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1 Dipartimento di Biologia e Patologia Cellulare e Molecolare, University ‘Federico II’ c/o Istituto di Endocrinologia ed Oncologia Sperimentale, Consiglio Nazionale delle Ricerche, Naples, Italy; 2 Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio; 3 Endocrinology and Metabolism Division, University of California at Los Angeles School of Medicine, Los Angeles, California; and 4 Cancer Discovery, Astra Zeneca Mereside, Alderley Park, Macclesfield, Cheshire, United Kingdom
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Targeting TKs’ enzymatic activity by small molecule inhibitors is a promising strategy in human cancer therapy (4)
. Several molecules are being successfully used in clinical trials, and one of them, STI571 (imatinib mesylate or Gleevec), is now a standard for the treatment of BCR-ABL-positive chronic myeloid leukemia (5)
. We have identified TK inhibitors belonging to two different chemical classes, the pyrazolopyrimidine PP1 and the 4-anilinoquinazoline ZD6474, with a strong activity toward RET kinase (IC50 
100 nM; Refs. 6
, 7
).
In this work, we have exploited a panel of newly characterized human PTC cell lines, spontaneously harboring the RET/PTC1 translocation, to get insights in biological effects of RET/PTC oncogene and consequences of RET/PTC chemical blockade. We show that RET/PTC reversibly reduces p27Kip1 and increases cyclin D1 and E levels through MAPK signaling in transformed thyroid cells. RET/PTC blockade opposes this pathway leading to G1 arrest.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
NPAs are RET/PTC-negative papillary thyroid carcinoma cells, whereas FB-2 and TPC1 are papillary thyroid carcinoma cell lines harboring the RET/PTC1 oncogene (8)
. BHP2-7, BHP5-16, BHP7-13, BHP10-3, BHP14-9, and BHP17-10 cell lines were established from six different PTC patients as described elsewhere (9)
. All human cell lines were grown in RPMI medium supplemented with 10% FCS (Life Technologies, Inc., Paisley, PA) and antibiotics. PC Cl 3s are normal rat thyroid follicular cells and were grown in Coon’s modified Ham’s F-12 medium supplemented with 5% FCS and a mixture of hormones as described previously (10)
. PC-RET/PTC1 and RET/PTC1(Y1062F) are mass populations of PC Cl 3 cells transfected with RET/PTC1 or its Y1062F mutant (11)
. The P5 primary culture of normal human thyroid follicular cells was a kind gift of Francesco Curcio.
Nucleic Acids Extraction and Analysis.
DNA extraction and Southern blot were performed according to standard procedures. RNA was isolated by the RNeasy kit (Qiagen, Crawley, West Sussex, United Kingdom) and subjected to on-column DNase digestion with the RNase-free DNase set (Qiagen) following manufacturer’s instructions. Random-primed first-strand cDNA was synthesized in a 50-µl reaction volume starting from 2 µg of RNA by using the GeneAmp RNA PCR Core kit (Applied Biosystems, Warrington, United Kingdom). PCR amplification was performed using the GeneAmp RNA PCR Core kit system. To exclude DNA contamination, each PCR reaction was also performed on untranscribed RNA. Levels of ß-actin transcripts were measured by reverse transcription-PCR (forward: 5'-TGCGTGACATTAAGGAGAAG-3' and reverse: 5'-GCTCGTAGCTCTTCTCCA-3') for normalization. PCR products were loaded on 2% agarose gel, stained with ethidium bromide, and the image saved by the Typhoon 8600 laser scanning system (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). The amplimers for RET/PTC1 were as follows: 5'-ATTGTCATCTCGCCGTTC-3' (forward, nucleotides 145–162) and 5'-CTTTCAGCATCTTCACGG-3' (reverse, nucleotides 434–451). To establish the expression levels of p27Kip1 mRNA, RNA (10 µg) was denatured and resolved by electrophoresis in a 1% (w/v) agarose/formaldehyde gel and transferred to Nylon filters (Hybond-N; Amersham Pharmacia Biotech). A [
-32P]dATP-labeled full-length human p27Kip1 cDNA (11)
was used as a probe. Equal loading was verified by ethidium bromide staining of 18S and 28S rRNA.
