Increased Serotonin Signaling Contributes to the Warburg Effect in Pancreatic Tumor Cells Under Metabolic Stress and Promotes Growth of Pancreatic Tumors in Mice.
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.



We measured the levels of proteins that regulate 5-HT synthesis, packaging, and degradation in pancreata from KrasG12D/+/Trp53R172H/+/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.



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.



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.
State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University, Shanghai, P.R. China; Department of Biliary-Pancreatic Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, P.R. China.
Shu-Heng
SH
Jiang
State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University, Shanghai, P.R. China.
Jun
J
Li
Department of Gastroenterology, Shanghai Key Laboratory of Clinical Geriatric Medicine, Huadong Hospital, Shanghai, PR China.
Fang-Yuan
FY
Dong
Department of Biliary-Pancreatic Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, P.R. China.
Jian-Yu
JY
Yang
Department of Biliary-Pancreatic Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, P.R. China.
De-Jun
DJ
Liu
State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University, Shanghai, P.R. China.
Xiao-Mei
XM
Yang
State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University, Shanghai, P.R. China.
Ya-Hui
YH
Wang
Department of Biliary-Pancreatic Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, P.R. China.
Min-Wei
MW
Yang
Department of Biliary-Pancreatic Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, P.R. China.
Xue-Liang
XL
Fu
State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University, Shanghai, P.R. China.
Xiao-Xin
XX
Zhang
State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University, Shanghai, P.R. China.
Qing
Q
Li
Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, China.
Xiu-Feng
XF
Pang
Department of Biliary-Pancreatic Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, P.R. China.
Yan-Miao
YM
Huo
Department of Biliary-Pancreatic Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, P.R. China.
Jiao
J
Li
Department of Biliary-Pancreatic Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, P.R. China.
Jun-Feng
JF
Zhang
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of Korea.
Ho-Young
HY
Lee
Department of Life Science, Research Institute for Nature Sciences, Hanyang University, Seoul, Republic of Korea.
Su-Jae
SJ
Lee
State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University, Shanghai, P.R. China.
Wen-Xin
WX
Qin
State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University, Shanghai, P.R. China.
Jian-Ren
JR
Gu
Department of Biliary-Pancreatic Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, P.R. China. Electronic address: syw0616@126.com.
Yong-Wei
YW
Sun
State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Shanghai Jiao Tong University, Shanghai, P.R. China. Electronic address: zzhang@shsci.org.
Zhi-Gang
ZG
Zhang
D000368:Aged; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D021441:Carcinoma, Pancreatic Ductal / Q000737:chemistry / Q000188:drug therapy / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D005260:Female; D020868:Gene Silencing; D005947:Glucose / Q000378:metabolism; D006019:Glycolysis / Q000187:drug effects; D006801:Humans; D007211:Indoles / Q000627:therapeutic use; D019344:Lactic Acid / Q000096:biosynthesis; D008297:Male; D051379:Mice; D008875:Middle Aged; D008995:Monoamine Oxidase / Q000032:analysis; D009368:Neoplasm Transplantation; D010179:Pancreas / Q000737:chemistry; D010190:Pancreatic Neoplasms / Q000737:chemistry / Q000188:drug therapy / Q000378:metabolism*; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D044403:Receptor, Serotonin, 5-HT2B / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology; D058826:Serotonin 5-HT2 Receptor Agonists / Q000494:pharmacology; D058830:Serotonin 5-HT2 Receptor Antagonists / Q000494:pharmacology / Q000627:therapeutic use; D015398:Signal Transduction; D013312:Stress, Physiological; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism; D046888:Tissue Array Analysis; D059467:Transcriptome; D014365:Tryptophan Hydroxylase / Q000032:analysis; D014508:Urea / Q000031:analogs & derivatives / Q000627:therapeutic use; D019061:src-Family Kinases / Q000378:metabolism
D007211:Indoles; C092756:N-(1-methyl-5-indolyl)-N'-(3-methyl-5-isothiazolyl)urea; D016271:Proto-Oncogene Proteins c-myc; D044403:Receptor, Serotonin, 5-HT2B; D058826:Serotonin 5-HT2 Receptor Agonists; D058830:Serotonin 5-HT2 Receptor Antagonists; D012701:Serotonin; D019344:Lactic Acid; D014508:Urea; C497467:TPH1 protein, human; D014365:Tryptophan Hydroxylase; D008995:Monoamine Oxidase; C579458:monoamine oxidase A, human; C546842:MTOR protein, human; D058539:Phosphatidylinositol 3-Kinase; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D005947:Glucose
5-HT2B; 5-hydroxytryptamine; Pancreatic Intraepithelial Neoplasia; Tryptophan Hydroxylase