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Molecular Biology and Genetics |
Departments of Developmental Neurobiology [R. H., R. F., C. C., A. J. F., R. J. G.] and Biochemistry [K. H. M.], St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, and School of Biological Sciences, Life Sciences Building, University of Liverpool, Liverpool L69 7ZB, United Kingdom [D. A., R. B.]
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ABSTRACT |
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INTRODUCTION |
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In addition to ERBB2, several other regulators of metastasis have been proposed to alter the cytoskeleton of tumor cells. These include the calcium binding protein S100A4 (19) . Elevated S100A4 expression is correlated with the metastatic potential of tumor cells in vivo (20 , 21) and with poor clinical outcome in breast and bladder cancer (22 , 23) . In addition, S100A4 accelerates metastasis in two independent transgenic models of breast cancer (24 , 25) .
Previously, we reported that ERBB2 overexpression is associated with poor clinical outcome and advanced metastatic disease stage in medulloblastoma (16 , 18 , 26 , 27) . Medulloblastoma is a highly invasive pediatric brain tumor that is frequently disseminated throughout the central nervous system at the time of diagnosis (28) . Conventional therapeutic approaches have not reduced the high mortality associated with metastatic medulloblastoma and little is known regarding the molecular mechanisms that promote invasion. Here, we demonstrate that ERBB2 promotes a metastatic phenotype in medulloblastoma by up-regulating a series of pro-metastatic genes, including S100A4, and by enhancing tumor cell invasion. We show that ERBB2 directs S100A4 expression via a signaling network that includes PI3k, AKT1, and ERK1/2. Finally, we demonstrate that this prometastatic phenotype can be reversed in vitro and in vivo using OSI-774 (Erlotinib), a small molecule inhibitor of ERBB2 signaling. Therefore, we identify a new prometastatic pathway that may provide a target for therapeutic intervention in medulloblastoma metastasis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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19 years) medulloblastoma from the St. Jude Children’s Research Hospital Tumor Bank. Samples were snap frozen at the time of primary surgical resection and stored at -80°C until use. Frozen sections from each sample were examined by light microscopy to ensure 
80% consisted of tumor. RNA was then extracted from samples by direct homogenization in the TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD).
Cell Culture.
We obtained the Daoy medulloblastoma cell line from the American Type Culture Collection. Cells were maintained as monolayer cultures in complete DMEM supplemented with 10% FBS. The effects of small molecule inhibitors on protein expression and S100A4 promoter activity were performed by culturing Daoy cells for the indicated times in the presence of the appropriate inhibitor (or 0.1% DMSO control) followed by Western blotting and luciferase analysis as described. PD158780, LY249002, and PD98059 were all from Calbiochem-Novabiochem (San Diego, CA). OSI-774 was generously provided by OSI Pharmaceuticals (Melville, NY).
Plasmids.
The complete human ERBB2 cDNA was inserted into the HindIII site of pcDNA3.1 to generate the construct pcDNA3.1/ERBB2. The first 1487, 1099, 697, and 322 bp of the human S100A4 promoter region, immediately 5' of the transcriptional start site, were inserted into the pGL-basic vector to generate plasmids pDA-L47, pDA-L49, pDA-L412, and pDA-L413, respectively. We generated the WT-AKT1 retroviral vector by inserting HA-tagged AKT1 into the EcoRI site of the MSCV retroviral vector. DN-AKT1 vectors were similarly prepared using kinase dead K179M or Myr
11-60 AKT1 sequences.
Stable Transfections and Retroviral Infections.
Daoy cells were transfected with 1 µg of pcDNA3.1 or pcDNA3.1/ERBB2 using the Lipofectamine reagent (Invitrogen Corporation, Carlsbad, CA) and separate clones of ERBB2 (designated Daoy.1–4), and empty vector-transfected (designated Daoy.V) cells selected under G418. Daoy.2 cells were infected with WT-AKT1 or DN-AKT1 retroviral vectors using standard procedures and expression confirmed by HA-specific Western blotting.
Tumor Cell Invasion.
