Estrogen Receptor Inhibits c-Jun-dependent Stress-induced Cell Death by Binding and Modifying c-Jun Activity in Human Breast Cancer Cells
2004; Elsevier BV; Volume: 279; Issue: 8 Linguagem: Inglês
10.1074/jbc.m311492200
ISSN1083-351X
AutoresXiao-Mei Qi, Stanley Borowicz, Rocky Pramanik, Richard M. Schultz, Jiahuai Han, Guan Chen,
Tópico(s)Stress Responses and Cortisol
Resumoc-Jun, a major component of the AP-1 transcription factor, is either pro- or anti-apoptotic with cellular determinants unknown. Nuclear estrogen receptor (ER), on the other hand, regulates gene expression through both estrogen response elements and AP-1. Here we show that stress stimulates c-Jun phosphorylation and AP-1 activity in both ER+ and ER- human breast cancer cells and only induces cell death in ER- cells, indicating a determinant role of ER in c-Jun/AP-1 activity. The inhibitory effect of ER in stress-induced cell death is confirmed by ER transfection into ER- cells. Furthermore, inhibition of c-Jun activation by a dominant negative c-Jun blocks AP-1 activity in ER+ cells and attenuates stress-induced cell death but not AP-1 activity in ER- cells, suggesting that the c-Jun/AP-1 activity has distinct properties depending on ER status. ER was shown to inhibit stress-induced cell death through its physical interaction with c-Jun. This is because ER binds c-Jun in breast cancer cells, stress treatment further increases the ER-bound phosphorylated c-Jun, and the c-Jun binding-deficient ER mutant fails to protect stress-induced cell death. Together, our studies reveal a novel function of ER in stress response by modification of c-Jun activity. c-Jun, a major component of the AP-1 transcription factor, is either pro- or anti-apoptotic with cellular determinants unknown. Nuclear estrogen receptor (ER), on the other hand, regulates gene expression through both estrogen response elements and AP-1. Here we show that stress stimulates c-Jun phosphorylation and AP-1 activity in both ER+ and ER- human breast cancer cells and only induces cell death in ER- cells, indicating a determinant role of ER in c-Jun/AP-1 activity. The inhibitory effect of ER in stress-induced cell death is confirmed by ER transfection into ER- cells. Furthermore, inhibition of c-Jun activation by a dominant negative c-Jun blocks AP-1 activity in ER+ cells and attenuates stress-induced cell death but not AP-1 activity in ER- cells, suggesting that the c-Jun/AP-1 activity has distinct properties depending on ER status. ER was shown to inhibit stress-induced cell death through its physical interaction with c-Jun. This is because ER binds c-Jun in breast cancer cells, stress treatment further increases the ER-bound phosphorylated c-Jun, and the c-Jun binding-deficient ER mutant fails to protect stress-induced cell death. Together, our studies reveal a novel function of ER in stress response by modification of c-Jun activity. c-Jun, a major component of AP-1 transcription factor, is activated by both mitogenic and stress signals downstream of ERK 1The abbreviations used are: ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK(s), mitogen-activated protein kinase(s); MEK1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-1; ER, estrogen receptor; ERE, estrogen responsive element; GST, glutathione S-transferase; FACS, fluorescence-activated cell sorter; ARS, arsenite; ad, adenovirus; Tet, tetracycline; WT, wild type. (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase), and p38 MAPKs (mitogen-activated protein kinases) through phosphorylation and trans-activation. AP-1 is composed of homodimers of the Jun family or heterodimers of a Jun family member with any of the Fos family members or other