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.
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.
Materials and methods
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.
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