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

Gastroenterology. 2017 Jul;153(1):277-291.e19. doi: 10.1053/j.gastro.2017.03.008. Epub 2017 Mar 15.

Abstract

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 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.

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.

Keywords: 5-HT2B; 5-hydroxytryptamine; Pancreatic Intraepithelial Neoplasia; Tryptophan Hydroxylase.

MeSH terms

  • Aged
  • Animals
  • Apoptosis / drug effects
  • Carcinoma, Pancreatic Ductal / chemistry
  • Carcinoma, Pancreatic Ductal / drug therapy
  • Carcinoma, Pancreatic Ductal / metabolism*
  • Cell Line, Tumor
  • Cell Proliferation / drug effects
  • Cell Survival / drug effects
  • Female
  • Gene Silencing
  • Glucose / metabolism
  • Glycolysis / drug effects
  • Humans
  • Indoles / therapeutic use
  • Lactic Acid / biosynthesis
  • Male
  • Mice
  • Middle Aged
  • Monoamine Oxidase / analysis
  • Neoplasm Transplantation
  • Pancreas / chemistry
  • Pancreatic Neoplasms / chemistry
  • Pancreatic Neoplasms / drug therapy
  • Pancreatic Neoplasms / metabolism*
  • Phosphatidylinositol 3-Kinase / metabolism
  • Proto-Oncogene Proteins c-akt / metabolism
  • Proto-Oncogene Proteins c-myc / metabolism
  • Receptor, Serotonin, 5-HT2B / genetics
  • Receptor, Serotonin, 5-HT2B / metabolism*
  • Serotonin / analysis
  • Serotonin / metabolism*
  • Serotonin / pharmacology
  • Serotonin 5-HT2 Receptor Agonists / pharmacology
  • Serotonin 5-HT2 Receptor Antagonists / pharmacology
  • Serotonin 5-HT2 Receptor Antagonists / therapeutic use
  • Signal Transduction
  • Stress, Physiological
  • TOR Serine-Threonine Kinases / metabolism
  • Tissue Array Analysis
  • Transcriptome
  • Tryptophan Hydroxylase / analysis
  • Urea / analogs & derivatives
  • Urea / therapeutic use
  • src-Family Kinases / metabolism

Substances

  • Indoles
  • N-(1-methyl-5-indolyl)-N'-(3-methyl-5-isothiazolyl)urea
  • Proto-Oncogene Proteins c-myc
  • Receptor, Serotonin, 5-HT2B
  • Serotonin 5-HT2 Receptor Agonists
  • Serotonin 5-HT2 Receptor Antagonists
  • Serotonin
  • Lactic Acid
  • Urea
  • TPH1 protein, human
  • Tryptophan Hydroxylase
  • Monoamine Oxidase
  • monoamine oxidase A, human
  • MTOR protein, human
  • Phosphatidylinositol 3-Kinase
  • lyn protein-tyrosine kinase
  • src-Family Kinases
  • Proto-Oncogene Proteins c-akt
  • TOR Serine-Threonine Kinases
  • Glucose