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Tumor Biology |
Departments of Microbiology and Cancer Center and Paul Mellon Prostate Cancer Research Institute [R. E. B., D. G., M. J. W.], Urology [R. A. S.], and Health Evaluation Sciences [E. A. B.], University of Virginia, Charlottesville, Virginia 22908
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, hepatocyte growth factor, transforming growth factor ß, the anti-inflammatory cytokines IL-4, IL-6, and IL-10, vascular endothelial growth factor, neuropeptides, and neuroendocrine hormones. The growth factor receptor c-HER-2/neu is overexpressed in 16–69% of prostate cancers (5, 6, 7, 8, 9, 10, 11, 12)
, and overexpression has been shown to promote steroid-independent growth in both prostate (13
, 14)
and breast cancer (15
, 16)
. Conversely, inhibition of growth factor receptors with monoclonal antibodies or tyrosine kinase inhibitors inhibits growth in androgen-dependent (17
, 18)
and androgen-independent (19
, 20)
prostate cancer xenografts. A change from paracrine to autocrine regulation of the EGF receptor has been observed in the progression of prostate cancer to androgen-independence (21)
. Taken together, these results demonstrate that up-regulated activation of mitogenic signaling pathways by autocrine and paracrine growth factor loops is closely correlated with acquisition of hormone refractory disease. Many of the growth factor pathways implicated in prostate cancer progression use the small GTPase c-Ras as a component of their signaling activities. Ras is the prototypical member of a class of low molecular weight GTP binding proteins involved in the regulation of many cellular functions including cell cycle progression (22) , apoptosis (23) , cytoskeletal organization (24) , membrane trafficking (25) , and differentiation. In response to a diverse array of extracellular stimuli, Ras recruits and activates downstream signaling cascades leading to the regulation of both nuclear and cytoplasmic processes (26) . Whereas activating Ras mutations are infrequent in prostate cancer (27, 28, 29) , overexpression of c-Ras is often found in breast cancer where it correlates positively with poor prognosis (30 , 31) . We propose that Ras is positioned uniquely to function as a common mediator of prostate cancer progression by coordinating mitogenic signals of disparate origins.
Ras is a multieffector signaling molecule that controls several signaling pathways (26) . One of the best-characterized effector pathways triggered by Ras activation is the MAPK pathway. Our laboratory has previously used a phosphorylation state-specific antibody that reacts with the activated form of MAPK and has demonstrated a correlation between elevated levels of active phospho-MAPK and prostate tumors of advanced stage and grade (32) . These findings provided the first evidence that a Ras effector, and presumably Ras itself, was chronically activated in at least some advanced prostate cancers.
Here we provide data showing that stable expression of activated Ras mutants promoting constitutive activation of the Ras/MAPK pathway is sufficient to sensitize hormone-responsive LNCaP prostate cancer cells to subphysiological levels of androgen with respect to endogenous PSA expression, anchorage-dependent and -independent growth, and increased tumorigenicity. We propose that chronic activation of c-Ras by autocrine and paracrine growth factor stimulation provides a common mechanism for prostate cancer progression driven by a broad array of mitogens.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Establishment of Ras Effector-Loop LNCaP Stable Cell Lines.
LNCaP cells were transfected with 5 µg of Ras vector {N-[1-(2,3-dioleoyloxyl)propyl]-N,N,N-trimethylammoniummethyl sulfate; Boehringer-Mannheim/Roche}, after 8 h were rinsed, grown with phenol red-free RPMI 1640 supplemented with 10% FBS for 48 h, and then selected with 600 µg/ml active G418. Multiple colonies from five 10-cm dishes were trypsinized, pooled, and replated in G418 containing medium. The pooled cells were amplified and shown to be >95% positive by immunofluorescence of either HA or FLAG epitope, with predominant membrane staining. Western blotting with a pan-Ras antibody showed RasT35S and RasE37G expression approximately equal to that of endogenous Ras, whereas c-Ras and RasY40C expression levels were significantly higher (data not shown). Cell lines were subjected to reselection every 2 weeks with 300 µg/ml active G418 to maintain expression of Ras effector-loop mutants.
Reagents.
