Cancer-Testis Antigen Lactate Dehydrogenase C4 in Human Cancers: Old and New Perspectives

Lactate dehydrogenase (LDH) isozymes are abundant in various
mammalian tissue types and accountable for the interconversion
of pyruvate and lactate during the last step of anaerobic glycolysis...


Introduction
Lactate dehydrogenase (LDH) isozymes are abundant in various mammalian tissue types and accountable for the interconversion of pyruvate and lactate during the last step of anaerobic glycolysis [1][2][3]. Human LDH consists of four subunits of two types, A and B, linking together to form five tetrameric isoenzymes (LDH-1 to LDH-5). Following these findings, the sixth isoenzyme, designated as LDH-X, was detectable in the mature human testis in the 1960s [4,5].
LDH-X is also termed LDH-C4/LDHC as it is a tetrameric protein composed of four identical C-subunits. It, therefore, exhibits utterly distinct enzymatic, physicochemical, and immunological properties from the other isoenzymes [6][7][8][9][10]. Intriguingly, the current progress of LDH-C4 in human and murine reveals that though sharing high amino acid identities, human and mouse LDH-C4 are biochemically different from each other regarding some properties, particularly one that LDH-C4 is confined to mature testis, spermatocytes, spermatids, and spermatozoa [4- 6,11]. LDH-C4 is associated with glucose metabolism and is essential in the maintenance of ATP concentrations in spermatozoa [11,[12][13][14][15].
Cancer/testis antigen (CTA) is, by definition, expressed only in the testis and tumor cells, absent in non-testis or noncancerous somatic cells [16]. LDH-C4 has been identified as a CTA molecule expressed in multiple malignancies, such as breast cancer (BC), lung cancer, and melanoma, with a high frequency of 47% in lung cancer, 44% in melanoma, 35% in breast carcinoma, and 15% in colon cancer. As suggested by an underlying mechanism, LDHC escapes from transcriptional repression, giving rise to significantly increased expression levels in a wide range of human tumor entities [17]. Our study series ascertained LDHC/LDH-C4 expressions in BC [18,19], liver cancer [20], nasopharyngeal carcinoma, lung cancer, and osteosarcoma and its predictive power as a biomarker [21].
Based on the latest discovery and our previous findings, emphasis will be given to the roles of LDH-C4 in human tumors.

LDH-C4 and Breast Cancer
Our BC research using an LDH-C4 inhibitor N-propyl oxamate has proven that endogenous LDH-C4 inhibition is crucial in the  [17]. But our results demonstrated much higher positive rates of serum and exosomal LDHC mRNA expressions of 91.66% (22/24) and 87.50% (21/24) in BC patients, respectively, versus the rates as low as 9.16% (11/120) and 6.67% (8/120) in healthy controls; the average LDH-C4 expression in the serum and exosomes of newly diagnosed BC cases were 9.79 and 8.87 folds of healthy controls [19]. Moreover, as the AUC values of serum and exosomal LDHC expression in discriminating BC from noncancerous specimens were 0.9587 and 0.9464, it could be used as a strong index for BC diagnosis [19].
Serum and exosomal LDHC levels are also associated with tumor size and HER2 and Ki-67 expressions in BC and with treatment efficacy and relapse, featuring a remarkable reduction in LDHC expression levels after the treatment or rebound LDHC levels since relapse [19]. By using high-throughput tissue microarray combined with the immunohistochemical assay, our finding demonstrated LDH-C4 protein expressions in BC tissues and their correlation with clinicopathological characteristics and BC prognosis [19]. LDH-C4 protein expression was predominantly detectable in the cytoplasm of BC cells but sparsely expressed in some nuclei. The positive rate of LDH-C4 in BC tissues was 91.55%, including low LDH-C4 expression (-/+) in 33.10% of BC tissues and high expression (+/++) in 66.90% of BC tissues. About 89% of adjacent normal tissues were free of LDH-C4, leaving only 11% with low LDH-C4 expression (-/+) [19]. Together with the survival analysis results that patients with high LDH-C4 levels were at higher risk of a poor prognosis, LDH-C4 is a useful indicator for prognosis prediction in BC.

