Alterations in signal transduction pathways implicated in tumour progression during multistage mouse skin carcinogenesis

Vassilis Zoumpourlis1, Sylvia Solakidi, Alexandra Papathoma and Dimitra Papaevangeliou

Laboratory of Gene Regulation, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, 48 Vas Constantinou Avenue, 11635 Athens, Greece

1 To whom correspondence should be addressed. Tel: +30 210 7273745; Fax: +30 210 7273677; Email: vzub{at}eie.gr


    Abstract
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 Abstract
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The multistage mouse skin carcinogenesis model, although an artificial one, is an ideal system to study the timing of qualitative and quantitative alterations which take place during the different stages of chemical carcinogenesis, allowing analysis of the events that lead to the transition from the stage of initiation to the stage of promotion and finally to the progression of carcinogenesis. In this review we focus on the role of the H-ras gene and its target molecules during mouse skin carcinogenesis. Besides H-ras, which is a critical target of chemical carcinogens, we report alterations in oncosuppressor genes. Finally, we examine the potential suppression of metastatic dynamics of spindle cells after biological or chemical inhibition of the signalling cascades involved in mouse skin carcinogenesis.

Abbreviations: AP-1; activated protein-1; ARF, alternative reading fragment; cdk, cyclin-dependent kinase; CRE, c-AMP response elements; DMBA, 7,12-dimethylbenz[a]anthracene; MAPK, mitogen activated protein kinase; MMPs, matrix metalloproteinases; PIN, prostate intraepithelial neoplasia; SRF, serum response factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; TRE, TPA response elements


    Introduction to the multistage mouse skin carcinogenesis model
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 Abstract
 Introduction to the multistage...
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Tumourigenesis is a multistep process in which genetic and epigenetic events determine the transition from a normal to a malignant cellular state. The rate of the process of tumour progression is accelerated by mutagenic agents (tumour initiators) and by non-mutagenic agents (tumour promoters) that affect gene expression and stimulate cell proliferation. Advances in the understanding of the genetic and cellular processes associated with tumourigenesis, as well as methodological progress in animal manipulation, have led to the development of more accurate and powerful mouse carcinogenesis models. Mouse skin has provided a paradigm for studies of multistage chemical carcinogenesis in epithelial cells. The study of the process of tumour initiation, promotion and progression in this model of mouse epidermis has provided insight into the mechanism of carcinogen–DNA interactions and the nature of mutations in the critical target genes.

The most common chemical carcinogenesis regimen is two-stage induction, which involves the administration of a single dose of the polycyclic aromatic hydrocarbon 7,12-dimethylbenz[a]anthracene (DMBA), followed by weekly applications of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). This treatment results in the development of numerous benign papillomas, some of which progress to malignant squamous cell carcinomas 20–40 weeks after the first exposure to carcinogens.


    Development and characterization of cell lines representing different stages of skin tumours
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Because of the problems associated with studying biological mechanisms of carcinogenesis using in vivo tumour material, a series of cell lines has been developed in Allan Balmain's laboratory. They represent the development of the three distinct stages in mouse carcinogenesis, initiation, promotion and progression, thus covering the full spectrum of mouse skin carcinogenesis.

In Figure 1 we describe the development of the C5N (1), P1 and P6 (2), B9 (3), A5 (3), CarB, PDV and PDVC57 (4,5) cell lines. Although the A5 spindle cell line was isolated from the same primary tumor as the B9 cell line, it has different morphological and growth properties. C5N, P1, P6 and B9 cells have a typical epithelial morphology, being cuboidal in shape and characterized by a cobblestone pattern of growth (3,6), while the A5 and CarB cell lines show a fibroblastic morphology. B9 cells give rise to well-differentiated tumours in nude mice, while the A5 and CarB cell lines exhibit aggressive metastatic tumor growth in vivo, giving rise to spindle tumours 4 and 2 weeks, respectively, after injection into nude mice (3). The C5N, P1, P6 and B9 cell lines have a typical pattern of keratin and E-cadherin expression, whereas the A5 cell line has an altered cytoskeleton and fails to express E-cadherin (7). The other spindle cell line, CarB, does not express keratin, but expresses vimentin (7). Compared with PDV cells, the PDVC57 cell line has a more heterogeneous morphology, is characterized by an increased number of giant cells and is more tumourigenic when re-injected into adult syngeneic mice, giving rise to carcinomas at all five injection sites (8). PDVC57 cells are 8-fold more invasive and secrete 2-fold more type IV collagenase compared with PDV cells, and are also more chemotactic.