The metastatic activity of Daoy.V, Daoy.1, and Daoy.2 cells was measured in vitro using the 24-well BD BioCoat FluoroBlok Invasion System as recommended by the manufacturer (BD Biosciences, Bedford, MA). Briefly, 3 x 104 cells were labeled for 20 min with 10 µM of the fluorescent cell probe CellTracker Green (Molecular Probes, Eugene, OR) in 10% FBS DMEM. Cells were then incubated for an additional 30 min in fresh 10% FBS DMEM. Baseline fluorescence analysis confirmed equivalent labeling of the three different Daoy-derived cell types. Ten percent FBS DMEM was then added as a chemoattractant to the lower chambers of the 24-well plates, and cells were seeded into the prehydrated upper wells of the BD BioCoat FluoroBlok chambers. Nonhydrated wells were included as controls. Cells were then cultured under standard conditions for the indicated times and invasion measured from below at excitation wavelength 485 nm and emission wavelength 530 nm using a Fusion Universal Microplate Analyzer (Perkin-Elmer Life Sciences, Boston, MA). The fluorescence of Daoy.1 and 2 cells that had invaded to the undersurface of the membrane was recorded relative to that of Daoy.V cells. Assays were conducted in quadruplicate. The influence of OSI-774 treatment on invasion was performed in an identical fashion using Daoy.2 cells that were preincubated in 30 nM of drug (or 0.1% DMSO only control) for 6 h before labeling with CellTracker Green.
Microarray Expression Analysis.
We determined the gene expression profiles of Daoy.V and Daoy.2 cells grown in vitro and in vivo using Affymetrix microarray analysis. Briefly, first- and second-strand cDNA was synthesized from 5–15 µg of total Daoy cell RNA using the SuperScript Double-Stranded cDNA Synthesis Kit (Life Technologies, Inc., Rockville, MD) and an oligo-dT24-T7 primer. This was then used to prepare cRNA that was labeled with biotinylated UTP and CTP by in vitro transcription using a T7 promoter-coupled double-stranded cDNA as template and the T7 RNA Transcript Labeling Kit (ENZO Diagnostics, Inc., Farmingdale, NY). After purification and precipitation at -20°C, 10 µg of this cRNA was fragmented by heat and ion-mediated hydrolysis at 95°C [200 mM Tris-acetate (pH 8.1), 500 mM KOAc, 150 mM MgOAc] and hybridized to the Human Genome U95Av2 oligonucleotide array chip (Affymetrix, Santa Clara, CA). U95Av2 contains 12,600 full-length annotated genes together with additional probe sets designed to represent EST sequences. Arrays were washed at 25°C with 6 x saline-sodium phosphate-EDTA (0.9 M NaCl, 60 mMNaH2PO4, 6 mM EDTA +0.01% Tween 20) followed by a stringent wash at 50°C with 100 mM MES, 0.1 M [Na+], 0.01% Tween 20. We then stained arrays with phycoerythrin-conjugated streptavidin (Molecular Probes), and the fluorescence intensities were determined using a laser confocal scanner (Hewlett-Packard, Palo Alto, CA). The scanned images were analyzed using Microarray software (Affymetrix). We standardized for sample loading and variations in staining by scaling the average of the fluorescent intensities of all genes on an array to a constant target intensity (2500) for all arrays used. The expression data were analyzed as described previously (29)
. The signal intensity for each gene was calculated as the average intensity difference, represented by [
(PM - MM)/(number of probe pairs)], where PM and MM denote perfect match and mismatch probes. Separate microarray experiments were conducted for Daoy.V and Daoy.2 clones. We constructed scatterplots comparing the average intensity differences of expression profiles for the two cell types. Genes whose expression significantly varied (positively or negatively 2-fold) in Daoy.2 versus Daoy.V cells were identified using Spotfire Decision Site 6.2 software (Spotfire, Somerville, MA).
Luciferase Reporter Assay.
