Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036, Cosenza, Rende, Italy.
Department of Biology, Ecology and Earth Sciences, University of Calabria, 87036, Rende, Italy.
J Exp Clin Cancer Res. 2019 Aug 1;38(1):335. doi: 10.1186/s13046-019-1337-2.
The chemical carcinogen 3-methylcholanthrene (3MC) binds to the aryl hydrocarbon receptor (AHR) that regulates the expression of cytochrome P450 (CYP) enzymes as CYP1B1, which is involved in the oncogenic activation of environmental pollutants as well as in the estrogen biosynthesis and metabolism. 3MC was shown to induce estrogenic responses binding to the estrogen receptor (ER) α and stimulating a functional interaction between AHR and ERα. Recently, the G protein estrogen receptor (GPER) has been reported to mediate certain biological responses induced by endogenous estrogens and environmental compounds eliciting an estrogen-like activity.
Molecular dynamics and docking simulations were performed to evaluate the potential of 3MC to interact with GPER. SkBr3 breast cancer cells and cancer-associated fibroblasts (CAFs) derived from breast tumor patients were used as model system. Real-time PCR and western blotting analysis were performed in order to evaluate the activation of transduction mediators as well as the mRNA and protein levels of CYP1B1 and cyclin D1. Co-immunoprecipitation studies were performed in order to explore the potential of 3MC to trigger the association of GPER with AHR and EGFR. Luciferase assays were carried out to determine the activity of CYP1B1 promoter deletion constructs upon 3MC exposure, while the nuclear shuttle of AHR induced by 3MC was assessed through confocal microscopy. Cell proliferation stimulated by 3MC was determined as biological counterpart of the aforementioned experimental assays. The statistical analysis was performed by ANOVA.
We first ascertained by docking simulations the ability of 3MC to interact with GPER. Thereafter, we established that 3MC activates the EGFR/ERK/c-Fos transduction signaling through both AHR and GPER in SkBr3 cells and CAFs. Then, we found that these receptors are involved in the up-regulation of CYP1B1 and cyclin D1 as well as in the stimulation of growth responses induced by 3MC.
In the present study we have provided novel insights regarding the molecular mechanisms by which 3MC may trigger a physical and functional interaction between AHR and GPER, leading to the stimulation of both SkBr3 breast cancer cells and CAFs. Altogether, our results indicate that 3MC may engage both GPER and AHR transduction pathways toward breast cancer progression.
化学致癌物 3-甲基胆蒽（3MC）与芳香烃受体（AHR）结合，调节细胞色素 P450（CYP）酶的表达，如 CYP1B1，其参与环境污染物的致癌激活以及雌激素的生物合成和代谢。已经表明 3MC 通过与雌激素受体（ER）α结合并刺激 AHR 和 ERα 之间的功能相互作用来诱导雌激素反应。最近，G 蛋白雌激素受体（GPER）已被报道介导内源性雌激素和环境化合物引起的某些生物学反应，产生类似雌激素的活性。
进行分子动力学和对接模拟，以评估 3MC 与 GPER 相互作用的潜力。SkBr3 乳腺癌细胞和源自乳腺癌患者的癌相关成纤维细胞（CAFs）用作模型系统。进行实时 PCR 和 Western blot 分析，以评估转导介质的激活以及 CYP1B1 和细胞周期蛋白 D1 的 mRNA 和蛋白水平。进行共免疫沉淀研究，以探索 3MC 触发 GPER 与 AHR 和 EGFR 结合的潜力。进行荧光素酶测定以确定 3MC 暴露后 CYP1B1 启动子缺失构建体的活性，同时通过共聚焦显微镜评估 3MC 诱导的 AHR 核穿梭。通过 3MC 刺激的细胞增殖来确定上述实验测定的生物学对应物。通过 ANOVA 进行统计分析。
我们首先通过对接模拟确定了 3MC 与 GPER 相互作用的能力。此后，我们确定 3MC 通过 AHR 和 GPER 在 SkBr3 细胞和 CAFs 中激活 EGFR/ERK/c-Fos 转导信号。然后，我们发现这些受体参与 CYP1B1 和细胞周期蛋白 D1 的上调以及 3MC 诱导的生长反应的刺激。
在本研究中，我们提供了关于 3MC 可能触发 AHR 和 GPER 之间物理和功能相互作用的分子机制的新见解，从而刺激 SkBr3 乳腺癌细胞和 CAFs。总之，我们的结果表明，3MC 可能参与 GPER 和 AHR 转导途径，促进乳腺癌的进展。
