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Fusion
Department of Molecular Medicine and Surgery, Karolinska University Hospital (T.F., G.W., J.G., L.F., W.-O.L., J.Z., C.L.), SE-171 76 Stockholm, Sweden; and Cancer Genetics Unit, Kolling Institute of Medical Research, University of Sydney, Royal North Shore Hospital (A.Y.M.A., R.C.-B., B.G.R.), Sydney, New South Wales 2065, Australia
Address all correspondence and requests for reprints to: Dr. Theodoros Foukakis, Department of Molecular Medicine and Surgery, Karolinska University Hospital, CMM L8:01, SE-171 76 Stockholm, Sweden. E-mail: theodoros.foukakis{at}ki.se.
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Abstract |
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Objective: The objective of this study was to investigate whether NORE1A is involved in follicular thyroid cancer (FTC) development.
Design: We analyzed NORE1A expression in 25 FTCs, eight follicular thyroid adenomas, and seven normal thyroid tissues by TaqMan quantitative RT-PCR. The results were evaluated in relation to RASSF1A expression, RAS mutations, and PAX8-PPAR
fusions assessed in the same material. NORE1A promoter methylation was assessed by the combined bisulfite restriction endonuclease assay.
Results: Although the NORE1A mRNA levels of the majority of the tumors were similar to those in the normal controls, the cases harboring a PAX8-PPAR translocation (n = 6) exhibited dramatically reduced NORE1A expression (P < 0.001). In contrast, RAS mutations (n = 5) and NORE1A down-regulation were mutually exclusive. A significant reduction in the expression of the NORE1A homolog and the bona fide tumor suppressor gene RASSF1A was observed, but with weak correlation to the respective NORE1A values. No NORE1A promoter methylation was detected in the 32 thyroid tumors analyzed.
Conclusions: Our experiments demonstrate the suppression of NORE1A, a known Ras effector, in PAX8-PPAR carrying FTCs.
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Introduction |
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NORE1 (RASSF5) is the founding member of the Ras association (RalGDS/AF-6) domain subfamily of proteins (RASSF1–6) that is characterized by its ability to bind selectively to active Ras and is considered to mediate tumor inhibitory effects (3). Two main isoforms of NORE1 are expressed from alternative transcription start sites and with alternate promoter usage, namely, NORE1A (RASSF5A) and NORE1B (RASSF5C) (4). NORE1A functions as a tumor suppressor in vitro and has been found to be underexpressed in a variety of cell lines and primary tumors (4, 5, 6). Moreover, promoter methylation has in several instances been proposed as the main mechanism for the observed reduction in NORE1A expression (4, 5, 6). RASSF1, another member of the RalGDS/AF-6 domain family of Ras effectors, shows almost 60% similarity to NORE1 at the protein level. It is known as a bona fide tumor suppressor that blocks cell cycle progression (7), mediates Ras-dependent apoptosis (8), and heterodimerizes with NORE1 (9).
Ras and its effectors are key players in human thyroid tumor development. Activating RAS point mutations are found at varying frequencies (0–85%) in all subtypes of thyroid tumors, including both benign and malignant forms (reviewed in Ref.10). Moreover, BRAF (B-type Raf kinase gene) mutations are often present in papillary thyroid cancer (PTC), varying from 36–69% in different series of adult patients (11). Reduced expression and promoter methylation of isoform A of RASSF1 have been reported in thyroid cancer cell lines and primary tumors, thus implicating RASSF1A as a tumor suppressor in thyroid (12, 13). To our knowledge, NORE1 has not been investigated in thyroid neoplasia. Another common genetic abnormality in follicular thyroid carcinoma (FTC) is the PAX8-PPAR fusion oncogene associated with a cytogenetic rearrangement t(2;3)(q13;p25) (14). It is found in 19–63% of FTCs in different series, but also occurs in follicular thyroid adenomas (FTA). Interestingly, activating RAS mutations and PAX8-PPAR translocation were reported to be distinct and nonoverlapping events in FTC (15).