Daoy.V and Daoy.2 cells were grown to 60–80% confluency and transfected with a 20:1 mix of the pDA S100A4 and pRL Renilla control luciferase reporter constructs using the Lipofectamine reagent (Invitrogen Corporation). After 48 h, cells were lysed in Passive Lysis Buffer (Promega, Madison, WI) and the firefly (pDA reporter plasmids) relative to Renilla (control) luciferase activity determined using the Dual-Luciferase reporter assay (Promega). We assessed the impact of ERBB2, PI3k, and MEK inhibition on the activity of the S100A4 promoter by repeating these assays in the presence of 5 µM PD158780, 50 µM LY249002, or 50 µM PD98059, respectively.
Western Blotting.
Expression of phospho-Y1248 ERBB2 (Upstate Biotechnology, Waltham, MA), phospho-Y204 ERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA), phospho-Ser473 of AKT1 (New England Biolabs, Beverly, MA), ERBB2 (Novacastra Ltd., Newcastle, United Kingdom), ERK1 (Santa Cruz Biotechnology), AKT1 (New England Biolabs), and S100A4 (Dako, Carpinteria, CA) was performed by Western blotting as previously described (16)
using commercially available primary antibodies (companies shown in parentheses). All blots were reprobed with an antibody for ß-actin (Sigma Chemicals, St. Louis, MO) to control for protein loading and transfer. We determined the expression level of each protein by densitometric analysis. Expression levels of phospho-specific proteins were normalized to those of the respective total protein.
Northern Blotting.
Northern blotting was performed using standard techniques. Briefly, 10 µg of total RNA was separated by formaldehyde gel electrophoresis and transferred to nylon membrane by capillary blotting. Membranes were then probed with a 500-bp, [
32P]dCTP-labeled PCR fragment generated from the COOH-terminal region of the ERBB2 cDNA. We then stripped and reanalyzed membranes using a probe generated from the entire S100A4 coding region. Finally, we probed membranes with a PCR-generated fragment of r18S to control for RNA loading and transfer. Expression level of each RNA transcript was determined using Phosphorimaging.
Xenografts and Animal Treatment Protocols.
A total of 1 x 107 Daoy.V or Daoy.2 cells was grown as s.c. xenografts in female CD-1 nu/nu mice. When tumors were 
900 mm3, we euthanized animals and resected tumors. We fixed half of the tumors in 10% buffered formalin and snap froze half in liquid nitrogen. We processed fixed tissue and performed IHC analysis as described previously (16)
. We extracted total RNA and protein from frozen tissue and performed Western blotting analysis as described above. For therapeutic studies, we treated tumor-bearing mice p.o. with 100 mg/kg (0.1 ml/10 g body weight) of the ERBB tyrosine kinase inhibitor OSI-774 or vehicle only twice daily for 5 days followed by tumor excision and analysis.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. As previously reported in other cell line systems (30, 31, 32)
, overexpression of ERBB2 in Daoy cells resulted in spontaneous receptor activation and signaling, secondary to receptor homodimerization (Fig. 1
and data not shown).
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Overexpression of ERBB2 in human and mouse mammary cancer has been shown to up-regulate a number of important prometastatic genes (34, 35, 36)
. Therefore, we next investigated whether ERBB2 overexpression similarly alters gene expression patterns in Daoy cells. The Daoy cell line is a valid model for studying gene expression in medulloblastoma because the global DNA methylation pattern (37)
and basal gene expression profile (33)
of this cell line are closely related to those of primary medulloblastoma. Paired expression profiles from four independent cultures of Daoy.2 and Daoy.V cells were compared using the Human Genome U95Av2 Affymetrix microarray containing 12,600 probe sets (Fig. 1B)
. Using scatter plot analysis, we identified 11 genes that significantly differed ( or 2-fold) in all four comparisons (Fig. 1B)
. As expected, ERBB2 was among the up-regulated genes. The remaining 10 genes have previously been associated with tumor progression and metastasis (Fig. 1B)
. In a recent Affymetrix array study of primary medulloblastoma, MacDonald et al. (33)
reported increased expression of several RAS/MAPK and cytokine signal pathway members and the metallothionein family to predict for metastatic medulloblastoma. Interestingly, six of the genes up-regulated by ERBB2 in Daoy cells are also members of these three groups of genes. The three remaining up-regulated genes included S100A4 (19)
, osteoblastic specific factor-2 (also known as Periostin; Ref. 38
), and Proto-cadherin-7 (39)
. Each of these has previously been implicated in controlling cell adhesion and invasion in adult cancers. Finally, one gene, IGFBP5, was reproducibly down-regulated by ERBB2 overexpression in Daoy cells. High concentrations of IGFBP5 have been reported to inhibit proliferation of neuroblastoma cells in culture (40)
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The novel finding that S100A4 is up-regulated by ERBB2 in Daoy cells is of particular interest because this gene has been suggested to function as a regulator of metastasis of human tumors in which ERBB2 plays a prominent role (19)
. Therefore, we further investigated the relationship between ERBB2 and S100A4 in Daoy cells (Fig. 1C)
. In agreement with the results of expression profiling, both S100A4 protein and mRNA levels were higher in Daoy cells transfected with ERBB2 relative to empty vector. Taken together, these data indicate that overexpression of ERBB2 increases the metastatic phenotype of medulloblastoma cells by increasing invasion and specifically up-regulating the expression of prometastatic genes that include S100A4.
