SLC25A22 Promotes Proliferation and Survival of Colorectal Cancer Cells With KRAS Mutations and Xenograft Tumor Progression in Mice via Intracellular Synthesis of Aspartate
SLC25A22 Promotes Proliferation and Survival of Colorectal Cancer Cells With KRAS Mutations and Xenograft Tumor Progression in Mice via Intracellular Synthesis of Aspartate
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SLC25A22 通过细胞内合成天冬氨酸促进小鼠 KRAS 突变结直肠癌细胞的增殖和存活以及异种移植肿瘤的进展
DOI：
10.1053/j.gastro.2016.07.011
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发表时间：
2016-11-01
期刊：
影响因子：
29.4
通讯作者：
Yu, Jun
中科院分区：
文献类型：
--
作者：
Wong, Chi Chun;Qian, Yun;Yu, Jun
背景与目的：许多结直肠癌（CRC）细胞含有KRAS突变。对APC或CTNNB 1和KRAS突变的CRC细胞的分析确定了编码线粒体谷氨酸转运蛋白的SLC 25 A22为合成致死基因。我们研究了SLC 25 A22在KRAS突变的CRC细胞中的功能。方法：我们测量了130例香港患者和17例中国患者的配对肿瘤和非肿瘤结肠组织中SLC 25 A22信使RNA和蛋白质的水平，并将蛋白质水平与患者生存时间进行了比较。在KRAS突变型CRC细胞系（DLD 1、HCT 116、LOVO、SW 480、SW 620和SW 1116）和KRAS无突变的CRC细胞系（CACO-2、COLO 205、HT 29和SW 48）中，SLC 25 A22的表达被敲低;在基质胶和聚合酶链反应阵列分析中，分析细胞的集落形成、增殖、β-氨基水解和天冬氨酸合成以及细胞凋亡。将SLC 25 A22敲减的DLD 1和HCT 116细胞作为异种移植肿瘤在裸鼠中生长;测量肿瘤生长和转移。SLC 25 A22在HCT 116细胞中异位表达，将其在体外分析并在裸鼠中作为异种移植瘤生长。研究结果：与匹配的非肿瘤结肠组织相比，结直肠肿瘤组织中SLC 25 A22信使RNA和蛋白水平升高;蛋白水平升高与患者生存时间缩短相关（P = 0.01）。与没有SLC 25 A22敲低的细胞相比，KRAS突变CRC细胞中SLC 25 A22的敲低降低了它们的体外增殖、迁移和侵袭，以及小鼠中的肿瘤形成和转移。SLC 25 A22的敲低减少了天冬氨酸的生物合成，导致细胞凋亡，降低了KRAS突变CRC细胞中的细胞增殖。用天冬氨酸敲减SLC 25 A22的KRAS突变CRC细胞的孵育增加了增殖并减少了细胞凋亡，这需要GOT 1，表明草酰乙酸是细胞存活所需的。在SLC 25 A22敲低的细胞中草酰乙酸水平的降低减少了氧化的烟酰胺腺嘌呤二核苷酸的再生和还原的烟酰胺腺嘌呤二核苷酸磷酸。还原型氧化烟酰胺腺嘌呤二核苷酸抑制糖酵解并降低三磷酸腺苷的水平，从而通过激活AMP活化的蛋白激酶来灭活促分裂原活化的蛋白激酶激酶和细胞外信号调节激酶信号传导。氧化型烟酰胺腺嘌呤二核苷酸磷酸与还原型烟酰胺腺嘌呤二核苷酸磷酸的比率增加诱导氧化应激和谷胱甘肽氧化，从而抑制细胞增殖。天冬酰胺合成酶介导天冬氨酸合成天冬酰胺促进细胞迁移。结论：SLC 25 A22促进具有突变KRAS的CRC细胞的增殖和迁移，以及小鼠中CRC异种移植肿瘤的形成和转移。SLC 25 A22表达水平增加的结直肠肿瘤患者的生存时间比肿瘤水平较低的患者短。SLC 25 A22诱导天冬氨酸的细胞内合成、丝裂原活化蛋白激酶激酶和细胞外信号调节激酶信号传导的活化，并降低氧化应激。
BACKGROUND & AIMS: Many colorectal cancer (CRC) cells contain mutations in KRAS. Analyses of CRC cells with mutations in APC or CTNNB1 and KRAS identified SLC25A22, which encodes mitochondrial glutamate transporter, as a synthetic lethal gene. We investigated the functions of SLC25A22 in CRC cells with mutations in KRAS. METHODS: We measured levels of SLC25A22 messenger RNA and protein in paired tumor and nontumor colon tissues collected from 130 patients in Hong Kong and 17 patients in China and compared protein levels with patient survival times. Expression of SLC25A22 was knocked down in KRAS mutant CRC cell lines (DLD1, HCT116, LOVO, SW480, SW620, and SW1116) and CRC cell lines without mutations in KRAS (CACO-2, COLO205, HT29, and SW48); cells were analyzed for colony formation, proliferation, glutaminolysis and aspartate synthesis, and apoptosis in Matrigel and polymerase chain reaction array analyses. DLD1 and HCT116 cells with SLC25A22 knockdown were grown as xenograft tumors in nude mice; tumor growth and metastasis were measured. SLC25A22 was expressed ectopically in HCT116 cells, which were analyzed in vitro and grown as xenograft tumors in nude mice. RESULTS: Levels of SLC25A22 messenger RNA and protein were increased in colorectal tumor tissues compared with matched nontumor colon tissues; increased protein levels were associated with shorter survival times of patients (P = .01). Knockdown of SLC25A22 in KRAS mutant CRC cells reduced their proliferation, migration, and invasion in vitro, and tumor formation and metastasis in mice, compared with cells without SLC25A22 knockdown. Knockdown of SLC25A22 reduced aspartate biosynthesis, leading to apoptosis, decreased cell proliferation in KRAS mutant CRC cells. Incubation of KRAS mutant CRC cells with knockdown of SLC25A22 with aspartate increased proliferation and reduced apoptosis, which required GOT1, indicating that oxaloacetate is required for cell survival. Decreased levels of oxaloacetate in cells with knockdown of SLC25A22 reduced regeneration of oxidized nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate. Reduced oxidized nicotinamide adenine dinucleotide inhibited glycolysis and decreased levels of adenosine triphosphate, which inactivated mitogen-activated protein kinase kinase and extracellular signal-regulated kinase signaling via activation of AMP-activated protein kinase. An increased ratio of oxidized nicotinamide adenine dinucleotide phosphate to reduced nicotinamide adenine dinucleotide phosphate induced oxidative stress and glutathione oxidation, which suppressed cell proliferation. Asparagine synthetase mediated synthesis of asparagine from aspartate to promote cell migration. CONCLUSIONS: SLC25A22 promotes proliferation and migration of CRC cells with mutations KRAS, and formation and metastasis of CRC xenograft tumors in mice. Patients with colorectal tumors that express increased levels of SLC25A22 have shorter survival times than patients whose tumors have lower levels. SLC25A22 induces intracellular synthesis of aspartate, activation of mitogen-activated protein kinase kinase and extracellular signal-regulated kinase signaling and reduces oxidative stress.