Alterations in Nucleolar Structure and Gene Expression Programs in Prostatic Neoplasia Are Driven by the MYC Oncogene
2011; Elsevier BV; Volume: 178; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2010.12.040
ISSN1525-2191
AutoresCheryl M. Koh, Bora Gürel, Siobhan Sutcliffe, Martin J. Aryee, Denise Schultz, Tsuyoshi Iwata, Motohide Uemura, Karen Zeller, Uzoma A. Anele, Qizhi Zheng, Jessica Hicks, William G. Nelson, Chi V. Dang, Srinivasan Yegnasubramanian, Angelo M. De Marzo,
Tópico(s)Cancer, Lipids, and Metabolism
ResumoIncreased nucleolar size and number are hallmark features of many cancers. In prostate cancer, nucleolar enlargement and increased numbers are some of the earliest morphological changes associated with development of premalignant prostate intraepithelial neoplasia (PIN) lesions and invasive adenocarcinomas. However, the molecular mechanisms that induce nucleolar alterations in PIN and prostate cancer remain largely unknown. We verify that activation of the MYC oncogene, which is overexpressed in most human PIN and prostatic adenocarcinomas, leads to formation of enlarged nucleoli and increased nucleolar number in prostate luminal epithelial cells in vivo. In prostate cancer cells in vitro, MYC expression is needed for maintenance of nucleolar number, and a nucleolar program of gene expression. To begin to decipher the functional relevance of this transcriptional program in prostate cancer, we examined FBL (encoding fibrillarin), a MYC target gene, and report that fibrillarin is required for proliferation, clonogenic survival, and proper ribosomal RNA accumulation/processing in human prostate cancer cells. Further, fibrillarin is overexpressed in PIN lesions induced by MYC overexpression in the mouse prostate, and in human clinical prostate adenocarcinoma and PIN lesions, where its expression correlates with MYC levels. These studies demonstrate that overexpression of the MYC oncogene increases nucleolar number and size and a nucleolar program of gene expression in prostate epithelial cells, thus providing a molecular mechanism responsible for hallmark nucleolar alterations in prostatic neoplasia. Increased nucleolar size and number are hallmark features of many cancers. In prostate cancer, nucleolar enlargement and increased numbers are some of the earliest morphological changes associated with development of premalignant prostate intraepithelial neoplasia (PIN) lesions and invasive adenocarcinomas. However, the molecular mechanisms that induce nucleolar alterations in PIN and prostate cancer remain largely unknown. We verify that activation of the MYC oncogene, which is overexpressed in most human PIN and prostatic adenocarcinomas, leads to formation of enlarged nucleoli and increased nucleolar number in prostate luminal epithelial cells in vivo. In prostate cancer cells in vitro, MYC expression is needed for maintenance of nucleolar number, and a nucleolar program of gene expression. To begin to decipher the functional relevance of this transcriptional program in prostate cancer, we examined FBL (encoding fibrillarin), a MYC target gene, and report that fibrillarin is required for proliferation, clonogenic survival, and proper ribosomal RNA accumulation/processing in human prostate cancer cells. Further, fibrillarin is overexpressed in PIN lesions induced by MYC overexpression in the mouse prostate, and in human clinical prostate adenocarcinoma and PIN lesions, where its expression correlates with MYC levels. These studies demonstrate that overexpression of the MYC oncogene increases nucleolar number and size and a nucleolar program of gene expression in prostate epithelial cells, thus providing a molecular mechanism responsible for hallmark nucleolar alterations in prostatic neoplasia. Alterations in the structure of nuclei are hallmarks of cancer cells. Among these, enlargement, increased number, or altered morphology of the nucleolus all serve as morphological cues for the diagnosis of many premalignant and malignant tumors.1Derenzini M. Montanaro L. Trere D. What the nucleolus says to a tumour pathologist.Histopathology. 