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[Cancer Research 66, 2700-2707, March 1, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

p44 Mitogen-Activated Protein Kinase (Extracellular Signal-Regulated Kinase 1)–Dependent Signaling Contributes to Epithelial Skin Carcinogenesis

Christine Bourcier1, Arnaud Jacquel2, Jochen Hess5, Isabelle Peyrottes4, Peter Angel5, Paul Hofman3, Patrick Auberger2, Jacques Pouysségur1 and Gilles Pagès1

1 Institute of Signaling, Developmental Biology and Cancer Research, UMR Centre National de la Recherche Scientifique; 2 Unité Institut National de la Sante et de la Recherche Medicale, Equipe labellisée Ligue Nationale contre le Cancer; 3 Equipe Institut National de la Sante et de la Recherche Medicale and Laboratory of Clinical and Experimental Pathology, Faculté de Médecine, University of Nice-Sophia Antipolis; 4 Service Anatomo Pathologie Centre Antoine Lacassagne, Nice, France; and 5 Division of Signal Transduction and Growth Control, Deutsches Krebsforschungszentrum, Heidelberg, Germany

Requests for reprints: Gilles Pagès, Institute of Signaling, Developmental Biology and Cancer Research UMR Centre National de la Recherche Scientifique 6543, University of Nice-Sophia Antipolis, 33 Avenue de Valombrose, Nice, France 06189. Phone: 33-492-03-1237; Fax: 33-492-03-1235; E-mail: gpages{at}unice.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Extracellular signal-regulated kinases (ERK) regulate cellular functions in response to a variety of external signals. However, the specific functions of individual ERK isoforms are largely unknown. Hence, we have investigated the specific function of ERK1 in skin homeostasis and tumorigenesis in ERK1 knockout mice. They spontaneously develop cutaneous lesions and hyperkeratosis with epidermis thickness. Skin hyperproliferation and inflammation induced by application of 12-O-tetradecanoylphorbol-13-acetate (TPA) is strongly reduced in mutant mice. ERK1–/– mice are resistant to development of skin papillomas induced by 7,12-dimethylbenz(a)anthracene (DMBA) and promoted by TPA. Tumor appearance was delayed, their formation was less frequent, and their number and size were reduced. Keratinocytes obtained from knockout mice showed reduced growth and resistance to apoptotic signals, accompanied by an impaired expression of genes implicated in growth control and invasiveness. These results highlight the importance of ERK1 in skin homeostasis and in the process of skin tumor development. (Cancer Res 2006; 66(5): 2700-7)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The p42/p44 mitogen-activated protein kinases [MAPK; extracellular signal-regulated kinase 2/1 (ERK2/1)] are implicated in a variety of biological processes, including cell proliferation, differentiation, and apoptosis. Therefore, both enzymes are considered attractive therapeutic targets for cancer therapy. Effectively, a drug targeting MAP/ERK kinase activity decreases the incidence of colon carcinoma in vivo (1). Moreover, inactivation of ERK pathway using the MAPK phosphatase 3 reverts the Ras-transformed phenotype (2). Although ERK2–/– mice are embryonic lethal (3), ERK1–/– mice are viable, fertile, and of normal size, which argues for a unique role of each isoform or a threshold of total ERK activity for normal viability (4). However, ERK1–/– thymocyte maturation and proliferation, in response to activation with TCR and phorbol esters, are severely reduced. ERK1–/– mice also show an unexpected enhancement of striatum-dependent long-term memory (5). Mice lacking ERK1 also have decreased adiposity and fewer adipocytes than wild-type animals and are resistant to obesity induced by high fat diet (6). These results indicate that ERK1 is implicated in specific biological functions, in particular in highly specialized organs. To confirm this hypothesis, we have specifically focused on the role of ERK1 activity in the skin, a tissue where the ERKs seem to be particularly fundamental (7, 8). Another goal of our study was to define whether ERK activity was required for the development of skin papilloma induced by the classic skin carcinogenesis protocol. Hence, the objective of our study was to show, for the first time in integrated animal model, the importance of ERK1 in the skin homeostasis and in the process of skin tumor formation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tumor induction experiments. Male ERK1–/– mice were generated as reported previously (4). Both wild-type and knockout animal were used for the skin carcinogenesis protocol. The number of mice for each experimental group was 8 to 10. The experiment was repeated twice. For tumor initiation, a single 200 nmol dose of 7,12-dimethylbenz(a)anthracene (DMBA; Sigma Chemical, St. Louis, MO) in 0.2 mL of acetone was applied topically to the shaved backs of the mice. Two weeks after initiation, 17 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA; Sigma Chemical) in 0.2 mL of acetone was applied twice weekly to the skin for the duration of the experiment. The tumor incidence and burden were observed each week starting at 5 weeks of TPA promotion. The papilloma incidence is expressed as the percentage of mice bearing one or more papillomas, and the tumor burden is expressed as the number of tumors per surviving mouse in mice with tumors.

