Role of the Nuclear Receptor Coactivator AIB1-Δ4 Splice Variant in the Control of Gene Transcription
2011; Elsevier BV; Volume: 286; Issue: 30 Linguagem: Inglês
10.1074/jbc.m110.216200
ISSN1083-351X
AutoresChristopher D. Chien, Alexander Kirilyuk, Jordan V. Li, Wentao Zhang, Tyler Lahusen, Marcel O. Schmidt, Annabell S. Oh, Anton Wellstein, Anna T. Riegel,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoThe oncogene amplified in breast cancer 1 (AIB1) is a nuclear receptor coactivator that plays a major role in the progression of various cancers. We previously identified a splice variant of AIB1 called AIB1-Δ4 that is overexpressed in breast cancer. Using mass spectrometry, we define the translation initiation of AIB1-Δ4 at Met224 of the full-length AIB1 sequence and have raised an antibody to a peptide representing the acetylated N terminus. We show that AIB1-Δ4 is predominantly localized in the cytoplasm, although leptomycin B nuclear export inhibition demonstrates that AIB1-Δ4 can enter and traffic through the nucleus. Our data indicate an import mechanism enhanced by other coactivators such as p300/CBP. We report that the endogenously and exogenously expressed AIB1-Δ4 is recruited as efficiently as full-length AIB1 to estrogen-response elements of genes, and it enhances estrogen-dependent transcription more effectively than AIB1. Expression of an N-terminal AIB1 protein fragment, which is lost in the AIB1-Δ4 isoform, potentiates AIB1 as a coactivator. This suggests a model whereby the transcriptional activity of AIB1 is squelched by a repressive mechanism utilizing the N-terminal domain and that the increased coactivator function of AIB1-Δ4 is due to the loss of this inhibitory domain. Finally, we show, using Scorpion primer technology, that AIB1-Δ4 expression is correlated with metastatic capability of human cancer cell lines. The oncogene amplified in breast cancer 1 (AIB1) is a nuclear receptor coactivator that plays a major role in the progression of various cancers. We previously identified a splice variant of AIB1 called AIB1-Δ4 that is overexpressed in breast cancer. Using mass spectrometry, we define the translation initiation of AIB1-Δ4 at Met224 of the full-length AIB1 sequence and have raised an antibody to a peptide representing the acetylated N terminus. We show that AIB1-Δ4 is predominantly localized in the cytoplasm, although leptomycin B nuclear export inhibition demonstrates that AIB1-Δ4 can enter and traffic through the nucleus. Our data indicate an import mechanism enhanced by other coactivators such as p300/CBP. We report that the endogenously and exogenously expressed AIB1-Δ4 is recruited as efficiently as full-length AIB1 to estrogen-response elements of genes, and it enhances estrogen-dependent transcription more effectively than AIB1. Expression of an N-terminal AIB1 protein fragment, which is lost in the AIB1-Δ4 isoform, potentiates AIB1 as a coactivator. This suggests a model whereby the transcriptional activity of AIB1 is squelched by a repressive mechanism utilizing the N-terminal domain and that the increased coactivator function of AIB1-Δ4 is due to the loss of this inhibitory domain. Finally, we show, using Scorpion primer technology, that AIB1-Δ4 expression is correlated with metastatic capability of human cancer cell lines. IntroductionGene transcription in eukaryotes is a complex and highly regulated process. One of the major controls of gene transcription is exerted by the coregulator family of proteins. These include both corepressors, which dampen transcription, and coactivators, which potentiate transcription. A subgroup of coactivators has been shown to be critical for the malignant progression of cancer and is known as the p160 steroid receptor coactivators (1Xu J. Wu R.C. O'Malley B.W. Nat. Rev. Cancer. 2009; 9: 615-630Crossref PubMed Scopus (373) Google Scholar). One member in particular was identified to be amplified in breast cancer. Amplified in breast cancer 1 (AIB1, SRC-3, NCOA3, ACTR, TRAM-1, pCIP, and RAC3) has been shown to be a gene amplified in breast cancer (2Anzick S.L. Kononen J. Walker R.L. Azorsa D.O. Tanner M.M. Guan X.Y. Sauter G. Kallioniemi O.P. Trent J.M. Meltzer P.S. Science. 1997; 277: 965-968Crossref PubMed Scopus (1423) Google Scholar) and is also overexpressed at the mRNA and protein level in various cancers (1Xu J. Wu R.C. O'Malley B.W. Nat. Rev. Cancer. 