Kidd Mark, Modlin Irvin M, Pfragner Roswitha, Eick Geeta N, Champaneria Manish C, Chan Anthony K, Camp Robert L, Mane Shrikant M
Department of Surgery, Yale University School of Medicine, New Haven, Connecticut 06520-8062, USA.
Cancer. 2007 Jun 15;109(12):2420-31. doi: 10.1002/cncr.22725.
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.
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.
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%).
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).
虽然已知小肠类癌起源于肠嗜铬（EC）细胞，但这些细胞的特征仍不清楚，对这些神经内分泌细胞的生长调节机制也知之甚少。转化生长因子β1（TGFβ1）细胞生长抑制程序的下调或缺失以及TGFβ介导的转录网络的激活与其他神经肿瘤（胶质母细胞瘤）的生长失控和转移有关。本研究调查了这种现象在小肠类癌肿瘤中是否常见。
使用MTT法评估TGFβ1对培养的正常EC细胞（通过荧光激活细胞分选法分离）和肿瘤性EC细胞系KRJ-I的影响。在肿瘤细胞中鉴定TGFβRII转录本和蛋白，并对TGFβ1对SMAD2磷酸化和核转位的影响进行定量。检测SMAD4、SMAD7、c-Myc和P21（WAF1/CIP1）蛋白表达以及c-Myc和p21（WAF1/CIP1）转录本对TGFβ1的时间依赖性反应，并评估候选下游靶点MTA1和E-钙黏蛋白的转录本表达。
TGFβ1抑制正常EC细胞增殖（IC50 = 17 pM），但刺激肿瘤性EC细胞增殖（EC50 = 22 pM）。在肿瘤细胞中，鉴定出TGFβRII转录本显著减少（P <.01），但未发现受体突变且蛋白表达明显。与正常EC细胞中的93%相比，TGFβ1（1 ng/mL）导致SMAD2磷酸化，核表达<7%。在肿瘤细胞中，TGFβ1（1 ng/mL）导致SMAD4减少（>16%，P <.05），而SMAD7和c-Myc转录本和蛋白分别增加>21%（P <.05）和约40%（P <.002）。TGFβ1（1 ng/mL）还使p21（WAF1/CIP1）转录本减少60%（P <.00），并且在24小时时蛋白无法检测到。c-Myc途径的下游靶点MTA1的表达增加（20%），E-钙黏蛋白减少（30%）。
肿瘤性EC细胞的特征是丧失TGFβ-1介导的生长抑制，并且与胶质母细胞瘤类似，利用TGFβ系统诱导与生长促进（c-Myc和ERK途径）、侵袭（E-钙黏蛋白）和转移（MTA1）相关的基因反应。
