S6 Kinase 1 Regulates Estrogen Receptor α in Control of Breast Cancer Cell Proliferation

The 40 S ribosomal S6 kinase 1 (S6K1) acts downstream of mTOR (mammalian target of rapamycin) and is sensitive to inhibition by rapamycin. The chromosomal region 17q23 containing the RPS6KB1 gene is frequently amplified in breast cancer cells, leading to S6K1 overexpression. The role of S6K1 in disease development and progression is supported by the observation that S6K1 overexpression is associated with poor prognosis in breast cancer patients. However, the identity of mammary cell-specific S6K1 targets is not well understood. In this study, we report that overexpression of S6K1 endows breast cancer cells with a proliferative advantage in low serum conditions and enhanced sensitivity to rapamycin. We investigate the molecular mechanism behind this observation to show that S6K1 regulates estrogen receptor α (ERα) by phosphorylating it on serine 167, leading to transcriptional activation of ERα. By contributing to the activation of ERα, S6K1 promotes ERα-mediated cell proliferation and may be a target of therapeutic intervention in breast cancer. The 40 S ribosomal S6 kinase 1 (S6K1) acts downstream of mTOR (mammalian target of rapamycin) and is sensitive to inhibition by rapamycin. The chromosomal region 17q23 containing the RPS6KB1 gene is frequently amplified in breast cancer cells, leading to S6K1 overexpression. The role of S6K1 in disease development and progression is supported by the observation that S6K1 overexpression is associated with poor prognosis in breast cancer patients. However, the identity of mammary cell-specific S6K1 targets is not well understood. In this study, we report that overexpression of S6K1 endows breast cancer cells with a proliferative advantage in low serum conditions and enhanced sensitivity to rapamycin. We investigate the molecular mechanism behind this observation to show that S6K1 regulates estrogen receptor α (ERα) by phosphorylating it on serine 167, leading to transcriptional activation of ERα. By contributing to the activation of ERα, S6K1 promotes ERα-mediated cell proliferation and may be a target of therapeutic intervention in breast cancer. mTOR (mammalian target of rapamycin) is a conserved protein kinase that is a key regulator of cell growth and proliferation in response to extracellular cues, including nutrient availability and growth stimuli. Rapamycin is a naturally derived inhibitor of mTOR that was revealed to be an inhibitor of cell proliferation, as manifested by its potent immunosuppressive properties and activity against solid tumors (1Sehgal S.N. Transplant Proc. 2003; 35: 7S-14SCrossref PubMed Scopus (588) Google Scholar). The 40 S ribosomal S6 kinase 1 (S6K1) 2The abbreviations used are: S6K1, S6 kinase 1; eIF4B, eukaryotic translation initiation factor 4B; ERα, estrogen receptor α; HA, hemagglutinin; NR, supravital dye neutral red; PMA, phorbol 12-myristate 13-acetate; WT, wild type; KD, kinase-dead; RR, rapamycin-resistant; ERE, estrogen response element; 4-HT, 4-hydroxytamoxifen; LAM, lymphangioleiomyomatosis; MAPK, mitogen-activated protein kinase; RSK, p90 ribosomal S6 kinase. is one of the best characterized downstream targets of mTOR. Rapamycin treatment results in rapid dephosphorylation and inactivation of S6K1 (2Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3454) Google Scholar). S6K1 is an important regulator of cell size control, protein translation, and cell proliferation (3Fingar D.C. Blenis J. Oncogene. 2004; 23: 3151-3171Crossref PubMed Scopus (1056) Google Scholar). The 40 S ribosomal protein S6 is the best characterized target of S6K1 (4Jeno P. Ballou L.M. Novak-Hofer I. Thomas G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 406-410Crossref PubMed Scopus (144) Google Scholar). Other targets have been reported, some of which include the apoptotic protein Bad, the eukaryotic elongation factor 2 kinase, the eukaryotic translation initiation factor 4B (eIF4B), the RNA-binding protein SKAR, and the translational inhibitor PDCD4 (5Harada H. Andersen J.S. Mann M. Terada N. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 9666-9670Crossref PubMed Scopus (466) Google Scholar, 6Wang X. Li W. Williams M. Terada N. Alessi D.R. Proud C.G. EMBO J. 2001; 20: 4370-4379Crossref PubMed Scopus (626) Google Scholar, 7Richardson C.J. Broenstrup M. Fingar D.C. Julich K. Ballif B.A. Gygi S. Blenis J. Curr. Biol. 2004; 14: 1540-1549Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 8Raught B. Peiretti F. Gingras A.C. Livingstone M. Shahbazian D. Mayeur G.L. Polakiewicz R.D. Sonenberg N. Hershey J.W. EMBO J. 2004; 23: 1761-1769Crossref PubMed Scopus (361) Google Scholar, 9Dorrello N.V. Peschiaroli A. Guardavaccaro D. Colburn N.H. Sherman N.E. Pagano M. Science. 2006; 314: 467-471Crossref PubMed Scopus (560) Google Scholar). It appears that S6K1 regulates its targets to increase the biosynthetic capacity of the cell that is necessary for cell division (9Dorrello N.V. Peschiaroli A. Guardavaccaro D. Colburn N.H. Sherman N.E. Pagano M. Science. 2006; 314: 467-471Crossref PubMed Scopus (560) Google Scholar, 10Holz M.K. Ballif B.A. Gygi S.P. Blenis J. Cell. 2005; 123: 569-580Abstract Full Text Full Text PDF PubMed Scopus (898) Google Scholar, 11Ma X.M. Yoon S.O. Richardson C.J. Julich K. Blenis J. Cell. 2008; 133: 303-313Abstract Full Text Full Text PDF PubMed Scopus (241) Google Scholar). Data suggest that S6K1 is implicated in breast cancer. S6K1 is encoded by the RPS6KB1 gene localized to the chromosomal region 17q23. Region 17q23 is amplified in several breast cancer cell lines and in ∼30% of primary tumors (12Brugge J. Hung M.C. Mills G.B. Cancer Cell. 2007; 12: 104-107Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), whereas S6K1 is overexpressed in the majority of cell lines and primary tumors with this amplification (13Barlund M. Forozan F. Kononen J. Bubendorf L. Chen Y. Bittner M.L. Torhorst J. Haas P. Bucher C. Sauter G. Kallioniemi O.P. Kallioniemi A. J. Natl. Cancer Inst. 2000; 92: 1252-1259Crossref PubMed Scopus (250) Google Scholar, 14Couch F.J. Wang X.Y. Wu G.J. Qian J. Jenkins R.B. James C.D. Cancer Res. 1999; 59: 1408-1411PubMed Google Scholar, 15Monni O. Barlund M. Mousses S. Kononen J. Sauter G. Heiskanen M. Paavola P. Avela K. Chen Y. Bittner M.L. Kallioniemi A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 5711-5716Crossref PubMed Scopus (212) Google Scholar, 16Sinclair C.S. Rowley M. Naderi A. Couch F.J. Breast Cancer Res. Treat. 2003; 78: 313-322Crossref PubMed Scopus (121) Google Scholar, 17Wu G.J. Sinclair C.S. Paape J. Ingle J.N. Roche P.C. James C.D. Couch F.J. Cancer Res. 2000; 60: 5371-5375PubMed Google Scholar). Furthermore, the role of S6K1 in disease development and progression is supported by the observation that RPS6KB1 amplification and S6K1 overexpression are associated with poor prognosis in breast cancer patients (13Barlund M. Forozan F. Kononen J. Bubendorf L. Chen Y. Bittner M.L. Torhorst J. Haas P. Bucher C. Sauter G. Kallioniemi O.P. Kallioniemi A. J. Natl. Cancer Inst. 2000; 92: 1252-1259Crossref PubMed Scopus (250) Google Scholar, 18van der Hage J.A. van den Broek L.J. Legrand C. Clahsen P.C. Bosch C.J. Robanus-Maandag E.C. van de Velde C.J. van de Vijver M.J. Br. J. Cancer. 2004; 90: 1543-1550Crossref PubMed Scopus (80) Google Scholar). Interestingly, whereas RPS6KB1 is amplified in several cancer types, high level (multicopy) amplification of RPS6KB1 is limited to breast cancer (16Sinclair C.S. Rowley M. Naderi A. Couch F.J. Breast Cancer Res. Treat. 