Histology. The tumor was excised promptly after euthanasia and placed in 10% formol. It was fixed for at least 1 hour in formol and then embedded in paraffin. Sections of 4 µm were cut for H&E staining. The same protocol was applied to skin samples.

Western blot analysis. The dorsal skin of the mice was shaved 24 hours before TPA treatment. The dorsal skin was removed and immediately placed on dry ice. Proteins from skin samples were obtained as previously described (9). Cell extracts were obtained by lysing the cells in Laemmli sample buffer. Protein extracts were resolved on a 12% SDS-PAGE gel and transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Bedford, MA). The levels of phosphorylated and total ERKs, which serve as loading control, were selectively measured by Western blot using specific antibodies (respectively from Zymed, South San Francisco, CA and Sigma, St. Louis, MO). Loading controls were also obtained by determining the presence of actin using a specific antibody from Sigma. The antibody-bound proteins were revealed using an enhanced chemiluminescence system (Amersham Biosciences, Piscataway, NJ). Quantification of the Western blot was done by using the MacBAS software. The total ERK activity was evaluated by adding the signals for phospho-ERK1 and phospho-ERK2 or phospho-ERK2 alone for knockout skin or knockout cells. These values were divided by the signals of ERK1 and ERK2 or ERK2 alone or the amounts of actin.

Statistics. Linear regression was used to determine the rate of tumor development between wild-type and ERK1–/– mice. For comparison between groups, one-way ANOVA was used to determine significant differences. P < 0.05 was considered significant.

Generation of keratinocytes cell lines and thymidine incorporation. Both wild-type and knockout keratinocytes were obtained from neonatal epidermis by CELLnTEC Advanced Cell Systems (Bern, Switzerland). They were cultured in specific keratinocyte medium supplied by the company (CnT-02). Both cell types were tested for normal expression of keratin 14, plakoglobin, and ß-catenin. For thymidine incorporation experiments, cells were seeded at 105 per 12-well culture plate. At confluency, cells were deprived of growth factors for 24 hours. They were then stimulated with the keratinocytes growth medium in the absence or presence of TPA (10 ng/mL) in a medium containing 0.25 µCi/mL and 3 µmol/L [methyl-3H]thymidine (Amersham, Arlington Heights, IL). After 24 hours of stimulation, radioactivity incorporated in acid-precipitable material was measured by liquid scintillation spectrometry. For cell growth experiments, cells were seeded at a concentration of 80,000 per six-well culture plate. Each day, cells were trypsinized and counted in a Coulter counter.