2009; 54: 753-762Crossref PubMed Scopus (178) Google Scholar, 2Derenzini M. Trere D. Pession A. Montanaro L. Sirri V. Ochs R.L. Nucleolar function and size in cancer cells.Am J Pathol. 1998; 152: 1291-1297PubMed Google Scholar For example, it has been documented since at least the 1920s that neoplastic prostate cells are characterized by enlarged, prominent nucleoli.3Scholl A.J. Histology and mortality in tumors of the prostate, bladder, and kidney.Cal West Med. 1927; 26: 185-189PubMed Google Scholar Further, this is considered a diagnostic morphological alteration in both PIN and adenocarcinoma of the prostate,4Bostwick D.G. Pacelli A. Lopez-Beltran A. Molecular biology of prostatic intraepithelial neoplasia.Prostate. 1996; 29: 117-134Crossref PubMed Scopus (174) Google Scholar and nucleolar number and size increase with increasing degrees of prostatic malignancy.5Hansen A.B. Ostergard B. Nucleolar organiser regions in hyperplastic and neoplastic prostatic tissue.Virchows Arch A Pathol Anat Histopathol. 1990; 417: 9-13Crossref PubMed Scopus (45) Google Scholar Although a number of potential oncogenic events that are implicated in prostate cancer have been proposed as potential causes of these nucleolar alterations, there is little evidence that known specific molecular changes in prostate cancer drive nucleolar structural and functional alterations in this disease.6Fischer A.H. Bardarov Jr., S. Jiang Z. Molecular aspects of diagnostic nucleolar and nuclear envelope changes in prostate cancer.J Cell Biochem. 2004; 91: 170-184Crossref PubMed Scopus (53) Google Scholar For example, it has been suggested that loss of PTEN or the activation of Ets family transcription factors in the mouse prostate results in nucleolar enlargement,7Tomlins S.A. Laxman B. Dhanasekaran S.M. Helgeson B.E. Cao X. Morris D.S. Menon A. Jing X. Cao Q. Han B. Yu J. Wang L. Montie J.E. Rubin M.A. Pienta K.J. Roulston D. Shah R.B. Varambally S. Mehra R. Chinnaiyan A.M. Distinct classes of chromosomal rearrangements create oncogenic ETS gene fusions in prostate cancer.Nature. 2007; 448: 595-599Crossref PubMed Scopus (652) Google Scholar, 8Carver B.S. Tran J. Chen Z. Carracedo-Perez A. Alimonti A. Nardella C. Gopalan A. Scardino P.T. Cordon-Cardo C. Gerald W. Pandolfi P.P. ETS rearrangements and prostate cancer initiation.Nature. 2009; 457 (discussion E2-E3): E1Crossref PubMed Scopus (89) Google Scholar, 9Shin S. 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Interphase FISH analysis of PTEN in histologic sections shows genomic deletions in 68% of primary prostate cancer and 23% of high-grade prostatic intra-epithelial neoplasias.Cancer Genet Cytogenet. 