Medulloblastoma Sensitivity to 17-Allylamino-17-demethoxygeldanamycin Requires MEK/ERK
2003; Elsevier BV; Volume: 278; Issue: 27 Linguagem: Inglês
10.1074/jbc.m211600200
ISSN1083-351X
AutoresChristopher Calabrese, Adrian Frank, Kirsteen H. Maclean, Richard J. Gilbertson,
Tópico(s)Cancer therapeutics and mechanisms
ResumoERBB2 increases the sensitivity of breast cancer cells to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). This has been attributed to the disruption of ERBB3/ERBB2 heterodimers that maintain a crucial cell survival signal via phosphatidylinositol 3-kinase/AKT. ERBB2 confers a poor clinical outcome in medulloblastoma, the most common malignant pediatric brain tumor. Here, we show that medulloblastoma cell sensitivity to 17-AAG is directly related to ERBB2 expression level. Furthermore, overexpression of exogenous ERBB2 in these cells induces spontaneous homodimerization, further enhancing cell sensitivity to 17-AAG. In contrast to breast cancer cells, this increased sensitivity to 17-AAG does not result from cell dependence on AKT1 activity. Rather, we show that 17-AAG generates a dose- and time-dependent increase in MEK/ERK signaling that is required for the drug to inhibit the proliferation of medulloblastoma cells and that ERBB2 sensitizes medulloblastoma cells to 17-AAG by up-regulating basal MEK/ERK signaling. We further show that down-regulation of MEK1 activity markedly reduces the sensitivity of medulloblastoma, breast, and ovarian cancer cells to 17-AAG, whereas expression of a constitutively active MEK1 potentiates the activity of 17-AAG against these cells. Therefore, intact MEK/ERK signaling may be required for optimal 17AAG activity against a variety of tumor cell types. These data identify a new mechanism by which 17-AAG inhibits the proliferation of cancer cells. Defining the precise mode of action of these agents within specific tumor cell types will be crucial if this class of drugs is to be efficiently developed in the clinic. ERBB2 increases the sensitivity of breast cancer cells to the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). This has been attributed to the disruption of ERBB3/ERBB2 heterodimers that maintain a crucial cell survival signal via phosphatidylinositol 3-kinase/AKT. ERBB2 confers a poor clinical outcome in medulloblastoma, the most common malignant pediatric brain tumor. Here, we show that medulloblastoma cell sensitivity to 17-AAG is directly related to ERBB2 expression level. Furthermore, overexpression of exogenous ERBB2 in these cells induces spontaneous homodimerization, further enhancing cell sensitivity to 17-AAG. In contrast to breast cancer cells, this increased sensitivity to 17-AAG does not result from cell dependence on AKT1 activity. Rather, we show that 17-AAG generates a dose- and time-dependent increase in MEK/ERK signaling that is required for the drug to inhibit the proliferation of medulloblastoma cells and that ERBB2 sensitizes medulloblastoma cells to 17-AAG by up-regulating basal MEK/ERK signaling. We further show that down-regulation of MEK1 activity markedly reduces the sensitivity of medulloblastoma, breast, and ovarian cancer cells to 17-AAG, whereas expression of a constitutively active MEK1 potentiates the activity of 17-AAG against these cells. Therefore, intact MEK/ERK signaling may be required for optimal 17AAG activity against a variety of tumor cell types. These data identify a new mechanism by which 17-AAG inhibits the proliferation of cancer cells. Defining the precise mode of action of these agents within specific tumor cell types will be crucial if this class of drugs is to be efficiently developed in the clinic. ERBB2 (HER-2/neu) is a potent oncogene in cell culture (1Bargmann C.I. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 5394-5398Google Scholar, 2Di Marco E. Pierce J.H. Knicley C.L. Di Fiore P.P. Mol. Cell. Biol. 1990; 10: 3247-3252Google Scholar, 3Di Fiore P.P. Pierce J.H. Kraus M.H. Segatto O. King C.R. Aaronson S.A. Science. 1987; 237: 178-182Google Scholar, 4Zhang K. Sun J. Liu N. Wen D. Chang D. Thomason A. Yoshinaga S.K. J. Biol. Chem. 1996; 271: 3884-3890Google Scholar) and transgenic models of cancer (5Andrechek E.R. Hardy W.R. Siegel P.M. Rudnicki M.A. Cardiff R.D. Muller W.J. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3444-3449Google Scholar, 6Li B. Rosen J.M. McMenamin-Balano J. Muller W.J. Perkins A.S. Mol. Cell. Biol. 1997; 17: 3155-3163Google Scholar), and overexpression of this receptor tyrosine kinase is associated with a poor clinical outcome in a number of human malignancies (7Yarden Y. Sliwkowski M.X. Nat. Rev. Mol. Cell Biol. 2001; 2: 127-137Google Scholar). Therefore, compounds that inhibit ERBB2 function are being developed in the clinic as anti-cancer drugs (7Yarden Y. Sliwkowski M.X. Nat. Rev. Mol. Cell Biol. 2001; 2: 127-137Google Scholar, 8Slichenmyer W.J. Fry D.W. Semin. Oncol. 2001; 28: 67-79Google Scholar). These include the ansamycin 17-allylamino-17-demethoxygeldanamycin (17-AAG), 1The abbreviations used are: 17-AAG, 17-allylamino-17-demethoxygeldanamycin; HSP, heat shock protein; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; HA, hemagglutinin. 1The abbreviations used are: 17-AAG, 17-allylamino-17-demethoxygeldanamycin; HSP, heat shock protein; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ERK, extracellular signal-regulated kinase; HA, hemagglutinin. which binds to the molecular chaperone heat shock protein (HSP)-90 and inhibits its function (9Neckers L. Trends Mol. Med. 2002; 8: 555-561Google Scholar).HSP90 plays a key role in the conformational maturation of important cell signaling proteins, including ERBB2, AKT1, and RAF1 (9Neckers L. Trends Mol. Med. 2002; 8: 555-561Google Scholar, 10Pratt W.B. Proc. Soc. Exp. Biol. Med. 1998; 217: 420-434Google Scholar). Inhibition of HSP90 targets these proteins for proteasomal degradation (11An W.G. Schnur R.C. Neckers L. Blagosklonny M.V. Cancer Chemother. Pharmacol. 1997; 40: 60-64Google Scholar, 12An W.G. Schulte T.W. Neckers L.M. Cell Growth Differ. 2000; 11: 355-360Google Scholar, 13Stancato L.F. Silverstein A.M. Owens-Grillo J.K. Chow Y.H. Jove R. Pratt W.B. J. Biol. Chem. 1997; 272: 4013-4020Google Scholar, 14Supko J.G. Hickman R.L. Grever M.R. Malspeis L. Cancer Chemother. Pharmacol. 1995; 36: 305-315Google Scholar, 15Whitesell L. Sutphin P. An W.G. Schulte T. Blagosklonny M.V. Neckers L. Oncogene. 1997; 14: 2809-2816Google Scholar), resulting in growth inhibition and apoptosis of tumor cells (16Hostein I. Robertson D. DiStefano F. Workman P. Clarke P.A. Cancer Res. 2001; 61: 4003-4009Google Scholar, 17Munster P.N. Srethapakdi M. Moasser M.M. Rosen N. Cancer Res. 2001; 61: 2945-2952Google Scholar, 18Munster P.N. Marchion D.C. Basso A.D. Rosen N. Cancer Res. 2002; 62: 3132-3137Google Scholar, 19Basso A.D. Solit D.B. Munster P.N. Rosen N. Oncogene. 2002; 21: 1159-1166Google Scholar, 20Solit D.B. Zheng F.F. Drobnjak M. Munster P.N. Higgins B. Verbel D. Heller G. Tong W. Cordon-Cardo C. Agus D.B. Scher H.I. Rosen N. Clin. Cancer Res. 2002; 8: 986-993Google Scholar, 21Kelland L.R. Sharp S.Y. Rogers P.M. Myers T.G. Workman P. J. Natl. Cancer Inst. 1999; 91: 1940-1949Google Scholar). Although 17-AAG down-regulates a large number of cellular proteins (9Neckers L. Trends Mol. Med. 2002; 8: 555-561Google Scholar), the anti-tumor properties of this drug may be attributed to effects on specific signal pathways, most notably