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[Cancer Research 65, 572-578, January 15, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Induction of Ornithine Decarboxylase Activity Is a Necessary Step for Mitogen-Activated Protein Kinase Kinase–Induced Skin Tumorigenesis

David J. Feith1, David K. Bol2, Joan M. Carboni2, Mark J. Lynch2, Suzanne Sass-Kuhn1, Paula L. Shoop1 and Lisa M. Shantz1

1 Department of Cellular and Molecular Physiology, The Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania and 2 Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey

Requests for reprints: Lisa M. Shantz, Department of Cellular and Molecular Physiology H166, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033. E-mail: lms17{at}psu.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A transgenic mouse line overexpressing a constitutively active mutant of MEK1, a downstream effector of Ras, driven by the keratin 14 (K14) promoter, has been used to test the hypothesis that ornithine decarboxylase (ODC) induction during tumor promotion following a single initiating event [i.e., the activation of the Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway], is a necessary step in skin carcinogenesis. K14-MEK mice exhibit moderate hyperplasia, with spontaneous skin tumor development within 5 weeks of birth. Analysis of epidermis and dermis showed induction of MEK protein and ERK1/ERK2 phosphorylation, but no change in Akt-1, suggesting that the PI 3-kinase pathway, another pathway downstream of ras, is not activated. Examination of tumors revealed high levels of ODC protein and activity, indicating that activation of signaling cascades dependent on MEK activity is a sufficient stimulus for ODC induction. When K14-MEK mice were given {alpha}-difluoromethylornithine (DFMO), a suicide inactivator of ODC, in the drinking water from birth, there was a dramatic delay in the onset of tumor growth (~6 weeks), and only 25% of DFMO-treated mice developed tumors by 15 weeks of age. All untreated K14-MEK mice developed tumors by 6 weeks of age. Treatment of tumor-bearing mice with DFMO reduced both tumor size and tumor number within several weeks. Tumor regression was the result of both inhibition of proliferation and increased apoptosis in tumors. The results establish ODC activation as an important component of the Raf/MEK/ERK pathway, and identify K14-MEK mice as a valuable model with which to study the regulation of ODC in ras carcinogenesis.

Key Words: ornithine decarboxylase • MEK • ras • skin tumorigenesis • transgenic mice


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The vast majority of skin tumors arising from the classic 7,12-dimethylbenz(a)anthracene/tetradecanoyl phorbol acetate mouse skin tumorigenesis protocol possess an A-T transversion at codon 61 of Ha-ras, resulting in a constitutively activated Ras protein (1) that is thought to be the result of a direct interaction between Ha-ras and the initiating carcinogen (2). Thus, activation of ras is an essential step in tumor initiation. Ras is a crucial component of receptor-mediated signal transduction pathways regulating growth and differentiation, and constitutively active Ras mutants have been implicated in many types of cancer.

Ornithine decarboxylase (ODC), the first enzyme in the biosynthesis of cellular polyamines, is dramatically induced in response to ras activation in a variety of in vitro models (3, 4), and blocking ODC activity reverts the transformed phenotype of cells overexpressing ras (5). Induction of ODC is known to occur in response to tetradecanoyl phorbol acetate treatment in 7,12-dimethylbenz(a)anthracene/tetradecanoyl phorbol acetate models of skin tumorigenesis, and ODC is constitutively elevated in the resulting skin tumors (6, 7). Transgenic mice overexpressing ODC in hair follicle keratinocytes (K6-ODC mice and K5-ODC mice) were shown to be much more sensitive than littermate controls to 7,12-dimethylbenz(a)anthracene–induced carcinogenesis and did not require treatment with a tumor promoter to develop tumors, suggesting that ODC overexpression is a sufficient promoting stimulus in this model (8). Double transgenic mice targeting ODC overexpression to the hair follicles in conjunction with an activated v-Ras protein (K6-ODC/Ras mice) develop spontaneous skin carcinomas without the need for initiation or promotion (9).

Of the known Ras effector pathways, the Raf/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) cascade is the most extensively characterized and is thought to be necessary for the transforming activity of Ras (reviewed in ref. 10). When ras is activated by mutation, it is hypothesized that all downstream components of this pathway are elevated in activity, mimicking the constitutive activation of growth factor receptors, and corresponding aberrant expression of genes that drive proliferation. We have shown that ODC is a target of the Raf/MEK/ERK pathway in vitro, and this pathway acts to regulate ODC by controlling the transcription of ODC RNA and its translation into protein (11).

