Baldwin Rae Lynn, Tran Hang, Karlan Beth Y
Division of Gynecologic Oncology, Cedars-Sinai Medical Center, Los Angeles, California 90048, USA.
Cancer Res. 2003 Mar 15;63(6):1413-9.
Many epithelial carcinomas, including ovarian, are refractory to the antiproliferative effects of transforming growth factor (TGF) beta. In some cancers, TGF-beta resistance has been linked to TGF-beta receptor II (TbetaR-II) and Smad4 mutations; however, in ovarian cancer, the mechanism of resistance remains unclear. Primary ovarian epithelial cell cultures were used as a model system to determine the mechanisms of TGF-beta resistance. To simulate in vivo responses to TGF-beta, primary cultures derived from normal human ovarian surface epithelium (HOSE) and from ovarian carcinomas (CSOC) were grown on collagen I gel, the predominant matrix molecule in the ovarian tumor milieu. When treated with 5 ng/ml TGF-beta for 72 h, HOSE (n = 11) proliferation was inhibited by 20 +/- 21% on average. In contrast, CSOC (n = 10) proliferation was stimulated 5 +/- 10% in response to TGF-beta (a statistically significant difference in response when compared with HOSE; P = 0.001). To dissect the TGF-beta/Smad signaling pathway we used a quantitative RNase protection assay (RPA) for measuring mRNA levels of TGF-beta pathway components in 20 HOSE and 20 CSOC cultures. Basal mRNA levels of TGF-beta receptors I and II, downstream signaling components Smad2, 3, 4, 6, 7, and the transcriptional corepressors Ski and SnoN did not show a statistically significant difference between HOSE and CSOC, and cannot explain their differential susceptibility to TGF-beta-induced cell cycle arrest. To assess functional differences of the TGF-beta pathway in TGF-beta-sensitive HOSE and TGF-beta-resistant CSOC, we measured Smad2/4 and 3/4 complex induction after TGF-beta treatment. HOSE and CSOC showed equivalent Smad2/4 and 3/4 complex induction after TGF-beta exposure for 0, 0.5, 2, and 4 h. It has been proposed that SnoN and Ski are corepressors of the TGF-beta/Smad pathway and undergo TGF-beta-induced degradation followed by reinduction of SnoN mRNA. However, our data show equivalent SnoN degradation in HOSE and CSOC, and equivalent SnoN mRNA induction after TGF-beta treatment. Surprising, TGF-beta-induced Ski degradation was not observed in HOSE or CSOC, suggesting that Ski may not function as a TGF-beta/Smad corepressor in ovarian epithelial cells. These data implied that the TGF-beta/Smad pathway remains functional in CSOC, although CSOC cells are resistant to antimitogenic TGF-beta effects. CSOC resistance to TGF-beta coincided with the loss of c-myc down-regulation. These data suggest that TGF-beta/Smad signaling is blocked downstream of Smad complex formation or that an alternate signaling pathway other than TGF-beta/Smad may transmit TGF-beta-induced cell cycle arrest in the ovarian epithelium.
包括卵巢癌在内的许多上皮癌对转化生长因子（TGF）β的抗增殖作用具有抗性。在某些癌症中，TGF-β抗性与TGF-β受体II（TβR-II）和Smad4突变有关；然而，在卵巢癌中，抗性机制仍不清楚。原发性卵巢上皮细胞培养物被用作模型系统来确定TGF-β抗性的机制。为了模拟体内对TGF-β的反应，将源自正常人卵巢表面上皮（HOSE）和卵巢癌（CSOC）的原代培养物在I型胶原凝胶上培养，I型胶原凝胶是卵巢肿瘤环境中的主要基质分子。用5 ng/ml TGF-β处理72小时后，HOSE（n = 11）的增殖平均被抑制20±21%。相比之下，CSOC（n = 10）的增殖在TGF-β刺激下增加了5±10%（与HOSE相比，反应有统计学显著差异；P = 0.001）。为了剖析TGF-β/Smad信号通路，我们使用定量核糖核酸酶保护分析（RPA）来测量20个HOSE和CSOC培养物中TGF-β通路成分的mRNA水平。TGF-β受体I和II、下游信号成分Smad2、3、4、6、7以及转录共抑制因子Ski和SnoN的基础mRNA水平在HOSE和CSOC之间没有显示出统计学显著差异，无法解释它们对TGF-β诱导的细胞周期停滞的不同敏感性。为了评估TGF-β敏感的HOSE和TGF-β抗性的CSOC中TGF-β通路的功能差异，我们测量了TGF-β处理后Smad2/4和3/4复合物的诱导情况。在TGF-β暴露0、0.5、2和4小时后，HOSE和CSOC显示出等效的Smad2/4和3/4复合物诱导。有人提出SnoN和Ski是TGF-β/Smad通路的共抑制因子，会经历TGF-β诱导降解，随后SnoN mRNA重新诱导。然而，我们的数据显示HOSE和CSOC中SnoN降解等效，TGF-β处理后SnoN mRNA诱导也等效。令人惊讶的是，在HOSE或CSOC中未观察到TGF-β诱导的Ski降解，这表明Ski可能在卵巢上皮细胞中不作为TGF-β/Smad共抑制因子发挥作用。这些数据表明，尽管CSOC细胞对TGF-β的抗有丝分裂作用具有抗性，但TGF-β/Smad通路在CSOC中仍然起作用。CSOC对TGF-β的抗性与c-myc下调的丧失一致。这些数据表明，TGF-β/Smad信号在Smad复合物形成的下游被阻断，或者除TGF-β/Smad之外的另一条信号通路可能在卵巢上皮中传递TGF-β诱导的细胞周期停滞。
