Complex Regulation of the Cyclin-Dependent Kinase Inhibitor p27kip1 in Thyroid Cancer Cells by the PI3K/AKT Pathway
2005; Elsevier BV; Volume: 166; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)62295-x
ISSN1525-2191
AutoresMaria Letizia Motti, Daniela Califano, Giancarlo Troncone, Carmela De Marco, Ilenia Migliaccio, Emiliano A. Palmieri, Luciano Pezzullo, Lucio Palombini, Alfredo Fusco, Giuseppe Viglietto,
Tópico(s)Ubiquitin and proteasome pathways
ResumoFunctional inactivation of the tumor suppressor p27kip1 in human cancer occurs either through loss of expression or through phosphorylation-dependent cytoplasmic sequestration. Here we demonstrate that dysregulation of the PI3K/AKT pathway is important in thyroid carcinogenesis and that p27kip1 is a key target of the growth-regulatory activity exerted by this pathway in thyroid cancer cells. Using specific PI3K inhibitors (LY294002, wortmannin, and PTEN) and a dominant active AKT construct (myrAKT), we demonstrated that the PI3K/AKT pathway controlled thyroid cell proliferation by regulating the expression and subcellular localization of p27. Results obtained with phospho-specific antibodies and with transfection of nonphosphorylable p27kip1 mutant constructs demonstrated that PI3K/AKT-dependent regulation of p27kip1 mislocalization in thyroid cancer cells occurred via phosphorylation of p27kip1 at T157 and T198 (but not at S10 or T187). Finally, we evaluated whether these results were applicable to human tumors. Analysis of 100 thyroid carcinomas indicated that p27kip1 phosphorylation at T157/T198 and cytoplasmic mislocalization were preferentially associated with activation of the PI3K/AKT pathway. Thus the PI3/AKT pathway and its effector p27kip1 play major roles in thyroid carcinogenesis. Functional inactivation of the tumor suppressor p27kip1 in human cancer occurs either through loss of expression or through phosphorylation-dependent cytoplasmic sequestration. Here we demonstrate that dysregulation of the PI3K/AKT pathway is important in thyroid carcinogenesis and that p27kip1 is a key target of the growth-regulatory activity exerted by this pathway in thyroid cancer cells. Using specific PI3K inhibitors (LY294002, wortmannin, and PTEN) and a dominant active AKT construct (myrAKT), we demonstrated that the PI3K/AKT pathway controlled thyroid cell proliferation by regulating the expression and subcellular localization of p27. Results obtained with phospho-specific antibodies and with transfection of nonphosphorylable p27kip1 mutant constructs demonstrated that PI3K/AKT-dependent regulation of p27kip1 mislocalization in thyroid cancer cells occurred via phosphorylation of p27kip1 at T157 and T198 (but not at S10 or T187). Finally, we evaluated whether these results were applicable to human tumors. Analysis of 100 thyroid carcinomas indicated that p27kip1 phosphorylation at T157/T198 and cytoplasmic mislocalization were preferentially associated with activation of the PI3K/AKT pathway. Thus the PI3/AKT pathway and its effector p27kip1 play major roles in thyroid carcinogenesis. Disruption of cell cycle control is frequent in human cancer.1Sherr CJ Cancer cell cycles.Science. 1996; 274: 1672-1677Crossref PubMed Scopus (5016) Google Scholar Dysregulation of cell proliferation and failure to suppress tumor growth often result from alterations in the activity of Cdk inhibitors.2Sherr CJ Roberts JM CDK inhibitors: positive and negative regulators of G1-phase progression.Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5202) Google Scholar Ink4 Cdk inhibitors are lost through deletion, point mutations, and/or promoter methylation in a variety of human neoplasms and are thus true tumor-suppressor genes.3Ortega S Malumbres M Barbacid M Cyclin D-dependent kinases, INK4 inhibitors and cancer.Biochim Biophys Acta. 2002; 1602: 73-87Crossref PubMed Scopus (665) Google Scholar Differently, the Cip/Kip Cdk inhibitor p27kip1 does not fit the classic tumor-suppressor paradigm because mutations in the gene encoding p27kip1 are rare.4Sgambato A Cittadini A Faraglia B Weinstein IB Multiple functions of p27Kip1 and its alterations in tumor cells: a review.J Cell Physiol. 2000; 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8: 1145-1152Crossref PubMed Scopus (686) Google Scholar, 14Viglietto G Motti ML Bruni P Melillo RM D'Alessio A Califano D Vinci F Chiappetta G Tsichlis P Bellacosa A Fusco A Santoro M Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27Kip1 by AKT/AKT-mediated phosphorylation in breast cancer.Nat Med. 2002; 8: 1136-1144Crossref PubMed Scopus (610) Google Scholar Here we report that the PI3K/AKT pathway is activated in ∼50% of thyroid carcinomas, and that this signaling cascade contributes to inactivation of p27kip1 through AKT-dependent cytoplasmic sequestration of p27kip1. Five human thyroid carcinoma cell lines were used in this study: TPC-1 and NPA (derived from PTCs), WRO (derived from FTCs), and FRO and FB1 (derived from anaplastic carcinomas).10Baldassarre G Belletti B Bruni P Boccia A Trapasso F Pentimalli F Barone MV Chiappetta G Vento MT Spiezia S Fusco A Viglietto G Overexpressed cyclin D3 contributes to retaining the growth inhibitor p27Kip1 in the cytoplasm of thyroid tumor cells.J Clin Invest. 1999; 104: 865-874Crossref PubMed Scopus (120) Google Scholar, 34Halachmi N Halachmi S Evron E Cairns P Okami K Saji M Westra WH Zeiger MA Jen J Sidransky D Somatic mutations of the PTEN tumor suppressor gene in sporadic follicular thyroid tumors.Genes Chromosom Cancer. 1998; 23: 239-243Crossref PubMed Scopus (117) Google Scholar All cell lines were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. LY294002 and cycloheximide were from Sigma-Aldrich (St. Louis, MO). Wortmannin was from Calbiochem (Merck KGaA, Darmstadt, Germany). Thyroid carcinomas were collected from the Surgery B section of the National Cancer Institute “Fondazione G. Pascale” (Naples, Italy) or retrieved from the files of the Department of Functional and Biomorphological Sciences at the University of Naples Federico II. Diagnosis was based on standard histological criteria. We selected paraffin blocks that were free of oxyphilic (Hurtle) changes, which are sources of aspecific cytoplasmic staining, and that included both the tumor and the rim of normal thyroid tissue around it; the latter serving as control of immunohistochemical staining. Serial sections were stained using monoclonal anti-p27kip1 antibody (dilution 1:4000; Transduction Laboratories, Lexington, KY) and the polyclonal antibody anti-phospho-AKT (Ser473) from Cell Signaling Technology (Beverly, MA) at a dilution of 1:250. After incubation with primary antibodies, the sections were incubated with biotinylated anti-mouse/rabbit immunoglobulins, and with peroxidase-labeled streptavidin (LSAB-DAKO, Glostrup, Denmark). Diaminobenzidine was used to visualize the signal. Sections incubated without the specific antibody and sections incubated with unrelated antibodies served as controls of the technique. Antibody specificity was assessed by competition with antigens used for antibody production (full-length p27kip1 and phospho-AKT-blocking peptides, respectively). Tumors were scored as p27kip1-positive or p27kip1-negative depending on a staining cutoff of 50%, as described elsewhere.10Baldassarre G Belletti B Bruni P Boccia A Trapasso F Pentimalli F Barone MV Chiappetta G Vento MT Spiezia S Fusco A Viglietto G Overexpressed cyclin D3 contributes to retaining the growth inhibitor p27Kip1 in the cytoplasm of thyroid tumor cells.J Clin Invest. 