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Fig. 1. Development of the cell lines of the mouse skin carcinogenesis system.

 
An important criterion for choosing the described cell lines for our studies is that all except C5N were obtained from tumours initiated in vivo with DMBA and, consequently, carry the H-ras mutation at codon 61, which is induced by this initiation agent (see below). They also harbour mutations in the p53 gene, except for the CarB cell line. Furthermore, the pairs B9:A5 and PDV:PDVC57 represent the clonal expansion of cancer cells in mouse skin and are useful for the in vivo examination of alterations in genetic and signal transduction pathway members during progression and the epithelial to mesenchymal transition.

Genetic alterations in the H-ras oncogene
Seminal experiments demonstrating the presence of activated oncogenes in tumour cell lines (9,10) showed for the first time that mutations in specific genes could endow them with transforming properties. Further studies led to the identification of a single member of the ras family, the H-ras gene, as the transforming oncogene found to be consistently activated in mouse skin carcinomas initiated with DMBA (11).

Papillomas and carcinomas initiated with different carcinogens exhibit distinct spectra of point mutations in the H-ras gene. Over 90% of tumours initiated with DMBA showed an A->T point mutation at the middle adenosine nucleotide of codon 61 (CAA), which resulted in the introduction of an activating missense mutation (12). The alkylating agents N-methyl-N'-nitro-N-nitrosoguanidine and methylnitrosourea induced exclusively G->A transitions at codon 12, predominantly in papillomas (13). 3-Methylcholanthrene initiation produced both codon 13 G->T and codon 61 A->T transversions in papillomas, but only the G->T mutation was found in carcinomas (13). The high frequency of A->T mutations at codon 61 in the DMBA-initiated tumours can be explained by the fact that the diol epoxide, resulting from metabolic activation of DMBA, forms adducts primarily with adenosine nucleotides (14).

The causal nature of H-ras mutations in the initiation stage of carcinogenesis was demonstrated by experiments proving that the Harvey murine sarcoma retrovirus (which carries activated H-ras allelles) could in vivo replace chemical initiation by DMBA, when applied to mouse skin (15). Furthermore, targeted deletion of the H-ras gene decreased tumour formation in DMBA-treated mouse epidermis. Nevertheless, more than half of the tumours formed in these H-ras null mice carried mutations in the K-ras gene. The H-ras gene is therefore the initiator gene in mouse skin carcinogenesis, but it can be replaced in vivo by mutated K-ras (16).

Furthermore, H-ras plays an important role in more advanced stages of carcinogenesis, in which the mutant allele is further duplicated or amplified. Analysis at the chromosomal, DNA, mRNA and protein levels has demonstrated that the squamous and spindle cell lines studied differ in the ratio of normal to mutant H-ras alleles (Table I) (A.Balmain, personal communication; 1720). The availability of cell lines with a well-characterized H-ras endogenous status has been critical for carrying out a more detailed analysis of downstream signals and to correlate our results with the stage of malignant progression.


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Table I. Normal and mutant H-ras and p53 genes in mouse skin carcinogenesis cell lines

 
Downstream mediators of Ras
Ras-mediated tumourigenesis depends on signalling pathways that act preferentially through cyclin D1, a cell cycle-associated nuclear protein which is active in the G1 phase (21). Co-localization of the H-ras and cyclin D1 genes on the distal part of chromosome 7 may ensure that both genes are duplicated, with possible synergistic effects in tumour development, when trisomy 7 occurs in papillomas.

