The novel function of tumor protein D54 in regulating pyruvate dehydrogenase and metformin cytotoxicity in breast cancer.

The role of tumor protein D54 in breast cancer has not been studied and its function in breast cancer remains unclear. In our previous pharmacogenomic studies using lymphoblastoid cell line (LCL), this protein has been identified to affect metformin response. Although metformin has been widely studied as a prophylactic and chemotherapeutic drug, there is still a lack of biomarkers predicting the response to metformin in breast cancer. In this study, we revealed the novel function of TPD54 in breast cancer through understanding how TPD54 altered the cancer cell sensitivity to metformin. The role of TPD54 in altering cellular sensitivity to metformin treatment was carried out by either knockdown or overexpression of TPD54, followed by measuring cell viability and reactive oxygen species (ROS) production in MCF7 breast cancer cell line and breast cancer patient-derived xenografts. Functional analysis of TPD54 in breast cancer cells was demonstrated by studying TPD54 protein localization and identification of potential binding partners of TPD54 through immunoprecipitation followed by mass spectrometry. The effect of TPD54 on pyruvate dehydrogenase (PDH) protein regulation was demonstrated by western blot, immunoprecipitation, and site-directed mutagenesis. TPD54 inhibited colony formation and enhanced cellular sensitivity to metformin treatment in MCF7 cells and breast cancer patient-derived xenografts. Mechanistic study indicated that TPD54 had mitochondrial localization, bound to and stabilized pyruvate dehydrogenase E1α by blocking pyruvate dehydrogenase kinase 1 (PDK1)-mediated serine 232 phosphorylation. TPD54 knockdown increased PDH E1α protein degradation and led to decreased PDH enzyme activity, which reduced mitochondrial oxygen consumption and reactive oxygen species (ROS) production, thus contributing to the resistance of breast cancer cells to metformin treatment. We have discovered a novel mechanism by which TPD54 regulates pyruvate dehydrogenase and affects the sensitivity of breast cancer to metformin treatment. Our findings highlight the important post-translational regulation of PDK1 on PDH E1α and the potential application of TPD54 as a biomarker for selecting tumors that may be sensitive to metformin therapy. These provide new insights into understanding the regulation of PDH complexes and the resistance mechanisms of cancer cells to metformin treatment.