Fluorescence in Situ Hybridization Analysis.
The P1 probes used for dual color fluorescence in situ hybridization analysis were RMC10P013, corresponding to the RET gene, and 29F6, corresponding to the H4/D10S170 gene, as described previously (8)
. The RET probe was labeled with SpectrumGreen-dUTP and the H4 probe with SpectrumRed-dUTP (Vysis, Inc., Richmond, United Kingdom) using the Nick Translation kit (Vysis, Inc.). In addition, an centromeric probe specific for chromosome 10 (CEP-10; Vysis, Inc.) was used.
Western Blot Analysis.
Lysates containing the same amount of proteins, estimated by a modified Bradford assay (Bio-Rad, Munchen, Germany), were immunoprecipitated with the required antibody or immunoblotted. Immune complexes were detected by enhanced chemiluminescence kit (Amersham Pharmacia Biotech). Anti-RET is a polyclonal antibody raised against the TK protein fragment of human RET, anti-pY1062, anti-pY1015, and anti-pY905 are affinity-purified polyclonal antibodies raised against RET peptides containing phosphorylated Y1062, Y1015, or Y905 (6
, 7)
. Anti-phosphotyrosine (4G10) is a mouse monoclonal antibody from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-MAPK (no. 9101) and anti-phospho-MAPK (no. 9102) were from New England Biolabs (Beverly, MA). Polyclonal and monoclonal antibodies anti-cyclin E (M-20), anti-p21Waf1 (C-19), anti-cyclin D1 (A-12), and anti-Shc (H-108) were from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal anti--tubulin was from Sigma Chemical Co., and monoclonal anti-p27Kip1 was from Cell Signaling (Beverly, MA). Secondary antibodies coupled to horseradish peroxidase were from Amersham Pharmacia Biotech.
Antisense p27Kip1 Oligonucleotides.
p27Kip1 antisense and mismatch control oligonucleotides (Ref. 11
; final concentration, 200 nM) were heated for 5 min at 65°C in serum-free medium, then mixed with oligofectamine reagent (Invitrogen Corporation, Carlsbad, CA) and added to the cells according to manufacturer’s instructions. After 4 h, fresh medium was added to each dish, and the cells were incubated in the absence or presence of RET/PTC kinase inhibitors for 48 additional hours before harvesting proteins or measuring bromodeoxyuridine (BrdUrd) incorporation.
BrdUrd Incorporation.
DNA synthesis was measured by the 5'-bromo-3'-deoxyuridine Labeling and Detection kit from Boehringer Mannheim. Cells were seeded on glass coverslips and treated or not with the compounds for 24 h. Then, cells were incubated for 1 h with BrdUrd (final concentration of 10 µM), fixed with paraformaldehyde (4%), and permeabilized with Triton X-100 (0.2%). Coverslips were incubated with anti-BrdUrd mouse monoclonal antibody and with a Texas red-conjugated antimouse antibody (Jackson ImmunoResearch Laboratories, Philadelphia, PA). All coverslips were counterstained in PBS containing Hoechst 33258 (final concentration, 1 mM; Sigma Chemical Co.), rinsed in PBS, and mounted in Moviol on glass slides. The fluorescent signal was visualized with an epifluorescent microscope (Axiovert 2, Zeiss; equipped with a x100 lens) interfaced with the image analyzer software KS300 (Zeiss).
Cell Cycle Analysis.
Cells were grown to subconfluence and subjected or not to RET kinase inhibitors treatment for extra 12 or 36 h. After harvesting, cells were fixed in cold 70% ethanol in PBS, then washed with PBS. Propidium iodide (50 µg/ml) was added to the cells in the dark, and samples were analyzed with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) interfaced with a Hewlett Packard computer (Palo Alto, CA).
