A Reduction in Pten Tumor Suppressor Activity Promotes ErbB-2-Induced Mouse Prostate Adenocarcinoma Formation through the Activation of Signaling Cascades Downstream of PDK1
2009; Elsevier BV; Volume: 174; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.080859
ISSN1525-2191
AutoresOlga Rodriguez, Edwin W. Lai, Sarada Vissapragada, Caroline Cromelin, Maral Avetian, Patricia C. Salinas, Hida Ramos, Bhaskar Kallakury, Mathew C. Casimiro, Michael P. Lisanti, Herbert B. Tanowitz, Karel Pacák, Robert I. Glazer, Maria Laura Avantaggiati, Chris Albanese,
Tópico(s)Cancer Mechanisms and Therapy
ResumoLoss of function at the Pten tumor-suppressor locus is a common genetic modification found in human prostate cancer. While recent in vivo and in vitro data support an important role of aberrant ErbB-2 signaling to clinically relevant prostate target genes, such as cyclin D1, the role of Pten in ErbB-2-induced prostate epithelial proliferation is not well understood. In the Pten-deficient prostate cancer cell line, LNCaP, restoration of Pten was able to inhibit ErbB-2- and heregulin-induced cell cycle progression, as well as cyclin D1 protein levels and promoter activity. Previously, we established that probasin-driven ErbB-2 transgenic mice presented with high-grade prostate intraepithelial neoplasia and increased nuclear cyclin D1 levels. We show that mono-allelic loss of pten in the probasin-driven-ErbB-2 model resulted in increased nuclear cyclin D1 and proliferating cell nuclear antigen levels and decreased disease latency compared to either individual genetic model and, unlike the probasin-driven-ErbB-2 mice, progression to adenocarcinoma. Activated 3-phosphoinositide-dependent protein kinase-1 was observed during cancer initiation combined with the activation of p70S6K (phospho-T389) and inactivation of the 4E-binding protein-1 (phosphorylated on T37/46) and was primarily restricted to those cases of prostate cancer that had progressed to adenocarcinoma. Activation of mTOR was not seen. Our data demonstrates that Pten functions downstream of ErbB-2 to restrict prostate epithelial transformation by blocking full activation of the PDK1 signaling cascade. Loss of function at the Pten tumor-suppressor locus is a common genetic modification found in human prostate cancer. While recent in vivo and in vitro data support an important role of aberrant ErbB-2 signaling to clinically relevant prostate target genes, such as cyclin D1, the role of Pten in ErbB-2-induced prostate epithelial proliferation is not well understood. In the Pten-deficient prostate cancer cell line, LNCaP, restoration of Pten was able to inhibit ErbB-2- and heregulin-induced cell cycle progression, as well as cyclin D1 protein levels and promoter activity. Previously, we established that probasin-driven ErbB-2 transgenic mice presented with high-grade prostate intraepithelial neoplasia and increased nuclear cyclin D1 levels. We show that mono-allelic loss of pten in the probasin-driven-ErbB-2 model resulted in increased nuclear cyclin D1 and proliferating cell nuclear antigen levels and decreased disease latency compared to either individual genetic model and, unlike the probasin-driven-ErbB-2 mice, progression to adenocarcinoma. Activated 3-phosphoinositide-dependent protein kinase-1 was observed during cancer initiation combined with the activation of p70S6K (phospho-T389) and inactivation of the 4E-binding protein-1 (phosphorylated on T37/46) and was primarily restricted to those cases of prostate cancer that had progressed to adenocarcinoma. Activation of mTOR was not seen. Our data demonstrates that Pten functions downstream of ErbB-2 to restrict prostate epithelial transformation by blocking full activation of the PDK1 signaling cascade. The proto-oncogene ErbB-2 (Neu