Loss of Lactate/Proton Monocarboxylate Transporter 4 Induces Ferroptosis via the AMPK/ACC Pathway and Inhibition of Autophagy on Human Bladder Cancer 5637 Cell Line.

Abstract

Ferroptosis and autophagy have an important role in the occurrence and development of cancer, and lactate in cells and microenvironment is one of the influencing factors of ferroptosis and autophagy. The lactate/proton monocarboxylate transporter 4 (MCT4), which is expressed in the cell membrane, regulates the transport of intracellular lactic acid and lactate. The knockout of MCT4 can affect intracellular and extracellular lactic acid levels, thereby affecting the growth, proliferation, and metastasis of tumor cells via regulation of the oxidative stress in cells. However, whether MCT4 affects ferroptosis and autophagy in bladder cancer cells remains unclear. Colony formation assay and bladder cancer xenograft animal model were used to assess the effect of MCT4 on the growth in 5637 cells. Reactive oxygen species (ROS) assay, lipid ROS assay, lipid peroxidation assay (MDA), and transmission electron microscopy were performed to assess the level of lipid peroxidation in 5637 cells. RNA-sequence, RT-PCR, and Western Blot were used to analyze the mechanism of MCT4 of ferroptosis and autophagy. AdPlus-mCherry-GFP-LC3B reporter system was used to detect the effect of MCT4 on autophagy in 5637 cells, and the effect of knockdown of MCT4 on apoptosis was analyzed by flow cytometry. The mRNA level of MCT4 was significantly upregulated in patients with bladder cancer, which was associated with a poor prognosis. In vivo and in vitro studies demonstrated that knockdown of MCT4 could inhibit the proliferation of bladder cancer cells. Furthermore, knockdown of MCT4 led to the significant increase of ROS and MDA levels in 5637 cells and ferroptosis in 5637 cells induced by ferroptosis inducers including RSL3 (APExBIO) and erastin (APExBIO) via inhibition of AMPK-related proteins. Moreover, knockdown of MCT4 inhibited autophagy in 5637 cells, while siMCT4 promoted inhibition of autophagy by CQ (an autophagy inhibitor), which increased the level of apoptosis. This study confirmed that knockdown of MCT4 could affect oxidative stress and induce ferroptosis and inhibition of autophagy, thus suggesting that MCT4 may be a potential target for the treatment of bladder cancer.