Luciferase Assay.
Luciferase assay was performed as described previously (12)
. The reporter plasmid was the –1745CD1LUC, containing an 1882-bp PvuI fragment of the human cyclin D1 promoter subcloned into the vector pA3LUC (13)
. A total of 5 x 105 cells was plated 48 h before transfection in 60-mm tissue culture dishes. The medium was changed to DMEM (Life Technologies, Inc.) containing 5% FCS. Three h later, calcium phosphate DNA precipitates were incubated with the cells for 1 h. DNA precipitates were removed, and cells were washed with serum-free DMEM and incubated with 15% glycerol in HEPES-buffered saline for 2 min. Finally, cells were washed with DMEM and incubated in complete medium; luciferase activity was determined 48 h after transfection by using an Autolumat LB 953 (EG&G, Berthold, Bad Wildbad, Germany). ZD6474 or U0126 treatment was performed 24 h before harvesting. Activity was reported as fold change with respect to untreated cells or cells transfected with the empty vector; results were the average ± SD of three independent experiments performed in duplicate.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows that BHP2-7, BHP7-13, and BHP10-3 cell lines expressed RET/PTC1. We digested genomic DNA from the various cell lines with EcoRI or BamHI and used as a probe a 1-kbp BglII-BamHI DNA fragment mapping in RET intron 11 (8)
. BHP2-7, BHP7-13, and BHP 10-3 cells showed extra bands corresponding to rearranged RET alleles (asterisks in Fig. 1B
). Finally, dual-color fluorescence in situ hybridization analysis showed fusion of H4 and RET signals consistent with H4-RET recombination events in BHP2-7, BHP7-13, and BHP10-3 (Fig. 1C
and data not shown).
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Treatment with two different RET kinase inhibitors (PP1 and ZD6474) elicited a strong reduction of DNA synthesis rate, measured by BrdUrd incorporation, in the five RET/PTC1-positive cell lines (Fig. 2A
and data not shown). These effects were specific because they were neither observed in BHP17-10 cells nor in normal thyroid follicular PC Cl 3 cells. Accordingly, flow cytometry analysis showed a sharp increase of the G0-G1 fraction and a marked reduction of the S fraction upon ZD6474 treatment of the five RET/PTC1-positive cell lines but not of BHP17-10 cells (Fig. 2B
and data not shown).
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and data not shown). ZD6474 treatment was also followed by reduced levels of G1 cyclins D1 and E (Fig. 3A)
. Identical findings were obtained upon PP1 treatment (data not shown).
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. After 6 h of treatment, cyclin D1 and E levels were already significantly decreased (Fig. 3B)
.
We used antisense oligonucleotides to investigate whether p27Kip1 was required for ZD6474-mediated G1 arrest. Incubation of BHP2-7, BHP10-3, or TPC1 cells with antisense p27Kip1 oligonucleotides, but not mismatch oligonucleotides, greatly attenuated ZD6474-mediated DNA synthesis inhibition (Fig. 3D
and data not shown) in parallel to reduced p27Kip1 protein levels (Fig. 3C
and data not shown). Identical findings were obtained upon PP1 treatment (data not shown). These findings indicate a critical role played by p27Kip1 in the G1 arrest that follows RET/PTC1 kinase blockade.
Interruption of RET/PTC1 Signaling to MAPK by ZD6474 and PP1.
