Shehwana Huma, Keskus Ayse G, Ozdemir Sila E, Acikgöz Azer A, Biyik-Sit Rumeysa, Cagnan Ilgin, Gunes Damla, Jahja Ermira, Cingir-Koker Sahika, Olmezer Gizem, Sucularli Ceren, Konu Ozlen
Department of Molecular Biology and Genetics, Faculty of Science, Bilkent University, Ankara, Turkey.
Department of Biological Sciences, National University of Medical Sciences, Rawalpindi, 46000, Pakistan.
Cell Oncol (Dordr). 2021 Apr;44(2):453-472. doi: 10.1007/s13402-020-00581-x. Epub 2021 Jan 19.
Cholinergic signals can be important modulators of cellular signaling in cancer. We recently have shown that knockdown of nicotinic acetylcholine receptor subunit alpha 5, CHRNA5, diminishes the proliferative potential of breast cancer cells. However, modulation of CHRNA5 expression in the context of estrogen signaling and its prognostic implications in breast cancer remained unexplored.
Meta-analyses of large breast cancer microarray cohorts were used to evaluate the association of CHRNA5 expression with estrogen (E2) treatment, estrogen receptor (ER) status and patient prognosis. The results were validated through RT-qPCR analyses of multiple E2 treated cell lines, CHRNA5 depleted MCF7 cells and across a breast cancer patient cDNA panel. We also calculated a predicted secondary (PS) score representing direct/indirect induction of gene expression by E2 based on a public dataset (GSE8597). Co-expression analysis was performed using a weighted gene co-expression network analysis (WGCNA) pipeline. Multiple other publicly available datasets such as CCLE, COSMIC and TCGA were also analyzed.
Herein we found that CHRNA5 expression was induced by E2 in a dose- and time-dependent manner in breast cancer cell lines. ER breast tumors exhibited higher CHRNA5 expression levels than ER tumors. Independent meta-analysis for survival outcome revealed that higher CHRNA5 expression was associated with a worse prognosis in untreated breast cancer patients. Furthermore, CHRNA5 and its co-expressed gene network emerged as secondarily induced targets of E2 stimulation. These targets were largely downregulated by exposure to CHRNA5 siRNA in MCF7 cells while the response of primary ESR1 targets was dependent on the direction of the PS-score. Moreover, primary and secondary target genes were uncoupled and clustered distinctly based on multiple public datasets.
Our findings strongly associate increased expression of CHRNA5 and its co-expression network with secondary E2 signaling and a worse prognosis in breast cancer.
胆碱能信号可能是癌症细胞信号传导的重要调节因子。我们最近发现，敲除烟碱型乙酰胆碱受体α5亚基（CHRNA5）可降低乳腺癌细胞的增殖潜能。然而，雌激素信号背景下CHRNA5表达的调节及其在乳腺癌中的预后意义仍未得到探索。
对大型乳腺癌微阵列队列进行荟萃分析，以评估CHRNA5表达与雌激素（E2）治疗、雌激素受体（ER）状态和患者预后之间的关联。通过对多个E2处理的细胞系、CHRNA5缺失的MCF7细胞以及一组乳腺癌患者cDNA样本进行RT-qPCR分析，验证了结果。我们还根据一个公共数据集（GSE8597）计算了一个预测二级（PS）评分，该评分代表E2对基因表达的直接/间接诱导作用。使用加权基因共表达网络分析（WGCNA）流程进行共表达分析。还分析了多个其他公开可用的数据集，如CCLE、COSMIC和TCGA。
在此我们发现，在乳腺癌细胞系中，CHRNA5表达以剂量和时间依赖性方式被E2诱导。ER阳性乳腺癌肿瘤的CHRNA5表达水平高于ER阴性肿瘤。对生存结果的独立荟萃分析显示，在未经治疗的乳腺癌患者中，较高的CHRNA5表达与较差的预后相关。此外，CHRNA5及其共表达基因网络是E2刺激的二级诱导靶点。在MCF7细胞中，这些靶点在很大程度上因CHRNA5 siRNA的作用而下调，而ESR1主要靶点的反应则取决于PS评分的方向。此外，根据多个公共数据集，主要和次要靶点基因是解耦的且明显聚类。
我们的研究结果有力地表明，CHRNA5及其共表达网络的表达增加与E2二级信号传导以及乳腺癌较差的预后相关。
