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The Journal of Clinical Endocrinology & Metabolism 87(5):2150 –2159 Copyright © 2002 by The Endocrine Society
Isozyme-Specific Abnormalities of PKC in Thyroid Cancer: Evidence for Post-Transcriptional Changes in PKC Epsilon JEFFREY A. KNAUF, LAURA S. WARD, YURI E. NIKIFOROV, MARINA NIKIFOROVA, EFISIO PUXEDDU, MARIO MEDVEDOVIC, TAMAR LIRON, DARIA MOCHLY-ROSEN, AND JAMES A. FAGIN Division of Endocrinology and Metabolism (J.A.K., L.S.W., Y.E.N., M.N., E.P., J.A.F.), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; Department of Environmental Health (M.M.), University of Cincinnati, Cincinnati, Ohio 45267; and Department of Molecular Pharmacology (T.L., D.M.-R.), Stanford University School of Medicine, Stanford, California 94305 PKC isozymes are the major binding proteins for tumorpromoting phorbol esters, and PKC activity is abnormal in a number of different human cancers. Less is known about putative structural and functional changes of specific PKC isozymes in human neoplasms. A single-point mutation of PKC␣ at position 881 of the coding sequence has been observed in human pituitary adenomas and up to 50% of thyroid follicular neoplasms, and a rearrangement of PKC⑀ was reported in a thyroid follicular carcinoma cell line, suggesting that these signaling proteins may play a role in thyroid tumorigenesis. To explore this possibility, we examined thyroid neoplasms for mutations and changes in expression levels of
P
KC IS A SERINE-THREONINE kinase initially discovered on the basis of its activation in vitro by Ca2⫹, phospholipids, and diacylglycerol (1). PKC consists of a family of 12 isozymes that differ in their structure, cofactor requirements for activation, subcellular localization, and substrate specificity. PKC isozymes play a proven role in signal transduction pathways regulating cell growth (2–5), differentiation (6, 7), and apoptosis (8). The distribution of expression of PKC isozymes differs between cell types, as do their biological properties, allowing for considerable functional diversity (9). There has been considerable interest in the potential role of PKC isozymes in the multistage process of carcinogenesis (10, 11). This relates in part to the discovery that phorbol esters such as phorbol 12-myristate 13-acetate (PMA), which act by substituting for diacylglycerol as activators of PKC isozymes, also serve as powerful tumor promoters for mouse keratinocytes (12, 13). The skin cancer model illustrates some of the unresolved issues regarding PKC signaling in tumor development. Although PMA alone is not carcinogenic in skin, it increases the sensitivity for papilloma formation in response to other tumor initiators. PMA results in activation of most PKC isozymes; however, it is not clear which of these is primarily responsible for
Abbreviations: AC, Anaplastic carcinoma; FA, follicular adenoma; FC, follicular carcinoma; HRP, horseradish peroxidase; PC, papillary carcinoma; PMA, phorbol 12-myristate 13-acetate; SAOH, specific allele oligonucleotide hybridization; SSCP, single-strand conformation polymorphism analysis.
PKC⑀ or ␣. None of the 57 follicular adenomas, 26 papillary carcinomas (PCs), 7 follicular carcinomas, or the anaplastic carcinoma harbored the PKC␣ 881A>G mutation. Moreover, none of 15 PCs, 10 follicular adenomas, or 6 follicular carcinomas showed evidence of mutations of PKC⑀. However, 8 of 11 PCs had major isozyme-specific reductions of the PKC⑀ protein, which occurred through either translational or posttranslational mechanisms. These data indicate that post-transcriptional changes in PKC⑀ are highly prevalent in thyroid tumors and may play a significant role in their development. (J Clin Endocrinol Metab 87: 2150 –2159, 2002)
tumor promotion. For example, other PKC activators such as bryostatin and 12-deoxyphorbol-13-phenylacetate are either inactive or even inhibitory as tumor promoters in skin, which is thought to be due to a distinct profile of activation and down-regulation of the individual PKC isozymes (14 –17). Indeed, selective modulation of individual PKC isozymes in keratinocytes of transgenic mice resulted in quite distinct phenotypes. Whereas overexpression of PKC␦ decreased both incidence and promotion of skin tumors (18), overexpression of PKC⑀ under the control of the same promoter decreased papilloma burden and accelerated progression to carcinomas (19). The complexity of the system is compounded by the fact that chronic activation of PKC isozymes is followed by their down-regulation, making it difficult to conclude whether it is the unregulated activation or the subsequent loss of function that is important for tumor promotion. Although the expression and function of PKC isozymes have been studied in many cell types, their role in tumor initiation or progression is still conjectural. In general, there is a propensity for unregulated activation of PKC␣ and PKCII to promote transformation in vitro, whereas PKC␦ induces apoptosis (20). By contrast, the effects of PKC⑀ appear to vary according to the cell type or conditions of activation, with some reports implicating PKC⑀ in tumor promotion (21) and others in tumor suppression or apoptosis (22–25). Given the potential of PKC isozymes to regulate signal transduction pathways in ways that can either promote or