Preparation of RNA, Northern blots, and reverse transcription-PCR. Skin samples or cells were lysed in Trizol Reagent buffer (Life Technologies Bethesda Research Laboratories, Gaithersburg, MD). RNAs were prepared according to the manufacturer's protocol. Twenty micrograms of RNA were used for Northern blot analysis and hybridized with the different probes. c-Fos, Fra-1 probes were a generous gift from Dr. Jean Claude Chambard (CNRS UMR 6543, France). Calgranulin A8, A9 (10), and Bssp (11) cDNA were described previously. For Adam 8, tenascin C, CCL 27, and hypoxanthine phosphoribosyltransferase (HPRT) total cDNA was prepared by using a reverse transcription-PCR (RT-PCR) kit from Qiagen (Chatsworth, CA), using the following oligonucleotides for RT-PCR reactions: Adam 8 (5'-GGTCTTGGTGATCCTGGTGG-3', 5'-CCAGCTCCCTGAGTTGCTCC-3'); tenascin C (5'-CTGCTGGGTTTGGAGACCGC-3', 5'-GATTTCGGAAGTTGCTGGGTCTCAG-3'); CCL27 (5'-CTCTCCCCCGCCAGCAGCCTCCC-3', 5'-GAGTACATTTTCTTTTGTAG-3'); HPRT (5'-GCTGGTGAAAAGGACCTCT-3', 5'-CACAGGACTAGAACACCTGC-3').

Measurement of caspase activity. Cells were lysed as previously described (12). Each assay (in triplicate) was done with 50 µg of protein prepared from control cells or cells stimulated for 16 hours in the presence of staurosporine, or following UV irradiation (400 Mj/cm2 and lysed 16 hours after) or growth factor deprivation (16 hours) or stimulated by tumor necrosis factor-{alpha} (TNF-{alpha}; 10 nmol/L) or cycloheximide (10 mg/mL) or a combination of both factors for 16 hours. Briefly, cellular extracts were incubated in a 96-well plate with 0.2 mmol/L Ac-DEVD-pNa. Specific caspase activity was measured at 405 nm in the presence or the absence of 1 µmol/L Ac-DEVD-CHO. Caspase activity is expressed in nanomoles of paranitroaniline released per minute and per mg of protein. The same lysates were used in determining poly(ADP-ribose) polymerase (PARP) cleavage using a polyclonal antibody (sa-250, TEBU).

Bromodeoxyuridine labeling. Bromodeoxyuridine (BrdUrd) pulse labeling was done after 1 week of daily application of TPA. One hundred twenty minutes before the end of the experiment, BrdUrd (Sigma) was injected (50 mg/kg). The skin was fixed in formol, and immunohistochemistry was done with a monoclonal antibody against BrdUrd (Sigma). Skin from three mice (unstimulated conditions) or five mice (TPA treated) was analyzed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Spontaneous skin lesions in ERK1–/– mice. To focus on the implication of ERK in the skin homeostasis, we have carefully examined the structure of ERK1–/– mice skin. Surprisingly, the skin of adult ERK1–/– animals exhibited local cutaneous lesions associated with ortho-keratosis and para-keratosis as well as an increase in the thickness of the epidermis and dermis (Fig. 1A, a and a'). These lesions have been observed in different localizations (dorsal or ventral) and are associated with Escherichia coli infection and a discrete inflammatory infiltrate of mononuclear cells (lymphocytes and plasmocytes) in the skin (data not shown), which is probably caused by the immunologic defect that we described previously (4). No histologic lesions were observed in the spleen, the liver, the lungs, lymphatic nodes, the intestine, adipose tissue, the brain, and vessels (data not shown). To study the involvement of major cytokines implicated in skin inflammation, we tested mRNA levels of TNF-{alpha}, transforming growth factor-ß, interleukin-1ß (IL-1ß), IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-16, IL-17, and vascular endothelial growth factor-A. None of them was affected by inactivation of ERK1 except basal expression of CCL27, a cytokine implicated in skin inflammation in response to allergens or highly expressed within acute or chronic skin lesions of atopic dermatitis (Fig. 1B; ref. 13). These results suggest that ERK1 activity is indispensable for normal skin homeostasis.


