Dados do Trabalho

Título

Characterization of the role of protein pyruvate dehydrogenase kinase 3 (PDK3) in melanoma

Introdução

Pyruvate dehydrogenase kinase (PDK) plays a relevant role in metabolic reprogramming in cancer by inhibiting the pyruvate dehydrogenase complex (PDC) and redirecting pyruvate flux towards lactate production even in the presence of oxygen. The PDK3 isoform is described as overexpressed in melanomas and associated with increased cell survival and chemotherapy resistance. Despite the increased survival using vemurafenib, a BRAFV600E inhibitor, the prognosis of melanoma patients remains unfavorable due to developed resistance.

Objetivo

To evaluate, in vitro, the expression of PDK3 and the effect of PDK3 knockdown (KD) in functional assays (viability, proliferation, colony formation capacity, apoptosis, migration, and cellular invasion) and metabolic parameters (glucose consumption and lactate efflux).

Métodos

Both parental and vemurafenib-resistant melanoma cell lines A375 and SK-MEL-19 were used. PDK3 expression was assessed by Western blotting and PDK3 KD was achieved through reverse transfection using siRNA. Following PDK3 KD, cell viability was assessed using the Sulforhodamine B assay, cell proliferation through BrdU incorporation, and metabolic parameters as glucose and lactate extracellular levels using commercial kits - Glucose Colorimetric Assay (SpinReact) and Lactate Assay (SpinReact).

Resultados

An increased PDK3 expression was observed in both resistant cell lines when compared to their respective parental cell lines. PDK3 KD using siRNA reduced approximately 50% of PDK3 expression in both parental and resistant cell lines, 72 hours after reverse transfection. Subsequently, it was observed that PDK3 KD did not influenced cell viability, but led to a significant increase in cell proliferation in the vemurafenib-resistant SK-MEL-19 cell line. Furthermore, PDK3 KD did not significantly influence glucose consumption, while it significantly reduced lactate efflux in the parental A375 cell line and significantly increased lactate efflux in the vemurafenib-resistant SK-MEL-19 cell line.

Conclusões

The PDK3 KD did not decrease cell viability and proliferation as expected, which could be explained by the activity of other isoforms of the PDK complex. Further functional assays following PDK3 KD and investigation into the activity of others PDKs will be performed.

Palavras-chave

Pyruvate dehydrogenase kinase 3; Melanoma; Metabolic reprogramming

Financiador do resumo

CAPES, FAPESP (2022/13443-7), PAIP (Hospital de Câncer de Barretos)