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inhibit transformation, it is plausible that somatic mutations that alter their function may occur during progression of human tumors including those of the thyroid. For example PKC (26, 27) and PKC␣ (28) have been reported to be overexpressed in thyroid neoplasms. Human thyroid neoplasms represent an appropriate model in which to explore the role of PKCs in tumorigenesis because they fit a paradigm of multistage tumor progression. Moreover, PKC isozymes act as both antagonists (29) and intermediates (5, 30) of the signaling network activated by TSH, the most significant thyroid cell growth and differentiation factor. Both the TSH receptor (31) and stimulatory GTP-binding regulatory protein of adenylyl cyclase (32) are targets of somatic activating mutations in benign thyroid neoplasms. The TSH receptor also couples to PLC (33) and induces PKC activity. Prevostel et al. (35) reported that a specific somatic mutation of PKC␣, originally discovered in invasive pituitary adenomas, was also present in 5 of 10 follicular thyroid neoplasms (35). This point mutation resulting in a glycine for aspartic acid substitution at position 294 did not appear to modify the enzymatic activity of the isozyme in vitro but was associated with altered subcellular distribution and greater growth potential in rat fibroblasts (36). This observation, which is of great potential significance, has not been verified in other thyroid tumor series. To our knowledge, the only other report of a spontaneously occurring somatic mutation of a PKC isozyme in human cancer is a complex rearrangement of PKC⑀ in a follicular thyroid carcinoma cell line (37). This mutation was identified by comparative genomic hybridization, followed by positional cloning of a locus on chromosome 2p21 found to be commonly amplified in thyroid neoplasms (28%), including the follicular thyroid cancer cell line WRO. The gene for PKC⑀ was found to lie within the 2p21 amplicon in WRO cells, and encoded for a C-terminal truncated form of the isozyme (amino acids 1–116). Functional studies of the truncated PKC⑀ demonstrated that it acted as a dominantnegative inhibitor of wild-type PKC⑀ translocation and conferred cells with resistance to apoptosis induced by a variety of stimuli (38). Moreover, the increase in survival was associated with a block in p53 induction and elevated MDM2 levels. Murine Double Minute Clone 2 (MDM2) is thought to function as an oncogene by forming heterodimers with p53 and targeting it to the proteasome for degradation (39). It is not known whether thyroid neoplasms are subject to similar or related PKC⑀ genetic defects. Here we explored a large
number of human thyroid neoplasms for mutations and changes in expression of PKC␣ and PKC⑀. Materials and Methods DNA isolation Isolation of DNA and RNA from frozen tissue. Human thyroid tissues were either collected at surgery and immediately frozen in liquid N2 or recovered from paraffin-embedded samples. Tissues were obtained through the Tissue Procurement Facilities of the Cedars-Sinai Medical Center and the University of Cincinnati General Clinical Research Center after appropriate informed consent. Whenever possible, samples from tumor and normal thyroid of the same patient were obtained. Tissues that were snap frozen in liquid N2 immediately after surgery were ground under liquid N2 and the DNA and RNA isolated using the guanadinium-CsCl procedure previously described (40). DNA and RNA concentrations were determined by absorbance at 260 nm. Isolation of DNA from paraffin-embedded tissue. Both normal and tumor tissues were carefully microdissected from the paraffin blocks. DNA was isolated from paraffin-embedded tissue as previously described (41).
Single-strand conformation polymorphism analysis (SSCP) SSCP analysis was performed using a previously reported method (42). Briefly, PCR mixtures were prepared with 200 ng genomic DNA [or 1.0 l cDNA reaction prepared as described (43)], 10 pmol of each primer, 100 m dNTPs, 1 Ci ␣32PdCTP, 0.5–2.0 mm MgCl2, 10 mm Tris HCl (pH 9.0), 50 mm KCl, and 1U Taq polymerase (Promega Corp., Madison, WI) in a final volume of 20 l. Amplifications were carried out for 35 cycles with annealing temperatures optimized for each primer pair. The reaction mixture was then diluted in DNA gel-loading buffer (95% formamide, 10 mm NaOH, 0.25% bromophenol blue, 0.25% xylene cyanol), denatured by incubating at 94 C for 5 min, placed on ice, and immediately loaded onto a 0.50%– 0.75% MDE (AT Biochem, Malvern, PA), 0.6⫻ TBE gel with or without 20 mm HEPES. Gels were run at 300 V for 12–18 h at room temperature. Autoradiography was performed with an intensifying screen at ⫺70 C for 4 – 48 h. Construction of positive controls for PKC␣ SSCP. We generated positive controls for the screening of genomic DNA for the PKC␣ A881G mutation, using an overlapping PCR approach. The genomic control for wild-type PKC␣ was obtained using the PKC␣-E primer pair to amplify genomic DNA (Table 1). The 722-bp genomic fragment containing part of PKC␣ exon 7, the entire intron 7, and part of exon 8 were cloned into the pCR vector using the TA cloning kit (Invitrogen, Carlsbad, CA). To convert the A at position 881 to a G, we first created fragment A using the sense primer from the PKC␣-E primer pair and the primer 5⬘TTCCTCGCCCCCTTCCGGAATGGGTACG3⬘ and the cloned PKC␣-E fragment as template. The latter primer converted the T to a C in the antisense strand. The fragment B was then created using the antisense primer from the PKC␣-E primer pair and the primer 5⬘GAAGGGGGCGAGGAAGGAAACATGGAAC3⬘. The latter primer converted the A to a G in the sense strand. The fragments A and B were then denatured, annealed, and amplified with the PKC␣-E primer pair. The resulting
TABLE 1. Primers used in PKC␣ mutation analysis
PKC␣-A PKC␣-E PKC␣-I PKC␣-J wt-PKC␣ probe mut-PKC␣ probe a
Primer sequences
Position in PKC␣ coding sequencea
GACCGACGACTGTCTGTAGA3⬘ TTCCTGTCTTCAGAGGGAC3⬘ 5⬘ TTCAGACAAAGACCGACGAC3⬘ 5⬘ ACTCAGGCAGAAATTCGA3⬘ 5⬘ GGCTTGTTAAACTTGCGGTGGTA3⬘ 5⬘ CTGCCTGAGTTCCATGTTTCCTTC3⬘ 5⬘ GTTGCTTAACCAAGAAGAAGG3⬘ 5⬘ ACTCAGGCAGAAATTCGA3⬘ 5⬘ AAGGGGACGAGGAA3⬘ 5⬘ AAGGGGGCGAGGAA3⬘
709 –728 956 –974 699 –718 900 –917 Intron 7 886 –909 828 – 848 900 –917 875– 888 875– 888
5⬘ 5⬘
Primer positions and amplified regions are according to the PKC␣ coding sequence (accession no. X52479).
