Abstract
ObjectiveUnder 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 designLactate 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.ResultsAbsent 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% O2), 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.ConclusionsLoss 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.
Highlights
Breast cancer is the most common cancer diagnosis and the leading cause of cancer-related deaths in women worldwide [1]
Exposure of breast cancer cells to hypoxia (1% O2), 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
Loss of Lactate Dehydrogenase (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
Summary
Breast cancer is the most common cancer diagnosis and the leading cause of cancer-related deaths in women worldwide [1]. Despite advances in early detection and treatment, around 30% of patients with early-stage breast cancer will develop recurrent disease, which often presents as metastasis and a poor prognosis [3]. Glucose enters into the cell and undergoes a series of steps to generate pyruvate, which enters into the mitochondria where it is metabolized to CO2 and water with the formation of ATP. This process is facilitated by the presence of oxygen which activates oxidative phosphorylation and inhibits glycolysis (known as the Pasteur effect), [5,6]. When oxygen is limited (anaerobic conditions), such as following prolonged exercise, pyruvate undergoes fermentation where it is converted to lactate by a reaction catalyzed by Lactate Dehydrogenase (LDH), [7,8]
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