Transgenic Overexpression of RasGRP1 in Mouse Epidermis Results in Spontaneous Tumors of the Skin

Abstract

RasGRP1 is a guanine nucleotide exchange factor for Ras and a receptor of the second messenger diacylglycerol and its ultrapotent analogues, the phorbol esters. We have recently shown expression of RasGRP1 in the epidermal keratinocytes where it can mediate Ras activation in response to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, a well-known mouse skin tumor promoter. To explore the participation of RasGRP1 in skin carcinogenesis, we targeted the overexpression of RasGRP1 to basal epidermal keratinocytes using the keratin 5 promoter. These transgenic mice were viable and indistinguishable from their littermates, with normal differentiation and skin architecture. However, a percentage of the adult transgenic population developed spontaneous skin tumors, mainly squamous cell papillomas. The transgene was detected in the tumors as well as in primary keratinocytes isolated from transgenic mice. The transgenic keratinocytes also displayed elevated levels of active, GTP-loaded Ras compared with the levels observed in keratinocytes derived from wild-type littermates. We noticed a correlation between tumor incidence and wounding, which suggests that RasGRP1 overexpression may confer sensitivity to promotional stimuli, like wound repair mechanisms. Interestingly, we also found elevated levels of granulocyte colony-stimulating factor in conditioned media derived from transgenic keratinocytes subjected to in vitro wounding. Taken together, these data are the first to provide evidence of a novel role for RasGRP1 in skin carcinogenesis and suggest that RasGRP1 may participate in tumorigenesis through modulation of Ras and autocrine pathways. [Cancer Res 2007;67(1):276-80]

RasGRP1 Overexpression in the Epidermis of Transgenic Mice Contributes to Tumor Progression during Multistage Skin Carcinogenesis

“…A sample of the transgenic tumors was analyzed for mutations in codon 61 of Ha-ras. As previously observed with the spontaneous K5.RasGRP1-derived tumors (19), no mutations were detected (Fig. 3D), suggesting that RasGRP1 overexpression could be responsible for initiation in the absence of mutations in the ras proto-oncogene.…”

“…The K5.RasGRP1 mice were generated as previously described on the FVB/N background strain (19). For the skin carcinogenesis experiments, the back of 6-to 8-week-old mice, both males and females, were shaved with electric clippers 2 days before the beginning of the protocol.…”

“…H&E-stained slides were used for descriptive histopathology. Immunohistochemical localization of the transgenic RasGRP1-HA protein was performed as previously described (19). Briefly, deparaffinized sections were subjected to heat-induced epitope retrieval.…”

“…To investigate the potential role of RasGRP1 in skin tumorigenesis, we recently developed a transgenic mouse model for the overexpression of RasGRP1 in the epidermis (K5.RasGRP1; ref. 19). These mice are prone to developing spontaneous papillomas and squamous cell carcinomas (SCCs), implying a role for RasGRP1 in tumor initiation (19).…”

“…19). These mice are prone to developing spontaneous papillomas and squamous cell carcinomas (SCCs), implying a role for RasGRP1 in tumor initiation (19). To gain further insight into the effect of RasGRP1 during skin carcinogenesis, particularly TPA-induced tumor promotion, we subjected K5.RasGRP1 mice to the classic two-stage chemical carcinogenesis protocol using 7,12-dimethylbenz(a)anthracene (DMBA) as an initiator.…”