Anti-phospho MAPK antibody was from Calbiochem, anti-PSA antibody from Bio Genex, and anti-MAPK antibody 1B3B9 from Upstate Biotechnology. Casodex (Bicalutamide) was a generous gift from Zeneca, Inc. (Wilmington, DE). The synthetic androgen R1881 was purchased from DuPont/NEN, U0126 from Calbiochem, charcoal/dextran-treated FBS from HyClone, Inc., and FBS (certified) and RPMI 1640 were from Life Technologies, Inc.
Data Quantitation.
Relevant Western and Northern data were quantified using Alpha Innotech AlphaEase FC analysis software.
Agarose Colony Assay.
Cells (25,000) of each stable cell line were trypsinized and resuspended in 1 ml of 0.5% soft agarose, and layered on top of a solidified bottom layer. Cultures were then placed in a 37° incubator for 3 weeks undisturbed. Triplicate agarose colony plates were scanned on a flatbed scanner (Hewlett Packard ScanJet 4c/T) and quantified using EagleEye colony counting software (Stratagene). Results are presented as mean colony number of four separate experiments. Agarose assays were repeated via transient transfection using 1 µg each Ras effector-loop mutants per 200,000 cells.
Cell Proliferation Assays.
Viable cells (30,000) of each Ras effector-loop stable cell line were plated per well in a 24-well plate (Primaria; Becton Dickinson) and allowed to adhere in 10% FBS RPMI 1640 for 48 h. Viability of cells was determined by visual inspection under a microscope. Medium was then aspirated, rinsed once with serum-free RPMI 1640, then replaced with either 5% csFBS or 5% nFBS RPMI 1640 back supplemented with R1881 or ethanol vehicle control. Cell number was assayed via MTT according to the manufacturer’s protocol (Sigma).
Tumor Immunohistochemistry.
Xenograft subcellular localization of either HA-tagged or FLAG-tagged Ras proteins were characterized by addition of either biotin-conjugated polyclonal anti-HA (Santa Cruz) or biotin-conjugated monoclonal M5 anti-FLAG antibody (Sigma) followed by secondary streptavidin-horseradish peroxidase. The antibody specific for the dually phosphorylated MAPKs, ERK1 and ERK2, has been described previously (32
, 33)
. Anti-pan Ras monoclonal antibody RAS10 was from Upstate Biotechnology.
Northern Analysis.
Total RNA was isolated using Qiagen RNeasy kit. Ten µg of total RNA was separated on a 1% formaldehyde-agarose gel, transferred to a nylon membrane, and UV cross-linked (UV Crosslinker; Fisher Scientific). Membrane was stained with methylene blue to ensure equivalent sample loading as well as RNA integrity, prehybridized at 68°C for 30 min in ExpressHyb Solution (Clontech), hybridized with 32P-labeled probe (2 x 106 cpm/ml) for 1 h at 68°C, washed 3 x 15 min at room temperature in 2x SSC/0.05% SDS and 3 x 15 min at 50°C in 0.1x SSC/0.1% SDS. After exposure to X-ray film, the blot was stripped in boiling 0.5% SDS solution for 15 min and subsequently reprobed with 32P-labeled GAPDH to reaffirm equivalent RNA loading. A 500-bp EcoRI fragment of the PSA coding sequence and a 1-kb SacI/KpnI restriction fragment of GAPDH coding sequence were [32P]dCTP labeled using random priming (Amersham).
Animal Studies.
Male BALB/caNcRL-nuBR mice (5–6 weeks old; Charles River) were bilaterally inoculated s.c. with 2 x 106 cells in Matrigel (Becton Dickinson) at 50:50 volume for a total volume of 100 µl/injection site. Average tumor burden was calculated with calipers in millimeters as the mean tumor diameter measured in two dimensions. To minimize unnecessary animal use, included in the final data were a proportion of control mice harboring C4–2 xenografts from separate parallel experiments treated with the antibiotic doxycycline. Doxycycline administration was shown to have no observable effects on either in vivo or in vitro growth.
Bilateral castration was performed on Ketamine/Xylazine/Acepromazine anesthetized mice having tumor burdens at least 5 mm in diameter using standard surgical techniques. Mice were sacrificed using carbon dioxide asphyxiation, placed on ice, and tumors were excised. Tumor samples were fixed in Zn2+ buffered formalin for 48 h then paraffin embedded for immunohistochemistry. Alternatively, tumor samples were snap frozen in liquid nitrogen, pulverized with mortar and pestle, resuspended in 0.1% SDS-PAGE lysis buffer containing protease and phosphatase inhibitors, then sonicated on ice with eight 1-s bursts. Samples were subsequently microcentrifuged at 4° for 1 min, lysate removed from residual precipitated tissue, and subjected to standard SDS-PAGE on a 10% polyacrylamide gel.