In order to ascertain a difference between tumorigenesis mediated by activated Ras and by downstream elements of the Ras signal transduction cascade, the experiments described here use a transgenic mouse line that expresses a constitutively active mutant of MEK1 (S218D) under control of the keratin 14 (K14) promoter (K14-MEK mice), resulting in overexpression of MEK in the outer root sheath of the hair follicle and the basal cell layer of the interfollicular epidermis. Mice predisposed to spontaneous skin tumors have also been generated using Ras transgene overexpression driven by keratin promoters targeting either the interfollicular epidermis (12, 13) or the basal layer of the epidermis (14). Those studies confirmed that mutations in ras are sufficient to provide the initiating event leading to tumors in this model system. Similar evidence is lacking, however, for the tumorigenic potential of components of signal transduction cascades that connect the membrane-localized Ras with nuclear transcription effectors. Use of the K14-MEK mice thus allows in vivo studies that isolate an important Ras effector pathway and provides a model to examine both the in vivo effects of an activated MEK protein kinase on proliferation and transformation in mouse epidermis and the role of ODC in this process.

The results show that K14-MEK mice exhibit moderate hyperplasia and spontaneous skin tumor development within 5 weeks of birth. Molecular analysis of the tumors from these mice reveals no mutations in either K-ras or H-ras, suggesting no other Ras effector pathways are activated, and unregulated activation of MEK is a potent oncogenic stimulus. To test the hypothesis that ODC induction following the activation of this Ras-controlled pathway is a necessary component in the multistep process of skin tumorigenesis, we have inhibited ODC in the K14-MEK mice using {alpha}-difluoromethylornithine (DFMO). Inhibition of ODC from birth dramatically reduces tumor incidence and tumor burden, and reduction of ODC activity in established tumors causes rapid regression with reduced proliferation and increased apoptosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
DNA Construct Engineering. DNA manipulations for the cloning and expression of MEK were done by standard molecular cloning techniques. The cDNA for human MEK [MKK1a, ATCC 79572(32)] was obtained from the American Type Culture Collection, Manassas, VA. Wild-type human MEK cDNA was mutated at residue 218 by oligonucleotide-directed mutagenesis designed to change the serine to aspartic acid, resulting in the introduction of a negatively charged amino acid that would mimic the phosphorylation and activation of the normal MEK protein (15, 16). The coding region for MEK(S218D) was subcloned into the mammalian expression vector pZipNeo(SVX) (17) and the transforming activity of the cDNA was confirmed by focus forming assay on Rat-1 cells (data not shown).

Once cell-based MEK hyperactivity was confirmed, the mutated MEK cDNA was subcloned into the transgene vector. To create that construct, a 2.27 kb HindIII/SpeI fragment containing the regulatory elements of the human K14 gene necessary for tissue specific expression (18) was cloned into the Bluescript KS vector (Stratagene, La Jolla, CA). Subsequently, a BamHI/SacII cassette containing rabbit ß-globin intron and Poly(A) signal sequences (19) was directionally inserted 3' of the K14 promoter to make pK14.197. Transgene constructs were made by subcloning the MEKS218D cDNA into the SnaB1 site on pK14.197, placing the activated MEK cDNA between the ß-globin intron and polyadenylation sequences. The orientation and integrity of the insert were determined by sequencing.

Identification of Transgenic Mice. DNA was microinjected into the pronucleus of one cell fertilized B6D2F2 embryos. After incubation overnight, two cell embryos were transferred to the oviducts of pseudopregnant ICR female mice for full-term gestation. Matings of the founder animal for further characterization were on the ICR background. Genomic DNA was isolated from the tails of potential transgenic mice and subjected to PCR analysis to identify mice bearing the transgene. Oligonucleotides for PCR analysis were 5'-GCAAAGAATTCGCGGCCGCCTCGA-3', complimentary to the ß-globin intron, and 5'-GCTCCCTTATGATCTGGTTCCGGATTG-3', specific for the MEK cDNA. The amplified 490-bp product was detected only in mice bearing the transgene. The transgene was maintained in the heterozygous state by breeding of heterozygous males with ICR females (Charles River Laboratories, Wilmington, MA).

