title: Silencing KRAS overexpression in arsenic-transformed prostate epithelial and stem cells partially mitigates malignant phenotype.
abstract: Inorganic arsenic is a human carcinogen that likely targets the prostate. Chronic arsenic exposure malignantly transforms the RWPE-1 human prostate epithelial line to chronic arsenic exposed-prostate epithelial (CAsE-PE) cells, and a derivative normal prostate stem cell (SC) line, WPE-stem to arsenic-cancer SCs (As-CSCs). The KRAS oncogene is highly overexpressed in CAsE-PE cells and activation precedes transformation, inferring mechanistic significance. As-CSCs also highly overexpress KRAS. Thus, we hypothesize KRAS activation is key in causing and maintaining an arsenic-induced malignant phenotype, and hence, KRAS knockdown (KD) may reverse this malignant phenotype. RNA interference using shRNAmirs to obtain KRAS KD was used in CAsE-PE and As-CSC cells. Cells analyzed 2 weeks post transduction showed KRAS protein decreased to 5% of control after KD, confirming stable KD. KRAS KD decreased phosphorylated ERK, indicating inhibition of RAS/ERK signaling, a proliferation/survival pathway activated with arsenic transformation. Secreted metalloproteinase (MMP) activity was increased by arsenic-induced malignant transformation, but KRAS KD from 4 weeks on decreased secreted MMP-9 activity by 50% in As-CSCs. Colony formation, a characteristic of cancer cells, was decreased in both KRAS KD transformants. KRAS KD also decreased the invasive capacity of both cell types. KRAS KD decreased proliferation in As-CSCs, consistent with loss of rapid tumor growth. Genes predicted to impact cell proliferation (eg, Cyclin D1, p16, and p21) changed accordingly in both KD cell types. Thus, KRAS silencing impacts aspects of arsenic-induced malignant phenotype, inducing loss of many typical cancer characteristics particularly in As-CSCs.
authors.affiliation: Inorganic Toxicology Group, National Toxicology Program Laboratory, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
authors.forename: Ntube N O
authors.initials: NN
authors.lastname: Ngalame
authors.affiliation: Inorganic Toxicology Group, National Toxicology Program Laboratory, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
authors.forename: Erik J
authors.initials: EJ
authors.lastname: Tokar
authors.affiliation: Inorganic Toxicology Group, National Toxicology Program Laboratory, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
authors.forename: Rachel J
authors.initials: RJ
authors.lastname: Person
authors.affiliation: Inorganic Toxicology Group, National Toxicology Program Laboratory, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709 waalkes@niehs.nih.gov.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Waalkes
mesh_terms: D000818:Animals; D018053:Arsenites / Q000633:toxicity*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D002471:Cell Transformation, Neoplastic / Q000187:drug effects* / Q000235:genetics; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D008297:Male; D008819:Mice, Nude; D014411:Neoplastic Stem Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology*; D011467:Prostate / Q000378:metabolism / Q000473:pathology; D011471:Prostatic Neoplasms / Q000139:chemically induced / Q000235:genetics* / Q000473:pathology; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D017670:Sodium Compounds / Q000633:toxicity*; D023041:Xenograft Model Antitumor Assays; D018631:ras Proteins / Q000235:genetics*
chemical_list: D018053:Arsenites; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D017670:Sodium Compounds; C017947:sodium arsenite; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
keywords: KRAS; arsenic; cancer; prostate cells; shRNA; stem cells