Human Molecular Genetics Advance Access originally published online on November 30, 2005
Human Molecular Genetics 2006 15(1):97-103; doi:10.1093/hmg/ddi431
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Distinct expression profile in fumarate-hydratase-deficient uterine fibroids
1Department of Medical Genetics, PO Box 63 (Haartmaninkatu 8), Biomedicum Helsinki, 2Department of Obstetrics and Gynaecology, PO Box 22 (Haartmaninkatu 2), 3Department of Clinical Genetics, PO Box 140 (Haartmaninkatu 2B) and 4Department of Pathology, PO Box 21 (Haartmaninkatu 3), University of Helsinki, FIN-00014 Helsinki, Finland, 5Molecular and Population Genetics Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, UK and 6Molecular Diagnostic Laboratory, Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark
* To whom correspondence should be addressed. Tel: +358 91911; Fax: +358 919125105; Email: lauri.aaltonen{at}helsinki.fi
Received August 31, 2005; Accepted November 17, 2005
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ABSTRACT |
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Defects in mitochondrial enzymes predispose to severe developmental defects as well as tumorigenesis. Heterozygous germline mutations in the nuclear gene encoding fumarate hydratase (FH), an enzyme catalyzing the hydration of fumarate in the Krebs tricarboxylic acid cycle, cause hereditary leiomyomatosis and renal cell cancer; yet the connection between disruption of mitochondrial metabolic pathways and neoplasia remains to be discovered. We have used an expression microarray approach for studying differences in global gene expression pattern caused by mutations in FH. Seven uterine fibroids carrying FH mutations were compared with 15 fibroids with wild-type FH. The two groups showed markedly different expression profiles, and multiple differentially expressed genes were detected. The most significant increase in FH mutants was seen in the expression of carbohydrate metabolism- and glycolysis-related genes. Other significantly up-regulated gene categories in FH mutants were, for example, iron ion homeostasis and oxidoreduction. Genes with lower expression in FH-mutant fibroids belonged to groups such as extracellular matrix, cell adhesion, muscle development and cell contraction. We show that FH mutations alter significantly the expression profiles of fibroids, most strikingly increasing the expression of genes involved in glycolysis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALREFERENCES |
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Life is fundamentally based on order maintained by metabolism (1
), an important part of which in eukaryotic cells is carried out in mitochondria. A key mitochondrial metabolic pathway that maintains the constant flow of order from nutrients into cellular functions is the Krebs tricarboxylic acid (TCA) cycle, and homozygous mutations in genes encoding the enzymes involved in this process result in severe developmental defects early in life (2).
Recent studies show, however, that heterozygous germline mutations in genes encoding two sequential TCA cycle enzymes, succinate dehydrogenase (SDH) and fumarate hydratase (FH), predispose to tumor syndromes (2). Mutations in the SDH subunit genes SDHB, SDHC and SDHD, but not in SDHA, are associated with familial and sporadic pheochromocytoma and paraganglioma (2), and in the case of SDHB, also with renal cell carcinoma (3). Heterozygous mutations in FH cause hereditary leiomyomatosis and renal cell cancer (HLRCC) (2). FH somatic inactivation has been reported also in non-syndromic uterine fibroids (4) and sarcomas (5). The only tumor type common to SDH- and FH-associated neoplasia is renal cell carcinoma. The mechanisms leading from defects in mitochondrial metabolism to tumorigenesis are still far from well understood, although some hypotheses related to increased reactive oxygen species production (6) and stabilization of HIF-1
(7) have been proposed. So far, changes in gene expression in TCA-cycle-related tumors have not been studied. We have applied the expression microarray technique to study the transcriptome of uterine fibroids carrying mutations in FH and tested whether these tumors significantly differ from fibroids with wild-type FH.
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RESULTS |
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All tumors were histologically defined as typical leiomyomas. The Finnish fibroids (Table 1) had a mitotic rate of <1 per 10 high-power fields, and in the semi-quantitative grading of cellularity, most of them were classified into group 2 (73%), then to 1 (22%) and 3 (5%) (Fig. 1); all FH-mutant fibroids belonged to group 2 or 3. Both sporadic FH mutants, 4m3 and 32m1, FH wild-type 51m1 and three of the five Finnish HLRCC fibroids, B7m2, B7m5 and B7m6, showed loss of heterozygosity (LOH) on chromosome 1q, but no somatic FH mutations were detected in B7m1 and B7m3. All HLRCC fibroids from the UK showed LOH on chromosome 1q. Thus, 10 out of 12 fibroids carrying FH mutations showed robust genetic evidence for loss of FH in our analyses; in two cases, the loss of FH was not genetically confirmed.