The current study was undertaken to examine the role of NORE1A in FTC and explore its relation to RASSF1A expression, RAS mutation status, and PAX8-PPAR fusion. We show that NORE1A expression is significantly reduced in FTCs harboring a PAX8-PPAR fusion, is not coupled to promoter methylation, and occurs only in the context of wild-type RAS.
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Materials and Methods |
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The breast cancer cell line MCF-7 was provided by Dr. Dan Grandér (Karolinska Institute, Stockholm, Sweden). Cell culture was performed using standard reagents and procedures (16), and cells were harvested for DNA extraction. HeLa cells were in grown in DMEM (Invitrogen Life Technologies, Inc., Carlsbad, CA) supplemented with 10% fetal calf serum (Trace Biosciences, Castle Hill, Australia).
Tissue samples
The study includes 25 primary FTCs collected from patients who underwent thyroidectomy at Karolinska Hospital or Royal North Shore Hospital (Sydney, Australia). From seven of the FTCs treated with total thyroidectomy, histopathologically verified normal thyroid tissue was obtained from the contralateral lobe. For comparison, eight FTAs were also included. All tumors were classified according to the criteria of the World Health Organization committee (17). The tissues were snap-frozen in liquid nitrogen immediately after surgical removal and stored at –70 C until required. For each frozen sample used in this study, a representative section was cut and subjected to histopathological evaluation to confirm the high purity of tumor cells (>70%) in the tumor samples and the lack of neoplastic cells in the normal thyroid tissues. All samples were obtained with informed consent from the patients, and the study of tissue material was approved by the local ethics committee.
Extraction of DNA, RNA, and protein
High molecular weight DNA was extracted from all tumor and normal tissues and from the MCF-7 cell line using a standard method based on proteinase K digestion, phenol-chloroform purification, and ethanol precipitation. Total RNA was isolated from all tumors and normal thyroid tissues using the TRIzol reagent (Invitrogen Life Technologies, Inc.). After column purification with the RNeasy kit (QIAGEN, Valencia, CA) and deoxyribonuclease treatment (QIAGEN), the A260/A280 (interval, 1.9–2.1) of the RNA was determined by spectrophotometry. The integrity of the RNA was confirmed by the demonstration of sharp 28S and 18S bands at electrophoresis in denaturing agarose gels. Total protein was extracted from tumors FTC-2 and FTC-3 and from normal thyroid tissue of the same patients as well as from tumor FTC-17 using standard procedures.
Mutation screening of RAS genes
The mutation hot spot exons of the H-RAS, K-RAS, and N-RAS oncogenes were screened by direct DNA sequencing. Exons 1 and 2 of each gene were amplified using the Advantage-HF 2 PCR Enzyme System (BD Clontech, Palo Alto, CA) and the primers reported by Vasko et al. (10). The amplifications were performed using the following conditions: an initial denaturation step at 94 C for 1 min; 35 step cycles of denaturing at 94 C for 10 sec, annealing at 55–57 C for 10 sec, and extension at 72 C for 20 sec; and a final extension step at 72 C for 7 min. The PCR products were directly sequenced using Big Dye Terminator version 1.1 chemistry (Applied Biosystems, Foster City, CA) and analyzed on a 377XL automated DNA sequencer (Applied Biosystems). All suggested mutations were verified by repeated analysis and sequencing of both strands.
Assessment of PAX8-PPAR fusions
All tumor samples were previously screened for the presence of a PAX8-PPAR fusion (18, 19). To exclude the possibility of heterogeneity within a tumor, the presence or absence of the PAX8-PPAR transcript in the RNA sample that was used in the present study was confirmed by RT-PCR, using methods previously described (18). Furthermore, all tumors were analyzed by an interphase FISH (fluorescence in situ hybridization) split assay for PPAR that excluded translocations of PPAR with other fusion partners in the PAX8-PPAR-negative cases. An exception was tumor FTC-6, which was previously shown to carry a balanced translocation (3;7)(p25;q34) (20) and in which a CREB3L2-PPAR fusion has been detected (Lui, W.-O., unpublished observations; accession no. AY222643).
