Johnston S R, Lu B, Scott G K, Kushner P J, Smith I E, Dowsett M, Benz C C
Department of Medicine, Royal Marsden Hospital and Institute of Cancer Research, London, England.
Clin Cancer Res. 1999 Feb;5(2):251-6.
Human breast tumors that are initially responsive to tamoxifen (TAM) eventually relapse during treatment. Estrogen receptor (ER) expression and function are often preserved in these tumors, and clinical evidence suggests that this relapse may be related to TAM's known agonistic properties. ER can interact with the activator protein-1 (AP-1) transcription factor complex through protein-protein interactions that are independent of ER DNA binding and, in certain ER-positive cells, this may allow TAM to exert an agonist response on AP-1-regulated genes. We, therefore, assessed both AP-1 DNA binding and the known AP-1 activating enzyme, c-Jun NH2-terminal kinase (JNK), in a panel of 30 ER-positive primary human breast tumors with acquired TAM resistance, as compared to a matched panel of 27 untreated control ER-positive breast tumors and a separate control set of 14 primary tumors, which included 7 ER-positive tumors that were growth-arrested by 3 months of preoperative TAM. AP-1 DNA binding activity was measured from cryopreserved tumor extracts using a labeled oligonucleotide probe containing a consensus AP-1 response element by electrophoretic mobility shift assay. JNK was first extracted from the tumor lysates by incubation over a Sepharose-bound c-Jun(1-89) fusion protein, and its activity was then measured by chemiluminescent Western blot by detection of the phosphorylated product using a phospho-Jun(Ser-63)-specific primary antibody. The set of control ER-positive breast tumors growth arrested by TAM showed no significant difference from untreated control tumors in their AP-1 DNA binding and JNK activities. In contrast, there was a significant (P < 0.001) increase in mean AP-1 DNA binding activity for the panel of ER-positive TAM-resistant (TAM-R) tumors as compared to its matched control panel of untreated tumors. Mean JNK activity in the TAM-R tumors was also significantly higher than that found in the untreated tumors (P = 0.038). Overall, there was no significant correlation between JNK activity and AP-1 DNA binding; however, regression analysis showed that, for any given level of JNK activity, the TAM-R tumors possessed a 3.5-fold increase in AP-1 DNA binding activity as compared to the untreated tumors. These findings indicate that, when compared to untreated ER-positive primary breast tumors, TAM-R tumors demonstrate significantly increased levels of AP-1 DNA binding and JNK activity, consistent with experimental models suggesting that TAM-stimulated ER-positive tumor growth may be mediated by enhanced AP-1 transcriptional activity. These observations support the need for further evaluation of these markers in breast tumors as predictors of TAM resistance.
最初对他莫昔芬（TAM）有反应的人类乳腺肿瘤最终在治疗期间会复发。雌激素受体（ER）的表达和功能在这些肿瘤中通常得以保留，临床证据表明这种复发可能与TAM已知的激动剂特性有关。ER可通过独立于ER与DNA结合的蛋白质 - 蛋白质相互作用与激活蛋白 - 1（AP - 1）转录因子复合物相互作用，并且在某些ER阳性细胞中，这可能使TAM对AP - 1调节的基因产生激动剂反应。因此，我们评估了30例获得性TAM耐药的ER阳性原发性人类乳腺肿瘤中AP - 1与DNA的结合以及已知的AP - 1激活酶c - Jun氨基末端激酶（JNK），并与一组27例未经治疗的对照ER阳性乳腺肿瘤以及另一组14例原发性肿瘤进行比较，后者包括7例经术前3个月TAM治疗后生长停滞的ER阳性肿瘤。通过电泳迁移率变动分析，使用含有共有AP - 1反应元件的标记寡核苷酸探针从冷冻保存的肿瘤提取物中测量AP - 1与DNA的结合活性。首先通过与琼脂糖结合的c - Jun（1 - 89）融合蛋白孵育从肿瘤裂解物中提取JNK，然后使用磷酸化Jun（Ser - 63）特异性一抗通过化学发光免疫印迹检测磷酸化产物来测量其活性。经TAM治疗生长停滞的对照ER阳性乳腺肿瘤组在AP - 1与DNA的结合以及JNK活性方面与未经治疗的对照肿瘤组无显著差异。相比之下，与匹配的未经治疗的肿瘤对照组相比，ER阳性TAM耐药（TAM - R）肿瘤组的平均AP - 1与DNA的结合活性显著增加（P < 0.001）。TAM - R肿瘤中的平均JNK活性也显著高于未经治疗的肿瘤（P = 0.038）。总体而言，JNK活性与AP - 1与DNA的结合之间无显著相关性；然而，回归分析表明，对于任何给定水平的JNK活性，与未经治疗的肿瘤相比，TAM - R肿瘤的AP - 1与DNA的结合活性增加了3.5倍。这些发现表明，与未经治疗的ER阳性原发性乳腺肿瘤相比，TAM - R肿瘤表现出AP - 1与DNA的结合水平和JNK活性显著增加，这与实验模型一致，表明TAM刺激的ER阳性肿瘤生长可能由增强的AP - 1转录活性介导。这些观察结果支持进一步评估这些标志物作为乳腺肿瘤中TAM耐药预测指标的必要性。
