Ets1 Induces Dysplastic Changes When Expressed in Terminally-Differentiating Squamous Epidermal Cells

Sinha S (2009) Ets1 Induces Dysplastic Changes When Expressed in Terminally-Differentiating Squamous Epidermal Cells. PLoS ONE 4(1): e4179. doi:10.1371/journal.pone.0004179 Ets1 Induces Dysplastic Changes When Expressed in Terminally-Differentiating Squamous Epidermal Cells Priyadharsini Nagarajan 0 Neha Parikh 0 Lee Ann Garrett-Sinha 0 Satrajit Sinha 0 Juha Klefstrom, University of Helsinki, Finland 0 Department of Biochemistry, State University of New York at Buffalo, Center for Excellence in Bioinformatics and Life Sciences , Buffalo, New York , United States of America Background: Ets1 is an oncogene that functions as a transcription factor and regulates the activity of many genes potentially important for tumor initiation and progression. Interestingly, the Ets1 oncogene is over-expressed in many human squamous cell cancers and over-expression is highly correlated with invasion and metastasis. Thus, Ets1 is believed to mainly play a role in later stages of the oncogenic process, but not early events. Methodology/Principal Findings: To better define the role of Ets1 in squamous cell carcinogenesis, we generated a transgenic mouse model in which expression of the Ets1 oncogene could be temporally and spatially regulated. Upon Ets1 induction in differentiating cells of stratified squamous epithelium, these mice exhibited dramatic changes in epithelial organization including increased proliferation and blocked terminal differentiation. The phenotype was completely reversed when Ets1 expression was suppressed. In mice where Ets1 expression was re-induced at a later age, the phenotype was more localized and the lesions that developed were more invasive. Many potential Ets1 targets were upregulated in the skin of these mice with the most dramatic being the metalloprotease MMP13, which we demonstrate to be a direct transcriptional target of Ets1. Conclusions/Significance: Collectively, our data reveal that upregulation of Ets1 can be an early event that promotes preneoplastic changes in epidermal tissues via its regulation of key genes driving growth and invasion. Thus, the Ets1 oncogene may be important for oncogenic processes in both early and late stages of tumor development. - Funding: These studies were supported by grants from the American Cancer Society and the National Institutes of Dental and Craniofacial Research. Neither funder was involved in design or conduct of the study nor in data collection and analysis or manuscript preparation/review/approval. Competing Interests: The authors have declared that no competing interests exist. The stratified squamous epithelium of the skin forms a barrier between the underlying tissues and the outer milieu to prevent the passage of water and other substances between these compartments. Keratinocytes are the principal cell type found in stratified squamous epithelia and generate biomolecules that are necessary for the stability and resistance of the epithelial layer to mechanical stress. The innermost layer of this stratified epithelium, known as the basal layer (stratum basale), consists of a proliferative compartment of undifferentiated cells. Basal cells periodically withdraw from the cell cycle, detach from the basement membrane and migrate outwards to enter the suprabasal compartment. Differentiation of keratinocytes can be monitored by the morphological appearance of the cells and by the expression of particular marker proteins. Based on these criteria, the differentiated layers of the epidermis can be visualized as three separate regions: the spinous or prickle cell layer (stratum spinosum), the granular layer (stratum granulosum) and the cornified layer (stratum corneum). Squamous cell carcinoma (SCC) is a malignant tumor of skin keratinocytes that frequently arises in response to excessive sun exposure or to chronic irritation. Numerous mouse models of squamous cell cancer have been developed including carcinogeninduced SCC, ultraviolet (UV) light-induced SCC and spontaneous SCC in various transgenic and knockout mouse strains. Several common genetic alterations have been detected in squamous cell tumors, including upregulation of oncogenes and mutation of tumor suppressor genes [1,2]. In addition, several recent studies have reported upregulation of the Ets1 protooncogene in human SCC arising in skin [3] and other stratified epithelia [4,5,6,7,8]. Moreover, upregulation of Ets1 expression has also been detected in animal models of oral SCC [9,10]. Expression of high levels of Ets1 is correlated with increased invasiveness and metastatic potential in both human SCC and animal models of SCC. Ets1 is a transcription factor that regulates the expression of many key genes that are involved in cell growth, survival and invasion. Importantly, Ets1 is thought to regulate the expression of proteases such as urokinase plasminogen activator (uPA) and various members of the matrix metalloprotease family (MMP1, MMP2, MMP3, MMP9 and MMP13) [11,12,13,14,15,16]. Expression of these proteases is likely required for tumor cells to degrade the surrounding extracellular matrix, invade nearby tissues and eventually metastasize to distant sites. In addition, to these proteases, Ets1 is also implicated in upregulation of a widevariety of genes that may promote tumorigenesis including genes that control cellular proliferation or survival (bax, bcl-2, caspase-1, cmyc, CDK11, Fas ligand, GADD153, JunB, mdm2, p16, p21 and p53) [17,18,19,20,21,22,23,24,25,26,27,28] and genes involved in responses to a hypoxic environment [29]. Furthermore, Ets1 has been reported to directly interact with the tumor suppressor protein p53 and modulate its transcriptional activity [30,31,32,33,34]. Because Ets1 regulates the expression of a large cohort of target genes that influence cellular proliferation and survival, Ets1 might play an important role in early stages of the carcinogenic process in addition to its better known role in tumor invasion and metastasis. To investigate the ability of Ets1 to drive neoplastic or pre-neoplastic changes in stratified squamous epithelial cells, we developed a transgenic mouse model that allows us to inducibly express high levels of Ets1 protein in skin epithelium. Using this inducible model system, we have explored the role of Ets1 in SCC. Ets1 is expressed predominantly in the proliferative basal layer of the stratified squamous epithelium Although Ets1 has previously been reported to be expressed in embryonic mouse skin [35], the exact expression pattern of this transcription factor during epidermal keratinocyte differentiation has not been determined. We performed immunostaining for Ets1 protein in the skin epithelium of newborn and adult mice, which showed that expression of Ets1 is predominantly restricted to the nuclei of proliferative basal layer keratinocytes of the epidermis and hair follicles of newborn mice (Fig. 1A). In contrast, more differentiated suprabasal cells expressed little or no Ets1 p (...truncated)