Genomic profile of the estrogen induced neoplastic transformation of the human breast epithelial cell MCF10F. Homo sapiens

Estrogen is a risk factor for breast cancer, yet its mechanism in the initiation of this disease is not clear. Here we use a model of estrogen-mediated malignant transformation of MCF-10F cells to identify the temporal acquisition of changes in genome structure and gene expression that correspond to the progressive transformed phenotype culminating in tumorigenesis. Genomic DNA and total RNA were isolated from MCF10F, trMCF (MCF10F transformed by 70 nM 17-beta estradiol), bsMCF (trMCF selected by Boyden chamber, tumorigenic), bcMCF(clones of bsMCF), and caMCF (cells from tumors of bsMCF grown in SCID mice). The Affymetrix 100k SNP and HG-U133_Plus_2 chips were used for genotyping and gene expression analyses. Changes in chromosomal copy number and loss of heterozygosity were progressive. Gross changes were rarely observed in the trMCF10F cells; the earliest was a gain in chromosome 1p, 1p36.12-1p36.21. In the bsMCF and their sub-clones bcMCF, additional gains were seen in chromosomes 1p, 1p36.12-1pter, 5q, 5q21.1-5q35.3, and 13q, 13q21.32-13q34; losses were detected in chromosomes 4 and 8p, 8p11.1-8p23.1. In the caMCF, additional losses were seen in chromosomes 3p, 3p12.1-3p14.1, 9p, 9p22.1-9p24.3, and 18q, 18q11.2-18q23. Using microarray analysis we were able, in some cases, to narrow the regions of interest and their associated candidate cancer genes. The observation of progressive genomic changes, along with the tumor phenotype of a poorly differentiated adenocarcinoma, indicate an underlying mechanism common to breast cancer development in humans, and adds to the evidence indicating that estrogens are mutagenic and contribute early to the process of breast cancer. Keywords: Cell type comparison Overall design: Human MCF-10F cells were transformed by 70nM 17-beta-estradiol. The transformed cells were selected by Boyden chamber. The selected cells were injected into SCID mice and also cloned and expanded. The thus grown tumors were harvested for cell culture. Genomic DNA and total RNA were isolated from cell lines in each step for 100K SNP and gene expression microarray assay.

雌激素是乳腺癌的危险因素，但其在该疾病发生中的具体机制尚未阐明。本研究借助雌激素介导的MCF-10F细胞（MCF-10F cells）恶性转化模型，解析伴随进行性转化表型、最终导向致瘤过程的基因组结构与基因表达时序性变化。
本研究从MCF10F、trMCF（经70nM 17β-雌二醇（17-beta estradiol）转化的MCF-10F细胞）、bsMCF（经博登小室（Boyden chamber）筛选的致瘤性trMCF细胞）、bcMCF（bsMCF的克隆株）以及caMCF（在重症联合免疫缺陷（Severe Combined Immunodeficiency, SCID）小鼠体内生长的bsMCF来源的肿瘤细胞）中提取基因组DNA与总RNA。
本研究采用Affymetrix 100k单核苷酸多态性（Single Nucleotide Polymorphism, SNP）芯片与HG-U133_Plus_2芯片分别开展基因分型与基因表达谱分析。
染色体拷贝数改变与杂合性缺失呈进行性发展。trMCF10F细胞中极少观察到显著基因组改变，最早的改变为1号染色体短臂1p36.12-1p36.21区域的拷贝数增加。
在bsMCF及其亚克隆bcMCF中，额外出现的拷贝数增加区域包括1号染色体短臂1p36.12-1pter、5号染色体长臂5q21.1-5q35.3以及13号染色体长臂13q21.32-13q34；同时检测到4号染色体与8号染色体短臂8p11.1-8p23.1区域的拷贝数缺失。
在caMCF细胞中，额外出现的拷贝数缺失区域包括3号染色体短臂3p12.1-3p14.1、9号染色体短臂9p22.1-9p24.3以及18号染色体长臂18q11.2-18q23。
通过微阵列分析，本研究在部分案例中得以缩小目标区域范围，并明确其关联的候选癌基因。
本研究观察到的进行性基因组改变，结合低分化腺癌的肿瘤表型，提示存在一条与人类乳腺癌发生相关的共同潜在机制，同时为雌激素具有诱变作用并早期参与乳腺癌发生进程提供了新的佐证。
关键词：细胞类型比较
实验设计概述：本研究采用70nM 17β-雌二醇转化人MCF-10F细胞，通过博登小室筛选获得转化细胞；将筛选后的细胞接种至SCID小鼠体内，同时进行克隆扩增。收获小鼠体内生长的肿瘤并建立细胞培养体系。从各阶段的细胞系中提取基因组DNA与总RNA，用于100K SNP基因分型与基因表达微阵列检测。