Mechanisms of acquired androgen independence during arsenic-induced Malignant transformation of human prostate epithelial cells.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 MAPK Mitogen-Activated Protein Kinase
MAPK Map Kinase ) 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 oncogene
Gene that can cause cancer. It is a sequence of DNA that has been altered or mutated from its original form, the proto-oncogene (see mutation). Proto-oncogenes promote the specialization and division of normal cells. 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 MEK Noun 1. MEK - a terrorist organization formed in the 1960s by children of Iranian merchants; sought to counter the Shah of Iran's pro-western policies of modernization and opposition to communism; following a philosophy that mixes Marxism and Islam it now attacks the 1/2 and Elk1 in the transformant cells. The MEK1/2 inhibitor, U0126, blocked PSA (Professional Services Automation) An information system designed to organize, track and manage all opportunities, work, resources, costs, revenues and invoices to improve the productivity and efficiency of the workforce. 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 sen·si·tize
To make hypersensitive or reactive to an antigen, such as pollen, especially by repeated exposure. 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.
KEY WORDS: androgen-independent, androgen receptor, arsenic, cancer progression, hormone refractory, malignant transformation, MAP kinase, prostate, Ras. Environ Health Perspect 115:243-247(2007). doi:10.1289/ehp.9630 available via http://dx.doi.org/ [Online 20 November 2006]
Prostate cancer is a common malignancy and a leading cause of cancer death in the United States (Crawford 2003). The molecular mechanisms underlying its development and progression remain poorly understood. Prostate cancer usually begins as an androgen-dependent tumor that may undergo clinical regression in response to pharmacologic or surgical strategies that reduce circulating testosterone concentrations or block androgen actions (Kyprianou and Isaacs 1988). Despite this treatment, prostate tumors often reappear as androgen-independent or hormone-refractory cancers (Westin and Bergh 1998). The molecular basis for this acquired androgen independence is poorly defined.
Most androgen-independent prostate tumors overexpress androgen receptor (AR) as well as androgen-activated gene products such as prostate-specific antigen (PSA) (Kim and Coetzee 2004). In fact, hormone-refractory prostate cancer cells often can maintain functional AR signaling despite greatly reduced levels of circulating testosterone as, for instance, with orchiectomy orchiectomy /or·chi·ec·to·my/ (or?ke-ek´tah-me) excision of one or both testes. If bilateral it is called also castration.
or·chi·ec·to·my or or·chi·dec·to·my
n. (Deutsch et al. 2004) because of AR overexpression. High levels of AR can correlate with prostatic cancer progression in many cases (Lee and Tenniswood 2004), and AR gene amplification is observed in 20-30% of all hormone-refractory prostate cancers (Linja et al. 2001). A role for AR in prostate cancer progression is supported by the correlation between its overexpression and the expression of androgen-regulated genes (Gregory et al. 1998). Up-regulation of AR clearly increases sensitivity to low androgen levels, leading to ligand-dependent downstream androgen-regulated gene expression and prostate tumor recurrence. In fact, the term "androgen-independent" prostate cancer is somewhat of a misnomer, because it often involves androgen hypersensitivity hypersensitivity, heightened response in a body tissue to an antigen or foreign substance. The body normally responds to an antigen by producing specific antibodies against it. The antibodies impart immunity for any later exposure to that antigen. rather than true independence (Gregory et al. 2001).
There are also mechanisms for AR activation in recurrent or advanced prostate cancers in which overexpression is not obligatory. These include altered growth factor-induced phosphorylation phosphorylation, chemical process in which a phosphate group is added to an organic molecule. In living cells phosphorylation is associated with respiration, which takes place in the cell's mitochondria, and photosynthesis, which takes place in the chloroplasts. (Craft et al. 1999; Sadar et al. 1999) and AR mutations that broaden ligand specificity (Newmark et al. 1992; Tan et al. 1997). Indeed, some hormone-refractory prostate cancers appear to recruit non-steroidal signal transduction pathways that activate AR even in the face of severe androgen deprivation (Taplin and Balk balk
the action of a horse when it refuses to obey a command to which it usually responds. See also jibbing. 2004). Thus, a "by-pass" of the requirement for androgen to interact with AR for activation of downstream events can occur in prostate cancer with acquired androgen independence (Feldman and Feldman 2001; Gleave et al. 1999). Indeed, HER-2/neu, a member of the EGF-receptor family of receptor tyrosine kinase Receptor tyrosine kinases (RTK)s are the high affinity cell surface receptors for many polypeptide growth factors, cytokines and hormones. Of the ninety unique tyrosine kinase genes idenitified in the human genome, 58 encode receptor tyrosine kinase proteins. , can activate AR-dependent genes in the absence of AR-ligand interactions (Culig et al. 1994; Yeh et al. 1999). In fact, HER-2/neu, when overexpressed, not only activates the AR pathway in the absence of ligand but also synergizes with low levels of androgen to "superactivate" this pathway (Craft et al. 1999). HER-2/neu overexpression often occurs in advanced prostate cancers (Ady et al. 2004), and stimulates growth and PSA overproduction o·ver·pro·duce
tr.v. o·ver·pro·duced, o·ver·pro·duc·ing, o·ver·pro·duc·es
To produce in excess of need or demand.
o (Craft et al. 1999). Thus, increased expression of HER-2/neu in prostate cancer cells allows androgen-independent growth (Craft et al. 1999; Culig and Bartsch 2006).
