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We previously showed that rasH2 transgenic mice carrying the human c-Ha-ras protooncogene are highly susceptible to chemical skin carcinogenesis. In the dermis of rasH2 mice, mast cells are recruited constitutively, and the number of mast cells increases more than in wild-type mice in response to treatment with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate. To determine whether enhanced skin tumor development in rasH2 mice is dependent on the recruitment of mast cells, we generated rasH2 KITW/Wv mice by crossing rasH2 mice and W or Wv KIT mutants, and examined the chemical skin carcinogenesis. In rasH2 KITW/Wv mice, mast cells were not found in the dermis either before or after treatment with 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate. Papilloma multiplicity was up to 4.6-fold higher in rasH2 KIT+/+ mice compared with their rasH2 KITW/Wv siblings. At 12 weeks after the experiment began, the volumes of tumors were significantly smaller in rasH2 KITW/Wv relative to rasH2 KIT+/+ mice (rasH2 KITW/Wv: 29.2 ± 19.9 mm3 versus rasH2 KIT+/+: 179.6 ± 726.6 mm3; P = 0.0153). There was no difference in the latency or multiplicity of papillomas between mice without the rasH2 transgene, KITW/Wv mice and their wild-type littermates. Western blot analysis showed that expression of H-RAS protein in the skin was equivalent in rasH2 KITW/Wv and rasH2 KIT+/+ mice. In conclusion, the inhibition of c-kit decreased H-ras-induced skin carcinogenesis. The suppression of c-kit may be a unique and effective target as a preclinical model of cancer treatment where the activation of H-ras has a significant role. Targeting mast cells could also be a potential strategy for treating malignancies. (Cancer Sci 2007; 98: 1549–1556)
Human cancers develop through a multistep process that involves the accumulation of genetic mutations.(1) Based on the results of work in experimental animal model systems, the carcinogenesis process can be divided into the initiation, promotion and progression stages.(2,3) The initiation stage is an irreversible event in which carcinogens damage DNA and induce mutations in critical genes in target stem cells. During the promotion stage, initiated cells undergo selective clonal expansion due to the acquisition of a proliferative advantage, or the ability to evade growth inhibitory or apoptotic signals. An example of this is the activating mutations of c-Ha-ras.(4) Three members of the RAS family – H-RAS, K-RAS and N-RAS – are activated by mutations in human tumors.(5) Almost all RAS activation in tumors is accounted for by mutations at codons 12, 13 and 61.(6) These mutations all compromise the GTPase activity of RAS, preventing GAP from promoting hydrolysis of the GTP binding to RAS and therefore causing RAS to accumulate in the GTP-bound, active form.
The mouse skin carcinogenesis model is one of the most well-defined in vivo models of experimental carcinogenesis. In DMBA-initiated mouse skin, repeated applications of the tumor promoter TPA promotes papillomas and eventually carcinomas.(7) DMBA induces a mutation at either codon 12 or 61 of the Ha-ras gene. Consequent addition of TPA has a promotion effect, activating Stat3.(8)
RasH2 mice carry the human c-Ha-ras protooncogene with its own promoter region,(9) and are highly susceptible to chemical carcinogenesis.(10–12) We previously reported that rasH2 mice had enhanced chemical skin carcinogenesis in response to treatment with DMBA and TPA.(9) In rasH2 mice, the number and total volume of papillomas were far greater than those of their wild-type littermates, presumably because of the increased abundance of initiated cells in rasH2 mice. Furthermore, the latency of the formation of both squamous cell papilloma and squamous cell carcinoma after treatment with DMBA was shorter in rasH2 mice. This shorter duration of latency in tumor development compared with wild-type mice suggests that the activated rasH2 gene functions as a tumor promoter.(9)
Ras oncogene expression promotes and sustains the tumor–host interactions that are essential for neoplastic development. Constitutive Ras activity has been shown to contribute to increased tumor cell invasiveness through the activation of matrix metalloproteinases.(13) During tumor progression, cancer cells recruit immune cells, which remodel the tumor stroma and initiate angiogenesis.(14–16) Indeed, mast cells are present at the periphery of both experimentally induced rodent tumors(17,18) and human neoplasms.(19–21) Coussens et al.(14) investigated the importance of mast cells as tumor promoters in the skin carcinogenesis of K14-HPV16 mice. They found that infiltrating mast cells turn on and progressively intensify angiogenesis by releasing sequestered angiogenic activators in the premalignant early phase of hyperplasia and dysplasia in K14-HPV16 mice. These workers generated a K14-HPV16 KITW/KITWv mouse, which has a mast cell deficiency. Severe attenuation of early neoplasia is observed in these mice, which demonstrates the essential role of mast cells in the development of premalignant lesions.(14) However, the role of mast cells in late carcinogenesis events is not clear. In the current study, we generated rasH2 mice that were deficient in mast cells by crossing rasH2 and W and Wv KIT mutant mice, and conducted experiments using chemical skin carcinogenesis.