or HER2) is an 185-kDa receptor tyrosine kinase with no known ligand and is the preferential dimerization partner for all members of the epidermal growth factor receptor family. Enhanced ErbB-2 signaling has been demonstrated in a number of cancers, including those of the breast, head and neck, pancreas, and colon, and recently, increased ErbB-2 levels in the absence of gene amplification was shown to correlate with poor prognosis in prostate cancer (PCa) patients with progressive disease.1Shi Y Chatterjee SJ Brands FH Shi SR Pootrakul L Taylor CR Datar R Cote RJ Role of coordinated molecular alterations in the development of androgen-independent prostate cancer: an in vitro model that corroborates clinical observations.BJU Int. 2006; 97: 170-178Crossref PubMed Scopus (24) Google Scholar, 2Dai B Kong YY Ye DW Ma CG Zhou XY Yao XD Human epidermal growth factor receptor type 2 protein expression in Chinese metastatic prostate cancer patients correlates with cancer specific survival and increases after exposure to hormonal therapy.Asian J Androl. 2008; 10: 701-709Crossref PubMed Scopus (8) Google Scholar In previous studies, we established that increased ErbB-2 membrane expression correlated with increased nuclear cyclin D1 staining in clinical PCa specimens.3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar We also established in human PCa cell lines that ErbB-2 induced both cell cycle proliferation and cyclin D1, and that small interfering RNA targeting cyclin D1 blocked a significant proportion of the ErbB-2 or heregulin-induced cell cycle progression. Furthermore, we established that probasin-driven ErbB-2 transgene mice (PB-ErbB-2) presented with high-grade prostate intraepithelial neoplasia, (PIN), a localized adenoma, and induced epithelial cyclin D1 expression; transformation to adenocarcinoma was not observed.3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar The phosphatidylinositol-3-kinase (PI3K) signaling pathway is a major mediator of receptor tyrosine kinase signaling and plays an important role in controlling cell proliferation and cell survival. Pten is a lipid phosphatase that catalyzes the dephosphorylation of phosphatidylinositol-3,4,5-tri-phosphate and phosphatidylinositol-3,4-bis-phosphate, thereby inhibiting PI3K signaling. Modifications at the pten locus are frequently found in human diseases, and pten is one of the most frequently mutated genes identified in human PCa.4Cantley LC Neel BG New insights into tumor suppression: PTEN suppresses tumor formation by restraining the phosphoinositide 3-kinase/AKT pathway.Proc Natl Acad Sci USA. 1999; 96: 4240-4245Crossref PubMed Scopus (1748) Google Scholar Pten levels were reduced in as many as 50% of the tumors examined,5Whang YE Wu X Suzuki H Reiter RE Tran C Vessella RL Said JW Isaacs WB Sawyers CL Inactivation of the tumor suppressor PTEN/MMAC1 in advanced human prostate cancer through loss of expression.Proc Natl Acad Sci USA. 1998; 95: 5246-5250Crossref PubMed Scopus (563) Google Scholar and haploinsufficiency of pten was associated with early stage PCa.6Di Cristofano A Pandolfi PP The multiple roles of PTEN in tumor suppression.Cell. 2000; 100: 387-390Abstract Full Text Full Text PDF PubMed Scopus (1036) Google Scholar Additionally, loss of heterozygosity at the pten locus (and thereby loss of Pten expression) has been associated with increased Gleason score and poor clinical outcome.7McMenamin ME Soung P Perera S Kaplan I Loda M Sellers WR Loss of PTEN expression in paraffin-embedded primary prostate cancer correlates with high Gleason score and advanced stage.Cancer Res. 1999; 59: 4291-4296PubMed Google Scholar Mutations in pten may also serve as a molecular marker for metastatic PCa progression in humans,8Rubin MA Gerstein A Reid K Bostwick DG Cheng L Parsons R Papadopoulos N 10q23.3 loss of heterozygosity is higher in lymph node-positive (pT2–3,N+) versus lymph node-negative (pT2–3,N0) prostate cancer.Hum Pathol. 