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PCR product was then cloned into the pCR vector using the TA cloning kit and the sequence verified using an ABI sequencing machine. Sequencing analysis. Aberrantly migrating bands were excised from the gel and boiled in 200 l H20 for 10 min. The PCR products were then reamplified and sequenced using an ABI sequencing machine. Alternatively, PCR products were subcloned into the pCR vector using the TA cloning kit (Invitrogen) and the inserts sequenced using an ABI sequencing machine. At least six inserts for each PCR product were sequenced.
Specific allele oligonucleotide hybridization (SAOH) Genomic DNA from all samples was amplified by PCR as described above. Twenty microliters of the PCR mixture was denatured by heating at 94 C for 2 min in 100 l of 0.4 N NaOH ⫹ 25 mm EDTA. The samples were immediately placed on ice, mixed with 100 l of 2 m Tris HCl (pH 7.4), and then applied to a prewetted Hybond-N⫹H nylon membrane (Amersham, Piscataway, NJ) under vacuum using a slot blot apparatus. Target DNA was immobilized with UV cross-linking followed by a 30-min incubation at 80 C. The membranes were then incubated overnight at 42 C in 10 ml of hybridization buffer [0.25 m NaH2PO4, (pH 7.2), 1 mm EDTA, 7% SDS, 1% BSA, and 15% formamide] containing a 32 P-labeled oligonucleotide complementary to the mutant PKC␣ (see Table 1). The membrane was washed three times with 20 mm Na2HPO4, 1 mm EDTA, and 1% SDS. The same membrane was subsequently stripped by boiling in a 0.1% SDS solution and then rehybridized with the oligonucleotide complimentary to wild-type PKC␣ (Table 1).
Southern blot analysis Ten micrograms genomic DNA from paired papillary carcinomas (PC) and corresponding normal thyroid tissue were digested with EcoRI, electrophoresed through a 1.0% agarose gel, and transferred to a nylon membrane (Micron Separation Inc., Westborough, MA). The membrane was probed with the full-length (2.2 kb) human PKC⑀ cDNA obtained by NheI digestion of the PKC⑀/pBluebac expression vector, American Type Culture Collection, Manassas, VA) (44). The probe was labeled with 32P-dCTP by random priming (Stratagene, San Diego, CA).
Semiquantitative RT-PCR Two micrograms total RNA were reverse transcribed with 200 U Superscript reverse transcriptase (Life Technologies, Inc.-BRL, Grand Island, NY) in the presence of 2.5 m random 9-mer primers, 20 m dNTP for 60 min at 37 C, followed by a 5-min heat inactivation at 95 C. Two microliters of the cDNA reaction mixture were then used as a template in a duplex PCR reaction containing 1 m of the primer pairs for amplification of -actin (5⬘ATGATATCGCCGCGCTCGTCGTC3⬘ and 5⬘ CATGGCTGGGGTGTTGAAGGTCTC3⬘) and either PKC⑀ (PKC⑀-13, see Table 4) or PKC␣ (PKC␣-A, see Table 1), 20 m dNTP, and 1.5 mm MgCl2. Other components in the PCR reaction were as suggested by the manufacturer (Perkin-Elmer Corp., Boston, MA). The PCR conditions were 94 C for 45 sec, 56 C for 60 sec, and 72 C for 30 sec for 25 cycles, followed by a 5-min extension at 72 C. Control experiments indicated that at 25 cycles, amplification of PKC⑀, PKC␣, and -actin was within the linear phase. Reactions performed in the absence of reverse transcription yielded no product, indicating that the primer pairs used are specific to cDNA. The PCR products were electrophoresed through a 1.5% agarose gel, transferred to nylon membranes (Micron Separation Inc.), and the membrane probed with 32P-labeled oligos complimentary to -actin, PKC␣, or PKC⑀ PCR product. Band intensity was determined using a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) and used to calculate the percent of PKC⑀ or ␣ mRNA in the tumor vs. the corresponding normal tissue, after normalization with -actin.
Lysate preparation and Western blotting Total tissue lysates. Frozen thyroid tissue was placed in ice-cold buffer A [10 mm Tris-HCl (pH 7.5), 5 mm EDTA, 100 g/ml PMSF, 4 mm EGTA, 1 g/ml aprotinin, 5 g/ml E-64, 1 g/ml leupeptin, and 1 g/ml pepstatin] containing 1% Triton X-100 and homogenized with a polytron. The homogenate was then centrifuged at 10,000 ⫻ g for 15 min at
Knauf et al. • Abnormalities of PKC Isozymes in Thyroid Cancer
4 C, the supernatant collected, and the protein concentration determined. An equal amount of protein from each sample was then subjected to SDS-PAGE. Preparation of soluble and particulate fractions. Frozen thyroid tissue was placed in ice-cold buffer A and homogenized with a polytron. Soluble and particulate fractions were then separated by centrifugation at 100,000 ⫻ g for 1 h at 4 C. The supernatant (soluble fraction) was removed and the pellet resuspended in buffer A containing 1% Triton X-100. The Triton X-100 insoluble material was removed by centrifugation at 100,000 ⫻ g for 1 h at 4 C and the supernatant collected (particulate fraction). The distribution of the PKC isozymes in the various fractions was then analyzed by Western blotting. Western blot analysis. Protein from total cell lysate or soluble/particulate fractions was subjected to SDS-PAGE and Western blotting as described (45, 46). Briefly, blots were hybridized with antibodies to the indicated proteins and then with their corresponding species-specific horseradish peroxidase (HRP)-conjugated secondary IgG and visualized using ECL (Amersham Pharmacia Biotech Inc., Piscataway, NJ) as directed by the manufacturer. The images were captured using x-ray film or the Image Station (Eastman Kodak Co., New Haven, CT). To confirm similar loading between normal and tumor samples, the membranes were stained with Ponceau S before hybridization with antibodies (fractionated cell extracts) or reprobed with anti--actin IgG (total cell extracts). To quantify PKC⑀ levels in Western blots containing the fractionated cell extracts, multiple-exposure x-rays were taken and the one judged to be the most representative was scanned using the Image Station (Kodak). To quantify PKC⑀ levels in Western blots containing the total cell extracts, the Image Station was used to capture the image. In both cases the 1D software (Kodak) was used to determine band intensity. The percent of PKC⑀ or ␣ in the tumor vs. corresponding normal tissue was calculated, after normalization with -actin (total cell extracts) or Ponceau S staining (fractionated cell extracts).