LDH-C4 and Lung Cancer
Grunwald, et al. detected the mRNA expression levels of 7 CTAs (including LDHC) in NSCLC tissues and cell lines, and the positive rate of LDHC expression reached 25% in NSCLC tissues and completely undetectable in normal pulmonary specimens [22]. Histopathological subtyping revealed that LDHC mRNA was expressed in a high proportion of adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. They recommended the paired biomarker analysis of LDHC and CG genes for greater diagnostic power in NSCLC and used LDHC as a new tool for monitoring immunotherapy [22]. A study by Yen, et al. connected LDHC expression to cigarette smoking habits in lung cancer patients, especially in male patients with pleural effusion; the property of the aberrant LDHC expression between tumor entities of lung cancer makes the gene sensitive in predicting early-onset types like smoking-related lung adenocarcinoma [23]. The upregulation of LDHC gene expression is such a critical risk factor of lung cancer that LDH-C4 can serve as a potential marker for its diagnosis [23].
Consistently, our study with tissue microarrays also revealed that a high positive rate of LDH-C4 expression in adenocarcinoma of the lung of above 80% (unpublished data). Based on risk stratification, Kaplan-Meier curves disclosed a poor prognosis in patients with LDH-C4 overexpression (unpublished data). Correspondingly, a newly published study also confirmed that LDHC expression was significantly correlated with a poor prognosis of the lung adenocarcinoma patients; moreover, LDHC could promote proliferation, migration, invasion, and EMT in lung adenocarcinoma cells by triggering the PI3K/Akt/GSK-3β pathway [24]. Other studies of screening proteins related to cancer cell invasion and metastasis reported that endogenous LDH-C4 enhanced matrix metalloproteinase-9 (MMP-9) expression and lactic acid production, increasing the invasion and migration of malignant RCC cells [21,27,28]. Also, tumor-associated fibroblasts induced by substantial lactic acid build-up may propel hyaluronic acid secretion, creating an environment for the invasion and migration of aggressive RCC cells as well as exuberant metastasis [21].

LDH-C4 and Nasopharyngeal Carcinoma (NPC)
LDHC mRNA expression was proved undetectable in patients with head and neck squamous carcinoma (HNSCC) based on the analysis of CTA expression data from 51 cases [29]. However, our

LDH-C4 and Other Malignant Tumors
LDHC gene is also differentially expressed in melanoma and prostatic cancer and noncancerous specimens, implying its role in the occurrence and development of the two cancer types [17]. A study reported LDHC expression inhibition in osteosarcoma and that its complete transcript and metabolic characteristics were conducive to the early detection and diagnosis of osteosarcoma with high accuracy [30]. Our data for LDHC gene expression in hepatocellular carcinoma (HCC) demonstrated that 68% and 60% LDHC-positive specimens were detectable in the serum and serum-derived exosomes, showing AUCs of serum and exosomal LDHC in differentiating HCC from noncancerous controls of 0.8382 and 0.9451, respectively [20]. Consistently, LDH-C4 protein overexpression was found sensitive in predicting a poor prognosis of HCC patients [20]. Some studies have disclosed the aberrant LDHC transcript amplified in spermatocytic seminomas, osteosarcoma, and some melanoma [30][31][32]. Generally, the differentially expressed LDHC gene is detectable across various cancer types, and the expression status as well as the biological functions were summarized in Table 1. Melanoma cell lines (A375M and C81-61) CpG island hypomethylation and transcription factor Sp1 and CREB were found to play a major role in LDHC transcription [30] Lung adenocarcinoma LDHC served as a candidate oncogene in the carcinogenesis of smoking-related lung adenocarcinoma [23] Non-small cell lung cancer 25% cases were LDHC-positive; LDHC could not be activated by genomic hypomethylation; LDH-C4 is a prognostic factor; LDHC enhanced proliferation, migration, invasion, and EMT in lung adenocarcinoma cells by triggering the PI3K/ Akt/GSK-3β pathway [22,24] Hepatocellular carcinoma 68% and 60% of serum and exosomal LDHC-positive cases; patients with high LDH-C4 expression (+ + / + + +) in HCC tissues accounted for 55.84%. LDHC/LDH-C4 as a useful biomarker for early diagnosis, efficacy evaluation, relapse monitoring, and prognosis prediction for HCC [20] Breast cancer 91.66% and 87.50% of serum and exosomal LDHC-positive cases and 91.55% of LDH-C4-positive patients. LDHC/LDH-C4 exhibited high discrimination power in diagnosis, efficacy evaluation, relapse monitoring, and prognosis prediction for BC [18,19] Spermatocytic seminomas LDHC was specifically expressed in spermatocytic seminomas [32] Osteosarcoma LDHC was differentially expressed in osteosarcoma and normal specimens [30]

Conclusion and Perspectives
Despite the conventional understanding of LDH-C4 as a key isoenzyme essential for sperm motility, capacitation, and fertilization, its encoding gene LDHC is a CTA family member and a potential therapeutic target of immunotherapy against several cancer types.