Tumorigenesis Experiments. The number of spontaneous tumors that developed (beginning at 5-6 weeks) was counted at weekly intervals. DFMO (1% w/v; ILEX Oncology, San Antonio, TX) was given in the drinking water. Tumors were measured with calipers and tumor volume was calculated as follows: cm3 = length x (width)2 x 0.52 (20). All animal protocols were approved by the Animal Care and Use Committee of the Pennsylvania State University College of Medicine.

Biochemical Analyses. ODC was assayed at 37°C by measuring the release of 14CO2 from L-[1-14C] ornithine (21). Samples were acid extracted using 10% TCA and analyzed for polyamines using reverse phase high-performance liquid chromatography analysis (22).

Isolation of Dermis and Epidermis. Dermis and epidermis were harvested and flash-frozen as described previously (23). The samples were resuspended in radioimmunoprecipitation assay buffer [20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L ß-glycerol phosphate, 1 mmol/L Na3VO4, 1 µg/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride] for subsequent analysis. Epidermal samples were sonicated on ice for 30 seconds. Dermal samples were homogenized for 30 seconds on ice using a Polytron homogenizer. When tumors were harvested, they were homogenized in the same manner as dermal samples. All samples were centrifuged at 30,000 x g for 30 minutes at 4°C. Each supernatant sample was assayed in duplicate for ODC activity and total protein. ODC protein was detected by Western blot using a purified rabbit polyclonal antibody against mouse ODC (23), and quantitated using a FluorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). MEK, phosphorylated and total ERK1/ERK2, and phosphorylated and total Akt1 were detected using 1:1,000 dilutions of rabbit polyclonal antibodies (Cell Signaling Technology, Beverly, MA) and quantitated using a chemiluminescent detection system (Cell Signaling Technology). Tumor samples were also analyzed for expression of cleaved poly ADP ribose polymerase (PARP) using a rabbit polyclonal antibody that recognizes both uncleaved PARP (112-kDa) and its 85-kDa cleaved product (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA).

Immunohistochemical Analysis. Tissues were fixed overnight in 10% neutral buffered formalin, embedded in paraffin and 5-µm sections were cut for immunohistochemistry. H&E staining of samples was done by routine methods. For determination of cell proliferation, mice were injected i.p. with 100 mg/kg bromodeoxyuridine (BrdUrd) and sacrificed 2 hours later. Paraffin sections were stained for BrdUrd using the In situ BrdUrd kit (BD Biosciences Clontech, Palo Alto, CA). The 3'-OH end labeling of apoptotic cell DNA was done by using an ApopTag Plus in situ peroxidase detection kit (Chemicon, Purchase, NY). Tumor differentiation was evaluated using an anti-keratin 1 antibody (BAbCo, Richmond, CA). Phosphorylated ERK was detected in tissue sections using an antibody specific for ERK1/ERK2 phosphorylated at Thr202/Tyr204 (Cell Signaling Technology).

Statistical Analysis. Tumor counts taken over a time course were compared using two-way ANOVA. Tumor incidence data were compared using a log-rank test of the curves.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Phenotypic Characteristics of Skin and Spontaneous Tumors in K14-MEK Mice. Expression of the activated MEK mutant MEKS218D targeted to the basal layer of the epidermis of transgenic mice was achieved using the human K14 gene promoter to generate the K14-MEK transgene. Eight founder mice were identified by genotyping DNA from tail tissue from both live mice and those found dead as a result of transgene expression. Of the eight confirmed positive founder animals, only two mice survived to adulthood, one of which showed no obvious skin abnormalities. Phenotypic characteristics distinguished seven of the eight positive founder mice from nontransgenic littermates and were displayed as wrinkled, flaky, or hyperkeratotic skin within 3 days of birth. Five of the founders were dead by 8 days of age and were confirmed by genotyping postmortem to carry the K14-MEK transgene. All founders that died at early ages were found by Northern blot analysis to have very high expression of the transgene (data not shown). One positive mouse, founder 66, survived only to 30 days of age and was observed to have extremely hyperplastic skin and lesions typical of ulcerative, invasive carcinomas of the epidermis (data not shown). The remaining phenotypic founder (number 6) showed moderately wrinkled, flaky skin by 3 days of age and survived to pass the transgene in a Mendelian fashion to offspring. Further analysis of the oncogenic potential of activated MEKS218D was done using the offspring from this animal.