Inorganic arsenic, a common environmental contaminant and human carcinogen [International Agency for Research on Cancer The International Agency for Research on Cancer (IARC, or CIRC in its French acronym) is an intergovernmental agency forming part of the World Health Organisation of the United Nations.
Its main offices are in Lyon, France. (IARC) 2004; National Research Council (NRC NRC
1. National Research Council
2. Nuclear Regulatory Commission
Noun 1. NRC - an independent federal agency created in 1974 to license and regulate nuclear power plants ) 1999; National Toxicology Program National Toxicology Program Environment A program that conducts toxicologic tests on substances frequently found at the EPA's National Priorities List sites, which have the greatest potential for human exposure (NTP (Network Time Protocol) A TCP/IP protocol used to synchronize the real time clock in computers, network devices and other electronic equipment that is time sensitive. It is also used to maintain the correct time in NTP-based wall and desk clocks. ) 2000], has been associated with human prostate cancer (Chen and Wang 1990; Lewis et al. 1999; NRC 1999; Wu et al. 1989). In addition, we have shown that arsenic can precipitate events leading to malignant transformation of human prostate epithelial cells in vitro (Achanzar et al. 2002) and can affect prostate cancer cell progression (Benbrahim-Tallaa et al. 2005b). In this regard, the immortalized, nontumorigenic human prostate epithelial cell line (RWPE-1) is malignantly transformed by chronic low-level arsenic to produce CAsE-PE (chronic arsenic-exposed prostate epithelial) cells which form tumors resembling aggressive prostate carcinoma on injection into nude mice (Achanzar et al. 2002) and show characteristics of advanced, androgen-independent prostate cancer (Benbrahim-Tallaa et al. 2005b). Arsenic-induced androgen independence in CAsE-PE cells is not associated with AR overexpression or any apparent loss of ligand specificity (Benbrahim-Tallaa et al. 2005b), indicating that arsenic impacts progression through other mechanisms. Importantly, wild-type K-Ras activation was strongly correlated with arsenic-induced transformation in CAsE-PE cells (Benbrahim-Tallaa et al. 2005a). In this regard, Ras is a component of virtually all the signaling pathways shown to be up-regulated in advanced prostate cancer (Hoa et al. 2002; Rizos et al. 1999; Yu et al. 1993). Although Ras is infrequently mutated in prostate cancer, overexpression of wild-type Ras can be sufficient to induce malignant transformation in human prostate epithelial cells (Weber and Gioeli 2004).
Thus, the present study investigated in detail the molecular mechanism by which arsenic impacts prostate tumor cell progression using CAsE-PE cells. The results indicate that overexpression of HER-2/neu and K-Ras, resultant mitogen-activated protein kinase Mitogen-activated protein (MAP) kinases (EC 22.214.171.124) are serine/threonine-specific protein kinases that respond to extracellular stimuli (mitogens) and regulate various cellular activities, such as gene expression, mitosis, differentiation, and cell survival/apoptosis. (MAPK) pathway activation and downstream Raf activation are prominent features. Thus, it appears arsenic-induced malignant transformation precipitates up-regulation of Ras, which in turn allows a by-pass of AR to induce androgen independence in human prostate epithelial cells.
Material and Methods
Chemicals and reagents. We purchased sodium arsenite (NaAs[O.sub.2]) from Sigma Chemical Co. (St. Louis, MO), and keratinocyte keratinocyte /ke·rat·i·no·cyte/ (ker-at´in-o-sit) the epidermal cell that synthesizes keratin, known in its successive stages in the layers of the skin as basal cell, prickle cell, and granular cell. serum-free medium (K-SFM), epidermal growth factor Epidermal growth factor or EGF is a growth factor that plays an important role in the regulation of cell growth, proliferation and differentiation. Human EGF is a 6045 Da protein with 53 amino acid residues and three intramolecular disulfide bonds. (EGF EGF
epidermal growth factor ), bovine pituitary extract (BPE BPE
Bachelor of Physical Education ), 100 X antibiotic-antimycotic mixture, and TRIzol Reagent from Life Technologies, Inc. (Grand Island, NY). The mouse monoclonal anti-K-Ras and the mouse monoclonal anti-actin were purchased from Oncogene Research Products (Cambridge, MA). We purchased the rabbit polyclonal polyclonal /poly·clo·nal/ (-klon´'l)
1. derived from different cells.
2. pertaining to several clones.
derived from different cells; pertaining to several clones. anti-HER-2/Erb2, anti-A-Raf, anti-B-Raf, anti-MEK1/2, anti-Elk1, and horseradish horseradish
Hardy perennial plant (Armoracia lapathifolia) of the mustard family, native to Mediterranean lands and grown throughout the temperate zones. Its hotly pungent, fleshy root is used as a condiment and is traditionally considered medicinal. peroxidase-conjugated secondary antibody from Cell Signaling Technology (Beverly, MA), and the Bradford Protein Assay Takadouyoi 04:21, 18 October 2007 (UTC)
This article is about a scientific procedure. See Bradford (disambiguation) for other entries about Bradford.
The Bradford Protein Assay from Bio-Rad Laboratories (Hercules, CA).