2006; 169: 128-137Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar Critical cellular functions performed within nucleoli include ribosomal RNA (rRNA) transcription, processing, and modification (eg, 2′-O-ribose methylation and pseudouridylation), and assembly of the ribosomal subunits.14Boisvert F.M. van Koningsbruggen S. Navascues J. Lamond A.I. The multifunctional nucleolus.Nat Rev Mol Cell Biol. 2007; 8: 574-585Crossref PubMed Scopus (1122) Google Scholar Ribosomal RNA is a limiting factor for ribosome production and protein synthesis, which are required for cell growth (as in mass) and cell division. The activation of pathways that stimulate protein synthesis are vital elements of tumor cells, and a number of components of the protein synthetic machinery are recognized as bona fide oncoproteins and as targets for cancer treatment. The nucleolus is also involved in the regulation of cell cycle progression and some cellular stress responses. Several nucleolar proteins, such as B23/nucleophosmin, have been associated with cancer, and patients with certain genetic syndromes in which the protein products of the genes involved localize to nucleoli, such as dyskeratosis congenita and Werner, Bloom, and Rothmund-Thomson syndromes, display an increased predisposition to cancer.15Montanaro L. Trere D. Derenzini M. Nucleolus, ribosomes, and cancer.Am J Pathol. 2008; 173: 301-310Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 16Ruggero D. Pandolfi P.P. Does the ribosome translate cancer?.Nat Rev Cancer. 2003; 3: 179-192Crossref PubMed Scopus (774) Google Scholar MYC is one of the most frequently activated oncogenes in human cancers, and its overexpression is commonly observed in various cancer types.17Meyer N. Penn L.Z. Reflecting on 25 years with MYC.Nat Rev Cancer. 2008; 8: 976-990Crossref PubMed Scopus (1155) Google Scholar MYC is located on 8q24, a genomic region that is amplified in a subset of aggressive prostate cancers,18Jenkins R.B. Qian J. Lieber M.M. Bostwick D.G. Detection of c-myc oncogene amplification and chromosomal anomalies in metastatic prostatic carcinoma by fluorescence in situ hybridization.Cancer Res. 1997; 57: 524-531PubMed Google Scholar, 19Bova G.S. Isaacs W.B. Review of allelic loss and gain in prostate cancer.World J Urol. 1996; 14: 338-346Crossref PubMed Scopus (62) Google Scholar and elevated MYC mRNA levels have been observed in prostate cancer.20Buttyan R. Sawczuk I.S. Benson M.C. Siegal J.D. Olsson C.A. Enhanced expression of the c-myc protooncogene in high-grade human prostate cancers.Prostate. 1987; 11: 327-337Crossref PubMed Scopus (149) Google Scholar, 21Fleming W.H. Hamel A. MacDonald R. Ramsey E. Pettigrew N.M. Johnston B. Dodd J.G. Matusik R.J. Expression of the c-myc protooncogene in human prostatic carcinoma and benign prostatic hyperplasia.Cancer Res. 1986; 46: 1535-1538PubMed Google Scholar Recent studies indicate that MYC protein is markedly overexpressed in the nuclei in most lesions of high-grade PIN, and localized and metastatic prostatic adenocarcinomas.22Gurel B. Iwata T. Koh C.M. Jenkins R.B. Lan F. Van Dang C. Hicks J.L. Morgan J. Cornish T.C. Sutcliffe S. Isaacs W.B. Luo J. De Marzo A.M. Nuclear MYC protein overexpression is an early alteration in human prostate carcinogenesis.Mod Pathol. 2008; 21: 1156-1167Crossref PubMed Scopus (314) Google Scholar In vitro, benign prostate cells have been transformed by MYC in a single step.23Gil J. Kerai P. Lleonart M. Bernard D. Cigudosa J.C. Peters G. Carnero A. Beach D. Immortalization of primary human prostate epithelial cells by c-Myc.Cancer Res. 2005; 65: 2179-2185Crossref PubMed Scopus (103) Google Scholar, 24Williams K. Fernandez S. Stien X. Ishii K. Love H.D. Lau Y.F. Roberts R.L. Hayward S.W. Unopposed c-MYC expression in benign prostatic epithelium causes a cancer phenotype.Prostate. 