Protein lysates were immunoblotted with antibodies specific for phosphorylated tyrosines 905, 1015, and 1062, three major autophosphorylation sites in RET (2)
. All of the five RET/PTC-positive cancer cell lines expressed p57 and p62 RET/PTC1 protein products constitutively phosphorylated on the three tyrosines (Fig. 4A
, left panel, and data not shown). In RET-transfected cells, phosphorylated tyrosine 1062 binds Shc, a docking protein implicated in triggering the Ras/MAPK cascade (14)
. We immunoprecipitated cell lysates with anti-Shc and blotted the immunocomplexes with anti-phosphotyrosine antibodies. Shc products were constitutively phosphorylated in all cell lines harboring RET/PTC but not in RET/PTC-negative cells (Fig. 4B
, left panel, and data not shown). Finally, MAPK (extracellular signal-regulated kinases 1 and 2) were constitutively phosphorylated in PTC cell lines but not in P5, a primary culture of normal human thyroid follicular cells (Fig. 4C
, left panel). In RET/PTC-negative cancer cell lines (see, for example, BHP17-10 in Fig. 4C
), constitutive phosphorylation of MAPK is likely explained by the expression of an oncogenically activated B-raf (V599E) allele.5
Indeed, activating point mutations in B-raf have been identified in a large fraction of human papillary thyroid carcinomas and cell lines. When mutated, the B-raf kinase activates MAPK. No overlap was observed between carcinomas harboring RET/PTC and B-raf mutations, suggesting that B-raf acts along the RET/PTC-B-raf-MAPK pathway in thyroid carcinoma cells (15)
.
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, right panels, and data not shown).
Interruption of RET/PTC1 Signaling Increases p27Kip1 Protein Stability.
Ras/MAPK signaling has been implicated in the regulation of p27Kip1 protein levels, directly and indirectly, by up-regulating cyclin D1/cdk4 and cyclin E/cdk2 complexes. We treated TPC1, BHP10-3, and BHP2-7 cells with the MEK1/2 inhibitor U0126. Parallel to reduction of MAPK phosphorylation and accumulation of cells in G1 (data not shown), p27Kip1 protein levels increased, although cyclin D1 and E levels decreased (Fig. 5A
and data not shown), pointing to a role for MAPK in regulation of cell cycle downstream RET/PTC. U0126 treatment resulted in increased levels of p27Kip1 and reduced levels of cyclin D1 also in BHP17-10 cells (Fig. 5A)
. This was consistent with the constitutive activation of MAPK, probably sustained by oncogenic activation of B-raf, also in BHP cell lines negative for RET/PTC.
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To understand level of p27Kip1 regulation, RNA was extracted from untreated and ZD6474- and U0126-treated cells and probed with a p27Kip1 cDNA. p27Kip1 mRNA levels were not changed upon ZD6474 (Fig. 5C)
or U0126 (data not shown) treatment, suggesting a posttranscriptional level of regulation. To analyze whether changes in protein stability accounted for p27Kip1 regulation, we used the protein synthesis inhibitor cycloheximide. Treatment with cycloheximide revealed a half-life for p27Kip1 of 
6 h in untreated BHP2-7 cells. In sharp contrast, p27Kip1 did not undergo degradation in cells treated with ZD6474 or U0126, even after 24 h of cycloheximide treatment, suggesting that chemical blockade of the RET/PTC-MAPK pathway increased p27Kip1 protein stability (Fig. 5D)
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To further address the mechanisms of RET/PTC-MAPK-dependent degradation of p27Kip1, we treated BHP2–7 cells with ZD6474 (Fig. 6A)
or U0126 (Fig. 6B)
for 24 h to induce p27Kip1 accumulation. Starting after 6 h of RET or MEK inhibitors withdrawal, a progressive reduction of p27Kip1 levels was observed. Such down-regulation was strongly slowed down by the addition of MG132, an inhibitor of the ubiquitin-proteasome pathway, indicating that, in BHP2-7 cells, p27Kip1 protein levels are kept under check by proteasome-mediated degradation. Taken together, these results indicate that in RET/PTC-positive thyroid cancer cells, MAPK decreases p27Kip1 expression by increasing the p27Kip1 protein turnover exerted by the 26S proteasome.