Statistical analysis To test for correlation between PKC⑀ and MDM2, we first applied Spearman’s rank correlation. As an additional strategy, we counted the number of patients who had either discordant or concordant measurements of these two proteins (concordance was defined as both values being either above or below the average levels for each protein in all tumors). With this approach, if there was no significant correlation, the chance for these two proteins in any one patient to be discordant is the same as the chance that they will be concordant.
Results SSCP analysis of PKC␣
To amplify the region of PKC␣ harboring the 881A⬎G mutation, previously referred to as position 908 of the nucleotide sequence (28), we initially selected primer pair PKC␣-A (Table 1), used by Prevostel et al. (28) in their initial description of this structural defect. Despite varying PCR conditions and techniques, we were unable to obtain a PCR product of the predicted size using genomic DNA as template. However, we did obtain the appropriate product with thyroid tissue cDNA, suggesting that this region contained one or more introns. These experiments were conducted before the availability of information from the Human Genome Sequencing Database, and we therefore empirically designed alternative sets of primers to map the location of the introns. The primer pair PKC␣-E consistently gave a largerthan-predicted PCR product with genomic DNA, which allowed us to map and sequence a 516-bp intron between bases 821 and 822. This intron was subsequently confirmed by a Blat search of the PKC␣ coding sequence vs. the UCSC Human Genome Project Working Draft (http://genome. ucsc.edu/) (Table 2). In addition, the Blat analysis identified
Knauf et al. • Abnormalities of PKC Isozymes in Thyroid Cancer
an additional 43642-bp intron between bases 918 and 919. Using the sequence from the PKC␣-E PCR product, we designed two primer pairs, PKC␣-I and PKC␣-J, which were then used to amplify the target region of PKC␣ from genomic DNA. To validate the SSCP screening of genomic DNA for the position 881A⬎G substitution (amino acid 294), we generated controls by cloning a genomic fragment of the wildtype PKC␣ gene flanking position 881 into the pCR vector. In addition, an A-to-G substitution at 881 was generated by site-directed mutagenesis. Analysis of PCR products generated by either primer pairs I or J demonstrated that, under the SSCP conditions employed, we could distinguish between the wild-type and mutant PKC␣ at position 881 (Fig. 1A). Primer pairs I and J were subsequently used to screen genomic DNA of 62 thyroid tumors: 42 follicular adenomas (FAs), 12 PCs, 7 follicular carcinomas (FCs), and 1 anaplastic carcinoma (AC). A representative SSCP gel using primer pair J is shown in Fig. 1B. Here all tumor samples present a similar pattern, in contrast to the faster migrating band seen with the mutant DNA control. Only five tumors displayed suspect mobility shifts. These bands were excised from the gel, DNA extracted, and product reamplified and sequenced. In all five cases, a wild-type sequence was found. In addition, we di-
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rectly sequenced three additional PCR products from tumor samples with normal SSCP patterns, and all were wild type. SAOH analysis of PKC␣. Because of the discrepancy between our findings and those of the previously reported study (28), we elected to repeat the screen for the 881A⬎G substitution using another methodology. SAOH was performed on 29 of the 62 neoplasms examined by SSCP as well as an additional 15 FAs and 14 PCs. Altogether 49 thyroid neoplasms (31 FAs, 10 PCs, 7 FCs, and 1 AC) were examined by SAOH. An oligonucleotide probe containing the wild-type sequence of PKC␣ hybridized to PCR amplified tumor genomic DNA products from each of the 49 cases (Fig. 2A), whereas only the PKC␣ mutant controls hybridized to the probe containing the 881A⬎G substitution (Fig. 2B). These results confirm that the 881A⬎G mutation is not present in this series of thyroid neoplasms. Analysis of PKC⑀ by SSCP. To investigate the occurrence of point mutations of PKC⑀ in thyroid neoplasms, we examined 15 PCs, 10 FAs, and 6 FCs for point mutations in the V1 region (mutated in the WRO cell line) or the region containing the phosphate transfer domain and activation loop. We determined the intron/exon junctions of the PKC⑀ gene in these
FIG. 1. SSCP analysis of PKC␣ mutations in thyroid neoplasms. A, Validation of the controls used for the SSCP screening. Primer pairs PKC␣-I (lanes 1–2) and PKC␣-J (lanes 3– 4) were used to amplify the wild-type (Wt) and mutant (881A⬎G) templates in the presence of ␣P (32) dCTP. The PCR products were electrophoresed through an SSCP gel, dried, and exposed to X-film at ⫺70 C with intensifying screen. B, Representative autoradiogram of an SSCP gel containing ␣P (32) dCTP-labeled PCR products generated by amplification of the indicated tumor type using primer pair PKC␣-J. The gel contained 6 FAs, 7 PCs, and 5 FCs. The only PCR product displaying an aberrantly migrating band (lane 1) was generated from the control template containing the PKC␣ 881A⬎G mutation.