2005; 63: 369-384Crossref PubMed Scopus (57) Google Scholar Strikingly, overexpression of MYC in the mouse prostate confers virtually all of the phenotypes associated with the very early stages of prostatic neoplasia, including the development of PIN and invasive adenocarcinomas.25Ellwood-Yen K. Graeber T.G. Wongvipat J. Iruela-Arispe M.L. Zhang J. Matusik R. Thomas G.V. Sawyers C.L. Myc-driven murine prostate cancer shares molecular features with human prostate tumors.Cancer Cell. 2003; 4: 223-238Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar, 26Iwata T. Schultz D. Hicks J. Hubbard G.K. Mutton L.N. Lotan T.L. Bethel C. Lotz M.T. Yegnasubramanian S. Nelson W.G. Dang C.V. Xu M. Anele U. Koh C.M. Bieberich C.J. De Marzo A.M. MYC overexpression induces prostatic intraepithelial neoplasia and loss of Nkx3.1 in mouse luminal epithelial cells.PLoS One. 2010; 5: e9427Crossref PubMed Scopus (105) Google Scholar These data suggest that MYC plays a critical oncogenic role in the initiation and progression of prostate cancer. Given the almost ubiquitous overexpression of MYC coinciding with development of PIN during prostate carcinogenesis, we hypothesized that the MYC oncogene may be directly involved in mediating nucleolar enlargement, number, and hyperactive nucleolar function. In support of this, previous reports have shown that overexpression of the Drosophila homolog dMyc causes nucleolar enlargement in fly salivary gland cells.27Grewal S.S. Li L. Orian A. Eisenman R.N. Edgar B.A. Myc-dependent regulation of ribosomal RNA synthesis during Drosophila development.Nat Cell Biol. 2005; 7: 295-302Crossref PubMed Scopus (304) Google Scholar Further, when targeted to mouse liver, MYC overexpression induced many cellular changes, including nucleolar enlargement.28Kim S. Li Q. Dang C.V. Lee L.A. Induction of ribosomal genes and hepatocyte hypertrophy by adenovirus-mediated expression of c-Myc in vivo.Proc Natl Acad Sci U S A. 2000; 97: 11198-11202Crossref PubMed Scopus (152) Google Scholar Also, MYC is known to directly activate transcription of a number of genes whose protein products localize to and function primarily in the nucleolus.29Dai M.S. Lu H. Crosstalk between c-Myc and ribosome in ribosomal biogenesis and cancer.J Cell Biochem. 2008; 105: 670-677Crossref PubMed Scopus (96) Google Scholar, 30Ruggero D. The role of Myc-induced protein synthesis in cancer.Cancer Res. 2009; 69: 8839-8843Crossref PubMed Scopus (133) Google Scholar, 31Schlosser I. Holzel M. Murnseer M. Burtscher H. Weidle U.H. Eick D. A role for c-Myc in the regulation of ribosomal RNA processing.Nucleic Acids Res. 2003; 31: 6148-6156Crossref PubMed Scopus (142) Google Scholar, 32van Riggelen J. Yetil A. Felsher D.W. MYC as a regulator of ribosome biogenesis and protein synthesis.Nat Rev Cancer. 2010; 10: 301-309Crossref PubMed Scopus (1) Google Scholar, 33Zeller K.I. Jegga A.G. Aronow B.J. O'Donnell K.A. Dang C.V. An integrated database of genes responsive to the Myc oncogenic transcription factor: identification of direct genomic targets.Genome Biol. 2003; 4: R69Crossref PubMed Google Scholar However, whether MYC activation directly affects nucleolar architecture and activation of nucleolar gene expression programs in prostate cancer initiation or progression has not been established. In the current study, we report that MYC activation in vivo is coincident with increased nucleolar size and number at the onset of PIN. In neoplastic prostate cells in vitro, MYC regulates nucleolar number and architecture, and drives a nucleolar program of gene expression. Focusing on FBL, a crucial component of this nucleolar gene expression program involved in rRNA processing, we show that MYC directly regulates the expression of this gene by binding to its