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Supplementary Figure 3 from OSU-HDAC42, a Histone Deacetylase Inhibitor, Blocks Prostate Tumor Progression in the Transgenic Adenocarcinoma of the Mouse Prostate Model

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]

Supplementary Figure 1 from OSU-HDAC42, a Histone Deacetylase Inhibitor, Blocks Prostate Tumor Progression in the Transgenic Adenocarcinoma of the Mouse Prostate Model

Supplementary Figure 2 from OSU-HDAC42, a Histone Deacetylase Inhibitor, Blocks Prostate Tumor Progression in the Transgenic Adenocarcinoma of the Mouse Prostate Model

Supplementary Figure Legends 1-3 from OSU-HDAC42, a Histone Deacetylase Inhibitor, Blocks Prostate Tumor Progression in the Transgenic Adenocarcinoma of the Mouse Prostate Model

Supplementary Table 1 from OSU-HDAC42, a Histone Deacetylase Inhibitor, Blocks Prostate Tumor Progression in the Transgenic Adenocarcinoma of the Mouse Prostate Model

The transgenic adenocarcinoma of the mouse prostate (TRAMP) model is well established and offers several advantages for the study of chemopreventive agents, including its well-defined course of disease progression and high incidence of poorly differentiated carcinomas within a relatively short length of time. However, there is no consensus on the grading of prostatic lesions in these mice. In particular, agreement is lacking on the criteria for differentiating prostatic intraepithelial neoplasia (PIN) from well-differentiated adenocarcinoma, specifically as it relates to evidence of invasion. This differentiation is critical for evaluating the effects of putative chemopreventive agents on progression to neoplasia. Moreover, only one of the published grading schemes assigns numerical grades to prostatic lesions, which facilitate statistical analysis. Here, we review five currently available grading schemes and propose a refined scheme that provides a useful definition of invasion for the differentiation of PIN from well-differentiated adenocarcinoma and includes a numerical scoring system that accounts for both the most severe and most common histopathological lesions in each of the lobes of the prostate and their distributions. We expect that researchers will find this refined grading scheme to be useful for chemoprevention studies in TRAMP mice.

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.

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.

Purpose: HER-2/neu overexpression is a molecular subtype of breast cancer associated with poorer clinical outcome. The association of low-grade inflammation with increased risk for cancer and correlation of HER-2/neu overexpression and immune cell infiltration in human breast tumors suggest that an inflammatory mammary microenvironment is critical to early events in HER-2/neu carcinogenesis. Of interest are nutritional, complementary interventions that may suppress mammary gland inflammation, such as dietary ω-3 fatty acids. This study is testing the hypothesis that dietary fat content can modulate inflammation during early stages of HER-2/neu mammary carcinogenesis, with inhibition of mammary macrophage infiltration and activation by fish oil/ω-3 fatty acids relative to corn oil/ω-6 fatty acids. Methods: Female MMTV-HER-2/neu transgenic mice were fed semi-purifed diets with 25% energy as menhaden fish oil or corn oil x 29 weeks or corn oil x 19 weeks then fish oil x 10 weeks. Mammary glands were harvested at 35 weeks for F480, CD3, and LY6G immunostaining to identify macrophages, T cells, and granulocytes, respectively. A novel MMTV-HER-2/neu;c-fms-YFP double transgenic mouse model was generated to facilitate isolation of mammary gland macrophages. HER-2/neu;c-fms-YFP and control c-fms-YFP mice were fed fish or corn oil based diets x 19 weeks. YFP+ cells were sorted using high-speeed digital FACS and RNA isolated from the enriched cells. Differential gene expression based on diet and genotype was determined by Affymetrix microarray. Results: Diets were well tolerated by the mice without toxicity. By immunostaining, MMTV-HER-2/neu mammary glands of fish oil fed mice had a lower number of macrophages/area of mammary duct relative to the corn oil fed mice (p Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5426. doi:1538-7445.AM2012-5426

Introduction: Testosterone is a critical regulator of prostate function and carcinogenesis. Inhibition of testosterone action is an approach for prostate cancer prevention. We hypothesize that consumption of tomato phytochemicals may have a protective impact during prostate carcinogenesis via inhibition of androgen signaling in the prostate epithelium. Methods: Four week-old C57/BL6 male mice (n=84) were fed either: AIN93-G diet as a control, AIN93-G with10% tomato powder (0.02 g LYC/Kg diet) or AIN93-G with 10% LYC beadlets (0.40 g LYC/Kg diet). At eight weeks of age, mice were randomized among three endocrine treatments: intact control, castration, or castration + testosterone repletion (2.5 mg testosterone propionate/Kg body weight/mouse/day by subcutaneous osmotic minipumps starting 7 days after castration). At 10 weeks of age, blood was collected for carotenoid analysis by HPLC and tissues were collected for evaluating prostate epithelial cell proliferation index (Ki67), apoptosis index (ApopTag), and other biomarkers. Results: Dietary intakes did not differ among groups. However, the estimated LYC consumption was 0.00 mg/d for controls, 1.13 mg/d in LYC fed mice, and 0.06 mg/d in tomato fed mice (P< 0.001). Interestingly, serum LYC levels did not differ between mice fed the tomato and LYC diets (366 and 371 nmol/L, respectively). As anticipated, castration decreased proliferative indices in control mice (1.8% vs. 4.7%) and testosterone repletion restored and further enhanced the proliferative index (15.8%) (P Conclusion: LYC absorption in mice is limited and similar blood concentrations are achieved over a wide range of dietary intake from either pure LYC or tomato components. Mice consuming tomato powder or LYC show reduced prostate epithelial proliferation in response to testosterone. Tomato powder is a more potent enhancer of apoptotic cascades in response to androgen deprivation. Bioactive components in tomatoes in addition to LYC may influence prostate biology and participate in the inhibition of prostate carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 963.