Aberrant Ras and Wnt signaling are emerging as key events in the multistep nature of prostate tumorigenesis and progression. Here, we report the generation of a compound model of prostate cancer to define the synergism of activated K-ras (K-ras+/V12) and dominant stabilized β-catenin (Catnb+/lox(ex3)) in the murine prostate. Recombination of floxed alleles and subsequent expression of oncogenic transgenes was mediated by Cre recombinase expression governed by the composite Probasin (PB) promoter (termed PBCre). Concomitant with elevated mitogen-activated protein kinase (MAPK) signaling, PBCre+K-ras+/V12 mice developed AH at 100 days (100% incidence) and low-grade prostate intraepithelial neoplasia and adenocarcinoma (60% and 7% incidence) by 500 days. PBCre+Catnb+/lox(ex3) mice showed reduced longevity (average 428 days) and were predisposed to PIN-like keratinized squamous metaplasia at 100 days (100% incidence) and adenocarcinoma (100% incidence) at end-point. These lesions displayed elevated Wnt signaling and basal levels of MAPK signaling. Synchronous activation of K-ras and β-catenin significantly reduced survival (average 189 days), reflecting accelerated tumorigenesis and the development of invasive carcinoma that displayed activated Wnt and MAPK signaling. Notably, expression of the basal cell marker p63 negatively correlated with tumor grade, resembling human prostate adenocarcinoma. Taken together, our data show that combinatorial oncogenic mutations of K-ras and β-catenin drive rapid progression of prostate tumorigenesis to invasive carcinoma, characterized by the synergistic elevation of androgen receptor, cyclooxygenase-2, and c-Myc. [Cancer Res 2009;69(1):94–101]
- prostate cancer
Cancer development is a multistep process through which cells accumulate genetic mutations ( 1). Synchronous activation of Ras and Wnt signaling has been identified in several transgenic mouse tumor models, including the colon ( 2), intestine ( 3), kidney ( 3), breast ( 4), and liver ( 5). Homeostasis of the intestine is not affected in mice expressing a monoallelic K-ras activating mutation but can promote tumorigenesis in Apc-deficient mice ( 3). The convergence of the K-ras and Wnt signaling cascades to up-regulate the expression of genes that promote tumorigenesis, such as cyclooxygenase-2 (COX-2; ref. 6) and c-Myc ( 7, 8), indicates a direct synergy between these two pathways.
Between 17% and 25% of all human cancers are said to harbor an activating K-Ras mutation ( 9), with colonic (50%; ref. 2), pancreatic (90%; ref. 10), and lung (25–50%; ref. 11) malignancies exhibiting a high prevalence. However, H-Ras activating mutations are less abundant (<1%; ref. 9). In prostate cancer, Ras mutations are relatively uncommon ( 12). However, tissue microarray studies revealed Ras effector pathways to be up-regulated in prostate cancer, such as the mitogen-activated protein kinase (MAPK) and PI3K/AKT cascades ( 13, 14). Indeed, elevated p-ERK and p-AKT expression have been shown to correlate with tumor grade ( 13, 14). Up-regulation of Ras-mediated signaling cascades in the absence of oncogenic Ras mutations is considered to reflect the overexpression of autocrine and paracrine factors such as epidermal growth factor and transforming growth factor α, common to human prostate cancer ( 15).
Ras effector pathways are emerging as prime potential therapeutic targets for treating androgen-independent prostate cancer. The MAPK pathway has been shown to regulate an androgen receptor (AR)-sensitive reporter in human prostate cancer cell lines ( 16), whereas p-ERK has been shown to mediate androgen signaling by phosphorylating AR coactivators, including the steroid receptor coactivator and ARA70 ( 16).
To date, in vivo prostate cancer models expressing activating mutations in Ras have generated a variety of low-grade prostate phenotypes, considered to reflect differences in the transgenes and the genetic background used ( 17). Transgenic mice expressing activated H-Ras from the human T24 human bladder carcinoma cell line (Ha-RasT24G12V) under control of the PB promoter developed atypical hyperplasia (AH) in the dorsolateral and ventral lobes of the prostate between ages 6 to 12 months ( 18). More recently, Scherl and colleagues ( 17) showed that mice expressing a G12V point mutation in cH-Ras (termed H-RasV12), under the control of the minimal PB promoter, developed low-grade prostate intraepithelial neoplasia (LG-PIN) that displayed intestinal metaplasia by 3 months. At 12 months, the H-RasV12 transgene was not detected in this model, coinciding with a decrease in PIN incidence. This suggests that Ras signaling was necessary to maintain the phenotype. Together, these models show that activated Ras can facilitate prostate tumorigenesis and early stage tumor development.