High Levels of ERBB2 Correlate with Metastasis and with the Level of S100A4 in Primary Human Medulloblastoma.
To establish whether expression levels of ERBB2 and S100A4 are correlated in clinical medulloblastoma, we studied a large cohort of primary tumors using northern blotting (Fig. 2, A and B)
. In keeping with our observations in the Daoy cell line, expression levels of ERBB2 and S100A4 were closely correlated in patient samples (r2 = 0.67, P < 0.0001). Furthermore, in agreement with our previous studies (9)
, significantly higher levels of ERBB2 were expressed in metastatic (M3) compared with localized medulloblastoma (Fig. 2C
, P < 0.05). Together with our in vitro studies, these data support the hypothesis that ERBB2 signaling is prometastatic in primary human medulloblastoma and up-regulates expression of S100A4.
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. The pDA-L47 construct, which includes the first 1487 bp 5' of the S100A4 transcription start site, demonstrated 40-fold greater activity in Daoy.2 compared with empty vector control cells (Fig. 3B)
. In contrast, significantly reduced activity was associated with constructs in which bp -1487 to -1099 were deleted (Fig. 3B)
. Therefore, the human S100A4 promoter likely contains an ERBB2 response element 1099–1487 bp upstream of the transcription start site. Additional studies are under way to identify potential transcription factor binding sites within this region.
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. As expected, treatment with the ERBB tyrosine kinase inhibitor PD158780 significantly reduced the phosphorylation of ERBB2, AKT1, and ERK1/2 and inhibited both S100A4 expression and promoter activity (Fig. 4, A and B)
. We next treated Daoy.2 cells with specific inhibitors of MEK1 (PD98059) and PI3k (LY249002) activity. These also abrogated S100A4 expression and promoter activity. In addition to decreasing AKT1 phosphorylation, PI3k blockade also significantly reduced ERK1/2 phosphorylation. This observation was confirmed using Wortmannin, a second inhibitor of PI3k (data not shown). These data are in agreement with previously published reports that the RAF/MEK/ERK signal cassette is regulated by PI3k/AKT (41
, 42) .
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. In contrast, kinase dead K179M-AKT1 significantly increased S100A4 promoter activity and expression. Infection of cells with Myr11-60 AKT1 also significantly up-regulated S100A4 expression and promoter activity (data not shown).
Taken together, these data indicate that ERBB2 signaling via PI3k and ERK1/2 increases expression of S100A4 in Daoy medulloblastoma cells (Fig. 4C)
. Conversely, AKT1 appears to negatively regulate this signal, possibly by inhibiting ERK1/2 activation.
The ERBB Tyrosine Kinase Inhibitor OSI-774 Inhibits the Metastatic Phenotype in Daoy Cells in Vitro and in Vivo.
ERBB2 is central to the pathogenesis of a number of human cancers, therefore many novel agents that target this receptor are currently under development (8
, 43)
. These include small molecule inhibitors of the ERBB TKIs. The ERBB TKIs that are most advanced in clinical development are designed to target ERBB1, e.g., ZD1839 (Iressa) and OSI-774 (Erlotinib). However, there is increasing evidence that these agents may also inhibit ERBB2 signaling (44)
. Therefore, we investigated the ability of OSI-774 to inhibit ERBB2-directed medulloblastoma cell migration and prometastatic gene expression.