Cyclin D1 mRNA and protein levels are generally higher in mouse skin carcinomas than in papillomas and introduction of a mutant human ras construct into immortalized keratinocytes can induce expression of cyclin D1 (22). Furthermore, cyclin D1 deficiency results in up to an 80% decrease in the development of squamous tumours generated through two-stage chemical carcinogenesis (21).

Cyclin D1 forms a complex with cyclin-dependent kinase (cdk) 4 and cdk6, which are also up-regulated in keratinocytes in response to oncogenic ras. The assembly and catalytic activity of cyclin D1–cdk complexes are negatively regulated by a group of cdk inhibitors, such as the INK4 family members (2325). The INK4 locus was homozygously deleted in most spindle mouse skin carcinoma cell lines, but in none of the squamous cell lines studied (Table II) (26). Mice lacking INK4a/alternative reading fragment (ARF) or ARF alone are highly tumour prone (27). The results summarized above indicate that over-expression of cyclin D1 and deletion of the INK4 locus are significant molecular abnormalities that could be involved in the process of mouse skin tumour progression.


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Table II. Expression of signal transduction molecules in the mouse carcinogenesis cell lines

 
Ras target molecules
Ras activates members of the JNK group of mitogen activated protein kinase (MAPKs), which are the major mediators of c-Jun and ATF-2 terminal phosphorylation. We demonstrated that the content of JNK1 and JNK2 isoforms, as well as JNK activity, was increased in the malignant mouse skin cell lines, with JNK2 elevated to a lesser extent than JNK1 in the spindle cell lines A5 and CarB (Table II).

Ras also targets the Raf/MEK/ERK branch of the MAPK pathway (28). The ERK1/2 isoforms were preferentially activated in advanced tumour stages, since phosphorylated ERK1 and ERK2 were elevated in the A5 and CarB spindle cells, compared with the P1 and B9 epithelial cell lines. Studies in the PDV:PDVC57 pair revealed increased ERK1/2 phosphorylation in the PDVC57 cells, which are more aggressive than the PDV cell line. This finding suggests that the biological characteristics of the squamous phenotype may depend on the activation of ERK1/2 (20; Table II). The introduction of a human activated H-ras allele into PDV cells resulted in increased phosphorylation of ERK1/2, compared with the parental PDV cell line. Therefore, the gradual increase in ERK1/2 activation observed in the PDV:PDVC57 and B9:A5 cell line pairs is likely to arise from alterations in Ras protein levels and/or activity (20).

Activated protein-1 (AP-1) is a collection of dimeric basic region–leucine zipper (bzip) transcription factors that belong to the Jun (c-Jun, JunB and JunD), Fos (c-Fos, FosB, Fra-1 and Fra-2), Maf (c-Maf, MafB, MafG/F/K and Nrl) and ATF (ATF2 and LRF1/ATF3) subfamilies, recognizing either TPA response elements (TRE, 5'-TGAG/CTCA-3') or c-AMP response elements (CRE, 5'-TGACGTCA-3') (29), which are located in the promoter regions of genes encoding transcription factors, matrix-degrading enzymes, cell adhesion molecules, cyclins and cytokines. Jun/Fos heterodimers preferentially bind to the heptameric consensus TRE, while ATF2/ATF2 and c-Jun/ATF2 dimers prefer the octameric consensus CRE (30). Oncogenic Ras proteins regulate AP-1 activity at the transcriptional and post-translational levels, mainly through persistent activation of the ERK and JNK pathways (31,32). We (19) and others (33,34) have implicated transactivation of AP-1 in tumour promotion and/or progression. Studies in transgenic mice suggested that c-fos and c-jun expression is involved in benign to malignant tumour progression, but is not required for papilloma genesis (34,35). However, transgenic overexpression of c-jun in the absence of additional oncogenic stimulation did not increase tumour incidence (36).