promoter region, and that fibrillarin is required for prostate cancer cell proliferation and mature rRNA accumulation. Taken together, these results indicate that MYC overexpression represents a critical mediator of increased nucleolar size, number, and function in PIN and prostatic adenocarcinoma lesions. The human prostate cancer cell lines LNCaP, CWR22rv1, DU145, and PC3, were obtained from the American Type Culture Collection (ATCC, Rockville, MD). MYC-CaP cells were a generous gift from Charles Sawyers.34Watson P.A. Ellwood-Yen K. King J.C. Wongvipat J. Lebeau M.M. Sawyers C.L. Context-dependent hormone-refractory progression revealed through characterization of a novel murine prostate cancer cell line.Cancer Res. 2005; 65: 11565-11571Crossref PubMed Scopus (110) Google Scholar They were maintained at 37°C and 5% CO2, and supplemented with RPMI 1640 or Dulbecco's modified Eagle's medium with 10% serum. Cells were transfected using Oligofectamine or Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Pools containing four small-interfering RNA (siRNAs) against MYC (L-003282; Dharmacon) and fibrillarin (L-011269; Dharmacon, Lafayette, CO) were transfected at a final concentration of 50 nmol/L. As a control in each transfection experiment, cells were transfected with siCONTROL Non-Targeting siRNA pool #1 (D-001810; Dharmacon). Following transfection with siRNA, cells were seeded at low density (500 to 1000 cells/well) using standard plastic (untreated polystyrene) six-well tissue culture plates and incubated for 10 to 14 days. Colonies were fixed with 0.5% crystal violet/glutaraldehyde. Colonies with more than 50 cells were counted. Four independent knockdown experiments were performed, with each condition done in triplicate each time. Cells and human prostate tissue specimens (20 frozen sections, each 10 μm in thickness) were lysed in radioimmunoprecipitation assay buffer. Proteins were electrophoresed and transferred to polyvinylidene fluoride membranes for immunoblotting. Membranes were probed with the antibodies as follows: MYC (#1472-1; 1:5000 dilution; Epitomics, Burlingame, CA); fibrillarin (ab5821; 1:2000; Abcam, Cambridge, MA); tubulin (CP06; 1:2000; Calbiochem, Gibbstown, NJ); nucleolin (ab13541; 1:2000; Abcam); UBF (sc13125; 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA); and B23/nucleophosmin (#18-7288; 1:5000; Zymed, Carlsbad, CA). RNA was isolated using Trizol Reagent (Invitrogen, Carlsbad, CA), and cDNA synthesized using the RetroScript kit (Ambion, Austin, TX). Quantitative RT-PCR was performed using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). The primers used are as follows: TBP forward: 5′-CACGAACCACGGCACTGATT-3′; TBP reverse: 5′-TTTTCTTGCTGCCAG-TCTGGAC-3′; fibrillarin forward: 5′-TGGACCAGATCCACATCAAA-3′; fibrillarin reverse: 5′-GACTAGACCATCCGGACCAA-3′; pre-rRNA forward: 5′-GAACGGTGGTGTGTCGTTC-3′; pre-rRNA reverse: 5′-GCGTCTCGTCTCGTCTCACT-3′; 5.8s rRNA forward: 5′-ACTCGGCTCGTGCGTC-3′; 5.8s rRNA reverse: 5′-GCGACGCTCAGACAGG-3′; 18s rRNA forward: 5′-GCCTGCTGCCTTCCTTGG-3′; 18s rRNA reverse: 5′-GATGGTAGTCGCCGTGCC-3′; 28s rRNA forward: 5′-AGAGGTAAACGGGTGGGGTC-3′; 28s rRNA reverse: 5′-GGGGTCGGGAGGAACGG-3′. The relative amount of the gene of interest was determined using the ΔΔCt method, relative to TBP and to the control cells transfected with the nontargeting pool of siRNA. Three independent knockdown experiments were performed, and triplicate quantitative PCR experiments were done for each condition. Forty-eight hours after transfection with siRNA against MYC, samples were processed by the Sidney Kimmel