Increased Serotonin Signaling Contributes to the Warburg Effect in Pancreatic Tumor Cells Under Metabolic Stress and Promotes Growth of Pancreatic Tumors in Mice
Increased Serotonin Signaling Contributes to the Warburg Effect in Pancreatic Tumor Cells Under Metabolic Stress and Promotes Growth of Pancreatic Tumors in Mice
复制标题
血清素信号传导增加有助于代谢应激下胰腺肿瘤细胞的 Warburg 效应，并促进小鼠胰腺肿瘤的生长
DOI：
10.1053/j.gastro.2017.03.008
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发表时间：
2017-07-01
期刊：
影响因子：
29.4
通讯作者：
Zhang, Zhi-Gang
中科院分区：
文献类型：
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作者：
Jiang, Shu-Heng;Li, Jun;Zhang, Zhi-Gang
背景与目的：胰腺导管腺癌（PDACs）的结缔组织增生和血管不良导致严重的代谢应激。5-羟色胺（5-HT）是一种具有神经递质和神经内分泌功能的神经调节剂，有助于肿瘤的发生。我们研究了5-HT信号在胰腺肿瘤生长中的作用。方法：我们测量了Kras(G12D/+)/Trp53(R172H/+)/Pdx1-Cre （KPC）小鼠胰腺中调节5-HT合成、包装和降解的蛋白质水平，这些小鼠发生胰腺肿瘤，以及PDAC细胞系和包含81个人类PDAC样本的组织微阵列。我们还通过对包含311个PDAC标本的组织芯片进行免疫组织化学分析，分析了参与5-HT合成和降解的蛋白质的表达水平，以及与患者生存时间相关的表达水平。采用ELISA法分析14例匹配的PDAC肿瘤和非肿瘤组织中5-HT水平。用5-HT孵育PDAC细胞株，观察细胞存活和凋亡情况。我们分析了5-HT受体HTR2B在PDAC细胞中的表达、受体激动剂和拮抗剂的作用，以及HTR2B被小发夹rna敲低的情况。我们测定了5-HT刺激对BxPC-3细胞基因表达谱的影响。通过免疫荧光和免疫沉淀分析，以及测定细胞外酸比、葡萄糖消耗和乳酸产量，评估通过HTR2B介导的5-HT信号对糖酵解的调节。原发pdac，有或没有暴露于SB204741 （HTR2B的选择性拮抗剂），作为异种移植肿瘤在小鼠体内生长，SB204741给药于荷瘤KPC小鼠；通过影像学分析测量肿瘤生长和代谢。结果：在PDAC标本的组织芯片免疫组化分析中，调节5-羟色胺合成和降解的TPH1水平升高，MAOA水平降低，与PDAC的分期和大小以及患者生存时间缩短相关。我们发现，与非肿瘤胰腺组织相比，人类PDAC组织中的5-HT水平升高，PDAC细胞系与未转化的胰腺细胞相比，5-HT水平升高。PDAC细胞株经5-HT孵育后，增殖增加，凋亡减少。HTR2B的激动剂，而不是其他5-HT受体，促进PDAC细胞增殖和防止凋亡。在PDAC细胞中敲低HTR2B，或用HTR2B抑制剂孵育细胞，可减少其作为小鼠异种移植肿瘤的生长。我们观察到PDAC细胞中5-HT与糖酵解通量之间的相关性；5-HT刺激后，PDAC细胞中参与糖酵解、磷酸戊糖途径和己糖胺生物合成途径的代谢酶水平显著升高。5-HT刺激导致HTR2B-LYN-p85复合物的形成，通过增加MYC和HIF1A蛋白水平增加PI3K-Akt-mTOR信号和Warburg效应。给KPC小鼠注射SB204741可减缓已建立胰腺肿瘤的生长和代谢，延长小鼠的生存期。结论：人PDAC细胞中5-HT水平升高，PDAC细胞中5-HT受体HTR2B表达增加。这些增加允许代谢应激下的肿瘤糖酵解，并促进小鼠胰腺肿瘤和PDAC异种移植肿瘤的生长。
BACKGROUND & AIMS: Desmoplasia and poor vascularity cause severe metabolic stress in pancreatic ductal adenocarcinomas (PDACs). Serotonin (5-HT) is a neuromodulator with neurotransmitter and neuroendocrine functions that contributes to tumorigenesis. We investigated the role of 5-HT signaling in the growth of pancreatic tumors. METHODS: We measured the levels of proteins that regulate 5-HT synthesis, packaging, and degradation in pancreata from Kras(G12D/+)/Trp53(R172H/+)/Pdx1-Cre (KPC) mice, which develop pancreatic tumors, as well as in PDAC cell lines and a tissue microarray containing 81 human PDAC samples. We also analyzed expression levels of proteins involved in 5-HT synthesis and degradation by immunohistochemical analysis of a tissue microarray containing 311 PDAC specimens, and associated expression levels with patient survival times. 5-HT level in 14 matched PDAC tumor and non-tumor tissues were analyzed by ELISA. PDAC cell lines were incubated with 5-HT and cell survival and apoptosis were measured. We analyzed expression of the 5-HT receptor HTR2B in PDAC cells and effects of receptor agonists and antagonists, as well as HTR2B knockdown with small hairpin RNAs. We determined the effects of 5-HT stimulation on gene expression profiles of BxPC-3 cells. Regulation of glycolysis by 5-HT signaling via HTR2B was assessed by immunofluorescence and immunoprecipitation analyses, as well as by determination of the extracellular acid ratio, glucose consumption, and lactate production. Primary PDACs, with or without exposure to SB204741 (a selective antagonist of HTR2B), were grown as xenograft tumors in mice, and SB204741 was administered to tumor-bearing KPC mice; tumor growth and metabolism were measured by imaging analyses. RESULTS: In immunohistochemical analysis of a tissue microarray of PDAC specimens, increased levels of TPH1 and decreased level of MAOA, which regulate 5-HT synthesis and degradation, correlated with stage and size of PDACs and shorter patient survival time. We found levels of 5-HT to be increased in human PDAC tissues compared with non-tumor pancreatic tissues, and PDAC cell lines compared with non-transformed pancreatic cells. Incubation of PDAC cell lines with 5-HT increased proliferation and prevented apoptosis. Agonists of HTR2B, but not other 5-HT receptors, promoted proliferation and prevented apoptosis of PDAC cells. Knockdown of HTR2B in PDAC cells, or incubation of cells with HTR2B inhibitors, reduced their growth as xenograft tumors in mice. We observed a correlation between 5-HT and glycolytic flux in PDAC cells; levels of metabolic enzymes involved in glycolysis, the phosphate pentose pathway, and hexosamine biosynthesis pathway increased significantly in PDAC cells following 5-HT stimulation. 5-HT stimulation led to formation of the HTR2B-LYN-p85 complex, which increased PI3K-Akt-mTOR signaling and the Warburg effect by increasing protein levels of MYC and HIF1A. Administration of SB204741 to KPC mice slowed growth and metabolism of established pancreatic tumors and prolonged survival of the mice. CONCLUSIONS: Human PDACs have increased levels of 5-HT, and PDAC cells increase expression of its receptor, HTR2B. These increases allow for tumor glycolysis under metabolic stress and promote growth of pancreatic tumors and PDAC xenograft tumors in mice.