Histologic Characteristics of Constitutive MEK Activation in Mouse Epidermis. Skin from line 6 K14-MEK adult mice was moderately hyperplastic compared with control skin (Fig. 1A and B). Tumors began to develop spontaneously at about 5 weeks of age. Transgenic lesions appeared as normal exophytic papillomas that arose in all locations on the mouse skin and were similar in pathologic appearance to papillomas generated by initiation and promotion protocols in the two-stage mouse skin model (Fig. 1C). In full-thickness wounding experiments, only 1 in 10 transgenic mice formed tumors at the wound site (data not shown), suggesting that the events leading to tumorigenesis in these mice are unrelated to the induction of growth factors produced during wounding (13, 24). The most common sites of tumor development were the crown of the head, directly between the ears, and immediately beneath the front legs. Whereas patches of thickened skin and hyperkeratosis were obvious on the tails of a significant proportion of these mice, the site of tail and toe clippings (used for genotyping) or ear punches used for identification rarely gave rise to tumors. This pattern of tumor development is similar to that observed in mice expressing the ras oncogene driven by a truncated K5 promoter (14).



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  		Figure 1. Histological analysis of skin and tumors from K14-MEK transgenic mice. H&E stained sections of skin and tumors from offspring of founder 6 revealed (A) moderate hyperplasia of the epidermis in K14-MEK mice compared with (B) nontransgenic littermates. C, typical keratinocyte-derived exophytic lesions of the skin in K14-MEK mice, similar to those seen as a result of two stage initiation-promotion tumorigenesis treatment regimens. D, tumors were evaluated at 9 weeks of age for Keratin 1 expression as a marker of differentiation.

 
Tumor incidence in K14-MEK mice was 100%, but no tumors were observed to progress to carcinomas up to about 20 weeks of age. Most mice were sacrificed before this age due to large tumor burdens. Tumor differentiation was analyzed in K14-MEK mice by immunohistochemical analysis of Keratin 1 expression. Mouse skin papillomas with a high risk for malignant conversion have reduced Keratin 1 and 10 expression (25). Figure 1D shows that tumors from K14-MEK mice strongly express Keratin 1, indicating well-differentiated papillomas.

Large chronic wound sites developed on the dorsal skin of line 6 animals that were never observed in control littermates. At any given time, as many as 10% of the mice in the colony can be seen carrying these lesions. These abnormal patches of skin were characterized by highly hyperplastic epithelia at the edges, typical of newly formed full thickness wounds, as well as areas totally lacking epidermis (data not shown). Very high levels of infiltrated inflammatory cells were observed in the dermis of these lesions, contributing to the shiny, weeping appearance of the gross lesion. Whereas the initiation of these lesions is presumed to be a wounding stimulus, these lesions were progressive, with no observed healing noted.

Biochemical Characterization of Skin and Tumors from K14-MEK Mice. Epidermis and dermis from non tumor-bearing skin of K14-MEK mice and littermate controls (8-10 weeks old) analyzed by Western blot showed an induction of MEK protein in both the epidermis and dermis of K14-MEK mice (Fig. 2A). This is consistent with the expression pattern of the K14 promoter, which directs expression to the basal layer of the epidermis and the bulge region of the hair follicle, which resides in the dermal fraction of these samples (25). MEK protein was also induced in tumors from K14-MEK mice (Fig. 2A). The increased levels of MEK in the transgenic dermis and tumors were accompanied by an increased phosphorylation of ERK1/ERK2 (Fig. 2B). The ratio of phosphorylated ERK1/2 to total ERK1/2 protein is lower in the tumor samples than in epidermis or dermis from K14-MEK mice (Fig 2B). However, extensive phosphorylation of ERK in tumors was confirmed by immunohistochemical analysis, which revealed large patches of cells that were heavily stained in both in the nucleus and cytoplasm (Fig. 2C). Phosphorylation of Akt-1, which is induced by Ras but not by MEK, and can also act to induce ODC, was not changed in skin or tumors (data not shown).



