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The mechanisms whereby cyclooxygenase-2 (COX-2) overexpression may contribute to bladder carcinogenesis remain unknown. We recently developed a transgenic mouse model overexpressing COX-2 under the control of a bovine keratin 5 (BK5) promoter causing a high incidence of transitional cell hyperplasia (TCH) in the bladder with a proportion of lesions progressing to invasive carcinoma. Microarray gene analysis was employed to determine the effects of COX-2 overexpression on gene expression profiles in the urinary bladder. Statistical analysis revealed that 70 genes were upregulated and 60 were downregulated by twofold or more in bladders from transgenic compared to wild-type mice. Expression Analysis Systematic Explorer (EASE) analysis revealed that genes associated with Immune/Stress Response and Cell Cycle/Proliferation biological processes were overexpressed in the transgenic mice. Relevant downregulated genes included three transforming growth factor (TGF)-beta related genes, Tgfb2, Tgfb3, and Tgfbi. The growth factor epiregulin was the most highly induced gene among those validated by qRT-PCR in TCH of BK5.COX-2 mouse bladders in parallel with increased staining for Ki67. Prostaglandin E(2) (PGE(2)) directly induced the expression of epiregulin mRNA in bladders from wild-type FVB mice ex vivo. We further determined that recombinant epiregulin increased both cell proliferation and Erk phosphorylation in UMUC-3 bladder cancer cells. These results indicate that the response of the mouse urinary bladder to elevated COX-2 expression includes enhanced inflammatory response and induction of cell proliferation. The growth factor epiregulin may play a role in bladder carcinogenesis and may serve as a novel target for the prevention and treatment of bladder cancer.

Expression of the WWOX gene, encompassing the common chromosome fragile site FRA16D, is altered in a large fraction of cancers of various types, including prostate cancer. We have examined expression and biological functions of WWOX in prostate cancer. WWOX mRNA and protein expression were significantly reduced in prostate cancer-derived cells (LNCaP, DU145, and PC-3) compared with noncancer prostate cells (PWR-1E), and WWOX expression was reduced in 84% of prostate cancers, as assessed by immunohistochemical staining. Down-modulation of WWOX expression in the prostate cancer-derived cells is due to DNA hypermethylation in the WWOX regulatory region. Treatment with 5-aza-2′-deoxycytidine (AZA), a DNA methyltransferase inhibitor, and trichostatin A, a histone deacetylase inhibitor, led to increased WWOX mRNA and protein expression in prostate cancer-derived cells, most strikingly in DU145 cells. Transfection-mediated WWOX overexpression in DU145 cells suppressed colony growth (P = 0.0012), and WWOX overexpression by infection with Ad-WWOX virus induced apoptosis through a caspase-dependent mechanism and suppressed cell growth. Lastly, ectopic expression of WWOX by Ad-WWOX infection suppressed tumorigenicity of xenografts in nude mice, and intratumoral AZA treatment halted tumor growth. The data are consistent with a role for WWOX as a prostate cancer tumor suppressor and suggest that WWOX signal pathways should be further investigated in normal and cancerous prostate cells and tissues. (Cancer Res 2006; 66(13): 6477-81)

The ability to modify the expression of specific genes in the mouse through genetic engineering technologies allows for the generation of previously unavailable models for prostate cancer prevention research. Although animal models have existed for some time for the study of prostate cancer prevention (primarily in the rat), it is uncertain if the mechanisms that drive prostate carcinogenesis in these models are relevant to those in human prostate cancer. Cell culture studies are of limited usefulness because the conditions are inherently artificial. Factors such as relevant physiologic concentrations and metabolism of putative chemoprevention compounds are difficult to model in an in vitro system. These studies also preclude the types of interactions known to occur between multiple cell types in vivo. In addition, all prostate cancer cell lines are already highly progressed and are not representative of the type of cells to which most preventive strategies would be targeted. Due to the advent of genetically engineered mouse (GEM) models, we now have models of prostate cancer that are dependent on molecular mechanisms already implicated in human prostate carcinogenesis. With these models we can perform a variety of experiments that could previously only be done in cell culture or in prostate cancer cell line xenografts. The currently available GEM models of prostate cancer have been extensively reviewed therefore, this review will focus on the types of models available and their usefulness for various types of preclinical studies relevant to prostate cancer prevention.

