ESTRATEGIAS DE COMUNICACIÓN Y DIVULGACIÓN

The expression pattern of FOXM1 in normal oral epithelium in vivo is consistent with that of a proliferation associated protein as it is predominantly expressed in the parabasal, most proliferative, layers of the oral epithelium. Its expression was not seen in areas of intense p75NTR expression suggesting that FOXM1 is possibly down-regulated in the more immature populations of oral keratinocytes. This hypothesis was confirmed after analysis of the molecular gene expression profile of putative OKSC in vitro.

When keratinocyte populations are expanded in vitro under optimized conditions, they are expected to contain the slow cycling immature population, the more rapidly proliferating transit amplifying keratinocytes as well as terminally differentiated keratinocytes. This heterogeneity was previously shown to exist in ex vivo human epidermal keratinocyte cultures (Eisinger et al., 1979). By isolating each separate population, it is therefore expected that putative keratinocyte stem cells will display features of quiescence.

The evidence presented here, as well as in previous studies, demonstrate that the expression and activity of FOXM1 is mainly restricted to the G2/M phases of the cell cycle (Leung et al., 2001; Laoukili et al., 2008b). Therefore, it is possible that the suppression of FOXM1 in OKSC is a requirement for the maintenance of their slow cycling status. Others have found that the majority of isolated p75NTR+ human oesophageal keratinocytes, are mainly in a G0/G1 state, and are characterized by a slow cell cycle profile in vitro (Okumura et

al., 2003). Expression analysis of human epidermal keratinocyte stem cells in

vitro revealed that the maintenance of their quiescent state is partly attributed

to a negative regulator of c-MYC, namely LRIG1 (Jensen and Watt, 2006). As there is evidence that c-MYC transactivates FOXM1 (Blanco-Bose et al., 2008), it is possible, that LRIG1-induced suppression of c-MYC, indirectly contributed to the observed repression of FOXM1, in OKSC populations. It should be noted however, that while this may be a possible mechanism in normal physiological conditions, it is not necessarily applicable in cases of pathological activation of FOXM1. For example, FOXM1B is highly overexpressed in basal cell carcinomas (BCCs), despite the fact that these tumours exhibit reduced levels of c-MYC protein (Bonifas et al., 2001).

Although keratinocyte stem cells are slow cycling, they are capable of sustained proliferation and are solely responsible for the expansion and replenishment of the keratinocyte population. This can explain the finding that after prolonged culture, FOXM1 protein was present only in the originally isolated p75NTR+ cells. Given that FOXM1 promotes cellular proliferation by regulating the expression of G1/S and G2/M transition specific genes (Wierstra and Alves, 2007), its expression pattern represents the expansion of OKSCs, which give rise to the more rapidly dividing transit amplifying keratinocytes.

During the course of the experiments, it was observed that the initial expression levels of FOXM1 in freshly isolated primary keratinocytes, positively correlated with their clonogenic and proliferative potential in culture (personal observation). FOXM1 is broadly expressed in all embryonic tissues, while in adult organs its expression is restricted to tissues of a high proliferative index (e.g. testis, thymus and bone marrow), where it is important for replenishing the post-mitotic differentiated cells and for tissue expansion in response to injury (Ye et al., 1997). Hepatic deletion of Foxm1b in mice can result in diminution of mitotic entry during liver regeneration, while ectopic Foxm1b is alone sufficient to re-establish proliferation of aged mouse hepatocytes, following partial hepatectomy (Wang et al., 2002b). More recently, Foxm1 was shown to be critical for the maintenance and expansion

of pancreatic β-cell mass and pancreatic-wide deletion of Foxm1 resulted in impaired glucose tolerance and diabetes in mice (Zhang et al., 2006). In the context of epithelial carcinogenesis, a recent report provided in vivo evidence, suggesting that the presence of Foxm1 is essential for the successful expansion of tumourigenic progenies in chemically induced carcinogenesis in mice (Wang et al., 2009). Overall, these observations led to the hypothesis that the initial expression levels of FOXM1 in the keratinocyte stem cell pool may be an important determinant of their total proliferative output, and the successful expansion of their progeny. This is further supported by data presented here, where specific overexpression of FOXM1B in putative OKSC (p75NTR+), increased the ability of keratinocytes for clonal expansion.

The suppression of FOXM1 following prolonged culture of p75NTR- cells, suggests that FOXM1 is down-regulated in the progeny of keratinocytes undergoing terminal differentiation. This is consistent with the finding that FOXM1 protein and mRNA expression levels decline during confluence- induced differentiation of a human epithelial colon carcinoma cell line (Caco- 2) toward the enterocyte cell lineage (Ye et al., 1997). Although the onset of replicative senescence could also contribute to a the downregulation of FOXM1 in primary normal oral keratinocytes, FOXM1 expression levels were diminished in differentiating keratinocytes, well before they entered a state of replicative senescence, which became evident at approximately PD~23-28. Furthermore, FOXM1 protein is absent from the differentiated layers of normal oral epithelium (Figures 3.1, 5.1). The down-modulation of the proliferative effects of FOXM1 may allow exit from the cell cycle and signify the onset of keratinocyte terminal differentiation. The p75NTR- population consists of a good model of in vitro keratinocyte differentiation, in that it is a committed keratinocyte progeny with an early passage history, which allows a clear demonstration of the differentiation programme, devoid of the ‘contaminating’ effects of replicative senescence.