Figure 1
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  		Figure 1. Histopathologic analysis of untreated or TPA-treated skin from wild-type (WT) or ERK1–/– mice. A, H&E-stained sections of wild-type and ERK1–/– mouse skin 7 days after topical application of acetone (cont) or 17 nmol TPA in 0.2 mL of acetone. Original magnification, x20. Arrow, representative abscess. Left, overall differences observed without any treatment. B, RT-PCR analysis of CCL-27 expression of mouse skin after 6 hours of acetone (–) or TPA application. Representative of three different skin samples. HPRT was monitored on the same samples and was used as an invariant gene, which serves as a loading control.

 
ERK1–/– mice are insensitive to TPA-induced skin inflammation. To induce ERK activation within the epidermis, we challenged the skin of wild-type and ERK1–/– animals with TPA (14, 15). In response to TPA, intense inflammatory lesions associated with a great number of apoptotic cells characterized by picnotic or fragmented nuclei (data not shown) appeared in wild-type animals. Such a dense inflammatory infiltrate destroyed the epidermis and formed numerous microabscesses with infiltration of the superficial dermis (Fig. 1A, d). Lesions in the knockout mice had a more focal localization and were less intense (Fig. 1A, b', c', and d') with a significant reduction of apoptotic cells (data not shown). Moreover, whereas expression of CCL27 was highly increased in the skin of wild-type animals, it already reached maximal levels under unchallenged conditions in the skin of ERK1–/– animals (Fig. 1B). Proliferation of basal keratinocytes, measured by BrdUrd incorporation, in unstimulated conditions, was low and comparable in wild-type or knockout animals (3% of labeled nuclei). However, in response to TPA, proliferation of basal keratinocytes was decreased in ERK1–/– mice (15% of labeled nuclei) compared with wild-type skin (30% of labeled nuclei).

ERK1 deficiency is not compensated by ERK2 in response to TPA on the skin of ERK1–/– mice. As sustained ERK activity is required for mitogenic potential, we measured total ERK activity (ERK1 + ERK2) in the skin of wild-type and ERK1–/– animals following TPA application by analyzing the levels of the phosphorylated forms of ERKs. Figure 2A-C shows that in knockout samples of untreated skin, the ERK2 isoform presents with a higher activity, which is likely to compensate for the loss of ERK1. However, following long-term TPA stimulation, total ERK activity in ERK1–/– mice skin is decreased by 50% compared with wild-type skin and is accompanied by a drastic down-regulation of the mRNA abundance of an indicative end point of ERK signaling, c-fos (Fig. 2D). This difference in total ERK activity is principally due to the absence of ERK1 because ERK2 activity in the skin of both animals is almost equivalent following TPA stimulation. This result highlights the fact that in this specific tissue, ERK2 cannot compensate for the loss of ERK1, which is different of the result obtained in thymocytes (4). Both reduced ERK activity and impaired expression of c-fos could be considered as molecular signs of a reduced proliferation capacity.


Figure 2
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  		Figure 2. A, time course of TPA-stimulated ERK1 and ERK2 activities in wild-type (WT) and ERK1-deficient skins. A, the dorsal skin of wild-type (wt) and ERK1–/– mice treated for the indicated times with acetone or acetone-TPA (17 nmol) were biopsied and the samples extracted in SDS sample buffer (see Materials and Methods). Equal amounts of protein were analysed by Western blotting using antibodies directed against phosphorylated ERKs or total ERKs (see Materials and Methods). B, quantification of the experiment shown in (A). ERK activity is presented as the ratio between the phospho-ERK signal and the signal for total ERK. Representative of three independent experiments. C, total ERK activity measured in skin samples of wild-type and ERK1–/– mice treated with acetone or TPA for 3 hours (17 nmol). Here again, ERK activity represents the ratio between the phospho-ERK signal and the signal for total ERK. The ERK activity was measured in three different skin samples treated or not for 3 hours with TPA. Columns, average; bars, SE. D, c-fos mRNA was detected by Northern blotting following 3 hours of TPA stimulation. For each condition, 15 µg of RNA from two independent skin samples were analyzed. Representative of three different experiments. 18S ribosomal RNA is shown as a loading control.