2009; 9: 615-630Crossref PubMed Scopus (373) Google Scholar, 3Lahusen T. Henke R.T. Kagan B.L. Wellstein A. Riegel A.T. Breast Cancer Res. Treat. 2009; 116: 225-237Crossref PubMed Scopus (76) Google Scholar, 4List H.J. Reiter R. Singh B. Wellstein A. Riegel A.T. Breast Cancer Res. Treat. 2001; 68: 21-28Crossref PubMed Scopus (124) Google Scholar). Its role in tumorigenesis is attributed to its ability to coactivate both steroid hormone- and growth factor-dependent transcription (3Lahusen T. Henke R.T. Kagan B.L. Wellstein A. Riegel A.T. Breast Cancer Res. Treat. 2009; 116: 225-237Crossref PubMed Scopus (76) Google Scholar, 5Oh A. List H.J. Reiter R. Mani A. Zhang Y. Gehan E. Wellstein A. Riegel A.T. Cancer Res. 2004; 64: 8299-8308Crossref PubMed Scopus (72) Google Scholar, 6Lahusen T. Fereshteh M. Oh A. Wellstein A. Riegel A.T. Cancer Res. 2007; 67: 7256-7265Crossref PubMed Scopus (53) Google Scholar, 7York B. O'Malley B.W. J. Biol. Chem. 2010; 285: 38743-38750Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). In several oncogene-driven mouse models (8Fereshteh M.P. Tilli M.T. Kim S.E. Xu J. O'Malley B.W. Wellstein A. Furth P.A. Riegel A.T. Cancer Res. 2008; 68: 3697-3706Crossref PubMed Scopus (81) Google Scholar, 9Kuang S.Q. Liao L. Wang S. Medina D. O'Malley B.W. Xu J. Cancer Res. 2005; 65: 7993-8002Crossref PubMed Scopus (97) Google Scholar, 10Kuang S.Q. Liao L. Zhang H. Lee A.V. O'Malley B.W. Xu J. Cancer Res. 2004; 64: 1875-1885Crossref PubMed Scopus (158) Google Scholar, 11Qin L. Liao L. Redmond A. Young L. Yuan Y. Chen H. O'Malley B.W. Xu J. Mol. Cell. Biol. 2008; 28: 5937-5950Crossref PubMed Scopus (157) Google Scholar), reduction of AIB1 levels leads to a decrease in tumorigenesis, and overexpression of AIB1 leads to the formation of various tumors (12Torres-Arzayus M.I. Font de Mora J. Yuan J. Vazquez F. Bronson R. Rue M. Sellers W.R. Brown M. Cancer Cell. 2004; 6: 263-274Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Clinically, AIB1 expression in breast cancer cases is correlated with high HER2 levels, larger tumor size, higher tumor grade, and shorter disease-free survival (13Bautista S. Vallès H. Walker R.L. Anzick S. Zeillinger R. Meltzer P. Theillet C. Clin. Cancer Res. 1998; 4: 2925-2929PubMed Google Scholar, 14Harigopal M. Heymann J. Ghosh S. Anagnostou V. Camp R.L. Rimm D.L. Breast Cancer Res. Treat. 2009; 115: 77-85Crossref PubMed Scopus (41) Google Scholar, 15Thorat M.A. Turbin D. Morimiya A. Leung S. Zhang Q. Jeng M.H. Huntsman D.G. Nakshatri H. Badve S. Histopathology. 2008; 53: 634-641Crossref PubMed Scopus (10) Google Scholar). Also, high levels of AIB1 in conjunction with high HER2 levels coincide with reduced disease-free survival in patients treated with tamoxifen, suggesting a role for AIB1 in tamoxifen resistance (16Osborne 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. J. Natl. Cancer Inst. 2003; 95: 353-361Crossref PubMed Scopus (695) Google Scholar).We had previously identified a splice variant of AIB1, where exon 3 was spliced from the mature mRNA and the resulting protein named AIB1-Δ3 (17Reiter R. Wellstein A. Riegel A.T. J. Biol. Chem. 2001; 276: 39736-39741Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). More recently, an additional 5′ exon 81,164 bases upstream of the known 5′UTR was identified. Thus, the deleted exon is now exon 4, and we now refer to the splice variant as AIB1-Δ4. We had reported that AIB1-Δ4 mRNA results in an N-terminally truncated isoform of the AIB1 protein that was found to be a more potent coactivator of steroid-dependent transcription on a per mol basis when compared with the full-length AIB1 protein. AIB1-Δ4 mRNA expression was elevated in breast tumor tissue relative to normal breast tissue (17Reiter R. Wellstein A. Riegel A.T. J. Biol. Chem. 2001; 276: 39736-39741Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). It was also shown to increase the efficacy of estrogenic compounds and the agonist effects of the selective estrogen receptor modulator tamoxifen in breast and endometrial tumor cells (17Reiter R. Wellstein A. Riegel A.T. J. Biol. Chem. 2001; 276: 39736-39741Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 18Reiter R. Oh A.S. Wellstein A. Riegel A.T. Oncogene. 2004; 23: 403-409Crossref PubMed Scopus (37) Google Scholar). Overexpression of AIB1-Δ4 in mice leads to ductal ectasia in the mammary gland with an increased expression of proliferative markers such as proliferating cell nuclear antigen, phospho-histone H3, and cyclin D1 (19Tilli M.T. Reiter R. Oh A.S. Henke R.T. McDonnell K. Gallicano G.I. Furth P.A. Riegel A.T. Mol. Endocrinol. 