transcription factors such as ATF-2, CREB, and NFAT (1Shaulian E. Karin M. Nat. Cell Biol. 2002; 5: E131-E136Crossref Scopus (2284) Google Scholar, 2Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2265) Google Scholar). c-Jun is phosphorylated on Ser-63, Ser-73, Thr-91, and/or Thr-93 within its N-terminal portion by JNK in response to a variety of stimuli (2Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2265) Google Scholar). ERK, on the other hand, activates AP-1 through phosphorylation of ternary complex factors, leading to the induction of fos genes in response to mitogenic signaling (2Karin M. J. Biol. Chem. 1995; 270: 16483-16486Abstract Full Text Full Text PDF PubMed Scopus (2265) Google Scholar). p38 MAPK stimulates c-jun gene expression by activation of ATF-2 or MEF2s (3Angel P. Hattori K. Smeal T. Karin M. Cell. 1988; 55: 875-885Abstract Full Text PDF PubMed Scopus (1002) Google Scholar, 4Han J. Jiang Y. Li Z. Kravchenko V.V. Ulevitch R.J. Nature. 1997; 386: 296-299Crossref PubMed Scopus (689) Google Scholar), and our recent results demonstrated that the p38 pathways can also regulate c-Jun expression and phosphorylation in human breast cancer cells (5Qi X. Pramank R. Wang J. Schultz R.M. Maitra R.K. Han J. DeLuca H.F. Chen G. J. Biol. Chem. 2002; 277: 25884-25892Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 6Pramanik R. Qi X. Borowicz S. Choubey D. Schultz R.M. Han J. Chen G. J. Biol. Chem. 2003; 278: 4831-4839Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). c-Jun thus serves as a central transcription factor downstream of three major MAPK pathways (ERK, JNK, and p38) and converts various extracellular signals into target gene expressions and distinct biological responses. c-Jun phosphorylation in neurons and fibroblasts is linked to apoptosis activated by JNK and p38 stress MAPKs (7Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5064) Google Scholar, 8Tournier C. Hess P. Yang D.D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N. Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1557) Google Scholar, 9Kolbus A. Herr I. Schreiber M. Debatin K. Wagner E.F. Angel P. Mol. Cell. Biol. 2000; 20: 575-582Crossref PubMed Scopus (146) Google Scholar). In human glioma cells, however, inhibition of c-Jun activation by a dominant negative c-Jun (S63A/S73A) increases sensitivity to apoptosis induced by DNA-damaging agents (10Potapova O. Basu S. Mercola D. Holbrook N.J. J. Biol. Chem. 2001; 276: 28546-28553Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), indicating an anti-apoptotic activity of c-Jun. A recent study further showed that primary hepatocytes lacking c-Jun are more sensitive to tumor necrosis factor-α-induced cell death (11Eferi R. Ricci R. Kenner L. Zenz R. David J. Rath M. Wagner E.F. Cell. 2003; 112: 181-192Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar). Thus, c-Jun activity can be either pro- or anti-apoptotic in a manner dependent on cell and tissue type. Identification of the determinants that specify the outcome of c-Jun activation remains a major task in cell biology. Estrogens are decisive factors in driving normal mammary epithelial and breast cancer cell proliferation, but the precise mechanisms involved remain unknown (12Clark R. Dickson R.B. Lippman M.E. Crit. Rev. Oncol. Hematol. 