TY - JOUR
T1 - Lactate dehydrogenase-B is silenced by promoter methylation in a high frequency of human breast cancers
AU - Brown, Nicola J
AU - Higham, Sue E
AU - Perunovic, Branko
AU - Arafa, Mohammad
AU - Balasubramanian, Sabapathy
AU - Rehman, Ishtiaq
PY - 2013
Y1 - 2013
N2 - 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.
AB - 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.
KW - Adult
KW - Aged
KW - Aged, 80 and over
KW - Antimetabolites, Antineoplastic/pharmacology
KW - Azacitidine/pharmacology
KW - Breast Neoplasms/enzymology
KW - Carcinoma, Ductal/enzymology
KW - Case-Control Studies
KW - Cell Line, Tumor
KW - DNA Methylation
KW - Female
KW - Gene Expression Regulation, Neoplastic/drug effects
KW - Gene Silencing
KW - Glycolysis/genetics
KW - Humans
KW - Hypoxia/enzymology
KW - Isoenzymes/genetics
KW - L-Lactate Dehydrogenase/genetics
KW - Middle Aged
KW - Neoplasm Proteins/genetics
KW - Promoter Regions, Genetic
KW - Sequence Analysis, DNA
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U2 - 10.1371/journal.pone.0057697
DO - 10.1371/journal.pone.0057697
M3 - Article
C2 - 23437403
SN - 1932-6203
VL - 8
SP - e57697
JO - PLoS One
JF - PLoS One
IS - 2
M1 - e57697
ER -