Research Article: Arsenite malignantly transforms human prostate epithelial cells in vitro by gene amplification of mutated KRAS

Date Published: April 22, 2019

Publisher: Public Library of Science

Author(s): B. Alex Merrick, Dhiral P. Phadke, Meredith A. Bostrom, Ruchir R. Shah, Garron M. Wright, Xinguo Wang, Oksana Gordon, Katherine E. Pelch, Scott S. Auerbach, Richard S. Paules, Michael J. DeVito, Michael P. Waalkes, Erik J. Tokar, Kamaleshwar P Singh.

http://doi.org/10.1371/journal.pone.0215504

Abstract

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.

Partial Text

Environmental exposure to arsenic increases the risks of skin, lung, kidney, liver and urinary-bladder cancers [1, 2]. Although the mode of action for arsenic-induced tumors is unclear, many animal and human studies suggest arsenic can act as a carcinogen [3, 4], co-carcinogen [5, 6], or transplacental carcinogen [7]. Arsenite and arsenate, the inorganic tri- and pentavalent forms of arsenic, are considered non-mutagenic in bacterial and human cells [8, 9]. However, arsenic may indirectly cause DNA damage, chromosomal abnormalities, and generation of reactive oxygen species (ROS) like superoxide or hydrogen peroxide [10, 11]. Other transformational effects of arsenic may involve disruption of signaling pathways, miRNA dysregulation, inhibition of DNA repair, or formation of cancer stem cells or polycomb proteins [12–19]. Arsenite and other trivalent species can be acutely cytotoxic by readily binding to intracellular thiols (e.g. GSH) and sulfhydryl sites on macromolecules to inhibit critical biochemical processes [17]. Persistent cytotoxicity from prolonged arsenic exposure and subsequent regenerative proliferation may contribute to carcinogenesis as well [3]. Biotransformation of arsenic involves S-adenosylmethione (SAM), methyltransferases and sulfur redox metabolism so that arsenic-induced interference of methyl-donor pathways could lead to abnormal DNA methylation and histone modification patterns and epigenetic transformation [14, 15, 17, 20–24].

A major finding of this study was the highly mutated nature of a KRAS allele as well as its substantial overexpression. Missense mutations at codon 12 (p.G12S) and codon 59 (p.A59T) are both pathogenic as documented in the COSMIC database of human cancer mutations [54]. Codon 12 and codon 59 KRAS mutations are most commonly found in the large intestine tumors and to a lesser extent in lung, hematopoietic and stomach tumors. The sequence variant at codon p.D132E has not yet been reported for KRAS in human tumors. It is notable the other sequence variants result in silent mutations, most of which have not been annotated. KRAS mutations likely accumulate through clonal selection over months of arsenite exposure. Importantly, the concurrence of wt and amplified mutant KRAS DNA in CAsE-PE cells is consistent with the genomic aberration, ‘mutant allele specific imbalance’, known as MASI, that is frequently observed for oncogenes in many primary tumors [55]. The incidence of KRAS MASI genotypes range from about 12 to 18% of clinical cases that include colorectal [56, 57] and lung [58], and pancreatic [59] cancers involving mutations in codons 12 or 13. One study that examined over 400 human tumors, cell lines and xenografts of various tumor types reported that KRAS MASI was often present in primary tumors and malignantly transformed cell lines and that the combination of oncogene mutation, copy number gains and MASI may have a greater role in development and maintenance of malignancy than any individual alteration alone [47]. A more recent study involving more than 1,110 KRAS mutant tumors found an incidence of allelic imbalance at 55% [60]. Even though malignantly transformed cells may contain multiple gene mutations, sequencing studies suggest that mutations in driver oncogenes are often mutually exclusive, meaning the actions of one oncogene may predominate over others [61]. In recent work, knockdown of KRAS expression in CAsE-PE cells greatly reduced several malignant characteristics including anchorage independent growth and metalloprotease activity [38]. Thus, the high level of KRAS gene amplification, transcript expression and activating mutations suggest it is the lead oncogene driving the malignantly transformed CAsE-PE phenotype.

CAsE-PE cells comprise a well-studied in vitro human epithelial malignant transformation model produced by continuous low level arsenite exposure. Expression analysis showed >3,000 upregulated and about 4,000 downregulated transcripts that support increased cell growth, motility, survival and tumorigenic pathways consistent with prostate cancer. KRAS transcript was highly expressed in CAsE-PE cells, consistent with our prior work [25, 38], and is driven in part by an increase in KRAS copy number. Genomic analysis revealed a KRAS allelic imbalance with high expression of a mutated transcript carrying oncogenic mutations at codons 12 and 59 and many silent mutations, accompanied by relatively low expression of a wt allele. KRAS4a is the predominant transcript variant in CAsE-PE cells compared to KRAS4b in parental RWPE-1 epithelia and compared to normal primary prostate epithelium. These data are consistent with KRAS driven proliferation pathways found in spontaneous tumors or cell lines. Future work will focus on how KRAS becomes amplified in the in vitro arsenic transformation model to provide further insight into this important environmental carcinogen.

 

Source:

http://doi.org/10.1371/journal.pone.0215504

 

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