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

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.

Oncogenic KRAS–expressing organoids with biliary epithelial stem cell properties give rise to biliary tract cancer in mice

“…Gene amplification for mutant KRAS has been detected in a substantial fraction of lung 52 and prostate 53 cancers, and it is apparent in many cases of pancreatic cancer in The Cancer Genome Atlas (TCGA) 54 . In addition, such cases have been found to be clinically aggressive.…”

Targeting KRAS in Cancer: Promising Therapeutic Strategies

“…Mutations in KRAS account for 7% of prostate cancer cases and have been linked to effector proteins such as RAF, PI3K and GDP/GTP Ral exchange factor, contributing to downstream signaling responses. Mutant KRAS is known to be a transformative factor in prostate cancer and found to promote cancer stemness and bone metastasis [ 74 , 75 , 76 ]. In addition, KRAS rearrangements have been shown to promote the metastatic progression of prostate cancer [ 77 ].…”

The Multifaceted Role of Aldehyde Dehydrogenases in Prostate Cancer Stem Cells

“…The study of Merrick and colleagues modeled the changes of ALDH1A2 expression during the progression from a healthy to malignant prostate. The comparison of the ALDH1A2 transcript level in non-tumorigenic RWPE1 cells versus a malignant subline CAsE-PE (generated by long-term exposure to arsenite) revealed its more than 300-fold downregulation in neoplastic cells [ 73 ]. In addition, similarly, decreased ALDH1A2 transcript levels were shown to be part of a signature of genes able to predict the metastatic lethal outcome of patients that underwent radical prostatectomy [ 74 ].…”