Mechanisms of acquired androgen independence during arsenic-induced malignant transformation of human prostate epithelial cells

Abstract

Background: Prostate cancer progression often occurs with overexpression of growth factors and receptors, many of which engage the Ras/mitogen-activated protein MAP kinase (MAPK) pathway.

Objectives: In this study we used arsenic-transformed human prostate epithelial cells, which also show androgen-independent growth, to study the possibility that chronic activation of Ras/MAPK signaling may contribute to arsenic-induced prostate cancer progression.

Methods: Control and chronic arsenic-transformed prostate epithelial cells (CAsE-PE) were compared for Ras/MAPK signaling capacities using reverse transcription-polymerase chain reaction and Western blot analyses.

Results: We found activation of HER-2/neu oncogene in transformed CAsE-PE cells, providing molecular evidence of androgen independence in the transformed cells. CAsE-PE cells displayed constitutively increased expression of unmutated K-Ras (6-fold), and the downstream MAP kinases A-Raf and B-Raf (2.2-fold and 3.2-fold, respectively). There was also increased expression of phosphorylated MEK1/2 and Elk1 in the transformant cells. The MEK1/2 inhibitor, U0126, blocked PSA overexpression in CAsE-PE cells.

Conclusion: Thus, arsenic-induced malignant transformation and acquired androgen independence are linked to Ras signaling activation in human prostate epithelial cells. Chronic activation of this pathway can sensitize the androgen receptor to subphysiologic levels of androgen. This may be important in arsenic carcinogenesis and provide a mechanism that may be common for prostate cancer progression driven by diverse agents.

Figure 1

The effect of flutamide on the PSA mRNA expression of control RWPE-1 and CAsE-PE cells. Control and CAsE-PE were grown in complete medium. After 48 hr in steroid-reduced medium defined as K-SFM without BPE and EGF, cells were fed with fresh steroid-reduced medium with or without 5 μg/mL flutamide in the presence or absence of 0.1 μM DHT. RNA was isolated and subjected to RT-PCR analysis using a set of primers designed to amplify PSA and β -actin gene products. PCR products were separated on a 1.7% agarose gel. The data shown are expressed as means (n = 3); error bars represent SE. *Significantly different from untreated, cell-line–matched cells. **Significantly different from control cells treated with DHT or DHT plus flutamide.

Figure 2

Analysis of the effect of chronic arsenic exposure of prostate epithelial cells on HER-2/neu protein expression. CAsE-PE cells were first derived by exposing RWPE-1 cells to 5 μM sodium arsenite for 30 weeks. Proteins were isolated from control and arsenic-transformed CAsE-PE cells and subjected to Western blot analysis. Upper panel is a representative blot, whereas the lower panel is densitometric analysis normalized to β -actin. Densitometric data are given as fold-control and expressed as means (n = 3); error bars represent SE. *Significantly different from control.

Figure 3

Analysis of the effect of chronic arsenic exposure of prostate epithelial cells on K-ras protein expression. CAsE-PE cells were first derived by exposing RWPE-1 cells to 5 μM sodium arsenite for 30 weeks. Proteins were isolated from control and arsenic-transformed CAsE-PE cells and subjected to Western blot analysis. Upper panel is a representative blot, whereas the lower panel is densitometric analysis normalized to β -actin. Densitometric data are given as fold-control and expressed as means (n = 3); error bars represent SE. *Significantly different from control.

Figure 4

MAPK pathway. Abbreviations: GPCR, G protein coupled receptors; GTPase, guanosine triphosphatases. MAPK are a family of serine–threonine protein kinases widely conserved among eukaryotes and are involved in many cellular programs such as cell proliferation, cell differentiation, cell movement, and cell death. MAPK signaling cascades are organized hierarchically into three-tiered modules. MAPKs (Erk) are phosphorylated and activated by MAPK-kinases (MAPKKs), (MEK1/2), which in turn are phosphorylated and activated by MAPKK-kinases (MAPKKKs), (Raf). The MAPKKKs are in turn activated by interaction with the family of small GTPases and/or other protein kinases (Ras, Rap1), connecting the MAPK module to cell surface receptors or external stimuli. An activated Erk dimer can regulate targets in the cytosol and also translocate to the nucleus where it phosphorylates a variety of transcription factors regulating gene expression.

Figure 5

Analysis of the effect of chronic arsenic exposure of prostate epithelial cells on MAPK activation. ELK, E-26-like protein-1. CAsE-PE cells were first derived by exposing RWPE-1 cells to 5 μM sodium arsenite for 30 weeks. Proteins were isolated from control and arsenic-transformed CAsE-PE cells and subjected to Western blot analysis. (A) A- and B-Raf protein expression. (B) MEK1/2 and ELK-1 MAPK activation. Upper panel is a representative blot, whereas the lower panel is densitometric analysis normalized to β -actin. Densitometric data are given as fold-control and expressed as means (n = 3); error bars represent SE. *Significantly different from control.

Figure 6

The effect of U0126, a MEK inhibitor, on PSA expression in control RWPE-1 and CAsE-PE cells. Control and CAsE-PE were grown in complete medium in the presence or absence of 20 μM U0126. RNA was isolated and subjected to RT-PCR analysis using a set of primers designed to amplify PSA and β -actin gene products. PCR products were separated on a 1.7% agarose gel. Results are normalized to β -actin. The data shown are expressed as means (n = 3); error bars represent SE. *Significantly different from untreated, cell-line–matched cells. **Significantly different from control cells treated with U0126.