BACKGROUND. Prostate cancer (PCa) is the leading cancer related death in America. Gleasongradingiscurrentlythepredominantmethodforprediction,withonlyfewbiomarkers available. More biomarkers, especially as they relate to cancer progression are desirable. METHODS. The abundance of several important proteins in prostate tissue was compared between wild-type mouse dorsal prostate and well-differentiated transgenic adenocarcinoma mouse prostate (TRAMP) mouse dorsal prostates, and between wild-type mouse dorsal prostate and poorly-differentiated TRAMP mouse tumor tissue. 2DIGE method in conjunction with MALDI-ToF and Western blots was used to determine differential expression. RESULTS. In TRAMP dorsal prostates with well-differentiated adenocarcinoma, there were fewsignificantchangesintheproteinabundancescomparedtowild-typedorsalprostates,with the exception of increases in proliferating cell nuclear antigen (PCNA) and beta tubulin, two proteins implicated in cell proliferation, and a more than 2-fold increase in Hsp60, a protein involved in the suppression of apoptosis. In the poorly-differentiated tumors, the changes in protein abundance were substantial. While some of those changes could be related to the disappearance of stromal tissue or the appearance of epithelial tissue, other changes in protein abundanceweremoresignificanttothecancerdevelopmentitself.Mostnotablewastheoverall decrease in calcium homeostasis proteins with a 10-fold decrease in calreticulin and Hsp70 and a 40-fold decrease in creatine kinase bb in the cancerous tissue. CONCLUSIONS. ProteomicsofTRAMPmiceprovideanexcellentmethodtoobservechanges in protein abundance, revealing changes in pathways during cancer progression. Prostate 64: 128–138, 2005. # 2005 Wiley-Liss, Inc.

The inducible form of cyclooxygenase (COX), COX-2, is up-regulated in many epithelial cancers and its prostaglandin products increase proliferation, enhance angiogenesis, and inhibit apoptosis in several tissues. Pharmacologic inhibition and genetic deletion studies showed a marked reduction of tumor development in colon and skin. COX-2 has also been strongly implicated in urinary bladder cancer primarily by studies with nonselective COX- and COX-2-selective inhibitors. We now show that forced expression of COX-2, under the control of a keratin 5 promoter, is sufficient to cause transitional cell hyperplasia (TCH) in 17% and 75% of the heterozygous and homozygous transgenic lines, respectively, in an age-dependent manner. TCH was strongly associated with inflammation, primarily nodules of B lymphocytes; some T cells and macrophage infiltration were also observed. Additionally, transitional cell carcinoma was observed in ∼10% of the K5.COX-2 transgenic mice; no TCH or transitional cell carcinoma was observed in wild-type bladders. Immunohistochemistry for vascular proliferation and vascular endothelial growth factor showed significant increases above that in wild-type urinary bladders. Our results suggest that overexpression of COX-2 is sufficient to cause hyperplasia and carcinomas in the urinary bladder. Therefore, inhibition of COX-2 should continue to be pursued as a potential chemopreventive and therapeutic strategy.

Histone deacetylase (HDAC) inhibitors suppress tumor cell growth via a broad spectrum of mechanisms, which should prove advantageous in the context of cancer prevention. Here, we examined the effect of dietary administration of OSU-HDAC42, a novel HDAC inhibitor, on prostate tumor progression in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model. Based on a series of pilot studies, an AIN-76A diet was formulated containing 208 ppm OSU-HDAC42, which was estimated to deliver ∼25 mg/kg of drug per day to each mouse and found to cause a suppression of PC-3 xenograft tumor growth equivalent to that achieved by gavage administration of a similar dose. At 6 weeks of age, TRAMP mice received this drug-containing or control diet for 4 or 18 weeks and were evaluated for prostatic intraepithelial neoplasia (PIN) and carcinoma development, respectively. OSU-HDAC42 not only decreased the severity of PIN and completely prevented its progression to poorly differentiated carcinoma (74% incidence in controls versus none in drug-treated mice), but also shifted tumorigenesis to a more differentiated phenotype, suppressing absolute and relative urogenital tract weights by 86% and 85%, respectively, at 24 weeks of age. This tumor suppression was associated with the modulation of intraprostatic biomarkers, including those indicative of HDAC inhibition, increased apoptosis and differentiation, and decreased proliferation. With the exception of completely reversible hematologic alterations and testicular degeneration, no significant changes in body weight or other indicators of general health were observed in drug-treated mice. These results suggest that OSU-HDAC42 has value in prostate cancer prevention. [Cancer Res 2008;68(10):3999–4009]

The phosphoinositide-dependent kinase 1 (PDK1)/Akt pathway is an important regulator of multiple biological processes including cell growth, survival, and glucose metabolism. In light of the mechanistic link between Akt signaling and prostate tumorigenesis, we evaluated the chemopreventive relevance of inhibiting this pathway in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model with OSU03012, a celecoxib-derived, but COX-2-inactive, PDK1 inhibitor. Beginning at ten weeks of age when prostatic intraepithelial neoplasia (PIN) lesions are well developed, TRAMP mice received OSU03012 via daily oral gavage for 8 weeks. The drug treatment significantly decreased the weight of all 4 prostate lobes as well as the grade of epithelial proliferation in the dorsal and lateral lobes compared to vehicle-treated control mice. The incidences of carcinoma and metastasis were decreased, although not to statistically significant levels. Treated mice lost body fat and failed to gain weight independent of food intake. This change and periportal hepatocellular atrophy can be linked to sustained PDK1 inhibition through downstream inactivation of glycogen synthase. Centrilobular hepatocellular hypertrophy and necrosis of Type II skeletal myofibers were also compound-related effects. We conclude that targeting of the PDK1/Akt pathway has chemopreventive relevance in prostate cancer and causes other in vivo effects mediated in part by an alteration of bioenergetic signaling.