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First, we analyzed the microarray data from the set of Finnish samples. In hierarchical clustering, the fibroids separated into two major branches, one of which comprised both sporadic FH mutants and all but one HLRCC fibroid (Fig. 1). A clear separation of the fibroids according to their FH status was also evident in the principal component analysis plot (Fig. 2). Altogether, 360 probe sets showed differential expression between FH-mutant and wild-type fibroids, 181 of which had lower expression in FH mutants (Supplementary Material, Table S1). The differentially expressed probe sets represented 297 genes, 41 of which were represented by more than one probe set showing consistent fold changes (FCs). The genes with the lowest relative expression in FH mutants were apolipoprotein D (APOD), Purkinje cell protein 4 (PCP4) and latent transforming growth factor beta binding protein 2 (LTBP2) with FCs of 0.21, 0.27 and 0.29, respectively. Other genes with lower expression in FH mutants were, for example, cyclin-dependent kinase inhibitor 1C (p57, Kip2 and CDKN1C), neuroblastoma suppression of tumorigenicity 1 (NBL1), connective tissue growth factor (CTGF), estrogen receptor 1 (ESR1), B-cell CLL/lymphoma 2 (BCL2) and FH. The genes with the highest expression in FH mutants compared with FH wild-type fibroids were NAD(P)H dehydrogenase quinone 1 (NQO1), aldo-keto reductase family 1 member C1 (AKR1C1) and transketolase (TKT) with FCs of 8.1, 7.2 and 5.1, respectively. Among the genes showing higher expression in FH-mutant fibroids were also malic enzyme 1 (ME1), peroxiredoxins 1, 4 and 6 (PRDX1, PRDX4 and PRDX6), cyclin-dependent kinase inhibitor 1A (p21, Cip1 and CDKN1A) and apoptosis antagonizing transcription factor (AATF and CHE-1). To evaluate the microarray data, we performed quantitative RT–PCR analyses; the higher expression of TKT (P=0.006), LDHA (P=0.023), CDKN1A (P=0.043) and AATF (P=0.004) in FH-mutant fibroids compared with FH wild-types was confirmed.
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Of the 297 differentially expressed genes, 214 had a gene ontology annotation and they belonged to 1519 different gene ontology categories. In the functional group enrichment test, 50 of them had a P-value <0.01 (Table 2; Supplementary Material, Table S2). Genes belonging to hexose metabolism category were the most significantly enriched group within the differentially expressed genes (P=1.7x10–9) and, for example, all 10 differentially expressed genes involved in glycolysis (P=1.1x10–8) had higher expression in FH mutants. In fact, of the 50 categories, 14 were directly involved in carbohydrate metabolism, and all but a few of the genes, such as FH, had higher expression in FH mutants. Additional categories with higher expression in FH mutants included iron ion homeostasis, oxidoreductase activity, membrane lipid catabolism, integral to endoplasmic reticulum membrane and electron transporter activity. Genes with lower expression in FH mutants, in contrast, tended to belong to functional categories such as extracellular matrix, cell motility, muscle contraction, organogenesis, muscle development, cell adhesion and plasma membrane.
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We performed the expression analysis also with FH-mutant fibroids compared with normal myometrium; the most differentially expressed genes are listed in the Supplementary Material, Table S3. Increased expression of carbohydrate metabolism genes was detected in FH-mutant fibroids compared with normal myometrium and again glycolysis-related genes were highly enriched within the differentially expressed genes (P=1.3x10–7). Similarly, many of the genes showing higher expression in FH-mutant fibroids when compared with FH wild-type fibroids, such as NQO1, AKR1C1, TKT, CDKN1A, ME1, PRDX1, PRDX4, PRDX6 and LDHA, showed higher expression also when compared with normal myometrium.
