Thomas G. O'Brien

Pronounced reduction in adenoma recurrence associated with aspirin use and a polymorphism in the ornithine decarboxylase gene

Most sporadic colon adenomas acquire mutations in the adenomatous polyposis coli gene (APC) and show defects in APC-dependent signaling. APC influences the expression of several genes, including the c-myc oncogene and its antagonist Mad1. Ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, is a transcriptional target of c-myc and a modifier of APC-dependent tumorigenesis. A single-nucleotide polymorphism exists in intron 1 of the human ODC gene, which lies between two myc-binding domains. This region is known to affect ODC transcription, but no data exist on the relationship of this polymorphism to risk of colorectal neoplasia in humans. We show that individuals homozygous for the minor ODC A-allele who reported using aspirin are ≈0.10 times as likely to have an adenoma recurrence as non-aspirin users homozygous for the major G-allele. Mad1 selectively suppressed the activity of the ODC promoter containing the A-allele, but not the G-allele, in a human colon cancer-derived cell line (HT29). Aspirin (≥10 μM) did not affect ODC allele-specific promoter activity but did activate polyamine catabolism and lower polyamine content in HT29 cells. We propose that the ODC polymorphism and aspirin act independently to reduce the risk of adenoma recurrence by suppressing synthesis and activating catabolism, respectively, of colonic mucosal polyamines. These findings confirm the hypothesis that the ODC polymorphism is a genetic marker for colon cancer risk, and support the use of ODC inhibitors and aspirin, or other nonsteroidal antiinflammatory drugs (NSAIDs), in combination as a strategy for colon cancer prevention.

Tumor promoters: Effects on proliferation and differentiation of cells in culture

Abstract Tumor promoters enhance tumor formation when administered after an initiating action by a carcinogen. The phorbol diester class of tumor promoters has been shown to affect many biochemical and biological processes in mouse skin and cell culture. The effects of these compounds on the proliferation and differentiation of cells in culture are reviewed herein; the possible relation of these effects to the mechanism of tumor promotion are discussed.

A Definitive Role of Ornithine Decarboxylase in Photocarcinogenesis

Excessive exposure of solar ultraviolet (UV) radiation, particularly its UVB component, to human skin is the major cause for more than a million new cases of cutaneous malignancies diagnosed annually in the United States. Photocarcinogenesis, like other cancers, is a multistep process that includes initiation and promotion. A proper understanding of the molecular events occurring during the tumor promotion phase of photocarcinogenesis could lead to the development of novel approaches for the management of skin cancer. Using a transgenic mouse model (K5/ODC mice), which overexpresses the enzyme ornithine decarboxylase (ODC) in hair follicle keratinocytes, we studied the role of this gene in photocarcinogenesis. A single UVB-exposure of 180 mJ/cm(2) to the transgenic mice resulted in a minimal increase in bifold skin thickness and ODC activity. However, in SKH-1 hairless mice, the most common and highly sensitive model for photocarcinogenesis, and in littermate nontransgenic mice, increases in skin thickness and ODC activity were substantial. In long-term experiments, mice were exposed to 180 mJ/cm(2) of UVB radiation three times a week for 2 weeks (tumor-initiating dose). At 30 weeks after this treatment, in two independent experiments, 40% of the K5/ODC transgenic mice exposed to UVB were found to develop epidermal tumors. The tumors were histologically verified as benign papillomas and squamous cell carcinomas. Interestingly, 100% of the transgenic mice also developed >20 pigmented cysts/mouse, which contained keratinocyte material with increased keratinocytic melanization. Under similar UVB-exposure protocol, the nontransgenic littermates or SKH-1 hairless mice did not develop tumors or pigmented cysts for up to 50 weeks. Oral consumption of alpha-difluoromethylornithine, an irreversible specific inhibitor of ODC, in the drinking water (1% w/v) to the transgenic mice resulted in complete prevention of UVB-mediated tumorigenesis and a substantial decrease in the formation of pigmented cysts (<10 per mouse). These data establish a definitive role of ODC in the promotion phase of photocarcinogenesis.