2005; 19: 644-656Crossref PubMed Scopus (40) Google Scholar) as well as increased cross-talk with ERα 2The abbreviations used are: ERαestrogen receptor-αEGFRepidermal growth factor receptorNLSnuclear localization sequencebHLHbasic helix loop helixFAKfocal adhesion kinaseANOVAanalysis of varianceIPimmunoprecipitationIMEMIscove's modified Eagle's mediumCCScharcoal-stripped serumEREestrogen-responsive elementSsenseASantisenseHMEChuman mammary epithelial cellMMTVmouse mammary tumor virusPASPer Arnt Sim domain. in epithelial and stromal responses (20Nakles R.E. Shiffert M.T. Díaz-Cruz E.S. Cabrera M.C. Alotaiby M. Miermont A.M. Riegel A.T. Furth P.A. Mol. Endocrinol. 2011; 25: 549-563Crossref PubMed Scopus (16) Google Scholar). More recently, AIB1-Δ4 was shown to act as a molecular bridge between epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) in the cytoplasm, and its overexpression increased the invasiveness of the MDA-MB-231 metastatic breast cancer cell line (21Long W. Yi P. Amazit L. LaMarca H.L. Ashcroft F. Kumar R. Mancini M.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. 2010; 37: 321-332Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar).Because AIB1-Δ4 lacks a nuclear localization sequence (NLS), any function for this protein in cancer to date has been attributed predominantly to its role in the cytoplasm (21Long W. Yi P. Amazit L. LaMarca H.L. Ashcroft F. Kumar R. Mancini M.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. 2010; 37: 321-332Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). We now show that AIB1-Δ4 can enter the nucleus by a noncanonical nuclear import mechanism. AIB1-Δ4 is recruited to estrogen-response elements of endogenous estrogen-regulated genes and increases their expression. We also determined that the N-terminal region absent from the AIB1-Δ4 protein contains an inhibitory domain. Through the use of Scorpion primer technology, we have created the first quantitative assay for the AIB1-Δ4 transcript and found a correlation between AIB1-Δ4 expression and the metastatic phenotype of human cancer cell lines. These data suggest that the nuclear activities of AIB1-Δ4 can contribute to its function in malignancy.DISCUSSIONWe show in this study that AIB1-Δ4 enters the nucleus and has a nuclear function. In accordance with previous data that the NLS is contained in the N terminus of AIB1 (26Li C. Wu R.C. Amazit L. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. Biol. 2007; 27: 1296-1308Crossref PubMed Scopus (38) Google Scholar, 27Qutob M.S. Bhattacharjee R.N. Pollari E. Yee S.P. Torchia J. Mol. Cell. Biol. 2002; 22: 6611-6626Crossref PubMed Scopus (38) Google Scholar, 28Yeung P.L. Zhang A. Chen J.D. Biochem. Biophys. Res. Commun. 2006; 348: 13-24Crossref PubMed Scopus (13) Google Scholar), we found that the majority of AIB1-Δ4, which lacks an NLS, is predominantly in the cytoplasm at steady state levels. This is in contrast to the localization of AIB1, which resides mostly in the nucleus. Coactivation occurs in the nucleus, which raised the question how the mainly cytoplasmic AIB1-Δ4 had such potent effects on transcription. The first question to address was if AIB1-Δ4 enters the nucleus. We observed that AIB1-Δ4 accumulated in the nucleus after blockade of nuclear export suggesting that it was indeed being imported into the nucleus. The next question to address was whether AIB1-Δ4 had a functional role in the nucleus. We saw that AIB1-Δ4 was recruited as efficiently as AIB1 to ERE in estrogen-regulated genes in the nucleus despite significantly lower steady state nuclear levels of AIB1-Δ4. The mainly cytoplasmic location of AIB1-Δ4 is potentially due to either an inefficient nuclear import mechanism or through a rapid nuclear export mechanism. We argue for the former scenario given that AIB1-Δ4 lacks the N-terminal NLS and there is no alteration in the nuclear export sequence of AIB1-Δ4. It is known that molecules larger than 40 kDa have to be actively transported through the nuclear pore complex through interaction of the NLS with nuclear importins (46Stewart M. Nat. Rev. Mol. Cell Biol. 2007; 8: 195-208Crossref PubMed Scopus (640) Google Scholar). Another potential mechanism for nuclear import termed "piggybacking" was demonstrated for various proteins such as eIF4E, IκBα, and CDK2, and BRCA1 (47Dostie J. Ferraiuolo M. Pause A. Adam S.A. Sonenberg N. EMBO J. 2000; 19: 3142-3156Crossref PubMed Scopus (155) Google Scholar, 48Fabbro M. Rodriguez J.A. Baer R. Henderson B.R. J. Biol. Chem. 2002; 277: 21315-21324Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 49Jans D.A. Hübner S. Physiol. Rev. 1996; 76: 651-685Crossref PubMed Scopus (384) Google Scholar, 50Moore J.D. Yang J. Truant R. Kornbluth S. J. Cell Biol. 1999; 144: 213-224Crossref PubMed Scopus (171) Google Scholar, 51Thompson M.E. FEBS J. 2010; 277: 3072-3078Crossref PubMed Scopus (48) Google Scholar, 52Turpin P. Hay R.T. Dargemont C. J. Biol. Chem. 1999; 274: 6804-6812Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). BRCA1 has a naturally occurring splice variant that lacks an NLS, and it is able to localize to the nucleus through interaction with another protein BARD1, which contains a canonical NLS. We propose that AIB1-Δ4 is able to similarly piggyback onto p300/CBP and/or other NLS-containing proteins and thus enter the nucleus. Alternatively, NLS-containing coactivators could up-regulate genes involved in Ran-independent nuclear import mechanisms such as via calmodulin (53Hanover J.A. Love D.C. Prinz W.A. J. Biol. Chem. 2009; 284: 12593-12597Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Whatever the mechanism, it appears to be an inefficient process likely accounting for the largely cytoplasmic distribution of AIB1-Δ4 in the cell. Also consistent with this hypothesis is the difference in the kinetics of recruitment of AIB1 and AIB1-Δ4. We saw a delay in the recruitment of AIB1-Δ4 to the ERE relative to AIB1 suggesting that the mechanism of nuclear import of AIB1-Δ4 is less efficient than that of AIB1.The fact that we saw more effective coactivation by AIB1-Δ4 despite having much more AIB1 in the nucleus than AIB1-Δ4 suggests that there is a regulation of AIB1 coactivator activity that does not exist for AIB1-Δ4. Because we observed that the levels of AIB1-Δ4 interacting with p300 were higher than the levels of AIB1 associated with p300 (Fig. 4a) and p300 is generally found in active transcriptional complexes, we believe that AIB1-Δ4 is highly recruited to sites of active transcription. The increased coactivator activity of AIB1-Δ4 was confirmed by a higher increase in endogenous estrogen-regulated gene expression in cells transfected with AIB1-Δ4. These data suggest that the large amount of AIB1 that resides in the nucleus is not in active transcriptional complexes, and the reason why AIB1-Δ4 is a more potent coactivator than AIB1 can best be explained by the presence of an inhibitory domain in the N-terminal 223 amino acids of AIB1. Alternatively, loss of the N terminus could cause steric changes that increase the affinity for other coactivators. We conjectured that this N-terminal fragment containing the bHLH and PAS A domains would be able to bind N-terminal repressors of AIB1. We found that the N-terminal fragment containing the bHLH and PAS A domains of AIB1 when cotransfected with AIB1 is able to relieve repression of the coactivator function of the AIB1 protein. This effect was dose-dependent, and the more AIB1 N-terminal fragment added to the cells the less repression there was on the AIB1 protein. Interestingly, transfection of the fragment alone showed an increase in luciferase activity, which is probably not due to an inherent coactivator function of this fragment because most of the transcriptional activity of the AIB1 and AIB1-Δ4 proteins resides in their recruitment of p300/CBP in the C terminus (36Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalsky M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1253) Google Scholar). This increase in transcription is most likely due to a relief of repression of the endogenous AIB1 in the COS-7 cells because we are able to detect endogenous AIB1 protein by Western blot (17Reiter R. Wellstein A. Riegel A.T. J. Biol. Chem. 2001; 276: 39736-39741Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Previous studies from our group and others have suggested that the N-terminal region containing the bHLH and PAS A domains contains an inhibitory domain that represses activity in both the nucleus and cytoplasm (17Reiter R. Wellstein A. Riegel A.T. J. Biol. Chem. 2001; 276: 39736-39741Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 18Reiter R. Oh A.S. Wellstein A. Riegel A.T. Oncogene. 