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FIG. 2. SAOH analysis of thyroid neoplasms. Representative autoradiogram of a membrane dotted with PCR products generated by amplification of 35 tumor samples or the indicated control templates using primer pair PKC␣-J. The membrane was hybridized with 32P-labeled oligonucleotide complementary to the PKC␣ 881A⬎G (left) or 32P-labeled oligonucleotide complementary to wild-type PKC␣ (right) (see Table 1). The arrows indicate the location of PCR products generated from the following templates: (N) wild-type PKC␣, (M) PKC␣ 881A⬎G mutation, and (N/M) 1:4 mixture of PKC␣ 881A⬎G to wild type. TABLE 2. Exon/intron junctions of the PKC␣ gene Exon no.
Exon size
Position in PKC␣ coding sequence
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
173 32 83 112 129 157 135 97 138 174 92 63 139 81 108 141 165
1–173 174 –205 206 –288 289 – 400 401–529 530 – 686 687– 821 822–918 919 –1056 1057–1230 1231–1322 1323–1385 1386 –1524 1525–1605 1606 –1713 1714 –1854 1855–2019
TABLE 3. Exon/intron junctions of the PKC gene Intron size
Exon no.
Exon size
Position in PKC coding sequence
3073 188444 145070 3918 41601 1036 516 43642 2665 3103 2805 902 31228 12801 1866 14895
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
348 64 160 35 86 130 143 97 200 174 155 139 189 147 147
1–348 349 – 412 413–572 573– 607 608 – 693 694 – 823 824 –966 967–1063 1064 –1263 1264 –1437 1438 –1592 1593–1731 1732–1920 1921–2067 2068 –2214
The information on the intron/exon junctions of PKC␣ was obtained by BLAT search of the PKC coding sequence vs. the UCSC Human Genome Draft Sequence (http://genome.ucsc.edu/).
two regions using a Blat search of the PKC⑀ coding sequence vs. the UCSC Human Genome Project Working Draft (http://genome.ucsc.edu). This showed that the V1 region was part of a single exon, whereas the phosphate transfer domain and activation loop was encoded in four different exons (Table 3). With this information we designed primer pairs 1–5 within the respective flanking introns to amplify the indicated exons using genomic DNA as template (Table 4). Because the size of exon 1 (nucleotides 1–348) is beyond the optimal range for SSCP analysis, we used two overlapping primer pairs for this region. Sequencing of PCR products from shifted bands identified the following changes: a 1761C⬎T silent polymorphism in 7 of 31 samples, and an A-to-G substitution 23 bp into intron 1 in 10 of 31 samples. The 1761C⬎T substitution was confirmed to be a germline variation by sequencing corresponding normal tissue. The A-to-G intron substitution was identified as a single nucleotide polymorphism in the NCBI database. No other changes were found. To determine whether other regions of PKC⑀
Intron size
191546 133367 2389 1287 4171 16725 2997 2825 2684 75692 58732 5811 8378 24984
The information on the intron/exon junctions of PKC was obtained by BLAT search of the PKC coding sequence vs. the UCSC Human Genome Draft Sequence (http://genome.ucsc.edu/).
were mutated, we examined the entire coding sequence of PKC⑀ in 3 FCs and 13 PCs. Twelve overlapping pairs of primers spanning the entire length of the PKC⑀ coding sequence were used to screen these samples using cDNA as a template (Table 4). Besides additional confirmation of the 1761C⬎T polymorphism, no other changes were found. Analysis of PKC⑀ by Southern blotting. The mutation of PKC⑀ previously reported in WRO cells was a complex rearrangement resulting in expression of a truncated gene product (37). To investigate whether similar changes were present in PCs, Southern blots of EcoRI digested DNA from nine normal/ tumor pairs were probed with 32P-labeled full length PKC⑀ cDNA. None of the tumors exhibited restriction fragments that varied in size or intensity from those seen in the corresponding normal tissues (Fig. 3), indicating that large-scale rearrangements were not present. Analysis of PKC⑀ and ␣ in PC by Western blotting. In a previous report, PKC␣ was found to be elevated in human follicular thyroid neoplasms, compared with paired normal thyroid controls by Western blotting (28). There is also evidence that