Comprehensive Cancer Center's Microarray Core Facility using Agilent's 44k whole genome array platform (Santa Clara, CA). Analysis was performed using R and Bioconductor open source software. The gene expression microarray data were preprocessed using Lowess normalization to correct for dye bias. Using gene set enrichment analysis (GSEA), all Gene Ontology (GO) and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway gene sets were tested for statistically significant enrichment of differentially expressed genes following ranking of individual probes by a moderated t-statistic as described.35Haffner M.C. Aryee M.J. Toubaji A. Esopi D.M. Albadine R. Gurel B. Isaacs W.B. Bova G.S. Liu W. Xu J. Meeker A.K. Netto G. De Marzo A.M. Nelson W.G. Yegnasubramanian S. Androgen-induced TOP2B-mediated double-strand breaks and prostate cancer gene rearrangements.Nat Genet. 2010; 42: 668-675Crossref PubMed Scopus (456) Google Scholar Microarray slides were hydrated, steamed for 40 minutes in Target Retrieval Solution (Dako, Glostrup, Denmark), and incubated in 1% dithiothreitol (Invitrogen) for 15 minutes. Slides were stained in the dark at 37°C for 9 minutes with 5% silver nitrate (Sigma, St Louis, MO) dissolved in 1% formic acid/0.33% gelatin (Sigma). The experimental protocol was approved by the Animal Care and Use Committee at Johns Hopkins University, and the animals were cared for in accordance with institutional guidelines. The Lo-MYC transgenic mice used in this study were obtained from the Mouse Repository of the National Cancer Institute Mouse Models of Human Cancer Consortium at NCI Frederick, MD, and were maintained as previously described.26Iwata T. Schultz D. Hicks J. Hubbard G.K. Mutton L.N. Lotan T.L. Bethel C. Lotz M.T. Yegnasubramanian S. Nelson W.G. Dang C.V. Xu M. Anele U. Koh C.M. Bieberich C.J. De Marzo A.M. MYC overexpression induces prostatic intraepithelial neoplasia and loss of Nkx3.1 in mouse luminal epithelial cells.PLoS One. 2010; 5: e9427Crossref PubMed Scopus (105) Google Scholar Tissues for electron microscopy were obtained from the ventral prostates of Lo-MYC and FVB wild-type mice that were approximately 10 months of age and were fixed in glutaraldehyde and osmium tetroxide and embedded into Epon-Araldite. After routine processing, regions of high-grade PIN and normal were identified after toluidine blue staining of “thick” (1 μm) sections, and these regions were subjected to “thin” sectioning and standard staining with lead citrate/uranyl acetate. Thin sections were examined and imaged on a Hitachi7600 (Tokyo, Japan) transmission electron microscope (TEM) equipped with a Dual AMT CCD camera system. Immunohistochemistry was done with the Power Vision+ poly-HRP IHC Kit (ImmunoVision Inc, Springdale, AZ). Slides were steamed for 40 minutes in EDTA solution (Zymed) and incubated with rabbit polyclonal anti-fibrillarin antibody (1:50 dilution) for 45 minutes. Poly-HRP–conjugated anti-mouse/rabbit IgG antibody was used as secondary antibody. Staining was visualized using 3,3′-diaminobenzidine (Sigma), and slides were counterstained with hematoxylin. Chromatin immunoprecipitation (ChIP) was performed using the rabbit polyclonal anti-MYC antibody (sc-764; Santa Cruz Biotechnology), as previously described.33Zeller K.I. Jegga A.G. Aronow B.J. O'Donnell K.A. Dang C.V. An integrated database of genes responsive to the Myc oncogenic transcription factor: identification of direct genomic targets.Genome Biol. 2003; 4: R69Crossref PubMed Google Scholar The primers used are as follows: ChIP fibrillarin forward: 5′-GAGAATCCAGGCTCACTGC-3′; ChIP