Tumour suppressor EP300, a modulator of paclitaxel resistance and stemness, is downregulated in metaplastic breast cancer.
Tumour suppressor EP300, a modulator of paclitaxel resistance and stemness, is downregulated in metaplastic breast cancer.
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肿瘤抑制因子 EP300 是紫杉醇耐药性和干性的调节剂，在化生性乳腺癌中表达下调
DOI：
10.1007/s10549-017-4202-z
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发表时间：
2017-06
影响因子：
3.8
通讯作者：
Yagüe E
中科院分区：
文献类型：
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作者：
Asaduzzaman M;Constantinou S;Min H;Gallon J;Lin ML;Singh P;Raguz S;Ali S;Shousha S;Coombes RC;Lam EW;Hu Y;Yagüe E
目的我们之前描述了一种控制乳腺癌细胞耐药性、上皮间质转化（EMT）和干性的新途径。在该通路的上游，三个 miR（miR-106b、miR-93 和 miR-25）以 EP300（E-钙粘蛋白的转录激活剂）为目标。这些 miR 的上调导致 EP300 和 E-钙粘蛋白下调，并启动 EMT。然而，miR调节许多基因的表达，并且不能排除EP300以外的miR靶点对EMT的贡献。方法我们使用表达EP300靶向shRNA的慢病毒来下调其在MCF-7细胞以及EP300敲除的结肠癌细胞系中的表达。使用 EP300 表达质粒上调其在 basal 样 CAL51 和 MDA-MB-231 乳腺癌细胞中的表达。通过短期增殖和长期集落形成测定来确定耐药性。通过软琼脂和液体培养物中肿瘤球的形成以及 CD44/CD24/ALDH 标记物的表达来确定干性。在缺乏 EP300 的 MCF-7 细胞中进行基因表达微阵列分析。通过免疫组织化学分析 17 个化生性乳腺癌样本中的 EP300 表达。结果缺乏 EP300 的细胞对紫杉醇的耐药性更强，而 EP300 过度表达则增加了对药物的敏感性。癌症干细胞标志物的表达以及肿瘤球的形成在EP300耗尽的细胞中也增加，而在EP300过表达的细胞中减少。 EP300 调控的基因特征突出显示与粘附 (CEACAM5)、细胞骨架重塑 (CAPN9)、干性 (ABCG2)、细胞凋亡 (BCL2) 和转移 (TGFB2) 相关的基因。该特征中的一些基因也在之前使用最低限度转化的乳腺上皮细胞生成的 EP300 耗尽的乳腺癌模型中得到了验证。重要的是，细胞凋亡和干性中的两个关键基因BCL2和ABCG2在EP300敲除的结肠癌细胞及其紫杉醇抗性衍生物中也上调。免疫组织化学分析表明，EP300 在化生性乳腺癌中表达较低，这是一种罕见但具有侵袭性的疾病，预后不良，其特征是 EMT 的形态和生理特征。结论 EP300 在重编程事件中发挥重要作用，导致更恶性的表型，并获得耐药性和细胞可塑性，这是化生性乳腺癌的特征。
PurposeWe have previously described a novel pathway controlling drug resistance, epithelial-to-mesenchymal transition (EMT) and stemness in breast cancer cells. Upstream in the pathway, three miRs (miR-106b, miR-93 and miR-25) target EP300, a transcriptional activator of E-cadherin. Upregulation of these miRs leads to the downregulation of EP300 and E-cadherin with initiation of an EMT. However, miRs regulate the expression of many genes, and the contribution to EMT by miR targets other than EP300 cannot be ruled out.MethodsWe used lentiviruses expressing EP300-targeting shRNA to downregulate its expression in MCF-7 cells as well as anEP300-knocked-out colon carcinoma cell line. An EP300-expression plasmid was used to upregulate its expression in basal-like CAL51 and MDA-MB-231 breast cancer cells. Drug resistance was determined by short-term proliferation and long-term colony formation assays. Stemness was determined by tumour sphere formation in both soft agar and liquid cultures as well as by the expression of CD44/CD24/ALDH markers. Gene expression microarray analysis was performed in MCF-7 cells lacking EP300. EP300 expression was analysed by immunohistochemistry in 17 samples of metaplastic breast cancer.ResultsCells lacking EP300 became more resistant to paclitaxel whereas EP300 overexpression increased their sensitivity to the drug. Expression of cancer stem cell markers, as well as tumour sphere formation, was also increased in EP300-depleted cells, and was diminished in EP300-overexpressing cells. The EP300-regulated gene signature highlighted genes associated with adhesion (CEACAM5), cytoskeletal remodelling (CAPN9), stemness (ABCG2), apoptosis (BCL2) and metastasis (TGFB2). Some genes in this signature were also validated in a previously generated EP300-depleted model of breast cancer using minimally transformed mammary epithelial cells. Importantly, two key genes in apoptosis and stemness,BCL2andABCG2, were also upregulated in EP300-knockout colon carcinoma cells and their paclitaxel-resistant derivatives. Immunohistochemical analysis demonstrated that EP300 expression was low in metaplastic breast cancer, a rare, but aggressive form of the disease with poor prognosis that is characterized by morphological and physiological features of EMT.ConclusionsEP300 plays a major role in the reprogramming events, leading to a more malignant phenotype with the acquisition of drug resistance and cell plasticity, a characteristic of metaplastic breast cancer.