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TABLE 4. Primers used in PKC mutation analysis Primer pair b
PKC-1a
Region amplifieda
Primer sequences 5⬘
3⬘
CGGGCCGTCGGTTCTTCATTC AGGCCGGGCTGTTGGTCTTCTG3⬘ 5⬘ CCCGGCCTGGCACGACGAGTT3⬘ 5⬘ CCGGAGCCCTACGATCAAGACCAG3⬘ 5⬘ AGAGAAGAGGCTACCTGTA3⬘ 5⬘ GTGGGCAGCTGCAATCAAATG3⬘ 5⬘ GGTCTTACCTCAGGAGCTATGTAG3⬘ 5⬘ CTGGCCTATCTTGCAGGGATTTG3⬘ 5⬘ GGGCAGTGACTCACAGCTTTCAAG3⬘ 5⬘ ATGGTGCCTGACATTGCTGGTTT3⬘ 5⬘ AACGTGCACGGGGACCAACTTA3⬘ 5⬘ GCTGGCCAGGCCTTTGTCACTA3⬘ 5⬘ CCCATAGGCTACGACGACTT3⬘ 5⬘ TGGGCTGCCGAAGATAGGTG3⬘ 5⬘ GAAGCCCCTAAAGACAATGAAGAG3⬘ 5⬘ ACGCAGGTGCAGACTTGACA3⬘ 5⬘ GGAAAGCAGGGATACCAGTGTCAA3⬘ 5⬘ GCCGCAAGAGTCCCCAGAGCAG3⬘ 5⬘ GGGGACTCTTGCGGCAGGGTTTG3⬘ 5⬘ GCGGGGACTCGGCACCAG3⬘ 5⬘ CAACAGCGGCCAGAGAAGGAAAAA3⬘ 5⬘ CTTGCCGGACTTCGCCATTCTCAC3⬘ 5⬘ AGGAGCACCGGGCAGCATCGTCT3⬘ 5⬘ TTCCGTGCCAGAGCCAAAAT3⬘ 5⬘ AGGATGATGACGTGGACTGC3⬘ 5⬘ TCGGGAGCGCTGAATCTGAAACA3⬘ 5⬘ GAGCCTCGTTCACGGTTCTATGC3⬘ 5⬘ CCCCAGGGCCCACCAGTC3⬘ 5⬘ CCTGCAGGAGTTGGAGTATGG3⬘ 5⬘ CAGGCGCTTGTGGGGATTCTT3⬘ 5⬘ AGCAGCACCCATTCTTCAA3⬘ 5⬘ GTGGGCTCTCAGGGCATCAGGTCT3⬘
1–193
5⬘
PKC-1bb PKC-2b PKC-3b PKC-4b PKC-5b PKC-6 PKC-7 PKC-8 PKC-9 PKC-10 PKC-11 PKC-12 PKC-13 PKC-14 PKC-15 a b
186 –348 1438 –1592 1593–1731 1732–1920 1921–2067 262–541 409 – 620 582– 805 791–991 942–1192 1121–1394 1337–1506 1516 –1781 1734 –1950 1988 –2214
Primer positiona
⫺72 to ⫺52 172–193 186 –206 Intron 1 Intron 10 Intron 11 Intron 11 Intron 12 Intron 12 Intron 13 Intron 13 Intron 14 262–281 522–541 409 – 432 601– 620 582– 606 784 – 805 791– 813 974 –991 942–965 1169 –1192 1121–1143 1375–1394 1337–1356 1484 –1506 1516 –1538 1765–1781 1734 –1754 1930 –1950 1988 –2006 2199 –3⬘UTR
Primer positions and amplified regions are according to the PKC coding sequence (accession no. X65293). Genomic DNA used as a template.
FIG. 3. Southern blot analysis of PKC⑀ in PCs. Southern blot containing 10 g EcoRI-digested DNA from paired normal (N) and PC tumor tissue samples (T). The blot was hybridized with a 32Plabeled full length PKC⑀ cDNA and exposed to x-ray film at ⫺70 C with intensifying screen.
the subcellular distribution of 881A⬎G PKC␣ is abnormal in Rat 6 cells in vitro. In addition, we have previously reported that expression of a truncated mutant of PKC⑀ containing the V1 domain of the protein abrogates activation-induced translocation of endogenous PKC⑀ (38). To determine whether similar alterations in abundance or localization of either PKC⑀ or PKC␣ were present in PCs, Western blots of par-
ticulate and soluble fractions from six normal/tumor paired specimens of thyroid PCs were sequentially probed with anti-PKC⑀ or anti-PKC␣ IgGs. As shown in Fig. 4A, there was no consistent difference in subcellular distribution of PKC⑀ or PKC␣ between the tumor and normal tissues from the same patients. However, in four of the six tumors, PKC⑀ expression was markedly reduced, compared with corre-
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Knauf et al. • Abnormalities of PKC Isozymes in Thyroid Cancer
FIG. 4. Western blot analysis of PKC␣, PKC⑀, and MDM2 in PC. A, Fifty micrograms of protein from either the soluble (S) or particulate (P) fractions of PC or corresponding normal thyroid tissue samples were electrophoresed in a 7.5% SDS-PAGE and transferred to a nitrocellulose membrane. B, Fifty micrograms of protein from total cell lysates of PCs or corresponding normal thyroid tissue were electrophoresed in a 7.5% SDS-PAGE and transferred to a nitrocellulose membrane. PKC⑀ and PKC␣ were detected using rabbit polyclonal anti-PKC⑀ anti-PKC␣ IgG, respectively (Santa Cruz Biotechnology, Santa Cruz, CA), and a HRP-conjugated goat antirabbit IgG. -Actin was detected using monoclonal anti--actin IgG (Santa Cruz Biotechnology) and a HRP-conjugated goat antimouse IgG. C, Membrane from panel B was reprobed with mouse monoclonal anti-MDM2 IgG (Santa Cruz Biotechnology) and a HRP-conjugated goat antimouse IgG.