cDNA synthesis and quantitative real-time RT-PCR (qRT-PCR)
First-strand cDNA was synthesized from 1 µg of each purified RNA sample using MultiScribe reverse transcriptase primed with random hexamers (high-capacity cDNA archive kit, Applied Biosystems) according to the protocol recommended by the manufacturer. The gene expression levels of NORE1A and RASSF1A were then quantified using TaqMan technology on an ABI PRISM 7700 sequence detection system (Applied Biosystems). Gene-specific primers and probes were available as TaqMan gene expression assays (Applied Biosystems). For the NORE1, assay Hs00417514_m1 was used, which detects isoform A (accession no. NM_182663), but not isoform B (accession no. NM_182665). Similarly, assay Hs00945257_m1 was chosen as specific for RASSF1A analysis. The 18S ribosomal RNA was amplified and used as an endogenous control in the quantifications (assay Hs99999901_s1). All assays had a 6-carboxyfluorescein reporter dye at the 5' end of the TaqMan MGB probe and a nonfluorescent quencher at the 3' end of the probe. The qRT-PCR was performed in 25-µl reactions containing 1x TaqMan Universal Master Mix, 1x Target Assay Mix (Applied Biosystems), and 10 µl first-strand cDNA from each sample as a template, using MicroAmp optical 96-well plates covered with MicroAmp optical caps (Applied Biosystems). The thermocycling conditions were 2 min at 50 C, 10 min at 95 C, and 40 cycles of 15 sec at 95 C and 1 min at 60 C. All qRT-PCR experiments included a no-template control and were performed in duplicate. Serial dilutions of cDNA from normal thyroid tissue were amplified in parallel as a control of amplification efficiency within each experiment and for the establishment of a standard curve for relative quantification.
qRT-PCR data analysis
Initial analysis of the raw data was performed using the Sequence Detection System (SDS version 1.9.1, Applied Biosystems) following the manufacturer’s recommendations. The efficiency of each PCR was validated with the threshold cycle slope method described in detail previously (21). All reactions had amplification efficiencies higher than 90% in the samples’ quantities range. After arbitrary quantification in relation to the generated standard curves, the data were exported to Microsoft Excel (Redmond, WA) for spreadsheet analysis. The expression of 18S was consistent across the samples when equal starting amounts of RNA were used, indicating that it is a good endogenous control for our material. Moreover, no differences were seen in 18S expression among normal thyroid, FTA, and FTC, excluding this as a source of bias in the analysis. The expression values of NORE1A and RASSF1A were therefore normalized to the respective 18S values for each sample.
Western analysis
The NORE1 protein expression was determined in selected samples by Western analysis. Equal amount of total protein extracts from each sample were size fractionated in a polyacrylamide gel and subsequently transferred to a nitrocellulose membrane. The filter was incubated with a rabbit polyclonal antibody to NORE1 (ab16757, Abcam Ltd., Cambridge, UK) at a 1:750 dilution. ß-Actin was used as a loading control.
Combined bisulfite restriction endonuclease assay (CoBRA)
The NORE1A promoter methylation status was analyzed using CoBRA, according to previously published methodology (5). In brief, genomic DNA was treated with sodium bisulfite at 55 C for 16 h using a standard protocol, purified using Wizard DNA clean-up system (Promega Corp., Madison, WI), and desulfonated by adding NaOH to a final concentration of 0.3 M and incubating for 15 min at 37 C. After neutralization with 3 M ammonium acetate (pH 7) and ethanol precipitation, the DNA was eluted in water and stored at –70 C until use. The NORE1A promoter was amplified from the modified DNA using nested PCR with primers and conditions described previously (5). The PCR products were subjected to restriction endonuclease cleavage by TaqI (Invitrogen Life Technologies, Inc.) and visualized in 3% agarose gels. Cleavage of the initial PCR product (202-, 123-, and 10-bp fragments) indicated that the NORE1A promoter is methylated, whereas the lack of cleavage (single 335-bp fragment) corresponds to unmethylated promoter. DNA from MCF-7 cells was processed in parallel as a positive control for the methylated status (4).
