LDHA (Lactate Dehydrogenase A)
The LDHA gene encodes the A subunit, which is also often called M subunit, of lactate dehydrogenase, an enzyme which catalyzes the interconversion of pyruvate to lactate and NADH to NAD+. This enzyme was first reported as having five isozymes in 1959, where it was also shown that they were tissue-specific (Markert and Moller 1959). Tsujibo and cols first described the nucleotide sequence of the human LDHA in 1984, and its predicted amino acid sequence showed 92% homology with pig LDHA (Tsujibo et al. 1985). In 2001, Read and cols determined the crystal structure of the two main LDH isoforms, which provided evidence for the hypothesis that the different activity of the isoforms is likely to be due to the variation of charged residues peripheral to the active site, since both isoforms have very similar crystal structures (Read et al. 2001).
Regulation of LDHA synthesis is complex and not yet completely understood. It is known to be regulated by major transcription factors hypoxia-inducible factor 1 (HIF1) and c-Myc, in addition to forkhead box protein M1 (FOXM1) and Kruppel-like factor 4 (KLF4). Also, several factors may influence its transcription, such as lactate, cyclic adenosine monophosphate (cAMP), estrogen, ErbB2, and heat shock factor 1 (Valvona et al. 2015). Post-transcription regulation is made by phosphorylation and acetylation of amino acid residues, by factors like FGFR1 and SIRT2, and this kind of regulation is associated with cancer (Fan et al. 2011; Zhao et al. 2013).
A pathology associated with the LDHA gene is LDH deficiency, also called glycogen storage disease XI, where patients present a deletion in exon 6 of the gene, resulting in a frameshift that causes the LDHA subunit to contain 259 instead of 331 amino acids, leading to rapid degradation by immunological detection (Maekawa et al. 1990). The first case of LDHA deficiency was reported on an article where a family was studied after an 18-year-old male complained of exertional myoglobinuria and easy fatigue 10–12 h after hard exercise. A complete lack of the LDHA subunit was revealed in his erythrocytes, leukocytes, and in the intermediate vastus muscle, and the same was demonstrated in three of his five siblings. Furthermore, the H/M ratio suggested a partial absence of the M subunit in two siblings and in the parents (Kanno et al. 1980). After that, Maekawa and cols reported a case of a homozygous woman with LDHA deficiency on a screening for the disease on a province in Japan. A family analysis showed that the parents of the propositus are heterozygous individuals with LDHA deficiency, and her sister is also homozygous. In this case, the patient was asymptomatic, but she did not perform strenuous exercise (Maekawa et al. 1984). Later on, Kanno and cols analyzed the carbohydrate metabolism in these two families of the cases stated above and one other family, where the propositus presented excessive fatigue after exercise, severe cramps, and myoglobinuria, and his sister also presented fatigue and muscle stiffness. In all three families, the response to ischemic forearm work was evaluated, and they did not present an increase of venous lactate concentration after ischemic work, showing a significant increase of venous pyruvate. Also, glycolysis was significantly retarded in the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) step in the patient’s muscle, and for this reason, ATP production was severely impaired on those cells, causing the release of proteins like creatine kinase (CK) and myoglobin into the bloodstream (Kanno et al. 1988). Furthermore, in another study, a 16-year-old Japanese girl was reported presenting asymptomatic cutaneous lesions, and the electrophoretic analysis showed a major activity band of LDH1 and a very small band of LDH2. Moreover, erythrocyte LDH analysis revealed a single isozyme pattern, and similar analysis on the patient’s parent erythrocytes showed a noteworthy increase in the H-subunit/M-subunit ratio (H/M ratio). Skin LDH analysis showed that the epidermis of the diseased skin and scalp hair follicles were virtually devoid of LDH activity (Takayasu et al. 1991). All these reports show that this disease can be asymptomatic, and symptoms may only appear after strenuous exercise, such as fatigue and myoglobinuria, or it can show skin eruptions. Importantly, however, there is a latent risk of acute renal failure due to the myoglobinuria.
Altered energetic metabolism is one of the hallmarks of cancer. Tumor cells present a preference for glycolysis over oxidative phosphorylation even on the presence of oxygen, the so-called Warburg effect. This process can be understood as a selective growth advantage, where glycolysis is enhanced to produce energy, and other metabolic pathways are altered to promote protein and lipid synthesis, enabling rapid cell proliferation (Gatenby and Gillies 2004). Hence, enzymes that catalyze reactions of those pathways present a key role in this process, where alterations on enzyme’s isoforms can redirect the metabolic flux of its pathway, leading to an aberrant metabolism. Therefore, since LDHA is a key enzyme on the regulation of glycolysis, it plays an important role on cancer cells’ metabolism. Immunohistochemistry studies demonstrated overexpression of LDHA on cancer cells on several types of cancer, such as prostate cancer, pancreatic cancer, and breast cancer (He et al. 2015; Xian et al. 2015; Xiao et al. 2016). For prostate cancer patients, this overexpression was found to be concomitant to c-Myc overexpression, and correlated with TNM stage and tumor size, indicating poor prognosis. Reducing the c-Myc-LDHA signaling decreased tumor growth and metastasis (He et al. 2015). For breast cancer patients, the overexpression of LDHA was associated with cell proliferation, metastasis, and poor patient overall survival and disease-free survival (Xiao et al. 2016). Therefore, LDHA has been studied as a biomarker for many malignancies, including lymphoma, prostate cancer, renal cell carcinoma, and melanoma (Miao et al. 2013).
LDHA has also been extensively studied as a possible target for cancer therapy. Inhibition of LDHA by short hairpin RNA resulted on an increased oxygen consumption and oxidative phosphorylation, concomitant to a decrease of mitochondrial membrane potential and reduced rate of ATP production, especially under hypoxia (Fantin et al. 2006). Furthermore, inhibition of LDHA by the small-molecule inhibitor FX11 was found to decrease cell proliferation, migration, and invasion, as well as promoting apoptosis of prostate cancer cell lines (Xian et al. 2015). Similar results were found on osteosarcoma cell lines and also by genetic silencing of the enzyme (Gao et al. 2016). Interestingly, a role for LDHA on chemotherapy resistance was also observed by Feng and cols, where it was demonstrated that cisplatin-resistant oral cancer cells presented lower levels of LDHA, and Taxol-resistant cancer cells presented increased levels of LDHA. On this study, it was also shown that knockdown of LDHA sensitized cells to Taxol, but desensitized them to cisplatin treatment, whereas exogenous expression of LDHA sensitized cells to cisplatin, but desensitized them to Taxol (Feng et al. 2015).
LDHA has a pivotal function on energetic metabolism regulation. The conversion of NADH to NAD+ is fundamental for glycolysis maintenance on the absence of oxygen under physiological conditions. The absence of this enzyme can lead to LDH deficiency, an important pathology of metabolism, where patients present a risk of having acute renal failure. Furthermore, because of its importance on the metabolic reprogramming of cancer cells, intense research has revealed its possible role as a cancer biomarker, where it has been associated with poor prognosis. Also, this enzyme has been studied as a possible therapeutic target, where not only its inhibition decreased cancer cell proliferation, but it was also associated with chemotherapy resistance. Further studies are necessary to better evaluate LDHA role on cancer therapy and diagnosis, but several studies pointed out its potential. Since cancer remains a very lethal disease and current therapies are still inefficient, a new light brought by the Warburg effect theory provided a new view on carcinogenesis, and targeting cancer metabolism and its altered enzymes might be a more efficient approach on cancer treatment.
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