An Extensive Tumor Array Analysis Supports Tumor Suppressive Role for Nucleophosmin in Breast Cancer
2011; Elsevier BV; Volume: 179; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2011.04.009
ISSN1525-2191
AutoresPiia‐Riitta Karhemo, Antti Rivinoja, Johan Lundin, Maija Hyvönen, Anastasiya Chernenko, Johanna Lammi, Harri Sihto, Mikael Lundin, Päivi Heikkilä, Heikki Joensuu, Petri Bono, Pirjo Laakkonen,
Tópico(s)Advanced biosensing and bioanalysis techniques
ResumoNucleophosmin (NPM) is a multifunctional protein involved in a complex network of interactions. The role of NPM in oncogenesis is controversial. The NPM gene (NPM1) is mutated or rearranged in a number of hematological disorders, but such changes have not been detected in solid cancers. However, experiments with cultured NPM-null cells and with mice carrying a single inactivated NPM allele indicate a tumor suppressor function for NPM. To resolve the role of NPM in solid cancers, we examined its expression and localization in histologically normal breast tissue and a large array of human breast carcinoma samples (n = 1160), and also evaluated its association with clinicopathological variables and patient survival. The intensity and localization (nucleolar, nuclear, cytoplasmic) of NPM varied across clinical samples. No mutations explaining the differences were found, but the present findings indicate that expression levels of NPM affected its localization. Our study also revealed a novel granular staining pattern for NPM, which was an independent prognostic factor of poor prognosis. In addition, reduced levels of NPM protein were associated with poor prognosis. Furthermore, luminal epithelial cells of histologically normal breast displayed high levels of NPM and overexpression of NPM in the invasive MDA-MB-231 cells abrogated their growth in soft agar. These results support a tumor suppressive role for NPM in breast cancer. Nucleophosmin (NPM) is a multifunctional protein involved in a complex network of interactions. The role of NPM in oncogenesis is controversial. The NPM gene (NPM1) is mutated or rearranged in a number of hematological disorders, but such changes have not been detected in solid cancers. However, experiments with cultured NPM-null cells and with mice carrying a single inactivated NPM allele indicate a tumor suppressor function for NPM. To resolve the role of NPM in solid cancers, we examined its expression and localization in histologically normal breast tissue and a large array of human breast carcinoma samples (n = 1160), and also evaluated its association with clinicopathological variables and patient survival. The intensity and localization (nucleolar, nuclear, cytoplasmic) of NPM varied across clinical samples. No mutations explaining the differences were found, but the present findings indicate that expression levels of NPM affected its localization. Our study also revealed a novel granular staining pattern for NPM, which was an independent prognostic factor of poor prognosis. In addition, reduced levels of NPM protein were associated with poor prognosis. Furthermore, luminal epithelial cells of histologically normal breast displayed high levels of NPM and overexpression of NPM in the invasive MDA-MB-231 cells abrogated their growth in soft agar. These results support a tumor suppressive role for NPM in breast cancer. Nucleophosmin (NPM) is a ubiquitously expressed multifunctional nucleolar phosphoprotein. It localizes mainly to the nucleoli, but also shuttles in and out of the nucleolus, and between the nucleus and the cytoplasm.1Borer R.A. Lehner C.F. Eppenberger H.M. Nigg E.A. Major nucleolar proteins shuttle between nucleus and cytoplasm.Cell. 1989; 56: 379-390Abstract Full Text PDF PubMed Scopus (918) Google Scholar, 2Wang D. Umekawa H. Olson M.O. Expression and subcellular locations of two forms of nucleolar protein B23 in rat tissues and cells.Cell Mol Biol Res. 1993; 39: 33-42PubMed Google Scholar NPM belongs to the nucleoplasmin family of nuclear chaperone proteins. It is involved in a complex network of interactions and has multiple functions. In addition to its chaperone activity,3Okuwaki M. Matsumoto K. Tsujimoto M. Nagata K. Function of nucleophosmin/B23, a nucleolar acidic protein, as a histone chaperone.FEBS Lett. 2001; 506: 272-276Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 4Szebeni A. Olson M.O. Nucleolar protein B23 has molecular chaperone activities.Protein Sci. 1999; 8: 905-912Crossref PubMed Scopus (206) Google Scholar NPM is involved in centrosome duplication,5Okuda M. The role of nucleophosmin in centrosome duplication.Oncogene. 