Dose response evaluation of gene expression profiles in the skin of K6/ODC mice exposed to sodium arsenite

Chronic drinking water exposure to inorganic arsenic and its metabolites increases tumor frequency in the skin of K6/ODC transgenic mice. To identify potential biomarkers and modes of action for this skin tumorigenicity, we characterized gene expression profiles from analysis of K6/ODC mice administered 0, 0.05, 0.25, 1.0 and 10 ppm sodium arsenite in their drinking water for 4 weeks. Following exposure, total RNA was isolated from mouse skin and processed to biotin-labeled cRNA for microarray analyses. Skin gene expression was analyzed with Affymetrix Mouse Genome 430A 2.0 GeneChips, and pathway analysis was conducted with DAVID (NIH), Ingenuity Systems and MetaCores GeneGo. Differential expression of several key genes was verified through qPCR. Only the highest dose (10 ppm) resulted in significantly altered KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, including MAPK, regulation of actin cytoskeleton, Wnt, Jak-Stat, Tight junction, Toll-like, phosphatidylinositol and insulin signaling pathways. Approximately 20 genes exhibited a dose response, including several genes known to be associated with carcinogenesis or tumor progression including cyclin D1, CLIC4, Ephrin A1, STAT3 and DNA methyltransferase 3a. Although transcription changes in all identified genes have not previously been linked to arsenic carcinogenesis, their association with carcinogenesis in other systems suggests that these genes may play a role in the early stages of arsenic-induced skin carcinogenesis and can be considered potential biomarkers.

Haploinsufficiency for Odc Modifies Mouse Skin Tumor Susceptibility

Numerous studies have linked overexpression of ornithine decarboxylase (Odc) gene with enhanced susceptibility to mouse skin tumorigenesis. However, there is little experimental evidence suggesting that modest reductions in Odc expression might reduce tumor susceptibility. To address this issue, here we report the use of the Odc(+/-) haploinsufficiency model, in which one copy of the murine Odc gene has been inactivated by a homologous recombination. Compared with Odc(+/+) mice, Odc(+/-) mice exhibit reduced epidermal ODC enzyme activity and polyamine accumulation following treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Furthermore, following chronic TPA treatment, the characteristic hyperplastic response of the epidermis was diminished in Odc(+/-) mice. Finally, when subjected to a two-stage initiation-promotion protocol, substantially fewer skin papillomas developed in Odc(+/-) mice compared with wild-type littermates. These results support the concept that differences in tissue polyamine levels, resulting from either overexpression or reductions in ODC, are important modifiers of tumor susceptibility.

Lipophilic lysine-spermine conjugates are potent polyamine transport inhibitors for use in combination with a polyamine biosynthesis inhibitor.

Cancer cells can overcome the ability of polyamine biosynthesis inhibitors to completely deplete their internal polyamines by the importation of polyamines from external sources. This paper discusses the development of a group of lipophilic polyamine analogues that potently inhibit the cellular polyamine uptake system and greatly increase the effectiveness of polyamine depletion when used in combination with DFMO, a well-studied polyamine biosynthesis inhibitor. The attachment of a length-optimized C(16) lipophilic substituent to the epsilon-nitrogen atom of an earlier lead compound, D-Lys-Spm (5), has produced an analogue, D-Lys(C(16)acyl)-Spm (11) with several orders of magnitude more potent cell growth inhibition on a variety of cultured cancer cell types including breast (MDA-MB-231), prostate (PC-3), melanoma (A375), and ovarian (SK-OV-3), among others. These results are discussed in the context of a possible membrane-catalyzed interaction with the extracellular polyamine transport apparatus. The resulting novel two-drug combination therapy targeting cellular polyamine metabolism has shown exceptional efficacy against cutaneous squamous cell carcinomas (SCC) in a transgenic ornithine decarboxylase (ODC) mouse model of skin cancer. A majority (88%) of large, aggressive SCCs exhibited complete or nearly complete remission to this combination therapy, whereas responses to each agent alone were poor. The availability of a potent polyamine transport inhibitor allows, for the first time, for a real test of the hypothesis that starving cells of polyamines will lead to objective clinical response.