Expression plasmids
The human NORE1A promoter (–261 to 159 bp relative to the transcription start site) was amplified from genomic DNA and cloned into the pGL3-basic vector (Promega Corp.). The presence and accuracy of the NORE1A promoter sequence upstream of the luciferase gene were confirmed by sequencing. The PAX8 and PPAR plasmids were provided by Dr. V. K. K. Chatterjee (University of Cambridge, Cambridge, UK), and the PAX8-PPAR fusion plasmid was provided by Dr. T. G. Kroll (University of Chicago, Chicago, IL).
Luciferase reporter assays
HeLa cells were grown to approximately 80% confluence in 24-well plates and transient transfected with 0–2 µg transcription factor expression plasmid (PAX8, PPAR, PAX8-PPAR, or pcDNA3 empty vector), 1 µg NORE1A luciferase promoter, and 1 µg ß-galactosidase plasmid. Cells were incubated overnight and replenished with normal growing medium with 5% calf serum. After 24 h, cells were lysed and assayed for luciferase reporter activity (Promega Corp.). ß-Galactosidase activity was used for normalization of reporter activity. The transcriptional response was expressed relative to that seen with empty vector, and data shown represent the mean activity of triplicate wells.
Statistical analysis
Log-transformed expression values of NORE1A were compared between groups of samples with different diagnoses, presence or absence of PAX8-PPAR fusion/PPAR rearrangement, and/or presence or absence of RAS mutation using unpaired t test. RASSF1A expression values were highly skewed and the nonparametric Mann-Whitney U test was applied for the same comparisons between groups. A paired t test was used for the comparison of RASSF1A expression in the matched FTC-normal thyroid samples. Finally, the correlation between NORE1A and RASSF1A expression was investigated by Spearman’s rank correlation statistic. All analyses were performed in STATISTICA data analysis software system version 6 (Statsoft, Inc., Tulsa, OK), and for all tests, P < 0.05 was considered significant.
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rearrangements, especially PAX8-PPAR. RAS mutations
Sequencing of exons 1 and 2 of H-, K-, and N-RAS revealed a total of five mutations in the 33 tumors analyzed (Table 1
). All mutations were present in heterozygous form and were previously characterized for their activating effect on Ras proteins. Four mutations were identified in N-RAS and one in K-RAS, whereas no mutations were found in H-RAS. Four of the cases harboring a RAS mutation were FTCs (four of 25; 16%) and one was an FTA (one of seven; 14%). FTC-7 exhibited a single base substitution (GGT
GTT) in codon 12 of K-RAS, predicted to give an amino acid shift from glycine to valine (G12V). The remaining four mutations were found in codon 61 of N-RAS. In FTC-8, the substitution (CAAAAA) resulted in an amino acid change from glutamine to lysine (Q61K). In the other three cases (FTC-9, FTC-10, and FTA-8) the same missense alteration (CAACGA) was found, resulting in a change from glutamine to arginine (Q61R). No overlap was seen between the tumors with a RAS mutation and the tumors with a PPAR rearrangement in either the FTC or FTA group (Table 1).
