TY - JOUR
T1 - Arsenite malignantly transforms human prostate epithelial cells in vitro by gene amplification of mutated KRAS
AU - Alex Merrick, B.
AU - Phadke, Dhiral P.
AU - Bostrom, Meredith A.
AU - Shah, Ruchir R.
AU - Wright, Garron M.
AU - Wang, Xinguo
AU - Gordon, Oksana
AU - Pelch, Katherine E.
AU - Auerbach, Scott S.
AU - Paules, Richard S.
AU - DeVito, Michael J.
AU - Waalkes, Michael P.
AU - Tokar, Erik J.
N1 - Funding Information:
This work was supported by funds from the Division of the National Toxicology Program (DNTP). The funder is the federal government at the National Institute of Environmental Health Sciences (NIEHS) https://www.niehs.nih.gov/index. cfm] and has provided support in the form of salaries for DHMRI authors, MAB, GMW, XW, OG, under NIEHS contract HHSN273201100016C; and salaries for Sciome, LLC authors DPP and RRS under NIEHS contract HHSN273201700001C. The website for Sciome, LLC is https://www.sciome. com and for DHMRI are https://transforming-science.com/dhmri/. The Sciome, LLC and DHMRI (David H. Murdoch Research Institute) authors did not have any additional role in the study design, decision to publish, or preparation of the manuscript. Sciome, LLC provided support in the form of salary for authors DPP,RRS, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of these authors is articulated in the ‘author contributions’ section.
Publisher Copyright:
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
PY - 2019/4
Y1 - 2019/4
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85064816353&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0215504
DO - 10.1371/journal.pone.0215504
M3 - Article
C2 - 31009485
AN - SCOPUS:85064816353
SN - 1932-6203
VL - 14
JO - PLoS ONE
JF - PLoS ONE
IS - 4
M1 - e0215504
ER -