To validate the results obtained from the initial set of Finnish samples, we analyzed a separate set of UK samples. In an unsupervised clustering, the UK HLRCC fibroids clustered together with the Finnish FH mutants and the UK sporadic fibroids clustered with the Finnish FH wild-types (data not shown). The up-regulation of several genes involved in glycolysis, such as LDHA (FC 2.3, unadjusted P-value=0.04), ENO1 (FC 2.5, P=0.0006), PKM2 (FC 2.0, P=0.026), PFKP (FC 3.0, P=0.01) and PGAM1 (FC 1.6, P=0.01), was confirmed. Similarly, the up-regulation of TKT (FC 2.5, P=4.8x10–7), NQO1 (FC 2.5, P=0.005) and CDKN1A (FC 4.3, P=0.016) was also confirmed.
We also tested whether a small set of genes (<10) could be used to predict the FH status of fibroids. A 7-gene predictor consisting of LDHA, NQO1, LAMA2, BNIP3, MYO15B, CDKN1C and COL6A2 gave the best results in the leave-one-out cross-validation performed on the Finnish sample set, and thus it was subsequently validated with the independent set of UK fibroids. Our classifier correctly predicted the FH mutation status of all seven UK fibroids.
As none of the Finnish HLRCC fibroids had a second somatic mutation in FH and only three showed LOH on 1q, no second inactivating hit was detectable in B7m1 and B7m3. Also, two FH wild-type fibroids as determined by genetic analysis, 44m2 and 44m3, grouped together with FH mutants in the hierarchical clustering. FH protein levels were therefore measured in seven samples, normal myometrium B7n and 44n, and fibroids B7m1, B7m2, B7m3, 44m2 and 44m3. B7m1 had clearly the highest FH level of the HLRCC fibroids and no reduction of FH was observed in 44m2 or 44m3 (Fig. 3).
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DISCUSSION |
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In this study, we show that FH-mutant fibroids differ significantly from their FH wild-type counterparts in their overall expression profile. We also provide data on the specific changes caused by the loss of wild-type FH in fibroids, thus providing insight into the pathogenesis of TCA-cycle-related tumors.
In comparing the expression profiles of seven FH-mutant tumors to 15 FH wild-types, significant differences were already evident in the unsupervised clustering of the samples. The FH mutants, both sporadic and HLRCC, separated into their own branch, except for B7m1, which had an overall expression profile resembling that of FH wild-types. B7m1 had the highest FH protein level among the HLRCC fibroids and showed neither LOH in 1q nor somatic mutations in FH. This lesion may thus represent a non-syndromic fibroid in an HLRCC patient, which is possible given the high prevalence of uterine fibroids (8). In addition, fibroid 51m1, which clustered together with the FH wild-types, showed 1q LOH but no FH mutations. Together these findings suggest that two hits are indeed required for the FH-mutant expression profile to develop.
Fibroids 44m2 and 44m3 clustered together with FH mutants, although no FH mutations, 1q LOH, or reduced FH protein levels were observed. It is possible that some mutations could have escaped our analysis, and thus the apparently normal protein level of FH in these samples would still have reduced FH activity. Another explanation could be that these fibroids harbor mutations in some as-yet-undiscovered gene(s) on the FH-fibroid pathway.
The genes differentially expressed between FH-mutant and wild-type fibroids revealed several functional categories which had higher expression in FH mutants. The most striking increase was in genes related to carbohydrate metabolism, particularly in glycolysis. The glycolytic phenotype is selected in the progression of several malignant tumors and is a hallmark of invasive cancer (9). A recent report suggests that over-expression of phosphoglycerate mutase, a glycolytic enzyme we found to be over-expressed in FH-mutant fibroids, can immortalize mouse embryonic stem cells and make them resistant to ras-induced arrest (10). Increased glycolysis also makes the tumor environment more acidic, thus hindering the survival of adjacent normal cells (9). In addition to increased glycolysis, FH-mutant fibroids also seem to have become adapted to the acidic environment, as the expression of ATP6V0D1, a gene encoding one subunit of H+ ATPase maintaining intracellular pH, had a significantly higher expression in FH mutants. Moreover, FH-mutant fibroids tended to have higher levels of cellularity, a well-known feature of neoplasia. As 
10% of Finnish female HLRCC patients develop early-onset leiomyosarcoma (11), it is of interest that FH-mutant fibroids display these expression features. The increased expression of genes involved in glycolysis could directly contribute to the malignant transformation, although additional factors are also most probably necessary.
