- Short Report
- Open Access
No evidence for promoter region methylation of the succinate dehydrogenase and fumarate hydratase tumour suppressor genes in breast cancer
© Fox et al; licensee BioMed Central Ltd. 2009
- Received: 23 May 2009
- Accepted: 25 September 2009
- Published: 25 September 2009
Succinate dehydrogenase (SDH) and fumarate hydratase (FH) are tricarboxylic acid (TCA) cycle enzymes that are also known to act as tumour suppressor genes. Increased succinate or fumarate levels as a consequence of SDH and FH deficiency inhibit hypoxia inducible factor-1α (HIF-1α) prolyl hydroxylases leading to sustained HIF-1α expression in tumours. Since HIF-1α is frequently expressed in breast carcinomas, DNA methylation at the promoter regions of the SDHA, SDHB, SDHC and SDHD and FH genes was evaluated as a possible mechanism in silencing of SDH and FH expression in breast carcinomas.
No DNA methylation was identified in the promoter regions of the SDHA, SDHB, SDHC, SDHD and FH genes in 72 breast carcinomas and 10 breast cancer cell lines using methylation-sensitive high resolution melting which detects both homogeneous and heterogeneous methylation.
These results show that inactivation via DNA methylation of the promoter CpG islands of SDH and FH is unlikely to play a major role in sporadic breast carcinomas.
- Breast Carcinoma
- Promoter Methylation
- Invasive Breast Carcinoma
- Prolyl Hydroxylase
- Fumarate Hydratase
The hypoxia-inducible factor (HIF-1) transcription factor plays a pivotal role in breast tumour progression [1–4] by activating genes involved in angiogenesis, cell proliferation and survival [1, 2, 5]. Levels of HIF-1 α subunits (HIF-1α) are tightly regulated with rapid degradation via hydroxylation by prolyl hydroxylases (PHDs) 1, 2 and 3 and proteasomal degradation by the von Hippel-Lindau (VHL) protein . Increased levels of fumarate and succinate inhibit PHD activity via product inhibition as well as by direct inhibition by competing with α-ketoglutarate at the PHD α-ketoglutarate binding site [6–8]. Thus any mechanism whereby the level of succinate dehydrogenase (SDH) or fumarate hydratase (FH) is reduced may result in tumorigenesis [9, 10]. Indeed, the SDH and FH genes have been demonstrated to be tumour suppressor genes (TSG) via this pseudohypoxic drive in paraganglioma , hereditary leiomyomatosis and renal cell carcinomas . In view of this potential mechanism to enhance HIF-1α levels and in view of the association of HIF-1α levels with breast cancer prognosis and resistance to treatment, we hypothesised that epigenetic silencing by promoter methylation for the SDH and FH genes may be a mechanism underlying upregulated HIF-1 in a proportion of breast carcinomas.
DNA was obtained from 72 invasive breast carcinomas from the John Radcliffe Hospital, Oxford, UK (Ethics committee approval: JR C02.216) and from the following cancer cell lines: breast: MCF10A, MCF7, BT20, SkBr3, Hs578T, T47D, MDA-MB 153, MDA-MB 468, MDA-MB 453, MDA-MB 231; colorectal: Colo205, HCT116, SW948, SW48; leukaemia: HL60, KG1, RPMI8226, CCRF-CEM; ovarian: 2008; neuroblastoma: SK-N-SH, SH-SY5Y, Be(2)c, IMR32; and prostate: PC3.
DNA from samples were bisulfite modified as described previously . CpGenome™ Universal Methylated DNA (Chemicon/Millipore, Billerica, MA) and DNA from peripheral blood mononuclear cells were used as the methylated and unmethylated controls, respectively. Standards (5, 10, 25 and 50% methylation) were generated by diluting Universal Methylated DNA in the unmethylated DNA. Whole-genome amplification (WGA) DNA was used as an alternative unmethylated control .
Methylation-sensitive high resolution melting (MS-HRM) and methylation-specific PCR (MSP)
Primer sequences, annealing temperature and amplicon information for the MS-HRM assays.
