Chaperone-mediated autophagy promotes breast cancer angiogenesis via regulation of aerobic glycolysis

PLOS ONE RESEARCH ARTICLE Chaperone-mediated autophagy promotes breast cancer angiogenesis via regulation of aerobic glycolysis Rui Chen1☯, Peng Li2☯, Yan Fu3☯, Zongyao Wu1, Lijun Xu1, Junhua Wang4, Sha Chen5, Mingzhen Yang5, Bingjie Peng5, Yao Yang3, Hongwei Zhang4, Qi Han4,5*, Shuhui Li ID5* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Tibetan Traditional Medical College, Lhasa, China, 2 Yantai Mountain Hospital, Yantai, Shandong, China, 3 General Hospital, Western Theater Command, Chengdu, Sichuan, China, 4 General Hospital of Tibet Area Military Command, Lhasa, China, 5 Army Medical University (Third Military Medical University), Chongqing, China ☯ These authors contributed equally to this work. * (SL); (QH) Abstract OPEN ACCESS Citation: Chen R, Li P, Fu Y, Wu Z, Xu L, Wang J, et al. (2023) Chaperone-mediated autophagy promotes breast cancer angiogenesis via regulation of aerobic glycolysis. PLoS ONE 18(3): e0281577. https://doi.org/10.1371/journal. pone.0281577 Editor: Masuko Ushio-Fukai, Medical College of Georgia at Augusta University, UNITED STATES Received: May 5, 2022 Accepted: January 26, 2023 Published: March 13, 2023 Copyright: © 2023 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting information files. Funding: This work was supported by National Natural Science Foundation of China (82072946) and Natural Science Foundation of Tibet (XZ202101ZR0109G). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Evidence shows that chaperone-mediated autophagy (CMA) is involved in cancer cell pathogenesis and progression. However, the potential role of CMA in breast cancer angiogenesis remains unknown. We first manipulated CMA activity by knockdown and overexpressing of lysosome-associated membrane protein type 2A (LAMP2A) in MDA-MB-231, MDA-MB436, T47D and MCF7 cells. We found that the tube formation, migration and proliferation abilities of human umbilical vein endothelial cells (HUVECs) were inhibited after cocultured with tumor-conditioned medium from breast cancer cells of LAMP2A knockdown. While the above changes were promoted after cocultured with tumor-conditioned medium from breast cancer cells of LAMP2A overexpression. Moreover, we found that CMA could promote VEGFA expression in breast cancer cells and in xenograft model through upregulating lactate production. Finally, we found that lactate regulation in breast cancer cells is hexokinase 2 (HK2) dependent, and knockdown of HK2 can significantly reduce the ability of CMAmediated tube formation capacity of HUVECs. Collectively, these results indicate that CMA could promote breast cancer angiogenesis via regulation of HK2-dependent aerobic glycolysis, which may serve as an attractive target for breast cancer therapies. Introduction Breast cancer is the most common cancer and the leading cause of cancer death in women worldwide, many factors contribute to initiation and progression of breast cancer [1, 2]. As angiogenesis is essential for tumor growth and metastasis, inhibiting tumor-associated angiogenesis has long been a promising target in limiting cancer progression [3, 4]. Vascular endothelial growth factor A (VEGFA) is a major player in angiogenesis among the VEGF family that promotes angiogenesis through binding and activating the tyrosine kinase receptor VEGFR2 [5]. Tumor cells always activate and initiate angiogenesis by releasing VEGFA PLOS ONE | https://doi.org/10.1371/journal.pone.0281577 March 13, 2023 1 / 14 PLOS ONE Chaperone-mediated autophagy promotes breast cancer angiogenesis via regulation of aerobic glycolysis to improve their microenvironment in many physiological and pathological conditions, such as hypoxia and high levels of lactate [3, 6]. Lactate, the obligatory product of aerobic glycolysis, plays an important role in angiogenesis, immune escape, cell migration and metastasis [7]. Cells undergo degradation of intracellular components by lysosomes, also known as autophagy, which is crucial for cellular homeostasis and adaptation to various forms of stress [8]. Of the three different kinds of autophagic pathways, chaperone-mediated autophagy (CMA) is unique for the selective degradation of damaged soluble cytosolic proteins by recognizing peptide sequence motif (KFERQ) via heat shock cognate protein of 70 kDa (HSC70) [9], then the targeted substrate binds to lysosomes through the lysosome-associated membrane protein type 2A (LAMP2A), key protein in the CMA [10, 11], and undergoes degradation. Recently, there has been growing interest in how CMA impacts cancer cell pathogenesis and progression. Blockage of CMA reduces tumor growth by reducing glycolytic flux and/or the accumulation of glycolytic intermediates in lung cancer cells and melanoma [12, 13]. CMA can also promote lung cancer cell survival through selective stabilization of the pro-survival protein, MCL1 [14]. Inhibition of LAMP2A also induces cell death by AKT1 and ROS pathway in breast cancer cells [15]. Evidence shows that key glycolytic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and pyruvate kinase muscle isozyme (PKM), aldolase, and HIF1A are all identified as substrate of CMA [16, 17]. However, the link among CMA, glycolysis and breast cancer angiogenesis has not been explored. Thus, we hypothesized that CMA regulating breast cancer angiogenesis may be associated with aerobic glycolysis. Materials and methods Animal study 20 four-six-week-old female BALB/c nude mice in total were randomly divided into four groups, and each group had five mice (n = 5). Mice were held at the Experimental Animal Center of Army Medical University for one week before injection, and the health status and well-being of the mice were checked twice a day by the animal care staff. All animal studies were approved by the Ethics Committee of Army Medical University and all the procedures were performed according to the regulations made by the Experimental Animal Center of Army Medical University. Mice were injected with MDA-MB-436 cells (in 200 μl of serumfree RPMI-1640 medium at 1 × 107 cells/mouse) after LAMP2A knockdown or overexpression at the right flank. When tumor volumes of negative and control groups reached about 150 mm3 (tumor size = length × width × width × 0.5), animals were sacrificed by CO2 euthanasia, and all efforts were made to minimize suffering, then the subcutaneous tumors were collected and used for Western blot. Cell culture Human breast cell lines of MCF7, MDA-MB-231, T47D and MDA-MB-436 were obtained from the cell bank of the Com (...truncated)