An increase in the production of reactive oxygen species has been proposed as a consequence of defects in mitochondrial metabolic pathways (6). Landis et al. (12) showed that oxidative stress enhances the expression of genes encoding proteins with antioxidant activity, and genes involved in iron homeostasis also play a role in protection against oxidative stress (13). In our experiment, we observed increased expression of iron ion homeostasis and oxidoreduction genes in FH-mutant tumors.
Genes with lower expression in FH mutants tended to belong to functional categories such as muscle development, extracellular matrix, cell adhesion and cell motility. The lower expression of extracellular matrix genes also appeared in the fibroid histology, as the FH mutants tended to have higher cellularity levels than the wild-types. The roughly 50% reduction in the expression of the genes on 1q, MEF2D, S100A4, TUFT1, S100A13, TXNIP, FLJ21919, RGL1, GLUL, CD34, TOMM20 and LGALS8, is also of particular interest, as this could result solely from the 1q-deletions in FH-mutant fibroids.
Among the differentially expressed genes, several have been proposed as playing a role in the formation of human tumors. Although it is hard to predict the result of deregulation of some individual genes, it is tempting to hypothesize that some of them might play a direct role in tumorigenesis related to loss of wild-type FH. Apoptosis-antagonizing transcription factor (AATF) prevents DAPK3 (DLK/ZIPK)-induced apoptosis by sequestering DAPK3 in the nucleus (14). It also enhances the expression of CDKN1A (p21) (15). DAPK1, in contrast, a member of the same kinase family as DAPK3, has a role in apoptosis induced by the detachment of the cell from the extracellular matrix (14). We detected higher expression of AATF and CDKN1A and lower expression of genes involved in cell adhesion in FH-mutant fibroids, suggesting AATF over-expression as a mechanism for FH-mutant fibroids in cell survival.
Transketolase (TK) had one of the highest relative expression levels in FH-mutant fibroids. TK is a thiamine-dependent enzyme in the pentose phosphate pathway, which produces ribose-5-phosphate from glucose. Ribose-5-phosphate is then used for the production of nucleic acids and of some coenzymes involved in nucleic acid synthesis (16). Inhibitors of TK cause cell cycle arrest (17) and recently new more specific small molecule inhibitors of TK have been identified (16). As the expression of TK is high in FH-mutant fibroids, a therapeutic intervention targeting TK could be effective, especially in HLRCC tumors.
In conclusion, our results show that mutations in FH significantly affect the global expression pattern of fibroids. That multiple differentially expressed genes were discovered suggests new pathways in TCA-cycle-related tumorigenesis and consequently we propose possible targets for therapeutic interventions in HLRCC-related tumors. We show that FH-mutant fibroids tend to have higher cellularity than their FH wild-type counterparts, and the former also display a glycolytic phenotype frequently seen in invasive and meta-static cancers (9). This could directly contribute to the increased risk for uterine leiomyosarcoma in HLRCC patients. Mutations in a large number of genes playing a role in mitochondrial energy metabolism are likely to produce a similar end result, including enhanced potential for tumorigenesis. The impact of such mutations in human cancer burden should be vigorously investigated.
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MATERIALS AND METHODS |
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Patient material
The Finnish fibroid and myometrium samples (Table 1) were collected at the Helsinki University Central Hospital with the full approval of the local ethics committee. Of the 22 fresh-frozen fibroid samples, seven carried FH mutations: two non-syndromic sporadic lesions, 32m1 carrying a missense mutation 586G>A (Ala196Thr) and 4m3 carrying a splice-site mutation IVS4+3A>G, both showing loss of the wild-type allele (4
), and five separate myoma samples from an HLRCC patient carrying a truncating germline mutation 541delAG. Eleven samples of normal myometrium were available, and the 15 wild-type FH samples came from nine different patients (18), two of whom were the same as those carrying the two sporadic fibroids with FH mutations. Two patients thus had both FH-mutant and FH wild-type fibroids. Frozen sections from the tumors were hematoxylin-eosin-stained and the diagnosis was confirmed, followed by assessment of the mitotic rate and cellularity. The cellularity was semi-quantitatively graded from 1 to 3, with 1 defined as high, 2 as medium and 3 as low stroma/nuclei ratio. Subsequent DNA and RNA extractions were performed as described (18). A separate sample set was obtained in the UK with full Research Ethics Committee approval. The UK samples comprised: three samples of normal myometrium; two sporadic, FH wild-type fibroids and five FH-mutant fibroids from an HLRCC patient carrying a germline FH mutation K187R. RNA was extracted as described (18).