OSI-774 significantly inhibited ERBB2 phosphorylation and S100A4 expression in Daoy cells a time- and dose-dependent manner (Fig. 5A)
. No apparent effect of OSI-774 treatment on ERBB2 activation or S100A4 expression was seen before 4 h. Subsequently, a gradual decrease in the level of phosphorylated ERBB2 was observed in a dose-dependent fashion. This effect was maximal at 8 h. As expected, the inhibition and recovery of ERBB2 phosphorylation was paralleled by changes in S100A4 expression (Fig. 5A)
. We next investigated the ability of OSI-774 to inhibit ERBB2-directed tumor cell invasion. To ensure that ERBB2 receptor signaling was effectively but specifically inhibited, we preincubated Daoy.2 cells for 6 h with the minimum concentration of drug required to generate receptor blockade (30 nM). This treatment significantly inhibited the migration of Daoy.2 cells in vitro (Fig. 5B)
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. However, oral dosing of Daoy.2 tumor-bearing animals with OSI-774 markedly reduced the level of phospho-ERBB2 and S100A4 expression by xenografts (Fig. 6, A and B)
. Therefore, the control of S100A4 expression by ERBB2 is functionally active in vivo and can be effectively targeted with small molecule inhibitors of ERBB2 signaling.
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. Affymetrix gene expression profiling of xenografts demonstrated that five of these nine genes were also up-regulated by ERBB2 in vivo (Table 1)
. In agreement with our IHC and Western blotting studies (Fig. 6)
, these included S100A4. We next compared the gene expression-profiles of Daoy.2 tumors derived from mice treated with OSI-774 or vehicle alone (Table 1)
. Only the five genes that were up-regulated by ERBB2 in xenografts were significantly down-regulated by treating host animals with OSI-774. Therefore, up-regulation of prometastatic genes by ERBB2 in vivo can be selectively inhibited using an oral small molecule inhibitor of ERBB2 signaling.
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DISCUSSION |
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The molecular mechanisms responsible for metastasis of medulloblastoma are largely unknown. Recently, members of the RAS/MAPK and cytokine signal pathways and the metallothionein family were identified as predictors of medulloblastoma metastasis (33) . In this study, we demonstrate that 6 of 10 genes identified as up-regulated by ERBB2 in cultured medulloblastoma cells also belong to these three groups of genes. Therefore, we propose that these genes are key regulators of medulloblastoma metastasis and that ERBB2 signaling promotes their expression during metastasis.
There is increasing evidence that the cytokine network, including the chemokine group of chemoattractant cytokines, contributes to human tumor growth and progression (46, 47, 48) . Chemokines released by solid tumors recruit monocytes and macrophages, which in turn secrete a number of tumor cell growth factors, angiogenic growth factors, and extracellular matrix-degrading enzymes (47 , 48) . Tumor infiltration by monocytes and macrophages is frequently seen in solid tumors, including medulloblastoma (49) , and is associated with a poor clinical outcome in certain adult cancers (47 , 48) . In this study, we show that expression of the CC chemokine RANTES and the IFN-inducible genes p56 and p78 are up-regulated by ERBB2 in Daoy cells. RANTES is a T cell and monocyte attractant that is produced by several tumors, including carcinomas of the breast and ovary (50 , 51) . Recent evidence was presented implicating RANTES in promoting angiogenesis and the production of matrix metalloproteinase 9 by monocytes (50) . Additional analyses will be required to establish the precise role of cytokine signaling in medulloblastoma metastasis and whether this may be targeted therapeutically.
Metallothionein If was also up-regulated by ERBB2 overexpression in Daoy cells. The metallothioneins, which are preferentially overexpressed in metastatic medulloblastoma and other poor prognosis human tumors (33 , 52) , are involved in many cellular processes, including metal homeostasis and detoxification, cell proliferation, and apoptosis (53 , 54) . The metallothioneins may play a permissive role in metastasis by promoting tumor cell resistance to chemotherapeutic agents (53 , 54) . However, it remains to be determined whether they play a more direct role in invasion and migration.