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. Transient expression of RET/PTC1, but not its Y1062F mutant, caused a significant up-regulation of the activity of the cyclin D1 promoter in PC Cl 3 cells, confirming that RET/PTC effects on cyclin D1 are mainly at the transcriptional level (Fig. 7B)
. Restoration of p27Kip1 protein levels was observed when PC-RET/PTC1 cells were treated with MG132 or U0126 (Fig. 7C)
, confirming that RET/PTC is able to promote proteasome-dependent p27Kip1 down-regulation through MAPK.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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An increased half-life of p27Kip1 protein was detected upon RET/PTC inhibition, whereas adoptive RET/PTC expression in normal thyrocytes caused a reduced half-life of p27Kip1 that was reversed by proteasome block. PTC cell lines exerted a RET/PTC-dependent constitutive phosphorylation of Shc and activation of MAPK. Upon RET/PTC blockade, this pathway was rapidly turned off. Furthermore, p27Kip1 accumulated parallel to MEK1/2 inhibition. Finally, expression of a RET/PTC mutant unable to stimulate MAPK in normal thyrocytes failed to cause p27Kip1 degradation. All together, these data indicate that MAPK triggering by RET/PTC accounts for p27Kip1 down-regulation. Activation of the MAPK pathway is a common finding in most PTC cell lines (Fig. 4C)
. Indeed, also those carcinomas that are negative for RET/PTC rearrangements often feature activating mutations in B-raf (15)
, a direct upstream regulator of MEK. Thus, the MAPK-p27Kip1 pathway could be a promising therapeutic target to achieve cytostatic effects in PTCs initiated by different (RET/PTC or B-raf) oncogenic events.
p27Kip1 protein degradation is a three-step process that requires phosphorylation at threonine 187, recognition by the F-box protein SKP2, ubiquitylation, and degradation by the 26S proteasome (19) . MAPK can affect p27Kip1 protein levels primarily by triggering cyclin E-Cdk2 that, in turn, phosphorylates p27Kip1 on T187 and promotes its degradation (19, 20, 21, 22, 23) . Up-regulation of cyclin D1/cdk4 secondary to MAPK-triggering by stimulating cyclin E transcription may contribute to p27Kip1 obstruction as well. In addition, MAPK activation, according to some studies (24 , 25) , could reduce p27Kip1 protein stability by directly phosphorylating it. Therefore, we speculate that the increase of p27Kip1 induced by RET/PTC kinase inhibitors could be secondary to: (a) abrogation of MAPK-mediated destabilization of p27Kip1; and (b) reduction of cyclin D1/cdk2 and cyclin E/cdk2 levels.
Our findings indicate that reduction of the levels of p27Kip1 protein is necessary, but they do not prove that it is sufficient for obstruction of p27Kip1 in thyroid cancer cells. Subcellular localization is another important level of regulation of p27Kip1. Up-regulated cyclin D/cdk complexes may sequester p27Kip1. Furthermore, interaction of p27Kip1 with JAB1/CNS5 or phosphorylation at serine 10 (26 , 27) promotes its export from the nucleus. Finally, phosphorylation of threonine 157 by PKB/AKT impairs p27Kip1 nuclear import (28, 29, 30) . It is feasible that RET/PTC triggers at least some these additional pathways that could therefore synergistically obstruct p27Kip1 by cooperating with MAPK-dependent down-regulation.
In conclusion, p27Kip1 down-regulation mediates subversion of the G1-S checkpoint in RET/PTC-positive thyroid carcinoma cell lines, the reversal of this phenomenon being critical for growth arrest mediated by RET/PTC kinase inhibitors. We hypothesize that failure in retrieving p27Kip1 expression or block of p27Kip1 activity at different levels such as for instance delocalization can cause escape of thyroid cancer cells from the therapeutic effects of RET/PTC chemical blockade.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Massimo Santoro, Dipartimento di Biologia e Patologia Cellulare e Molecolare, University ‘Federico II,’ via. S. Pansini 5, 80131 Naples, Italy. Phone: 39-081-7463056; Fax: 39-081-7463037; E-mail: masantor{at}unina.it
5 G. Salvatore and M. Santoro, unpublished observations. 
Received 12/15/03. Revised 3/11/04. Accepted 3/17/04.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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