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- Materials and Methods
In chemical carcinogenesis, the process of skin tumor development can be subdivided into three stages: initiation, promotion and progression. Initiation is typically induced by the topical application of the carcinogen DMBA. DMBA treatment causes mutations within Ha-ras in epidermal cells. These mutations, however, are not sufficient to induce de novo transformation. Promotion of tumorigenesis is effected by the topical application of phorbol esters such as TPA to the skin, leading to epithelial cell proliferation and inviting inflammation. Accumulated epidemiological studies support the idea that chronic inflammatory diseases are frequently associated with increased risk of cancers.(30–32) It has been realized that the development of cancers from inflammation might be a process driven by inflammatory cells as well as a variety of mediators, including cytokines, chemokines and enzymes, which together establish an inflammatory microenvironment.(32)
Current evidence suggests that mast cells contribute to tumorigenesis through several mechanisms. Much evidence indicates that mast cells likely induce tumor progression by providing mitogenic stimulation or inducing angiogenesis.(33–38) Data from experimental studies support the hypothesis that neovascularization is induced by mast cell-derived angiogenic mediators or growth factors.(14) Mast cells release several mitogenic mediators, including fibroblast growth factor-2 and interleukin-8,(39) and mast cell proteases reorganize the stroma to facilitate endothelial cell migration. Via the action of their own proteases, and indirectly via interactions with other cells, mast cells participate in degradation of the matrix, which is a process required for tumor spread. In several malignancies, mast cell density has been found to correlate with increased risk of metastasis and poor prognosis.(40–43) These studies emphasize the role of mast cells in accelerating the progression of cancers by providing scaffolds in the pro-progressive microenvironment. In a recent study it was found that allograft tolerance cannot be induced in mast-cell-deficient mice, showing that mast cells are crucial for allograft tolerance. Thus, in addition to secreting various cytokines that induce angiogenesis and remodeling stroma, mast cells may play a role in the immune tolerance of tumor cells.(44)
In the present study, we showed that the introduction of KITW/Wv suppresses H-ras-induced skin carcinogenesis in rasH2. Contrary to our findings, Tanooka et al. reported that mast cell-deficient KITW/Wv mice had an increased tumor incidence after subcutaneous treatment with 3-methylcholanthrene (MCA) compared with that in normal congenic mice treated in the same way.(45) Their paper is indeed a landmark paper that recognizes the involvement of mast cells in carcinogenesis in the microenvironment. Their study showed that deletion of mast cells in KITW/Wv mice stimulated early carcinogenesis without changing the latency of tumor formation. The effect of mast cell depletion was restored by the reconstitution of bone marrow transplant. However, the experimental conditions are different between the two studies. First, different carcinogens could activate disparate signaling pathways in chemical carcinogenesis. Second, the cell types of developed tumors were different between their study and ours. Their study and others show that skin carcinogenesis induced by DMBA + TPA primarily develops papillomas, and then carcinomas develop subsequently among papilomas,(7–9) meanwhile MCA develops mainly sarcomas. A recent study using a conditional knockout of p53 shows that restoration of p53 expression after the deletion of p53 induces apoptosis in developed lymphoma, but does not induce apoptosis in sarcoma.(46) The involvement of immune-responsible cells can work differently in different tumor cell types, even in the same genetic background. Thus, it may not be contradictory to see the different role of mast cells in sarcoma induced by MCA and papilloma by DMBA + TPA. Third, their study saw a difference in the number of mice that gave rise to tumors, but our study saw a difference in the latency and number of tumors in individual mice. Thus, it is likely that immune-responsible cells located at the interface of tumor and normal mesenchyme can act to both stimulate and inhibit carcinogenesis. However, the role of mast cells in the present study lacks robust evidence as proved by the study of bone marrow reconstitution.
Moreover, in chronic inflammation, mast cells are not unique among inflammatory cells in potentiation of neoplastic processes. Polymorphonuclear cells (PMN), macrophages and activated T lymphocytes can also contribute to malignancies by releasing proteases, angiogenic factors and chemokines.(47,48) Wershil et al. reported that the inflammatory response per se is relatively weak in the skin of KITW/Wv mice compared with wild type upon treatment with PMA.(28) Introduction of W/Wv KIT alleles might have changed the immune response to PMA in addition to the deficiency of mast cells.
Many mutations have been described for the dominant-white spotting (W) locus on mouse chromosome 5, which encodes the c-kit receptor. These mutations are known to induce pleiotropic effects, such as sterility, macrocytic anemia and depletion of melanocytes and mast cells.(49) In W/Wv double heterozygous mutant transgenic mice, c-kit activity is decreased to less than 10% of the levels in wild-type mice.(50) In addition to causing mast cell deficiency, impaired c-kit kinase in mutant mice could interfere with skin carcinogenesis via several signal transduction pathways, given that numerous Kit interactions lead to activation of multiple signal transduction pathways, including those mediated by mitogen-activated protein kinase(51) and phosphatidylinositol 3-kinase.(44) In the current study, because there was no difference in the tumor latency and the number of papillomas between wild-type and W/Wv mice, we infer that impairment of c-kit kinase in W/Wv mice did not significantly affect chemical skin carcinogenesis per se. Biochemical and genetic evidence shows that cyclin D1 is a critical target for oncogenic ras in mouse skin. Ras-mediated skin tumorigenesis is substantially reduced in a cyclin D1-deficient background.(29) Indeed, in rasH2 KITW/Wv mice, expression of cyclin D1 in the keratinocytes was equivalent to that in rasH2 KIT+/+ mice. Thus, expression of cyclin D1 was not dependent on the presence of accumulated mast cells. Impairment of c-kit kinase in the W/Wv mutant did not affect the expression of cyclin D1 via activation of the ras signaling cascade in rasH2 mice. These data indicate that the deficiency of mast cells interfered with carcinogenesis at downstream points in cascades mediated by the expression of cyclin D1.
In conclusion, the current study was the first, to our knowledge, to show that inhibition of c-kit decreases H-ras-induced skin carcinogenesis. The suppression of c-kit may be a unique and effective target as a preclinical model of cancer treatment where the activation of H-ras has a significant role. Targeting mast cells could also be a potential strategy for treating malignancies that are dependent on the activation of H-ras.