Silencing KRAS Overexpression in Cadmium-Transformed Prostate Epithelial Cells Mitigates Malignant Phenotype

Abstract

Cadmium (Cd) is a potential human prostate carcinogen. Chronic Cd exposure malignantly transforms RWPE-1 human prostate epithelial cells into CTPE cells by an unclear mechanism. Previous studies show that RWPE-1 can also be malignantly transformed by arsenic, and KRAS activation is key to causation and maintenance of this phenotype. Although Cd and arsenic can both transform prostate epithelial cells, it is uncertain whether their mechanisms are similar. Thus, here we determined whether KRAS activation is critical in causing and maintaining Cd-induced malignant transformation in CTPE cells. Expression of KRAS, miRNAs, and other genes of interest was analyzed by Western blot and RT-PCR. Following stable KRAS knockdown (KD) by RNA interference using shRNAmir, the malignant phenotype was assessed by various physical and genetic parameters. CTPE cells greatly overexpressed KRAS by 20-fold, indicating a likely role in Cd transformation. Thus, we attempted to reverse the malignant phenotype via KRAS KD. Two weeks after shRNAmir transduction, KRAS protein was undetectable in CTPE KD cells, confirming stable KD. KRAS KD reduced stimulated RAS/ERK and PI3K/AKT signaling pathways and markedly mitigated multiple physical and molecular malignant cell characteristics including: hypersecretion of MMP-2, colony formation, cell survival, and expression of cancer-relevant genes (reduced proliferation and cell cycle-related genes; activated tumor suppressor PTEN). However, KRAS KD did not reverse miRNA expression originally down-regulated by Cd transformation. These data strongly suggest KRAS is a key gene in development and maintenance of the Cd-induced malignant phenotype, at least in the prostate. It is not, however, the only genetic factor sustaining this phenotype.

Potential Ameliorative Effects of Qing Ye Dan Against Cadmium Induced Prostatic Deficits via Regulating Nrf-2/HO-1 and TGF-β1/Smad Pathways

“…Several studies report that Cd exposure produces harmful effects on prostate, testicle and sperm [6, 8, 13]. The orally LD 50 of CdCl 2 is suggested to be 90 mg/kg [14].…”

“…When compared to other heavy metals, Cd can stimulate the emergence of organ disorders at a relatively lower dose [3]. Especially for prostate, Cd requires much less time than other toxicant to produce the malignantly transformation on normal prostate epithelial cells [8]. Moreover, Cd triggers the prostatic local inflammatory responses and fibrillar rearrangement, damages the prostatic balance of collagen deposition and degradation, and disturbs the homeostasis of proliferation and apoptosis [7, 9, 10].…”

Toxic-Metal-Induced Alteration in miRNA Expression Profile as a Proposed Mechanism for Disease Development

“…Evidence supporting the toxic metal modification of miRNA expression and cancer development is increasing, with Cd and As being two primary mediators [60,67]. Exposure to Cd leads to the transformation of prostate epithelial cells and a significant upregulation of KRAS (>20-fold) as well as the downregulation of 12 miRNAs, with miR-373 exhibiting the largest (11-fold) downregulation, and only a slight (6-fold) increase in miR-9 [68]. Cd exposure also can upregulate miR-372, which affects p21Cip1/WAF-1 expression in hepatic cell lines [69].…”

Micro-RNAs: Crossroads between the Exposure to Environmental Particulate Pollution and the Obstructive Pulmonary Disease

“…Normal human prostate cells (RWPE-1) exposed to Cd undergo malignant transformation in vitro and, once inoculated in mice, induce highly invasive adenocarcinoma. Ngalame and colleagues demonstrated an alteration of miRNA expression profiles in Cd-transformed RWPE-1 such as miR-155, miR-205, and miR-134 [ 97 ]. Bioinformatics analysis identified the potential targets involved in oncogenesis and cancer progression, and their expression levels were further analyzed.…”