Analysis of LOH and genomic sequencing
For studying loss of heterozygosity (LOH) on chromosome 1q in the Finnish HLRCC fibroids, we used microsatellite markers and methods described previously (4). For the sequencing of FH, we used identical PCR primers and conditions to those used by Kiuru et al. (5) and the sequencing was performed as described (4). In the UK samples, LOH was assessed by microarray comparative genomic hybridization as described (18) and, where possible, by direct inspection of FH sequence around the site of the germline mutation.
Expression microarray procedures and data analysis
The sample preparation for microarray analysis, the subsequent hybridization and scanning of the GeneChip® HG-U133A oligonucleotide chips (Affymetrix, Inc., Santa Clara, CA, USA) and the data normalization were performed as described (18). The principal component analysis was performed by utilizing the TIGR MeV software (19) with the method described by Raychaudhuri et al. (20).
For hierarchical clustering, gene vectors with standard deviation <0.65 were filtered out resulting in a set of 990 probe sets most differentially expressed across all the samples. The data were median polished and normalized, i.e. the row-wise and column-wise median values were close to zero, and all column and row magnitudes were close to 1. The arrays were organized with self-organizing maps and hierarchically clustered using the Pearson correlation coefficient as the similarity metric (21); the cluster dendrogram was produced using TreeView 1.60 (21).
The genes differentially expressed between the Finnish FH wild-type and mutant fibroids were identified using a permutation-based method that estimates the false discovery rate (FDR), i.e. significance analysis of microarrays (22). A minimal FC of 1.5 and an FDR smaller than 5% were requirements. To classify the differentially expressed genes into biologically meaningful groups, we implemented a functional enrichment test (23), which groups the genes according to gene ontology annotations and calculates a P-value (Fisher's exact test) for the enrichment of differentially expressed genes in each category. Categories with fewer than five genes consistently detected across all samples were excluded. All microarray analyses were repeated with FH-mutant fibroids only compared with normal myometrium.
A molecular classifier was built to predict the FH status of fibroids based on their gene expression profile. Gene Cluster software (24) was utilized to first test the performance of predictors consisting of 1–10 different genes in a leave-one-out cross-validation of the Finnish sample set as described by Golub et al. (25). The cross-validation results then guided the selection of the number of genes for the predictor that was further validated on the independent set of UK samples. For each step, the class assigned for tested samples was determined by three of its nearest-neighbors using the Cosine distance metric.
Quantitative real-time PCR
The relative TKT, LDHA, CDKN1A and AATF expression levels in the Finnish fibroids were determined by quantitative real-time PCR as described (18); ß-actin (ACTB) was used as an endogenous control, as its expression did not vary significantly among the fibroids. A Mann–Whitney U-test was performed to compare the expression of all four genes between FH wild-type and mutant fibroids.
Western blotting
Protein was extracted with the T-PERTM Tissue Protein Extraction Reagent (Pierce, Rockford, IL, USA) protocol. The protein concentration was measured by BCA Protein Assay Reagent Kit (Pierce). Five micrograms of protein were separated on 10% polyacrylamide gel (Bio-Rad Laboratories, Hercules, CA, USA) and blotted onto polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA). The membranes were probed with purified polyclonal rabbit anti-porcine FH antibody (Nordic Immunological Laboratories, Tilburg, the Netherlands) at a 1 : 500 dilution. Monoclonal mouse -tubulin antibody (Sigma-Aldrich, St Louis, MO, USA) was used as a loading control at a 1 : 10 000 dilution. Both FH and -tubulin were detected by ECL plus Western Blotting Detection System (Amersham Biosciences UK Ltd, Buckinghamshire, UK).
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Supplementary Material is available at HMG Online. Microarray data will be available in Gene Expression Omnibus at http://www.ncbi.nih.gov/geo/.
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ACKNOWLEDGEMENTS |
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We thank Sini Marttinen for helping with the samples and Mikko Aho for technical support. This work was supported by grants from the Finnish Cancer Society, the Helsinki University Central Hospital, the Sigrid Juselius Foundation, the Maud Kuistila Foundation, The Emil Aaltonen Foundation and the Academy of Finland (grant 44870, Finnish Center of Excellence Programme 2000–2005).
Conflict of Interest statement. None declared.
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