Studies using mouse prostate reconstitution (MPR) models have provided evidence that further mutations/genetic events are required for the development of advanced prostate lesions in the context of an activating Ras mutation. Introduction of activated vHa-RasV12 or c-Myc into the fetal urogenital sinus (UGS) by recombinant retroviral vectors caused dysplasia and hyperplasia, respectively, when transplanted into the renal capsule of an adult isogenic male host ( 19). However, in combination, activated Ras and c-Myc cooperate to facilitate progression to carcinoma ( 19). The multistep nature of tumorigenesis was further shown in the MPR model, where loss of p53 and activation of Ras and c-Myc induced prostate carcinoma that metastasised to the lung, liver, small intestine, bone, and mesentery ( 20).
Activating mutations and overexpression of β-catenin have been detected in a variety of human malignancies including colorectal, hepatocellular, ovarian, and prostate cancer ( 21). The β-catenin (CTNNB1) oncogene is a critical regulator of the Wnt pathway, essential for normal mammalian development, polarity, and migration, as well as forming adherens junctions at the cell surface membrane ( 22). Recently, a growing body of evidence has implicated aberrant Wnt signaling and its convergence with the androgen signaling pathway as critical events in (human or mouse) prostate tumorigenesis ( 23, 24).
The role of deregulated Wnt signaling in prostate tumorigenesis has been shown in several conditional transgenic mouse prostate models. Mouse mammary tumor virus (MMTV)-Cre mediated dominant stabilization of β-catenin in the prostate been shown to predispose to PIN-like keratinized squamous metaplasia ( 25, 26), whereas deletion of the β-catenin regulator APC, mediated by PBCre, results in androgen-independent prostate adenocarcinoma ( 27), highlighting the importance of the Wnt cascade during tumorigenesis and disease progression.
To determine whether the Wnt and K-ras pathways synergize during prostatic tumorigenesis, the Cre-loxP system was used to conditionally activate K-ras (K-ras+/V12) and/or dominant stabilized β-catenin (Catnb+/Δex3) in the prostate. We show for the first time that K-ras activation causes a prostate phenotype (AH and LG-PIN), resembling activated H-rasV12 prostate cancer models ( 17– 19). PBCre+Catnb+/Δex3 mice displayed high-grade PIN-like keratinized squamous metaplasia and adenocarcinoma that were phenotypically distinct to PBCre+K-ras+/V12 prostate lesions. In double mutants, disease progression was accelerated to invasive carcinoma that displayed elevated COX-2, c-Myc, and AR, which are regulated by both the Ras and Wnt pathways. Together, these data support the concept that Ras and Wnt signaling cooperate to promote prostate tumorigenesis.
Materials and Methods
Experimental animals. All animal studies and breeding were carried out under UK Home Office regulations. The PB-Cre4 (ARR2PB) mice were obtained from the Mouse Models of Human Cancers Consortium (National Cancer Institute-Frederick). K-rasLSLV12 and Catnb+/lox(ex3) mice have been described previously ( 28, 29). Mice were backcrossed six times onto a C57 Bl/6 background. The PBCre transgene was incorporated into cohorts using male mice, as PBCre+ female mice have been shown to recombine in the ovaries ( 30). Cohorts were aged to 500 d or sacrificed upon signs of failing health.
Genotyping. Mice were genotyped from DNA isolated from tail biopsies as described previously for K-rasLSLV12 floxed/recombined alleles ( 28) and Cre Recombinase ( 31). Floxed β-catenin alleles were detected using Catnb-F 5′-CTGCGTGGACAATGGCTACT-3′ and Catnb-R 5′-TCCATCAGGTCAGCTGTAAAAA-3′ (324 bp and 500 bp products produced for wild-type and floxed alleles, respectively). Detection of the dominant stabilized β-catenin–recombined allele has been described previously ( 29).
Tissue isolation and histology. Histologic analysis of the genitourinary (GU) tract was done in accordance with the consensus report from the Bar Harbor meeting of the mouse models of human cancer consortium prostate pathology committee ( 32). Tissue was harvested as described previously ( 33) and fixed for no longer than 24 h in 10% neutral buffered formaldehyde at 4°C, embedded in paraffin, and sectioned at 5 μm. Sections were stained with H&E for histologic analysis.