We also identified four ERBB2-regulated genes that have previously been implicated in controlling cell adhesion and invasion but not medulloblastoma metastasis. These include osteoblastic specific factor-2 (55) and PCDH7 (39) that encode cell adhesion molecules, IGFBP5 (40) and S100A4 (19) . Although S100A4 has been shown to synergize with ERBB2 in the development of metastatic breast cancer (19 , 22 , 24 , 25) , it was not know whether ERBB2 signaling directly controls the expression of S100A4. Our data show that ERBB2 signaling via PI3k and ERK1/2 does activate S100A4 expression, at least in part, by enhancing transcription. We also show that the expression of ERBB2 and S100A4 are strictly concordant in primary human medulloblastoma samples and medulloblastoma xenografts, demonstrating that this pathway is active in vivo. The observation that ERBB2 up-regulates the expression of two members of the RAS/MAPK pathway (MAPK KKK4 and MEK5) as well as directing S100A4 expression via this pathway is intriguing. These data suggest that ERBB2 may promote the metastasis of medulloblastoma through a complex series of collaborating events in which this receptor up-regulates both the expression of prometastatic genes, e.g., S100A4, and the signal pathways required to do this. Our analyses of the S100A4 promoter region indicate that ERBB2 signaling controls the expression of this gene via a response element located between bp -1487 and -1099. Cis-acting response elements have been identified within the S100A4 gene. These include a composite enhancer element within the first intron (56) and a GC-factor recognition sequence located 1300 bp upstream of the rodent S100A4 transcriptional start site (57) . We are therefore investigating whether this later sequence coordinates the ERBB2 signaling response. Our data also show that ERBB2/PI3k signaling may abrogate S100A4 expression through interaction between AKT1 and the MEK/ERK pathway. Interaction between these pathways has been described in a number of cell signal systems, including the glial cell line-derived neurotrophic factor signaling system in neuroectodermal cells (42) . Therefore, interplay between the ERK1/2 and PI3k systems may be of general importance in neuroectodermal cell growth factor signaling.
Selective targeting of the molecular abnormalities responsible for medulloblastoma metastasis may represent a more effective and less toxic means of treatment than conventional chemo- and radiotherapies. Here, we provide proof of principal that small molecule inhibitors of ERBB2 tyrosine kinase activity may be used to inhibit the prometastatic phenotype in medulloblastoma. Furthermore, our comparative expression profile analyses of tumors resected from OSI-774 and vehicle only-treated animals provide the first evidence to date that these inhibitors selectively reduce the expression of ERBB2 up-regulated genes. Therefore, we propose ERBB2 to be a new therapeutic target for metastatic medulloblastoma. We are currently conducting additional preclinical studies of the antitumor and antimetastatic properties of ERBB2 inhibitors against medulloblastoma. Additional analysis of the other prometastatic genes and pathways identified in this study may also provide additional targets for the development of novel therapeutic approaches. In this regard, MacDonald et al. (33) recently reported the platelet-derived growth factor receptor signaling system to be up-regulated in metastatic medulloblastoma and demonstrated the ability of selective inhibitors of this pathway to prevent Daoy tumor cell invasion in vitro. Combined ERBB2 and platelet-derived growth factor receptor blockade may therefore prove a particularly effective therapeutic strategy for metastatic medulloblastoma. Finally, it will also be important to determine whether ERBB2 signaling controls S100A4 expression in other human cancers, particularly breast cancer, given the significant role identified for both of these proteins in this disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Cancer Center Support Grant CA 21765, the American Lebanese Syrian Associated Charities, and grants from the Simon Trory Brain Tumour Fund, the American Brain Tumour Association, the Medical Research Council of the United Kingdom, and the Cancer and Polio Research Fund. 
2 To whom requests for reprints should be addressed, at E-mail: Richard.Gilbertson{at}stjude.org
3 The abbreviations used are: PI3k, phosphatidylinositol 3'-kinase; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; HA, Hemagglutinin; WT, wild type; DN, dominant negative; MAPK, mitogen-activated protein kinase; MEK, MAP/ERK kinase; IHC, immunohistochemical; IGFBP5, insulin-like growth factor binding protein-5; STAT, signal transducers and activators of transcription; ERBB TKI, ERBB receptor tyrosine kinase.