1992; 12: 1-23Crossref PubMed Scopus (123) Google Scholar). Most of the biological effects of estrogen are mediated through an estrogen receptor (ER), a member of a superfamily of nuclear hormone receptors that act as ligand-activated transcription factors (13Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6150) Google Scholar, 14Hall J.M. Couse J.F. Korach K.S. J. Biol. Chem. 2001; 276: 36869-36872Abstract Full Text Full Text PDF PubMed Scopus (1011) Google Scholar). The classical model for estrogen action is that upon ligand binding, ER binds to specific estrogen responsive elements (EREs) within target genes that thereby trans-regulate their expression (15Glass C.K. Rose D.W. Rosenfeld M.G. Curr. Opin. Cell Biol. 1997; 9: 222-232Crossref PubMed Scopus (602) Google Scholar). Recent studies, however, showed that the ER and AP-1 signaling frequently affect each other in response to mitogenic signaling. Estrogens, for example, induce c-Jun expression through an estrogen-inducible enhancer in the c-Jun promoter (16Hyder S.M. Nawaz Z. Chiappetta C. Yokoyama K. Stancel G.M. J. Biol. Chem. 1995; 270: 8506-8513Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). AP-1 activity, on the other hand, can be induced by estrogen and inhibited by anti-estrogens without affecting c-Jun and c-Fos expressions (17Philips A. Chalbos D. Rochefort H. J. Biol. Chem. 1993; 268: 14103-14108Abstract Full Text PDF PubMed Google Scholar). Moreover, overexpression of c-Jun in ER+ cells not only increases AP-1 activity but also decreases ERE-dependent transcription (18Smith L. Wise S.C. Hendricks D.T. Sabichi A.L. Bos T. Reddy P. Brown P.H. Birrer M. Oncogene. 1999; 18: 6063-6070Crossref PubMed Scopus (190) Google Scholar). Furthermore, progression from the hormone-responsive to the anti-estrogen-resistant phenotype is frequently linked to a decrease in ER expression and an increase in c-Jun phosphorylation and/or AP-1 activity (19Johnston S.R. Lu B. Scott G.K. Kushner P.J. Smith I.E. Dowsett M. Benz C.C. Clin. Cancer Res. 1999; 5: 251-256PubMed Google Scholar, 20Schiff R. Reddy P. Ahotupa M. Coronado-Heinsohn E. Grim M. Hilsenbeck S.G. Lawrence R. Deneke S. Herrera R. Chammenss G.C. Fuqua S.A. Brown P.H. Osborne C.K. J. Natl. Cancer Inst. 2000; 92: 1926-1934Crossref PubMed Scopus (176) Google Scholar). Perhaps most intriguing, ER regulates the expression of genes that contain no ERE but contain an AP-1 site in response to estrogen (non-classical) (14Hall J.M. Couse J.F. Korach K.S. J. Biol. Chem. 2001; 276: 36869-36872Abstract Full Text Full Text PDF PubMed Scopus (1011) Google Scholar, 21Jakacka M. Ito M. Weiss J. Chien P. Gehm B.D. Jameson J.L. J. Biol. Chem. 2001; 276: 13615-13621Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). This has been speculated to occur through protein-protein interactions as suggested by GST pull down and overexpression experiments, and the significance of this interaction under physiological conditions has not been explored (21Jakacka M. Ito M. Weiss J. Chien P. Gehm B.D. Jameson J.L. J. Biol. Chem. 2001; 276: 13615-13621Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar, 22Webb P. Lopez G.N. Uht R.M. Kushner P.J. Mol. Endocrinol. 1995; 9: 443-456Crossref PubMed Google Scholar). In addition to proliferative signaling, stress also activates c-Jun/AP-1 (1Shaulian E. Karin M. Nat. Cell Biol. 2002; 5: E131-E136Crossref Scopus (2284) Google Scholar), and a possible coordinating role of ER and c-Jun in processing the stress signal has not been explored. Stress signaling may play an equally important role as mitogenic signaling in breast cancer progression. Clinical breast cancers, for example, may typically grow under stress conditions such as hypoxia (23Helezynska K. Kronblad A. Jogi A. Nilsson E. Beckman S. Landberg G. Pahlman S. Cancer Res. 2003; 63: 1441-1444PubMed Google Scholar) and/or repeated exposure to genotoxic agents like radiation and chemotherapeutic drugs (24Liu X. Gupta A.K. Corry P.T. Lee J.L. J. Biol. Chem. 1997; 272: 11690-11693Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Investigation