sponding normal tissue (Fig. 4A and Table 5). In contrast, PKC␣ levels were similar or slightly higher in the six tumors (Fig. 4A, Table 5). To exclude potential confounding effects of losses during fractionation, Western blots of total lysates from an additional five normal/tumor pairs of PCs were examined. PKC⑀ levels were markedly reduced in four of five PCs, whereas only one of five had lower PKC␣ immunoreactivity (Fig. 4B and Table 5). Thus, altogether 8 of 11 PCs had markedly decreased levels of PKC⑀. To determine whether the reduced abundance of PKC⑀ was due to a decrease in PKC⑀ mRNA, RT-PCR was used to quantify PKC⑀ mRNA levels in seven PCs with reduced and two with normal PKC⑀ protein levels. As summarized in Table 5, tumors with reduced PKC⑀ protein did not have a corresponding reduction
in PKC⑀ mRNA. These results suggest that a translational, or more likely a posttranslational mechanism, is responsible for the reduced level of PKC⑀ protein seen in the PC tissues (Table 5). Increase in MDM2 expression is associated with reduced expression of PKC⑀. In a previous report, we showed that PKC⑀ may be involved in signal transduction pathways regulating MDM2 expression or stability (38). We investigated whether the reduction in PKC⑀ levels observed in the PCs was associated with changes in abundance of MDM2. Western blots containing total cell extracts demonstrated a 1.6- to 4-fold increase in expression of MDM2 in four of five PCs (Fig. 4C). Western blots containing insoluble and soluble fraction (data
Knauf et al. • Abnormalities of PKC Isozymes in Thyroid Cancer
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TABLE 5. Analysis of PKC␣ and in thyroid papillary carcinomas Western blots (% of normal)
Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient
1b 2b 3b 4b 5b 6b 7 8 9 10 11 12 13 14 15 16
RT-PCR (% of normal)
Mutational analysis of the PKC coding sequence
PKC
PKC␣
PKC
PKC␣
V1a
C4a
Remaining regions
6.2 3.5 5.2 43.8 101.6 90.6 56.1 40.6 9.7 45.9 91.8 ND ND ND ND ND
145.6 158.6 87.6 132.6 95.8 126.2 90.4 103.7 19.6 82.3 91.6 ND ND ND ND ND
92.8 71.6 86.2 ND ND 147.3 141.3 85.7 116.4 71.6 86.5 72.3 115.8 ND ND ND
80.4 116.0 71.6 ND ND 127.3 88.2 80.1 87.7 87.7 147.0 80.5 100.8 ND ND ND
Wt Polc Wt ND Wt Wt Wt Wt Wt Wt Wt Polc Wt Polc Wt Wt
Wt Wt Wt ND Pold Pold Wt Wt Wt Wt Wt Pold Wt Pold Wt Wt
Wt Wt Wt ND Wt Wt Wt Wt ND Wt Wt Wt Wt Wt ND Wt
Western blots for PKC and ␣ were performed as described. Membranes were stained with Ponceau S or rehybridized with -actin to normalize for difference in loading between the normal/tumor pairs. Quantification of PKC and ␣ mRNA levels in paired normal and tumor tissue was determined by semiquantitative RT-PCR using primers for -actin and either PKC or PKC␣. Values represent the percentage of PKC or ␣ in the tumor compared with normal tissue and were calculated as described in Materials and Methods. Wt, Wild type; ND, not determined. a Regions of the PKC cDNA analyzed included the following domains: V1, 1–363 bp, and C4, 1451–2082 bp. This latter region contains the activation loop and kinase domain. b Total PKC and ␣ levels were determined by combining the OD from autoradiograms of soluble and particulate fractions. c A-to-G polymorphism 23 bp into intron 1. d C1761T silent polymorphism.
not shown) identified an additional three of six PCs with elevated MDM2 levels. Interestingly, all tumors with elevated MDM2 expression also had reduced expression of PKC⑀. The inverse relationship between PKC⑀ and MDM2 levels was first examined with Spearman’s rank correlation. The correlation coefficient between PKC⑀ and MDM2 was ⫺0.57 with a P value of 0.0679, whereas the correlation coefficient between PKC␣ and MDM2 was ⫺0.45 with a P value of 0.1466. This was highly suggestive but not conclusive evidence for a negative correlation between PKC⑀ and MDM2 levels in thyroid papillary carcinomas. However, this statistical tool assumes a linear relationship, which may not be the case. To further investigate whether there is a negative correlation between PKC⑀ and MDM2, we counted the number of patients that had discordant levels of these two proteins (see Materials and Methods). When we categorized the data as described, 9 of 11 patients had discordant levels of PKC⑀ and MDM2. The probability of this happening under the assumption that concordant cases are equally probable as discordant ones (P value) is less than 0.01, supporting the negative correlation between PKC⑀ and MDM2 expression in thyroid papillary carcinomas. Analysis of PKC⑀ and ␣ in follicular adenomas. To determine whether follicular adenomas also had similar reduction in PKC⑀, Western blots of total lysates from normal/tumor pairs of FAs were performed. We found that two of the three neoplasms had reduced PKC⑀ protein levels (51.9% and 65.1%), compared with corresponding normal tissue. Fresh frozen samples of FCs were not available for Western blot analysis. Discussion
The purpose of this study was to explore the possible role of genetic or epigenetic changes in PKC␣ and ⑀ in thyroid
tumorigenesis. The rationale was based in part on the fact that PKC isozymes are positioned in the effector pathway of several known thyroid growth factors and oncogenes. In addition, a specific role for putative somatic mutations of PKC␣ has been proposed in thyroid follicular neoplasms. A role for gain-of-function mutations in PKC␣ was initially proposed by Megidish and Mazurek (47), who identified abnormal subcellular distribution of PKC␣ in UV-induced fibrosarcoma cell lines. PKC␣ cDNA from one of these cell lines was found to contain four somatic mutations leading to three amino acid substitutions in the regulatory domain and one in the catalytic domain (47– 49). Mutant, but not wildtype, PKC␣ evoked transformation of Balb/c 3T3 fibroblasts, although Borner et al. (50) were unable to reproduce these observations. In light of this, the identification of spontaneously arising somatic point mutations of PKC␣ in human pituitary and thyroid neoplasms was of major interest. In the case of human thyroid tumors, their presence in both benign and malignant neoplasms appeared to place PKC␣ mutations as one of the early events in thyroid tumor progression. One notable feature was the presence of an identical A-to-G transition at base pair 881 in all tumors, a rare occurrence in tumor genetics. Here we were unable to confirm the presence of the PKC␣ 881 mutation in any of the 91 thyroid neoplasms we tested with SSCP, SAOH, or by direct sequencing. We used multiple screening modalities because of initial difficulties in replicating the conditions used in the original report and for added verification. In the original report, the authors used primer pair PKC␣-A (Table 1) to generate genomic PCR products used to screen for mutations. However, we identified two introns within the region amplified by this primer pair (Table 2), suggesting that the Prevostel et al. study (28) was likely performed on cDNA. We cannot rule