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-rearranged tumors
All FTCs, FTAs, and normal thyroid tissues were analyzed for NORE1A mRNA expression by TaqMan quantitative RT-PCR. The raw expression values obtained were quantified in relation to a standard curve and normalized to the respective expression levels of 18S rRNA, which was used as an internal control. The results for each case are summarized in Table 1
and are graphically illustrated in Fig. 1. Overall, the expression levels showed a larger variation in the tumors compared with the normal thyroid, a pattern that was particularly pronounced in FTCs. Compared with the presence of PPAR rearrangement and RAS mutations, several significant associations were observed. NORE1A mRNA expression was significantly lower in FTCs with a PAX8-PPAR translocation compared with FTCs without this rearrangement (P < 0.001) or with normal thyroid (P < 0.001). Notably, NORE1A expression was also clearly reduced in case FTC-6, which harbored a CREB3L2-PPAR fusion. Finally, FTA-7, the PAX8-PPAR+ FTA, showed reduced NORE1A expression, albeit not as pronounced as in the PAX8-PPAR + FTCs. No significant differences were noted between NORE1A expression in PAX8-PPAR – FTCs, the PAX8-PPAR – FTAs and the normal thyroid samples included in the study. Furthermore, the NORE1A expression tended to be higher in cases with a RAS mutation (Fig. 1). To confirm the qRT-PCR findings on the protein level, Western analyses were carried out in representative samples. As illustrated in Fig. 2, PAX8-PPAR + FTCs showed very low NORE1A protein levels, compared with their matching normal thyroid tissues and a PAX8-PPAR – FTC.
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Transfection of HeLa cells with a PAX8-PPAR construct did not change NORE1A promoter activity using a luciferase reporter assay (Fig. 3), in contrast with other promoters that are either up-regulated (sodium-iodide symporter gene) or down-regulated (thyroglobulin gene) by this PAX8-PPAR construct in similar assay conditions (22).
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RASSF1A expression was assessed in all tumors and normal thyroid samples. In general, significantly lower levels of RASSF1A mRNA were detected in FTCs compared with normal thyroid (P < 0.01; Fig. 4A). This finding was also substantiated by the clear differences in RASSF1A expression observed for the seven matched pairs of FTCs and normal thyroid tissues (P < 0.01; Fig. 4B). However, RASSF1A did not differ significantly between PAX8-PPAR– and PAX8-PPAR+ FTCs (P = 0.18), or between tumors with and without RAS mutations (P = 0.66). No significant correlation was observed between NORE1A and RASSF1A expression levels in the 25 FTCs (Spearman’s r = 0.22).
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Discussion |
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fusion oncogenes and RAS mutations are nonoverlapping events in FTC. To approach these issues, we investigated whether Ras could have a similar central role in FTC, either directly or mediated by its effectors. In the current study we focused on the tumor-suppressing Ras effectors NORE1A and RASSF1A.
Both RASSF1A and NORE1A have been proposed to possess tumor inhibitory properties, and their expression is reduced in various cancer cell lines and primary tumors. Using TaqMan quantitative RT-PCR, we showed that a subgroup of the follicular thyroid tumors studied exhibited dramatically reduced NORE1A expression compared with normal thyroid tissues. Notably, a strong correlation between the expression of NORE1A and the presence of a PAX8-PPAR fusion oncogene was seen. The very low levels of NORE1A expression detected at the mRNA level using qRT-PCR were also demonstrated at the protein level using Western analysis. Furthermore, the majority of the thyroid tumors had reduced levels of RASSF1A compared with normal thyroid. This is in accordance with previously published data (12, 13) and confirms the importance of RASSF1A as a tumor suppressor in FTC. However, no significant correlations were made between RASSF1A reduced expression and the other genetic events evaluated, indicating a more general role for RASSF1A, not confined to a particular genetic subgroup.
Previous studies have shown that the inactivation of RASSF1A (and occasionally of NORE1A) is coupled to epigenetic events, whereas point mutations are seldom reported. Accordingly, RASSF1A promoter methylation is found in the majority of the tumors analyzed here (Foukakis, T., and C. Larsson, unpublished observations). In contrast, the promoter of NORE1A was not methylated in any of the tumors, FTA or FTC, thereby excluding promoter methylation as a regulatory mechanism for NORE1A in follicular thyroid tumors.