Immunohistochemistry. Immunohistochemistry (IHC) was carried out as described previously ( 33). Primary antibodies were obtained from the following sources: AR (#RB-1358-P0; Lab Vision Corporation), β-catenin (#C19220; BD Transduction Laboratories), CD44 (#550538; Pharmingen), Keratin-5 (#PRB-160P; Covance), Ki-67 (#VP-K452; Vector Laboratories), p63 (#MS-1081; LabVision), p-Erk1/2 (Thr 202/Tyr204; #4376, Cell Signaling Technology), p-Mek1/2 (Ser221; #2338; Cell Signaling Technology), COX-2 (#RP-9072-P0; LabVision), and c-Myc (Santa Cruz #SC-764).
Scoring. The percentage of basal (p63 positive), proliferating (Ki-67 positive), AR, COX-2, and c-Myc–positive cells was determined by counting the total positive and negative cells from 20 acini, or in the case of prostate lesions, from 20 random 2,500 μm2 regions using “AnalySIS” software (Olympus Soft Imaging System; GMBH) at ×40 magnification (a minimum of 1,000 cells per mouse were counted), where n = 3. Statistical analysis was carried out using the nonparametric Mann-Whitney Test (95% confidence interval).
PBCre+K-ras+/V12 mice develop prostate cancer. To investigate the role of activated Ras signaling within the prostate, we crossed the PBCre transgenic line to mice harboring a conditional oncogenic K-rasV12 allele ( 28). Male cohorts of wild-type (PBCre+K-ras+/+) and mutant (PBCre+K-ras+/V12) mice were generated and observed for signs of illness. To monitor disease progression we examined cohorts at 100 (n = 6), 200 (n = 6), and 500 (n ≥ 17) days.
No gross phenotype was observed in wild-type mice (n = 32), whereas the PBCre+K-ras+/V12 cohort (n = 17) was predisposed to prostate lesions in all 4 lobes of the prostate ( Fig. 1A ). At 100 days, PBCre+K-ras+/V12 mice displayed infrequent AH. AH composed of focal tufting with nuclear abnormalities, including enlargement and the increased prominence of nucleoli. At 200 days, PBCre+K-ras+/V12 mice displayed localized AH and progression to LG-PIN with 100% and 67% incidence, respectively. LG-PIN foci displayed small solid and cribriform intraluminal proliferation of markedly atypical epithelial cells, which was accompanied by nuclear atypia ( 32). Aged PBCre+K-ras+/V12 mice all survived to the 500 day end point and displayed frequent focal AH (93% incidence), LG-PIN (60% incidence), and in 1 case, diffuse adenocarcinoma.
Excision of the transcriptional STOP cassette within the K-ras+/V12 transgene ( 28) was confirmed within the PBCre+K-ras+/V12 prostate lesions (at 500 days) by PCR from tumor DNA ( Fig. 1B). Wild-type and PBCre+K-ras+/V12 tail DNA served as negative controls.
PBCre+K-ras+/V12 prostate lesions display aberrant Ras signaling. To validate the hyperactivation of K-ras, we used IHC to monitor the expression of MAPK signaling components. Nuclear and cytoplasmic levels of activated p-Erk1/2 (Thr202/Tyr204) and p-Mek1/2 (Ser 221) were significantly elevated in K-ras+/V12 prostate lesions compared with controls ( Fig. 1C), positively correlating with severity. These data indicate that stimulation of the MAPK pathway might be required for tumor progression.
To determine whether aberrant Ras signaling deregulated the Wnt pathway, we analyzed the expression of β-catenin ( Fig. 1D). Although β-catenin was predominantly expressed on the cell surface in control prostate epithelium and PBCre+K-ras+/V12 mutants, nuclear β-catenin was rarely detected ( Fig. 1D). These data strongly suggest that elevated Ras signaling in prostate epithelium does not deregulate canonical Wnt signaling. In accordance with this, the Wnt/β-catenin target CD44 is also not up-regulated in PBCre+K-ras+/V12 prostate lesions.
