Canagliflozin mediates tumor suppression alone and in combination with radiotherapy in non-small cell lung cancer (NSCLC) through inhibition of HIF-1a

Non-small cell lung cancer (NSCLC) has a poor prognosis and effective therapeutic strategies are lacking. The diabetes drug canagliflozin inhibits NSCLC cell proliferation and the mammalian target of rapamycin (mTOR) pathway, which mediates cell growth and survival, but it is unclear whether this drug can enhance response rates when combined with cytotoxic therapy. Here, we evaluated the effects of canagliflozin on human NSCLC response to cytotoxic therapy in tissue cultures and xenografts. Ribonucleic acid sequencing (RNA-seq), real-time quantitative PCR (RT-qPCR), metabolic function, small interfering ribonucleic acid (siRNA) knockdown and protein expression assays were used in mechanistic analyses. We found that canagliflozin inhibited proliferation and clonogenic survival of NSCLC cells, and augmented the efficacy of radiotherapy to mediate these effects and inhibit NSCLC xenograft growth. Canagliflozin treatment alone moderately inhibited mitochondrial oxidative phosphorylation and exhibited greater anti-proliferative capacity than specific mitochondrial complex-I inhibitors. The treament downregulated genes mediating hypoxia-inducible factor (HIF)-1a stability, metabolism and survival, activated adenosine monophosphate-activated protein kinase (AMPK) and inhibited mTOR, a critical activator of HIF-1a signaling. HIF-1a knockdown and stabilization experiments suggested that canagliflozin mediates anti-proliferative effects, in part, through suppression of HIF-1a. Transcriptional regulatory network analysis pinpointed histone deacetylase 2 (HDAC2), a gene suppressed by canagliflozin, as a key mediator of canagliflozin’s transcriptional reprogramming. HDAC2 knockdown eliminated HIF-1a levels and enhanced the anti-proliferative effects of canagliflozin. HDAC2-regulated genes suppressed by canagliflozin are associated with poor prognosis in several clinical NSCLC datasets. In addition, we include evidence that canagliflozin also improves NSCLC response to chemotherapy. In summary, canagliflozin may be a promising therapy to develop in combination with cytotoxic therapy in NSCLC. Total RNA was isolated from 3 replicates of A549 cells treated with either 10 uM canagliflozin, 5 Gy radiotherapy (RT) or vehicle (DMSO) for 24h. A total of 9 samples were analyzed.

非小细胞肺癌（Non-small cell lung cancer, NSCLC）预后不佳且缺乏有效的治疗策略。糖尿病药物卡格列净（canagliflozin）可抑制非小细胞肺癌细胞增殖及哺乳动物雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）通路——该通路介导细胞生长与存活，但目前尚不明确该药物联合细胞毒性治疗时能否提升应答率。本研究在细胞培养及异种移植模型中，评估了卡格列净对人非小细胞肺癌细胞毒性治疗应答的影响。机制分析采用了RNA测序（Ribonucleic acid sequencing, RNA-seq）、实时定量聚合酶链反应（real-time quantitative PCR, RT-qPCR）、代谢功能检测、小干扰核糖核酸（small interfering ribonucleic acid, siRNA）敲低及蛋白质表达分析等手段。本研究发现，卡格列净可抑制非小细胞肺癌细胞的增殖与集落形成存活能力，并增强放疗的相关效应，同时可抑制非小细胞肺癌异种移植瘤的生长。单独使用卡格列净可轻度抑制线粒体氧化磷酸化，且其抗增殖能力优于特异性线粒体复合体I抑制剂。该药物可下调介导缺氧诱导因子（hypoxia-inducible factor, HIF）-1α稳定性、代谢及存活的基因，激活腺苷酸活化蛋白激酶（adenosine monophosphate-activated protein kinase, AMPK），并抑制HIF-1α信号通路的关键激活因子mTOR。HIF-1α敲低及稳定化实验表明，卡格列净的抗增殖效应部分通过抑制HIF-1α实现。转录调控网络分析显示，组蛋白去乙酰化酶2（histone deacetylase 2, HDAC2）——一种可被卡格列净抑制的基因——是卡格列净转录重编程的关键介导因子。敲低HDAC2可消除HIF-1α的表达水平，并增强卡格列净的抗增殖能力。在多项临床非小细胞肺癌数据集当中，被卡格列净抑制的HDAC2调控基因与不良预后相关。此外，本研究还证实卡格列净可提升非小细胞肺癌对化疗的应答水平。综上，卡格列净有望成为与细胞毒性治疗联合应用的非小细胞肺癌潜在治疗方案。本研究从经10 μM卡格列净、5 Gy放疗（RT）或溶剂（Dimethyl sulfoxide, DMSO）处理24小时的A549细胞的3份重复样本中提取总RNA，共分析9份样本。