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  		Figure 2. Skin and tumor expression of MEK, ERK1/2, and ODC. Mice were sacrificed and epidermis, dermis, and tumors were harvested as described in Materials and Methods. Proteins were separated by SDS-PAGE and expression of MEK, ERK1/2, and ODC was analyzed by Western blot. A, MEK protein expression in nontumor-bearing skin from K14-MEK mice and littermate controls and in tumors from K14-MEK mice. For each sample, 100 µg of protein were used. Each band is expressed as a ratio of its density to that of control epidermis, which was assigned a density equal to 1. Skin samples were from two transgenic and two control mice and a representative tumor sample from a transgenic mouse. B, MEK activity assayed in skin and tumors using phospho-specific antibodies for ERK 1/2. For each sample, 30 µg of protein were used. The blots were stripped and reblotted with an antibody specific for total ERK 1/2. Blots were quantitated by dividing the absorbance of phosphorylated ERK1/2 by total ERK1/2. Skin and tumor samples were from three separate mice in each group. C, phosphorylation of ERK1/2 in tumors from K14-MEK mice. Paraffin sections of tumors were processed as described in Materials and Methods. Similar patterns of staining were found in five separate K14-MEK tumors isolated from different mice. D, ODC protein expression and activity in tumors from K14-MEK mice. For each sample, 100 µg of protein were used. ODC activity was assayed in tumor extracts as described in Materials and Methods. Tumors were from two separate mice. ODC protein and activity were undetectable in skin of K14-MEK mice and littermate controls.

 
Western blot analysis and activity assays showed no measurable ODC activity in the epidermal and dermal fractions from non tumor-bearing skin of K14-MEK mice. Examination of tumors from 10-week-old K14-MEK mice revealed high levels of both ODC protein and activity (Fig. 2D). This is consistent with the expression of MEK and corresponding induction of ODC in a limited population of cells in the hair follicle, which are then expanded as the tumors develop.

Analysis of Tumor Development in Mice with Reduced ODC Activity. To analyze whether inhibition of ODC activity with a specific inactivator of the enzyme would have a chemopreventive effect, K14-MEK mice were given DFMO in the drinking water from birth. DFMO was supplied in the drinking water of nursing females from the day the pups were born and then maintained in the drinking water after weaning. When ODC inhibition was present from birth, there was a dramatic delay in the onset of tumor growth (by ~6 weeks) in K14-MEK mice compared with water-drinking littermates, and only 25% of DFMO-treated mice developed tumors by 15 weeks of age. All untreated K14-MEK mice developed tumors by 6 weeks (Fig. 3A). In addition, the number of tumors per mouse was dramatically reduced by DFMO administration (Fig. 3B), and those tumors that did form were much smaller than in their untreated littermates. Treatment with DFMO from birth did not alter the pattern of Keratin 1 expression in K14-MEK tumors (data not shown). To quantitate the difference in tumor size, tumors were counted and tumor volume was measured weekly. In 9-week-old K14-MEK mice, 18 of 28 tumors (n = 6 mice) were 30 mm3 or larger, with five measuring >100 mm3 in volume. Mice receiving DFMO were analyzed at 15 weeks, when only 3 of 9 tumors (n = 13 mice) were 30 mm3 or larger.



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  		Figure 3. Formation of spontaneous tumors in untreated K14-MEK mice and in K14-MEK mice treated with DFMO from birth. Tumor incidence (A) and multiplicity (B) were determined for untreated mice ({bullet}, n = 13) and mice treated with 1% DFMO from birth ({blacksquare}, n = 8). Points, mean for each time point; bars, ±SD. P < 0.0001 for both tumor incidence and multiplicity.

 
Effect of ODC Inhibition on Tumor Maintenance in K14-MEK Mice. To determine the effect of reducing ODC activity on tumor maintenance, 10-week-old tumor-bearing mice (n = 5) were treated with 1% DFMO in the drinking water. Treatment with DFMO rapidly reduced tumor size compared with untreated mice. Figure 4 shows a representative 10-week old K14-MEK mouse (A) and the same mouse after 7 weeks of treatment with DFMO (B). Similar results were obtained with all treated mice.



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  		Figure 4. Regression of K14-MEK tumors with DFMO treatment. The same K14-MEK mouse is shown (10 weeks old on day 1 of DFMO treatment). A, no treatment; B, 7 weeks DFMO (1% w/v in drinking water).

 
To establish whether inhibition of ODC with DFMO decreases cell proliferation in K14-MEK tumors, tumor-bearing mice were given DFMO in the drinking water from ages 16 to 20 weeks. Mice were injected with BrdUrd, sacrificed 2 hours later, and tumors were analyzed for S-phase cells (Fig. 5A and B). Analysis shows that DFMO treatment results in a dramatic reduction in proliferation in established tumors derived from K14-MEK mice.