Cells and cell culture. Control (untransformed) RWPE-1 cells were originally derived from normal human prostate epithelial cells and are immortalized but nontumorigenic (Bello et al. 1997; Webber et al. 1997). Unless otherwise noted, cells were grown in K-SFM containing 50 [micro]g/mL BPE and 5 ng/mL EGF, supplemented with antibiotic/antimycotic mixture. K-SFM containing BPE and EGF is henceforth termed "complete medium." The steroid-reduced media is defined as a medium without complement. The BPE is likely the major source of steroids in complete medium. Cultures were incubated at 37[degrees]C in a humidified atmosphere containing 5% carbon dioxide and passaged weekly. Cells were exposed continuously to 5 [micro]M arsenite (as sodium arsenite). The arsenic-exposed cells were designated as CAsE-PE cells to distinguish them from the parental control cells. Parallel cultures grown in arsenic-free medium provided passage-matched controls. At 29 weeks of exposure, CAsE-PE cells produce malignant tumors after inoculation into nude mice (Achanzar et al. 2002). We previously showed that a clear transition from the androgen-sensitive to androgen-independent state occurs during arsenic-induced malignant transformation of human prostate epithelial cells (Benbrahim-Tallaa et al. 2005b).
RNA RNA: see nucleic acid.
in full ribonucleic acid
One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic extraction and RT-PCR RT-PCR
reverse transcriptase-polymerase chain reaction. See PCR1. . We isolated total RNA using TRIzol reagent by manufacturer's instructions. Reverse transcription-polymerase chain reaction (RT-PCR) was performed using a TITANIUM one-step kit (Clontech, San Jose, CA) and a GeneAmp PCR PCR polymerase chain reaction.
polymerase chain reaction
Polymerase chain reaction (PCR) system 9700 (Applied Biosystems, Foster City, CA) according to the kit's instructions. Amplification conditions were as follows: 60 min at 50[degrees]C and 5 min at 94[degrees]C followed by 35 cycles each for 1 min at 94[degrees]C, 1 min at 54[degrees]C, 1 min at 72[degrees]C. We used 1 [micro]g of total RNA in each amplification. Primers were designed for PSA and [beta]-actin and were synthesized by Invitrogen as follows: PSA (5'-GAGGTCCACACACTGAAGTT-3' and 5'-CCTCCTGAAGAATCGATTCCT-3'), product size: 214 bp; [beta]-actin (5'-AGAGATGGCCACGGCTGCTT-3' and 5'-ATTTGCGGTGGACGATGGAG-3'), product size: 460 bp. PCR products were electrophoresed on 1.7% agarose gels and the gel image captured and quantified with a Gel Doc 2000 System equipped with TDS TDS
total dissolved solids. Quantity One software (Bio-Rad Laboratories). The level of [beta]-actin was used to normalize results.
Western blot analysis West·ern blot analysis
An electrophoretic procedure for separating proteins. . We isolated total proteins using M-PER reagent (Pierce, Rockford, IL) as directed by the manufacturer. We determined protein concentration using the Bradford assay, and 20-40 [micro]g of each sample were electrophoresed and transferred to nitrocellulose nitrocellulose, nitric acid ester of cellulose (a glucose polymer). It is usually formed by the action of a mixture of nitric and sulfuric acids on purified cotton or wood pulp. membranes (Invitrogen). Immunoblotting immunoblotting,
n the immunologic methods for isolating and quantitatively measuring immunoreactive substances. When used with immune reagents such as monoclonal antibodies, the process is known generically as
Western blot analysis. was performed using the human K-Ras antibody at a 1: 1,000 dilution; horseradish peroxidase-conjugated anti-mouse secondary antibody at a 1: 5,000 dilution; HER-2/neu antibody at 1: 1,000 dilution; A-Raf antibody at 1: 1,000 dilution; B-Raf antibody at 1: 1,000 dilution; Phospho-MEK1/2 antibody at 1: 1,000 dilution; and phospho-Elk1 antibody at 1: 1,000 dilution; horseradish peroxidase-conjugated anti-rabbit secondary antibody at a 1: 5,000 dilution; and Super Signal West Pico Chemiluminescent chem·i·lu·mi·nes·cence
Emission of light as a result of a chemical reaction at environmental temperatures.
chem Substrate (Pierce). Signals were visualized by exposure to Hyperfilm (Amersham). Densitometric analysis was performed using Quantity One software (Bio-Rad).
Statistical analysis. All data are represented as mean [+ or -] SE derived from three or more independent experiments. Statistical significance was determined by the Student t-test or ANOVA anova
see analysis of variance.
ANOVA Analysis of variance, see there followed by Dunnett's t-test as appropriate, with p [less than or equal to] 0.05 considered statistically significant.