 
Keratinocytes of ERK1–/– mice present with a differential expression of genes implicated in growth control and invasiveness potential. To measure the intrinsic proliferation and apoptotic capacities of wild-type and ERK1–/– keratinocytes independent of TPA-induced inflammatory cytokines produced by fibroblasts and immune cells, we have derived keratinocytes cultures from wild-type and mutant mice. When cells were arrested in G0, mimicking unchallenged skin, basal ERK activity was slightly higher in ERK1–/– cells compared with wild-type cells (Fig. 3A). Following growth factor stimulation, total ERK activity remains higher in wild-type cells than in ERK1–/– keratinocytes throughout the time course of activation. Following TPA stimulation, total ERK activity is higher in wild-type cells after a short stimulation (5 minutes) but progressively declined to reach almost equivalent levels in cells of both genotypes after a long-term exposure (Fig. 3A and B). For growth factor or TPA stimulations, the difference in total ERK activity is principally due to the absence of ERK1 because ERK2 activity is equivalent in both cell types. Here again, ERK2 cannot compensate for the loss of ERK1. Under these conditions (TPA alone), in ERK1–/– cells, a down-regulation of many genes implicated in proliferation and invasion was observed, including c-Fos (as for total skin) and Fra-1 (Fig. 3C), which were already shown to be overexpressed in squamous cell carcinoma following TPA induction (16, 17). Moreover, Fra-1 accumulation is in part an ERK-mediated mechanism of transcriptional regulation (18). There is also in vivo evidence that ERK signaling and Fra-1 function are the major mediators of TPA-dependent neoplastic transformation in keratinocytes (19). The Calgranulin S100A8 and S100A9, which have already been shown to be stimulated by TPA, overexpressed all along multistage carcinogenesis and implicated in inflammatory processes, are also down-regulated in ERK1–/– keratinocytes following TPA treatment (Fig. 3C; ref. 10). Reduced induction of the metalloproteinase and disintegrin domain-containing transmembrane glycoproteins Adam 8 by TPA is also in favor of a reduction in the inflammatory process and in tumor invasiveness in ERK1–/– mice, as it was shown to be implicated in the degradation of the vascular basement membrane and in the shedding of active membrane bound molecules (20). As Adam 8 was reported to be involved in Fas ligand release from the membrane, decreased Adam 8 could be implicated in the reduction of keratinocyte apoptosis following TPA treatment (21). In fact, reduced apoptosis was also observed in primary keratinocytes in response to inflammation-associated signals (see below). Finally, altered expression was also observed for genes encoding extracellular matrix components. Tenascin C is a member of a family of large extracellular matrix proteins, which shows highest expression in connective tissue surrounding tumors, in wounds and in inflamed tissues, where it may regulate cell morphology, growth, and migration by activating diverse intracellular signaling pathways (22). Its impaired induction following TPA in knockout keratinocytes is also in accordance with reduced growth and invasive potential of mutant cells. The keratinocyte-specific serine protease Bssp, a protein highly expressed in hyperplastic epidermis and in malignant skin tumors is also down-regulated in ERK1–/– keratinocytes and could also explain the reduced size and delayed appearance of papillomas in ERK1–/– animals (see below; ref. 11). The expression of p21 mRNA, a gene implicated in the control of cell proliferation, is almost not affected by TPA.