2003; 78: 313-322Crossref PubMed Scopus (121) Google Scholar). This suggests that S6K1 may have a specific role in regulating the growth of breast cancer cells. Overexpression of S6K1 has been linked to rapamycin sensitivity of breast cancer cells (19Noh W.C. Mondesire W.H. Peng J. Jian W. Zhang H. Dong J. Mills G.B. Hung M.C. Meric-Bernstam F. Clin. Cancer Res. 2004; 10: 1013-1023Crossref PubMed Scopus (262) Google Scholar). However, the molecular mechanism explaining this observation was not determined. Therefore, identification of mammary cell-specific downstream effectors of S6K1 in control of proliferation can provide us with insight for the development of new anticancer strategies. In this study, we focus on estrogen receptor α (ERα) as a target of S6K1 in control of breast cancer cell proliferation. Clinically, up to 60% of breast cancers are ER-positive, making them a target of endocrine therapy. However, resistance to endocrine therapy develops in most cases. Thus, specifically targeting S6K1 in combination with endocrine therapy may be an important strategy to combat resistant breast tumors. The addition of the ERα ligand, 17β-estradiol, and growth factors leads to ERα hyperphosphorylation, DNA binding, and transcriptional activity. One of the phosphorylation sites, Ser167, is found within the sequence RERLAS167, which conforms to the S6K1 consensus phosphorylation motif. This residue has been previously shown to be regulated by RSK and Akt kinases (20Joel P.B. Smith J. Sturgill T.W. Fisher T.L. Blenis J. Lannigan D.A. Mol. Cell. Biol. 1998; 18: 1978-1984Crossref PubMed Scopus (312) Google Scholar, 21Campbell R.A. Bhat-Nakshatri P. Patel N.M. Constantinidou D. Ali S. Nakshatri H. J. Biol. Chem. 2001; 276: 9817-9824Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar). However, because both p90 ribosomal S6 kinase (RSK) and Akt are upstream activators of S6K1 via inhibition of TSC2, it is possible that S6K1 is the predominant kinase for Ser167 as a result of RSK and/or Akt signaling. There are precedents supporting this hypothesis. mTOR, PDCD4, and GSK3 are phosphorylated by S6K1 under conditions where Akt is not active (9Dorrello N.V. Peschiaroli A. Guardavaccaro D. Colburn N.H. Sherman N.E. Pagano M. Science. 2006; 314: 467-471Crossref PubMed Scopus (560) Google Scholar, 22Holz M.K. Blenis J. J. Biol. Chem. 2005; 280: 26089-26093Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, 23Zhang H.H. Lipovsky A.I. Dibble C.C. Sahin M. Manning B.D. Mol. Cell. 2006; 24: 185-197Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). eIF4B and ribosomal protein S6 are differentially phosphorylated by both S6K1 and RSK (8Raught B. Peiretti F. Gingras A.C. Livingstone M. Shahbazian D. Mayeur G.L. Polakiewicz R.D. Sonenberg N. Hershey J.W. EMBO J. 2004; 23: 1761-1769Crossref PubMed Scopus (361) Google Scholar, 24Shahbazian D. Roux P.P. Mieulet V. Cohen M.S. Raught B. Taunton J. Hershey J.W. Blenis J. Pende M. Sonenberg N. EMBO J. 2006; 25: 2781-2791Crossref PubMed Scopus (400) Google Scholar, 25Roux P.P. Shahbazian D. Vu H. Holz M.K. Cohen M.S. Taunton J. Sonenberg N. Blenis J. J. Biol. Chem. 2007; 282: 14056-14064Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar). Thus, we sought to investigate the role of S6K1 in ERα phosphorylation and breast cancer cell proliferation. Plasmids-Generation of the hemagglutinin (HA)-S6K1 constructs in the pRK7 expression vector has been described previously (26Cheatham L. Monfar M. Chou M.M. Blenis J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11696-11700Crossref PubMed Scopus (114) Google Scholar, 27Schalm S.S. Blenis J. Curr. Biol. 2002; 12: 632-639Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar, 28Schalm S.S. Tee A.R. Blenis J. J. Biol. Chem. 2005; 280: 11101-11106Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). VP16-ERα and pGL2–3xERE-TATA-luc were kindly provided by Donald P. McDonnell (Duke University, Durham, NC). pIS2 Renilla luciferase reporter was kindly provided by David Bartel (Michigan Institute of Technology, Cambridge, MA). Antibodies-Anti-HA and anti-phospho-ERK1/2 