Serum PSA levels were quantified using an automated Abbott Laboratories IM/X MEIA clinical assay machine.
Statistical Methods.
Profiles of tumor diameter were modeled using random coefficient regression models, which incorporated all of the available post-treatment data for each animal (34)
. Baseline was set at the first measurement before the animals in the treatment group were castrated. Models were fit separately within each cell line. When multiple tumors were available for an animal, both contributed curves to the analyses. The curves were estimated as restricted cubic spline functions of time since castration (in weeks) with three knots, which was deemed sufficient by Akaike’s information criterion. The random coefficient regression model, in essence, estimates and averages curves calculated for each tumor within each group, accounting for the correlation among the repeated measurements over time. The models also included a term for precastration tumor diameter, which served as a baseline adjustment. The model was fit with no main effects for treatment group and no intercept term. Curves shapes were not found to be dependent on size of tumor at baseline, although we had very limited power to test for this effect. Type 3 F tests were used to test for differences between the castrated and not castrated curves. The models were fit using the MIXED procedure in SAS Version 8.2 (SAS Institute, Cary, NC).
AUC was calculated using the Trapezoid rule and is presented as a graphical summary (Fig. 6)
of the growth curve analyses (Table 1)
. The last value was carried forward to the end of study for animals sacrificed more than halfway through the experiment. Two tumors from the C4–2 group were excluded from the AUC summary because of an animal death resulting in only two measurements (of a possible five). Confidence intervals (95%) for tumor takes and tumors remaining were generated using an exact binomial distribution.
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RESULTS |
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The Raf/MEK/ERK pathway is a major effector arm of Ras, and its activation has been correlated with disease progression in several cancer models (32
, 35, 36, 37, 38, 39)
. Western blotting with a phospho-specific MAPK antibody demonstrated constitutive phospho-MAPK activity in both RasT35S and RasE37G stable cell lines under all of the cell culture conditions tested (Fig. 1A)
. Importantly, constitutive MAPK activity was observed under serum-free culture conditions where media were changed frequently to minimize confounding autocrine effects. Both EGF and serum were capable of elevating MAPK levels in Ras T35S and RasE37G cell lines, indicating MAPK activity is still regulated in these cell lines. Alterations in levels of steroid did not affect phospho-MAPK profiles, as similar results were obtained using steroid depleted csFBS with or without R1881 supplementation (data not shown). Addition of either RasT35S or RasE37G conditioned medium to serum-starved parental LNCaP cells did not promote MAPK activation (data not shown) suggesting that activation of MAPK was not because of autocrine signaling.
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RasT35S and RasE37G Induce Androgen Hypersensitivity.
The correlation of hormone refractory prostate cancer with up-regulated growth factor signaling pathways, including MAPK, led us to ask whether LNCaP cell lines demonstrating constitutive phospho-MAPK activity would have a growth advantage under steroid-depleted culture conditions. MTT growth assays revealed that whereas all of the cell lines exhibited similar growth curves both in 10% nFBS supplemented medium as well as steroid depleted, csFBS back-supplemented with 1 nM R1881, only RasT35S and RasE37G cells retained the ability to proliferate under reduced levels of androgen (Fig. 2)
. In particular, significant cell growth was seen in RasT35S and RasE37G stable cell lines growing in charcoal-stripped serum back-supplemented with as little as 10 pM R1881, a steroid concentration 2 orders of magnitude lower than normal physiological levels (
1 nM). Importantly, RasT35S and RasE37G cell lines did not display significant growth when steroid levels fell below 10 pM, demonstrating that the cells had become hypersensitive to androgen rather than completely androgen independent.
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RasT35S and RasE37G Expression Is Sufficient for Androgen Hypersensitivity of Anchorage-independent Growth.
