Anaplastic thyroid carcinoma (ATC) is one of the most lethal and aggressive human malignancies with a high mortality rate and a median survival duration of 4 to 6 months. Current therapeutic approaches to ATC have limited efficacy, heightening the urgency for additional therapeutic options for this disease. Lactate dehydrogenase A (LDHA) is a critical and essential glycolytic enzyme responsible for conversion of pyruvate into lactate in anaerobic and aerobic glycolysis (Warburg effect). LDHA overexpression is a hallmark of aggressive malignancies and has been linked to tumor initiation, reprogramming, and progression in multiple tumor types. We established an inducible LDHA knockdown cell line to investigate the actual contribution of LDHA to tumorigenesis in vitro and in vivo.
Anaplastic thyroid cancer (ATC) cell lines were authenticated by short tandem repeat profiling. Inducible LDHA knockdown cell lines were established by using pINDUCER-EmGFP-shLDHA lentivirus system. LDHA and LDHB expression were determined by immunoblotting. LDHA enzyme activity and lactate production were determined by biochemical assay kits. Functional validation of LDHA knockdown was measured kinetically using hyperpolarized 13C-Pyruvate MRI. We assessed the functional contribution of LDHA in cell apoptosis, proliferation and colony formation following exposure to doxycycline (DOX) to induce LDHA knockdown. We assessed the anti-tumor effects of LDHA suppression in an orthotopic xenograft tumor mouse model. Xenograft tumor growth was monitored by serial bioluminescence imaging and physical measurement.
Anaplastic thyroid cancer cells demonstrated increased LDH enzyme activation. LDHA expression was decreased following exposure to DOX (0.01-5 µg/mL) for 24-96 hours in a dose-dependent manner with no changes in LDHB expression. Inhibition of LDHA by inducible shRNA led to decreased LDH enzyme activation and lactate production. LDHA suppression resulted in decreased nLac values and conversion ratio of 13C-pyruvate to lactate as measured by HP-MRI. Decreased LDHA activation decreased cell proliferation and colony formation following DOX treatment. Decreased LDHA protein levels and LDH activity were determined in ATC tumors following treatment with DOX. In vivo suppression of LDHA significantly reduced ATC orthotopic tumor growth compared with control tumors and significantly prolonged median survival time.
Targeting of LDHA in ATC affected tumor function and growth in vitro and in vivo. Decreased LDHA activation led to decreased cell proliferation and colony formation of ATC cell lines. LDHA activity is required for tumor proliferation and its suppression resulted in significant tumor growth delay. Understanding the functional role of LDHA supports the development of new anti-metabolic therapeutic strategies to treat aggressive carcinomas.