antibodies were purchased from Sigma. Anti-phospho-S6 Ser240/244, anti-phospho-ER Ser167, anti-S6K1, and anti-phospho-Akt Ser473 antibodies were purchased from Cell Signaling Technology. Anti-ER and anti-actin antibodies were purchased from Santa Cruz Biotechnology. For immunoblotting, horseradish peroxidase-conjugated anti-rabbit, anti-mouse, and anti-goat antibodies were purchased from Amersham Biosciences, Chemicon, and Santa Cruz Biotechnology, respectively. Cell Culture, Transfections, and Generation of Stable Cell Lines- ZR-75-1 cells were a gift from William Hahn (Dana Farber Cancer Institute, Boston, MA). MDA-MB-231 cells were from Joan Massague (Memorial Sloan-Kettering Cancer Center, New York, NY). MDA-MB-435 and T47D cells were generously provided by Jeffrey Segall (Albert Einstein College of Medicine, Bronx, NY). MCF10a, MCF7, MDA-MB-468, HEK293E, and BT-474 cells were a gift from John Blenis (Harvard Medical School, Boston). MDA-MB-361 and MDA-MB-436 were from ATCC. All cell lines were maintained according to the suppliers' instructions. For transfection studies, HEK293E cells were transfected using the calcium phosphate method as described previously (22Holz M.K. Blenis J. J. Biol. Chem. 2005; 280: 26089-26093Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar). T47D cells were transfected using Lipofectamine 2000 (Invitrogen) following the manufacturer's protocol. Lysates were prepared at 48 h posttransfection. To stably suppress S6K1, we used pLKO.1 lentiviral small hairpin RNA constructs generated by William Hahn. Lentiviral infections of the indicated cell lines were performed, and stable cell lines were selected in puromycin. Cell Proliferation Measurements- For proliferation assays, cells were seeded in quadruplicate at a density of 5000 cells/well in 96-well plates and grown overnight. Media were changed to assay media with or without the agents (as indicated in the figure legends). Cell proliferation was assayed after 96 h using the supravital dye neutral red (NR) incorporation (29Borenfreund E. Babich H. Martin-Alguacil N. In Vitro Cell. Dev. Biol. 1990; 26: 1030-1034Crossref PubMed Scopus Google Scholar). The was of containing was and was for h at were and with a of and the the cells was the with a of was at with a Cell density was and using Cell were grown in media and (as indicated in the figure and cells were in assay with 1 40 and at Lysates were of by at for or was of cell were by or as were to a and with the indicated were using enhanced and a with One and ERα or S6K1 was with or antibodies (as indicated in the figure and protein were in 1 of 1 1 2 40 as for and 1 and and in serum were with as by the addition of S6K1 in a containing protein kinase in a of phosphorylation for at The were using and the of was by with anti-phospho-ER Ser167 cells were transfected with ERα, luciferase under the control of estrogen response control Renilla and the of S6K1 or vector rapamycin and/or was as 48 h cells were using and luciferase activity was using the reporter assay and the We sought to S6K1 ERα in cells, leading to its ERα a S6K1 phosphorylation RERLAS167, to the S6K1 consensus phosphorylation where is we phosphorylation of Ser167 in cell T47D and MCF7, the with low S6K1 expression and the with high S6K1 shown in we found that were in in T47D cells. with rapamycin Ser167 phosphorylation in cells. Thus, Ser167 phosphorylation with S6K1 and is by Akt R.A. Bhat-Nakshatri P. Patel N.M. Constantinidou D. Ali S. Nakshatri H. J. Biol. Chem. 2001; 276: 9817-9824Abstract Full Text Full Text PDF PubMed Scopus (814) Google and RSK (20Joel P.B. Smith J. Sturgill T.W. Fisher T.L. Blenis J. Lannigan D.A. Mol. Cell. Biol. 1998; 18: 1978-1984Crossref PubMed Scopus (312) Google has been linked to Ser167 we to the leading to Ser167 phosphorylation we cells with which the and not We that under Ser167 was phosphorylated in