4 C. Calabrese, A. Frank, K. Maclean, Richard Gilbertson. Medulloblastoma sensitivity to 17-allylamino 17-demethoxygeldanamycin requires MEK/ERK, submitted for publication.
Received 7/10/02. Accepted 10/31/02.
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I. Shin, T. Miller, and C. L. Arteaga ErbB Receptor Signaling and Therapeutic Resistance to Aromatase Inhibitors Clin. Cancer Res., February 1, 2006; 12(3): 1008s - 1012s. [Abstract] [Full Text] [PDF]
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S. C. Garrett, K. M. Varney, D. J. Weber, and A. R. Bresnick S100A4, a Mediator of Metastasis J. Biol. Chem., January 13, 2006; 281(2): 677 - 680. [Full Text] [PDF]
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 D. Zhang, L. K. Tai, L. L. Wong, L.-L. Chiu, S. K. Sethi, and E. S. C. Koay Proteomic Study Reveals That Proteins Involved in Metabolic and Detoxification Pathways Are Highly Expressed in HER-2/neu-positive Breast Cancer Mol. Cell. Proteomics, November 1, 2005; 4(11): 1686 - 1696. [Abstract] [Full Text] [PDF]
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Y. Li, B. Lal, S. Kwon, X. Fan, U. Saldanha, T. E. Reznik, E. B. Kuchner, C. Eberhart, J. Laterra, and R. Abounader The Scatter Factor/Hepatocyte Growth Factor: c-Met Pathway in Human Embryonal Central Nervous System Tumor Malignancy Cancer Res., October 15, 2005; 65(20): 9355 - 9362. [Abstract] [Full Text] [PDF]
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 R. J. Gilbertson ERBB2 in Pediatric Cancer: Innocent Until Proven Guilty Oncologist, August 1, 2005; 10(7): 508 - 517. [Abstract] [Full Text] [PDF]
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I. Shin, J. Rotty, F. Y. Wu, and C. L. Arteaga Phosphorylation of p27Kip1 at Thr-157 Interferes with Its Association with Importin {alpha} during G1 and Prevents Nuclear Re-entry J. Biol. Chem., February 18, 2005; 280(7): 6055 - 6063. [Abstract] [Full Text] [PDF]
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 C. Thieblemont, V. Nasser, P. Felman, K. Leroy, S. Gazzo, E. Callet-Bauchu, B. Loriod, S. Granjeaud, P. Gaulard, C. Haioun, et al. Small lymphocytic lymphoma, marginal zone B-cell lymphoma, and mantle cell lymphoma exhibit distinct gene-expression profiles allowing molecular diagnosis Blood, April 1, 2004; 103(7): 2727 - 2737. [Abstract] [Full Text] [PDF]
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A. Gajjar, R. Hernan, M. Kocak, C. Fuller, Y. Lee, P. J. McKinnon, D. Wallace, C. Lau, M. Chintagumpala, D. M. Ashley, et al. Clinical, Histopathologic, and Molecular Markers of Prognosis: Toward a New Disease Risk Stratification System for Medulloblastoma J. Clin. Oncol., March 15, 2004; 22(6): 984 - 993. [Abstract] [Full Text] [PDF]
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 A. T. Kho, Q. Zhao, Z. Cai, A. J. Butte, J. Y.H. Kim, S. L. Pomeroy, D. H. Rowitch, and I. S. Kohane Conserved mechanisms across development and tumorigenesis revealed by a mouse development perspective of human cancers Genes & Dev., March 15, 2004; 18(6): 629 - 640. [Abstract] [Full Text] [PDF]
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C. Calabrese, A. Frank, K. Maclean, and R. Gilbertson Medulloblastoma Sensitivity to 17-Allylamino-17-demethoxygeldanamycin Requires MEK/ERK J. Biol. Chem., July 4, 2003; 278(27): 24951 - 24959. [Abstract] [Full Text] [PDF]
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