2002; 21: 6170-6174Crossref PubMed Scopus (150) Google Scholar ribosome biogenesis,6Herrera J.E. Savkur R. Olson M.O. The ribonuclease activity of nucleolar protein B23.Nucleic Acids Res. 1995; 23: 3974-3979Crossref PubMed Scopus (133) Google Scholar, 7Savkur R.S. Olson M.O. Preferential cleavage in pre-ribosomal RNA by protein B23 endoribonuclease.Nucleic Acids Res. 1998; 26: 4508-4515Crossref PubMed Scopus (192) Google Scholar and environmental stress responses.8Chan P.K. Aldrich M. Busch H. 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Human histone chaperone nucleophosmin enhances acetylation-dependent chromatin transcription.Mol Cell Biol. 2005; 25: 7534-7545Crossref PubMed Scopus (145) Google Scholar sumoylation,17Liu X. Liu Z. Jang S.W. Ma Z. Shinmura K. Kang S. Dong S. Chen J. Fukasawa K. Ye K. Sumoylation of nucleophosmin/B23 regulates its subcellular localization, mediating cell proliferation and survival.Proc Natl Acad Sci USA. 2007; 104: 9679-9684Crossref PubMed Scopus (67) Google Scholar, 18Tago K. Chiocca S. Sherr C.J. Sumoylation induced by the Arf tumor suppressor: a p53-independent function.Proc Natl Acad Sci USA. 2005; 102: 7689-7694Crossref PubMed Scopus (98) Google Scholar ubiquitinylation,19Sato K. Hayami R. Wu W. Nishikawa T. Nishikawa H. Okuda Y. Ogata H. Fukuda M. Ohta T. Nucleophosmin/B23 is a candidate substrate for the BRCA1-BARD1 ubiquitin ligase.J Biol Chem. 2004; 279: 30919-30922Crossref PubMed Scopus (127) Google Scholar and phosphorylation.20Beckmann R. Buchner K. Jungblut P.R. Eckerskorn C. Weise C. Hilbert R. Hucho F. Nuclear substrates of protein kinase C.Eur J Biochem. 1992; 210: 45-51Crossref PubMed Scopus (56) Google Scholar, 21Okuda M. Horn H.F. Tarapore P. Tokuyama Y. Smulian A.G. Chan P.K. Knudsen E.S. Hofmann I.A. Snyder J.D. Bove K.E. Fukasawa K. Nucleophosmin/B23 is a target of CDK2/cyclin E in centrosome duplication.Cell. 2000; 103: 127-140Abstract Full Text Full Text PDF PubMed Scopus (560) Google Scholar, 22Peter M. Nakagawa J. Doree M. Labbe J.C. Nigg E.A. Identification of major nucleolar proteins as candidate mitotic substrates of cdc2 kinase.Cell. 1990; 60: 791-801Abstract Full Text PDF PubMed Scopus (286) Google Scholar, 23Pfaff M. Anderer F.A. Casein kinase II accumulation in the nucleolus and its role in nucleolar phosphorylation.Biochim Biophys Acta. 1988; 969: 100-109Crossref PubMed Scopus (52) Google Scholar NPM has been heavily implicated in cancer pathogenesis, but its actual role in oncogenesis is controversial.24Grisendi S. Mecucci C. Falini B. Pandolfi P.P. Nucleophosmin and cancer.Nat Rev Cancer. 2006; 6: 493-505Crossref PubMed Scopus (661) Google Scholar NPM1 is mutated or rearranged in a number of hematological disorders,25Falini B. Nicoletti I. Bolli N. Martelli M.P. Liso A. Gorello P. Mandelli F. Mecucci C. Martelli M.F. Translocations and mutations involving the nucleophosmin (NPM1) gene in lymphomas and leukemias.Haematologica. 