Scopus (27) Google Scholar, 34Wang J. Duan Z. Nugent Z. Zou J.X. Borowsky A.D. Zhang Y. Tepper C.G. Li J.J. Fiehn O. Xu J. Kung H.J. Murphy L.C. Chen H.W. Reprograming metabolism by histone methyltransferase NSD2 drives endocrine resistance via coordinated activation of pentose phosphate pathway enzymes.Cancer Lett. 2016; 378 (27164560): 69-7910.1016/j.canlet.2016.05.004Crossref PubMed Scopus (33) Google Scholar). To investigate the role of miR-23b-3p/ZBTB1 in aerobic glycolysis, we decided to carry out a series of experiments on tamoxifen-resistant cells. Lactate production and glucose uptake increases were observed in MCF-7/TamR cells compared with MCF-7 cells (Fig. S9). More importantly, miR-23b-3p inhibition or ZBTB1 re-expression significantly reduced lactate production and glycose uptake in MCF-7/TamR and BT474 cells in the presence of tamoxifen (Fig. 7A–D, Fig. S10, A–D). Western blotting indicated that miR-23b-3p inhibition or ZBTB1 re-expression suppressed the expressions of HKII (hexokinase II) and LDHA (lactate dehydrogenase A), which are critical for the glycolytic pathway (Fig. 7E, Fig. S10E). Moreover, knockdown of ZBTB1 diminished the inhibitory effect of anti-miR-23b-3p oligo on lactate production and glucose uptake (Fig. 7, F and G). Our Western blotting assays also indicated that miR-23b-3p regulates HKII and LDHA expressions via targeting ZBTB1 (Fig. 7H). These results suggested the role of miR-23b-3p/ZBTB1 in aerobic glycolysis of tamoxifen-resistant cells. Through this study, we have identified a key role of the ZBTB1 protein in tamoxifen resistance through HE" @default.
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• W3044149721 date "2020-10-01" @default.
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• W3044149721 title "A novel tumor suppressor ZBTB1 regulates tamoxifen resistance and aerobic glycolysis through suppressing HER2 expression in breast cancer" @default.
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