2000; 31: 504-508Abstract Full Text PDF PubMed Scopus (51) Google Scholar further supporting the hypothesis that pten is a clinically important PCa tumor suppressor gene. In preclinical models of prostate disease, prostate intraepithelial neoplasia has been observed in mice deleted of candidate tumor suppressor genes, and combinatorial genetic manipulations allow for the accurate modeling of known human genetic lesions in vivo (reviewed in9Albanese C Rodriguez O Johnson MD Fricke S Models of prostate cancer.Drug Discov Today: Disease Models. 2005; 2: 7-13Crossref Scopus (6) Google Scholar). Mice harboring heterozygous deficiency at the pten locus (pten+/−) displayed intermittent PIN with long latency.10Di Cristofano A De Acetis M Koff A Cordon-Cardo C Pandolfi PP Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the mouse.Nat Genet. 2001; 27: 222-224Crossref PubMed Scopus (404) Google Scholar In some models where genetic modification induced PIN, but not adenocarcinoma, the extent of glandular involvement and PCa progression could be induced through the combination of pten haploinsufficiency and alterations in the function of key cell regulatory genes, such as p27Kip110Di Cristofano A De Acetis M Koff A Cordon-Cardo C Pandolfi PP Pten and p27KIP1 cooperate in prostate cancer tumor suppression in the mouse.Nat Genet. 2001; 27: 222-224Crossref PubMed Scopus (404) Google Scholar or nkx3.1.11Abate-Shen C Banach-Petrosky WA Sun X Economides KD Desai N Gregg JP Borowsky AD Cardiff RD Shen MM Nkx3.1; Pten mutant mice develop invasive prostate adenocarcinoma and lymph node metastases.Cancer Res. 2003; 63: 3886-3890PubMed Google ScholarPten haploinsufficiency has recently been shown to interact cooperatively with the overexpression of the mTOR regulatory protein Rheb, to induce PCa.12Nardella C Chen Z Salmena L Carracedo A Alimonti A Egia A Carver B Gerald W Cordon-Cardo C Pandolfi PP Aberrant Rheb-mediated mTORC1 activation and Pten haploinsufficiency are cooperative oncogenic events.Genes Dev. 2008; 22: 2172-2177Crossref PubMed Scopus (98) Google Scholar Additionally, the targeted homozygous ablation of pten induced latent PCa, which was dependent on the p110β catalytic subunit of PI3K,13Trotman LC Niki M Dotan ZA Koutcher JA Cristofano AD Xiao A Khoo AS Roy-Burman P Greenberg NM Dyke TV Cordon-Cardo C Pandolfi P Pten dose dictates cancer progression in the prostate.PLoS Biol. 2003; 1: E59Crossref PubMed Scopus (565) Google Scholar, 14Jia S Liu Z Zhang S Liu P Zhang L Lee SH Zhang J Signoretti S Loda M Roberts TM Zhao JJ Essential roles of PI(3)K-p110beta in cell growth, metabolism and tumorigenesis.Nature. 2008; 454: 776-779Crossref PubMed Scopus (596) Google ScholarPten ablation in a p53 knockout background resulted in the induction of early invasive PCa and the loss of cellular senescence,15Chen Z Trotman LC Shaffer D Lin HK Dotan ZA Niki M Koutcher JA Scher HI Ludwig T Gerald W Cordon-Cardo C Pandolfi PP Crucial role of p53-dependent cellular senescence in suppression of Pten-deficient tumorigenesis.Nature. 2005; 436: 725-730Crossref PubMed Scopus (1555) Google Scholar while modeling studies have further established that in FGF8b transgenic × pten+/− mice where prostate cancer is seen, the expression from both pten alleles was lost.16Zhong C Saribekyan G Liao CP Cohen MB Roy-Burman P Cooperation between FGF8b overexpression and PTEN deficiency in prostate tumorigenesis.Cancer Res. 2006; 66: 2188-2194Crossref PubMed Scopus (58) Google Scholar To better understand the role of Pten in regulating ErbB-2-induced tumorigenesis in the prostate epithelium, we analyzed the