Combination therapy with 2-difluoromethylornithine and a polyamine transport inhibitor against murine squamous cell carcinoma

Using a recently developed autochthonous mouse model of squamous cell carcinoma (SCC), a combination therapy targeting polyamine metabolism was evaluated. The therapy combined 2‐difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), and MQT 1426, a polyamine transport inhibitor. In 1 trial lasting 4 weeks, combination therapy with 0.5% DFMO (orally, in the drinking water) and MQT 1426 (50 mg/kg i.p., bid) was significantly more effective than with either single agent alone when complete tumor response was the endpoint. In the combination group, 72% of SCCs responded completely vs. 21 and 0% for DFMO and MQT 1426, respectively. A second trial involved a 4‐week treatment period followed by 6 weeks off‐treatment. With apparent cures as an endpoint, combination therapy was again more effective than either agent alone: a 50% apparent cure rate was observed in the combination group vs. 7.7% in the DFMO group. MQT 1426 had no inhibitory effect on SCC ODC activity nor did it enhance the inhibition by DFMO, but SCC polyamine levels declined more rapidly when treated with combination therapy vs. DFMO alone. The apoptotic index in SCCs was transiently increased by combination therapy but not by DFMO alone. Thus, targeting both polyamine biosynthesis and polyamine transport from the tumor microenvironment enhances the efficacy of polyamine‐based therapy in this mouse model of SCC.

Conversion of C57Bl/6 mice from a tumor promotion–resistant to a –sensitive phenotype by enhanced ornithine decarboxylase expression

A transgenic mouse model was developed in which ornithine decarboxylase (ODC) can be overexpressed in a tissue‐specific and regulated manner. Hair follicle keratinocytes were targeted by use of a bovine keratin 6 (K6) promoter/regulatory region, and regulation was accomplished by using the tetracycline‐regulated transactivator/tetracycline‐response element system. Double‐transgenic mice carrying both transgenes (K6/tetracycline‐regulatable transactivator protein (tTA) and tetracycline‐response element/Odc) on a C57Bl/6 background had no obvious phenotypic abnormalities in the absence (Odc transgene–expressed) of doxycycline (a tetracycline analog) in the drinking water. However, induction of K6‐driven tTA expression by the tumor promoter (12‐O‐tetradecanoylphorbol‐13‐acetate) (TPA) led to very high levels of epidermal ODC activity and robust hyperplasia, especially involving hair follicles. Both effects were abolished by inclusion of doxycycline in the drinking water to repress transgene expression. Finally, the number of papillomas that developed in a standard (7,12‐dimethybenz[a]anthracene) (DMBA)/TPA protocol was greatly reduced in mice in which transgenic Odc expression was repressed by doxycycline. Our results demonstrated that the higher levels of ODC expression produced in the transgenic model in the induced versus the repressed condition make the normally promotion‐resistant C57Bl/6 strain much more sensitive to the short‐term and long‐term (i.e., tumor‐promoting) effects of TPA. Mol. Carcinog. 26:32–36, 1999.

Analysis of ras gene mutational spectra in epidermal papillomas from K6/ODC transgenic mice

In standard mouse strains, a high proportion (more than 90%) of epidermal tumors produced by initiation with 7,12‐dimethylbenz[a]anthracene and promotion with a variety of chemical agents contain an activating mutation in codon 61 (A182→T) of the c‐Ha‐ras gene. We analyzed the ras mutational spectra in 69 tumors induced by DMBA in a unique transgenic model, the K6/ODC mouse. In this model, low‐dose DMBA treatment is sufficient per se for tumor induction, so tumor promotion with chemical agents is not required. In contrast to previous studies in standard mouse strains, our study showed that less than 50% of epidermal tumors from K6/ODC mice contained an activating codon 61 c‐Ha‐ras mutation (A182→T). This result was obtained in mice initiated either as newborns (when the transgene is not expressed) or as adults (when the transgene is fully expressed). Analysis of other codon hot‐spots and other ras genes revealed the presence of three codon 12 and 20 codon 61 (A182→T) mutations in the c‐Ki‐ras gene in the 36 tumors that did not have c‐Ha‐ras mutations. We concluded that promotion in this model, by means of constitutive ornithine decarboxylase expression, causes the clonal expansion of a population of initiated cells not promoted by chemical agents. Mol. Carcinog. 22:145–149, 1998.

Inhibition of adipose conversion of BALB/c 3T3 cells by interferon and 12-O-tetradecanoylphorbol-13-acetate

The adipose conversion of BALB/c 3T3 preadipose cells is inhibited by interferon; this inhibition is directly correlation with the interferon concentration. In cultures treated with low doses of interferon and the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, another inhibitor of adipose conversion (Diamond et al., 1977), the two compounds act synergistically to block differentiation. Several lines of evidence suggest that the compounds differ in the mechanism by which they inhibit adipose conversion.