We detected increased levels of total and phosphorylated c-Jun in the malignant cell lines B9, A5 and CarB (Table II). Maximum levels were observed in the spindle cell lines. High levels of Fra-2, hyperphosphorylated Fra-1 and total and phosphorylated ATF2 also characterized the malignant phenotypes (Table II; 19). This increase probably takes place due to Ras protein overexpression, also observed in these cells. These changes in expression and post-translational modification of the AP-1 family members resulted in enhanced AP-1 DNA activity at the collagenase I TRE and Jun2 TRE in the metastatic cell lines A5 and CarB. The major AP-1 components participating in the AP-1/DNA binding complex were c-Jun and ATF-2 (19). c-Jun and ATF2 are, therefore, likely to be involved in the progression of carcinogenesis in mouse epidermis (19).

Serum response factor (SRF) is a transcription factor first identified as a critical factor involved in mediating serum and growth factor-induced transcriptional regulation of the c-fos proto-oncogene (37). Its activation involves the Rho family GTPases pathway (38), which regulates, among other things, actin cytoskeleton rearrangement (39). Ras and Rho GTPases cross-talk, since transformation by oncogenic Ras leads to alterations in the cytoskeleton and GTPases of the Ras subfamily activate Rho family members (40).

In the mouse skin spindle cell lines we found increased SRF protein levels and SRF DNA binding activity to the c-fos serum response element oligonucleotide. In order to investigate the RhoA effect, we chose B9 and A5 cells, which represent the two distinct stages of epithelial to mesenchymal transition. It has been demonstrated that both total and active RhoA levels are significantly higher in A5 compared with B9 cells (Table II; 41).

In order to find out whether the increased SRF DNA binding activity was regulated by RhoA signalling, we transfected B9 and A5 cells with active and dominant negative forms of RhoA. Although we detected no changes in the expression levels of SRF protein, increased DNA binding activity in B9 cells transfected with the active form of RhoA and decreased DNA binding activity in A5 cells transfected with the dominant negative form of RhoA were demonstrated. SRF overexpression has, therefore, an important role in spindle phenotype formation and RhoA signalling regulates DNA binding activity of SRF in the mouse carcinogenesis system (41).

T lymphoma invasion and metastasis gene 1 (Tiam-1) is an invasion-inducing gene in T lymphoma cells, which functions as a guanine nucleotide exchange factor of the Rho GTPase Rac (42). In order to study the effect of Tiam-1 in cell migration and tumour progression Tiam-1+/+ mice were treated with DMBA and TPA. These mice developed a variety of skin tumours, predominantly benign papillomas, ~35% of which progressed to malignancy, whereas 80% of the tumours that arose in Tiam-1-/- mice after DMBA/TPA treatment acquired an invasive malignant phenotype. Tiam-1 deficiency seems therefore to increase the rate of malignant conversion in animals treated with the DMBA/TPA regimen. This role of Tiam-1 was proved by further analysis in mice treated only with DMBA (43).

Previous studies showed that increased Ras signalling, associated with advanced skin malignancies, was responsible for the reduction or loss of Tiam-1, as well as for the decrease in Rac activity in these lesions (44). These results demonstrated that A5 spindle cells (which are characterized by increased amounts of mutant H-Ras protein) did not express any Tiam-1 protein, in contrast to P1 cells, which expressed high levels of Tiam-1 (43). Moreover, loss of Tiam-1 protein in A5 cells was accompanied by a strong reduction in Rac basal activity (43).

Tiam-1 function appears therefore to be essential for the initiation and promotion of Ras-induced skin tumours, but the histological and biochemical data suggest that a subsequent loss of Tiam-1 increases the rate of malignant conversion of benign tumours (43).

Matrix metalloproteinases (MMPs) are members of the zinc-binding endopeptidases involved in the degradation of the extracellular matrix components. In the invasive A5 and CarB spindle cells we demonstrated high levels of MMP-9, which has been shown to be regulated by the AP-1 and Ets transcription factors, whereas MMP-2 levels were independent of tumourigenic or invasive properties of cells (Table II; 46).