of the effects of the signaling cross-talk at the ER and c-Jun level in response to stress thus constitutes an essential element in understanding roles of ER in breast cancer. Because the effects of c-Jun activation are biologically pleiotropic, and ER signaling and c-Jun/AP-1 activity are often interactive, we hypothesized that that stress-induced c-Jun activation would have different biological consequences in human breast cancer cells in the absence and in the presence of ER expression. Our results showed that stress selectively induces c-Jun-dependent cell death in ER- but not in ER+ human breast cancer cells, indicating a cell death inhibitory activity of ER, which is further confirmed by ER transfection in ER- cells. ER inhibition of stress-induced cell death was further shown to be independent of estrogen and ER transcription activity but dependent on ER-c-Jun complex formation. These studies thus suggest a dual function of ER in breast cancer progression. Under mitogenic conditions, such as in response to estrogen and/or growth factors, ER may function as a transcription factor to increase breast cancer cell proliferation through gene regulation. Under pathological or therapeutic stress conditions, however, ER may inhibit stress-induced and c-Jun-dependent cell death via a direct interaction with c-Jun, thereby facilitating breast cancer growth. cDNA Constructs and Other Reagents—Recombinant adenovirus vector containing HA-tagged MKK6 was constructed as described previously (25Huang S. Jiang Y. Li Z. Nishida E. Mathias P. Lin S. Ulevitch R.J. Nemerow G.R. Han J. Immunity. 1997; 6: 739-749Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 26Wang X. McGowan C.H. Zhao M. He L. Downey J.S. Fearns C. Wang Y. Huang S. Han J. Mol. Cell. Biol. 2000; 20: 4543-4552Crossref PubMed Scopus (241) Google Scholar). A dominant negative c-Jun (Tam67) that codes for c-Jun minus its activation domain (containing key Ser/Thr phosphorylation residues) was provided by Birrer and co-workers (27Brown P.H. Alani R. Preis L.H. Szabo E. Birrer M.J. Oncogene. 1993; 8: 877-886PubMed Google Scholar). ER cDNA and ERE luciferase reporter (ERE-Luc, four ERE repeats cloned upstream of a luciferase gene) were kindly provided by Zhang and Shapiro (28Zhang C.C. Shapiro D.J. J. Biol. Chem. 2000; 275: 479-486Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). A mouse c-Jun cDNA in HA-tagged pHM6 vector (6Pramanik R. Qi X. Borowicz S. Choubey D. Schultz R.M. Han J. Chen G. J. Biol. Chem. 2003; 278: 4831-4839Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) was used to generate pHM6-HA-Tam67 by PCR (forward primer GGT ACC AGC CAG AAC ACG CTT CCC AGT G and reverse primer CGG AAT TCT CAA AAT GTT TGC AAC TGC TGC G). A constitutively active c-Jun was generated by changing Ser-63, Ser-73, Thr-91, and Thr-93 of mouse c-Jun to aspartic acid (D) (c-Jun(ST/D)), whereas a non-phosphorylation inert mutant c-Jun was made by replacing these residues with alanine (A) (c-Jun(ST/A)) by PCR, as described previously (45Papavassiliou A.G. Treier M. Bohmann D. EMBO J. 1995; 14: 2014-2019Crossref PubMed Scopus (119) Google Scholar). An AP-1 luciferase construct (AP-1 Luc) was kindly provided by Craig Hauser, which was generated by cloning three AP-1 repeats into a luciferase reporter gene containing a minimal Fos promoter (29Galang C.K. Der C.J. Hauser C.A. Oncogene. 