those driven by ERBB2.ERBB2 is one of the most sensitive HSP90-dependent client proteins (22Neckers L. Clin. Cancer Res. 2002; 8: 962-966Google Scholar). Overexpression of this receptor by breast and ovarian cancer cells renders them acutely sensitive to growth inhibition by 17-AAG (18Munster P.N. Marchion D.C. Basso A.D. Rosen N. Cancer Res. 2002; 62: 3132-3137Google Scholar, 23Smith V. Hobbs S. Court W. Eccles S. Workman P. Kelland L.R. Anticancer Res. 2002; 22: 1993-1999Google Scholar). This enhanced sensitivity has been attributed to the disruption of ERBB2/ERBB3 heterodimers, which block activation of phosphatidylinositol 3-kinase/AKT, thereby inhibiting a crucial cell survival signal (18Munster P.N. Marchion D.C. Basso A.D. Rosen N. Cancer Res. 2002; 62: 3132-3137Google Scholar, 19Basso A.D. Solit D.B. Munster P.N. Rosen N. Oncogene. 2002; 21: 1159-1166Google Scholar). Although ERBB2-dependent phosphatidylinositol 3-kinase/AKT signaling appears to be an important target of 17-AAG, this drug also inhibits components of the RAS/mitogen-activated protein kinase pathway (20Solit D.B. Zheng F.F. Drobnjak M. Munster P.N. Higgins B. Verbel D. Heller G. Tong W. Cordon-Cardo C. Agus D.B. Scher H.I. Rosen N. Clin. Cancer Res. 2002; 8: 986-993Google Scholar, 22Neckers L. Clin. Cancer Res. 2002; 8: 962-966Google Scholar). Indeed, depletion of N-RAS, Ki-RAS, and RAF1 from colon carcinoma cells inhibits ERK1/2 activation, resulting in growth inhibition and apoptosis (16Hostein I. Robertson D. DiStefano F. Workman P. Clarke P.A. Cancer Res. 2001; 61: 4003-4009Google Scholar).Little progress has been made in the development of molecular targeted therapies for pediatric malignancies. Medulloblastoma is a highly invasive pediatric brain tumor. Conventional chemo- and radiotherapy achieves a cure in only a subset of patients, and there is a great need for new therapeutic approaches (24Packer R.J. Brain Dev. 1999; 21: 75-81Google Scholar, 25Zeltzer P.M. Boyett J.M. Finlay J.L. Albright A.L. Rorke L.B. Milstein J.M. Allen J.C. Stevens K.R. Stanley P. Li H. Wisoff J.H. Geyer J.R. McGuire-Cullen P. Stehbens J.A. Shurin S.B. Packer R.J. J. Clin. Oncol. 1999; 17: 832-845Google Scholar). Previously, we reported that overexpression of ERBB2 in medulloblastoma up-regulates the transcription of pro-metastatic genes (26Hernan R. Fasheh R. Calabrese C. Frank A.J. Maclean K.H. Allard D. Barraclough R. Gilbertson R.J. Cancer Res. 2003; 63: 140-148Google Scholar), promotes metastasis and tumor cell proliferation (27Gilbertson R.J. Clifford S.C. MacMeekin W. Meekin W. Wright C. Perry R.H. Kelly P. Pearson A.D. Lunec J. Cancer Res. 1998; 58: 3932-3941Google Scholar, 28Gilbertson R.J. Jaros E. Perry R.H. Kelly P.J. Lunec J. Pearson A.D. Eur. J. Cancer. 1997; 33: 609-615Google Scholar), and confers a poor clinical outcome (29Gilbertson R.J. Perry R.H. Kelly P.J. Pearson A.D. Lunec J. Cancer Res. 1997; 57: 3272-3280Google Scholar, 30Gilbertson R.J. Pearson A.D. Perry R.H. Jaros E. Kelly P.J. Br. J. Cancer. 1995; 71: 473-477Google Scholar, 31Gilbertson R. Wickramasinghe C. Hernan R. Balaji V. Hunt D. Jones-Wallace D. Crolla J. Perry R. Lunec J. Pearson A. Ellison D. Br. J. Cancer. 