5' - 3'
Annealing temperature (°C)
Amplified region (GenBank accession and nucleotide numbers)
Screened CpGs/amplicon size (bp)
F - CGGGGTTTTAAAAATGTTGGTGTT
R - CGAACCCCCGACATATCTACTATTACC
F - CGGGGGAAGTTAAATGGGTATG
R - CGCCCAACCTACATCCACTAAA
F - GCGGTTAGTGGGTTTTTAGTGGAT
R - CAAACAAACTCCGCCAAAAATTATAACC
F - TCGTTATATGATATTTTTAATTTCGATTTTTAGT
R - ATCTTAAATTCCGATCTAAACGAAAATAAC
F - CGGGTTGGTGGATGATTTTGAG
R - CCTCACCTCGACCTCCTAAAACAC
F - TTTGTTTTATTTGTCGGTGTGAGGT
R - AAAACTTAAATAAAATTTCTAAACGACTATAACCAC
Methylation of SDHA, SDHB, SDHC, SDHD and FH in cell lines and tumours
RASSF1A and MAL methylation frequencies in the breast carcinoma samples as determined by MS-HRM
Overexpression of HIF-1α has been previously reported to correlate with angiogenesis , an aggressive phenotype [3, 17] and poor outcome after conventional adjuvant therapy [18, 19] in breast cancer. Thus mechanisms that enhance HIF-1α expression are important in cancer development and would be potential targets for treatment [2, 20].
The tricarboxylic acid cycle enzymes, SDH and FH are involved in the conversion of succinate to fumarate and fumarate to malate, respectively. SDH also takes part in the electron transport chain as a functional complex II member.
Both SDH and FH can act as tumour suppressors, and germline mutations in their genes predispose to tumour development. Mutations in the genes coding for SDH subunits B, C and D predispose to familial paragangliomas and phaeochromocytomas [11, 21, 22], and mutations in FH cause hereditary leiomyomatosis and renal cell carcinomas .
Although the mechanisms that link SDH and FH mutations to tumour formation are unclear, it is likely that pseudohypoxia is a primary mechanism. Both Selak et al.  and Pollard et al.  have suggested that overexpression of HIF-1α in normoxic conditions is due to the accumulation of succinate, which then is able to inhibit the activity of HIF-1α prolyl hydroxylases via product inhibition. A recent study has also shown that disruption of mitochondrial metabolism using small interfering RNAs to silence SDHB resulted in up-regulation of HIF-1α. . Furthermore, microarray analysis has confirmed that genes involved in the hypoxic pathway are dysregulated when SDHB is silenced .
Since many tumour suppressor genes are known to be inactivated by DNA promoter methylation, we examined promoter methylation of SDH and FH in a cohort of breast carcinomas. However, we found no evidence of DNA methylation of the promoter regions of these genes in breast carcinomas cancer or a panel of cancer cell lines, including ten breast cancer cell lines, making it unlikely that methylation of the promoter regions of these genes is responsible for increased HIF expression in breast cancers. Although we cannot exclude the possibility that methylation of regions other than the proximal promoter may be involved, our findings are also in keeping with others who have been unable to demonstrate methylation of SDHD in neuroblastomas and FH in renal cell cancers [26, 27].
In conclusion, promoter methylation of the SDHA, SDHB, SDHC, SDHD and FH genes is unlikely to be an important mechanism in stabilising HIF-1 in breast carcinomas through the downregulation of the expression of SDH and FH genes.
We wish to thank the Molecular Pathology Research and Development group in Peter MacCallum Cancer Centre for their help and support and Ida Candiloro for proofreading the manuscript. We would like to thank Professor Adrian L. Harris for supplying us with the breast carcinoma samples. We would also like to thank Associate Professor David Ashley and Andrea Muscat for supplying us with the neuroblastoma cell lines. This work was funded by grants from the Victorian Breast Cancer Research Consortium and the Cancer Council of Victoria.