2007; 92: 519-532Crossref PubMed Scopus (165) Google Scholar and it is the most frequently mutated gene in acute myeloid leukemia. In addition, NPM protein is reported to be overexpressed in cancer cells, and it was originally proposed as a proto-oncogene. However, rapidly proliferating tumor cells could show elevated NPM levels, simply because NPM expression increases rapidly in early G1 phase during mitosis.26Feuerstein N. Spiegel S. Mond J.J. The nuclear matrix protein, numatrin (B23), is associated with growth factor-induced mitogenesis in Swiss 3T3 fibroblasts and with T lymphocyte proliferation stimulated by lectins and anti-T cell antigen receptor antibody.J Cell Biol. 1988; 107: 1629-1642Crossref PubMed Scopus (139) Google Scholar On the other hand, inactivation of NPM1 in the germline leads to embryonic lethality.27Feuerstein N. Chan P.K. Mond J.J. Identification of numatrin, the nuclear matrix protein associated with induction of mitogenesis, as the nucleolar protein B23 Implication for the role of the nucleolus in early transduction of mitogenic signals.J Biol Chem. 1988; 263: 10608-10612Abstract Full Text PDF PubMed Google Scholar, 28Subong E.N. Shue M.J. Epstein J.I. Briggman J.V. Chan P.K. Partin A.W. Monoclonal antibody to prostate cancer nuclear matrix protein (PRO:4–216) recognizes nucleophosmin/B23.Prostate. 1999; 39: 298-304Crossref PubMed Scopus (100) Google Scholar Moreover, experiments with cultured NPM-null cells27Feuerstein N. Chan P.K. Mond J.J. Identification of numatrin, the nuclear matrix protein associated with induction of mitogenesis, as the nucleolar protein B23 Implication for the role of the nucleolus in early transduction of mitogenic signals.J Biol Chem. 1988; 263: 10608-10612Abstract Full Text PDF PubMed Google Scholar, 28Subong E.N. Shue M.J. Epstein J.I. Briggman J.V. Chan P.K. Partin A.W. Monoclonal antibody to prostate cancer nuclear matrix protein (PRO:4–216) recognizes nucleophosmin/B23.Prostate. 1999; 39: 298-304Crossref PubMed Scopus (100) Google Scholar and mice carrying a single inactivated NPM allele indicate a tumor suppressor function for NPM.27Feuerstein N. Chan P.K. Mond J.J. Identification of numatrin, the nuclear matrix protein associated with induction of mitogenesis, as the nucleolar protein B23 Implication for the role of the nucleolus in early transduction of mitogenic signals.J Biol Chem. 1988; 263: 10608-10612Abstract Full Text PDF PubMed Google Scholar NPM1 function is required for the maintenance of genomic stability,27Feuerstein N. Chan P.K. Mond J.J. Identification of numatrin, the nuclear matrix protein associated with induction of mitogenesis, as the nucleolar protein B23 Implication for the role of the nucleolus in early transduction of mitogenic signals.J Biol Chem. 1988; 263: 10608-10612Abstract Full Text PDF PubMed Google Scholar, 28Subong E.N. Shue M.J. Epstein J.I. Briggman J.V. Chan P.K. Partin A.W. Monoclonal antibody to prostate cancer nuclear matrix protein (PRO:4–216) recognizes nucleophosmin/B23.Prostate. 1999; 39: 298-304Crossref PubMed Scopus (100) Google Scholar, 29Sportoletti P. Grisendi S. Majid S.M. Cheng K. Clohessy J.G. Viale A. Teruya-Feldstein J. Pandolfi P.P. Npm1 is a haploinsufficient suppressor of myeloid and lymphoid malignancies in the mouse.Blood. 2008; 111: 3859-3862Crossref PubMed Scopus (102) Google Scholar and NPM1 acts as a haploinsufficient tumor suppressor in the hematopoietic compartment.29Sportoletti P. Grisendi S. Majid S.M. Cheng K. Clohessy J.G. Viale A. Teruya-Feldstein J. Pandolfi P.P. Npm1 is a haploinsufficient suppressor of myeloid and lymphoid malignancies in the mouse.Blood. 2008; 111: 3859-3862Crossref PubMed Scopus (102) Google Scholar To shed light on the role of NPM in solid cancers, we investigated NPM expression levels and localization in a large array of human breast carcinoma samples (n = 1160) and evaluated its association with clinicopathological variables, patient survival, and molecular subtypes of breast cancer. We identified granular staining as a novel staining pattern for NPM. Both reduced NPM levels and granular staining pattern were associated with poor prognosis. All media were supplemented with 10% fetal calf serum, 1% glutamine, and penicillin-streptomycin, except as stated otherwise. MDA-MB-231 and MCF-7 cells were maintained in RPMI 1640 medium, MDA-MB-436 cells in Leibovitz's L-15 medium with 16 μg/mL glutathione, 10 μg/mL insulin, and MDA-MB-361 cells in Leibovitz's L-15 medium with 20% fetal calf serum. We used a monoclonal mouse-anti-NPM antibody (Zymed Laboratories, South San Francisco, CA). The specificity of the antibody has been