Elevated polyamine content and increased ornithine decarboxylase (ODC) activity have been associated with neoplastic growth in numerous animal models and human tissues. Antizyme (AZ) is a negative regulator of polyamine metabolism that inhibits ODC activity, stimulates ODC degradation, and suppresses polyamine uptake. Preliminary evidence, obtained from transgenic mice with tissue specific overexpression of AZ indicates that tumor development can be suppressed by AZ. To extend these studies, we have examined the effect of keratin 5 (K5)- or K6-driven AZ transgenes on 7,12-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) chemical carcinogenesis of the skin, in promotion-resistant C57BL/6 and promotion-sensitive DBA/2 mice. On both genetic backgrounds, K6-AZ mice showed a reduction in tumor multiplicity, with 85% fewer tumors than wild-type controls on the C57BL/6 background and 50% fewer tumors on the DBA/2 background. K5-AZ mice developed 50% fewer tumors than controls on both backgrounds. The percent of mice with tumors and tumor size were also reduced in the K5-AZ and K6-AZ groups. Tumor and TPA-treated skin sections from K6-AZ mice exhibited the strongest AZ expression, with localization mainly in suprabasal keratinocytes. K6-AZ mice also had slightly reduced cell proliferation rates in tumors and TPA-treated skin. The lack of a more pronounced effect on cell proliferation is probably explained by the observation that AZ staining did not colocalize with proliferating cell nuclear antigen (PCNA), a marker for the proliferative compartment. These studies demonstrate a tumor-suppressive effect of AZ in C57BL/6 and DBA/2 mice, and confirm the importance of ODC and polyamines in tumor development.

Numerous studies have demonstrated a link between elevated polyamine biosynthesis and neoplastic growth, but the specific contribution of spermine synthase to epithelial tumor development has never been explored in vivo. Mice with widespread overexpression of spermine synthase (CAG-SpmS) exhibit decreased spermidine levels, increased spermine and a significant rise in tissue spermine:spermidine ratio. We characterized the response of CAG-SpmS mice to two-stage skin chemical carcinogenesis as well as spontaneous intestinal carcinogenesis induced by loss of the Apc tumor suppressor in Apc (Min) (/+) (Min) mice. CAG-SpmS mice maintained the canonical increases in ornithine decarboxylase (ODC) activity, polyamine content and epidermal thickness in response to tumor promoter treatment of the skin. The induction of S-adenosylmethionine decarboxylase (AdoMetDC) activity and its product decarboxylated AdoMet were impaired in CAG-SpmS mice, and the spermine:spermidine ratio was increased 3-fold in both untreated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated skin. The susceptibility to 7,12-dimethylbenz[a]anthracene (DMBA)/TPA skin carcinogenesis was not altered in CAG-SpmS mice, and SpmS overexpression did not modify the previously described tumor resistance of mice with targeted antizyme expression or the enhanced tumor response in mice with targeted spermidine/spermine-N ( 1) -acetyltransferase expression. CAG-SpmS/Min mice also exhibited elevated spermine:spermidine ratios in the small intestine and colon, yet their tumor multiplicity and size was similar to Min mice. Therefore, studies in two of the most widely used tumorigenesis models demonstrate that increased spermine synthase activity and the resulting elevation of the spermine:spermidine ratio does not alter susceptibility to tumor development initiated by c-Ha-Ras mutation or Apc loss.

The objective of these studies was to utilize a novel transgenic mouse model with inducible and mammary gland-specific antizyme (AZ) expression to test the hypothesis that AZ will prevent mammary tumorigenesis and restrict the growth of established mammary tumors. AZ is a negative regulator of cellular polyamine levels that inhibits ornithine decarboxylase (ODC) activity, stimulates ODC degradation and suppresses exogenous polyamine uptake. Mouse lines were generated harboring a transgene construct (tetO-AZ) containing Tet operon repeat sequences upstream of an AZ cDNA followed by an internal ribosome entry site and a luciferase coding region. The tetO-AZ mice were crossed with MMTV-rtTA (MTB) mice to achieve AZ expression within the mammary gland that is induced by doxycycline (Dox). Bioluminescent imaging and in vitro assays of luciferase activity and AZ protein demonstrate tissue-specific and Dox-dependent transgene expression. Morphometric and immunohistochemical analysis of the mammary epithelial tree verified that normal development is unaffected by elevated levels of AZ. Next, we examined the ability of AZ to prevent tumor appearance in a widely used model of human breast cancer. TetO-AZ/MTB mice were crossed with MMTV-neu mice that develop spontaneous mammary tumors and Dox-treated animals were monitored for up to 1 year. Tumor-free survival was greatly extended in tetO-AZ/MTB/neu mice relative to controls (348 vs. 234 days, p=0.0003). In vivo imaging of the luciferase reporter revealed a pronounced absence of transgene expression within all 19 tumors detected in Dox-treated tetO-AZ/MTB/neu mice, therefore these tumors developed from cells that initially lacked AZ expression or lost AZ expression at a very early stage of tumor growth. The Dox-regulated transgenic model also allowed us to examine the effect of AZ on the maintenance and growth of established tumors in Dox-naïve mice. Once tumors reached 5 mm in diameter, Dox treatment was initiated to induce AZ expression in tetO-AZ/MTB/neu mice and tumor growth was evaluated. The established primary tumors from Dox-treated controls grew very rapidly and 34/45 (76%) exceeded 2000 mm3 within the monitoring period of 8 weeks, whereas only 6/16 (38%) of the tumors from tetO-AZ/MTB/neu mice reached that size. In vivo imaging of transgene-derived luciferase reporter expression allows us to compare transgene expression levels with tumor growth rate over time. This analysis has revealed several classes of response including a) reduced tumor growth in the presence of strong AZ expression, b) evolutionary loss of AZ expression and subsequent increased growth rate and c) occasional rapidly growing tumors that tolerate high levels of AZ expression. In summary, our studies indicate that elevated AZ expression prevents the appearance of HER2/neu-induced mammary tumors and suppresses the growth of established mammary tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 829. doi:10.1158/1538-7445.AM2011-829