2004; 23: 403-409Crossref PubMed Scopus (37) Google Scholar, 21Long W. Yi P. Amazit L. LaMarca H.L. Ashcroft F. Kumar R. Mancini M.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. 2010; 37: 321-332Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 26Li C. Wu R.C. Amazit L. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. Biol. 2007; 27: 1296-1308Crossref PubMed Scopus (38) Google Scholar, 36Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalsky M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1253) Google Scholar). Our studies and those from Chen et al. (36Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalsky M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1253) Google Scholar) have shown that the loss of the N-terminal region leads to potent coactivation of nuclear hormone receptor-mediated transcription. Expression of AIB1-Δ4 (loss of amino acids 1–223 of AIB1) or ACTR38 (loss of amino acids 1–447 of AIB1) leads to potent coactivation of nuclear receptor-dependent transcription from estrogen, progesterone, retinoic acid, thyroid, glucocorticoid, vitamin D, and retinoid X receptors. Interestingly, data from Li et al. (26Li C. Wu R.C. Amazit L. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. Biol. 2007; 27: 1296-1308Crossref PubMed Scopus (38) Google Scholar) showed expression of AIB1 constructs containing mutations in either or both NLS (NLS amino acids 16–19 and 35–38 of AIB1) or with the bHLH domain deleted (amino acids 16–88 of AIB1) had no coactivator activity presumably because of lack of import into the nucleus. The other possibility is that they still retained amino acids 88–224, which would support the evidence that there is an inhibitory domain in this region. Loss of these amino acids in AIB1-Δ4 and ACTR38 allows these proteins to be potent coactivators. A number of proteins have been shown to bind to the bHLH PAS domain of p160 SRC family members. These have been described as having coactivator, corepressor, and phosphatase activity (54Belandia B. Parker M.G. J. Biol. Chem. 2000; 275: 30801-30805Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 55Goel A. Janknecht R. J. Biol. Chem. 2004; 279: 14909-14916Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 56Kim J.H. Li H. Stallcup M.R. Mol. Cell. 2003; 12: 1537-1549Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 57Li C. Liang Y.Y. Feng X.H. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. 2008; 31: 835-849Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 58Wu X. Li H. Chen J.D. J. Biol. Chem. 2001; 276: 23962-23968Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 59Zhang A. Yeung P.L. Li C.W. Tsai S.C. Dinh G.K. Wu X. Li H. Chen J.D. J. Biol. Chem. 2004; 279: 33799-33805Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) depending on the context in which they are examined. It remains to be determined if these proteins play a role in repression or loss of activation of AIB1 on endogenous genes or conversely whether they have lost affinity or changed interaction with AIB1-Δ4.Both AIB1 and AIB1-Δ4 have been shown to have effects in the epidermal growth factor (EGF) signaling. Data from our laboratory has shown that loss of both AIB1 and AIB1-Δ4 proteins together can lead to a decrease in EGF receptor (EGFR) phosphorylation (6Lahusen T. Fereshteh M. Oh A. Wellstein A. Riegel A.T. Cancer Res. 2007; 67: 7256-7265Crossref PubMed Scopus (53) Google Scholar), and AIB1-Δ4 is able to potently coactivate EGF-stimulated transcription (17Reiter R. Wellstein A. Riegel A.T. J. Biol. Chem. 2001; 276: 39736-39741Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Long et al. (21Long W. Yi P. Amazit L. LaMarca H.L. Ashcroft F. Kumar R. Mancini M.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. 