with activation of S6K1 and The was not However, Ser167 phosphorylation was the that Akt was the kinase for this We with the phorbol phorbol 12-myristate which the and not the We Ser167 which with S6K1 and not with Ser167 phosphorylation was that the was not for the phosphorylation of Ser167 under Thus, S6K1 Ser167 in and cells. We S6K1 Ser167 in We an in kinase assay with ERα as a We ERα from T47D cells and a kinase assay by the addition of S6K1 from cells or and cells We phosphorylation of Ser167 that was after the addition of S6K1 and not S6K1 or the control We this observation by the kinase assay with transfected ERα and the wild containing a and containing an of with in a increase in Ser167 phosphorylation with the control, whereas phosphorylation was Thus, S6K1 appears to ERα. S6K1 is the kinase for Ser167, of its we the of S6K1 in cells on Ser167 phosphorylation of S6K1 expression in a in Ser167 overexpression of S6K1 in T47D cells In both in Ser167 phosphorylation were by that S6K1 is for the phosphorylation of both in cells. The transcriptional activity of ERα has been previously shown to be sensitive to rapamycin R.A. M. Clin. Cancer Res. 2004; 10: PubMed Scopus Google Scholar). To S6K1 regulates the transcriptional activity of ERα, we transfected HEK293E cells with ERα, a reporter that the luciferase gene under the control of a control Renilla luciferase under the control of the for luciferase activity and WT, KD, and of Overexpression of and of S6K1 in a increase in transcriptional with or vector expression rapamycin treatment in a in whereas was not by rapamycin. activity was by Ser167 phosphorylation Thus, S6K1 ERα and regulates its transcriptional activity. an active of is an of ERα. We the of rapamycin and in combination on the transcriptional activity of ERα using the luciferase reporter In HEK293E cells, transcriptional activity by However, in combination with the of was not that of rapamycin was overexpressed in cells, and rapamycin transcriptional activity by in the inhibition Thus, S6K1 and estrogen to ERα-mediated transcriptional activity. To the S6K1 and the proliferative capacity of mammary cells, we the of S6K1 in mammary cell shown in we that S6K1 was overexpressed in of cell lines with 17q23 specifically MCF7, and T47D cells, the S6K1 to to cells with 17q23 we the S6K1 overexpression and sensitivity to rapamycin in of cell It was previously that S6K1 overexpression may with rapamycin sensitivity (19Noh W.C. Mondesire W.H. Peng J. Jian W. Zhang H. Dong J. Mills G.B. Hung M.C. Meric-Bernstam F. Clin. Cancer Res. 2004; 10: 1013-1023Crossref PubMed Scopus (262) Google Scholar). we found that the growth of cell lines were sensitive to with S6K1 overexpression were at capacity with cells, whereas cells with low S6K1 expression were at capacity with cells Thus, mammary cells with S6K1 overexpression enhanced rapamycin We were to include MDA-MB-361 cells in the because the of cells with the cell an important regulator of cell growth and cell progression D.C. Richardson C.J. Tee A.R. L. C. Blenis J. Mol. Cell. Biol. 2004; 24: PubMed Scopus Google Scholar), S6K1 overexpression may a role in cancer and It has been that overexpression of S6K1 may cells to under conditions of low a of D.C. Richardson C.J. Tee A.R. L. C. Blenis J. Mol. Cell. Biol. 2004; 24: PubMed Scopus Google Scholar). We the S6K1 in mammary cell lines and to in low serum We found that cells with low S6K1 expression were at capacity with cells in in to cells that were to at capacity with cells in Thus, S6K1 overexpression a proliferative advantage to mammary cells in low serum contributing to we sought to the of of S6K1 expression on growth in low We generated cell lines in which the expression of S6K1 was by small hairpin RNA because of its role as a cell S6K1 in in cell proliferation of cell lines in serum However, low cells an whereas cell lines proliferative