Cell anchorage is required for efficient signal propagation between Ras and downstream effectors such as MAPK, and for growth of fibroblasts and epithelial cells (40)
. Anchorage-independent growth is a hallmark of malignant transformation of these cell types. Therefore, we asked whether Ras expression in LNCaP cells would alter the steroid dependence of anchorage-independent growth. Parental androgen-dependent LNCaP cells readily formed colonies in agarose medium supplemented with 5% nFBS containing endogenous levels of androgen (data not shown). However, LNCaP cells were unable to form colonies in agarose supplemented with steroid-depleted charcoal-stripped serum (Fig. 3A)
. By contrast, C4–2 cells, a fully hormone refractory and highly tumorigenic derivative of LNCaP (41)
, readily formed agarose colonies both in complete and in steroid-depleted charcoal-stripped serum conditions independent of R1881 concentration. Stable expression of either RasT35S or RasE37G rescued anchorage-independent colony formation under steroid-reduced conditions, in a manner similar to adherent growth. Additional reduction in androgen levels below 10 pM completely abolished colony formation.
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. Anchorage-independent growth of C4–2 cells, treated similarly, was not affected by short-term MEK inhibition. This makes it unlikely that the growth inhibition was because of generalized toxicity of transient MEK inhibition, and suggests that constitutive phospho-MAPK activity is likely necessary for (at least) the early phases of anchorage-independent growth induced by Ras effector loop mutants. In sum, expression of the mutants RasT35S and RasE37G served to bypass the anchorage requirement for efficient MAPK activation, rendering LNCaP cells hypersensitive to low androgen under anchorage-independent conditions. Qualitatively similar results were obtained using transient transfection assays (data not shown).
RasT35S and RasE37G Effector-Loop Stable Cell Lines Express High Levels of PSA at Low Levels of Androgen.
Serum PSA levels are used routinely as a clinical marker of tumor burden and disease progression after androgen ablation therapy. Because Ras/MAPK signaling can reduce the androgen requirement of prostate cancer cell growth, one would predict that this signaling might similarly hypersensitize androgen-responsive gene expression to low levels of androgen. We have measured endogenous PSA expression as an indicator of androgen regulated gene expression.
Both RasT35S and RasE37G cell lines growing in 10% nFBS displayed significantly elevated endogenous steady-state cellular PSA transcription relative to parental LNCaP, c-Ras, and RasY40C stable cell lines (Fig. 4A)
. Quantitation of PSA mRNA levels from RasT35S and RasE37G stable cell lines revealed an increase over parental LNCaP of 3-fold and 2-fold, respectively. RasT35S and RasE37G PSA protein levels demonstrated an enhancement of 8-fold and 6-fold, respectively (Fig. 4B)
. The antiandrogen Casodex significantly attenuated PSA protein levels in all of the stable cell lines, including RasT35S and RasE37G, confirming a necessary role of the AR in the enhanced expression of endogenous PSA in response to elevated Ras/MAPK signaling (Fig. 4B)
. Daily administration of a subtoxic 20-µM dose of U0126 over a 3-day period significantly reduced PSA protein levels, but not ERK2 levels in both the RasT35S and RasE37G cell lines, confirming the requirement of MAPK signaling in the elevation of endogenous PSA expression (Fig. 4C)
. In sum, constitutive MAPK activation can hyperinduce PSA expression in LNCaP cells under normal levels of androgen.
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. Similar PSA expression in parental LNCaP cells required an R1881 concentration 2 orders of magnitude higher (1 nM). No alterations in immunoreactive AR content were observed (data not shown). These data show that MAPK activity can enhance endogenous PSA expression under normal and reduced levels of steroid, but not in the absence of steroid, indicating coregulation of the PSA promoter by the Ras/MAPK pathway and the AR.
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. Moreover, the rate of tumor development was accelerated in both RasT35S and RasE37G cell lines, demonstrating measurable tumors within 21 and 29 days, respectively. The fully hormone-refractory LNCaP derivative cell line, C4–2, injected similarly, had a >90% tumor take occurring within 25 days.
The ability of prostate cancer to proliferate under castrate levels of androgen is a hallmark of disease progression. We asked whether tumors expressing RasT35S and RasE37G would demonstrate continued survival and growth in a reduced androgen environment. Tumor-bearing mice were either castrated or sham-castrated when tumor burdens achieved a minimal average diameter of 5 mm. As expected, castration of mice harboring androgen-dependent LNCaP parental tumors resulted in rapid regression of all of the tumors within 3 weeks (Fig. 6
; Table 1
). No evidence of tumor rebound or PSA in peripheral blood was observed in the time period after castration (16 weeks). The hormone-refractory derivative, C4–2, maintained 100% tumor burden postcastration with no observable tumor regression and significant serum PSA levels proportional to their volume.