2001; 85: 705-712Google Scholar). Therefore, we investigated whether 17-AAG might be a potential new treatment for this disease. In particular, we studied the role of ERBB2 signaling in modulating the sensitivity of medulloblastoma cells to 17-AAG.EXPERIMENTAL PROCEDURESReagents, Antibodies, and Western Blotting—17-AAG (NSC 330507) was obtained from the National Cancer Institute's Developmental Therapeutic Program. The MEK1/2 inhibitors PD98059 and U0126 were from Calbiochem. Phosphorothioated MEK1 antisense 5′-GCCGCCGCCGCCGCCAT-3′ and scrambled control 5′-CGCGCGCTCGCGCACCC-3′ oligonucleotides (32Robinson C.J. Scott P.H. Allan A.B. Jess T. Gould G.W. Plevin R. Biochem. J. 1996; 320: 123-127Google Scholar) were synthesized using an ABI™ 3900 High-Throughput DNA Synthesizer (Applied Biosystems, Foster City, CA). The antibodies used were HSP70 and HSP90 mouse monoclonal antibodies (StressGen Biotechnology, Victoria, Canada), TP53 mouse monoclonal (Oncogene Research Products, Boston, MA), RAF-1 rabbit polyclonal, total ERK1 (goat), and phospho-ERK1/2 (Y204) mouse monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA), total AKT1 and phospho-AKT-1 (Ser-473) rabbit polyclonal antibodies (Cell Signaling Technology, Beverly, MA), hemagglutinin (HA) mouse monoclonal antibody (Covance, Richmond, CA), ERBB1 mouse monoclonal antibody NCL-EGFR (NovaCastra, Newcastle-upon-Tyne, UK), phospho-Y1068 ERBB1 (Cell Signaling Technology), ERBB2 mouse monoclonal antibody NCL-CB11 (NovaCastra, Newcastle-upon-Tyne, UK), phospho-Y1248 ERBB2 (Cell Signaling Technology), ERBB3 rabbit polyclonal antibody (Santa Cruz Biotechnology), and ERBB4 rabbit polyclonal antibody (Santa Cruz Biotechnology). Western blotting was performed using standard techniques as described previously (29Gilbertson R.J. Perry R.H. Kelly P.J. Pearson A.D. Lunec J. Cancer Res. 1997; 57: 3272-3280Google Scholar).Cells, DNA Constructs, and Transfections—The MHH-MED-1 and MEB-MED-8A cells lines (33Pietsch T. Scharmann T. Fonatsch C. Schmidt D. Ockler R. Freihoff D. Albrecht S. Wiestler O.D. Zeltzer P. Riehm H. Cancer Res. 1994; 54: 3278-3287Google Scholar) were provided by Dr. Torsten Pietsch (University of Bonn, Bonn, Germany). The Daoy medulloblastoma, MCF-7 and SKBR3 breast cancer, and SKOV3 ovarian cancer cell lines were obtained from the American Type Culture Collection. Cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (BioWhittaker, Walkersville, MA). ERBB2-overexpressing Daoy cell clones (designated Daoy.1 and Daoy.2) were generated by stable transfection with human ERBB2 cDNA inserted into pcDNA3.1 as described previously (26Hernan R. Fasheh R. Calabrese C. Frank A.J. Maclean K.H. Allard D. Barraclough R. Gilbertson R.J. Cancer Res. 2003; 63: 140-148Google Scholar). Control cells transfected with empty vector alone were designated Daoy.V. We generated the AKT1 and green fluorescent protein retroviral vectors as described previously (26Hernan R. Fasheh R. Calabrese C. Frank A.J. Maclean K.H. Allard D. Barraclough R. Gilbertson R.J. Cancer Res. 2003; 63: 140-148Google Scholar). The pUSEamp/MEK1* expression vector that contains an HA-tagged constitutively activated (S218D/S222D) MEK1 was obtained from Upstate Biotechnology, Lake Placid, NY. Daoy cells were infected with retroviral vectors using standard procedures as described previously (26Hernan R. Fasheh R. Calabrese C. Frank A.J. Maclean K.H. Allard D. Barraclough R. Gilbertson R.J. Cancer Res. 2003; 63: 140-148Google Scholar). Tumor cells were transiently transfected with pUSEamp/MEK1* or empty vector using the LipofectAMINE reagent as detailed by the manufacturer (Invitrogen). Expression of exogenous AKT1 and activated MEK1 was confirmed by HA-specific Western blotting. Green fluorescent protein expression was detected by fluorescence microscopy (34Gilbertson R.J. Bentley L. Hernan R. Junttila T.T. Frank A.J. Haapasalo H. Connelly M. Wetmore C. Curran T. Elenius K. Ellison D.W. Clin. Cancer Res. 2002; 8: 3054-3064Google Scholar). MEK1 oligonucleotides were added to serum-free Dulbecco's modified Eagle's medium containing 16 μl of LipofectAMINE (Invitrogen) per 1 μg of DNA to give a final oligonucleotide concentration of 10 μm. Cells were incubated with oligonucleotides for 8 h before