described previously.8Chan P.K. Aldrich M. Busch H. Alterations in immunolocalization of the phosphoprotein B23 in HeLa cells during serum starvation.Exp Cell Res. 1985; 161: 101-110Crossref PubMed Scopus (60) Google Scholar For immunofluorescence staining, cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% NP-40 in PBS, blocked using 3% bovine serum albumin in PBS, and incubated with primary and secondary (Molecular Probes goat anti-mouse Alexa Fluor 594; Invitrogen, Carlsbad, CA) antibodies. DNA was visualized with DAPI (Vector Laboratories, Burlingame, CA). For the immunohistochemical (IHC) analyses, 4-μm sections were prepared and deparaffinized. After antigen retrieval (120°C autoclave, 2 minutes in 10 mmol/L sodium citrate buffer, pH 6.0), blocking of endogenous peroxidase activity (0.7% hydrogen peroxidase in methanol, 30 minutes), and blocking of unspecific antibody binding sites (normal horse serum in 0.3% bovine serum albumin-PBS, 30 minutes), sections were incubated with anti-NPM antibody (0.3% bovine serum albumin-PBS, overnight, 4°C). NPM antibody was detected with peroxidase staining (biotinylated horse anti-mouse immunoglobulins 1:200 and Vectastain ABC complex; Vector Laboratories) visualized with 3,3′-diaminobenzidine (Vector Laboratories). The sections were counterstained with Mayer's hematoxylin. c-Kit staining was performed using a polyclonal rabbit-anti-CD117 antibody (1:300, A4502; Dako, Carpinteria, CA) similarly to the NPM staining, with the following exceptions: 98°C water bath for 15 minutes was used in antigen retrieval and a PowerVision Novocastra preantibody blocking solution (Leica Microsystems, Wetzlar, Germany) was used for primary antibody incubation. Staining was detected using a PowerVision + Poly-HRP histostaining kit (DPVB + 110DAB; ImmunoVision Technologies, Daly City, CA; Springdale, AR) according to the manufacturer's instructions. Cytokeratin staining was performed with a Ventana Discovery IHC slide stainer (Ventana Medical Systems, Tucson, AZ) and a Ventana 3,3′-diaminobenzidine tetrahydrochloride biotin avidin detection kit using a polyclonal rabbit-anti-cytokeratin 7 antibody (1:100, ab52870; Abcam, Cambridge, UK). The NPM1 gene was cloned into a lentiviral pBOB\cag\green fluorescent protein (GFP) expression vector (a kind gift from Dr. Ylä-Herttuala, University of Eastern Finland, Kuopio, Finland) and was transfected into human embryonic kidney cells (293FT; 4 × 106) using Lipofectamine 2000 (Invitrogen). Virus-containing supernatants were collected at 72 hours after transfection. To remove cell debris, the medium was centrifuged briefly (2 minutes, 160 × g) and filtered through a 0.45-μm filter. The supernatant was concentrated with an Optima L-80 XP ultracentrifuge supplied with a swinging bucket rotor (SW28) (both Beckman Coulter, Brea, CA). MDA-M-231 and MDA-MB-361 cells (50% confluent) were transduced with concentrated NPM-GFP and GFP encoding viruses. We sorted the moderately transgene-positive cells by using a FACSAria fluorescence-activated cell sorter (purity mode; BD Biosciences, San Jose, CA). Expression of transgenes was verified using Western blot analysis. Efficiency of transduction was determined using immunofluorescence microscopy by counting the percentage of transgene-expressing cells. Cells grown to 60% to 70% confluency were starved for 48 hours in Phenol Red-free RPMI (Gibco, California, USA) supplemented with 2% charcoal-dextran filtrated fetal calf serum (HyClone; Thermo Scientific, Logan, UT), 1% glutamine, and penicillin-streptomycin. After starvation, cells were treated with 10 nmol/L 17β-estradiol diluted in dimethyl sulfoxide (DMSO) for 3 days, followed by either fixation for the immunofluorescence analysis or cell lysis in NP-40 buffer (1% NP-40, 50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl) for 10 minutes at 4°C. DMSO-treated cells served as control. Percentage of cells displaying even nuclear staining (no visible nucleoli) and nucleolar staining was determined from the microscopic images. NPM expression was quantified