AR activity and malignant transformation leading to acquired androgen independence. Arsenic precipitates events leading to rapid growth and greatly reduced androgen dependence during malignant transformation of human prostate epithelial cells in vitro (Benbrahim-Tallaa et al. 2005a, 2005b). AR expression or ligand specificity played a minimal role in this arsenic-induced prostate cancer cell progression (Benbrahim-Tallaa et al. 2005b). In fact, AR was expressed at essentially the same level in both control and CAsE-PE cells (Benbrahim-Tallaa et al. 2005b). To further assess the activity of AR in these cells, we examined androgen-induced gene expression through AR stimulation. In this case we examined PSA expression, which is activated by androgens through AR. As is typical with prostate malignancies, CAsE-PE cells expressed significantly more PSA (2.1-fold, p < 0.05) than control cells (Figure 1). However, compared with respective untreated cells, a marked 3.5-fold increase in cellular PSA mRNA levels occurred with dihydrotestosterone dihydrotestosterone /di·hy·dro·tes·tos·te·rone/ (DHT) (-tes-tos´te-ron) an androgenic hormone formed in peripheral tissue by the action of 5 on testosterone; thought to be the androgen responsible for development of male primary sex (DHT (Distributed Hash Table) A method for storing hash tables in geographically distributed locations in order to provide a failsafe lookup mechanism for distributed computing. ) treatment in control RWPE-1 cells, whereas levels increased only 0.8-fold in CAsE-PE cells. Indeed, DHT-induced increases in PSA were to a significantly lower maximal level in CAsE-PE cells compared with control cells (Figure 1). Thus, AR overexpression was clearly not required for the steroid-independent growth in CAsE-PE cells. Because androgen-independent prostate cancers often become resistant to antiandrogens, we tested the effect of the antiandrogen antiandrogen /an·ti·an·dro·gen/ (-an´dro-jen) any substance capable of inhibiting the biological effects of androgens.
n. flutamide on DHT-stimulated PSA mRNA levels in control and CAsE-PE cells. A DHT-stimulated PSA mRNA level was completely suppressed by flutamide in control cells (Figure 1). On the other hand, in CAsE-PE cells the androgen-stimulated PSA secretion was blocked only partially by flutamide. Collectively, CAsE-PE cells responded differently to DHT and flutamide, both of which are thought to act through direct interaction with AR.
HER-2/neu activation with arsenic-induced acquired androgen independence. Several reports have described the involvement of HER-2/neu tyrosine kinase--a type I growth factor receptor A growth factor receptor is a receptor which binds to growth factor. External links
• • tyrosine kinase--in activating the AR through the MAPK pathway (Ady et al. 2004; Craft et al. 1999), and HER-2/neu expression has been implicated in the progression of prostate cancer to a hormone independence (Yeh et al. 1999; Wen et al. 2000). Therefore, the level of HER-2/neu expression in control (RWPE-1) cells was compared with androgen-independent, arsenic-transformed CAsE-PE cells (Figure 2). HER-2/neu protein in transformed CAsE-PE cells was expressed at levels > 400% greater than control. This is consistent with the aggressive nature and androgen independence of CAsE-PE cells.
Arsenite enhances K-Ras gene expression during malignant transformation. Because Ras-related pathways are often related to androgen independence in prostate cancer, this potential oncogene was studied in detail. K-Ras protein level in CAsE-PE cells was greatly increased (6-fold) compared with control (Figure 3). We previously showed that K-Ras overexpression precedes and remains elevated with malignant transformation of CAsE-PE cells (Benbrahim-Tallaa et al. 2005a).
MAPK pathway correlates with prostate cancer cell progression. Ras is a critical signaling molecule that controls several signaling pathways in prostate cancer. One of the best-characterized effector effector /ef·fec·tor/ (e-fek´ter)
1. an agent that mediates a specific effect.
2. an organ that produces an effect in response to nerve stimulation. pathways trigged by Ras activation is the MAPK pathway (Figure 4). Thus, events downstream of Ras in CAsE-PE cells were compared with control RWPE-1. Clearly, proteins downstream of K-Ras, including A-Raf and B-Raf (both serine-threonine MAP kinases) showed greatly increased expression in CAsE-PE cells compared with control (2.6- and 3.1-fold, respectively) (Figure 5A). There was also an increased expression of phosphorylated MEK1/2 and ELK in CAsE-PE cells compared with control (Figure 5B). Thus, there is a correlation between elevated levels of active phospho-MAPK and arsenic-induced prostate cell transformation. Also, treatment with the specific MEK inhibitor, U0126, inhibited PSA expression up to 80% in CAsE-PE cells (Figure 6).
The normal development, growth, and survival of the prostate epithelium is regulated both by androgen and by the paracrine paracrine /para·crine/ (par´ah-krin)
1. denoting a type of hormone function in which hormone synthesized in and released from endocrine cells binds to its receptor in nearby cells and affects their function.
2. production of growth factors by the prostatic stroma stroma /stro·ma/ (stro´mah) pl. stro´mata [Gr.] the matrix or supporting tissue of an organ.stro´malstromat´ic
n. pl. stro·ma·ta
1. (Feldman and Feldman 2001). However, regulatory interactions between androgens and growth factors often become distorted in prostate cancer. Early-stage prostate cancer typically requires androgen for growth and thus responds to androgen ablation (Feldman and Feldman 2001). However, following such therapy, the disease often progresses to an androgen-independent state, rendering androgen ablation ineffective (Arnold and Isaacs 2002).