2010; 37: 321-332Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) have shown that AIB1-Δ4 acts as a bridging molecule between EGFR and FAK, and this interaction facilitates the motility and metastatic capability of MDA-MB-231 breast cancer cells. They also show that AIB1-Δ4 is phosphorylated by p21-activated kinase (PAK1), which increases the association of AIB1-Δ4 with EGFR and FAK. Intriguingly, in this latter study the N-terminal region of AIB1 inhibited the interaction of AIB1 with FAK and therefore was unable to stimulate the EGF-induced migration of cancer cells, suggesting that even in the cytoplasm the N-terminal region of AIB1 was repressive. Expression of an NLS mutant of AIB1, which resides predominantly in the cytoplasm, showed much weaker interaction with FAK in cells. Another recent publication by Cai et al. (60Cai D. Shames D.S. Raso M.G. Xie Y. Kim Y.H. Pollack J.R. Girard L. Sullivan J.P. Gao B. Peyton M. Nanjundan M. Byers L. Heymach J. Mills G. Gazdar A.F. Wistuba I. Kodadek T. Minna J.D. Cancer Res. 2010; 70: 6477-6485Crossref PubMed Scopus (49) Google Scholar) shows in non-small cell lung cancer that AIB1 expression is correlated with poor prognosis, and knockdown of AIB1 in non-small cell lung cancer cell line resistant to gefitinib treatment restored sensitivity to EGFR inhibition by gefitinib. Taken together, these data indicate that there is a signaling loop existing between AIB1/AIB1-Δ4 and EGFR where EGFR can affect AIB1-Δ4 through PAK1 activation and AIB1/AIB1-Δ4 can affect EGFR signaling and transcription as well. It is unclear if the siRNA used to target AIB1 in the gefitinib study also targeted AIB1-Δ4 as well, so the effects of AIB1-Δ4 on EGFR signaling are not currently known. Overall, the contribution of nuclear versus cytoplasmic function of AIB1-Δ4 to steroid and growth factor signaling needs to be further explored.Given that we have found that the expression of AIB1-Δ4 at the protein level is higher in breast and pancreatic cancer cells (17Reiter R. Wellstein A. Riegel A.T. J. Biol. Chem. 2001; 276: 39736-39741Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 61Henke R.T. Haddad B.R. Kim S.E. Rone J.D. Mani A. Jessup J.M. Wellstein A. Maitra A. Riegel A.T. Clin. Cancer Res. 2004; 10: 6134-6142Crossref PubMed Scopus (108) Google Scholar), we were interested to investigate if AIB1-Δ4 is regulated at the protein level. We have previously shown that AIB1 protein levels are greatly reduced in response to growth of cells at high confluence and the removal of growth factors (62Mani A. Oh A.S. Bowden E.T. Lahusen T. Lorick K.L. Weissman A.M. Schlegel R. Wellstein A. Riegel A.T. Cancer Res. 2006; 66: 8680-8686Crossref PubMed Scopus (58) Google Scholar). Interestingly, we found that the AIB1-Δ4 isoform is not regulated in the same fashion as AIB1 protein (supplemental Fig. 5). AIB1-Δ4 is resistant to proteasomal degradation induced by high confluence. This is probably due to loss of a site of regulation in the N-terminal 223 amino acids. The proteasomal regulation of AIB1 has been well characterized (57Li C. Liang Y.Y. Feng X.H. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. 2008; 31: 835-849Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 62Mani A. Oh A.S. Bowden E.T. Lahusen T. Lorick K.L. Weissman A.M. Schlegel R. Wellstein A. Riegel A.T. Cancer Res. 2006; 66: 8680-8686Crossref PubMed Scopus (58) Google Scholar, 63Li X. Lonard D.M. Jung S.Y. Malovannaya A. Feng Q. Qin J. Tsai S.Y. Tsai M.J. O'Malley B.W. Cell. 2006; 124: 381-392Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). Interestingly, regulation of a phospho-degron at Ser102 by protein phosphatase 1 (PP1) was shown to be important for regulating the activity of AIB1. PP1 overexpression was able to inhibit the reporter activity as well as the cell proliferative ability of AIB1. The Ser102 site is also a site of regulation by the ubiquitin ligase SPOP (64Li C. Ao J. Fu J. Lee D.F. Xu J. Lonard D. O'Malley B.W. Oncogene. 2011; (in press)Google Scholar). This site is lost in AIB1-Δ4 and could explain the high coactivator activity and stability of the AIB1-Δ4 protein.A major question that arises from these studies on AIB1-Δ4 is whether there are distinct biological functions for this isoform. To begin to investigate this, we took advantage of the unique splice junction sequence that exists in the AIB1-Δ4 transcript to develop a new technique to specifically measure the amounts of AIB1-Δ4 mRNA independent of AIB1 transcript. By utilizing Scorpion primer technology, we see higher expression of AIB1-Δ4 in cancer cell lines relative to normal cell lines and higher expression in more metastatic cancer cell lines. Observations on the role of AIB1 in disease do not make the distinction between the relative contribution of AIB1 or AIB1-Δ4 to the phenotype. Studies that analyze mRNA expression of AIB1 utilize primers that detect both AIB1 and AIB1-Δ4. We believe that it will be important to dissect out the contribution