effect of alterations in Pten levels on ErbB-2 signaling both in vitro and in vivo. Herein, we demonstrate that the heterozygous loss of pten when integrated into the PB-ErbB-2 mouse model (PB-ErbB-2 × Pten+/−) resulted in increased cyclin D1 and proliferating cell nuclear antigen (PCNA) nuclear positivity and decreased disease latency compared with either singly modified genetic model. Notably, the PB-ErbB-2 × Pten+/−mice also developed prostate adenocarcinomas while retaining Pten expression. Pten re-expression in the Pten-deficient prostate cancer cell line LNCaP inhibited ErbB-2-induced cyclin D1 promoter activity. Mechanistically, modest activation of phosphoinositide-dependent kinase (PDK)1 (phosphorylated at S241) was observed in PIN lesions, and which was further increased in adenocarcinomas. In contrast, the combined activation of 70S6K (phosphorylated at T389) and inactivation of the eIF4E-binding protein-1 (4E-BP1, phosphorylated at pT37/46) was primarily restricted to those glands that had progressed to adenocarcinoma, interestingly however, activation of mTOR was not observed. Collectively, these data indicate a role for Pten in the suppression of ErbB-2-induced prostate epithelial transformation through an inhibition of proteins that function downstream of PDK-1 that are involved in the regulation of cell proliferation and protein biosynthesis. The PB-ErbB-2 transgenic mice have been previously described.3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar, 17Fricke S Rodriguez O Vanmeter J Dettin L Casimiro M Chien C Newell T Johnson K Ileva L Johnson MD Albanese C In vivo magnetic resonance volumetric and spectroscopic analysis of mouse prostate cancer models.Prostate. 2006; 66: 708-717Crossref PubMed Scopus (40) Google Scholar Briefly the minimal rat probasin promoter was used to drive prostate-specific expression of an activated ErbB-2 growth factor receptor.3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar, 17Fricke S Rodriguez O Vanmeter J Dettin L Casimiro M Chien C Newell T Johnson K Ileva L Johnson MD Albanese C In vivo magnetic resonance volumetric and spectroscopic analysis of mouse prostate cancer models.Prostate. 2006; 66: 708-717Crossref PubMed Scopus (40) Google Scholar The pten+/− mice, which harbor hemizygous inactivation of one pten allele and PB-CRE × PtenPC1 mice, which delete both pten alleles in the prostate epithelium as previously reported13Trotman LC Niki M Dotan ZA Koutcher JA Cristofano AD Xiao A Khoo AS Roy-Burman P Greenberg NM Dyke TV Cordon-Cardo C Pandolfi P Pten dose dictates cancer progression in the prostate.PLoS Biol. 2003; 1: E59Crossref PubMed Scopus (565) Google Scholar, 18Di Cristofano A Pesce B Cordon-Cardo C Pandolfi PP PTEN is essential for embryonic development and tumour suppression.Nat Genet. 1998; 19: 348-355Crossref PubMed Scopus (1304) Google Scholar were kindly provided by Dr. Pier Paolo Pandolfi, Memorial Sloan-Kettering Cancer Center/Beth Israel Deaconess Medical Center. The compound-engineered PB-ErbB-2 × pten+/− mice in an FVBN background resulted from the repeated cross-breeding of the Pten+/− mice into the PB-ErbB-2 line for six or more generations. The genotypes were established as previously described.3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar, 17Fricke S Rodriguez O Vanmeter J Dettin L Casimiro M Chien C Newell T Johnson K Ileva L Johnson MD Albanese C In vivo magnetic resonance volumetric and spectroscopic analysis of mouse prostate cancer models.Prostate. 2006; 66: 708-717Crossref PubMed Scopus (40) Google Scholar, 18Di Cristofano A Pesce B Cordon-Cardo C Pandolfi PP PTEN is essential for embryonic development and tumour suppression.Nat Genet. 