The role of the p53 tumour suppressor gene in tumour progression
The p53 tumour suppressor protein functions in normal cells as a transcription factor. Its levels rise in response to DNA damage and/or other stresses and it triggers dramatic cellular responses, including cell cycle arrest, senescence and apoptosis (47). The Mdm2 protein is a key element controlling the p53 response through a negative feedback loop (48).

Mutations in the gene encoding the p53 protein are found in more than half of human cancers. In a study carried out on chemically induced mouse skin tumours, loss of heterozygosity at the p53 locus was detected in approximately one-third of carcinomas, but not in papillomas. Furthermore, no loss of heterozygosity was detected in PDV and PDVC57 cell lines. Sequencing analysis has revealed an ATG->GTG mutation at codon 231 (3). Sequencing analysis of the remaining allele of p53 in the clonally related B9 squamous and A5 spindle cell lines (which were derived from primary carcinoma MSC11) demonstrated two point mutations at codons 236 (TGC->TTC) and 246 (CTT->CTA) (3). Western blots performed on extracts from MSC11 and CarB cells, using monoclonal antibodies immunoprecipitating p53 either dependently or independently of normal or mutant conformation, demonstrated that mutant p53 protein was present in MSC11 but not in CarB cells. Based on these results, Burns et al. concluded that the p53 alterations arose before the transition from squamous to spindle phenotype (3). Kemp et al. demonstrated that p53-/- mice showed a reduced yield of papillomas, but these underwent much more rapid malignant progression, in comparison with wild-type mice (45). These results suggest that absence of p53 does not augment the frequency of initiation or the rate of promotion at the stage of malignant progression (45).

Mdm2 oncoprotein is deregulated in human tumours and has transforming properties. Ganguli et al. showed that Mdm2 in transgenic mice increases papilloma formation induced by chemical carcinogenesis and results in the appearance of premalignant lesions and squamous cell carcinomas (49). The major physiological role of Mdm2 is to inhibit the tumor suppressor p53. Although the results of Ganguli et al. and Kemp et al. seem to be contradictory, it should be noted that Mdm2 overexpression is not biologically equivalent to loss of p53. Overexpression of Mdm2 is able to diminish, but not completely abolish p53 activity (49). This may explain why in mdm2 transgenic mice an increased number of papillomas is observed. In contrast, loss of p53 results in bypassing this benign stage (low number of papillomas), whereas it leads to progression to the malignant stage, since an increased number of carcinomas is observed (45).

Inhibition of signalling cascades involved in mouse skin carcinogenesis
The members of signalling pathways reported to play a role in the process of carcinogenesis in mouse skin are candidate targets for reversion of the malignant phenotype by inhibition of their activity, which could be achieved in both chemical and biological ways. In this section we summarize the results of such studies carrried out in our laboratory.

The chemical approach
The key role of the Raf/MEK/ERK pathway in Ras-mediated cellular transformation has encouraged efforts to develop kinase inhibitors that would block the cascade leading to activation of nuclear transcription factors. We have already mentioned that a gradual elevation of ERK1/2 activation was observed from the papilloma to the squamous and spindle cell lines, as well as within the squamous PDV:PDVC57 pair. PDV cells have a 2:1 normal to mutant H-ras ratio, whereas this ratio in the PDVC57 cell line is 1:2. Furthermore, mutant H-ras is amplified and overexpressed in A5 cells. These cell lines represent, therefore, an in vivo model for studies of H-ras and ERK1/2 signalling in mouse tumourigenesis.

