Department of Pathology and Comprehensive Cancer Center, University of California at San Francisco, 513 Parnassus Avenue, San Francisco, California, 94143-0511, USA.
Breast Cancer Res. 2009;11(6):R87. doi: 10.1186/bcr2457. Epub 2009 Dec 8.
We have previously identified a rare subpopulation of variant human mammary epithelial cells (vHMEC) with repressed p16INK4A that exist in disease-free women yet display premalignant properties, suggesting that they have engaged the process of malignant transformation. In order to gain insight into the molecular alterations required for vHMEC to progress to malignancy, and to characterize the epigenetic events associated with early progression, we examined the effect of oncogenic stress on the behavior of these cells.
HMEC that express p16INK4A and vHMEC that do not, were transduced with constitutively active Ha-rasV12 and subsequently exposed to serum to determine whether signals from the cellular microenvironment could cooperate with ras to promote the malignant transformation of vHMEC. Epigenetic alterations were assessed using methylation-specific polymerase chain reaction (PCR).
vHMEC expressing Ha-rasV12 (vHMEC-ras) bypassed the classic proliferative arrest that has been previously documented in normal fibroblasts following oncogenic stress, and that we also observe here in normal HMEC. Moreover, vHMEC-ras cells exhibited many additional alterations that are observed during progression to malignancy such as the generation of chromosomal abnormalities, upregulation of telomerase activity, immortalization following exposure to serum, and anchorage-independent growth, but they did not form tumors following orthotopic injection in vivo. Associated with their early progression to malignancy was an increase in the number of genes methylated, two of which (RASSF1A and SFRP1) were also methylated in other immortalized mammary cell lines as well as in breast cancer cells and tissues.
We have characterized a mammary progression model that recapitulates molecular and methylation alterations observed in many breast cancers. Our data suggest that concomitant methylation of RASSF1A and SFRP1 marks an early event in mammary transformation and may thus have prognostic potential.
我们之前已经鉴定出一种罕见的变异型人乳腺上皮细胞（vHMEC）亚群，其 p16INK4A 被抑制，但存在于无疾病的女性中，却显示出恶性前特性，这表明它们已经开始恶性转化过程。为了深入了解 vHMEC 进展为恶性肿瘤所需的分子改变，并描述与早期进展相关的表观遗传事件，我们研究了致癌应激对这些细胞行为的影响。
表达 p16INK4A 的 HMEC 和不表达 p16INK4A 的 vHMEC 被转导了组成性激活的 Ha-rasV12，随后暴露于血清中，以确定细胞微环境中的信号是否可以与 ras 合作促进 vHMEC 的恶性转化。使用甲基化特异性聚合酶链反应（PCR）评估表观遗传改变。
表达 Ha-rasV12 的 vHMEC（vHMEC-ras）绕过了之前在致癌应激后正常成纤维细胞中已经记录到的经典增殖停滞，我们在正常 HMEC 中也观察到了这一点。此外，vHMEC-ras 细胞还表现出许多在向恶性转化过程中观察到的其他改变，如染色体异常的产生、端粒酶活性的上调、在暴露于血清后永生化以及锚定独立生长，但它们在体内原位注射后没有形成肿瘤。与它们早期向恶性转化相关的是甲基化基因数量的增加，其中两个基因（RASSF1A 和 SFRP1）在其他永生化乳腺细胞系以及乳腺癌细胞和组织中也被甲基化。
我们已经描述了一种乳腺进展模型，该模型重现了许多乳腺癌中观察到的分子和甲基化改变。我们的数据表明，RASSF1A 和 SFRP1 的同时甲基化标志着乳腺转化的早期事件，因此可能具有预后潜力。
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