1994; 9: 2913-2921PubMed Google Scholar). ER deletion and site-directed mutagenesis were carried out by a PCR-based technique using the QuikChange site-directed mutagenesis kit (Stratagene). These constructs were then cloned into a V5-tagged mammalian expression vector pcDNA3.ID/V5-His-Topo (Invitrogen). Primers for a previously described dominant negative ER (L540Q) (changing Leu-540 to Gln) (30Ince B.A. Zhuang Y. Wrenn C.K. Shapiro D.J. Katzenellenbogen B.S. J. Biol. Chem. 1993; 268: 14026-14032Abstract Full Text PDF PubMed Google Scholar): forward, 5′-CCC TCT ATG ACC TGC TGC AAG AGA TGC TGG ACG CC-3′; reversed, 5′-CGT CCA GCA TCT CTT GCA GCA GGT CAT AG-3′. Primers for an N-terminal deletion ER mutant (Δ313ER): forward, 5′-CAC CAT GGT CAG TGC CTT GTT GGA T-3′; reversed, 5′-CGT GGC AGG GAA ACC CTC TGC CTC-3′. All of the mutations generated were confirmed by DNA digestion with restriction enzymes and DNA sequencing. Reagents for cell culture were supplied by Invitrogen. Fetal bovine serum was obtained from BioWhittaker. Protein G-Sepharose and protein A-Sepharose 4B beads were purchased from Zymed Laboratories Inc. Rabbit polyclonal antibodies against c-Jun (sc-44-G to the C-terminal, H-79 to the N-terminal) and anti-ER polyclonal (HC-20) and monoclonal (F10) antibodies were purchased from Santa Cruz. An ECL plus kit for Western detection was purchased from Amersham Biosciences. Anti-phosphor-c-Jun (Ser-63) and anti-HA antibody were purchased from Cell Signaling and Roche, respectively. Mouse V5 monoclonal antibody was purchased from Invitrogen. Cell Culture, Transfection, Luciferase Assay, Gel Retardation Assay, and Data Analyses—Human ER+ breast cancer cell lines were obtained from ATCC. All of these cells were maintained in minimum Eagle's medium containing 10% fetal bovine serum and antibiotics at 37 °C with 5% CO2. For promoter analyses and stable transfection, the protocol of calcium phosphate-mediated transfection from Promega was followed. To increase transfection efficiency for ER-c-Jun interaction, the transfection reagent FuGENE 6 (Invitrogen) was used. To establish stable Tam67-expressing clones, cells were transfected with equal amounts of pCMV-Tam and pcDNA3 (neomycin-resistant gene expression plasmid) using the FuGENE 6 kit, followed by selection with 0.6 mg/ml G418 for 1 month (5Qi X. Pramank R. Wang J. Schultz R.M. Maitra R.K. Han J. DeLuca H.F. Chen G. J. Biol. Chem. 2002; 277: 25884-25892Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The resistant clones were pooled and early passages of these cells were used for experiments. For the luciferase assay, cells were collected 48 h later in the lysis buffer, and the luciferase activity of the promoter was assayed with a dual luciferase kit from Promega using pRL-TK (encoding Renilla luciferase) as an internal control in a TD-20/20 Luminometer (Turner Designs). To assess arsenite (ARS)-induced AP-1 activity, cells were treated with 2 mm ARS for 30 min 24 h before the luciferase assay. Preparation of nuclear extract and electrophoresis mobility shift assay were carried out as described previously (5Qi X. Pramank R. Wang J. Schultz R.M. Maitra R.K. Han J. DeLuca H.F. Chen G. J. Biol. Chem. 2002; 277: 25884-25892Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The results of all experiments, whenever possible, were analyzed using Student's t test for a statistically significant difference. Establishment of Tet-inducible ER-expressing Cell Lines—The inducible expression system (T-Rex) was purchased from Invitrogen. A full-length human ER cDNA was cloned into a pcDNA4 vector (pcDNA4-ER) by a PCR-based technique (primers: forward, CCC GAA TTC ACC ATG GCC ATG ACC CTC CAC; reversed, CCC CTC GAG TCA GAC CGT GGC AGG GAA) via EcoRI and XhoI sites. ER- 231 breast cancer cells were cotransfected with pcDNA4-ER and pcDNA6/TR at a ratio of 1:6 using a FuGENE 6 transfection kit and were selected with zeocin (50 μg/ml) and blasticidin (2.5 μg/ml) according to the Invitrogen manual. At least 20 of