Cyclooxygenase and lipoxygenase arachidonate products, including prostaglandins (PGs), leukotrienes (LTs), and hydroxyeicosatetraenoic acids (HETEs), are known to modulate inflammation within tissues and can serve as important etiologic factors in carcinogenesis. Eicosanoid content in tissues is typically determined either as a single molecular species through antibody-based assays or by high-performance liquid chromatography after addition of an exogenous substrate such as arachidonic acid. Unfortunately, the methods currently in use are either time-consuming or complicated. Here we report a method for simultaneously identifying eicosanoids appearing as endogenous bioactive lipids in in vivo settings using LC/MS/MS. The analyses indicate marked differences in endogenous eicosanoid content between malignant tissue types suggesting a need for selective therapeutic approaches. As a demonstration of the utility of the method, we present data to show that the technique can be used to distinguish eicosapentaenoic acid-derived formation of PGE(3) from PGE(2) in murine prostate tissue. The method has also been applied to an examination of endogenous eicosanoid metabolism in 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral cancer in hamsters demonstrating the inflammatory nature of this type of cancer with elevated levels of both PGE(2) and LTB(4). In addition, the concentration of the eicosanoid 13-hydroxyoctadecadienoic acid was 67.6% lower in DMBA treated specimens than in control specimens. Thus, our method provides a powerful tool for measuring modulation of eicosanoid metabolites in various preclinical and clinical tissues and may be useful in studies of the endogenous changes in eicosanoid metabolism at various stages of cancer development.

Ultraviolet (UV) irradiation is the primary environmental insult responsible for the development of most common skin cancers. To better understand the multiple molecular events that contribute to the development of UV-induced skin cancer, in a first study, serial analysis of gene expression (SAGE) was used to compare the global gene expression profiles of normal SKH-1 mice epidermis with that of UV-induced squamous cell carcinomas (SCCs) from SKH-1 mice. More than 200 genes were found to be differentially expressed in SCCs compared to normal skin (P < 0.0005 level of significance). As expected, genes related to epidermal proliferation and differentiation were deregulated in SCCs relative to normal skin. However, various novel genes, not previously associated with skin carcinogenesis, were also identified as deregulated in SCCs. Northern blot analyses on various selected genes validated the SAGE findings: caspase-14 (reduced 8.5-fold in SCCs); cathepsins D and S (reduced 3-fold and increased 11.3-fold, respectively, in SCCs); decorin, glutathione S-transferase omega-1, hypoxia-inducible factor 1 alpha, insulin-like growth factor binding protein-7, and matrix metalloproteinase-13 (increased 18-, 12-, 12-, 18.3-, and 11-folds, respectively, in SCCs). Chemokine (C-C motif), ligand 27 (CCL27), which was found downregulated 12.7-fold in SCCs by SAGE, was also observed to be strongly downregulated 6-24 h after a single and multiple UV treatments. In a second independent study we compared the expression profile of UV-irradiated versus sham-treated SKH-1 epidermis. Interestingly, numerous genes determined to be deregulated 8 h after a single UV dose were also deregulated in SCCs. For instance, genes whose expression was upregulated both after acute UV-treated skin and SCCs included keratins 6 and 16, small proline-rich proteins, and S100 calcium binding protein A9. Studies like those described here do not only provide insights into genes and pathways involved in skin carcinogenesis but also allow us to identify early UV irradiation deregulated surrogate biomarkers of potential use in chemoprevention studies.

A quantitative overview of 14 studies of rat colon carcinogenesis was undertaken to examine the relationship between fat intake, and fat intake by degree of saturation, on the incidence of colon carcinoma while controlling for calorie consumption. Calorie consumption was not recorded in 11 of the 14 studies. Hence, two types of analyses were conducted. The first examines carcinoma incidence as a function of percent fat (by weight), with calories controlled for by including weight gain per week in the analysis. The second estimates calories per day, for studies not providing such information, using weight gain per week and age at death, followed by a joint analysis of estimated fat calories and estimated total calories. With either approach, a rather strong positive relationship between colon carcinoma incidence and fat intake is indicated for Fischer 344 rats, but no association is apparent for Sprague-Dawley rats. This situation is somewhat clarified when the degree of saturation is taken into account: both strains gave results that suggest a negative relationship between colon cancer incidence and omega-3 fatty acids intake and a positive relationship with non-omega-3 polyunsaturated fat intake among Fischer 344 rats. These analyses suggest an important and specific role for dietary fat in the promotion of rat colon carcinoma.