Figure 3
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  		Figure 3. Keratinocytes obtained from wild-type or ERK1–/– mice show reduced ERK activity, reduced proliferation potential, and a different panel of gene expression. A, growth arrested keratinocytes were stimulated for the indicated times with keratinocytes growth medium or TPA (10 ng/mL). Phosphorylated or total forms of ERKs were detected by Western blotting. Actin was also detected by Western blotting and is shown as a loading control. B, quantification of the blots in (A). ERK activity corresponds to the ratio between phospho-ERK signal and the signal for actin. C, gene induction in wild-type or ERK1–/– keratinocytes following TPA stimulation. Wild-type or ERK1–/– keratinocytes were deprived of growth factors for 24 hours then stimulated for the indicated times with TPA. Expression of Adam 8, Calgranulin S100A8 (Cal A8), Calgranulin S100A9 (Cal A9), the cell cycle inhibitor p21 (p21), c-Fos, Tenascin C, Fos-related antigen 1 (Fra-1), and Brain and Skin–specific protease (Bssp) was determined by Northern blot using 15 µg of total RNA. 18S ribosomal RNA is shown as a loading control. D, the proliferation potential of wild-type or –/– keratinocytes. Top, both types of cells were seeded at a density of 80,000/20 cm2 at day 0 in triplicate. Each day, cells were trypsinized and counted for 4 days (1, 4) using a Coulter counter. Columns, average number of cells; bars, SE. Representative of three independent experiments. * and **, P < 0.05. Bottom, reinitiation of DNA synthesis in response to growth medium was measured as described in Materials and Methods. Fold increase compared with the basal values in absence of growth factors for wild-type or knockout (ko) keratinocytes. Representative of two independent experiments done in duplicate.

 
Impaired proliferation potential of ERK1–/– keratinocytes. As for total skin, a reduced proliferation capacity, evaluated by growth curves and thymidine incorporation, was also observed with keratinocytes obtained from knockout mice in normal growth medium, conditions mimicking TPA-challenged skin (presence of growth factors and cytokines; Fig. 3D). This result is in accordance with the fact that growth factors, compared with TPA, induced a more sustained ERK activity at long term stimulation (Fig. 3A-B). Curiously, TPA did not act as a mitogen on both cell types but rather decreased cell growth and thymidine incorporation induced by growth factors probably by desensitizing the PKC pathway (data not shown). One of the key genes regulated by the ERK pathway and responsible for the G1-S transition is cyclin D1 (23). In the presence or in the absence of growth factors, TPA stimulation up-regulates basal cyclin D1 expression in wild-type cells, whereas such induction is reduced in knockout cells (data not shown). This result indicates that eliminating ERK1 impairs the capacity to induce key genes implicated in growth control.

ERK1–/– keratinocytes are resistant to apoptosis. As we observed an impaired apoptosis rate in response to TPA in mutant mice, we evaluated the capacity of wild-type and knockout keratinocytes to undergo apoptosis in response to various apoptosis inducing stimuli. In agreement with the apoptosis response in vivo in total skin, ERK1–/– keratinocytes are poorly sensitive to UV irradiation, staurosporine, growth factor deprivation, and a combination of TNF-{alpha} plus cycloheximide as measured by the caspase-3 activity (Fig. 4A) or by PARP cleavage (Fig. 4B). As normal apoptosis participates in the elimination of fully differentiated keratinocytes, this result probably reflects the increased in epidermis thickness that we observed in vivo.


Figure 4
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  		Figure 4. Keratinocytes obtained from ERK1–/– mice are resistant to apoptosis. A, wild-type or ERK1–/– keratinocytes were untreated (Cont) or either treated with staurosporine (S, 10 nmol/L), UV illuminated (400 Mj/cm2), growth factor deprived for 16 hours (GF), treated with cycloheximide (Cy, 10 mg/mL), TNF-{alpha} (T, 10 nmol/L), or a combination of cycloheximide and TNF-{alpha} (T+Cy). Then the caspase activity was measured as described in Materials and Methods. B, cell lysates from wild-type or –/– keratinocytes, UV stimulated, deprived of growth factors for 16 hours or treated with cycloheximide, TNF-{alpha}, or a combination of both were analyzed for the presence of PARP. Arrow, PARP cleaved fragment (CF). Total ERK is shown as loading control. An asterisk represents an unspecific protein.