with ImageJ software version 1.45h (NIH, Bethesda, MD) against β-tubulin, detected from the same membranes. Samples were analyzed using SDS-PAGE and transferred to Immobilon-P membrane (Millipore, Billerica, MA) for immunoblotting. We used the following primary antibodies: anti-GFP (Molecular Probes A6455; Invitrogen), anti-NPM (Zymed Laboratories; Cell Signaling Technology, Danvers, MA), and anti-β-tubulin (BD Biosciences). Proteins were visualized with a SuperSignal West Pico kit (Pierce; Thermo Scientific, Rockford, IL). Histologically normal breast samples from reduction surgery were obtained from the Helsinki University Central Hospital. The Finprog breast cancer database used in the cancer tissues analysis contains samples from women diagnosed with breast cancer within five well-defined geographical regions in Finland in 1991 and 1992; approximately 50% of the Finnish population lives in these five regions.30Lundin J. Lundin M. Isola J. Joensuu H. A web-based system for individualised survival estimation in breast cancer.BMJ. 2003; 326: 29Crossref PubMed Scopus (34) Google Scholar Patients with in situ carcinoma, distant metastases at the time of diagnosis, synchronous or metachronous bilateral breast cancer, malignancy other than breast cancer in history (except for basal cell carcinoma or cervical carcinoma in situ), or women who did not undergo breast surgery were excluded from the study series, leaving a total of 2032 patients eligible. Tissue array core biopsies with an interpretable staining for NPM were available for 1160 of these patients. The median follow-up time for patients alive and without recurrence at the end of follow-up was 9.5 years. Adjuvant chemotherapy (typically a combination of cyclophosphamide, methotrexate, and 5-fluorouracil) was given to 160 of the 1160 patients (13.8%), adjuvant hormonal therapy (typically tamoxifen) to 278 patients (24%), and combined chemotherapy and hormonal therapy to 6 patients (0.5%). TMAs were prepared as described previously.31Joensuu H. Isola J. Lundin M. Salminen T. Holli K. Kataja V. Pylkkänen L. Turpeenniemi-Hujanen T. von Smitten K. Lundin J. Amplification of erbB2 and erbB2 expression are superior to estrogen receptor status as risk factors for distant recurrence in pT1N0M0 breast cancer: a nationwide population-based study.Clin Cancer Res. 2003; 9: 923-930PubMed Google Scholar Expression and localization of NPM was examined under a multiheaded Leica DM LB microscope by two independent observers (P.-R.K. and P.L.). All evaluations were performed with blinding to the clinical data. The expression level scoring of the TMA was performed as follows: 0, all tumor cells negative; 1, low expression in tumor cells (5% to 100%); 2, moderate expression in ≥50% of tumor cells; and 3, high expression in ≥50% of tumor cells. Nucleolar localization, nucleoplasmic staining pattern, and cytoplasmic staining of NPM were scored independently of the expression analysis. The following criteria were used for the nucleolar NPM staining: 0 (negative), no staining in the nucleoli or the nucleoplasmic intensity much higher than that found in the nucleoli; 1 (an intermediate staining), equal staining intensities in the nucleolus and in the nucleoplasm; and 2, staining only in the nucleoli. Nucleoplasmic staining pattern was scored as granular if the staining was observed at the nuclear periphery and appeared speckled. Cytoplasmic staining was scored as negative, low, or high. Because of nonrepresentative sample material, the nucleolar and the cytoplasmic localization could not be analyzed in 10% (112/1160) and in 4% (47/1160) of the tumors, respectively. Image acquisition was performed through an Olympus DP50 color camera and with Olympus Studio Lite software version 1.0. Immunofluorescence staining of cells was visualized with an Axioplan 2 epifluorescence microscope (Zeiss, Jena, Germany) with appropriate filters (Chroma Technology, Rockingham, VT). Image acquisition was performed using a Zeiss digital AxioCam camera and AxioVision software version 4.5. Soft agar assays