Lactate dehydrogenase inhibition affects homologous recombination repair independently of cell metabolic asset; implications for anticancer treatment

https://doi.org/10.1016/j.bbagen.2020.129760Get rights and content

Highlights

  • The increased glycolytic rate of cancer cells facilitates DNA repair and confers resistance to chemotherapeutic agents.

  • LDH is a key enzyme in maintaining the glycolytic flux of cancer cells

  • Inhibition of LDH was found to dampen homologous recombination repair and increase the antineoplastic efficacy of Olaparib

Abstract

Background

Cancer cells show highly increased glucose utilization which, among other cancer-essential functions, was found to facilitate DNA repair. Lactate dehydrogenase (LDH) activity is pivotal for supporting the high glycolytic flux of cancer cells; to our knowledge, a direct contribution of this enzyme in the control of DNA integrity was never investigated. In this paper, we looked into a possible LDH-mediated regulation of homologous recombination (HR) repair.

Methods

We identified two cancer cell lines with different assets in energy metabolism: either based on glycolytic ATP or on oxidative reactions. In cells with inhibited LDH, we assessed HR function by applying four different procedures.

Results

Our findings revealed an LDH-mediated control of HR, which was observed independently of cell metabolic asset. Since HR inhibition is known to make cancer cells responsive to PARP inhibitors, in both the cellular models we finally explored the effects of a combined inhibition of LDH and PARP.

Conclusions

The obtained results suggest for LDH a central role in cancer cell biology, not merely linked to the control of energy metabolism. The involvement of LDH in the DNA damage response could suggest new drug combinations to obtain improved antineoplastic effects.

General significance

Several evidences have correlated the metabolic features of cancer cells with drug resistance and LDH inhibition has been repeatedly shown to increase the antineoplastic power of chemotherapeutics. By shedding light on the processes linking cell metabolism to the control of DNA integrity, our findings also give a mechanistic explanation to these data.

Introduction

Cancer can be essentially considered a metabolic disease, characterized by aberrant and/or dysregulated glucose metabolism and bioenergetics [1]. Neoplastic cells typically show highly increased rates of glucose uptake and glycolysis, after which the obtained pyruvate is converted to lactate by lactate dehydrogenase (LDH). In cells with defective mitochondrial function or residing in hypoxic tissue niches, lactate is usually extruded in the microenvironment; otherwise, this metabolite can be reconverted to pyruvate in mitochondria, providing additional energy fuel [2].

The highly increased glucose consumption observed in cancer cells is likely dictated by their continuous need of biosynthetic precursors, required to build up new macromolecules and to sustain the unrestricted cell proliferation. As well-known and just as an example, the sugar backbone of nucleotides is obtained from glucose-6-phosphate through the pentose phosphate pathway, which is also an essential source of NADPH, the “reducing power” driving anabolic processes [3].

The enhanced glucose metabolism was also found to promote other cancer-essential functions, such as metastatic spread [4], resistance to apoptosis [5] and genome stability [6]. Among these, the maintenance of a certain level of DNA integrity is a crucial function for supporting active proliferation, even for cancer cells already bearing genetic mutations. Interestingly, elevation of glycolysis was found to facilitate DNA repair and to confer cancer cells resistance to ionizing radiation [7]. Furthermore, considerable amounts of evidences suggest that inhibition of glycolysis leads to compromised DNA repair [8], which is accompanied by energy depletion. However, the mechanisms linking the glycolysis-based metabolic program to radiation resistance have not been fully understood, although alteration in pH and lactate levels have been implicated [9].