fibrillarin reverse: 5′-GTTCACAACTCCACGAGTCC-3′; ChIP negative control forward: 5′-AACTCCACATTTCCTAAGTGACC-3′; ChIP negative control reverse: 5′-CCAACCCACACCAAGTACC-3′. This study was approved by the Johns Hopkins University School of Medicine institutional review board. Tissue microarrays (TMAs) were constructed and evaluated as described.22Gurel B. Iwata T. Koh C.M. Jenkins R.B. Lan F. Van Dang C. Hicks J.L. Morgan J. Cornish T.C. Sutcliffe S. Isaacs W.B. Luo J. De Marzo A.M. Nuclear MYC protein overexpression is an early alteration in human prostate carcinogenesis.Mod Pathol. 2008; 21: 1156-1167Crossref PubMed Scopus (314) Google Scholar Every scanned image from the TMAs was rediagnosed by both B.G. and A.M.D. For image analysis, we used FrIDA (Framework for Image Data Analysis) essentially as described.36Cornish T, DeMarzo AM, Gurel B, Morgan J: FRIDA. An open source framework for image dataset analysis. Presented at the 12th Annual International Conference on Advancing Practice, Instruction and Innovation Through Informatics. 2007 September 9–12, Pittsburgh, PAGoogle Scholar Briefly, TMA slides were scanned using the AperioScanScope CS (Vista, CA), and composite images for each TMA spot were generated and imported into the TMAJ software package. For each scanned TMA image, a region of interest mask was manually segmented using polygon tools in FrIDA/TMAJ, and hue, saturation, and value color space segmentation was used to identify the pixels and their intensity for IHC-positive (brown staining) and IHC-negative nuclei (blue staining). Only nuclear staining was considered, and the Fibrillarin Nuclear Area Ratio (brown nuclear area)/(brown + blue nuclear area) and Fibrillarin Score (the summed brown nuclear intensity)/(brown + blue nuclear area) were calculated. Statistical analyses for TMA data were performed using SAS (Cary, NC). P values were calculated by linear regression with robust variance estimation to account for the correlation between observations. MYC is known to be a key regulator of cell proliferation in both normal and cancer cells.37Wang H. Mannava S. Grachtchouk V. Zhuang D. Soengas M.S. Gudkov A.V. Prochownik E.V. Nikiforov M.A. c-Myc depletion inhibits proliferation of human tumor cells at various stages of the cell cycle.Oncogene. 2008; 27: 1905-1915Crossref PubMed Scopus (116) Google Scholar We performed siRNA-mediated depletion of MYC in four different human prostate cancer cell lines, two that are androgen responsive (LNCaP and CWR22Rv1) and two that are androgen nonresponsive (DU145 and PC3). We also studied a mouse cell line, MYC-CaP, obtained from a MYC-driven prostate cancer.34Watson P.A. Ellwood-Yen K. King J.C. Wongvipat J. Lebeau M.M. Sawyers C.L. Context-dependent hormone-refractory progression revealed through characterization of a novel murine prostate cancer cell line.Cancer Res. 2005; 65: 11565-11571Crossref PubMed Scopus (110) Google Scholar In all cell lines, MYC protein expression was reduced by approximately 50% to 90% by siRNA transfection (see Supplemental Figure S1A at http://ajp.amjpathol.org). Compared to control cells transfected with a nontargeting pool of “scrambled” siRNAs, MYC-depleted prostate cancer cells showed markedly suppressed cell proliferation (P < 0.001; see Supplemental Figure S1B at http://ajp.amjpathol.org). We also observed, as expected, similar results in MYC-CaP cells (see Supplemental Figure S1B at http://ajp.amjpathol.org). In four of the five cell lines, DNA synthesis was reduced, as measured by the incorporation of 5-ethynyl-2′-deoxyuridine (EdU) (P < 0.05; see Supplemental