 
ERK1 deficiency inhibits TPA-promoted tumor growth. All the results presented above strongly suggest that elimination of ERK1 affects skin homeostasis and modified the proliferation/apoptosis balance. As such equilibrium directly controls the potential to form tumors, we investigated the incidence of ERK1 gene knockout on the process of skin tumor formation.

For that purpose, we used the classic two-stage mouse skin carcinogenesis model in which DMBA was used as an initiator and TPA as a promoter. Wild-type and ERK1–/– mice were initiated by application of a single 200 nmol dose of DMBA and treated twice weekly with 17 nmol TPA. The first papillomas appeared after 5 weeks of TPA treatment in the wild-type group but were delayed by 2 weeks in the ERK1–/– group (Fig. 5). After 7 weeks of TPA treatment, 55% of wild-type male mice developed papillomas, whereas the papilloma incidence remained significantly low (25%) in mutant animals (Fig. 5A). One hundred percent incidence was achieved by week 19 of promotion in the wild-type group, whereas the incidence reached, at the maximum, 45% in the mutant group by week 21. In both groups, some tumors had a tendency to desiccate and regress with a higher frequency in knockout mice. Right from the start of the experiment, the average number of papillomas per mouse was significantly higher in wild-type animals when compared with the ERK1–/– mice (Fig. 5B). The number of large tumors (>1.5 mm) per tumor-bearing mouse was significantly enhanced in wild-type mice and mutant groups from week 7, but we noticed a striking difference after 15 weeks of promotion (Fig. 5C). At the end of the experiment, the average size of tumors in wild-type mice was 20 mm2 compared with 2 mm2 in ERK1–/– animals (Fig. 5D). Differences in morphology were observed after week 19. Most papillomas on wild-type male mice continued to grow rapidly and were well vascularized, whereas papillomas of ERK1–/– mice seemed growth arrested and desiccated. Examination by light microscopy showed polypoid tumors in both wild-type and knockout animals. The implantation base of the tumors was of a different size. The epidermis was greatly thickened without dysplasia but with hyperkeratosis. This hyperkeratosis was amplified in tumors of wild-type animals. The underlying connective tissue was fibrous with a slight nonspecific inflammatory infiltrate (Fig. 5E). These data strongly suggest that a deficiency in the ERK1 gene prevents formation and growth of skin tumors initiated by DMBA and promoted by TPA. It is also in accordance with the slight increased expression of IL-18 (2.7-fold) that we observed in knockout skin samples following TPA application (data not shown). IL-18 was shown to be a tumor suppressor and possesses strong antiangiogenic capacities (24).


