RasGRP1 Transgenic Mice Develop Cutaneous Squamous Cell Carcinomas in Response to Skin Wounding : Potential Role of Granulocyte Colony-Stimulating Factor

Models of epidermal carcinogenesis have demonstrated that Ras is a critical molecule involved in tumor initiation and progression. Previously, we have shown that RasGRP1 increases the susceptibility of mice to skin tumorigenesis when overexpressed in the epidermis by a transgenic approach, related to its ability to activate Ras. Moreover, RasGRP1 transgenic mice develop spontaneous papillomas and cutaneous squamous cell carcinomas, some of which appear to originate in sites of injury, suggesting that RasGRP1 may be responding to signals generated during the wound-healing process. In this study, we examined the response of the RasGRP1 transgenic animals to full-thickness incision wounding of the skin, and demonstrated that they respond by developing tumors along the wounded site. The tumors did not present mutations in the H-ras gene, but Rasgrp1 transgene dosage correlated with tumor susceptibility and size. Analysis of serum cytokines showed increased levels of granulocyte colony-stimulating factor in transgenic animals after wounding. Furthermore, in vitro experiments with primary keratinocytes showed that granulocyte colony-stimulating factor stimulated Ras activation, although RasGRP1 was dispensable for this effect. Since granulocyte colony-stimulating factor has been recently associated with proliferation of skin cancer cells, our results may help in the elucidation of pathways that activate Ras in the epidermis during tumorigenesis in the absence of oncogenic ras mutations.The role of Ras activation in non-melanoma skin cancer is well-documented, both from analysis of human squamous cell carcinomas (SCC)¹ as well as from studies using mouse models of skin carcinogenesis.^2,3 In particular, the multistage carcinogenesis protocol on mouse skin has identified activating mutations in the ras proto-oncogene as the initiation event in skin neoplasms.⁴ Interestingly, whereas Ras mutations are prevalent in mouse models of skin carcinogenesis, they have only been identified in 12% to 46% of sporadic human SCC samples, despite the fact that Ras is activated in the majority of the human SCC.^1,5 This suggests that other mechanisms of Ras activation play a role in the human disease. For example, epidermal growth factor receptor overexpression by amplification is known to occur in human SCC,^6,7 and epidermal growth factor receptor could lead to the biochemical stimulation of Ras. In animal models, overexpression of epidermal growth factor receptor ligands like transforming growth factor-α,⁸ or a dominant form of the Ras exchange factor Sos of seven less (SOS),⁹ act as an initiation event in the epidermis, further demonstrating that alterations in Ras upstream signals could lead to tumorigenesis in the skin via wild-type Ras activation.Biochemical activation of Ras in keratinocytes can be triggered by various extracellular stimuli, but in all cases it requires the participation of exchange factors that catalyze the GDP–GTP exchange. The best studied GDP–GTP exchange factor is SOS1, which is activated in response to receptor tyrosine kinase activation.^10,11 In recent years, we have identified a new GDP–GTP exchange factor in epidermal keratinocytes, RasGRP1, and demonstrated that it can transduce the activation of Ras by the diacylglycerol analog and potent skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA).¹² The initial studies prompted us to investigate the role of RasGRP1 in the tumor promotion effects of phorbol esters in the multistage carcinogenesis protocol using a transgenic mouse model for overexpression of RasGRP1 under the keratin 5 promoter (K5.RasGRP1). Surprisingly, RasGRP1 overexpression did not increase the susceptibility to TPA in animals initiated by the carcinogen 7, 12-Dimethylbenz[a]anthracene (DMBA), but caused more malignant tumors than those observed in the wild-type mice, suggesting a role of RasGRP1 in tumor progression.¹³ This effect appeared strongly related to increases in active Ras, as RasGRP1 overexpression clearly leads to elevated Ras^GTP levels in mouse epidermal keratinocytes, both under basal as well as TPA stimulated conditions.¹³ Interestingly, the K5.RasGRP1 mice also produced tumors in response to TPA alone, implying that RasGRP1 overexpression can act as an initiation event. In addition to the response to chemical carcinogens and tumor promoters, we observed that the RasGRP1 transgenic colony developed spontaneous skin tumors over time, more frequently seen in animals housed in groups or with previous episodes of skin abrasion or injury.¹⁴ This response resembled that of the Tg.Ac mice, which express v-H-ras under the ζ-globin promoter and generate tumors in response to tumor promoting stimuli like wounding and phorbol esters in absence of carcinogenic/initiation events.^15,16The in vivo studies with the K5.RasGRP1 mice have implicated RasGRP1 as a novel link for Ras activation in epidermal keratinocytes and skin cancer. However, the signaling mechanisms contributing to RasGRP1 activation and spontaneous tumor formation, as well as the direct evidence for a role of skin wounding as a tumor promoter stimulus in the context of RasGRP1 overexpression, remained to be established. To address these questions, we have now characterized the tumorigenic response of the K5.RasGRP1 to full-thickness incision wounding of the skin. Our findings show that RasGRP1 overexpression in the epidermis conferred sensitivity to wounding-induced promotional stimuli. Furthermore, upon wounding, there was a significant increase in the levels of granulocyte colony-stimulating factor (G-CSF) in the circulation of transgenic mice compared with the wild-type animals. In vitro, G-CSF was able to stimulate Ras activation in keratinocytes in a very rapid fashion, although RasGRP1 appeared dispensable for this effect. Since G-CSF has been associated to the growth and progression of skin carcinoma cells, the data presented here may have implications for the understanding of RasGRP1-Ras signaling in skin tumor biology.