Progression of prostate cancer to the frequently fatal androgen-independent disease is often associated with the elevation and autocrine autocrine /au·to·crine/ (-krin) denoting a mode of hormone action in which a hormone binds to receptors on and affects the function of the cell type that produced it.
adj. production of multiple polypeptide growth factors (Gioeli et al. 1999). For example, EGF, transforming growth factor [alpha], insulin-like growth factor insulin-like growth factor
one of the twenty or so substances, additional to the classic bone-regulating hormones, which exert an effect on bone cell metabolism. See also somatomedin C. 1, interleukin 6, keratinocyte growth factor The Keratinocyte Growth Factor (KGF) is a growth factor present in the epithelialization-phase of wound healing. In this phase, keratinocytes are covering the wound, forming the epithelium. , and other fibroblast growth factor Fibroblast growth factors, or FGFs, are a family of growth factors involved in wound healing and embryonic development. The FGFs are heparin-binding proteins and interactions with cell-surface associated heparan sulfate proteoglycans have been shown to be essential for FGF family members are expressed in advanced prostate cancers and are believed to be important in fueling androgen-independent growth (Culig et al. 1994; Yeh et al. 1999). Among compounds that activate AR function in the absence of ligand or in a synergistic manner with low androgen levels, it appears the EGF receptor-related molecule, HER-2/neu, plays a critical role (Craft et al. 1999). A type I growth factor receptor tyrosine kinase, HER-2/neu is over-expressed in most epithelial malignancies (Carles et al. 2004; Ren et al. 2005; Wu et al. 2004). HER-2/neu overexpression results in enhanced growth of prostate cancer and up-regulation of PSA (Craft et al. 1999; Shi et al. 2006; Veeramani et al. 2005). Our results showed that CAsE-PE cells greatly overexpress HER-2/neu compared with control in association with malignant transformation and acquired androgen independence. HER-2/neu production is positively regulated by androgens in androgen-dependent LNCaP prostate cancer cells (Zhau et al. 1992). A transmembrane transmembrane /trans·mem·brane/ (trans-mem´bran) extending across a membrane, usually referring to a protein subunit that is exposed on both sides of a cell membrane.
adj. tyrosine kinase, HER-2/neu, triggers intracellular signaling cascades such as MAP and Akt kinase pathways (Culig 2004; Wen et al. 2000). Importantly, HER-2/neu promotes phosphorylation of AR at multiple sites, which results in a highly active transcriptional unit even in the presence of low androgen levels (Culig 2004; Sugita et al. 2004). Thus, it appears HER-2/neu activation is a key factor in arsenic-induced acquired androgen independence in malignant human prostate epithelial cells.
Because Ras/MAPK signaling can reduce the androgen requirement of prostate cancer cells (Bakin et al. 2003b), one would predict that stimulation of this signaling pathway might allow androgen-regulated gene expression even at very low levels of androgen. Previous studies showed that flutamide inhibits the androgen-signaling pathway in androgen-dependent but not refractory tumors (Ilagan et al. 2005). We measured endogenous PSA expression as an indicator of androgen-regulated gene expression. The antiandrogen flutamide completely abolished PSA expression in control cells but only partially attenuated Attenuated
Alive but weakened; an attenuated microorganism can no longer produce disease.
Mentioned in: Tuberculin Skin Test
having undergone a process of attenuation. PSA production in transformed CAsE-PE cells. This finding is consistent with an androgen refractory status.
Overexpression of K-Ras and activation of MAPK correlate with progression in CAsE-PE cells (Benbrahim-Tallaa et al. 2005a, 2005b). Similarly, expression of activated Ras makes LNCaP epithelial prostate cancer cell line less dependent on androgens (Bakin et al. 2003b). Also, expression of dominant negative Ras restores androgen dependence to C4-2 cells (Bakin et al. 2003a). C4-2 is a hormone-refractory derivative of the LNCaP cell line (Wu et al. 1994). Our work in vitro shows that the common environmental contaminant arsenic can induce malignant transformation associated with androgen independence in human prostate epithelial cells (Achanzar et al. 2002; Benbrahim-Tallaa et al. 2005b). The increase in MAPK activation in androgen-independent CAsE-PE cells in the present study is consistent with the hypothesis that prostate cancer progression induced by arsenic is associated with chronic stimulation of the Ras signaling pathway.
Treatment of cells with the MAPK inhibitor U0126 totally abolished PSA expression in CAsE-PE cells but only partially in control cells. PSA expression is normally controlled by androgen through AR. Because androgen-refractory CAsE-PE cells greatly overexpress PSA, these results clearly show MAPK activity is critical to arsenic-induced acquired androgen independence. This fortifies the concept that events downstream of AR are of critical importance to arsenic-induced prostate cancer progression.
In summary, this study indicates that arsenic-induced malignant transformation and acquired androgen independence in human prostate epithelial cells is associated with an apparent "by-pass" of the androgen requirement for AR pathway activation. The most likely basis of this androgen independence is overexpression of HER-2/neu. Activation of the Ras/MAPK pathway in CAsE-PE cells correlates with this progression to androgen independence. Indeed, blockage of MAPK inhibited androgen-regulated gene expression, highlighting the role of Rasdirected signaling pathways in arsenic-induced androgen independence. These observations indicate that this important and common environmental contaminant can potentially stimulate both initiation and progression of human prostate cancer. Progression to androgen independence is a key factor in the eventual mortality from this all too common malignancy.