In a manner paralleling their in vitro phospho-MAPK activity, RasT35S and RasE37G tumors exhibited a significant degree of hormone refractory growth and survival after castration. Here, 62% of all of the T35S tumors and 50% of all of the E37G tumors either maintained tumor volume or rapidly rebounded to original tumor volume within 3 weeks (Fig. 6
; Table 1
). To our surprise, two of two c-Ras and four of five RasY40C tumors either maintained size or continued to grow and secrete PSA after castration.
As expected, all of the RasT35S and RasE37G tumors that survived androgen ablation displayed significant in vivo phospho-MAPK activity (Fig. 7A)
. Interestingly, although c-Ras and RasY40C cell lines did not display detectable constitutive phospho-MAPK activity in vitro, significant in vivo phospho-MAPK activity was observed in all of the c-Ras and RasY40C tumors after castration as illustrated by a representative Western analysis (Fig. 7A)
. Phospho-MAPK activity was not observed in parental LNCaP tumors before castration (data not shown). Complete regression of parental LNCaP tumors prevented analysis of phospho-MAPK levels after castration. Curiously, a small percentage of all of the Ras-expressing tumors spontaneously regressed in both castrated and noncastrated animals for unknown reasons. Like parental LNCaP tumors, complete tumor regression prevented the determination of the phospho-MAPK status of these tumors. Immunohistochemistry of tumor samples confirmed significant nuclear and cytoplasmic phospho-MAPK staining in the tumor epithelial cell population as well as membrane localized exogenous Ras expression (Fig. 7, B and C)
. We observed no significant alterations in expression or electrophoretic mobility of AR in the various cell lines either in vitro or in vivo (data not shown).
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DISCUSSION |
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We were unable to isolate stable cell lines expressing either mutationally activated MEK (S218/222D) or activated RasV12 without secondary effector loop mutations. In light of the observations that activated RasV12 is apoptotic in fibroblasts (46) whereas activated Raf-1 promotes cell cycle arrest in LNCaP cells (47) , we suspect that stable expression of either RasV12 or MEK 218/222 is incompatible with long-term survival of LNCaP cells. It is likely that stable expression of either of these two molecules can occur only with the appropriate balance of growth stimulatory and antiapoptotic signaling. This may help explain why fully activating Ras mutations are rarely encountered in clinical prostate cancer specimens.
Activation of the Ras/MAPK Pathway Correlates with Hormone Refractory Tumor Growth.
We found that the LNCaP cell lines with enhanced in vitro Ras/MAPK activity (e.g., RasT35S and RasE37G) had significantly higher tumor takes and decreased time to tumor establishment, in athymic nude mice. Indeed, the tumor take of the Ras T35S derivative approached the highly tumorigenic and hormone refractory LNCaP derivative C4–2.
To assess hormone dependence for the growth of these tumors, we initiated tumor formation in intact male mice followed by castration after tumors were established. This paradigm parallels the course of human prostate cancer initiation, progression, and subsequent clinical intervention. No tumor rebound was observed in parental LNCaP tumors during the course of this study (16 weeks after castration). In contrast, a significant proportion of both RasT35S- and E37G-expressing tumors demonstrated minimal regression, and marked hormone refractory tumor growth in castrated mice.
Interestingly, growth of all of the c-Ras and 80% of RasY40C-overexpressing tumors was refractory to hormone ablation. This was surprising, because these cell lines (in contrast to the T35S and E37G lines) did not display elevated basal MAPK activity in cell culture. However, because we observed that activation of MAPK in these cell lines was hyper-responsive to serum or EGF addition (Fig. 1A)
, we wondered whether comparable paracrine stimulation could induce MAPK activation in vivo. When we examined these tumors, either by Western blots or histochemistry, we found substantial phospho-MAPK. We speculate that in the c-Ras- and Y40C-overexpressing tumors, paracrine signaling from stroma is responsible for triggering in vivo Ras signaling, phospho-MAPK activity, and subsequent hormone refractory growth. Moreover, both c-Ras and RasY40C cells showed levels of Ras expression much higher than endogenous, as has also been observed in advanced breast cancer (30)
. Western blotting of tumors formed from LNCaP cells overexpressing c-Ras failed to detect the mobility shift commonly associated with activating Ras mutations (49)
, suggesting that mutational activation of c-Ras was not responsible for the acquired tumorigenicity of these cells (data not shown). Thus, it is plausible that chronic paracrine signaling may mediate activation of MAPK in vivo in cells overexpressing c-Ras.