the resistant clones were screened for ER expression by Western blot (and immunostaining in some cases) and two of these were chosen for our experiments. Cell Death Assay, Immunostaining, Immunoprecipitation, and Immunoblotting—For cell death assay, breast cancer cells were infected with either ad-MKK6 or the vector control for 5 h in serum-free medium, followed by an overnight incubation in 10% serum-containing medium, as described previously (5Qi X. Pramank R. Wang J. Schultz R.M. Maitra R.K. Han J. DeLuca H.F. Chen G. J. Biol. Chem. 2002; 277: 25884-25892Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). After an additional 24-h incubation, viable cells were determined with a hemocytometer by trypan blue exclusion assay, and the cell viability was calculated by dividing the viable cell number by that of total cells (dead + viable) (31Chen G. Shu J. Stacey D.W. Oncogene. 1997; 15: 1643-1651Crossref PubMed Scopus (55) Google Scholar). Portions of these cells after infection or ARS treatment also were washed with phosphate-buffered saline and resuspended in cold ethanol for flow cytometric analyses (FACS) to determine the apoptotic sub-G1 population. To determine the long term effects of stress treatment on cell survival, cells were incubated for 2 weeks for colony formation, as previously reported (32Chen G. Waxman D.J. Biochem. Pharmacol. 1994; 47: 1079-1087Crossref PubMed Scopus (77) Google Scholar). For immunostaining, cells were plated on coverslips 1 day prior and fixed in 3.7% formaldehyde immediately after stress treatment. After being permeabilized in a buffer containing 0.5% Triton X-100 and 0.5% Nonidet P-40, cells were blocked in 3% bovine serum albumin in phosphate-buffered saline. A rabbit polyclonal phosphor-c-Jun antibody (Ser-63, Cell Signaling) at 1:100 and a mouse monoclonal anti-ER (F10, Santa Cruz) at 1:50 in 3% bovine serum albumin in phosphate-buffered saline were used for phosphor-c-Jun and ER staining, respectively. After a 1-h incubation with the second antibody at room temperature (anti-rabbit Cy3 for phosphor-c-Jun, and anti-mouse fluorescein isothiocyanate for ER, both at 1:1000), the coverslips were mounted with a mounting medium from Vector Laboratories (containing 4,6-diamidino-2-phenylindole) and examined under a fluorescence microscope (Leica) for phosphor-c-Jun or ER signal. For immunoprecipitation, cells were washed with cold phosphate-buffered saline and lysed in modified radioimmune precipitation assay buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mm EGTA, 10 mm NaF, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, and 1 μg/ml aprotinin, leupeptin, and pepstatin). The same amount of cell lysates of each group (corresponding to about 300 μg of protein) was incubated overnight at 4 °C with different antibodies for immunoprecipitation. For Western blot analyses, cells were directly lysed in 1× loading buffer and separated on SDS-PAGE, as described previously (5Qi X. Pramank R. Wang J. Schultz R.M. Maitra R.K. Han J. DeLuca H.F. Chen G. J. Biol. Chem. 2002; 277: 25884-25892Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 6Pramanik R. Qi X. Borowicz S. Choubey D. Schultz R.M. Han J. Chen G. J. Biol. Chem. 2003; 278: 4831-4839Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Experiments in Athymic Mice—4-6-week-old female athymic mice were purchased from Harlan Sprague-Dawley, Inc. (Madison, WI). ER- 468 human breast cancer cells were infected with ad-vector or ad-MKK6 as described above for 5 h in non-serum-containing medium at 37 °C and 5% CO2. Thereafter, cells were washed and trypsinized, and 107 cells in 0.1 ml of medium were inoculated subcutaneously to the front flanks of