to phenotypes attributed to AIB1-Δ4 independent of AIB1 and vice versa in future clinical studies. We have also developed affinity-purified antibodies based on the knowledge of the unique N-acetylated N-terminal sequence of AIB1-Δ4, which will serve as another resource to determine the expression of AIB1-Δ4 at the protein level at various stages of tumorigenesis.The precise role of AIB1-Δ4 in tumorigenesis is not known. It is intriguing that AIB1-Δ4 is not degraded under conditions of low growth (e.g. high confluence), although full-length AIB1 is rapidly lost. AIB1-Δ4 protein expression may provide a selective advantage for a cancer cell to continue to grow in conditions unfavorable for proliferation for both its metastatic and transcriptional function. Therefore, further studies into the regulation and possible targets of AIB1-Δ4 in tumorigenesis need to be pursued. IntroductionGene transcription in eukaryotes is a complex and highly regulated process. One of the major controls of gene transcription is exerted by the coregulator family of proteins. These include both corepressors, which dampen transcription, and coactivators, which potentiate transcription. A subgroup of coactivators has been shown to be critical for the malignant progression of cancer and is known as the p160 steroid receptor coactivators (1Xu J. Wu R.C. O'Malley B.W. Nat. Rev. Cancer. 2009; 9: 615-630Crossref PubMed Scopus (373) Google Scholar). One member in particular was identified to be amplified in breast cancer. Amplified in breast cancer 1 (AIB1, SRC-3, NCOA3, ACTR, TRAM-1, pCIP, and RAC3) has been shown to be a gene amplified in breast cancer (2Anzick S.L. Kononen J. Walker R.L. Azorsa D.O. Tanner M.M. Guan X.Y. Sauter G. Kallioniemi O.P. Trent J.M. Meltzer P.S. Science. 1997; 277: 965-968Crossref PubMed Scopus (1423) Google Scholar) and is also overexpressed at the mRNA and protein level in various cancers (1Xu J. Wu R.C. O'Malley B.W. Nat. Rev. Cancer. 2009; 9: 615-630Crossref PubMed Scopus (373) Google Scholar, 3Lahusen T. Henke R.T. Kagan B.L. Wellstein A. Riegel A.T. Breast Cancer Res. Treat. 2009; 116: 225-237Crossref PubMed Scopus (76) Google Scholar, 4List H.J. Reiter R. Singh B. Wellstein A. Riegel A.T. Breast Cancer Res. Treat. 2001; 68: 21-28Crossref PubMed Scopus (124) Google Scholar). Its role in tumorigenesis is attributed to its ability to coactivate both steroid hormone- and growth factor-dependent transcription (3Lahusen T. Henke R.T. Kagan B.L. Wellstein A. Riegel A.T. Breast Cancer Res. Treat. 2009; 116: 225-237Crossref PubMed Scopus (76) Google Scholar, 5Oh A. List H.J. Reiter R. Mani A. Zhang Y. Gehan E. Wellstein A. Riegel A.T. Cancer Res. 2004; 64: 8299-8308Crossref PubMed Scopus (72) Google Scholar, 6Lahusen T. Fereshteh M. Oh A. Wellstein A. Riegel A.T. Cancer Res. 2007; 67: 7256-7265Crossref PubMed Scopus (53) Google Scholar, 7York B. O'Malley B.W. J. Biol. Chem. 2010; 285: 38743-38750Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). In several oncogene-driven mouse models (8Fereshteh M.P. Tilli M.T. Kim S.E. Xu J. O'Malley B.W. Wellstein A. Furth P.A. Riegel A.T. Cancer Res. 2008; 68: 3697-3706Crossref PubMed Scopus (81) Google Scholar, 9Kuang S.Q. Liao L. Wang S. Medina D. O'Malley B.W. Xu J. Cancer Res. 2005; 65: 7993-8002Crossref PubMed Scopus (97) Google Scholar, 10Kuang S.Q. Liao L. Zhang H. Lee A.V. O'Malley B.W. Xu J. Cancer Res. 2004; 64: 1875-1885Crossref PubMed Scopus (158) Google Scholar, 11Qin L. Liao L. Redmond A. Young L. Yuan Y. Chen H. O'Malley B.W. Xu J. Mol. Cell. Biol. 2008; 28: 5937-5950Crossref PubMed Scopus (157) Google Scholar), reduction of AIB1 levels leads to a decrease in tumorigenesis, and overexpression of AIB1 leads to the formation of various tumors (12Torres-Arzayus M.I. Font de Mora J. Yuan J. Vazquez F. Bronson R. Rue M. Sellers W.R. Brown M. Cancer Cell. 2004; 6: 263-274Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). Clinically, AIB1 expression in breast cancer cases is correlated with high HER2 levels, larger tumor size, higher tumor grade, and shorter disease-free survival (13Bautista S. Vallès H. Walker R.L. Anzick S. Zeillinger R. Meltzer P. Theillet C. Clin. Cancer Res. 1998; 4: 2925-2929PubMed Google Scholar, 14Harigopal M. Heymann J. Ghosh S. Anagnostou V. Camp R.L. Rimm D.L. Breast Cancer Res. Treat. 2009; 115: 77-85Crossref PubMed Scopus (41) Google Scholar, 15Thorat M.A. Turbin D. Morimiya A. Leung S. Zhang Q. Jeng M.H. Huntsman D.G. Nakshatri H. Badve S. Histopathology. 