Achanzar WE, Brambila EM, Diwan Noun 1. diwan - a Muslim council of state
privy council - an advisory council to a ruler (especially to the British Crown)
2. diwan - a collection of Persian or Arabic poems (usually by one author)
divan BA, Webber MM, Waalkes MP. 2002. Inorganic arsenite induced malignant transformation of human prostate epithelial cells. J Natl Cancer Inst 94(24):1888-1891.
Ady N, Morat L, Fizazi K, Soria J-C, Mathieu M-C, Prapotnich D, et al. 2004. Detection of HER 2/neu positive circulating epithelial cells in prostate cancer patients. Br J Cancer 90(2):443-448.
Arnold JT, Isaacs JT. 2002. Mechanisms involved in the progression of androgen independent prostate cancers: it is not only the cancer cell's fault. Endocr Relat Cancer 9(1):61-73.
Bakin RE, Gioeli D, Bissonette EA, Weber MJ. 2003a. Attenuation Loss of signal power in a transmission.
The reduction in level of a transmitted quantity as a function of a parameter, usually distance. It is applied mainly to acoustic or electromagnetic waves and is expressed as the ratio of power densities. of Ras signaling restores androgen sensitivity to hormone-refractory C4-2 prostate cancer cells. Cancer Res 63(8):1975-1980.
Bakin RE, Gioeli D, Sikes Sikes can refer to: People
Bello D, Webber MM, Kleinman HK, Wartinger DD, Rhim JS. 1997. Androgen responsive adult human prostatic epithelial cell lines immortalized by human papillomavirus 18. Carcinogenesis 18(6):1215-1223.
Benbrahim-Tallaa L, Waterland RA, Styblo M, Achanzar WE, Webber MM, Waalkes MP. 2005a. Molecular events associated with arsenic-induced malignant transformation of human prostatic epithelial cells: aberrant genomic DNA methylation methylation,
n a phase-II detoxification pathway in the liver; methyl groups combine with toxins to rid the body of various substances.
(meth´ and K-ras oncogene activation. Toxicol Appl Pharmacol 206(3):288-298.
Benbrahim-Tallaa L, Webber MM, Waalkes MP. 2005b. Acquisition of androgen independence by human prostate epithelial cells during arsenic-induced malignant transformation. Environ Health Perspect 113:1134-1139.
Carles J, Lloreta J, Salido M, Font A, Suarez M, Baena V, et al. 2004. Her-2/neu expression in prostate cancer: a dynamic process? Clin Cancer Res 10(14):4742-4745.
Chen CJ, Wang CJ. 1990. Ecological correlation between arsenic levels in well water and age adjusted mortality from malignant neoplasms. Cancer Res 50(17):5470-5474.
Craft N, Shostak Y, Carey M, Sawyers CL. 1999. A mechanism for hormone-independent prostate cancer through modulation of androgen receptor signaling by the HER-2/neu tyrosine kinase. Nat Med 5(3):280-285
Crawford ED. 2003. Epidemiology of prostate cancer. Urology 62(suppl 1):3-12.
Culig Z. 2004. Androgen receptor cross-talk with cell signaling pathways. Growth Factors 22(3):179-184.
Culig Z, Bartsch G. 2006. Androgen axis in prostate cancer. J Cell Biochem 99(2):373-381.
Culig Z, Hobisch A, Cronauer MV, Radmayr C, Trapman J, Hittmair A, et al. 1994. Androgen receptor activation in prostatic tumor cell lines by insulin-like growth factor-I, keratinocyte growth factor, and epidermal growth factor. Cancer Res 54(20):5474-5478.
Deutsch E, Maggiorella L, Eschwege P, Bourhis J, Soria JC, Abdulkarim B. 2004. Environmental, genetic, and molecular features of prostate cancer. Lancet Oncol 5(5):303-313.
Feldman BJ, Feldman D. 2001. The development of androgen-independent prostate cancer. Nat Rev Cancer 1(1):34-45.
Gioeli D, Mandell JW, Petroni GR, Frierson HF, Jr. Weber MJ. 1999. Activation of mitogen-activated protein kinase associated with prostate cancer progression. Cancer Res 59(2):279-284.
Gleave M, Tolcher A, Miyake H, Nelson C, Brown B, Beraldi E et al. 1999. Progression to androgen independence is delayed by adjuvant treatment with antisense Bcl-2 oligodeoxynucleotides after castration castration, removal of the sex glands of an animal, i.e., testes in the male, or ovaries and often the uterus in the female. Castration of the female animal is commonly referred to as spaying. in the LNCaP prostate tumor model. Clin Cancer Res 5(10):2891-2898.
Gregory CW, Hamil KG, Kim D, Hall SH, Pretlow TG, Mohler JL, et al. 1998. Androgen receptor expression in androgen-independent prostate cancer is associated with increased expression of androgen-regulated genes. Cancer Res 58(24):5718-5724.
Gregory CW, Johnson RT Jr, Mohler JL, French FS, Wilson EM. 2001. Androgen receptor stabilization in recurrent prostate cancer is associated with hypersensitivity to low androgen. Cancer Res 61(7):2892-2898.
Hoa M, Davis SL, Ames SJ, Spanjaard RA. 2002. Amplification of wild-type K-ras promotes growth of head and neck squamous cell carcinoma squamous cell carcinoma
A carcinoma that arises from squamous epithelium and is the most common form of skin cancer. Also called cancroid, epidermoid carcinoma. . Cancer Res 62(24):7154-7156.
