Lactate dehydrogenase-B is silenced by promoter methylation in a high frequency of human breast cancers

Nicola J Brown, Sue E Higham, Branko Perunovic, Mohammad Arafa, Sabapathy Balasubramanian, Ishtiaq Rehman

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44 Citations (Scopus)

Abstract

OBJECTIVE: Under normoxia, non-malignant cells rely on oxidative phosphorylation for their ATP production, whereas cancer cells rely on Glycolysis; a phenomenon known as the Warburg effect. We aimed to elucidate the mechanisms contributing to the Warburg effect in human breast cancer.

EXPERIMENTAL DESIGN: Lactate Dehydrogenase (LDH) isoenzymes were profiled using zymography. LDH-B subunit expression was assessed by reverse transcription PCR in cells, and by Immunohistochemistry in breast tissues. LDH-B promoter methylation was assessed by sequencing bisulfite modified DNA.

RESULTS: Absent or decreased expression of LDH isoenzymes 1-4, were seen in T-47D and MCF7 cells. Absence of LDH-B mRNA was seen in T-47D cells, and its expression was restored following treatment with the demethylating agent 5'Azacytadine. LDH-B promoter methylation was identified in T-47D and MCF7 cells, and in 25/25 cases of breast cancer tissues, but not in 5/5 cases of normal breast tissues. Absent immuno-expression of LDH-B protein (<10% cells stained), was seen in 23/26 (88%) breast cancer cases, and in 4/8 cases of adjacent ductal carcinoma in situ lesions. Exposure of breast cancer cells to hypoxia (1% O(2)), for 48 hours resulted in significant increases in lactate levels in both MCF7 (14.0 fold, p = 0.002), and T-47D cells (2.9 fold, p = 0.009), but not in MDA-MB-436 (-0.9 fold, p = 0.229), or MCF10AT (1.2 fold, p = 0.09) cells.

CONCLUSIONS: Loss of LDH-B expression is an early and frequent event in human breast cancer occurring due to promoter methylation, and is likely to contribute to an enhanced glycolysis of cancer cells under hypoxia.

Original languageEnglish
Pages (from-to)e57697
JournalPLoS One
Volume8
Issue number2
DOIs
Publication statusPublished - 2013

Keywords

  • Adult
  • Aged
  • Aged, 80 and over
  • Antimetabolites, Antineoplastic/pharmacology
  • Azacitidine/pharmacology
  • Breast Neoplasms/enzymology
  • Carcinoma, Ductal/enzymology
  • Case-Control Studies
  • Cell Line, Tumor
  • DNA Methylation
  • Female
  • Gene Expression Regulation, Neoplastic/drug effects
  • Gene Silencing
  • Glycolysis/genetics
  • Humans
  • Hypoxia/enzymology
  • Isoenzymes/genetics
  • L-Lactate Dehydrogenase/genetics
  • Middle Aged
  • Neoplasm Proteins/genetics
  • Promoter Regions, Genetic
  • Sequence Analysis, DNA

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