Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, United States of America.
Sciome, LLC, Research Triangle Park, North Carolina, United States of America.
PLoS One. 2019 Apr 22;14(4):e0215504. doi: 10.1371/journal.pone.0215504. eCollection 2019.
Inorganic arsenic is an environmental human carcinogen of several organs including the urinary tract. RWPE-1 cells are immortalized, non-tumorigenic, human prostate epithelia that become malignantly transformed into the CAsE-PE line after continuous in vitro exposure to 5μM arsenite over a period of months. For insight into in vitro arsenite transformation, we performed RNA-seq for differential gene expression and targeted sequencing of KRAS. We report >7,000 differentially expressed transcripts in CAsE-PE cells compared to RWPE-1 cells at >2-fold change, q<0.05 by RNA-seq. Notably, KRAS expression was highly elevated in CAsE-PE cells, with pathway analysis supporting increased cell proliferation, cell motility, survival and cancer pathways. Targeted DNA sequencing of KRAS revealed a mutant specific allelic imbalance, 'MASI', frequently found in primary clinical tumors. We found high expression of a mutated KRAS transcript carrying oncogenic mutations at codons 12 and 59 and many silent mutations, accompanied by lower expression of a wild-type allele. Parallel cultures of RWPE-1 cells retained a wild-type KRAS genotype. Copy number analysis and sequencing showed amplification of the mutant KRAS allele. KRAS is expressed as two splice variants, KRAS4a and KRAS4b, where variant 4b is more prevalent in normal cells compared to greater levels of variant 4a seen in tumor cells. 454 Roche sequencing measured KRAS variants in each cell type. We found KRAS4a as the predominant transcript variant in CAsE-PE cells compared to KRAS4b, the variant expressed primarily in RWPE-1 cells and in normal prostate, early passage, primary epithelial cells. Overall, gene expression data were consistent with KRAS-driven proliferation pathways found in spontaneous tumors and malignantly transformed cell lines. Arsenite is recognized as an important environmental carcinogen, but it is not a direct mutagen. Further investigations into this in vitro transformation model will focus on genomic events that cause arsenite-mediated mutation and overexpression of KRAS in CAsE-PE cells.
无机砷是一种环境人类致癌物，可致癌的器官包括泌尿系统。RWPE-1 细胞是永生化的、非致瘤性的人前列腺上皮细胞，在体外连续暴露于 5μM 亚砷酸盐数月后，连续体外暴露于 5μM 亚砷酸盐可恶性转化为 CAsE-PE 系。为了深入了解亚砷酸盐的体外转化，我们对差异基因表达进行了 RNA-seq 分析，并对 KRAS 进行了靶向测序。我们报告了 CAsE-PE 细胞与 RWPE-1 细胞相比，有 >7000 个差异表达的转录本，差异倍数 >2 倍，q<0.05。值得注意的是，CAsE-PE 细胞中 KRAS 表达显著升高，通路分析支持细胞增殖、细胞迁移、存活和癌症途径增加。对 KRAS 的靶向 DNA 测序显示，一种突变特异性等位基因不平衡（MASI），在原发性临床肿瘤中经常发现。我们发现携带 12 号和 59 号密码子致癌突变和许多沉默突变的突变 KRAS 转录本高表达，而野生型等位基因表达较低。RWPE-1 细胞的平行培养保留了野生型 KRAS 基因型。拷贝数分析和测序显示，突变 KRAS 等位基因扩增。KRAS 表达两种剪接变体，KRAS4a 和 KRAS4b，在正常细胞中变体 4b 比肿瘤细胞中更常见，而变体 4a 水平更高。454 Roche 测序测量了每种细胞类型的 KRAS 变体。我们发现 CAsE-PE 细胞中 KRAS4a 是主要的转录变体，而 RWPE-1 细胞和正常前列腺、早期传代、原代上皮细胞中主要表达 KRAS4b。总的来说，基因表达数据与自发肿瘤和恶性转化细胞系中发现的 KRAS 驱动的增殖途径一致。亚砷酸盐被认为是一种重要的环境致癌物，但它不是直接诱变剂。对这种体外转化模型的进一步研究将集中在导致 CAsE-PE 细胞中亚砷酸盐介导的突变和 KRAS 过表达的基因组事件上。