Acquisition of Androgen Hypersensitivity.
We found that activation of Ras/MAPK signaling diminished but did not replace the requirement of LNCaP cells for androgen. LNCaP cells expressing Ras T35S or E37G were able to grow robustly at concentrations of androgen that were 1–2 orders of magnitude lower than the 10-9 M that were required by the parental LNCaP cells. However, reduction of androgen to 10-12 M resulted in growth arrest even for the Ras-expressing cells. Comparable results were obtained whether measuring adherent or nonadherent growth. Thus, these cells have become hormone hypersensitive rather than hormone independent.
A similar phenomenon was observed when measuring PSA production, consistent with previous results showing that the PSA promoter can be dually regulated by signal transduction pathways in addition to androgens (50, 51, 52, 53, 54) . As expected, we found that steady state endogenous cellular PSA was dramatically elevated in the RasT35S and RasE37G stable cell lines. Northern analysis confirmed up-regulated PSA mRNA expression in both of these cell lines. However, as with the growth response, PSA expression diminished when androgen levels were reduced to 10-12 M or less. We additionally demonstrated that the antiandrogen Casodex and the MEK inhibitor U0126 were each able to attenuate the elevated endogenous PSA levels in RasT35S and RasE37G cell lines, highlighting the cross-talk between Ras directed signaling pathways and AR-dependent gene expression. Thus, Ras signaling did not bypass the requirement for steroid of this androgen-dependent gene expression. Rather, Ras-mediated signaling cascades apparently converted the normal regulatory machinery to an androgen hypersensitive state.
The Role of the AR in Ras-expressing Cells.
The fact that Ras signaling did not eliminate the steroid requirement of LNCaP cells is consistent with the concept that hormone refractory prostate cancer still requires the AR (55
, 56)
. This is reflected in the fact that retention, overexpression, and mutation of ARs are common in tumors that are unresponsive to hormone ablation. More recent reports have demonstrated that knockdown of the AR by hammerhead ribozyme, antibody (57)
, or antisense (58
, 59) blocks prostate cancer cell growth.
How could signal transduction sensitize the AR to steroids? In the case of the estrogen receptor, several reports demonstrate that signal-transducing kinases can directly phosphorylate the steroid receptor and decrease or eliminate the requirement for estrogen (60) . However, we recently have mapped the major phosphorylation sites on the AR (61) , and suspect that none of them is sufficient for regulating the transcriptional activation of the AR.
On the other hand, several transcriptional coregulators have been shown to be targets of Ras-related signal transduction. In addition, recent reports point to overexpressed transcriptional coactivators as regulators of androgen sensitivity in advanced disease (62, 63, 64, 65) . We suspect that these coactivators may be major targets of signaling pathways that regulate androgen dependence.
Clinical androgen ablation therapy reduces but does not eliminate androgen from the circulation (66) . Residual steroid creates conditions favorable for selection of cancer cells having the ability to proliferate under reduced levels of androgen. Compensatory mechanisms that allow growth in castrate levels of androgen include overexpression or mutation of the AR (55 , 56) , overexpression of transcriptional coregulators (62 , 65) , and, as shown by us (32) , activation of Ras signaling. These mechanisms are not mutually exclusive but are likely to be mutually reinforcing.
Taken together, our findings show that chronic activation of Ras by mutation, overexpression, or growth factor signaling can reduce the androgen requirement of prostate cancer cells with respect to proliferation, tumorigenicity, and gene expression. These findings provide a common mechanism for prostate cancer progression driven by diverse primary alterations, and thus identify the Ras to MAPK pathway as an attractive target for therapeutic intervention.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by a grant from CaPCURE and by National Cancer Institute Grants CA39076, CA40042, CA76465, and CA44579 (to M. J. W.); Supported by National Cancer Institute Grants, National Institutes of Health Grant T32 DK07320, and the American Foundation for Urologic Disease/Scott Fund (to D. G.). 
