Aryl hydrocarbon receptor/cytochrome P450 1A1 pathway mediates breast cancer stem cells expansion through PTEN inhibition and β-Catenin and Akt activation.
Aryl hydrocarbon receptor/cytochrome P450 1A1 pathway mediates breast cancer stem cells expansion through PTEN inhibition and β-Catenin and Akt activation.
复制标题
芳基碳氢化合物受体/细胞色素 P450 1A1 通路通过 PTEN 抑制以及 β-Catenin 和 Akt 激活介导乳腺癌干细胞扩增。
DOI：
10.1186/s12943-016-0570-y
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发表时间：
2017-01-19
期刊：
影响因子：
37.3
通讯作者：
Korashy HM
中科院分区：
文献类型：
--
作者：
Al-Dhfyan A;Alhoshani A;Korashy HM
乳腺癌干细胞（Breast cancer stem cells，CSCs）是乳腺癌细胞中的一个小的亚类，是肿瘤发生、发展和转移的主要动力。最近的研究已经证明了芳香烃受体（AhR）/细胞色素P4501 A1途径在CSCs扩增中的作用。然而，确切的分子机制仍不清楚。本研究旨在a）在体外和体内水平确定AhR活化和抑制对乳腺CSC发育、维持、自我更新和化学抗性的影响，和B）探索β-连环蛋白、PI 3 K/Akt和PTEN信号传导途径的作用。为了检验这一假设，使用Aldefluor测定、侧群和乳腺球形成来确定用AhR活化剂或抑制剂处理的五种人乳腺癌细胞SKBR-3、MCF-7和MDA-MB 231、HS 587 T和T47 D的CSC特征。采用RT-PCR、Western blot和免疫荧光等方法检测目的基因的mRNA、蛋白表达、细胞内含量和定位。在体内水平，用AhR/CYP 1A 1诱导剂处理雌性Balb/c小鼠，观察其组织病理学变化，并对靶蛋白进行免疫组化检测。CYP 1A 1和CYP 1B 1，AhR调节基因的组成型mRNA表达和细胞含量在CSC中明显高于5种不同人乳腺癌细胞的分化非CSC。在MCF-7细胞中，TCDD和DMBA（强AhR激活剂）激活AhR/CYP 1A 1，显著增加CSC特异性标志物、乳腺球形成、醛脱氢酶（ALDH）活性和侧群（SP）细胞百分比，而使用化学抑制剂α-萘甲酮（α-NF）或通过基因shRNA敲低灭活AhR/CYP 1A 1，显著抑制ALDH活性和SP细胞的上调。重要的是，AhR/CYP 1A 1的失活显著增加了CSC对化疗剂阿霉素的敏感性。TCDD和DMBA对AhR/CYP 1A 1的诱导与β-Catenin mRNA和蛋白表达、核转位及其下游靶细胞Cyclin D1的显著增加相关，而使用shRNA敲低AhR或CYP 1A 1则显著抑制β-Catenin细胞含量和核转位。这与PTEN的显著抑制和总Akt蛋白和磷酸化Akt蛋白表达的诱导有关。重要的是，LY 294002抑制PI 3 K/Akt通路完全阻断TCDD诱导的SP细胞扩增。在体内，未处理的和DMBA（30 mg/kg，IP）处理的小鼠的乳腺结构的IHC染色显示出PTEN表达的极大抑制，伴随着p-Akt、β-连环蛋白和干细胞标志物ALDH 1表达的增加。本研究首次证明AhR/CYP 1A 1信号通路通过抑制PTEN、激活β-Catenin和Akt通路来调控乳腺CSCs的增殖、发育、自我更新和化疗耐药性。
Breast cancer stem cells (CSCs) are small sub-type of the whole cancer cells that drive tumor initiation, progression and metastasis. Recent studies have demonstrated a role for the aryl hydrocarbon receptor (AhR)/cytochrome P4501A1 pathway in CSCs expansion. However, the exact molecular mechanisms remain unclear. The current study was designed to a) determine the effect of AhR activation and inhibition on breast CSCs development, maintenance, self-renewal, and chemoresistance at the in vitro and in vivo levels and b) explore the role of β-Catenin, PI3K/Akt, and PTEN signaling pathways. To test this hypothesis, CSC characteristics of five human breast cancer cells; SKBR-3, MCF-7, and MDA-MB231, HS587T, and T47D treated with AhR activators or inhibitor were determined using Aldefluor assay, side population, and mammosphere formation. The mRNA, protein expression, cellular content and localization of the target genes were determined by RT-PCR, Western blot analysis, and Immunofluorescence, respectively. At the in vivo level, female Balb/c mice were treated with AhR/CYP1A1 inducer and histopathology changes and Immunohistochemistry examination for target proteins were determined. The constitutive mRNA expression and cellular content of CYP1A1 and CYP1B1, AhR-regulated genes, were markedly higher in CSCs more than differentiating non-CSCs of five different human breast cancer cells. Activation of AhR/CYP1A1 in MCF-7 cells by TCDD and DMBA, strong AhR activators, significantly increased CSC-specific markers, mammosphere formation, aldehyde dehydrogenase (ALDH) activity, and percentage of side population (SP) cells, whereas inactivation of AhR/CYP1A1 using chemical inhibitor, α-naphthoflavone (α-NF), or by genetic shRNA knockdown, significantly inhibited the upregulation of ALDH activity and SP cells. Importantly, inactivation of the AhR/CYP1A1 significantly increased sensitization of CSCs to the chemotherapeutic agent doxorubicin. Mechanistically, Induction of AhR/CYP1A1 by TCDD and DMBA was associated with significant increase in β-Catenin mRNA and protein expression, nuclear translocation and its downstream target Cyclin D1, whereas AhR or CYP1A1 knockdown using shRNA dramatically inhibited β-Catenin cellular content and nuclear translocation. This was associated with significant inhibition of PTEN and induction of total and phosphorylated Akt protein expressions. Importantly, inhibition of PI3K/Akt pathway by LY294002 completely blocked the TCDD-induced SP cells expansion. In vivo, IHC staining of mammary gland structures of untreated and DMBA (30 mg/kg, IP)- treated mice, showed tremendous inhibition of PTEN expression accompanied with an increase in the expression p-Akt, β-Catenin and stem cells marker ALDH1. The present study provides the first evidence that AhR/CYP1A1 signaling pathway is controlling breast CSCs proliferation, development, self-renewal and chemoresistance through inhibition of the PTEN and activation of β-Catenin and Akt pathways.