The role of GLI2 in human basal cell carcinoma tumourigenesis

Abstract

Abnormal Sonic Hedgehog (SHH) signalling leads to increased transcriptional activation of its downstream effector, GLI2, which is implicated in the pathogenesis of a variety of human tumours, including human basal cell carcinoma (BCC). However, little is known about the molecular mechanisms underlying the tumourigenic role of GLI2 in human skin keratinocytes. This study examines the effects of inducible and stable expression of constitutively active GLI2 (GLI2:N) oncogenic transcription factor, on immortalised human epidermal keratinocytes. It is shown here that GLI2:N overexpressing N/TERT keratinocytes display gene expression patterns and phenotypic characteristics reminiscent of those observed in human BCC in vivo. It is also shown for first time, that expression of GLI2:N in N/TERT keratinocytes is sufficient to induce accumulation of binucleated/tetraploid cells as evidenced by an increase in G2/M phase of the cell cycle and binucleate cell counting, and to promote polyploidy and aneuploidy, in the absence of increased cell death or apoptosis. This cell cycle deregulation is accompanied by strong activation of anti-apoptotic protein BCL-2 and simultaneous suppression of important cell-cycle regulators such as 14-3-3σ and CDK inhibitor p21WAF1/CIP1, with no change in p53 protein levels, indicating uncontrolled proliferation of cells with ploidy abnormalities and/or DNA damage. Consistently, it is shown that p21WAF1/CIP1 protein is also absent in human BCC tumours and that forced overexpression of GLI2:N renders human keratinocytes resistant to apoptosis mediated by ultraviolet B (UVB, 290-320 nm), one of the most important etiological factors in BCC formation. Karyotype analysis of GLI2:N N/TERT keratinocytes further demonstrates that overexpression of GLI2:N induces numerical (tetraploidy, polyploidy, aneuploidy) and structural instability in N/TERT keratinocytes including chromosomal translocations and double minute chromosomes. Furthermore, β-catenin activation is the most common alteration observed during aberrant WNT signalling, and is often implicated in the development of human carcinogenesis and metastasis. In this study it is shown that GLI2:N induction induces nuclear accumulation of β-catenin in keratinocyte cell culture and in the basal layer of organotypic skin rafts, similar to human BCCs. In addition, several WNT genes were found to be upregulated upon GLI2:N induction, while β-catenin transcriptional activity is increased upon stable and conditional expression of GLI2:N. Overall these data give new insights for the possible mechanisms that mediate the tumourigenic potential of GLI2.