title: Chromogranin A and its fragments as regulators of small intestinal neuroendocrine neoplasm proliferation.
abstract: BACKGROUND
  Chromogranin A is a neuroendocrine secretory product and its loss is a feature of malignant NEN de-differentiation. We hypothesized that chromogranin A fragments were differentially expressed during NEN metastasis and played a role in the regulation of NEN proliferation.
  
  
  METHODS
  Chromogranin A mRNA (PCR) and protein (ELISA/western blot) were studied in 10 normal human mucosa, 5 enterochromaffin cell preparations, 26 small intestinal NEN primaries and 9 liver metastases. Cell viability (WST-1 assay), proliferation (bromodeoxyuridine ELISA) and expression of AKT/AKT-P (CASE ELISA/western blot) in response to chromogranin A silencing, inhibition of prohormone convertase and mTOR inhibition (RAD001/AKT antisense) as well as different chromogranin A fragments were examined in 4 SI-NEN cell lines.
  
  
  RESULTS
  Chromogranin A mRNA and protein levels were increased (37-340 fold, p<0.0001) in small intestinal NENs compared to normal enterochromaffin cells. Western blot identified chromogranin A-associated processing bands including vasostatin in small intestinal NENs as well as up-regulated expression of prohormone convertase in metastases. Proliferation in small intestinal NEN cell lines was decreased by silencing chromogranin A as well as by inhibition of prohormone convertase (p<0.05). This inhibition also decreased secretion of chromogranin A (p<0.05) and 5-HT (p<0.05) as well as expression of vasostatin. Metastatic small intestinal NEN cell lines were stimulated (50-80%, p<0.05) and AKT phosphorylated (Ser473: p<0.05) by vasostatin I, which was completely reversed by RAD001 (p<0.01) and AKT antisense (p<0.05) while chromostatin inhibited proliferation (~50%, p<0.05).
  
  
  CONCLUSIONS
  Chromogranin A was differentially regulated in primary and metastatic small intestinal NENs and cell lines. Chromogranin A fragments regulated metastatic small intestinal NEN proliferation via the AKT pathway indicating that CgA plays a far more complex role in the biology of these tumors than previously considered.
authors.affiliation: Department of Surgery, Yale University School of Medicine, New Haven, Connecticut, United States of America ; Laboratory of Translational Surgery-LURM, University of Verona, Verona, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Giovinazzo
authors.forename: Simon
authors.initials: S
authors.lastname: Schimmack
authors.forename: Bernhard
authors.initials: B
authors.lastname: Svejda
authors.forename: Daniele
authors.initials: D
authors.lastname: Alaimo
authors.forename: Roswitha
authors.initials: R
authors.lastname: Pfragner
authors.forename: Irvin
authors.initials: I
authors.lastname: Modlin
authors.forename: Mark
authors.initials: M
authors.lastname: Kidd
mesh_terms: D037282:Calreticulin / Q000378:metabolism / Q000494:pharmacology; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002470:Cell Survival; D053379:Chromogranin A / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D000068338:Everolimus; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007414:Intestinal Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000556:secondary; D018358:Neuroendocrine Tumors / Q000235:genetics* / Q000378:metabolism / Q000556:secondary; D010446:Peptide Fragments / Q000378:metabolism / Q000494:pharmacology; D010766:Phosphorylation; D043484:Proprotein Convertases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D020123:Sirolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D037282:Calreticulin; D053379:Chromogranin A; D010446:Peptide Fragments; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C121986:vasostatin; D000068338:Everolimus; C546842:MTOR protein, human; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases; D043484:Proprotein Convertases; D020123:Sirolimus