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  		Figure 5. DNA synthesis and apoptosis in K14-MEK tumors with and without DFMO treatment. Tumor sections from K14-MEK mice that received no treatment (A and C) or 1% DFMO in the drinking water from age 16 to 20 weeks (B and D) were analyzed. Arrows, representative positive cells. A and B, mice were injected with BrdUrd (100 mg/kg, i.p.) 2 hours before sacrifice and proliferating cells were stained with an anti-BrdUrd antibody as described in Materials and Methods. Results are representative of multiple tumors taken from three separate mice in each group. C and D, apoptotic cells were visualized by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay as described in Materials and Methods. Results are representative of multiple tumors taken from three separate mice in each group. E, Cleavage of PARP was measured by Western blot analysis as an independent test of apoptosis as described in Materials and Methods. Each tumor sample is from a separate mouse.

 
TUNEL assays were done as an indication of apoptosis in established tumors from K14-MEK mice with and without 4 weeks of DFMO treatment (Fig. 5C and D). The results show a clear increase in apoptosis in tumors from DFMO-treated mice, especially in the basal cell layer. Western blots were done to examine cleavage of PARP, another marker of apoptosis, in tumor extracts from K14-MEK mice that had received 1% DFMO or water. Activated caspase-3 cleaves the 112-kDa PARP protein to an 85-kDa fragment and a 25-kDa fragment. Consistent with the TUNEL results, an increase in the fraction of cleaved PARP protein (85-kDa fragment) was found in the MEK/DFMO tumors compared with tumors from MEK mice that had not received DFMO (Fig. 5E). Phosphorylation of ERK1/ERK2 as measured by immunohistochemical analysis of tumors was not affected by DFMO treatment, demonstrating that DFMO does not inhibit MEK activity directly (data not shown).

The increase in apoptosis and decrease in cell division in tumors from mice exposed to DFMO was accompanied by a 75% decrease in tumor putrescine content compared with mice drinking water alone (Table 1). These results are consistent with previous studies using K5-ODC mice, pointing to putrescine levels as an important regulator of tumor growth (8). Both spermidine and spermine levels were increased in mice receiving DFMO (Table 1).


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  		Table 1. Polyamine levels in tumors from K14-MEK mice with and without DFMO treatment

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The current studies extend previous results by showing for the first time that activation of a single pathway downstream of Ras (Raf/MEK/ERK) can lead to spontaneous skin tumorigenesis, and that induction of ODC is necessary for this process. Two recent studies using transgenic mice with inducible expression of either Raf or another constitutively active MEK1 mutant (R4F) in the skin of adult mice reported increased proliferation and decreased differentiation in the epidermis, but used a relatively short time frame of transgene induction and did not address tumorigenesis (26, 27). Although introduction of the S218D MEK1 mutant into Rat-1 fibroblasts in cell culture resulted in a transformed phenotype as measured by focus formation assay, a S218D/S222D double mutant (data not shown) or NH2-terminal deletion mutants (28, 29) showed greater transforming potential in vitro. We show here that constitutive expression of MEK S218D showed highly transforming properties in vivo. Transgenic mice expressing this MEK mutant from the K14 promoter frequently died within several days of birth. Histologic analysis confirmed the transforming potential of the mutated MEK, as neonates that succumbed to the lethality of this vector were found to have carcinomas as early as 6 days of age. Indeed, those that died were all found to have very high expression of the K14-MEK transgene. One line (line 6), used in the current experiments, expressed relatively low levels of the transgene that resulted in a viable phenotype, including mosaic areas of hyperplasia and hyperkeratosis and spontaneous tumorigenesis. A modest change in MEK protein was observed in the skin of these mice, accompanied by a small increase in ERK phosphorylation. Tumors that develop in line 6 animals show an ~4-fold increase in expression of the MEK protein and also high levels of ODC protein but fail to progress to advanced carcinomas.

The tumors that arise in K14-MEK mice are spontaneous and do not depend on wounding or tumor promotion. The focal papillomas observed arising from hyperplastic patches in line 6 K14-MEK transgenic mice showed no mutations in H-ras or K-ras (data not shown). These results suggest that sustained up-regulation of pathways controlled by MEK activation can provide both an initiating and a promoting stimulus when expression is directed to the outer root sheath of the hair follicle, where target cells for the formation of skin papillomas and carcinomas are postulated to reside (14). When expressing oncogenes in the skin, the cell type targeted is thought to be the critical determinant of both tumor development and tumor type (14). Mice predisposed to spontaneous tumors have also been generated in other models by directing transgene expression to the outer root sheath using either the K14 promoter or its partner K5, including mice with overexpression of a mutant H-ras, insulin-like growth factor-1, HPV16, or erbB2 (14, 30–32).