Using a luciferase reporter transactivation assay, we could not show a direct effect of PAX8-PPAR on the NORE1A promoter, implicating that the suppressed NORE1A expression in tumors harboring the fusion gene may not be a result of direct transcriptional effects on the NORE1A gene. No mechanistic link between PAX8-PPAR and NORE1A could therefore be demonstrated. It is possible that PAX8-PPAR response elements were not included in the 500-bp NORE1A promoter that we used in our transfection studies. Alternatively, it is also possible that an effect of PAX8-PPAR on NORE1A gene expression may require some as yet unidentified cofactors not present in the in vitro model, or indeed, that down-regulated NORE1A expression is an indirect result of other PAX8-PPAR transcriptional effects.
NORE1 and RASSF1 contain a Ras association domain at their carboxyl-terminal ends, and their action is, at least in part, Ras dependent. To investigate a possible relationship of Ras with NORE1A and RASSF1A expression, we screened the same material for RAS mutations by direct DNA sequencing. In total, RAS point mutations were present in 15% of the tumors. No overlap between RAS mutations and NORE1A reduced expression was observed, in analogy with PTC where RAS and BRAF mutations and RET/PTC rearrangements are essentially mutually exclusive. Taken together with our observation of NORE1A being indispensably and exclusively down-regulated in PAX8-PPAR + cases, our data support a model of follicular thyroid oncogenesis in which Ras pathways are targeted in different ways, either directly by RAS mutation or indirectly by NORE1A down-regulation after a PAX8-PPAR fusion.
To date, the pathways that lead to tumor formation due to a PAX8-PPAR fusion have not been clarified. Mechanistic in vitro experiments have shown that the PAX8-PPAR oncogene leads to transformation and increased cell growth due to increased cell cycle transit and decreased apoptosis (23). Interestingly, both cell cycle arrest and induction of apoptosis have been suggested as mechanisms of the tumor suppressor action of NORE1A (24). Considering the reduced NORE1A expression in the presence of the PAX8-PPAR fusion reported in this study, it seems plausible to assume a role for NORE1A in PAX8-PPAR signaling.
In conclusion, RAS mutations and PAX8-PPAR fusions are mutually exclusive events present in a considerable proportion of FTC, as shown both in this study and previously (15). We also demonstrated suppression of NORE1A, a known Ras effector, in PAX8-PPAR-carrying FTCs.
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Acknowledgments |
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Footnotes |
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Abbreviations: BRAF, B-type Raf kinase; CoBRA, combined bisulfite restriction endonuclease assay; FTA, follicular thyroid adenoma; FTC, follicular thyroid carcinoma; PPAR, peroxisomal proliferator-activated receptor ; PTC, papillary thyroid cancer; qRT-PCR, quantitative real-time RT-PCR.
This work was supported by grants from the Swedish Cancer Foundation, the Vera and Emil Cornell Foundation, the Åke Wiberg Foundation, the Nilsson-Ehle Foundation, the Cancer Society in Stockholm, the Swedish Society for Medical Research, the Knut and Alice Wallenberg Foundation, and the Stockholm County Council.
Received June 20, 2005.
Accepted December 7, 2005.
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References |
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1 fusion oncogene in human thyroid carcinoma. Science 289:1357–1360 rearrangement in thyroid tumors: evidence for distinct molecular pathways in thyroid follicular carcinoma. J Clin Endocrinol Metab 88:2318–2326 rearrangement in follicular thyroid tumors. J Clin Endocrinol Metab 88:4440–4445 fusion oncogene in both follicular thyroid carcinomas and adenomas. J Clin Endocrinol Metab 88:354–357 (PPAR) disrupts normal PAX8 or PPAR transcriptional function and stimulates follicular thyroid cell growth. Endocrinology 147:367–376 fusion oncoprotein transforms immortalized human thyrocytes through a mechanism probably involving wild-type PPAR inhibition. Oncogene 23:3634–3641[CrossRef][Medline]This article has been cited by other articles:
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  T. Foukakis, A. Gusnanto, A. Y. Au, A. Hoog, W.-O. Lui, C. Larsson, G. Wallin, and J. Zedenius A PCR-based expression signature of malignancy in follicular thyroid tumors Endocr. Relat. Cancer, June 1, 2007; 14(2): 381 - 391. [Abstract] [Full Text] [PDF]
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