PBCre+Catnb+/lox(ex3) mice develop PIN-like keratinized squamous metaplasia and prostate adenocarcinoma. To investigate the role of Wnt signaling in prostate tumorigenesis, PBCre+ transgenic mice were intercrossed with mice heterozygous for the dominant stabilized form of β-catenin (Catnb+/lox(ex3)). Male cohorts of wild-type and PBCre+Catnb+/lox(ex3) mice were examined at 100 (n = 6) and 200 (n = 6) days to monitor disease progression and at 500 days or when they became sick (n ≥ 18). The average survival of the PBCre+Catnb+/lox(ex3) cohort (n = 18) was significantly reduced to 428 days (χ2 = 34.56, P = 0.000; Fig. 3A).
Histologic analysis revealed that although wild-type prostate developed normal, ordered epithelium, PBCre+Catnb+/lox(ex3) mice developed lesions in all four prostate lobes ( Fig. 2A ). At 100 days, PBCre+Catnb+/lox(ex3) mice (n = 6) manifested diffuse high grade PIN-like keratinized squamous metaplasia ( Fig. 2Ai). These lesions displayed nuclear atypia, apoptotic bodies, mitotic figures, and extensive overcrowding of the lumen as multicellular disorganized layers form. IHC to detect cytokeratin-5 expression (a prostate basal cell marker) confirmed the development of keratinization within multiple foci of squamous metaplasia ( Fig. 2Aii). At 200 days, PBCre+Catnb+/lox(ex3) mice developed high-grade PIN-like keratinized squamous metaplasia and adenocarcinoma foci ( Fig. 2Aiii). Adenocarcinoma lesions displayed nuclear atypia and cells formed rosette-like structures indicating glandular differentiation is maintained ( 32). At end-point, hyperactivation of β-catenin led to diffuse and locally invasive adenocarcinomas with 100% incidence in PBCre+Catnb+/lox(ex3) mice ( Fig. 2Aiv). Adenocarcinomas showed increased severity compared with those at 200 days and keratinized squamous metaplasia was rarely detected. Notably, these lesions resembled the MMTV-Cre+Catnb+/lox(ex3) and PBCre+Apcfl/fl models described previously ( 25– 27). Indeed, prostate cancer progression in PBCre+Catnb+/lox(ex3) mice closely mirrors that of Apc mutant mice, consistent with Wnt signaling deregulation.
Monoallelic deletion of exon 3 (amino acids 5–80) and subsequent stabilization of β-catenin was confirmed in both PIN-like keratinized squamous metaplasia and adenocarcinoma lesions by PCR analysis of tumor DNA ( Fig. 2B).
PBCre+Catnb+/lox(ex3) mice display elevated Wnt signaling. To determine whether dominant stabilization of β-catenin drives tumorigenesis by stimulating the Ras and Wnt signaling pathways, IHC was carried out to detect the activated MAPK signaling molecules p-Erk1/2 and p-Mek1/2 ( Fig. 2C) and the Wnt signaling component β-catenin and its transcriptional target CD44 ( Fig. 2D). PBCre+Catnb+/Δex3 prostate lesions displayed elevated β-catenin expression in the nucleus, concomitant with up-regulated CD44 expression, strongly suggestive of activated Wnt signaling ( 34). The MAPK cascade is not deregulated in PBCre+Catnb+/Δex3 prostate lesions, as p-Erk1/2 and p-Mek1/2 expression was not markedly increased compared with wild-type prostate epithelium (shown previously in Fig. 1C). This strongly suggests that endogenous K-ras is not active in these mice and that K-ras hyperactivation alone (at 500 days) is unable to progress tumorigenesis to the squamous metaplasia observed in the PBCre+Catnb+/Δex3 mice.
PBCre+Catnb+/lox(ex3) male mice develop multiple GU tract squamous metaplasias. In addition to prostate lesions, the PBCre+Catnb+/lox(ex3) cohort was also predisposed to a number of additional GU tract phenotypes. Bulbourethral gland keratinized squamous metaplasia was observed with 100% incidence at all time points analyzed (Supplementary Fig. S1A). Keratinized squamous metaplasia of the preputial gland was observed at 100 days (67% incidence), 200 days and at end-point (100% incidence; Supplementary Fig. S1B), which is consistent with the MMTV-Cre+Catnb+/lox(ex3) model ( 26). In addition, urethral keratinized squamous metaplasia was observed at 200 days and at end-point (17% and 28% incidence, respectively; Supplementary Fig. S1C).