IARC (International Agency for Research on Cancer). 2004. Some Drinking Water Disinfectants and Contaminants, Including Arsenic. IARC Monogr Eval Carcinog Risk Hum 84:269-477.
Ilagan R, Zhang LJ, Pottratz J, Le K, Salas S, Iyer M, et al. 2005. Imaging androgen receptor function during flutamide treatment in the LAPC LAPC Loughborough Antennas & Propagation Conference (IEEE conference)
LAPC Local Air Pollution Control (UK)
LAPC Los Angeles Poker Classic
LAPC Licensed Associate Professional Counselor 9 xenograft xenograft /xeno·graft/ (zen´o-graft) a graft of tissue transplanted between animals of different species; it may be concordant, model. Mol Cancer Ther 4(11):1662-1669.
Kim J, Coetzee GA. 2004. Prostate specific antigen PSA (Prostate specific antigen)
A tumor marker associated with prostate cancer.
Mentioned in: Tumor Markers gene regulation by androgen receptor. J Cell Biochem 93(2):233-241.
Kyprianou N, Isaacs JT. 1988. Activation of programmed cell death pro·grammed cell death
programmed cell death
proposed system of cell death, often including poly(ADP)-ribosylation, ensures that a cell will not survive if it is so badly damaged that its recovery would harm the in the rat ventral prostate. Endocrinology 122(2):552-562.
Lee EC, Tenniswood MP. 2004. Emergence of metastatic hormone-refractory disease in prostate cancer after anti-androgen therapy. J Cell Biochem 91(4):662-670.
Lewis DR, Southwick JW, Ouellet-Hellstrom R, Rench J, Calderon RL. 1999. Drinking water arsenic in Utah: a cohort mortality study. Environ Health Perspect 107:359-365.
Linja MJ, Savinainen KJ, Saramaki OR, Tammela TL, Vessella RL, Visakorpi T. 2001. Amplification and overexpression of androgen receptor gene in hormone-refractory prostate cancer. Cancer Res 61(9):3550-3555.
Newmark JR, Hardy DO, Tonb DC, Carter BS, Epstein JL, Isaacs WB, et al. 1992. Androgen receptor gene mutations in human prostate cancer. Proc Natl Acad Sci USA 89(14):6319-6323.
NRC (National Research Council). 1999. Arsenic in Drinking Water. Washington, DC: National Academy Press.
NTP. 2000. Reports on Carcinogens. First and Subsequent 2nd-9th (1980-2000). Research Triangle Park Research Triangle Park, research, business, medical, and educational complex situated in central North Carolina. It has an area of 6,900 acres (2,795 hectares) and is 8 × 2 mi (13 × 3 km) in size. Named for the triangle formed by Duke Univ. , NC: National Toxicology Program.
Ren SH, Wang JW, Zhang L. 2005. Effects of Her-2/neu siRNA-mediated gene silencing on cell cycle and apoptosis of lung adenocarcinoma cells. Zhonghua Yi Xue Za Zhi 85(22):1530-1534.
Rizos E, Sourvinos G, Arvanitis DA, Velegrakis G, Spandidos DA. 1999. Low incidence of H-, K- and N-ras oncogene mutations in cytological specimens of laryngeal laryngeal /lar·yn·ge·al/ (lah-rin´je-al) pertaining to the larynx.
la·ryn·geal or la·ryn·gal
Of, relating to, affecting, or near the larynx. tumours. Oral Oncol 35(6):561-563.
Sadar MD, Hussain M, Bruchovsky N. 1999. Prostate cancer: molecular biology of early progression to androgen independence. Endocr Relat Cancer 6(4):487-502.
Shi Y, Chatterjee SJ, Brands FH, Shi SR, Pootrakul L, Taylor CR, et al. 2006. Role of coordinated molecular alterations in the development of androgen-independent prostate cancer: an in vitro model that corroborates clinical observations. BJU BJU Bob Jones University (Greenville, SC, USA)
BJU British Journal of Urology
BJU Beach Jumper Unit Int 97(1):170-178.
Sugita S, Kawashima H, Tanaka T, Kurisu T, Sugimura K, Nakatani T. 2004. Effect of type I growth factor receptor tyrosine kinase inhibitors on phosphorylation and trans-activation activity of the androgen receptor in prostate cancer cells: Ligand-independent activation of the N-terminal domain of the androgen receptor. Oncol Rep 11(6):1273-1279.
Tan J-A, Sharief Y, Hamil KG, Gregory CW, Zang D-Y, Sar M, et al. 1997. Dehydroepiandrosterone activates mutant androgen receptors expressed in the androgen-dependent human prostate cancer xenograft CWR CWR Cabinet War Rooms (UK)
CWR Continuous Welded Rail
CWR Case Western Reserve (College)
CWR California Western Railroad
CWR Central Western Railway
CWR Center for Whale Research 22 and LNCaP cells. Mol Endocrinol 11(4):450-459.
Taplin ME, Balk SP. 2004. Androgen receptor: a key molecule in the progression of prostate cancer to hormone independence. J Cell Biochem 91(3):483-490.
