The process of squamous differentiation is essential in order for keratinocytes to alter both biochemically and morphologically into specialised, stratified squamous epithelial structures. Keratinocytes derived from the epidermis are a well-characterised example of epithelial cells which undergo squamous differentiation. As keratinocytes migrate towards the outermost layer of the epidermis, expression of proliferation-specific genes, such as cdc2 and E2F1, are repressed and differentiation-specific genes are activated. The expression of these differentiation-specific genes, such as keratin 10, transglutaminase type 1, involucrin, and loricrin, all contribute to the highly keratinised structure of the epidermis. Consequently, deregulation of squamous differentiation can compromise the essential barrier functions of skin. Furthermore, disruptions to the balance between proliferation and differentiation of epithelial cells can result in the development of malignant squamous cell carcinomas.
The E2F family of transcription factors (E2Fs 1-7) are best known for their essential role in regulating cellular proliferation. By association and temporal dissociation from retinoblastoma pocket protein members, E2Fs drive the progression of a cell through the cell cycle. Because of this role, it is not surprising that E2Fs, in particular E2F1, have been found to be aberrant in cancer. Whilst deregulated E2F1 is known to contribute to the hyperplastic phenotype characteristic of cancer, the consequence of aberrant E2F to the process of differentiation is relatively unknown.
This study aimed to examine the possible role of E2F in squamous differentiation and how the deregulated E2F observed in squamous cell carcinomas may contribute to the aberrant terminal differentiation programme of these tumour cells. By using human epidermal keratinocytes as a model, we investigated the consequences of E2F overexpression and inhibition in these cells. Our study demonstrated for the first time that E2Fs can play a dual role in keratinocyte proliferation and squamous differentiation and are also able to act as modulators of squamous differentiation. In this role, E2Fs contribute to the proliferation of keratinocytes until the cells receive an anti-mitogenic signal, such as E2F inhibition. This growth arrest in keratinocytes is associated with the downregulation of E2F-mediated transcriptional regulation of proliferation-associated genes. Simultaneously, downregulation of E2F also causes the relief of E2F-induced repression of differentiation-specific genes and permits the keratinocytes to respond to the actions of differentiation-inducing agents. Once committed to undergo squamous differentiation, E2Fs then assume their alternative role as negative modulators of differentiation in keratinocytes.
Although the precise molecular pathway by which E2F represses squamous differentiation is yet to be deciphered, our studies indicate that E2F modulation of squamous differentiation is mediated through a downstream regulatory factor, Sp1. In this role, E2Fs inhibit the expression of the differentiation-promoting transcription factor Sp1, thus preventing transcriptional activation of differentiation-specific genes. We extend these findings in a squamous cell carcinoma cell line and demonstrate that E2F inhibition is able to alleviate the differentiation-incompetence associated with these cells. Furthermore, subsequent overexpression of Sp1 is sufficient to induce the expression and activity of differentiation-specific genes. Thus, this thesis reports that: (i) E2Fs are pro-proliferative and can inhibit the initiation of squamous differentiation, (ii) E2F inhibition sensitizes keratinocytes to the action of differentiation-inducing stimuli, (iii) an independent differentiation stimulus is required that acts, in part, through the activation of Sp1, (iv) Sp1-mediated differentiation activation is suppressed by E2Fs and (v) E2Fs can negatively regulate differentiation-specific gene expression.
Taken together, this study identifies a novel role for the E2F family of transcription factors in the regulation of squamous differentiation in keratinocytes. In addition, we identify that this role may be disrupted in squamous cell carcinomas thereby contributing to both the hyperproliferation and differentiation-incompetence characteristic of this disorder. Thus, it may be feasible to develop therapies targeting aberrant E2F in squamous cell carcinomas in the hope of down regulating hyperproliferation as well as reinstating a normal differentiation programme in neoplastic cells.