Small bowel carcinoid (enterochromaffin cell) neoplasia exhibits transforming growth factor-β1 mediated regulatory abnormalities including up-regulation of c-Myc and MTA1

BACKGROUND. Although it is known that small intestinal carcinoids are derived from enterochrontaffin (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-beta 1 (TGF beta 1) cytostatic program and activation of TGFP-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 TGF beta 1 on cultured normal EC cells (isolated by FACS sorting) and the neoplastic EC cell line, KRJ-1, was assessed using the MTT assay. The TGF beta RII transcript and protein were identified in tumor cells and the effects of TGF beta 1 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 TGF beta 1 and the transcript expression of candidate downstream targets, MTA1 and E-cadherin, were assessed. RESULTS. TGF beta 1 inhibited normal EC cell proliferation (IC50 = 17 pM) but stimulated neoplastic EC cell proliferation (EC50 = 22 pM). In tumor cells, significantly decreased transcript (P <.01) of TGF beta RII was identified, but no receptor mutations were identified and protein expression was evident. TGF beta 1 (1 ng/mL) resulted in SMAD2 phosphorylation and < 7% nuclear expression compared with 93% in normal EC cells. In neoplastic cells, TGF beta 1 (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 approximate to 40% (P <.002). TGF beta 1 (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 TGF beta-Imediated growth inhibition and, similar to glioblastomas, utilizes the TGF beta system to induce gene responses associated with growth promotion (c-Myc and the ERK pathway), invasion (E-cadherin), and metastasis (MTA1).