PBCre+Catnb+/lox(ex3):K-ras+/V12 mice show reduced longevity. To investigate the potential cooperative role between Ras and Wnt signaling in prostate tumorigenesis, we generated a cohort of male mice heterozygous for both activated K-rasV12 and the dominant stable form of β-catenin, using the PBCre transgene to mediate expression (n = 13). Double mutants displayed a significant reduction in longevity (average survival, 189 days) compared with other cohorts (χ2 = 48.66; P = 0.000; Fig. 3A ), indicating that K-ras and β-catenin synergize to accelerate tumorigenesis in the prostate.
PBCre+Catnb+/lox(ex3):K-ras+/V12 mice develop invasive prostate carcinoma. Histologic analysis revealed that PBCre+Catnb+/lox(ex3):K-ras+/V12 mice were predisposed to diffuse, locally invasive carcinoma (100% incidence) that developed from solid or sheet-like proliferations with occasional rosette structures, nuclear atypia, apoptotic bodies, and mitosis ( Fig. 3B). PCR analysis confirmed that recombination of the LoxP-flanked K-rasV12 and β-catenin alleles had taken place in PBCre+Catnb+/Δex3:K-ras+/V12 prostate lesions ( Fig. 3C).
In addition to prostate carcinoma, the double mutant cohort also developed bulbourethral, preputial, and urethral gland keratinized squamous metaplasias, mirroring PBCre+Catnb+/lox(ex3) mice. Synchronous activation of Ras and Wnt signaling was also detected in the testis, predisposing to male infertility (83% incidence). Double mutants displayed hypospermatogenesis and AH of the epididymis and ductus deferens, which corresponded to PBCre-mediated recombination, determined by the surrogate Rosa26 allele (Supplementary Fig. S2).
PBCre+Catnb+/lox(ex3):K-ras+/V12 invasive carcinoma displays synergy between activated Ras and Wnt signaling. IHC revealed that PBCre+Catnb+/Δex3:K-ras+/V12 invasive carcinomas displayed nuclear β-catenin and subsequent elevated CD44 expression, in addition to activated MAPK signaling proteins p-Erk1/2 and p-Mek1/2 ( Fig. 3D). This differs from the single mutants, where only one signaling pathway was active and provides a clear rationale for the observed synergy.
Activated K-ras and β-catenin cause phenotypes similar to human prostate cancer. To characterize the synergistic relationship between dominant stabilization of β-catenin and activation of K-ras, we monitored the expression of the proliferation marker Ki-67, the basal cell marker p63 and AR by IHC ( Fig. 4A ), and scored the percentage of positive cells ( Fig. 4B–D). Wild-type prostate showed a basal level of Ki-67–positive cells in the adult gland at 500 days (1.8%), which was increased in PBCre+K-ras+/V12 LG-PIN (3.7%) and more so in PBCre+Catnb+/Δex3 adenocarcinomas (19.5%) and PBCre+Catnb+/Δex3:K-ras+/V12 invasive carcinomas (18.3%; P = 0.0404, Mann-Whitney; Fig. 4B). Slightly reduced Ki-67 staining in PBCre+Catnb+/Δex3:K-ras+/V12 lesions, compared with PBCre+Catnb+/Δex3 mice (P = 0.0404, Mann-Whitney), probably reflects the increased incidence of necrotic cells associated with invasive carcinomas.
Notably, prostate lesions from all cohorts analyzed at end point showed clusters of enlarged, circular p63-positive cells, compared with the typical small, triangular-shaped basal cells of normal prostate epithelium ( Fig. 4Av–viii). Wild-type prostate epithelial cells were 10.8% p63 positive ( Fig. 4C), corresponding to previous work ( 35). Analysis of K-ras (LG-PIN), β-catenin (adenocarcinoma), and double transgenic (invasive carcinoma) mice at end point revealed that p63 expression decreases with prostate tumor progression (7.1%, 2.9%, and 0.1%, respectively). These data mimic human prostate cancer ( 27), bladder tumors ( 36), and head and neck squamous carcinoma ( 37).