However, the proliferation was in low serum low cells not a in proliferation following S6K1 the proliferative capacity of cells was by with the cells in serum and that of BT-474 cells by This may that S6K1 overexpression cells on the high level expression and of S6K1 to proliferative capacity under conditions of low We overexpression of S6K1 in cells with low S6K1 expression sensitivity to rapamycin and serum shown we stably overexpressed S6K1 in cells using low by in puromycin. were for 2 to them to to of We used vector control and the WT, KD, and of We proliferation of cell lines in media with low serum All cell lines a proliferation in However, in media with and cells the vector or grown in the of rapamycin cells a small in proliferation with vector and Interestingly, cells not were not by rapamycin with a proliferative advantage overexpression of Thus, a increase in S6K1 activity cells S6K1 overexpression cells to in low It has been previously shown that some cells are sensitive to that observation rapamycin sensitivity to of the mTOR as Akt activation or phosphorylation of R.A. M. Clin. Cancer Res. 2004; 10: PubMed Scopus Google Scholar, A. J. J. S. Chen S. Clin. Cancer Res. 2005; PubMed Scopus Google Scholar). We that rapamycin sensitivity of cells may be specifically to inhibition of S6K1 and in ERα Ser167 We MCF7, and ZR-75-1 cell lines in the of or a combination of the agents and this with proliferation in shown in and low MDA-MB-231 cells were not by a combination of rapamycin and rapamycin and low T47D cells were not by a combination of rapamycin and and and ZR-75-1 cells a inhibition by a combination of rapamycin and This indicated that the ERα to cell proliferation. The of endocrine therapy for breast cancer following the of de or resistance a Clin. Cancer Res. 2005; Google Scholar). the of resistance has revealed that estrogen receptor activation can growth factor and in the and used by breast cancer cells to endocrine R.A. Cancer. 1999; PubMed Scopus (144) Google Scholar). In this study, we that S6K1 is a kinase for Ser167 of ERα. S6K1 phosphorylation regulates ERα transcriptional contributing to proliferation of breast cancer cells we that S6K1 overexpression in breast cancer cells them on the activity of this kinase for proliferation. S6K1 expression with the of cells to in low serum whereas of S6K1 expression results in a in proliferation that is in cells. This on S6K1 is to of cancer cells or or This has important because are that the of inhibition of the mTOR in breast cancer. of the role of the mTOR in cell growth and proliferation, rapamycin and its and are the most in the anticancer mTOR have shown an in the response has been patients. an to patients to most from mTOR inhibitor a of patients S6K1 amplification with a poor and patients a to a Therefore, of rapamycin and its can the of and breast cancer to endocrine therapy. an low as immunosuppressive and are a with rapamycin and its J. Clin. 2008; 26: PubMed Scopus Google Scholar). The may to inhibition of the mTOR Therefore, we that by on the S6K1 of the mTOR we have S6K1 as a specific target of therapeutic inhibition may result in The be to and new of S6K1 in the treatment of tumors with S6K1 the role of S6K1 in the phosphorylation and activation of ERα may have in and treatment of One is in D. S. J. 2008; Full Text Full Text PDF PubMed Scopus Google and D. G. 2008; PubMed Scopus Google Scholar). is a or in with a of the The of is by the proliferation of cells in the of and in the of and a on this estrogen receptor expression has been in the cells of patients. It has been that cell growth is associated with S6K1 leading to the cell proliferation in disease D.A. A. M.K. D. Chou M.M. R.A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). Thus, it is important to investigate S6K1 to disease phosphorylation and activation of ERα. We John Blenis for of this