1999; 104: 865-874Crossref PubMed Scopus (120) Google Scholar p27kip1 expression was evaluated from both nuclear and cytoplasmic staining. p27kip1-positive tumors with cytoplasmic plus nuclear staining or with only cytoplasmic staining were designated “cytoplasmic” and tumors with only nuclear staining were designated “nuclear.” If 10% of cells were stained, the tumor was considered AKT-positive. For the 5-bromo-2′deoxyuridine-5′-monophosphate (BrdU) incorporation assay cells were grown to subconfluence on coverslips, incubated with 10 μmol/L BrdU for 2 hours, fixed in 3% paraformaldehyde, and permeabilized with 0.2% Triton X-100. We used Texas Red-conjugated secondary antibodies to reveal BrdU-positive cells, and fluorescein isothiocyanate-conjugated secondary antibodies to reveal p27kip1-positive cells. Cell nuclei were identified by Hoechst staining. Fluorescence was visualized with a Zeiss 140 epifluorescent microscope equipped with filters that discriminated between Texas Red and fluorescein. All assays were performed three times in duplicate. The antibodies used in this study were: anti-p27kip1 (Transduction Laboratories), anti-AKT and anti-phospho AKT (Ser473) (New England Biolabs, Lake Placid, NY), anti-phosphothreonine 157 (anti-P-T157),12Liang J Zubovitz J Petrocelli T Kotchetkov R Connor MK Han K Lee JH Ciarallo S Catzavelos C Beniston R Franssen E Slingerland JM AKT/AKT phosphorylates p27Kip1, impairs nuclear import of p27Kip1 and opposes p27Kip1-mediated G1 arrest.Nat Med. 2002; 8: 1153-1160Crossref PubMed Scopus (818) Google Scholar, 13Shin I Yakes FM Rojo F Shin NY Bakin AV Baselga J Arteaga CL AKT/AKT mediates cell-cycle progression by phosphorylation of p27Kip1 at threonine 157 and modulation of its cellular localization.Nat Med. 2002; 8: 1145-1152Crossref PubMed Scopus (686) Google Scholar, 14Viglietto G Motti ML Bruni P Melillo RM D'Alessio A Califano D Vinci F Chiappetta G Tsichlis P Bellacosa A Fusco A Santoro M Cytoplasmic relocalization and inhibition of the cyclin-dependent kinase inhibitor p27Kip1 by AKT/AKT-mediated phosphorylation in breast cancer.Nat Med. 2002; 8: 1136-1144Crossref PubMed Scopus (610) Google Scholar anti-phosphothreonine 198 (anti-P-T198),43Motti ML De Marco C Califano D Fusco A Viglietto G Akt-dependent T198 phosphorylation of cyclin-dependent kinase inhibitor p27(kip1) in breast cancer.Cell Cycle. 2004; 7: 1074-1080Google Scholar anti-phosphothreonine 187 (anti-P-T187), and anti-phosphoserine 10 (anti-P-S10) (Zymed Laboratories Inc., San Francisco, CA). Total proteins were prepared as described elsewhere.10Baldassarre G Belletti B Bruni P Boccia A Trapasso F Pentimalli F Barone MV Chiappetta G Vento MT Spiezia S Fusco A Viglietto G Overexpressed cyclin D3 contributes to retaining the growth inhibitor p27Kip1 in the cytoplasm of thyroid tumor cells.J Clin Invest. 1999; 104: 865-874Crossref PubMed Scopus (120) Google Scholar Nuclear or cytoplasmic proteins were extracted by lysing cells in ice-cold hypotonic buffer (0.2% Nonidet P-40, 10 mmol/L Hepes, pH 7.9, 1 mmol/L ethylenediaminetetraacetic acid, 60 mmol/L KCl). Nuclei were separated through a 30% sucrose cushion and lysed in hypertonic buffer (250 mmol/L Tris-HCl, pH 7.8, 60 mmol/L HCl). The immunoblotting procedure we used is described elsewhere.10Baldassarre G Belletti B Bruni P Boccia A Trapasso F Pentimalli F Barone MV Chiappetta G Vento MT Spiezia S Fusco A Viglietto G Overexpressed cyclin D3 contributes to retaining the growth inhibitor p27Kip1 in the cytoplasm of thyroid tumor cells.J Clin Invest. 