mice, as described previously (33Dixit M. Yang J. Poirierl M. Price J.O. Andrews P.A. Arteaga C.L. J. Natl. Cancer Inst. 1997; 89: 365-372Crossref PubMed Scopus (73) Google Scholar, 34Campbell I. Maglioco A. Moyana T. Zheng C. Xiang J. Cancer Gene Ther. 2000; 7: 1270-1278Crossref PubMed Scopus (24) Google Scholar). Each mouse received two injections; the left site was injected with ad-MKK6-infected cells, and the right site was injected with the same amount of cells infected with ad-vector as a control. Beginning 2 weeks after injection, tumor volume was measured weekly with a caliper for the next 2 months (data not shown). The picture shown in Fig. 1 was taken 2 months after tumor inoculation. Stress Induces Cell Death in ER- but Not in ER+ Human Breast Cancer Cells—To examine whether endogenous ER expression may affect stress-induced cell death, ER- human breast cancer 468 and ER+ MCF-7 cells were infected with adenovirus expressing a constitutively active p38 stress kinase activator MKK6 or the adenovirus vector. The adenovirus-mediated MKK6 delivery has been shown to activate both p38 and JNK stress pathways in these two cell lines (5Qi X. Pramank R. Wang J. Schultz R.M. Maitra R.K. Han J. DeLuca H.F. Chen G. J. Biol. Chem. 2002; 277: 25884-25892Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Cell death was assessed by morphological alterations, trypan blue staining, and flow cytometric analysis, as described previously (31Chen G. Shu J. Stacey D.W. Oncogene. 1997; 15: 1643-1651Crossref PubMed Scopus (55) Google Scholar). Infection with ad-MKK6 but not the vector induced significant cell death in ER- 468 cells at 48 h, whereas, surprisingly, ER+ MCF-7 cells remained unresponsive to the ad-MKK6 (Fig. 1A). A similar selective cell death inducing effect was also observed by treatment with a chemical JNK/p38 activator arsenite (Fig. 1A) (5Qi X. Pramank R. Wang J. Schultz R.M. Maitra R.K. Han J. DeLuca H.F. Chen G. J. Biol. Chem. 2002; 277: 25884-25892Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 35Chen G. Hitomi M. Han J. Stacey D.W. J. Biol. Chem. 2000; 275: 38973-38980Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Because the morphological alterations by either ad-MKK6 or ARS are associated with an increase of cells in sub-G1 populations by FACS (Fig. 1A, the number to the right of each panel), these results indicated that the cell death induced in ER- cells is likely apoptotic. To rule out cell line specific effects, the ER+ T47D and ER- 231 human breast cell lines were included to extend this observation. Again, ARS treatment has no substantial effects on the viability of ER+ cell lines (MCF-7 and T47D) but reduces the viability by about 80 and 50% in ER- 468 and ER- 231 cell lines, respectively (Fig. 1B). To further examine the in vivo therapeutic potentials of adenovirus-mediated MKK6 gene delivery in ER- tumor, an ex vivo tumor growth assay using ER- 468 cells was performed in Balb/c nude mice as described previously (34Campbell I. Maglioco A. Moyana T. Zheng C. Xiang J. Cancer Gene Ther. 2000; 7: 1270-1278Crossref PubMed Scopus (24) Google Scholar). After infection in the cell culture, the 468 cells were collected and subcutaneously injected into the left flank (ad-MKK6 cells) or right flank (ad-vector cells) of the animal with each mouse as its own control. As shown in Fig. 1C, injection of the control virus-infected cells produced a noticeable tumor in all cases about 2 months later, whereas no ad-MKK6-infected cells produced a tumor. ER+ cells were not included in these ex vivo assays as ER+ cells require supplemental estrogen. Together, these results demonstrated that stress selectively induces cell death and inhibits