Veeramani S, Yuan TC, Chen SJ, Lin FF, Petersen JE, Shaheduzzaman S, et al. 2005. Cellular prostatic acid phosphatase Prostatic acid phosphatase (PAP) is an enzyme produced by the prostate. It may be found in increased amounts in men who have prostate cancer or other diseases.
This is also the same enzyme that is found in significant amounts in female ejaculate. : a protein tyrosine phosphatase Protein tyrosine phosphatases (PTPs) are a group of enzymes that remove phosphate groups from phosphorylated tyrosine residues on proteins. Functions
Together with tyrosine kinases, PTPs regulate the phosphorylation state of many important signalling molecules, such as involved in androgen-independent proliferation of prostate cancer. Endocr Relat Cancer 12(4):805-822.
Webber MM, Bello D, Kleinman HK, Hoffman MP. 1997. Acinar acinar /ac·i·nar/ (as´i-nar) pertaining to or affecting one or more acini.
Relating to an acinus.
pertaining to or affecting an acinus or acini. differentiation by nonmalignant immortalized human prostatic epithelial cells and its loss by malignant cells. Carcinogenesis 18(6):1225-1231.
Weber MJ, Gioeli D. 2004. Ras signaling in prostate cancer progression. J Cell Biochem 91(1):13-25.
Wen Y, Hu MC, Makino K, Spohn B, Bartholomeusz G, Yan DH, et al. 2000. HER-2/neu promotes androgen-independent survival and growth of prostate cancer cells through the Akt pathway. Cancer Res 60(24):6841-6845.
Westin P, Bergh A. 1998. Apoptosis and other mechanisms in androgen ablation treatment and androgen independent progression of prostate cancer. Cancer Detect Prev 22(5):476-484.
Wu HC, Hsieh JT, Gleave ME, Brown NM, Patha S, Chung LW. 1994. Derivation of androgen-independent human LNCaP prostatic cancer cell sublines: role of bone stromal cells. Int J Cancer 57(3):406-412.
Wu MM, Kuo TL, Hwang YH, Chen CJ. 1989. Dose-response relation between arsenic concentration in well water and mortality from cancers and vascular diseases. Am J Epidemiol 130(6):1123-1132.
Wu Y, Soslow RA, Marshall DS, Leitao M, Chen B. 2004. Her-2/neu expression and amplification in early stage ovarian surface epithelial neoplasms. Gynecol Oncol 95(3):570-575.
Yeh S, Lin HK, Kang HY, Thin TH, Lin MF, Chang C. 1999. From HER2/neu signal cascade to androgen receptor and its coactivators: a novel pathway by induction of androgen target genes through MAP kinase in prostate cancer cells. Proc Natl Acad Sci USA 96(10):5458-5463.
Yu M, Leav BA, Leav I, Merk FB, Wolfe HJ, Ho SM. 1993. Early alterations in ras protooncogene mRNA expression in testosterone and estradiol-17 beta induced prostatic dysplasia of Noble rats. Lab Invest 68(1):33-44.
Zhau HE, Wan DS, Zhou J, Miller GJ, von Eschenbach AC. 1992. Expression of c-erb B-2/neu proto-oncogene in human prostatic cancer tissues and cell lines. Mol Carcinog 5(4):320-327.
Lamia Lamia (lā`mēə), in Greek mythology, grief-crazed woman whose name was used to frighten children. Her own children were killed by Hera, who was jealous of Zeus' love for her; thereafter Lamia, out of envy for happy mothers, stole and Benbrahim-Tallaa, (1) Mukta M. Webber, (2,3) and Michael P. Waalkes (1)
(1) Inorganic Carcinogenesis Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at the National Institute of Environmental Health Sciences The National Institute of Environmental Health Sciences (NIEHS) is one of 27 Institutes and Centers of the National Institutes of Health (NIH),which is a component of the Department of Health and Human Services (DHHS). The Director of the NIEHS is Dr. David A. Schwartz. , National Institutes of Health, Department of Health and Human Services Noun 1. Department of Health and Human Services - the United States federal department that administers all federal programs dealing with health and welfare; created in 1979
Health and Human Services, HHS , Research Triangle Park, North Carolina, USA; (2) Department of Medicine, and (3) Department of Zoology, Michigan State University Michigan State University, at East Lansing; land-grant and state supported; coeducational; chartered 1855. It opened in 1857 as Michigan Agricultural College, the first state agricultural college. , East Lansing, Michigan East Lansing is a city in the U.S. state of Michigan. The city is located directly east of Lansing, Michigan, the state's capital. Most of the city is within Ingham County, though a small portion lies in Clinton County. , USA
Address correspondence to M.P. Waalkes, NCI See Liberate. at NIEHS NIEHS National Institute of Environmental Health Sciences (NIH, DHHS) , P.O. Box 12233, Mail Drop F0-09, 111 Alexander Dr., Research Triangle Park, NC 27709, USA. Telephone: (919) 541-2328. Fax: (919) 541-3970. E-mail: email@example.com
The authors thank J.-F. Coppin and E. Tokar for their critical review of this manuscript.
This research was supported by the Intramural intramural /in·tra·mu·ral/ (-mu´r'l) within the wall of an organ.
Occurring or situated within the walls of a cavity or organ. Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
The authors declare they have no competing financial interests.
Received 18 August 2006; accepted 20 November 2006.