1998; 19: 348-355Crossref PubMed Scopus (1304) Google Scholar Male wild-type FVBN mice used in these studies were littermates to the genetically engineered animals. The human prostate cancer cell line, LNCaP, was maintained in RPMI with 10% fetal calf serum, 0.1 mmol/L non-essential amino acids, 100u/ml penicillin-streptomycin, and 1 mmol/L sodium pyruvate at 37°C in 5% CO2 as previously described.3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar, 19Rodriguez O Fricke S Chien C Dettin L Vanmeter J Shapiro E Dai HN Casimiro M Ileva L Dagata J Johnson MD Lisanti MP Koretsky A Albanese C Contrast-enhanced in vivo imaging of breast and prostate cancer cells by MRI.Cell Cycle. 2006; 5: 113-119Crossref PubMed Scopus (48) Google Scholar For heregulin 1β (HRG) stimulation studies, subconfluent (50%) LNCaP cells were placed in RPMI with 2.0% fetal calf serum, and HRG (R&D Systems, Minneapolis, MN) was added to a concentration of 1 ng/ml3. The chemical inhibitors PD98059 (30 μmol/L), the PI3K inhibitor LY24002 (20 μmol/L), the mTOR/raptor complex inhibitor, rapamycin (1 ng/ml) or vehicle (dimethyl sulfoxide) were added and the cells were cultured for an additional 30 minutes or 12 hours. The cyclin D1 promoter construct and transfection methodologies have been previously described by our laboratory.3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar, 20Albanese C Johnson J Watanabe G Eklund N Vu D Arnold A Pestell RG Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions.J Biol Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (761) Google Scholar, 21Albanese C D'Amico M Reutens AT Fu M Watanabe G Lee RJ Kitsis RN Henglein B Avantaggiati M Somasundaram K Thimmapaya B Pestell RG Activation of the cyclin D1 gene by the E1A-associated protein p300 through AP-1 inhibits cellular apoptosis.J Biol Chem. 1999; 274: 34186-34195Crossref PubMed Scopus (170) Google Scholar, 22Albanese C Wu K D'Amico M Jarrett C Joyce D Hughes J Hulit J Sakamaki T Fu M Ben-Ze'ev A Bromberg JF Lamberti C Verma U Gaynor RB Byers SW Pestell RG IKKalpha regulates mitogenic signaling through transcriptional induction of cyclin D1 via Tcf.Mol Biol Cell. 2003; 14: 585-599Crossref PubMed Scopus (133) Google Scholar The pcDNA3 and pcDNA3-ErbB-2 expression vectors have been previously described.3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar, 23Kwong KY Hung MC A novel splice variant of HER2 with increased transformation activity.Mol Carcinog. 1998; 23: 62-68Crossref PubMed Scopus (116) Google Scholar CMV5-Pten expresses the wild-type human Pten cDNA and was a generous gift from Dr. Todd Waldman. The co-transfection of reporter constructs and expression vector DNA was accomplished using Lipofectamine Plus or Lipofectamine 2000 (Invitrogen. Carlsbad, CA), following the manufacturers conditions. Luciferase activity was measured in a Bertold Autolumat 963 luminometer as previously described3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar, 22Albanese C Wu K D'Amico M Jarrett C Joyce D Hughes J Hulit J Sakamaki T Fu M Ben-Ze'ev A Bromberg JF Lamberti C Verma U Gaynor RB Byers SW Pestell RG IKKalpha regulates mitogenic signaling through transcriptional induction of cyclin D1 via Tcf.Mol Biol Cell. 2003; 14: 585-599Crossref PubMed Scopus (133) Google Scholar and was measured in arbitrary light units by calculating the light emitted during the initial 10 seconds of the reaction. Background activity from cell extracts was typically <100 arbitrary light units/10s. Co-transfection of Renilla luciferase (TK-renilla) was used to control for transfection efficiency.3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar, 22Albanese C Wu K D'Amico M Jarrett C Joyce D Hughes J Hulit J Sakamaki T Fu M Ben-Ze'ev A Bromberg JF Lamberti C Verma U Gaynor RB Byers SW Pestell RG IKKalpha regulates mitogenic signaling through transcriptional induction of cyclin D1 