Department of Medicine, Hematology and Oncology, University of Muenster, Muenster, Germany.
BMC Cancer. 2010 Mar 3;10:78. doi: 10.1186/1471-2407-10-78.
Increased transcription of oncogenes like the epidermal growth factor receptor (EGFR) is frequently caused by amplification of the whole gene or at least of regulatory sequences. Aim of this study was to pinpoint mechanistic parameters occurring during egfr copy number gains leading to a stable EGFR overexpression and high sensitivity to extracellular signalling. A deeper understanding of those marker events might improve early diagnosis of cancer in suspect lesions, early detection of cancer progression and the prediction of egfr targeted therapies.
The basal-like/stemness type breast cancer cell line subpopulation MDA-MB-468 CD44high/CD24-/low, carrying high egfr amplifications, was chosen as a model system in this study. Subclones of the heterogeneous cell line expressing low and high EGF receptor densities were isolated by cell sorting. Genomic profiling was carried out for these by means of SNP array profiling, qPCR and FISH. Cell cycle analysis was performed using the BrdU quenching technique.
Low and high EGFR expressing MDA-MB-468 CD44+/CD24-/low subpopulations separated by cell sorting showed intermediate and high copy numbers of egfr, respectively. However, during cell culture an increase solely for egfr gene copy numbers in the intermediate subpopulation occurred. This shift was based on the formation of new cells which regained egfr gene copies. By two parametric cell cycle analysis clonal effects mediated through growth advantage of cells bearing higher egfr gene copy numbers could most likely be excluded for being the driving force. Subsequently, the detection of a fragile site distal to the egfr gene, sustaining uncapped telomere-less chromosomal ends, the ladder-like structure of the intrachromosomal egfr amplification and a broader range of egfr copy numbers support the assumption that dynamic chromosomal rearrangements, like breakage-fusion-bridge-cycles other than proliferation drive the gain of egfr copies.
Progressive genome modulation in the CD44+/CD24-/low subpopulation of the breast cancer cell line MDA-MB-468 leads to different coexisting subclones. In isolated low-copy cells asymmetric chromosomal segregation leads to new cells with regained solely egfr gene copies. Furthermore, egfr regain resulted in enhanced signal transduction of the MAP-kinase and PI3-kinase pathway. We show here for the first time a dynamic copy number regain in basal-like/stemness cell type breast cancer subpopulations which might explain genetic heterogeneity. Moreover, this process might also be involved in adaptive growth factor receptor intracellular signaling which support survival and migration during cancer development and progression.
表皮生长因子受体 (EGFR) 等癌基因的转录增加通常是由于整个基因或至少调节序列的扩增引起的。本研究的目的是确定 EGFR 拷贝数增加过程中发生的机制参数，导致 EGFR 过表达稳定和对外界信号的高敏感性。对这些标记事件的深入了解可能会改善可疑病变中癌症的早期诊断、癌症进展的早期检测和 EGFR 靶向治疗的预测。
本研究选择基底样/干细胞型乳腺癌细胞系 MDA-MB-468 CD44high/CD24-/low 亚群作为模型系统，该亚群携带高 EGFR 扩增。通过细胞分选分离出表达低和高 EGF 受体密度的异质细胞系亚克隆。通过 SNP 芯片分析、qPCR 和 FISH 对这些细胞进行基因组分析。使用 BrdU 淬灭技术进行细胞周期分析。
通过细胞分选分离出的低 EGFR 表达和高 EGFR 表达 MDA-MB-468 CD44+/CD24-/low 亚群分别显示 EGFR 中间和高拷贝数。然而，在细胞培养过程中，中间亚群中仅 EGFR 基因拷贝数增加。这种转变是基于形成新细胞的方式，这些新细胞重新获得了 EGFR 基因拷贝。通过双参数细胞周期分析，可以排除携带更高 EGFR 基因拷贝数的细胞通过生长优势介导的克隆效应是驱动因素。随后，在 EGFR 基因远端检测到一个脆性位点，维持无端粒的端粒-less 染色体末端，染色体内 EGFR 扩增的梯状结构和更广泛的 EGFR 拷贝数范围支持这样的假设，即动态染色体重排，如断裂-融合-桥循环，而不是增殖，驱动 EGFR 拷贝数的增加。
乳腺癌细胞系 MDA-MB-468 的 CD44+/CD24-/low 亚群中进行的渐进性基因组调节导致不同共存的亚克隆。在分离的低拷贝细胞中，不对称染色体分离导致新细胞重新获得仅 EGFR 基因拷贝。此外，EGFR 恢复导致 MAP 激酶和 PI3 激酶途径的信号转导增强。我们在这里首次显示，基底样/干细胞样乳腺癌亚群中存在动态拷贝数恢复，这可能解释遗传异质性。此外，这个过程也可能参与适应性生长因子受体细胞内信号转导，支持癌症发展和进展过程中的存活和迁移。