the addition of 17-AAG and processed as described.Dimer Assay—Cultured cells were treated on ice with either phosphate-buffered saline or epidermal growth factor 50 ng/ml for 2 h. 1 mm bis-(sulfosuccinimidyl)-suberate (BS3) was then added, and cells were incubated for a further 45 min. Total protein lysates were then generated and analyzed by Western blotting as described.Drug Treatments—For growth inhibition studies, 3 × 103 cells were seeded into each well of a 96-well plate in 10% fetal bovine serum Dulbecco's modified Eagle's medium and incubated for 24 h. 17-AAG or vehicle only (0.1% Me2SO) was then added, and plates were incubated for the indicated time period, up to a maximum of 96 h. For drug exposures less than 96 h, drug-containing medium was replaced with drug-free medium, and incubation was continued for a total of 96 h. After incubation, cell growth was determined using an XTT-based assay (Roche Applied Science). IC50 was defined as the drug concentration that inhibited cell proliferation by 50% for a 96-h exposure. Morphological detection of apoptosis was performed by epifluorescence microscopy of Hoechst 33258-stained cells. One observer who was blinded to all preceding cell treatments performed the apoptotic counts using nuclei from triplicate experiments.For drug combination studies, cells were pretreated for 8 h with MEK1 oligonucleotide (or scrambled control) as described above or with the MEK1/2 inhibitors PD98059 or U0126 (or 0.1% Me2SO control) for 2 h. 17-AAG (or 0.1% Me2SO) was then added, and growth assays were conducted as described. To determine the impact of drug treatment on protein expression, cells were treated as described for growth assays, and the total protein lysates (29Gilbertson R.J. Perry R.H. Kelly P.J. Pearson A.D. Lunec J. Cancer Res. 1997; 57: 3272-3280Google Scholar) were analyzed by Western blotting.RESULTSERBB2 Expression Level Correlates with Medulloblastoma Cell Sensitivity to 17-AAG—Breast cancer cells that overexpress ERBB2 are dependent on ERBB3/ERBB2 heterodimer signaling. This renders them acutely sensitive to treatment with 17-AAG (18Munster P.N. Marchion D.C. Basso A.D. Rosen N. Cancer Res. 2002; 62: 3132-3137Google Scholar, 19Basso A.D. Solit D.B. Munster P.N. Rosen N. Oncogene. 2002; 21: 1159-1166Google Scholar, 23Smith V. Hobbs S. Court W. Eccles S. Workman P. Kelland L.R. Anticancer Res. 2002; 22: 1993-1999Google Scholar). We investigated whether ERBB2 similarly dictates 17-AAG activity against medulloblastoma cells. To do this, we determined the 17-AAG inhibitory concentration (IC) 50 values of 3 medulloblastoma cell lines after 96 h of continuous drug exposure and compared these to cell ERBB2 expression levels. Daoy cells that expressed low levels of ERBB2 (Fig. 1A) were relatively resistant to 17-AAG (Fig. 1B); indeed these cells displayed an IC50 value that was considerably higher than that previously reported for 40 human cancer cell lines treated under similar conditions (18Munster P.N. Marchion D.C. Basso A.D. Rosen N. Cancer Res. 2002; 62: 3132-3137Google Scholar, 20Solit D.B. Zheng F.F. Drobnjak M. Munster P.N. Higgins B. Verbel D. Heller G. Tong W. Cordon-Cardo C. Agus D.B. Scher H.I. Rosen N. Clin. Cancer Res. 2002; 8: 986-993Google Scholar, 21Kelland L.R. Sharp S.Y. Rogers P.M. Myers T.G. Workman P. J. Natl. Cancer Inst. 1999; 91: 1940-1949Google Scholar). In contrast, MHH-MED-1 and MEB-MED-8A cells, which express moderate to high levels of ERBB2, were relatively sensitive to 17-AAG (Fig. 1, A and B). Therefore, ERBB2 expression level correlates with medulloblastoma cell sensitivity to 17-AAG. However, in contrast to breast cancer cells, this enhanced sensitivity cannot be mediated via ERBB3/ERBB2 