2 To whom requests for reprints should be addressed, at Department of Microbiology, 2-16 Jordan Hall, University of Virginia, Charlottesville, VA 22908.
3 The abbreviations used are: EGF, epidermal growth factor; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase kinase; AR, androgen receptor; FBS, fetal bovine serum; csFBS, charcoal-stripped fetal bovine serum; nFBS, normal fetal bovine serum; R1881, methyltrienolone; PSA, prostate-specific antigen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IL, interleukin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; AUC, area under the curve.
Received 4/15/02. Accepted 2/27/03.
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T. Inoue, T. Yoshida, Y. Shimizu, T. Kobayashi, T. Yamasaki, Y. Toda, T. Segawa, T. Kamoto, E. Nakamura, and O. Ogawa Requirement of Androgen-Dependent Activation of Protein Kinase C{zeta} for Androgen-Dependent Cell Proliferation in LNCaP Cells and Its Roles in Transition to Androgen-Independent Cells Mol. Endocrinol., December 1, 2006; 20(12): 3053 - 3069. [Abstract] [Full Text] [PDF]
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 S. Chen, Y. Xu, X. Yuan, G. J. Bubley, and S. P. Balk Androgen receptor phosphorylation and stabilization in prostate cancer by cyclin-dependent kinase 1 PNAS, October 24, 2006; 103(43): 15969 - 15974. [Abstract] [Full Text] [PDF]
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X. Yuan, T. Li, H. Wang, T. Zhang, M. Barua, R. A. Borgesi, G. J. Bubley, M. L. Lu, and S. P. Balk Androgen Receptor Remains Critical for Cell-Cycle Progression in Androgen-Independent CWR22 Prostate Cancer Cells Am. J. Pathol., August 1, 2006; 169(2): 682 - 696. [Abstract] [Full Text] [PDF]
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 K. S. R. Sastry, A. J. Smith, Y. Karpova, S. R. Datta, and G. Kulik Diverse Antiapoptotic Signaling Pathways Activated by Vasoactive Intestinal Polypeptide, Epidermal Growth Factor, and Phosphatidylinositol 3-Kinase in Prostate Cancer Cells Converge on BAD J. Biol. Chem., July 28, 2006; 281(30): 20891 - 20901. [Abstract] [Full Text] [PDF]
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D. Gioeli, B. E. Black, V. Gordon, A. Spencer, C. T. Kesler, S. T. Eblen, B. M. Paschal, and M. J. Weber Stress Kinase Signaling Regulates Androgen Receptor Phosphorylation, Transcription, and Localization Mol. Endocrinol., March 1, 2006; 20(3): 503 - 515. [Abstract] [Full Text] [PDF]
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K-M Rau, H-Y Kang, T-L Cha, S A Miller, and M-C Hung The mechanisms and managements of hormone-therapy resistance in breast and prostate cancers Endocr. Relat. Cancer, September 1, 2005; 12(3): 511 - 532. [Abstract] [Full Text] [PDF]
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 C Hughes, A Murphy, C Martin, O Sheils, and J O'Leary Molecular pathology of prostate cancer J. Clin. Pathol., July 1, 2005; 58(7): 673 - 684. [Abstract] [Full Text] [PDF]
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Z Culig, H Steiner, G Bartsch, and A Hobisch Mechanisms of endocrine therapy-responsive and -unresponsive prostate tumours Endocr. Relat. Cancer, June 1, 2005; 12(2): 229 - 244. [Abstract] [Full Text] [PDF]
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A. Nur-E-Kamal, A. Zhang, S. M. Keenan, X. I. Wang, J. Seraj, T. Satoh, S. Meiners, and W. J. Welsh Requirement of Activated Cdc42-Associated Kinase for Survival of v-Ras-Transformed Mammalian Cells Mol. Cancer Res., May 1, 2005; 3(5): 297 - 305. [Abstract] [Full Text] [PDF]
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D.E. Clark, T.M. Errington, J.A. Smith, H.F. Frierson Jr., M.J. Weber, and D.A. Lannigan The Serine/Threonine Protein Kinase, p90 Ribosomal S6 Kinase, Is an Important Regulator of Prostate Cancer Cell Proliferation Cancer Res., April 15, 2005; 65(8): 3108 - 3116. [Abstract] [Full Text] [PDF]