via Tcf.Mol Biol Cell. 2003; 14: 585-599Crossref PubMed Scopus (133) Google Scholar Plasmid concentrations of the ErbB-2 and Pten expression vectors used in the dose response curves were 0.22, 0.45 and 0.9 μg per well. Statistical analyses were performed using the Student's t-test with significant differences established as at least P ≤ 0.05 on N ≥3 independent transfections. Data were plotted as average fold-induction ± SEM versus empty vector controls.3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar, 22Albanese C Wu K D'Amico M Jarrett C Joyce D Hughes J Hulit J Sakamaki T Fu M Ben-Ze'ev A Bromberg JF Lamberti C Verma U Gaynor RB Byers SW Pestell RG IKKalpha regulates mitogenic signaling through transcriptional induction of cyclin D1 via Tcf.Mol Biol Cell. 2003; 14: 585-599Crossref PubMed Scopus (133) Google Scholar LNCaP cells were collected by trypsinization, fixed in 10% citrate buffer and resuspended in PBS containing 20 mg/ml propidium iodide and 5U RNase A. DNA content measured using a FACStar Plus dual laser FACSort system as previously described.3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar, 21Albanese C D'Amico M Reutens AT Fu M Watanabe G Lee RJ Kitsis RN Henglein B Avantaggiati M Somasundaram K Thimmapaya B Pestell RG Activation of the cyclin D1 gene by the E1A-associated protein p300 through AP-1 inhibits cellular apoptosis.J Biol Chem. 1999; 274: 34186-34195Crossref PubMed Scopus (170) Google Scholar, 22Albanese C Wu K D'Amico M Jarrett C Joyce D Hughes J Hulit J Sakamaki T Fu M Ben-Ze'ev A Bromberg JF Lamberti C Verma U Gaynor RB Byers SW Pestell RG IKKalpha regulates mitogenic signaling through transcriptional induction of cyclin D1 via Tcf.Mol Biol Cell. 2003; 14: 585-599Crossref PubMed Scopus (133) Google Scholar Protein extracts were separated on 4% to 20% Tris-glycine gels and electro-blotted onto nitrocellulose.3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar ErbB-2 expression levels were assessed using the antibody OP15 (Calbiochem). Induction of signal transduction cascades was assessed using antibodies against total- and phospho-AKT (Cell Signaling, S473, 9271; total, 9272), PDK1 (Abcam, S241, ab32800: total, ab31406), S6Kinase (Cell Signaling T389, 9205), and 4E-BP1 (Cell Signaling T37/46, 9644). Cyclin D1 protein levels were assessed using an anti-cyclin D1 polyclonal antibody, AB3 (NeoMarker).3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar, 22Albanese C Wu K D'Amico M Jarrett C Joyce D Hughes J Hulit J Sakamaki T Fu M Ben-Ze'ev A Bromberg JF Lamberti C Verma U Gaynor RB Byers SW Pestell RG IKKalpha regulates mitogenic signaling through transcriptional induction of cyclin D1 via Tcf.Mol Biol Cell. 2003; 14: 585-599Crossref PubMed Scopus (133) Google Scholar β-actin (Cell Signaling, 4967) was used as loading control. Immunohistochemical staining was performed on prostate tissue using the following antibodies: PCNA (BD, 610664), Her2 (Calbiochem OP15),3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar cyclin D1 (Neomarkers, AB3),3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar Pten (Cell Signaling, 138G6),24Lai EW Rodriguez OC Aventian M Cromelin C Fricke ST Martiniova L Lubensky IA Lisanti MP Picard KL Powers JF Tischler AS Pacak K Albanese C ErbB-2 induces bilateral adrenal pheochromocytoma formation in mice.Cell Cycle. 