cell growth in ER- but not ER+ human breast cancer cells. Stress Phosphorylates c-Jun and Induces Cell Death in a c-Jun- and ER-dependent Manner—Because c-Jun activation is known to be apoptotic in many systems, we sought to examine whether the selective cell death induced by stress in ER- cells was because of a specific c-Jun activation event in these cells. Following the adenovirus infection, cell lysates were prepared and examined for c-Jun phosphorylation by Western blot using a specific antibody. As shown in Fig. 2A, ad-MKK6 increased c-Jun phosphorylations in both ER- 468 and ER+ MCF-7 cells with the amount of phosphorylated c-Jun generally correlated with the level of MKK6 expression. To examine the effects of ARS on c-Jun phosphorylation, ER+ and ER- cells were treated with ARS for 30 min, and c-Jun phosphorylation was examined by Western blot and indirect immunostaining analysis (Fig. 2, B and C). ARS again induced c-Jun phosphorylation in all cell lines examined regardless of their ER status. These results indicate that c-Jun phosphorylation appears to be a general response to stress signaling (at least to the active MKK6 and ARS) in human breast cancer cells, independent of ER expression. The opposite cell death-inducing effect of stress in ER+ and ER- cells, therefore, is apparently not because of a difference in the c-Jun activation. To assess whether c-Jun activation is responsible for the cell death induction by stress in ER- cells, a dominant negative c-Jun, Tam67, coding for c-Jun minus its activation domain, provided by Birrer and co-workers (27Brown P.H. Alani R. Preis L.H. Szabo E. Birrer M.J. Oncogene. 1993; 8: 877-886PubMed Google Scholar), was stably expressed in breast cancer cells (Fig. 2D). Tam67 has been widely applied to inhibit c-Jun activity in variety of systems (7Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5064) Google Scholar, 36Reuther J.Y. Kashatus D. Chen S. Li X. Westwick J. Davis R.J. Earp H.S. Wang C.Y. Baldwin A.S. Mol. Cell. Biol. 2002; 22: 8175-8183Crossref PubMed Scopus (78) Google Scholar, 37Alfranca A. Gutierrez M.D. Vara A. Aragones J. Vidal F. Landazuri M.O. Mol. Cell. Biol. 2002; 22: 12-22Crossref PubMed Scopus (102) Google Scholar, 38Ivanov V.N. Bhoumik A. Krasilnikov M. Raz R. Owen-Schaub L.B. Levy D. Horvath C.M. Ronai Z. Mol. Cell. 2001; 7: 517-528Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar) presumably through inhibition of transcription-competent endogenous c-Jun. The endogenous c-Jun at 39 kDa in both vector and Tam67-overexpressed cells was not detectable under these experimental conditions, consistent with limited c-Jun concentrations in these breast cancer cells (18Smith L. Wise S.C. Hendricks D.T. Sabichi A.L. Bos T. Reddy P. Brown P.H. Birrer M. Oncogene. 1999; 18: 6063-6070Crossref PubMed Scopus (190) Google Scholar). To determine effects of c-Jun inhibition on cell death, Tam67-expressing (468/Tam) and vector-transfected (468/Neo) cells were infected with advector or ad-MKK6, and cell death was analyzed by colony formation assay. As shown in Fig. 2E, the growth inhibitory effect of MKK6 was significantly reduced in 468/Tam cells in comparison with 468/Neo cells consistent with the cell death inhibitory activities of Tam67 previously observed in neural, vascular, and breast cancer cells (39Ham J. Babij C. Whitfield J. Pfarr C.M. Lallemand D. Yaniv M. Rubin L.L. Neuron. 1995; 14: 927-939Abstract Full Text PDF PubMed Scopus (758) Google Scholar, 40Wang N. Verna L. Hardy S. Zhu Y. Ma K. Birrer M. Stemerman M. Circ. Res. 1999; 85: 387-393Crossref PubMed Scopus (80) Google Scholar, 41Zhao B. Yu W. Qian M. Simmons-Menchaca M. Brown P. Birrer M. Sand
Referência(s)