- Harris AL: Hypoxia - A key regulatory factor in tumour growth. Nature Reviews Cancer. 2002, 2 (1): 38-47. 10.1038/nrc704.View ArticlePubMedGoogle Scholar
- Semenza GL: Targeting HIF-1 for cancer therapy. Nature Reviews Cancer. 2003, 3 (10): 721-732. 10.1038/nrc1187.View ArticlePubMedGoogle Scholar
- Yamamoto Y, Ibusuki M, Okumura Y, Kawasoe T, Kai K, Iyama K, Iwase H: Hypoxia-inducible factor 1 alpha is closely linked to an aggressive phenotype in breast cancer. Breast Cancer Research and Treatment. 2008, 110 (3): 465-475. 10.1007/s10549-007-9742-1.View ArticlePubMedGoogle Scholar
- Unruh A, Ressel A, Mohamed HG, Johnson RS, Nadrowitz R, Richter E, Katschinski DM, Wenger RH: The hypoxia-inducible factor-1 alpha is a negative factor for tumor therapy. Oncogene. 2003, 22 (21): 3213-3220. 10.1038/sj.onc.1206385.View ArticlePubMedGoogle Scholar
- Fox SB, Generali DG, Harris AL: Breast tumour angiogenesis. Breast Cancer Research. 2007, 9 (6): 11-10.1186/bcr1796.View ArticleGoogle Scholar
- King A, Selak MA, Gottlieb E: Succinate dehydrogenase and fumarate hydratase: linking mitochondrial dysfunction and cancer. Oncogene. 2006, 25 (34): 4675-4682. 10.1038/sj.onc.1209594.View ArticlePubMedGoogle Scholar
- Gottlieb E, Tomlinson IP: Mitochondrial tumour suppressors: a genetic and biochemical update. Nat Rev Cancer. 2005, 5 (11): 857-866. 10.1038/nrc1737.View ArticlePubMedGoogle Scholar
- Selak MA, Armour SM, MacKenzie ED, Boulahbel H, Watson DG, Mansfield KD, Pan Y, Simon MC, Thompson CB, Gottlieb E: Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-alpha prolyl hydroxylase. Cancer Cell. 2005, 7 (1): 77-85. 10.1016/j.ccr.2004.11.022.View ArticlePubMedGoogle Scholar
- Eng C, Kiuru M, Fernandez MJ, Aaltonen LA: A role for mitochondrial enzymes in inherited neoplasia and beyond. Nat Rev Cancer. 2003, 3 (3): 193-202. 10.1038/nrc1013.View ArticlePubMedGoogle Scholar
- Pollard PJ, Wortham NC, Tomlinson IPM: The TCA cycle and tumorigenesis: the examples of fumarate hydratase and succinate dehydrogenase. Annals of Medicine. 2003, 35 (8): 634-635. 10.1080/07853890310018458.View ArticleGoogle Scholar
- Baysal BE: On the association of succinate dehydrogenase mutations with hereditary paraganglioma. Trends Endocrinol Metab. 2003, 14 (10): 453-459. 10.1016/j.tem.2003.08.004.View ArticlePubMedGoogle Scholar
- Kristensen LS, Mikeska T, Krypuy M, Dobrovic A: Sensitive Melting Analysis after Real Time-Methylation Specific PCR (SMART-MSP): high-throughput and probe-free quantitative DNA methylation detection. Nucleic Acids Research. 2008, 36 (7): e42-10.1093/nar/gkn113.PubMed CentralView ArticlePubMedGoogle Scholar
- Wojdacz TK, Dobrovic A: Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Research. 2007, 35 (6): e41-10.1093/nar/gkm013.PubMed CentralView ArticlePubMedGoogle Scholar
- Astuti D, Morris M, Krona C, Abel F, Gentle D, Martinsson T, Kogner P, Neumann HP, Voutilainen R, Eng C, et al: Investigation of the role of SDHB inactivation in sporadic phaeochromocytoma and neuroblastoma. Br J Cancer. 2004, 91 (10): 1835-1841. 10.1038/sj.bjc.6602202.PubMed CentralView ArticlePubMedGoogle Scholar
- Dammann R, Yang G, Pfeifer GP: Hypermethylation of the cpG island of Ras association domain family 1A (RASSF1A), a putative tumor suppressor gene from the 3p21.3 locus, occurs in a large percentage of human breast cancers. Cancer Res. 2001, 61 (7): 3105-3109.PubMedGoogle Scholar