were performed with MDA-MB-231 human breast carcinoma cells infected with lentiviruses encoding NPM-GFP. Cells infected with lentiviruses encoding GFP served as control. Cells (5 × 103 cells per 35-mm well) were resuspended in complete medium containing 0.35% agarose. Cells were grown on tissue culture dishes containing a 2-mL layer of solidified 0.7% agar in a complete medium. After 14 days, number of colonies was quantified using ImageJ software from two randomly taken micrographs per well (original magnification, ×20). For visualization, foci were methanol-fixed and stained with 0.005% crystal violet. Cells (5 × 103) were grown on 96-well plates for 2 or 3 days, after which 10 μL MTT (5 mg/mL) was added. After incubation for 2 hours, cells were lysed (10% SDS, 10 mmol/L HCl) overnight. Absorbance was measured at 540 nm using Multiskan Ascent software version 2.6 (Thermo Labsystems, Vantaa, Finland). Total RNA was isolated from paraffin-embedded sections with a High Pure RNA paraffin kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions. A QuantiTect reverse transcription kit (Qiagen, Hilden, Germany) was used for the cDNA synthesis. A 206-bp C-terminal fragment was amplified with FastStart TaqDNA polymerase (Roche Diagnostics) using the following primers: 5′-CTTCCCAAAGTGGAAGCC-3′ and 5′-GGAAAGTTCTCACTCTGC-3′.32Calvo K.L. Ojeda M.J. Ammatuna E. Lavorgna S. Ottone T. Targovnik H.M. Lo-Coco F. Noguera N.I. Detection of the nucleophosmin gene mutations in acute myelogenous leukemia through RT-PCR and polyacrylamide gel electrophoresis.Eur J Haematol. 2009; 82: 69-72Crossref PubMed Scopus (9) Google Scholar The amplified fragment was sequenced using the 5′-GGAAAGTTCTCACTCTGC-3′ primer and the Dye Terminator kit version 3.1 with an ABI 3100 genetic analyzer (Applied Biosystems, Foster City, CA). Expression of NPM in the histologically normal breast tissue was investigated from cDNA samples by quantitative PCR (qPCR), using a DyNAmo HS SYBR Green qPCR kit (Finnzymes, Vantaa, Finland). The cDNA used as a template was prepared from the total RNA extracts as described above. Presence of NPM cDNA was detected using primers 5′-GCGCCAGTGAAGAAATCTATACG-3′ and 5′-GAAGGATTCTTGTCCTTTTGATCTTG-3′ and was normalized against the cDNA level of glyceraldehyde-3-phosphate dehydrogenase as the reference gene, which was detected using primers 5′-GAAGGTGAAGGTCGGAGTCAAC-3′ and 5′-CAGAGTTAAAAGCAGCCCTGGT-3′. Relative quantification of expression levels between the samples was performed according to the Pfaffl method.33Pfaffl M.W. A new mathematical model for relative quantification in real-time RT-PCR.Nucleic Acids Res. 2001; 29: e45Crossref PubMed Scopus (25413) Google Scholar The χ2 test was used to test for associations between factors and the odds ratio to examine the strength of the relationships. Life tables were calculated according to the Kaplan-Meier method. Distant disease-free survival (DDFS) was calculated from the date of diagnosis to the occurrence of either metastases outside the locoregional area or death from breast cancer. Survival curves were compared using the log-rank test. Multivariate survival analyses were performed with the Cox proportional hazards model, entering the following covariates: NPM intensity as a categorical variable (with the highest intensity value as the reference) and the number of metastatic lymph nodes, tumor size in centimeters, grade (well differentiated versus moderately versus poorly differentiated), estrogen receptor (ER) and progesterone receptor (PgR) status, and the HER2 test results as binary variables (negative versus positive). Cox regression was performed using a backward stepwise selection of variables and a P value of 0.05 was adopted as the limit for inclusion of a covariate. Separate Cox models were fitted with NPM staining pattern (granular versus not granular) instead of NPM intensity and with adjuvant chemotherapy, as well as adjuvant hormonal therapy, as covariates (no adjuvant versus adjuvant). To test for interaction, product terms between NPM staining pattern and chemotherapy and hormonal therapy, respectively, were added to the models. The assumption of proportional hazards was ascertained with complementary log plots. NPM expression level and localization varies among different normal organs and between embryonic and adult tissues.34Yun J.P. Miao J. Chen G.G. Tian Q.H. Zhang C.Q. Xiang J. Fu J. Lai P.B. Increased expression of nucleophosmin/B23 in hepatocellular carcinoma and correlation with clinicopathological parameters.Br J Cancer. 