2007; 6: 1946-1950Crossref PubMed Scopus (16) Google Scholar phospho-p70S6K T389 (Abcam, ab32359), phospho-PDK1 S241 (Abcam, ab32800), phospho-4E-BP1 (Cell Signaling, 2855), and phospho-mTOR (Cell Signaling, 2976). The slides were blocked for 20 minutes, and incubated overnight at 4° with the primary antibody. Detection was performed using DakoCytomation kits (Dako, Carpinteria, CA). Statistical analyses were performed using the Student's t-test with significant differences established as at least P ≤ 0.05. Semiquantitative immunohistochemical analyses of anti-ErbB-2 or anti-Pten with diaminobenzidine (DAB)- and hematoxylin-stained sections were performed by multispectral analysis using a Nikon E600 upright microscope system fitted with a Nuance 2 spectral imaging system (CRi Inc, MA) running Nuance 2.4 software. To perform semiquantitative image analysis, individual spectral databases or "spectral libraries" for DAB and hematoxylin were generated using a ×60 lens and transmitted light at wavelengths from 440 to 680 nm in 10-nm steps. Background staining data for DAB was established using prostate tissue slides incubated with the secondary antibody (in the absence of the primary antibody) followed by treatment with DAB. The Nuance software and spectral libraries were used to separate, or "unmix" the individual signals that represent DAB (antigen staining) and hematoxylin (nuclei). Quantification of the DAB staining per exposure (in milliseconds) was performed and the average ± SD for numerous regions of interest in multiple mice was calculated. Staining associated with ductal secretions or areas of the slide devoid of tissue were not used in the analyses. Previously, we reported that PB-ErbB-2 transgenic mice develop widespread PIN within the dorsal prostate, dorsolateral prostate (DLP), and ventral prostate (VP), and that approximately 50% of the mice exhibited moderate to high-grade PIN, but no progression to adenocarcinoma by 18 months of age.3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar In addition, we demonstrated that heterozygous deletion of the tumor suppressor gene, pten in the context of PB-ErbB-2 resulted in an increase in total prostate volume and an alteration in the prostatic choline to citrate ratio, as measured by magnetic resonance imaging and magnetic resonance spectroscopy, respectively, commensurate with induction of prostate disease.9Albanese C Rodriguez O Johnson MD Fricke S Models of prostate cancer.Drug Discov Today: Disease Models. 2005; 2: 7-13Crossref Scopus (6) Google Scholar To more fully investigate the pathobiology of prostate disease in these models, comprehensive pathological and immunohistochemical analyses were performed on over 25 mice from the compound engineered (PB-ErbB-2 × pten) line. The latency of initiation of prostate disease in both the DLP and VP was found to be greatly reduced in the PB-ErbB-2 × pten+/− model versus the singly modified PB-ErbB-2 model,3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar with 100% of the animals presenting with prostate disease by 16 months of age. Importantly, adenocarcinomas of the DLP and VP were found in 15% of the PB-ErbB-2 × pten+/− mice, some occurring as early as 8 months of age (Figure 1C). The pten+/− mice in an FVB background, but lacking the PB-ErbB-2 transgene, primarily presented with sporadic, low-grade PIN (Figure 1, A–B). We had previously shown that approximately 25% of the cells in PB-ErbB-2 PIN IV lesions stained for nuclear cyclin D1.3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar Immunostaining for cyclin D1 performed on DLP and VP sections from the various mouse models (Figure 2A) revealed that cyclin D1 nuclear positivity was less than 5% in the nontransgenic (Figure 2A and Casimiro et al3Casimiro MC Rodriguez O Pootrakul L Fricke ST Ostrowski M Rabbani S Datar R Cote RJ Pestell R Albanese C ErbB-2 induces the cyclin D1 gene in prostate epithelial cells in vitro and in vivo.Cancer Res. 2007; 67: 4364-4372Crossref PubMed Scopus (32) Google Scholar) and less than 20% in the pten+/− PIN lesions (not shown). In addition, there was a statistically significant increase in cyclin D1 nuclear positivit
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