In the recent years, inhibition of the metabolic features of cancer cells began to be viewed as an attractive therapeutic option. In this context, LDH appears to be one of the most interesting potential targets for designing an anti-metabolic cancer treatment [10]. This enzyme is constantly overexpressed in cancer cells and, because of its position at the end of glycolytic pathway, it is considered not necessary for the viability of normal cells, which mainly catabolize pyruvate via the TCA cycle. Recently, the search of LDH inhibitors with drug-like properties has involved a number of pharmaceutical industries and research laboratories worldwide [11]. These efforts led to the identification of a number of small molecule inhibitors, which gave promising results in some preclinical settings and recently, a research team from the NCI announced the identification of a novel LDH inhibitor which was found to reduce tumor growth in vivo [12]. During these studies, impairment of energy metabolism obtained through LDH inhibition or downregulation was found to dampen some key features linked with the dysregulated cancer cell proliferation, such as the enhanced clonogenic potential and the metastatic spread [13,14]. Recent experiments addressing the function of LDH in cancer cell biology also evidenced for this enzyme a possible role beyond the mere management of pyruvate / lactate balance in glucose metabolism. In fact, similar to other glycolytic enzymes, LDH was found to be a protein “moonlighting” in cell nucleus [15], where it takes part in transcriptional complexes regulating gene expression [16]. In some experimental settings, the LDH-related antineoplastic effect was also found to be linked to LDH protein depletion rather than to glycolysis inhibition [14]. Furthermore, increasing evidences suggest for lactate a role as signaling factor, involved in the modulation of gene expression [9,17,18]. This metabolite was found to decrease chromatin compactness and enhance gene expression, an effect linked to histone hyperacetylation [17]. In some contexts, the increased gene expression was also found to be mediated by the stimulation of a membrane receptor (HCA1/Gpr81) caused by the released metabolite. As a result, enhanced DNA repair capacity was observed [18].

Based on the above exposed premises, the experiments reported in the present paper were aimed at exploring a possible direct role of LDH in the DNA damage response (DDR). Our attention was focused on homologous recombination (HR) repair, a critical pathway to restore with high fidelity non-replication-associated double strand breaks (DSBs) and to mend collapsed replication forks. Intriguingly, cancer cells with defective HR show increased sensitivity to chemotherapeutic agents [19,20] and HR inhibition was suggested as a possible strategy to improve cancer cell response to therapy [21].

For these experiments we identified two human cancer cell models characterized by a different asset in energy metabolism: high reliance on glycolytic ATP production (BxPC-3 pancreatic adenocarcinoma) and, at the opposite, energy metabolism mainly based on oxidative reactions (SW620 colon adenocarcinoma). By using oxamate (OXA), a well-known LDH inhibitor with consolidated use [22], we tried to shed light on a possible LDH contribution in HR repair.

Section snippets

Cell cultures and treatments

BxPC3 and SW620 cells were grown in RPMI 1640 and DMEM medium, respectively. Both media were supplemented with 100 U/ml penicillin/streptomycin, 2 mM glutamine and 10% FBS. All the materials used for cell culture and reagents were obtained from Sigma-Aldrich, unless otherwise specified. Oxamate (OXA) and Olaparib (OLA, Selleckchem) were administered in culture medium. In OLA-including experiments, media were supplemented with 0.6% DMSO.

ATP and lactate levels

For ATP assay, 2 × 104 cells were plated in clear bottom

Metabolic characterization of SW620 and BxPC-3 cultures

To characterize the metabolic asset of SW620 and BxPC-3 cells we evaluated the expression of the two LDH isoforms (LDH-A and -B); furthermore, we assessed the levels of lactate and ATP in cultures exposed to oxamate (OXA), a well-studied LDH inhibitor, active in the millimolar range. Results are shown in Fig. 1.

The bar graph in Fig. 1A shows the relative levels of LDH-A and -B measured in the two cultures, normalized on the LDH-A expression of SW620 cells. Both cultures showed a clear

Discussion

Poor drug response is a major hitch limiting the success of anticancer therapy. This feature was found to be correlated with cell metabolic asset [8], but the role of LDH in this phenomenon was never extensively studied, although, as stated in the Introduction, this enzyme is a major player in cancer cell metabolism [10]. The data of our preliminary investigation suggest an LDH-mediated control on HR, the most critical DDR pathway for restoring chromosome integrity and, hence, potentially

Funding

This work was supported by Cornelia and Roberto Pallotti's Legacy for Cancer Research, Associazione Italiana per la Ricerca sul Cancro AIRC (Progetto IG 2018, id 21386), and by University of Bologna (RFO funds).

Declaration of Competing Interest

The authors declare that there are no conflicts of interest.

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