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out the possibility that our inability to find the 881 mutation of PKC␣ is because of regional differences in prevalence of this anomaly. We did not specifically study autonomously functioning thyroid nodules or undifferentiated carcinomas, and it is conceivable, although unlikely, that mutations may be confined to these histotypes. Of all PKC isozymes, PKC⑀ has proven to be the most consistently transforming when transfected into murine fibroblasts (2, 3). When overexpressed in rat 6 embryonal fibroblasts, PKC⑀ produced malignant transformation in the absence of treatment with phorbol esters. In the presence of 12-O-Tetradecanoyl Phorbol 13-acetate, PKC⑀-transfected cells exhibited a rearranged actin cytoskeleton and were growth inhibited, probably due in part to interference of the overexpressed isozyme with the translocation and activation of other PKC isozymes. We focused our attention on the role of this isozyme in thyroid cancer following the discovery of a rearrangement in the V1 region of PKC⑀ in the thyroid carcinoma cell line WRO. This rearrangement encoded a truncated PKC⑀ (amino acid 1–116) that acted as a dominantnegative inhibitor of translocation of the wild-type form of the isozyme (38). However, we found no large-scale rearrangements in the PKC⑀ gene by Southern blotting of nine normal/tumor pairs of papillary carcinomas. In addition to the loss of function found in cells expressing the truncated V1 domain, others have shown that kinase-dead mutants of PKC⑀ (51) or mutations in the activation loop of PKC⑀ (52) also act as dominant-negative inhibitors. Because mutants in these three domains have functional consequences, we focused our screening efforts in these regions. The two SSCP conditions we employed (with and without 20 mm HEPES) have been reported to be up to 96% efficient in detecting point mutations (53). Although it is likely that our sensitivity may be lower than this, we believe that our inability to find mutations in the V1, kinase domain, or activation loop of PKC⑀ in 30 thyroid neoplasms indicates these regions are rarely if ever altered in thyroid neoplasms. Although we cannot exclude the possibility that other regions of PKC⑀ may occasionally harbor somatic changes, it seems unlikely because we found no mutations in the 3 FCs and 13 PCs in which the entire PKC⑀ coding sequence was examined. In this report, we demonstrate by Western blotting that PKC⑀ levels are significantly and often strikingly reduced in the majority of the thyroid PCs we examined. Our data suggest that the reduced expression of PKC⑀ is likely a posttranscriptional event affecting translation or stability of the PKC⑀ protein. Evidence for this includes: (1) comprehensive screening of the entire coding sequence in six PCs with reduced expression of PKC⑀ did not identify mutations altering the PKC⑀ amino acid sequence; and (2) lack of correspondence between PKC⑀ mRNA as detected by semiquantitative RT-PCR and immunoreactive PKC⑀ levels in papillary carcinomas. Of note, PKC␣ protein levels were not decreased in the majority of the tumors examined and indeed tended to be higher, as described initially by Prevostel et al. (28) in a small number of follicular neoplasms. Differences in cellular content or tissue architecture between normal and tumor tissues can alter protein representation in Western blots. However, this is unlikely to account for the reduction in PKC⑀ seen in the tumor tissues. As shown in Fig. 4, B and C,
Knauf et al. • Abnormalities of PKC Isozymes in Thyroid Cancer
all but one of the tumor samples had PKC␣ and MDM2 levels that were either similar or greater than those found in normal tissue. Furthermore, one would predict that if protein representation changes were due to differences in tissue architecture (i.e. more acellular colloid in normal samples), then similar alterations in mRNA levels would also be observed, which was not the case. The cause of the isozyme-specific decrease in PKC⑀ abundance remains to be clarified. However, one explanation may be that the presence of a sustained upstream activation signal leads to down-regulation of the isozyme. In this regard, we have recently observed that acute expression of the oncogenes RET/PTC1 or RET/PTC3 in rat thyroid PCCL3 cells results in isozyme-specific translocation of PKC⑀. Furthermore, a reduction in PKC⑀ levels is found in cells chronically expressing RET/PTC3 (Knauf, J.A., and J.A. Fagin, unpublished data). The MDM2 gene has been classified as an oncogene based on its behavior in human tumors (39, 54). Activating mutations of MDM2 are rare in cancers; however, overexpression of MDM2 is found in a wide variety of human tumors (55), including thyroid cancers (56). In a previous report, we found that expression of a dominant-negative PKC⑀ mutant protects thyroid cells from doxorubicin-induced apoptosis, which was associated with increased degradation of p53, likely because of higher MDM2 levels (38). Accordingly, here we demonstrate that in all seven PC with overexpression of MDM2, there was also a reduction in PKC⑀ levels. These data raise the possibility that PKC⑀ may either directly or indirectly regulate levels of MDM2 in thyroid cells, thus impacting the ability of cells to mount p53-mediated stress responses. We conclude that post-transcriptional changes resulting in decreased abundance of PKC⑀ are frequent in thyroid neoplasms. The significance of the isozyme-specific decrease in PKC⑀ abundance remains to be clarified. However, based on functional studies in thyroid cells in culture (38), we propose that decreased abundance of this PKC isozyme may promote tumor progression by prolonging cellular life span. Acknowledgments Received April 2, 2001. Accepted December 6, 2001. Address all correspondence and requests for reprints to: Jeffrey A. Knauf, Ph.D., Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, P.O. Box 670547, Cincinnati, Ohio 452670547. E-mail:
[email protected]. J.A.K. and L.S.W. contributed equally to this work. This work was supported in part by NIH Grants CA50706 and CA72597 (to J.A.F.), K01DK02781 (to J.A.K.), and M01-RR08084.
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