Quite a number of lung cancer patients undergo tumor recurrence and metastasis after radiotherapy, and almost one half of loco-regional failure occur within the radiation field, indicating that radioresistance is widely existed in NSCLC patients[19]. Many factors might be involved in the poor response to radiotherapy, including hypoxia, cancer stem cell phenotype, cell cycle redistribution and activated DNA repair ability[20, 21]. Cancer cells exhibited higher glucose intake and enhanced glycolysis, which providing energy and nutrients rapidly, known as Warburg effect[22]. LDHA also known as LDH5, is a key enzyme involving in the glycolysis, and play a vital role in tumor initiation, maintenance, progression, and metastasis[10]. LDHA is reported to be up-regulated in many kinds of cancer, and associated with poor prognosis, including breast cancer[23], colorectal cancer[7], liver cancer[24], bladder cancer[8] and prostate cancer[5, 9], etc,. Especially, in the era of immune therapy treatment, serum LDH levels seem also to be associated with the efficiency of PD1/PDL1 efficiency [25].
In our study, we found that LDHA were up-regulated in NSCLC cells using online data, verified by IHC in tumor tissues. Then, we further confirmed that LDHA overexpression was associated with worse survival in NSCLC patients and those received radiotherapy. Using GESA analysis, LDHA expression is positively related to enhanced glucose intake and glycolysis, which promote cancer cell growth and progression. In fact, cancer cell mitochondria and metabolism, also take part in mediating the response to irradiation in tumor, as they regulate multiple processes involved in DNA damage and repair[26]. LDHA overexpression also remodels tumor microenvironment with activated HIF-1 signaling pathways, which is associated with resistance to radiotherapy and resulted in poorer clinical outcomes [21, 27]. Therefore, LDHA up-regulation in NSCLC leads to increased levels of glycolysis and hypoxic microenvironment, which further contribute to development of resistance to radiotherapy.
The mechanism underlying the radiosensitive effect of LDHA inhibition might involve multiple biological processes, for example, reactive oxygen species (ROS) accumulation, cell cycle redistribution, increased DNA damage and DNA repair repression. As we know, LDHA inhibition drives cancer cells metabolism from glycolysis to mitochondrial respiration, which results in enhanced oxygen consumption and increased mitochondrial ROS production[14]. High levels of ROS not only induce cell apoptosis or autophagy, but also significantly lead to DNA injury and influence DNA damage response[17]. In our study, the synergistic effect of LDHA inhibition and irradiation are compensated to some extent after NAC treatment, indicating that ROS played a pivotal role in the effect.
Generally, DNA damage repair efficiency is considered as a major determinant of cell radiosensitivity. Especially, double-strand breaks (DSBs) tend to trigger genomic instability and always induce cell-killing effects[28]. In our study, reduced capacity of DNA repair was observed by COMET assay when LDHA expression was inhibited, and further theoretically verified using GSEA analysis. According to previous studies[29, 30], the process might be mediated by PI3K-Akt signaling pathway, which both involves in metabolic reprogramming and tumor cell responsiveness to radiation. Consequently, up-regulation of PI3K-Akt signaling pathway results in acquired radioresistance by enhancing aerobic glycolysis[31]. In fact, PI3K-Akt signaling pathway was indeed down-regulated in A549 cells treated with oxamate in our earlier study[32]. In addition, LDHA controls a most potent pathway of rapid ATP production in cancer cells and its blockage deprives cancer cells from a major energy pathway, which may shift cell metabolism from glycolysis to mitochondrial oxidative phosphorylation(OXPHOS) and generate more ROS[33]. Combination of oxamate with radiation becomes more efficient in suppressing ATP formation and reducing DNA repair ability[34, 35]. Of particular interest, one recent study revealed that tumor metabolites, including 2-hydroxyglutarate, fumarate hydratase, and α-ketoglutarate, hindered DNA repair by disrupting local chromatin signaling[36], demonstrating the importance of metabolism in DNA repair activity. Yet, the molecular crosstalk between glycolysis and DNA repair still needs to be further explored.
As for cell cycle distribution, we found that in both A549 and H1975 cells, after oxamate pretreatment, more cells entered G0/G1 cell cycle and fewer cells were found at S phase, while the ratios of cells at G2/M phase were not significantly influenced. As reported by our previous study, G0/G1 arrest was dependent on the activation of GSK-3β, accompanied by the inhibition of PI3K-Akt pathway[12]. Generally speaking, cells at S phase are the most resistant to radiation, while cells at G2/M phase are the most radiosenstive, and G0/G1 redistribution usually favors radioresistance [37]. Thus, the reduction of cells at radiation-resistant S phase might contribute to increased radiosensitivity in our study. Moreover, using GSEA analysis, we found that LDHA inhibition was associated with inactivation of G1/S DNA Damage Checkpoints signaling pathway[38], which further validated our hypothesis. Similarly, glycolysis inhibitor dichloroacetate was also reported to induce G0/G1 arrest and increase cell sensitivity to the X-ray irradiation in A549 cells [39, 40]. Of note, G2/M arrest was also observed in other cancer cell lines exposed to glycolysis inhibitors and reported to be associated with increased cell radiosensitivity[12, 41]. Therefore, the role of cell cycle re-distribution induced by glycolysis inhibition in modulation of radiosensitivity is still unclear, it’s might be an accompanying consequence, instead of upstream signaling initiation, and less critical than the sensitizing effects of energy deprivation[42].
In summary, LDHA inhibition by oxamate suppressed glycolysis and reduced cellular ATP levels, shifted cell metabolism from glycolysis to mitochondrial oxidative phosphorylation (OXPHOS) and generated more ROS, which might increase DNA injury and hinder DNA repair activity. LDHA inhibition also induced cell apoptosis and autophagy accompanied by cell cycle alternations. All these factors contributed to enhanced radiosensitivity in NSCLC cells (Fig. 7).
Of note, although inhibition of LDHA mainly induces cell apoptosis by generating ROS, different responses to glycolysis inhibition were observed in different cancer cells[12, 43, 44]. Many efforts are being made to identify biomarkers to predict the sensitivity of glycolysis inhibitors(including LDHA inhibitors), several genetic mutations have been reported to be associated with the efficiency of glycolysis inhibitors, including Kras[45], PI3K[46], AMPK-mTOR[47],and mitochondrial DNA (mtDNA ) mutations[48]. Our results showed the A549 cells with Kras mutation was less sensitive to the combination of LHDA inhibition and radiotherapy than H1975, reflecting the heterogeneity of lung cancer. In addition, it’s recently reported that only a part of NSCLC cell lines may benefit from the combination of radiotherapy and metabolic inhibition[49]. Thus, the radiosensitive effects of LDHA inhibition seems to be different in different cancer cells, efforts are still needed to identify predictive biomarkers and tested by clinical trials.