The Role of Odc in Myc Induced Tumorigenesis

Studies evaluating the role of Odc in tumorigenesis have principally relied on the selective Odc enzyme inhibitor a-difluoromethylornithine (DFMO), which impairs tumor development in several mouse models of cancer, and appears to hold promise in the clinic (116). However, in general these studies have been correlative, and they have not addressed the mechanism by which DFMO impairs tumor development. To determine the role of Odc in Myc-driven tumors we used the immunoglobulin heavy chain enhancer (E|i)-Myc transgenic mouse, which overexpresses the c-Myc gene in B-cells by virtue of an E|i. These mice, after a somewhat protracted precancerous phase, develop an oligoclonal, aggressive, and lethal B-cell lymphoma, and the mice usually succumb to their disease between 3 and 6 mo of age (117). Although the transgenic construct faithfully recapitulates the t(8:14) translocation occurring in human Burkitt lymphoma (5), the mice do not develop Burkitt lymphoma, but an aggressive pre-B/mature B-cell lymphoma (117). Nevertheless, many of the secondary genetic hits that occur during tumorigenesis in this mouse model have now also been shown to occur in human Burkitt lymphoma (118-121).

In the precancerous phase of the disease, the B-cells of E^,-Myc mice exhibit accelerated rates of proliferation, yet this is kept in check by high rates of cell turnover through apoptosis (117,122). Gene expression profiling established that a large number of Myc targets are turned on during this precancerous phase, and among these are Odc, Amdl, and Srm. In addition, Satl, which encodes spermidine/spermine-^-acetyltransferase that directs polyamine catabolism is repressed in these cells (123). The net result is an elevation in the levels of all polyamines in the B-cells of these mice. Importantly, by breeding the E|i-Myc mice to mice lacking one allele of Odc, or by treating E|i-Myc mice with DFMO, we established a critical role for Odc in Myc-induced lymphomagenesis because E|i-Myc; Odc+/- mice and DFMO-treated animals had a greatly protracted course of disease and a threefold increase in their lifespan (123). This is quite remarkable, given that Odc is just one of well over 1000 targets regulated by Myc. These findings thus underscore the pivotal roles polyamines play during Myc-driven tumorigenesis, and they also suggest that targeting other metabolic enzymes regulated by Myc may also be effective in cancer chemoprevention.

A curiosity coming from these studies was the selective effects of DFMO on the polyamine levels of B-cells from E|i-Myc mice. Specifically, DFMO effectively reduced putrescine levels, and to some extent spermidine content, back to those levels found in the B-cells of wild-type mice. By contrast, there was essentially no effect of DFMO on the polyamine levels of wild-type B-cells. Normal cells typically respond to DFMO treatment by increasing polyamine import, which is directed by a dedicated (but as yet uncharacterized) active transporter (124). Strikingly, unlike its effect in normal cells, DFMO treatment reduced polyamine uptake in precancerous E|i-Myc B-cells (123). This finding suggests that precancerous cells that overexpress Myc have an Achilles' heel—they cannot recoup polyamines—and this explains why DFMO is so effective as a chemopreventative agent in cancer.

Mechanistically very few studies have addressed why Odc is required for tumori-genesis. DFMO studies in immortal myeloid cells had suggested that Odc was a mediator of Myc-induced apoptosis after the withdrawal of survival factors (125). If this were universally true, then in the E|i-Myc model DFMO treatment, and perhaps Odc heterozygosity, should impair Myc-induced apoptosis and be thus expected to accelerate disease, which is clearly the exact opposite of their effects (123). Indeed, in vivo in primary B-cells having intact apoptotic checkpoints, DFMO and Odc heterozygosity did not affect Myc-induced apoptosis. Rather, the preventative effects of DFMO or Odc heterozygosity squarely centered on their ability to impair Myc's proliferative response (123).

Myc accelerates the rates of cell cycle traverse, at least in part, by inhibiting the expression of the Cdk inhibitors p21Cip1 and p27Kip1. This occurs at two levels, where Myc represses p27Kip1 promoter activity (126) and inhibits Miz-1-mediated transactiva-tion of p21Cip1 (75,79,82), and at the level of the protein, where Myc promotes degradation of p27Kip1 by the proteasome (127-130). Interestingly, DFMO and Odc heterozygosity selectively blocked Myc's effects on p21Cip1 and p27Kip protein, but not their RNA, levels (123). Turnover of p21Cip1 and especially p27Kip1 protein is mediated by the SCFSkp2 complex that ubiquitylates these proteins to mark them for degradation

Fig. 5. Proposed mechanism of chemoprevention of Myc-induced cancers by targeting polyamine biosynthesis. In normal cells, Myc levels are tightly controlled, resulting in balanced rates of cell growth and proliferation. In various cancers, Myc levels are elevated as the consequence of mutations in upstream regulatory pathways or through direct genetic events involving Myc genes. This results in accelerated rates cell-cycle traverse, which puts the cell at risk for additional mutations and genomic instability. In precancerous cells, where one can perturb polyamine levels, Myc's ability to drive uncontrolled cell proliferation is hampered, which contributes to "genome-protection" and chemoprevention.

Fig. 5. Proposed mechanism of chemoprevention of Myc-induced cancers by targeting polyamine biosynthesis. In normal cells, Myc levels are tightly controlled, resulting in balanced rates of cell growth and proliferation. In various cancers, Myc levels are elevated as the consequence of mutations in upstream regulatory pathways or through direct genetic events involving Myc genes. This results in accelerated rates cell-cycle traverse, which puts the cell at risk for additional mutations and genomic instability. In precancerous cells, where one can perturb polyamine levels, Myc's ability to drive uncontrolled cell proliferation is hampered, which contributes to "genome-protection" and chemoprevention.

by the proteasome (131-142), and p27Kip1 loss (but not p21Cip1 loss) markedly accelerates Myc-induced tumorigenesis in the E^,-Myc mouse by selectively augmenting Myc's proliferative response (143). These findings are thus consistent with a model where DFMO or Odc heterozygosity leads to a reduction in polyamine levels in E|i-Myc B-cells that then somehow disrupts the functions of the SCFSkp2 complex. In turn, this leads to increases in these Cdk inhibitors and to the inhibition of cyclin-E/Cdk2 complexes and cell proliferation, which impair Myc-induced tumorigenesis (Fig. 5 and 6).

A second rather surprising finding coming from the analyses of E|i-Myc; Odc+/- mice and DFMO-treated transgenics was that targeting this pathway also alters the route of transformation that usually accompanies lymphoma development (123). A hallmark of Myc-induced malignancies is inactivation of the Arf-p53 tumor suppressor pathway, which is inactivated in most Burkitt lymphomas (119,121); in the E^,-Myc mouse, this involves biallelic deletions in Arf and missense "hot-spot" mutations in p53 that generate dominant-negative forms of p53 protein (118,120). Interestingly, lymphomas arising in E|i-Myc;Odc+/- mice and in DFMO-treated transgenics selectively lacked deletions in

tp21c*1 p27Kif> p53 K I

Apoptosis«-*Cell Growth

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