Small bowel carcinoid (enterochromaffin cell) neoplasia exhibits transforming growth factor-beta1-mediated regulatory abnormalities including up-regulation of C-Myc and MTA1.
BACKGROUND
Although it is known that small intestinal carcinoids are derived from enterochromaffin (EC) cells, these cells remain poorly characterized and little is known of the growth regulatory mechanisms of these neuroendocrine cells. Down-regulation or loss of the transforming growth factor-beta1 (TGFbeta1) cytostatic program and activation of TGFbeta-mediated transcriptional networks is associated with uncontrolled growth and metastasis in other neural tumors, glioblastomas. Whether this phenomenon is common to small intestinal carcinoid tumors was investigated.


METHODS
The effects of TGFbeta1 on cultured normal EC cells (isolated by FACS sorting) and the neoplastic EC cell line, KRJ-I, was assessed using the MTT assay. The TGFbetaRII transcript and protein were identified in tumor cells and the effects of TGFbeta1 on SMAD2 phosphorylation and nuclear translocation quantified. The time-dependent response of SMAD4, SMAD7, c-Myc, and P21(WAF1/CIP1) protein expression and c-Myc and p21(WAF1/CIP1) transcript was measured in response to TGFbeta1 and the transcript expression of candidate downstream targets, MTA1 and E-cadherin, were assessed.


RESULTS
TGFbeta1 inhibited normal EC cell proliferation (IC(50) = 17 pM) but stimulated neoplastic EC cell proliferation (EC(50) = 22 pM). In tumor cells, significantly decreased transcript (P < .01) of TGFbetaRII was identified, but no receptor mutations were identified and protein expression was evident. TGFbeta1 (1 ng/mL) resulted in SMAD2 phosphorylation and <7% nuclear expression compared with 93% in normal EC cells. In neoplastic cells, TGFbeta1 (1 ng/mL) caused a decrease in SMAD4 (>16%, P < .05), whereas SMAD7 and c-Myc transcript and protein were respectively increased >21% (P < .05) and approximately 40% (P < .002). TGFbeta1 (1 ng/mL) also decreased p21(WAF1/CIP1) transcript by 60% (P < .001) and protein that was undetectable at 24 hours. Expression of the downstream targets of the c-Myc pathway, MTA1, was increased (20%) and E-cadherin decreased (30%).


CONCLUSIONS
The neoplastic EC cell is characterized by loss of TGFbeta-1-mediated growth inhibition and, similar to glioblastomas, utilizes the TGFbeta system to induce gene responses associated with growth promotion (c-Myc and the ERK pathway), invasion (E-cadherin), and metastasis (MTA1).
Kidd
Mark
M
Department of Surgery, Yale University School of Medicine, New Haven, Connecticut 06520-8062, USA.
Modlin
Irvin M
IM
Pfragner
Roswitha
R
Eick
Geeta N
GN
Champaneria
Manish C
MC
Chan
Anthony K
AK
Camp
Robert L
RL
Mane
Shrikant M
SM
D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism; D002276:Carcinoid Tumor / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D004759:Enterochromaffin Cells / Q000187:drug effects; D015972:Gene Expression Regulation, Neoplastic; D006655:Histone Deacetylases / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007414:Intestinal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics* / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction; D051785:Smad Proteins / Q000378:metabolism; D015534:Trans-Activators; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D015820:Cadherins; D050759:Cyclin-Dependent Kinase Inhibitor p21; C089320:MTA1 protein, human; D016271:Proto-Oncogene Proteins c-myc; D012097:Repressor Proteins; D051785:Smad Proteins; D015534:Trans-Activators; D053773:Transforming Growth Factor beta1; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D006655:Histone Deacetylases