tumors as well as various other tumor types [51]. This association with invasion and metastasis is likely to reflect the ability of Ets1 to control the expression of MMP enzymes that function to degrade extracellular matrix components. However, Ets1 is also thought to regulate genes involved in cellular proliferation and survival, suggesting that it might also play a role in the earlier stages of carcinogenesis. To determine the precise biological role of Ets1 in tumor formation and progression, we developed a transgenic mouse model that allows us to induce the expression of an Ets1 transgene in a spatially and temporally-restricted manner. Ets1 drives dysplastic changes in stratified squamous epithelium Using an inducible transgenic model system, we have demonstrated that expression of the Ets1 oncogene in the differentiating layers of mouse stratified squamous epithelium leads to a dramatic epithelial phenotype, visibly apparent by the appearance of crusting, flaky skin and open sores. Dysregulated Ets1 expression induced a wide spectrum of progressive prema- lignant changes such as those induced by chemical carcinogens, activated oncogenes, or loss of tumor suppressors in well- established mouse models of squamous cell carcinogenesis. The dysplastic phenotype includes increased keratinocyte proliferation, decreased keratinocyte terminal differentiation and the presence of mitoses (some of which were abnormal) in the suprabasal layers of the epidermis. The dermal responses include increased vascular- ization and leukocytic infiltration. The inhibition of expression of terminal differentiation markers such as involucrin and loricin caused by Ets1 induction likely leads to a severe defect in epidermal barrier formation. Indeed, the phenotype induced by Ets1 transgene expression is similar to that seen in the adult skin of other strains of mice reported to develop barrier defects (e.g., transglutaminase-1 deficient skin or matrip- tase over-expressing skin) [52,53]. Like these strains, Ets1 BT mice develop extensive skin hyperplasia, abnormal terminal differenti- ation and hyperkeratosis. However, Ets1 BT mice also develop deep down-growths of the epidermis into the dermal compart- ment, which have not been reported in these other strains. These down-growths may be related to the high expression of several MMP family members by Ets1 BT mice. The defect in epidermal barrier function in the BT mice could trigger a hyper-proliferative state as an attempt to restore normal barrier function. This would be consistent with the observation that the majority of Ki67+ and PCNA+ cells are found in the basal layer rather than the suprabasal layers, where Ets1 is expressed. In contrast, dysplastic changes including abnormal terminal differentiation and the presence of abnormal mitoses were associated with the supra- basal layers where the Ets1 transgene is expressed, suggesting a direct effect of Ets1 on these parameters. Together these results indicate that Ets1 may have an early role in cancer progression in addition to its more established role in tumor metastasis. Despite the extensive alterations in cutaneous architecture, the molecular and the consequent structural changes in the inter-follicular epidermis are completely reversible when Ets1 expression is suppressed by re-instating Dox treatment. This reversal of the dysplastic phenotype is reminiscent of human SCC precursor lesions such as actinic keratosis and Bowen’s disease, which can undergo spontaneous regression. Importantly, these changes in the epidermis occur in the absence of any tumor promoting chemicals or UV radiation. Moreover, the fact that the entire skin epidermis is affected (in young animals), that the phenotype develops very rapidly after induction (within 3–6 weeks) and that the phenotype is completely reversible suggest that additional genetic hits to other oncogenes or Ets1 in Squamous Neoplasia tumor suppressor genes are not required. Hence, over-expression of Ets1 alone is sufficient to cause severe epidermal dysplasia. The dysplastic phenotype induced by Ets1 expression was stronger in animals induced at a young age (less than 3 months of age). The reason for this age-dependent affect is not yet clear, however, it may potentially be due to lower levels of expression of the transgene in older animals or to changes in the physiology of the epidermis between young and old mice. When we induced BT mice at a young age followed by suppression of Ets1 expression and re-induction at an older age, we noted that the phenotype in the second induction was also overall weaker. This weaker phenotype allowed a longer induction period after which discrete ulcerative or papillomatous lesions arose in these re-induced mice. The appearance of discrete lesions in an otherwise weakly affected epithelium and the need for a long induction period strongly suggests that additional genetic hits must occur in these tissues to promote lesion formation. Alternatively, it is possible that stochastic down-regulation of the Ets1 transgene expression via epigenetic changes could result in the expression of the transgene in discrete patches in the adult. Cells already committed to differentiation are targets of Ets1 action Oncogenic mutations in stem cells have been implicated in the development of skin tumors [54,55]. Epidermal stem cells are thought to reside in the hair follicle bulge region as well as being scattered throughout the basal layer of the interfollicular epidermis. Interestingly, in our transgenic model system, Ets1 overexpression is not targeted to the epidermal stem cells, but rather to keratinocytes that have already committed to differen- tiation. This is in contrast to most other transgenic models of SCC, where transgene expression is targeted to the basal proliferative layer (e.g., oncogenic Ras, [56]). Indeed, when the Ets1 transgene is expressed in the basal compartment of the epidermis (including epidermal stem cells) using a keratin 5 (K5) tTA transgenic line, no phenotype is evident in adult BT mice (our unpublished data). Interestingly expression of c-myc in the differentiated layers of the skin also results in hyperproliferation and formation of papillomas [57], whereas targeting c-myc to the basal proliferative layer does not promote keratinocyte proliferation or papilloma formation [58]. Thus, Ets1 and c-myc appear to share a property that allows them to induce pre-neoplastic changes in epidermal cells that have already committed to terminal differentiation. Ets1 regulates the expression of a variety of genes implicated in tumorigenesis Mice expressing the Ets1 transgene exhibit upregulation of many known and potential Ets1 target genes involved in proliferation, survival and apoptosis. These genes include proteases, cell cycle regulators, anti-apoptotic proteins and transcription factors. The phenotype arising in the induced BT mice likely reflects the combined action of several of these genes. For instance, upregulation of Myc in the suprabasal epidermal layers has been shown to lead to a similar epidermal phenotype characterized by reversible epidermal papillomatosis and seba- ceous gland enlargement [57,59]. Thus, upregulation of Myc could contribute to multiple aspects of the dysplastic phenotype observed in Ets1 BT mice. Another group of proteins that could potentially contribute to Ets1 induced dysplastic epidermal changes is the AP-1 family. AP-1 proteins control transcriptional activation or suppression of a number of genes involved in proliferation, differentiation and transformation. Of the AP-1 proteins that are upregulated in Ets1 BT skin, Jun and Fos have PLoS ONE | www.plosone.org 11 January 2009 | Volume 4 | Issue 1 | e4179