Key Laboratory of Laboratory Medical Diagnostics, Chinese Ministry of Education, Chongqing Medical University, Chongqing, 400016, China.
Department of Cell Biology and Medical Genetics, Basic Medical School, Chongqing Medical University, Chongqing, 400016, China.
J Exp Clin Cancer Res. 2021 May 14;40(1):168. doi: 10.1186/s13046-021-01933-7.
Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with poor prognosis and limited treatment options. Hypoxia is a key hallmark of TNBC. Metabolic adaptation promotes progression of TNBC cells that are located within the hypoxic tumor regions. However, it is not well understood regarding the precise molecular mechanisms underlying the regulation of metabolic adaptions by hypoxia.
RNA sequencing was performed to analyze the gene expression profiles in MDA-MB-231 cell line (20% O and 1% O). Expressions of Slc6a8, which encodes the creatine transporter protein, were detected in breast cancer cells and tissues by quantitative real-time PCR. Immunohistochemistry was performed to detect SLC6A8 protein abundances in tumor tissues. Clinicopathologic correlation and overall survival were evaluated by chi-square test and Kaplan-Meier analysis, respectively. Cell viability assay and flow cytometry analysis with Annexin V/PI double staining were performed to investigate the impact of SLC6A8-mediated uptake of creatine on viability of hypoxic TNBC cells. TNBC orthotopic mouse model was used to evaluate the effects of creatine in vivo.
SLC6A8 was aberrantly upregulated in TNBC cells in hypoxia. SLC6A8 was drastically overexpressed in TNBC tissues and its level was tightly associated with advanced TNM stage, higher histological grade and worse overall survival of TNBC patients. We found that SLC6A8 was transcriptionally upregulated by p65/NF-κB and mediated accumulation of intracellular creatine in hypoxia. SLC6A8-mediated accumulation of creatine promoted survival and suppressed apoptosis via maintaining redox homeostasis in hypoxic TNBC cells. Furthermore, creatine was required to facilitate tumor growth in xenograft mouse models. Mechanistically, intracellular creatine bolstered cell antioxidant defense by reducing mitochondrial activity and oxygen consumption rates to reduce accumulation of intracellular reactive oxygen species, ultimately activating AKT-ERK signaling, the activation of which protected the viability of hypoxic TNBC cells via mediating the upregulation of Ki-67 and Bcl-2, and the downregulation of Bax and cleaved Caspase-3.
Our study indicates that SLC6A8-mediated creatine accumulation plays an important role in promoting TNBC progression, and may provide a potential therapeutic strategy option for treatment of SLC6A8 high expressed TNBC.
三阴性乳腺癌（TNBC）是乳腺癌中侵袭性最强的亚型，预后较差，治疗选择有限。缺氧是 TNBC 的一个关键标志。代谢适应促进了位于缺氧肿瘤区域内的 TNBC 细胞的进展。然而，对于缺氧如何精确调节代谢适应的分子机制尚不清楚。
通过 RNA 测序分析 MDA-MB-231 细胞系（20% O 和 1% O）中的基因表达谱。通过定量实时 PCR 检测乳腺癌细胞和组织中编码肌酸转运蛋白的 Slc6a8 的表达。免疫组织化学检测肿瘤组织中 SLC6A8 蛋白的丰度。通过卡方检验和 Kaplan-Meier 分析评估临床病理相关性和总生存率。通过细胞活力测定和 Annexin V/PI 双重染色的流式细胞术分析研究 SLC6A8 介导的肌酸摄取对缺氧 TNBC 细胞活力的影响。使用 TNBC 原位小鼠模型评估体内肌酸的作用。
SLC6A8 在缺氧的 TNBC 细胞中异常上调。SLC6A8 在 TNBC 组织中明显过表达，其水平与晚期 TNM 分期、较高的组织学分级和 TNBC 患者的总生存率差密切相关。我们发现 SLC6A8 由 p65/NF-κB 转录上调，并在缺氧时介导细胞内肌酸的积累。SLC6A8 介导的肌酸积累通过维持氧化还原稳态促进缺氧 TNBC 细胞的存活并抑制凋亡。此外，肌酸有助于在异种移植小鼠模型中促进肿瘤生长。在机制上，细胞内肌酸通过降低线粒体活性和耗氧量来增强细胞抗氧化防御，减少细胞内活性氧的积累，最终激活 AKT-ERK 信号通路，该通路通过介导 Ki-67 和 Bcl-2 的上调以及 Bax 和 cleaved Caspase-3 的下调来保护缺氧 TNBC 细胞的活力。
我们的研究表明，SLC6A8 介导的肌酸积累在促进 TNBC 进展中起着重要作用，并且可能为治疗 SLC6A8 高表达的 TNBC 提供一种潜在的治疗策略选择。
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