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

Background 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. Methods 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. Results 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. Conclusion 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.

[1]  Yue Wang,et al.  ZEB1 induces ROS generation through directly promoting MCT4 transcription to facilitate breast cancer. , 2022, Experimental cell research.

[2]  Hui Xiao,et al.  Britanin relieves ferroptosis-mediated myocardial ischaemia/reperfusion damage by upregulating GPX4 through activation of AMPK/GSK3β/Nrf2 signalling , 2021, Pharmaceutical biology.

[3]  Wei-Hua Yan,et al.  Integrative Analysis Identified MCT4 as an Independent Prognostic Factor for Bladder Cancer , 2021, Frontiers in Oncology.

[4]  P. Blanchette,et al.  AMPK-dependent phosphorylation is required for transcriptional activation of TFEB and TFE3 , 2021, Autophagy.

[5]  Qingwei Zhu,et al.  Lactate-induced M2 polarization of tumor-associated macrophages promotes the invasion of pituitary adenoma by secreting CCL17 , 2021, Theranostics.

[6]  J. Witjes.  Follow-up in non-muscle invasive bladder cancer: facts and future , 2020, World Journal of Urology.

[7]  A. Lenis,et al.  Bladder Cancer: A Review. , 2020, JAMA.

[8]  C. Booth,et al.  Trimodality Therapy for Muscle-Invasive Bladder Cancer: Concurrent Chemotherapy is Not Enough. , 2020, Journal of Clinical Oncology.

[9]  Xiaoying Lin,et al.  The AMPK-MFN2 axis regulates MAM dynamics and autophagy induced by energy stresses , 2020, Autophagy.

[10]  Jitendra Kumar Meena,et al.  Energy stress-mediated AMPK activation inhibits ferroptosis , 2020, Nature Cell Biology.

[11]  D. Klionsky,et al.  Ferroptosis is a type of autophagy-dependent cell death. , 2020, Seminars in cancer biology.

[12]  Shufang Zhang,et al.  [Research progress in mechanisms for drug-resistance and recurrence in bladder cancer]. , 2019, Zhong nan da xue xue bao. Yi xue ban = Journal of Central South University. Medical sciences.

[13]  J. Manzo-Merino,et al.  Lactate in the Regulation of Tumor Microenvironment and Therapeutic Approaches , 2019, Front. Oncol..

[14]  Guanbin Song,et al.  Targeted inhibition of MCT4 disrupts intracellular pH homeostasis and confers self-regulated apoptosis on hepatocellular carcinoma. , 2019, Experimental cell research.

[15]  M. Ruscica,et al.  Lactate Buildup at the Site of Chronic Inflammation Promotes Disease by Inducing CD4+ T Cell Metabolic Rewiring , 2019, Cell metabolism.

[16]  D. Krysko,et al.  Ferroptosis at the crossroads of cancer-acquired drug resistance and immune evasion , 2019, Nature Reviews Cancer.

[17]  Yingyu Chen,et al.  AMPK and Autophagy. , 2019, Advances in experimental medicine and biology.

[18]  Maheedhara R. Guda,et al.  An overview of MCT1 and MCT4 in GBM: small molecule transporters with large implications. , 2018, American journal of cancer research.

[19]  Yuzhuo Wang,et al.  Selective Inhibition of the Lactate Transporter MCT4 Reduces Growth of Invasive Bladder Cancer , 2018, Molecular Cancer Therapeutics.

[20]  E. Messing,et al.  Effect of Intravesical Instillation of Gemcitabine vs Saline Immediately Following Resection of Suspected Low-Grade Non–Muscle-Invasive Bladder Cancer on Tumor Recurrence: SWOG S0337 Randomized Clinical Trial , 2018, JAMA.

[21]  C. Fankhauser,et al.  Prevention of bladder cancer incidence and recurrence: nutrition and lifestyle , 2017, Current opinion in urology.

[22]  R. Shaw,et al.  AMPK: Mechanisms of Cellular Energy Sensing and Restoration of Metabolic Balance. , 2017, Molecular cell.

[23]  S. Lowe,et al.  Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function , 2017, Molecular cell.

[24]  B. Stockwell,et al.  Ferroptosis: Death by Lipid Peroxidation. , 2016, Trends in cell biology.

[25]  D. Tang,et al.  Ferroptosis: process and function , 2016, Cell Death and Differentiation.

[26]  A. Ballabio,et al.  Lysosomal calcium signaling regulates autophagy via calcineurin and TFEB , 2015, Nature Cell Biology.

[27]  M. R. Lamprecht,et al.  Ferroptosis: An Iron-Dependent Form of Nonapoptotic Cell Death , 2012, Cell.

[28]  D. Hardie,et al.  AMPK: a nutrient and energy sensor that maintains energy homeostasis , 2012, Nature Reviews Molecular Cell Biology.

[29]  B. Viollet,et al.  AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1 , 2011, Nature Cell Biology.

[30]  Hong-Gang Wang,et al.  Abstract 4834: Energy-dependent AMPK association with the ULK1-mTORC1 complex regulates autophagy , 2010 .