Decreased lactate dehydrogenase B expression enhances claudin 1-mediated hepatoma cell invasiveness via mitochondrial defects

Abstract

Aerobic lactate production of which the final step is executed by lactate dehydrogenase (LDH) is one of the typical phenotypes in invasive tumor development. However, detailed mechanism of how LDH links to cancer cell invasiveness remains unclear. This study shows that suppressed LDHB expression plays a critical role in hepatoma cell invasiveness by inducing claudin-1 (Cln-1), a tight junction protein, via mitochondrial respiratory defects. First, we found that all the SNU human hepatoma cells with increased glycolytic lactate production have the defective mitochondrial respiratory activity and the Cln-1-mediated high invasive activity. Similar results were also obtained with human hepatocellular carcinoma tissues. Unexpectedly, the increased lactate production was due to LDH isozyme shifts to LDH5 by LDHB down-expression rather than LDHA induction, implying the importance of LDHB modulation. Second, LDHB knockdown did not only trigger Cln-1 induction at the transcriptional level, but also induced respiratory impairment. Interestingly, most respiratory inhibitors except KCN induced Cln-1 expression although complex I inhibition by rotenone was most effective on Cln-1 induction. Respiratory defect-mediated Cln-1 induction was further confirmed by knockdown of NDUFA9, one of complex I subunits. Finally, ectopic expression of LDHB attenuated the invasiveness of both SNU 354 and 449 cells whereas LDHB knockdown significantly augmented the invasiveness of Chang cells with Cln-1induction. The increased invasive activity by LDHB modulation was clearly reversed by knocking-down Cln-1. Taken together, our results suggest that LDHB suppression plays an important role in triggering or maintaining the mitochondrial defects and then contributes to cancer cell invasiveness by inducing Cln-1 protein.

Introduction

Increased aerobic glycolysis, continuous conversion of glucose to lactic acid in the presence of oxygen (i.e. the Warburg effect), is a distinctive hallmark of solid tumors [1], [2]. Because it is closely associated with increased metastasis in some cancers [3], [4], aerobic glycolysis is further considered a metabolic signature for invasive cancer. The glycolytic phenotype may be the result of adaptation to environmental constraints such as intermittent hypoxia in premalignant lesions. This hypothesis well corresponds to the concept termed ‘Pasteur effect,’ which glycolysis is inhibited by the presence of oxygen [5]. However, enhanced glycolysis persists in metastatic malignant cancers, even after increased angiogenesis restores a normoxic environment. This suggests that increased glycolysis is not just a passive response, but an active cellular strategy to confer a selective advantage for malignant progression by affecting acid-mediated matrix degradation, immune protection, and the dominant growth of cancer cells in an altered cellular environment [2], [6], [7], [8]. In addition to the metastatic ability, cell growth of malignant tumor persistently depends on glycolysis in normoxic culture conditions [9]. This suggests that augmented glycolysis may positively suppress or impair aerobic mitochondrial ATP production machinery, resulting in cellular dependence on glycolytic ATP. Evidence supporting this active strategy is that many glycolytic enzymes are upregulated by oncogenes such as Ras, Src, and Her2/Neu [10], [11]. Therefore, glycolytic activation and consequent aerobic glycolysis may also play a critical role in tumor progression associated with genetic alterations [12]. However, the underlying mechanisms are poorly understood.

Cultured cells derived from tumors maintain this altered metabolism when cultivated under normoxic conditions [2], [3], indicating that aerobic glycolysis is not just a transient result due to in vivo tumor microenvironment, but may be constitutively upregulated through stable genetic or epigenetic changes. Regulation of lactate dehydrogenase (LDH) plays a key role in this, since LDH is the alternative supplier of NAD⁺ in the absence of mitochondrial oxidation. Moreover, LDH produces lactate: lactate causes chronic acidification of the intratumoral microenvironment, which in turn helps drive cancer cell metastasis [13], [14].

Enzymatically functional LDH consists of four subunits, and there are two types of subunits designated M (muscle-type; LDHA gene product) and H (heart-type; LDHB gene product). Normal cells can contain five different LDH isozymes with different substrate reactivities as a result of the five different combinations of the two different subunits: LDH1 (H4); LDH2 (MH3); LDH3 (M2H2); LDH4 (M3H); LDH5 (M4) [15]. The expression levels of LDHA and LDHB expression determine the cell's isozyme pattern. LDH5 effectively catalyzes the conversion of pyruvate to lactate [16], and an isozyme shift to LDH5 has been linked with metastatic cancer [17], [18]. Furthermore, the importance of LDHA modulation in cancer progression is highlighted by reports that oncogenes can upregulate LDHA in tumor cells. For example, LDHA transcription is directly activated by cMyc and is indirectly induced by other oncogenes via hypoxia-inducing factor α (HIF-1α) stabilization [19], [20], [21]. However, it is not clear whether LDHA upregulation fully explains the stable and persistent isozyme shift toward LDH5 in cancer cells.

Hepatocellular carcinoma (HCC) is an increasingly common malignant tumor, with worldwide incidence of 5.5–14.9 per 100,000 inhabitants [22], [23]. Currently, liver transplantation and tumor resection are the most effective standard therapies. Although these procedures provide 5-year survival rates of 70%, this rate is only for patients within the Milan criteria (single tumor ≤ 5 cm in size or up to three tumors ≤ 3 cm in size) [24]. More than 60% of patients do not fall into these criteria because HCC is often diagnosed only after the size of tumor becomes larger, thereby resulting in survival time raging from 3 to 16 months [23]. Moreover, these therapeutic procedures are not complete cure, as half of the treated patients experience tumor recurrence within 3 years. Thus, alternative drug-based therapies for HCC are currently being tested and applied despite their low survival rates. High lethality with drug-based therapies is mainly due to its resistance to existing anticancer agents [25]. Glycolysis inhibition has recently been proposed to overcome drug resistance [9], but it is unknown whether this strategy is reasonable and would also be effective in treating HCC. Present study demonstrated the link of LDH isozyme shift via LDHB suppression to mitochondrial respiratory defects and claudin-1 induction, and their contribution to hepatoma cell invasiveness. Our results provide a new insight into the metabolic and molecular backgrounds of hepatocellular carcinoma for developing a novel drug based on glycolysis inhibition.