- Horne HN, Lee PS, Murphy SK, Alonso MA, Olson JA, Marks JR: Inactivation of the MAL gene in breast cancer is a common event that predicts benefit from adjuvant chemotherapy. Mol Cancer Res. 2009, 7 (2): 199-209. 10.1158/1541-7786.MCR-08-0314.PubMed CentralView ArticlePubMedGoogle Scholar
- Bos R, Zhong H, Hanrahan CF, Mommers ECM, Semenza GL, Pinedo HM, Abeloff MD, Simons JW, van Diest PJ, Wall van der E: Levels of hypoxia-inducible factor-1 alpha during breast carcinogenesis. Journal of the National Cancer Institute. 2001, 93 (4): 309-314. 10.1093/jnci/93.4.309.View ArticlePubMedGoogle Scholar
- Lundgren K, Holm C, Landberg G: Hypoxia and breast cancer: prognostic and therapeutic implications. Cellular and Molecular Life Sciences. 2007, 64 (24): 3233-3247. 10.1007/s00018-007-7390-6.View ArticlePubMedGoogle Scholar
- Trastour C, Benizri E, Ettore F, Ramaioli A, Chamorey E, Pouyssegur J, Berra E: HIF-1 alpha and CA IX staining in invasive breast carcinomas: Prognosis and treatment outcome. International Journal of Cancer. 2007, 120 (7): 1451-1458. 10.1002/ijc.22436.View ArticleGoogle Scholar
- Patiar S, Harris AL: Role of hypoxia-inducible factor-1 alpha as a cancer therapy target. Endocrine-Related Cancer. 2006, 13: S61-S75. 10.1677/erc.1.01290.View ArticlePubMedGoogle Scholar
- Niemann S, Muller U: Mutations in SDHC cause autosomal dominant paraganglioma, type 3. Nat Genet. 2000, 26 (3): 268-270. 10.1038/81551.View ArticlePubMedGoogle Scholar
- Astuti D, Douglas F, Lennard TW, Aligianis IA, Woodward ER, Evans DG, Eng C, Latif F, Maher ER: Germline SDHD mutation in familial phaeochromocytoma. Lancet. 2001, 357 (9263): 1181-1182. 10.1016/S0140-6736(00)04378-6.View ArticlePubMedGoogle Scholar
- Tomlinson IP, Alam NA, Rowan AJ, Barclay E, Jaeger EE, Kelsell D, Leigh I, Gorman P, Lamlum H, Rahman S, et al: Germline mutations in FH predispose to dominantly inherited uterine fibroids, skin leiomyomata and papillary renal cell cancer. Nat Genet. 2002, 30 (4): 406-410. 10.1038/ng849.View ArticlePubMedGoogle Scholar
- Pollard PJ, Briere JJ, Alam NA, Barwell J, Barclay E, Wortham NC, Hunt T, Mitchell M, Olpin S, Moat SJ, et al: Accumulation of Krebs cycle intermediates and over-expression of HIF1alpha in tumours which result from germline FH and SDH mutations. Hum Mol Genet. 2005, 14 (15): 2231-2239. 10.1093/hmg/ddi227.View ArticlePubMedGoogle Scholar
- Cervera AM, Apostolova N, Crespo FL, Mata M, McCreath KJ: Cells silenced for SDHB expression display characteristic features of the tumor phenotype. Cancer Res. 2008, 68 (11): 4058-4067. 10.1158/0008-5472.CAN-07-5580.View ArticlePubMedGoogle Scholar
- De Preter K, Vandesompele J, Hoebeeck J, Vandenbroecke C, Smet J, Nuyts A, Laureys G, Combaret V, Van Roy N, Roels F, et al: No evidence for involvement of SDHD in neuroblastoma pathogenesis. BMC Cancer. 2004, 4: 55-10.1186/1471-2407-4-55.PubMed CentralView ArticlePubMedGoogle Scholar
- Dulaimi E, Ibanez de Caceres I, Uzzo RG, Al-Saleem T, Greenberg RE, Polascik TJ, Babb JS, Grizzle WE, Cairns P: Promoter hypermethylation profile of kidney cancer. Clin Cancer Res. 2004, 10 (12 Pt 1): 3972-3979. 10.1158/1078-0432.CCR-04-0175.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.