IHC to detect AR revealed that as tumor stage progresses, nuclear AR accumulates in the neoplastic cells ( Fig. 4Aix–xii). Counting AR-positive cells revealed that nuclear AR is not highly expressed in wild-type prostate at 500 days (5.3%). Transgenic mice displayed a significant elevation in AR expression with tumor progression; K-ras mutant LG-PIN (35.8%), β-catenin mutant adenocarcinoma (52.6%), and double mutant invasive carcinoma (63.9%; Fig. 4D). This coincides with human prostate cancer studies that have observed a similar elevation in AR expression, thus providing the basis for antiandrogen hormone therapy ( 38). The fact that PBCre+Catnb+/Δex3:K-ras+/V12 mice display the highest level of AR expression may reflect cooperativity between these two oncogenes, possibly owing to MAPK and Wnt signaling mediated AR regulation ( 16, 39). AR antibody specificity was confirmed by staining sections with additional AR antibodies (Supplementary Fig. S3).
Together, this evidence suggests a synergistic relationship between activating K-rasV12 and dominant stable β-catenin mutations in the mouse prostate. To address this, the expression of c-Myc ( Fig. 5A ) and COX-2 ( Fig. 5B) were monitored by IHC. The Ras/MAPK and Wnt signaling pathways are known to drive transcription of both c-Myc and COX-2 ( 6– 8). Scoring the percentage of cells positive for nuclear c-Myc expression revealed that PBCre+K-ras+/V12, PBCre+Catnb+/Δex3, and PBCre+Catnb+/Δex3:K-ras+/V12 lesions show a progressive increase in nuclear c-Myc (12.03%, 46.22%, and 78.01%, respectively), when compared with wild-type prostate epithelium (0.28%; Fig. 5Avi). COX-2 expression at the cell surface of prostate epithelial cells was shown to increase in K-ras and β-catenin mutants (17.81% and 9.83%, respectively) compared with wild-type prostate (0.02%; Fig. 5Bvi). These data indicate that activated K-ras in prostate epithelial cells may drive more COX-2 expression than activated β-catenin. Double mutants displayed a further increase in COX-2 expression (30.14%), establishing the synergistic relationship between K-ras and β-catenin oncogenic mutations. Together, this evidence presents a direct mechanism for accelerated prostate tumor progression in double mutants.
Here, we show that K-ras+/V12 alone plays a role in the onset and progression of prostate cancer using a conditional transgenic approach. Activation of K-ras resulted in development of AH and LG-PIN at 100 and 200 to 500 days, respectively, although longevity was not decreased. Consistent with this model, mice expressing H-RasV12 driven by the minimal PB promoter also developed LG-PIN by 3 months, albeit in conjunction with intestinal metaplasia ( 17). Given that the PBCre+K-ras+/V12 model did not exhibit intestinal metaplasia, it is possible that the Ras isoform, genetic background, and the level of cellular stress may play a role in determining the observed phenotype ( 40). The fact that PBCre+K-ras+/V12 mice showed recombination of the targeted K-rasV12 allele at 500 days, whereas Scherl and colleagues ( 17) reported the H-RasV12 transgene was not detected in PB-H-rasV12 mice ≥12 months, suggests that the method of driving transgene expression may also contribute to the phenotypic differences observed.
Consistent with previous work, dominant stabilization of β-catenin predisposed to PIN-like keratinized squamous metaplasia and prostate adenocarcinoma, as well as keratinized squamous metaplasia of the preputial, bulbourethral, and urethral glands ( 25, 26). PBCre+Catnb+/lox(ex3) mice also displayed reduced survival, which was further reduced in PBCre+Catnb+/Δex3:K-ras+/V12 transgenic mice ( Fig. 3A). Double mutants showed a synergistic relationship between the activation of K-ras and Wnt signaling pathways, accelerating tumor progression to invasive carcinoma ( Fig. 3B). The PIN-like keratinized squamous metaplasia observed in the PBCre+Catnb+/lox(ex3) mice was not seen in the K-ras mutants, suggesting that this phenotype is specifically generated by aberrant Wnt signaling in the prostate, or that tumor progression beyond the 500 day end-point might be required. Squamous metaplasia was also absent from the double mutant at time of death (average, 189 days); however, we cannot rule out that squamous metaplasia may have occurred in these mice during earlier stages of tumor progression.
Previous work has shown that both Ras and Wnt signaling are able to regulate AR ( 16, 39). Here, we show an in vivo cooperation between K-ras and Wnt leading to synergistic up-regulation of AR in mouse prostate carcinoma in PBCre+Catnb+/Δex3:K-ras+/V12 mice. A 2- to 5-fold increase in AR mRNA is both necessary and sufficient for hormone-refractory progression in animal models, and subsequently, cells can become supersensitive to androgens rather than being independent of them ( 41). This evidence suggests that the observed increase in cells expressing AR in the PBCre+Catnb+/Δex3 and PBCre+Catnb+/Δex3;K-ras+/V12 prostate tumors may be sufficient to promote androgen-independent prostate growth, correlating to human disease progression. Consistent with this notion, castration studies revealed that PBCre+Apcfl/fl prostate tumors are androgen independent ( 27).
