Lactate Dehydrogenase-B Is Silenced by Promoter Methylation in a High Frequency of Human Breast Cancers

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.

Loss of NAPRT1 Expression by Tumor-Specific Promoter Methylation Provides a Novel Predictive Biomarker for NAMPT Inhibitors

“…It is not clear why loss of NAPRT1 expression would be advantageous to some cancers as the gene contributes to the NAD synthetic capacity of the cell. However, it is interesting to note that frequent hypermethylation of another metabolic enzyme, LDHB, has recently been reported in breast cancer (31), suggesting that alteration of metabolic pathways by DNA methylation may be a more general phenomenon in cancer. …”

Inhibition of Lactate Dehydrogenase Activity as an Approach to Cancer Therapy

“…A further interesting observation is that when LDH-A expression was silenced in noncancer cultured cells, proliferation and protein synthesis were not impaired [91,92].On the contrary, according to the data presently found in literature, the function of LDH-B in tumor development remains elusive and even in the same tumor form, conflicting results have often been obtained by the different authors. For example, the loss of LDH-B expression due to promoter methylation was recently described as an early and frequent event contributing to the development of breast cancer [93]; on the other hand, a screening study conducted on patients with the most aggressive form of breast cancer revealed higher LDH-B expression in lesions having poor clinical outcome [94]. Moreover, while LDH-B was described as an essential factor for mTOR mediated tumorigenesis [95] and for the growth of K-Ras dependent lung adenocarcinomas [96] and of maxillary sinus squamous carcinoma [97], its reduced expression was also associated with increased claudin-1 mediated invasiveness of hepatocellular carcinoma cells [98].…”

Inhibition of the Warburg effect with a natural compound reveals a novel measurement for determining the metastatic potential of breast cancers

“…In contrast to LDH-A expression, LDH-B is highly expressed in non-malignant tissues relative to tumors [17]. In malignant tumors, LDH-B is silenced by promoter hypermethylation; this occurs at a high frequency in primary breast tumors (100%, 25/25) and in primary prostate tumors (45%, 14/31) [17, 18].…”

“…In malignant tumors, LDH-B is silenced by promoter hypermethylation; this occurs at a high frequency in primary breast tumors (100%, 25/25) and in primary prostate tumors (45%, 14/31) [17, 18]. Although most reports indicate that LDH-B expression is decreased in breast tumors [17], integrative genomic analysis found that LDH-B expression is higher in the basal-like versus luminal subtype of cells in triple-negative breast cancers (TNBCs), and silencing of LDH-B expression in MDA-MB-231 cells decreases tumor growth [19]. While these findings appear contradictory, one caveat is that, in most of these studies, LDH-A and LDH-B were studied individually, thus hampering a broader view of their roles in cancer metabolism.…”