Figure S1C at http://ajp.amjpathol.org). There was no increase in apoptosis, as determined by morphological observation. Additionally, the ability to form colonies using clonogenic survival assays was reduced by 65% to 85% in cells with siRNA-mediated forced reductions in MYC, as compared to the control in DU145, PC3, and LNCaP cells (P < 0.005 for each), with less of a reduction (28%) in CWR22Rv1 cells (see Supplemental Figure S1D at http://ajp.amjpathol.org). These results indicate that androgen receptor–positive/androgen-dependent cells (LNCaP, MYC-CaP), androgen receptor–positive/androgen-independent cells (CWR22Rv1), and androgen receptor–negative/androgen-independent cells (PC3, DU145) all require MYC for cell replication, and help establish the relevance of these model systems in studying downstream effects of MYC expression. MYC is known to regulate several nucleolar genes,29Dai M.S. Lu H. Crosstalk between c-Myc and ribosome in ribosomal biogenesis and cancer.J Cell Biochem. 2008; 105: 670-677Crossref PubMed Scopus (96) Google Scholar, 30Ruggero D. The role of Myc-induced protein synthesis in cancer.Cancer Res. 2009; 69: 8839-8843Crossref PubMed Scopus (133) Google Scholar, 31Schlosser I. Holzel M. Murnseer M. Burtscher H. Weidle U.H. Eick D. A role for c-Myc in the regulation of ribosomal RNA processing.Nucleic Acids Res. 2003; 31: 6148-6156Crossref PubMed Scopus (142) Google Scholar, 32van Riggelen J. Yetil A. Felsher D.W. MYC as a regulator of ribosome biogenesis and protein synthesis.Nat Rev Cancer. 2010; 10: 301-309Crossref PubMed Scopus (1) Google Scholar, 33Zeller K.I. Jegga A.G. Aronow B.J. O'Donnell K.A. Dang C.V. An integrated database of genes responsive to the Myc oncogenic transcription factor: identification of direct genomic targets.Genome Biol. 2003; 4: R69Crossref PubMed Google Scholar and marked enlargement of subnuclear structures characteristic of nucleoli are observed using light microscopy in high-grade PIN and adenocarcinoma lesions in the Lo-MYC and Hi-MYC murine models of prostate cancer.25Ellwood-Yen K. Graeber T.G. Wongvipat J. Iruela-Arispe M.L. Zhang J. Matusik R. Thomas G.V. Sawyers C.L. Myc-driven murine prostate cancer shares molecular features with human prostate tumors.Cancer Cell. 2003; 4: 223-238Abstract Full Text Full Text PDF PubMed Scopus (606) Google Scholar, 26Iwata T. Schultz D. Hicks J. Hubbard G.K. Mutton L.N. Lotan T.L. Bethel C. Lotz M.T. Yegnasubramanian S. Nelson W.G. Dang C.V. Xu M. Anele U. Koh C.M. Bieberich C.J. De Marzo A.M. MYC overexpression induces prostatic intraepithelial neoplasia and loss of Nkx3.1 in mouse luminal epithelial cells.PLoS One. 2010; 5: e9427Crossref PubMed Scopus (105) Google Scholar The most definitive means to determine whether nuclear structures are nucleoli is by TEM. Thus, we performed TEM on a number of Lo-MYC and wild-type mice. Indeed, we observed markedly enlarged nucleoli in PIN cells from Lo-MYC mice, as compared with wild-type mice (Figure 1A). Since the onset of detectable levels of nuclear MYC protein corresponds precisely with increased apparent nucleolar size in Lo-MYC and Hi-MYC mice,26Iwata T. Schultz D. Hicks J. Hubbard G.K. Mutton L.N. Lotan T.L. Bethel C. Lotz M.T. Yegnasubramanian S. Nelson W.G. Dang C.V. Xu M. Anele U. Koh C.M. Bieberich C.J. De Marzo A.M. MYC overexpression induces prostatic intraepithelial neoplasia and loss of Nkx3.1 in mouse luminal epithelial cells.PLoS One. 2010; 5: e9427Crossref PubMed Scopus (105) Google Scholar these findings verify that MYC overexpressi
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