2008; 53: 634-641Crossref PubMed Scopus (10) Google Scholar). Also, high levels of AIB1 in conjunction with high HER2 levels coincide with reduced disease-free survival in patients treated with tamoxifen, suggesting a role for AIB1 in tamoxifen resistance (16Osborne 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. J. Natl. Cancer Inst. 2003; 95: 353-361Crossref PubMed Scopus (695) Google Scholar).We had previously identified a splice variant of AIB1, where exon 3 was spliced from the mature mRNA and the resulting protein named AIB1-Δ3 (17Reiter R. Wellstein A. Riegel A.T. J. Biol. Chem. 2001; 276: 39736-39741Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). More recently, an additional 5′ exon 81,164 bases upstream of the known 5′UTR was identified. Thus, the deleted exon is now exon 4, and we now refer to the splice variant as AIB1-Δ4. We had reported that AIB1-Δ4 mRNA results in an N-terminally truncated isoform of the AIB1 protein that was found to be a more potent coactivator of steroid-dependent transcription on a per mol basis when compared with the full-length AIB1 protein. AIB1-Δ4 mRNA expression was elevated in breast tumor tissue relative to normal breast tissue (17Reiter R. Wellstein A. Riegel A.T. J. Biol. Chem. 2001; 276: 39736-39741Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). It was also shown to increase the efficacy of estrogenic compounds and the agonist effects of the selective estrogen receptor modulator tamoxifen in breast and endometrial tumor cells (17Reiter R. Wellstein A. Riegel A.T. J. Biol. Chem. 2001; 276: 39736-39741Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 18Reiter R. Oh A.S. Wellstein A. Riegel A.T. Oncogene. 2004; 23: 403-409Crossref PubMed Scopus (37) Google Scholar). Overexpression of AIB1-Δ4 in mice leads to ductal ectasia in the mammary gland with an increased expression of proliferative markers such as proliferating cell nuclear antigen, phospho-histone H3, and cyclin D1 (19Tilli M.T. Reiter R. Oh A.S. Henke R.T. McDonnell K. Gallicano G.I. Furth P.A. Riegel A.T. Mol. Endocrinol. 2005; 19: 644-656Crossref PubMed Scopus (40) Google Scholar) as well as increased cross-talk with ERα 2The abbreviations used are: ERαestrogen receptor-αEGFRepidermal growth factor receptorNLSnuclear localization sequencebHLHbasic helix loop helixFAKfocal adhesion kinaseANOVAanalysis of varianceIPimmunoprecipitationIMEMIscove's modified Eagle's mediumCCScharcoal-stripped serumEREestrogen-responsive elementSsenseASantisenseHMEChuman mammary epithelial cellMMTVmouse mammary tumor virusPASPer Arnt Sim domain. in epithelial and stromal responses (20Nakles R.E. Shiffert M.T. Díaz-Cruz E.S. Cabrera M.C. Alotaiby M. Miermont A.M. Riegel A.T. Furth P.A. Mol. Endocrinol. 2011; 25: 549-563Crossref PubMed Scopus (16) Google Scholar). More recently, AIB1-Δ4 was shown to act as a molecular bridge between epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK) in the cytoplasm, and its overexpression increased the invasiveness of the MDA-MB-231 metastatic breast cancer cell line (21Long W. Yi P. Amazit L. LaMarca H.L. Ashcroft F. Kumar R. Mancini M.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. 2010; 37: 321-332Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar).Because AIB1-Δ4 lacks a nuclear localization sequence (NLS), any function for this protein in cancer to date has been attributed predominantly to its role in the cytoplasm (21Long W. Yi P. Amazit L. LaMarca H.L. Ashcroft F. Kumar R. Mancini M.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. 2010; 37: 321-332Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). We now show that AIB1-Δ4 can enter the nucleus by a noncanonical nuclear import mechanism. AIB1-Δ4 is recruited to estrogen-response elements of endogenous estrogen-regulated genes and increases their expression. We also determined that the N-terminal region absent from the AIB1-Δ4 protein contains an inhibitory domain. Through the use of Scorpion primer technology, we have created the first quantitative assay for the AIB1-Δ4 transcript and found a correlation between AIB1-Δ4 expression and the metastatic phenotype of human cancer cell lines. These data suggest that the nuclear activities of AIB1-Δ4 can contribute to its function in malignancy.
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