To test the hypothesis that suppression of ornithine decarboxylase (ODC) activity blocks the promotion of target cells in the outer root sheath of the hair follicle initiated by Raf/MEK/ERK activation, we crossed mice overexpressing an activated MEK mutant in the skin (K14-MEK mice) with two transgenic lines overexpressing antizyme (AZ), which binds to ODC and targets it for degradation. K14-MEK mice develop spontaneous skin tumors without initiation or promotion. These mice on the ICR background were crossed with K5-AZ and K6-AZ mice on both the carcinogenesis-resistant C57BL/6 background and the sensitive DBA/2 background. Expression of AZ driven by either the K5 or K6 promoter along with K14-MEK dramatically delayed tumor incidence and reduced tumor multiplicity on both backgrounds compared with littermates expressing the MEK transgene alone. The effect was most remarkable in the MEK/K6-AZ mice from the ICR/D2 F1 cross, where double transgenic mice averaged less than one tumor per mouse for more than 8 weeks, while K14-MEK mice averaged over 13 tumors per mouse at this age. Putrescine was decreased in MEK/AZ tumors, while spermidine and spermine levels were unaffected, suggesting that the primary role played by AZ in this system is to inhibit putrescine accumulation. MEK/AZ tumors did not show evidence of apoptosis, but there was a 15-20% decrease in S-phase cells and a 40-60% decrease in mitotic cells in MEK/AZ tumors. These results indicate that the principal effect of AZ may be to slow cell growth primarily by increasing G2/M transit time.

SYNOPSIS Upon ras activation, ornithine decarboxylase (ODC) is markedly induced, and numerous studies suggest that ODC expression is controlled by Ras effector pathways. ODC is therefore a potential target in the treatment and prevention of Ras-driven tumors. We compared ODC mRNA translation profiles and stability in normal and Ras12V-transformed rat intestinal epithelial (RIE-1) cells. While translation initiation of ODC increased modestly in Ras12V cells, ODC RNA was stabilized 8-fold. Treatment with the specific mTORC1 inhibitor rapamycin or siRNA knockdown of mTOR destabilized the ODC message, but rapamycin had only a minor effect on ODC translation initiation. Inhibition of mTORC1 also reduced the association of the mRNA binding protein HuR with the ODC transcript. We have shown previously that HuR binding to the ODC 3′UTR results in significant stabilization of the ODC mRNA, which contains several AU-rich regions within its 3′UTR that may act as regulatory sequences. Analysis of ODC 3′UTR deletion constructs suggests that cis-acting elements between bases 1969 and 2141 of the ODC mRNA act to stabilize the ODC transcript. These experiments thus define a novel mechanism of ODC synthesis control. Regulation of ODC mRNA decay could be an important means of limiting polyamine accumulation and subsequent 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.