heterodimers, since ERBB3 was not detected in any of the medulloblastoma cell lines (Fig. 1A). Furthermore, whereas Daoy and MHH-MED-1 cells express low level ERBB1 and high level ERBB4, respectively, ERBB2 is expressed in isolation in MEB-MED-8A cells (Fig. 1A).ERBB2 Homodimerization Activates AKT1 and ERK1/2 Signaling in Medulloblastoma Cells and Is Associated with Increased Sensitivity to 17-AAG—ERBB2 homodimers spontaneously arise when the receptor is expressed at high levels in cells (3Di Fiore P.P. Pierce J.H. Kraus M.H. Segatto O. King C.R. Aaronson S.A. Science. 1987; 237: 178-182Google Scholar, 35Olayioye M.A. Neve R.M. Lane H.A. Hynes N.E. EMBO J. 2000; 19: 3159-3167Google Scholar). Therefore, we reasoned that ERBB2 homodimer signaling might increase the sensitivity of medulloblastoma cells to 17-AAG. To investigate this we induced spontaneous ERBB2 homodimerization in Daoy cells by overexpressing exogenous ERBB2 (Fig. 2A, second lane). This resulted in activation of AKT1 and ERK1/2, but not p38, JNK, STAT3, or STAT5 (Fig. 2B and data not shown). Activation of phosphatidylinositol 3-kinase/AKT signaling by spontaneous ERBB2 homodimers in the absence of growth factors has been described in a number of cell types including NIH3T3 fibroblasts (36Zhou B.P. Hu M.C. Miller S.A. Yu Z. Xia W. Lin S.Y. Hung M.C. J. Biol. Chem. 2000; 275: 8027-8031Google Scholar). However, the precise mechanism by which ERBB2 homodimers activate phosphatidylinositol 3-kinase/AKT remains to be determined. Our data indicate that overexpression of ERBB2 did not increase cell signaling via ERBB1/ERBB2 heterodimerization, since ERBB1 was not phosphorylated (Fig. 2B) and ERBB1/ERBB2 heterodimers could only be detected after treatment with exogenous epidermal growth factor (EGF) (Fig. 2A, third lane).Fig. 2Overexpression of exogenous ERBB2 in the 17-AAG-resistant Daoy cell line induces spontaneous ERBB2 homodimerization, activation of AKT1, and ERK1/2 signaling and increased sensitivity to 17-AAG.A, Daoy cells transfected with empty vector (Daoy.V) or pcDNA3.1/ERBB2 (Daoy.2) were treated on ice with either phosphate-buffered saline (–) or 50 ng/ml epidermal growth factor (EGF)(+) for 2 h. 1 mm bis-(sulfosuccinimidyl)-suberate (BS3) was then added, and cells were incubated for a further 45 min. Total protein lysates were generated and analyzed by Western blotting using an antibody specific for phospho-Tyr-1248 of ERBB2. Note that spontaneous ERBB2 homodimers (D1) were only detected in ERBB2-overexpressing Daoy.2 cells (compare first and second lanes). Epidermal growth factor induced the formation of a distinct ERBB1/ERBB2 heterodimer complex (D2). M, monomers. B, total protein lysates were prepared from exponentially growing cultures of Daoy.V and two independent ERBB2-transfected Daoy clones (Daoy.1 and Daoy.2). Fifty micrograms of each lysate were analyzed by phospho (p)-specific Western blotting for activation of ERBB1, ERBB2, ERK1/2, and AKT1 signaling. Levels of phosphorylated proteins were compared with total amounts of the respective protein. Expression levels of RAF1, TP53, and the key chaperone components HSP90, HSP70, and GRP94 were also determined. C, Daoy.V, Daoy.1, and Daoy.2 cells were seeded in 96-well plates and exposed to the indicated concentrations of 17-AAG (or vehicle alone) for 96 h. The percentage growth inhibition of 17-AAG relative to vehicle-treated cells was then determined using an XTT-based assay.View Large Image Figure ViewerDownload (PPT)We next investigated the impact of ERBB2 homodimer signaling on cell sensitivity to 17-AAG. After 96 h of continuous exposure to 17-AAG, two independent ERBB2-transfected Daoy clones were significantly more sensitive to growth