The experiments described here use the K14-MEK mice as a model to study the importance of the observed ODC induction in skin tumorigenesis brought about by activation of this Ras effector pathway. Treatment of K14-MEK mice with DFMO from birth represents a chemopreventive approach to ODC inhibition, which results in life-long reduction of ODC activity in mice that are genetically predisposed to spontaneous skin tumor development. Treatment with DFMO after tumors have formed examines the role of continued high ODC activity in tumor maintenance. Therefore, this combined transgenic and pharmacologic approach provides valuable information on the role ODC plays in ras carcinogenesis and on the validity of ODC as a target for cancer therapy and prevention. The results show clearly that inhibition of ODC both inhibits tumor formation and causes tumor regression in K14-MEK mice.

In other skin tumorigenesis models, ODC induction was observed in the epidermis of K14-HPV16 mice (31) and of K14-PKC{varepsilon} mice (33). Treatment of mice with DFMO had a chemopreventive effect in both of these models. Squamous cell carcinomas or spindle cell carcinomas were present in both of these models, as well as in K6-ODC/Ras mice (9), and carcinoma formation was also prevented or reversed by DFMO. No conversion to carcinomas was observed in K14-MEK mice in the current studies. However, the need to sacrifice many mice at young ages due to large tumor burden makes analysis of malignant conversion difficult. In addition, several original founders were observed to have carcinomas at the time of death, confirming that expression of MEK from this promoter is capable of producing carcinomas.

The mechanism of tumor regression upon inhibition of ODC with DFMO has been analyzed in several transgenic carcinogenesis models. DFMO stimulated apoptosis in tumors from transgenic K6-ODC/Ras mice, suggesting that polyamines play a role in epithelial cell survival (9). DFMO administration did not reduce proliferation in K6-ODC/Ras tumors. In contrast, DFMO did not induce apoptosis in tumors from K5-ODC mice treated with 7,12-dimethylbenz(a)anthracene, but reduced proliferation (8). The current results show that in K14-MEK mice, DFMO both reduced cell proliferation and increased apoptosis in tumors. These results perhaps reflect the difference in polyamine profiles that result from transgenic overexpression of ODC versus the more modest endogenous overexpression of ODC that occurs in response to an upstream stimulus during tumor formation induced by Ras or MEK. Treatment of K14-MEK mice with DFMO caused an increase in the ratio of both spermine to spermidine and spermine to putrescine in tumors, whereas neither spermidine nor spermine was changed in transgenic ODC mice treated with DFMO (8). Our results agree more closely with recent experiments using the zinc-deficient mouse model of forestomach carcinogenesis, in which administration of DFMO reversed cell proliferation and counteracted N-nitrosomethylbenzylamine tumor initiation by stimulating apoptosis (34). Changes in polyamines have been associated with changes in cell proliferation and apoptosis using in vitro models as well (35, 36).

In summary, mice overexpressing a constitutively active mutant of MEK in the skin have been generated and characterized. These mice form spontaneous skin tumors without the need for chemical initiation or promotion, showing for the first time that tumorigenesis can be driven by downstream effectors of Ras. These studies also emphasize the importance of the Raf/MEK/ERK signal transduction pathway in Ras tumorigenesis and provide a direct link between MEK activation and ODC induction. The K14-MEK mice provide a valuable model in which to study the changes in ODC activity and polyamine levels that are associated with constitutive activation of this pathway and the mechanism by which ODC induction promotes tumorigenesis. Inhibition of ODC with DFMO resulted in both a decrease in proliferation and an increase in apoptosis in K14-MEK–derived tumors. These results add to our understanding of ODC as a downstream target for Ras, and stress the importance of current chemoprevention trials of DFMO in skin carcinogenesis (37).


   Acknowledgments
 
Grant support: National Cancer Institute grant CA-82768 (L.M. Shantz).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Louise Y.Y. Fong for help with the TUNEL assays and Krisna Duong-Ly for performing the Keratin 1 analysis.


   Footnotes
 
Note: D.K. Bol is currently at Avalon Pharmaceuticals, Germantown, MD and M.J. Lynch is currently at Bayer Healthcare, Pharmaceuticals Division, West Haven, CT.

Received 10/ 5/04. Revised 11/ 2/04. Accepted 11/17/04.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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