In order to examine whether the Ras/MEK/ERK pathway is required for manifestation of the tumourigenic properties of PDVC57 cells, PDVras transfectants and A5 cells, we used PD98059, a small, cell-permeable molecule which acts as a potent MEK inhibitor. After a 6–24 h treatment of squamous cells and a prolonged, up to 48 h treatment, of A5 spindle cells with PD98059, ERK1/2 phosphorylation was reduced. These results suggest that in A5 cells both the ERK1 and ERK2 isoforms are phosphorylated at high levels, explaining why A5 cells remain refractile to pharmacological inhibition by PD98059 when compared with squamous carcinoma cells. Proliferation and tumourigenicity of squamous and spindle carcinomas of mouse skin are therefore Raf/MEK/ERK pathway dependent (20). A similar approach was employed in vivo by Sebolt-Leopold et al., who achieved up to 80% inhibition of tumour growth in mice inoculated with human or mouse colon carcinoma cells by using the MEK-1 inhibitor PD184352 (50).

A5 spindle cells have a fibroblastic phenotype. In these cells the ERK1/2 cascade is activated as a result of the high content of mutant H-ras. To test whether autocrine signals present in A5 cells activate the Raf/MEK/ERK pathway and if they are required for spindle phenotype, the morphology and ERK1/2 phosphorylation status of serum-starved A5 cells were examined. Serum starvation, however, had no effect on either of these two parameters. When A5 cells grew for 48 h in serum-free medium supplemented with PD98059, they displayed a flat phenotype (similar to that of normal immortalized fibroblasts, such as Rat-1 cells), as well as reduced ERK1/2 phosphorylation levels. The induction of these changes by PD98059, in both the presence and absence of serum, suggests that inhibition of the Raf/MEK/ERK pathway is sufficient to elicit this effect. We have, therefore, demonstrated for the first time that the degree of progression in the mouse skin carcinogenesis model correlates with ERK1/2 signalling (20).

The biological approach
We have already mentioned that ATF2 is involved in the progression of carcinogenesis in mouse epidermis. Preliminary results from studies in our laboratory suggest that transfection of the A5 cell line with a dominant negative ATF2 phosphorylation mutant results in reversion of the spindle phenotype to squamous-like. This promising finding leads us to further investigation of the mechanism of this modification of morphology, as well as to future studies using dominant negative mutants of other signal transduction pathway members.


    Future prospects
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 Abstract
 Introduction to the multistage...
 Development and characterization...
 Future prospects
 References
 
The immortalized, benign and malignant cell lines comprising the mouse skin carcinogenesis system have proved to be an ideal model for the study of multistage carcinogenesis, since they have been valuable tools in investigations that succeeded in correlating specific genetic alterations with specific stages of carcinogenesis (Figure 2). These data could serve as a background for the identification of genes having a critical role in stage-to-stage transition in human multistage cancers.



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Fig. 2. Summary of key alterations regulating stage-to-stage transition during mouse skin carcinogenesis.

 
The multistage nature of human cancer has been well documented, especially in the case of colon cancer, in which well-defined stages (hyperplastic epithelium, adenomatous polyp and adenocarcinoma) have been identified in vivo in tumor samples from patients. Another example is human prostate cancer, in which progression from the prostate intraepithelial neoplasia (PIN) to the androgen-dependent and finally to the androgen-independent stage has been defined. Molecular studies in these multistage human cancers have shown a degree of genetic and biological similarity with the mouse skin carcinogenesis model. The particular genetic events we have reviewed for mouse skin carcinogenesis are also observed, with a few exceptions, in human cancers (51): ras gene family point mutations are a common genetic event in both murine and human cancers, but mouse squamous carcinomas have predominantly H-ras mutations, while colon adenocarcinomas exhibit preferentially K-ras mutations. Genetic alterations in the cyclin D1, p53 and p16 genes are also observed in human multistage cancers (51).

Such genes may be useful for the prediction of individual cancer risk, as well as potential novel targets for cancer prevention and therapy. Our studies leading to the reversion of biologically aggressive phenotypes, using both chemical and biological approaches, are suggestive in this direction.


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Received January 21, 2003; revised April 10, 2003; accepted April 16, 2003.