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G. Pandini, R. Mineo, F. Frasca, C. T. Roberts Jr., M. Marcelli, R. Vigneri, and A. Belfiore Androgens Up-regulate the Insulin-like Growth Factor-I Receptor in Prostate Cancer Cells Cancer Res., March 1, 2005; 65(5): 1849 - 1857. [Abstract] [Full Text] [PDF]
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C.-S. Yang, M. J. Vitto, S. A. Busby, B. A. Garcia, C. T. Kesler, D. Gioeli, J. Shabanowitz, D. F. Hunt, K. Rundell, D. L. Brautigan, et al. Simian Virus 40 Small t Antigen Mediates Conformation-Dependent Transfer of Protein Phosphatase 2A onto the Androgen Receptor Mol. Cell. Biol., February 15, 2005; 25(4): 1298 - 1308. [Abstract] [Full Text] [PDF]
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 B. Nicholson, K. Gulding, M. Conaway, S. R. Wedge, and D. Theodorescu Combination Antiangiogenic and Androgen Deprivation Therapy for Prostate Cancer: A Promising Therapeutic Approach Clin. Cancer Res., December 15, 2004; 10(24): 8728 - 8734. [Abstract] [Full Text] [PDF]
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E. Unni, S. Sun, B. Nan, M. J. McPhaul, B. Cheskis, M. A. Mancini, and M. Marcelli Changes in Androgen Receptor Nongenotropic Signaling Correlate with Transition of LNCaP Cells to Androgen Independence Cancer Res., October 1, 2004; 64(19): 7156 - 7168. [Abstract] [Full Text] [PDF]
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A. Biroccio and C. Leonetti Telomerase as a new target for the treatment of hormone-refractory prostate cancer Endocr. Relat. Cancer, September 1, 2004; 11(3): 407 - 421. [Abstract] [Full Text] [PDF]
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R. Foley, D. Hollywood, and M. Lawler Molecular pathology of prostate cancer: the key to identifying new biomarkers of disease Endocr. Relat. Cancer, September 1, 2004; 11(3): 477 - 488. [Abstract] [Full Text] [PDF]
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H. Li, T. J. Ahonen, K. Alanen, J. Xie, M. J. LeBaron, T. G. Pretlow, E. L. Ealley, Y. Zhang, M. Nurmi, B. Singh, et al. Activation of Signal Transducer and Activator of Transcription 5 in Human Prostate Cancer Is Associated with High Histological Grade Cancer Res., July 15, 2004; 64(14): 4774 - 4782. [Abstract] [Full Text] [PDF]
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 C. A. Heinlein and C. Chang Androgen Receptor in Prostate Cancer Endocr. Rev., April 1, 2004; 25(2): 276 - 308. [Abstract] [Full Text] [PDF]
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L. Jia, C. S-Y. Choong, C. Ricciardelli, J. Kim, W. D. Tilley, and G. A Coetzee Androgen Receptor Signaling: Mechanism of Interleukin-6 Inhibition Cancer Res., April 1, 2004; 64(7): 2619 - 2626. [Abstract] [Full Text] [PDF]
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M.-T. Ling, X. Wang, D. T. Lee, P.C. Tam, S.-W. Tsao, and Y.-C. Wong Id-1 expression induces androgen-independent prostate cancer cell growth through activation of epidermal growth factor receptor (EGF-R) Carcinogenesis, April 1, 2004; 25(4): 517 - 525. [Abstract] [Full Text] [PDF]
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M. Rahman, H. Miyamoto, and C. Chang Androgen Receptor Coregulators in Prostate Cancer: Mechanisms and Clinical Implications Clin. Cancer Res., April 1, 2004; 10(7): 2208 - 2219. [Full Text] [PDF]
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represent animals that were surgically castrated. Dashed horizontal lines are medians whereas solid lines are means.
5 mm were scored as positive. Tumors postcastration were scored positive if the tumors maintained size or continued to grow. Confidence intervals (95%) are provided. Tumor invasion into the abdominal cavity was determined at necropsy. Data are from two independent experiments. Western analysis was used to assess in vitro MAPK phosphorylation on cell cultures grown in serum-free medium, and in vivo MAPK phosphorylation on postcastration tumors.
