Aberrant microRNA expression likely controls RAS oncogene activation during malignant transformation of human prostate epithelial and stem cells by arsenic

Abstract

Inorganic arsenic (iAs), a human carcinogen, potentially targets the prostate. iAs malignantly transforms the RWPE-1 human prostate epithelial line to CAsE-PE cells, and a derivative normal stem cell (SC) line, WPE-stem, to As-Cancer SC (As-CSC) line. MicroRNAs (miRNA) are noncoding but exert negative control on expression by degradation or translational repression of target mRNAs. Aberrant miRNA expression is important in carcinogenesis. A miRNA array of CAsE-PE and As-CSC revealed common altered expression in both for pathways concerning oncogenesis, miRNA biogenesis, cell signaling, proliferation, and tumor metastasis and invasion. The KRAS oncogene is overexpressed in CAsE-PE cells but not by mutation or promoter hypomethylation, and is intensely overexpressed in As-CSC cells. In both transformants, decreased miRNAs targeting KRAS and RAS superfamily members occurred. Reduced miR-134, miR-373, miR-155, miR-138, miR-205, miR-181d, miR-181c, and let-7 in CAsE-PE cells correlated with increased target RAS oncogenes, RAN, RAB27A, RAB22A mRNAs, and KRAS protein. Reduced miR-143, miR-34c-5p, and miR-205 in As-CSC correlated with increased target RAN mRNA, and KRAS, NRAS, and RRAS proteins. The RAS/ERK and PI3K/PTEN/AKT pathways control cell survival, differentiation, and proliferation, and when dysregulated promote a cancer phenotype. iAs transformation increased expression of activated ERK kinase in both transformants and altered components of the PI3K/PTEN/AKT pathway including decreased PTEN and increases in BCL2, BCL-XL, and VEGF in the absence of AKT activation. Thus, dysregulated miRNA expression may be linked to RAS activation in both transformants.

Keywords: KRAS; arsenic; cancer; microRNA; prostate cells; stem/progenitor cells.

FIG. 1.

Validation of some miRNA predicted and/or confirmed targets in arsenic transformed CAsE-PE and As-CSCs. mRNA expression of genes associated with: (A) cell adhesion and metastasis; (B) miRNA biogenesis, mitogenesis, and SC pluripotency. Data represent mean ± SEM (n = 3). *p < 0.05 compared with time-matched controls.

FIG. 2.

Expression of RAS oncogenes in CAsE-PE and As-CSC cells. (A) Transcript levels of RAS mRNAs in CAsE-PE and As-CSC cells. (B) RAS protein levels in CAsE-PE and As-CSC cells. Data represent mean ± SEM (n = 3). *p < 0.05 compared with time-matched controls.

FIG. 3.

Analysis of the RAS/ERK signaling pathway in CAsE-PE and As-CSC cells. (A) Transcript levels of some genes downstream of RAS. (B) Western blot analysis of p-ERK (activated form). Data represent mean ± SEM (n = 3). *p < 0.05.

FIG. 4.

PTEN protein expression in arsenic-transformed CAsE-PE and As-CSC cells. Data represent mean ± SEM (n = 3). *p < 0.05 compared with time-matched controls.

FIG. 5.

Expression of genes associated with and further downstream of the PI3K/PTEN/AKT pathway. Data represent mean ± SEM (n = 3). *p < 0.05 compared with time-matched controls.