inhibition by 17-AAG than control cells (Fig. 2C). These data confirm that ERBB2 sensitizes medulloblastoma cells to 17-AAG and support the hypothesis that this is mediated by ERBB2 homodimer signaling via AKT1 and/or ERK1/2. Importantly, the expression of key chaperone proteins was not affected by ERBB2 transfection and, thus, did not account for differences in 17-AAG sensitivity (Fig. 2B).ERBB2 Homodimers Sensitize Medulloblastoma Cells to 17-AAG by Up-regulating ERK1/2 Signaling—We reasoned that ERBB2 homodimer signaling could increase medulloblastoma cell sensitivity to 17-AAG through two alternative mechanisms. Up-regulation of AKT1 or ERK1/2 activity might provide a crucial survival signal for medulloblastoma cells. Thus, analogous to the disruption of ERBB3/ERBB2 heterodimers by 17-AAG in breast cancer cells (18Munster P.N. Marchion D.C. Basso A.D. Rosen N. Cancer Res. 2002; 62: 3132-3137Google Scholar, 19Basso A.D. Solit D.B. Munster P.N. Rosen N. Oncogene. 2002; 21: 1159-1166Google Scholar), down-regulation of ERBB2 homodimers could inhibit a pro-survival signal. Conversely, AKT1 or ERK1/2 could play an active role in 17-AAG-mediated growth inhibition. In this situation, up-regulation of one or both of these pathways by ERBB2 homodimers could sensitize medulloblastoma cells to the drug.To investigate these alternative hypotheses we exposed Daoy cells to a range of 17-AAG concentrations and studied the impact of this treatment on cell protein expression levels. Continuous exposure of control cells to 17-AAG (48 h) abolished ERBB2 expression and generated a dose-dependent depletion of phosphorylated AKT1 (Fig. 3A). 17-AAG also caused a decrease in total AKT1 protein expression in these cells, albeit to a lesser extent than the phosphorylated form. In contrast, 17-AAG was relatively ineffective at inhibiting AKT1 in ERBB2-transfected cells (Fig. 3A). Indeed, phosphorylation of AKT1 was unaffected by 250 nm 17-AAG, which inhibited the growth of these cells by ≥75% (Fig. 2C). This signaling was maintained by active ERBB2 homodimers that could still be detected in the presence of 250 nm 17-AAG (Fig. 3A and data not shown). Therefore, we concluded that inhibition of ERBB2/AKT1 signaling is unlikely to account for the increased sensitivity of ERBB2-overexpressing Daoy cells to 17-AAG.Fig. 3Characterization of 17-AAG activity against the activation and expression status of signal pathway and chaperone proteins in Daoy, MHH-MED-1, and MEB-MED-8A medulloblastoma cells.A, exponentially growing cultures of Daoy.V, Daoy.1, and Daoy.2 cells were exposed to the indicated concentrations of 17-AAG (or vehicle alone, Control) for 48 h. Total cell lysates were then prepared, and expression of the indicated proteins was determined by Western blot analysis. B, the MHH-MED-1 and MEB-MED-8A medulloblastoma cell lines were subject to the same treatment described in A, and expression of ERBB2, RAF-1, and pERK1/2 was determined by Western blot analysis.View Large Image Figure ViewerDownload (PPT)Unexpectedly, 17-AAG generated a dose-dependent increase in ERK1/2 phosphorylation in Daoy cells (Fig. 3A). This was not affected by ERBB2 expression status and occurred despite concurrent degradation of RAF1. As previously reported (37Piatelli M.J. Doughty C. Chiles T.C. J. Biol. Chem. 2002; 277: 12144-12150Google Scholar, 38Schulte T.W. Blagosklonny M.V. Romanova L. Mushinski J.F. Monia B.P. Johnston J.F. Nguyen P. Trepel J. Neckers L.M. Mol. Cell. Biol. 1996; 16: 5839-5845Google Scholar, 39Schulte T.W. An W.G. Neckers L.M. Biochem. Biophys. Res. Commun. 