Figure 5
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  		Figure 5. Skin tumorigenesis in wild-type (wt) and ERK1 knockout (ko) (–/–) mice. Mice (n = 8-13) were subjected to the two-stage DMBA + TPA chemical tumorigenesis protocol. Tumors were counted and measured every week. A, papilloma incidence (the percentage of animals having one or more papillomas). B, papilloma multiplicity (the number of papillomas/mouse in mice with papillomas). C, tumor size (the number of tumours > 1.5 mm in mice with tumours). D, at the end of the experiment, tumours from wild-type (WT) or –/– mice were measured (length and width). Their sizes were plotted and the average size is indicated. E, histopathologic analysis of skin tumors from wild-type or ERK1–/– mice. At the end of the experiment, tumors >1.5 mm were prepared and stained with H&E and analyzed. Bottom, general appearance of skin papillomas in wild-type or –/– mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Taken together, our results show that in highly specialized cells, which participate in the constant renewal of the skin epithelium, a normal amount of ERK activity is absolutely indispensable for normal homeostasis. A reduction in the ERK activity provokes focal skin inflammation by two potential combined mechanisms. First, the immunologic defect that we have described probably favored bacterial infection, as soon as a small wound appeared. Hence, local inflammation, inflammatory cytokine overproduction, and scars, characterized by skin thickness, were observed. Second, a reduction in the apoptotic potential of keratinocytes prevents physiologic desquamation, participates in epidermis thickness, and perhaps decreases the capacity of mutagens to affect basal stem cells. Our results provide another example of the apoptotic potential of ERK already described for neurons (25, 26) and also highlight the ambivalence in the ERK activity for tumor promotion that was already described in different tumor cell lines (27). Another striking evidence of this ambivalence is the fact that chemotherapeutic agents mediate their effects through the activation of ERK (28–30). Moreover, the ERK pathway is inactivated in different tumor cells (31, 32). Some of them overexpress the MAPK phosphatases 1 and 2 that are considered as bad prognosis markers (33). However, in response to chronic inflammation induced by TPA, the absence of ERK1 is beneficial because it prevents skin tumor development. Two mechanisms may account for these results. First, a decrease in the total ERK activity reduced proliferation of basal keratinocytes that prevents intensive tumor growth. Moreover, the lack of ERK1 also affects skin inflammation probably because of abnormal recruitment of immune cells. The absence of an intense inflammatory response probably avoids the local massive production of inflammatory cytokines that induces, in wild-type mice, destruction of the epidermis. Such destruction favored the proliferation of basal stem cells for epidermis renewal and allows induction of Ras mutations (34) that lead to selection of cells with a high growth potential. Absence of intense desquamationin the superficial epidermis layers in knockout mice strongly reduced epidermis renewal and the probability to acquire mutations in basal stem cells. Such a phenomenon is emphasized by the intrinsic apoptotic insensibility of knockout keratinocytes. Hence, even it is generally admitted that the activation of the ERK pathway is linked to antiapoptotic signals that lead to tumor development (35), in that specific case, reduction of the apoptotic potential via ERK1 mutation provokes an "antitumoral" effect. ERK1 may possess specific substrates. Alternatively, the observed phenotype may depend on a certain threshold in total ERK activity. At the moment, we cannot favor one hypothesis over the other because ERK2 knockout is embryonic lethal. Conditional ERK2 knockout in the skin would clarify this question. At the molecular level, reduction of total ERK activity (ERK1 + ERK2) is accompanied by a drastic reduction of many genes following TPA treatment, which are potential candidates for ERK1 action. In this case, our results would highlight a specific role of ERK1 at least in specialized cells. Hence, targeting ERK activity for the treatment of skin cancers is quite promising, even if it has to be considered with extreme prudence. The effect on the proliferation capacity would probably reduce tumor growth and invasiveness. Such an effect on tumor development by targeting ERK was already suggested by using an inhibitor of the ERK pathway in the case of colon carcinomas (1). However, ERK inhibition has to be combined with additional drugs that circumvent the problem of apoptosis resistance.


   Acknowledgments
 
Grant support: Centre National de la Recherche Scientifique/Université de Nice-Sophia Antipolis, Association pour la Recherche Contre le Cancer, Fondation de France, Ligue Nationale Contre le Cancer, Ortho-Biotech Society/Division of Janssen-Cilag S.A.S., German Ministry for Education and Research/National Genome Research Network (NGFN-2), Association for International Cancer Research, and Ministère de la Recherche.

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. M.C. Brahimi-Horn for critical reading of the article and Dr. D. Aberdam for his advice.

Received 9/13/05. Revised 11/28/05. Accepted 12/16/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sebolt-Leopold JS, Dudley DT, Herrera R, et al. Blockade of the MAP kinase pathway suppresses growth of colon tumors in vivo. Nat Med 1999;5:810–6.[CrossRef][Medline]
  2. Marchetti S, Gimond C, Roux D, Gothie E, Pouysségur J, Pagès G. Inducible expression of a MAP kinase phosphatase-3-GFP chimera specifically blunts fibroblast growth and ras-dependent tumor formation in nude mice. J Cell Physiol 2004;199:441–50.[CrossRef][Medline]
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