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

PLoS One. 2013;8(2):e57697. doi: 10.1371/journal.pone.0057697. Epub 2013 Feb 21.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • Antimetabolites, Antineoplastic
  • Isoenzymes
  • Neoplasm Proteins
  • L-Lactate Dehydrogenase
  • lactate dehydrogenase 1
  • Azacitidine

Grant support

Sheffield Breast Cancer support group funding to NJB is acknowledged (Grant code X/001592). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.