1999; 104: 865-874Crossref PubMed Scopus (120) Google Scholar Cell cycle distribution was analyzed by flow cytometry as described previously.44Baldassarre G Barone MV Belletti B Sandomenico C Bruni P Spiezia S Boccia A Vento MT Romano A Pepe S Fusco A Viglietto G Key role of the cyclin-dependent kinase inhibitor p27kip1 for embryonal carcinoma cell survival and differentiation.Oncogene. 1999; 18: 6241-6251Crossref PubMed Scopus (46) Google Scholar In brief, cells were harvested in phosphate-buffered saline (PBS) containing 2 mmol/L ethylenediaminetetraacetic acid, washed once with PBS, and fixed for 30 minutes in cold ethanol (70%). Fixed cells were washed once in PBS and permeabilized with 0.2% Tween 20 and 1 mg/ml RNase A in PBS for 30 minutes. They were then washed once in PBS and stained with 50 μg/ml of propidium iodide (Roche, Basel, Switzerland). Stained cells were analyzed with a fluorescence-activated cell sorter (FACS) Calibur (Becton-Dickinson, Franklin Lakes, NJ), and the data were analyzed using a mod-fit cell cycle analysis program. Total cellular RNA was isolated from cultured cell lines as described previously.45Sambrook J Fritsch EF Maniatis T Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor1992Google Scholar For RT-PCR analysis, RNA extracted from thyroid cancer cell lines was digested with RNase-free DNase RQ1 (Promega). mRNA was reverse-transcribed using random hexamers as primers and Mo-MLV RT at 37°C for 10 minutes according to the Perkin-Elmer user manual. The PCRs were performed in the same tube using p27kip1- or actin-specific primers with 1.25 U Taq polymerase. Reactions were performed for 20 cycles as follows: 1 minute at 94°C for denaturation, 2 minutes at 55°C for annealing, and 2 minutes at 72°C for extension. Amplified DNA fragments were fractionated on a 2% polyacrylamide gel and hybridized with a 32P-labeled human p27kip1 probe. p27kip1 primers were chosen to amplify a full-length transcript resulting in a DNA fragment of 595 bp (forward: 5′-ATGTCAAACGTGCGAGTGTCTAAC-3′; reverse, 5′-ACGTTTGACGTCTTCTGAGGCCAG-3′). Human actin primers were chosen to amplify a cDNA fragment of 220 bp (forward 2329 to 2345, 5′-ACTTCGAGCAAGAGATG-3′; reverse 2611 to 2630, 5′-GCGGATGTCCACGGTCACACT-3′). All cDNA probes were radiolabeled with a random prime synthesis kit (Multi-Prime; Amersham Biosciences). Hybridization reactions were performed at 42°C in 50% formamide, 5% Denhardt's, 5× SSPE 0.2% sodium dodecyl sulfate (SDS), and 100 μg/ml of denatured sonicated salmon sperm DNA, with 2 × 106 cpm/ml of hybridization solution. Filters were washed at 60°C twice in 2× standard saline citrate, 0.2% SDS, twice for 30 minutes and subsequently, for the stringent washes, twice for 30 minutes each in 0.2× standard saline citrate, 0.1% SDS. AKT constructs,46Bellacosa A Testa JR Staal SP Tsichlis PN A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region.Science. 1991; 254: 274-277Crossref PubMed Scopus (805) Google Scholar the EGFP-PTEN construct,33Bruni P Boccia A Baldassarre G Trapasso F Santoro M Chiappetta G Fusco A Viglietto G PTEN expression is reduced in a subset of sporadic thyroid carcinomas: evidence that PTEN-growth suppressing activity in thyroid cancer cells is mediated by p27Kip1.Oncogene. 2000; 19: 3146-3155Crossref PubMed Scopus (130) Google Scholar and the wild-type p27kip1 construct44Baldassarre G Barone MV Belletti B Sandomenico C Bruni P Spiezia S Boccia A Vento MT Romano A Pepe S Fusco A Viglietto G Key role of the cyclin-dependent kinase inhibitor p27kip1 for embryonal carcinoma cell survival and differentiation.Oncogene. 1999; 18: 6241-6251Crossref PubMed Scopus (46) Google Scholar are described elsewhere. The p27kip1-T187A, p27kip1-T157A, p27kip1-T198A, p27kip1-S10A, and p27kip1-T157A/T198A mutants were generated by site-specific mutagenesis (Stratagene, La Jolla, CA)
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