2007; 96: 477-484Crossref PubMed Scopus (72) Google Scholar We therefore analyzed expression of NPM in 14 histologically normal breast tissue samples originating from breast reduction surgery. NPM was expressed at high levels (scored 3) in the luminal epithelial cells of all samples. Two different locations were observed: uniform nuclear localization (10/14, 71%; Figure 1A) and nucleolar localization (4/14, 29%; Figure 1B). c-Kit35Simon R. Panussis S. Maurer R. Spichtin H. Glatz K. Tapia C. Mirlacher M. Rufle A. Torhorst J. Sauter G. KIT (CD117)-positive breast cancers are infrequent and lack KIT gene mutations.Clin Cancer Res. 2004; 10: 178-183Crossref PubMed Scopus (91) Google Scholar (Figure 1, C and D) and cytokeratin-7 (Figure 1F) staining verified the luminal epithelial type of the NPM-positive cells. The two different localizations correlated with age at time of surgery (≤42 versus >42 years, P = 0.001), suggesting a possible hormonal regulation of NPM expression. We therefore quantified NPM expression levels from the histologically normal breast tissue samples using qPCR analysis. We were able to obtain RNA for this analysis from 7/14 samples; of these, 4 samples were from women ≤42 years of age and 3 samples were from women >42 years of age (Figure 1G). The qPCR analysis revealed that the younger women expressed approximately twofold more NPM than the older women (Figure 1G). This difference in expression was detected also at the protein level in the IHC stainings (Figure 1, A and B). To further study whether the expression level of NPM could affect its localization, we infected MDA-MB-361 breast carcinoma cells, which normally show nucleolar NPM staining, with lentiviruses encoding NPM-GFP. NPM overexpression in these cells affected its localization. Cells that overexpressed NPM displayed a uniform NPM staining throughout the nuclei (Figure 1, H and I), supporting the idea that the nucleolar localization of NPM in the histologically normal breast samples might be due to reduced NPM protein levels. To investigate whether the change in NPM expression in the luminal epithelial cells of histologically normal breast tissue was due to estrogen regulation, we treated serum-starved ER+ (MDA-MB-361 and MCF7) and ER− (MDA-MB-231 and MDA-MB-436) human breast cancer cells with 10 nmol/L 17β-estradiol for 3 days, followed by extraction with NP-40 buffer and analysis of NPM protein levels using Western blot. NP-40 extraction solubilizes cytoplasmic and nucleoplasmic but not nucleolar NPM, and allows analysis of extranucleolar NPM levels. The 17β-estradiol treatment led to approximately 2.5-fold and 2.0-fold up-regulation of NPM protein levels in the ER+ MDA-MB-361 and MCF-7 cells, respectively. No effect was detected in the ER− cell lines (MDA-MB-231 and MDA-MB-436), indicating regulation of NPM protein levels by estrogen (Figure 2, A and B). In agreement with results obtained from the qPCR analysis and with NPM overexpression in the MDA-MB-361 cells, increased NPM levels in the 17β-estradiol-treated MCF-7 and MDA-MB-361 cells showed more NPM immunoreactivity in the nucleoplasm, compared with the vehicle DMSO-treated control cells (Figure 2, C–F). We determined the number of cells displaying even nuclear staining (no visible nucleoli) and nucleolar staining from the microscopic images of MCF-7 and MDA-MB-361 cells. Even nuclear staining was significantly more frequent in the 17β-estradiol-treated cells (62.6% in MCF-7 and 60% in MDA-MB-361 cells) than in the vehicle (DMSO) treated co
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