Given that tumor microenvironments can cause genetic instability and therefore gene mutations, we cannot rule out the possibility that the lesions in our prostate models do not harbor an activating mutation in AR. Indeed, it will be interesting to perform mutational analysis of AR in these lesions to determine whether activated Ras/Wnt signaling in the prostate can generate an environment capable of inducing mutations in AR.
Progressive loss of p63 expression in parallel with tumor progression might reflect elevated MAPK signaling, which has been postulated to regulate p63 expression through Src ( 42). Kurita and colleagues ( 42) grafted the UGS from p63 null mice (which are basal cell deficient) into adult nude males, revealing that prostate development can occur in the absence of basal cells. In this model, luminal cells displayed elevated Ras signaling after increased expression of c-Src in an androgen-dependent manner. Additionally, ΔNp63 has been shown to block β-catenin phosphorylation and nuclear accumulation in squamous cell carcinoma cell lines, suggesting that down-regulation of the ΔNp63 isoform may play a role in further enhancing Wnt signaling ( 43). Interestingly, p63 expression is suppressed by Notch1 activation in human and mouse keratinocytes ( 44). This suggests that aberrant Notch signaling may also play a role in promoting prostate tumorigenesis in PBCre+K-ras+/V12, PBCre+Catnb+/Δex3, and PBCre+Catnb+/Δex3;K-ras+/V12 mice. Our data shows that both the Wnt and MAPK signaling transduction cascades effect upon the p63 expressing population. The direct relevance of this to tumor progression remains unclear but is speculated to reflect both the activation of prostate stem cells and the development of androgen-independent prostate growth.
Importantly, the Wnt pathway was not deregulated in PBCre+K-ras+/V12 lesions, suggesting that the augmented tumor multiplicity and malignant behavior in compound transgenic mutants is not related to activated K-ras–mediated induction of the Wnt/β-catenin pathway, as shown previously in intestinal studies ( 2). Expression of activated K-ras (pVillin-K-rasV12G) was shown to up-regulate β-catenin nuclear translocation in intestinal tumors, which was further increased in combination with an Apc deficiency (Apc+/1638N; ref. 2). The observation that activated K-ras did not induce Wnt signaling in our PBCre+K-ras+/V12 prostate cancer model suggests that this is probably a tissue specific event.
The synergistic relationship between activated K-ras and dominant stabilization of β-catenin in prostate epithelium was further shown by IHC to detect c-Myc and COX-2. c-Myc is a regulator of cell growth and mutations in this gene are among the most common genetic lesions found in a wide variety of human cancers ( 45). Recently, conditional deletion of c-Myc from the murine intestine was shown to rescue all the phenotypes of induced AhCre+Apcfl/fl mice ( 46), demonstrating that c-Myc is required for canonical Wnt-induced tumorigenesis in the intestine. Furthermore, c-Myc expression has also been shown to confer androgen independence in human prostate cancer cells in vitro ( 45). We observe high c-Myc expression in the lesions of the double mutant mice, suggesting these tumors may also be androgen independent. Our data are consistent with previous MPR models, in which activating vHa-Ras and c-Myc mutations show accelerated tumor progression, demonstrating the cooperativity of these oncogenes in prostate carcinogenesis ( 19).
To conclude, the PBCre+Catnb+/lox(ex3):K-ras+/V12 model further establishes the link between aberrant Ras and Wnt signaling in the multistep nature of prostate tumorigenesis and progression, suggesting that a similar interaction may occur in human prostate cancer tumorigenesis. Ultimately, the cooperative up-regulation of AR, c-Myc, and COX-2 via the Ras and Wnt pathways may present a series of novel targets for chemotherapeutic intervention.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Grant support: Tenovus.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Mark Bishop, Lucie Pietzca, Luke Piggott, and Derek Scarborough at Cardiff University for their technical assistance and Brindi Niranjan and Gail Risbrider for the gift of AR antibody.
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
- Received July 31, 2008.
- Revision received October 14, 2008.
- Accepted October 30, 2008.
- ©2009 American Association for Cancer Research.