1997; 239: 655-659Google Scholar), 17-AAG did not affect expression of either total MEK1 or ERK1. To investigate whether up-regulation of ERK1/2 activity by 17-AAG is a general feature of medulloblastoma cells, we analyzed the impact of 17-AAG treatment on ERBB2, RAF-1, and pERK1/2 expression levels in MHH-MED-1 and MEB-MED-8A cells. 17-AAG generated a dose-dependent increase in ERK1/2 phosphorylation independent of ERBB2 and RAF1 in these cells (Fig. 3B). In other cell line systems, ansamycins have been reported to decrease MEK/ERK signaling by depleting upstream components and activators of the mitogen-activated protein kinase pathway (37Piatelli M.J. Doughty C. Chiles T.C. J. Biol. Chem. 2002; 277: 12144-12150Google Scholar, 38Schulte T.W. Blagosklonny M.V. Romanova L. Mushinski J.F. Monia B.P. Johnston J.F. Nguyen P. Trepel J. Neckers L.M. Mol. Cell. Biol. 1996; 16: 5839-5845Google Scholar, 40Schulte T.W. Neckers L.M. Cancer Chemother. Pharmacol. 1998; 42: 273-279Google Scholar). Conversely, our data indicate that 17-AAG can positively regulate MEK/ERK signaling independent of RAF-1 in medulloblastoma cells, presumably by inducing alternative activators of this signal cassette. Furthermore, these data support the hypothesis that induction of ERK1/2 plays a positive role in the anti-proliferative activity of 17-AAG. Therefore, although the induction of ERK1/2 by 17-AAG appears independent of ERBB2 and RAF1 (Fig. 3, A and B), up-regulation of basal ERK1/2 signaling by ERBB2 homodimers (Fig. 2B) may nonetheless sensitize medulloblastoma cells to 17-AAG. Indeed, the high ERBB2-expressing, 17-AAG-sensitive Daoy.1, Daoy.2, MHH-MED-1, and MEB-MED-8A cells all demonstrate detectable basal pERK1/2, which is absent in low ERBB2-expressing, 17-AAG-resistant Daoy.V cells (Fig. 3, A and B).To investigate further the role of ERK1/2 signaling in 17-AAG activity, we used the isogenic empty vector and ERBB2-transfected Daoy cells to study the temporal relationship between drug-mediated activation of ERK1/2 and growth inhibition. As expected, phosphorylation of ERK1/2 was undetectable in untreated Daoy.V control cells (Fig. 4A). However, exposure of these cells to 250 nm 17-AAG for 16 h activated ERK1/2 (Fig. 4A). This coincided exactly with the duration of drug exposure required to inhibit the growth of these cells (Fig. 4B). In contrast, ERK1/2 activation was readily detected in vehicle and 17-AAG-treated ERBB2-transfected cells (Fig. 4A), and only 8 h of drug exposure were required to significantly inhibit cell growth (Fig. 4B). Similar results were observed in the other ERBB2-transfected clone (Daoy.1). In contrast, AKT1 activity in both control and ERBB2-transfected cells was largely unaffected by drug exposures of less than 24 h (Fig. 4A).Fig. 417-AAG-induced ERK1/2 activity and growth inhibition are correlated in Daoy cells.A, Daoy.V and Daoy.2 cells were exposed to 250 nm 17-AAG for the indicated times. Total protein lysates were then prepared and analyzed by Western blotting for expression of the indicated phosphorylated (p) proteins. Actin was employed as a loading and transfer control. B, cells were seeded into 96-well plates and exposed to 250 nm 17-AAG or vehicle-only for the indicated times. Medium was then replaced with drug-free medium, and incubation was continued for a total of 96 h. The percentage growth inhibition of 17-AAG relative to vehicle-treated cells was then determined using an XTT-based assay.View Large Image Figure ViewerDownload (PPT)These data support the hypothesis that inhibition of medulloblastoma cell growth by 17-AAG involves the induction of ERK1/2 signaling and that up-regulation of basal ERK1/2 activity by ERBB2 homodimers sensitizes these cells to 17-AAG. Furthermore, they provide additional evidence that inhibition of AKT1 is not required for 17-AAG-mediated growth inhib
Referência(s)