Abstract
Abnormal Sonic Hedgehog signalling leads to increased transcriptional activation of its downstream effector, glioma 2 (GLI2), which is implicated in the pathogenesis of a variety of human cancers. However, the mechanisms underlying the tumorigenic role of GLI2 remain elusive. We demonstrate that overexpression of GLI2-β isoform, which lacks the N-terminal repressor domain (GLI2ΔN) in human keratinocytes is sufficient to induce numerical and structural chromosomal aberrations, including tetraploidy/aneuploidy and chromosomal translocations. This is coupled with suppression of cell cycle regulators p21WAF1/CIP1 and 14-3-3σ, and strong induction of anti-apoptotic signalling, resulting in a reduction in the ability to eliminate genomically abnormal cells. Overexpression of GLI2ΔN also rendered human keratinocytes resistant to UVB-mediated apoptosis, whereas inhibition of B-cell lymphoma 2 (BCL-2) restored endogenous (genomic instability (GIN)) and exogenous (UVB) DNA damage-induced apoptosis. Thus, we propose that ectopic expression of GLI2 profoundly affects the genomic integrity of human epithelial cells and contributes to the survival of progenies with genomic alterations by deregulating cell cycle proteins and disabling the apoptotic mechanisms responsible for their elimination. This study reveals a novel role for GLI2 in promoting GIN, a hallmark of human tumors, and identifies potential mechanisms that may provide new opportunities for the design of novel forms of cancer therapeutic strategies.
Highlights
Basal cell carcinoma (BCC) of the skin is the most common malignancy in humans
Human telomerase-immortalised newborn epidermal (N/TERT) keratinocytes stably expressing enhanced green fluorescent protein (EGFP; SINCE) or enhanced green fluorescent protein-glioma 2 (GLI2)-b isoform, which lacks the N-terminal repressor domain (EGFP-GLI2DN; SINEG2) were generated and expression of the fusion product was confirmed by mRNA expression, fluorescent microscopy, flow cytometry and western blot analysis (Supplementary Figure S1)
The effect of GLI2DN on N/TERT cell proliferation was analysed by the Alamar blue and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) cell viability assays, revealing significantly less SINEG2 cells compared with parental N/TERT or N/TERT keratinocytes stably expressing EGFP (SINCE) cells after 7 days in culture
Summary
Basal cell carcinoma (BCC) of the skin is the most common malignancy in humans. Several studies have provided evidence of GIN in BCCs, highlighting their multiclonal nature and the presence of numerical chromosomal alterations, gene amplification, multinucleated cells and chromosome polysomy.[9,10,11,12,13,14,15] The development of BCC is associated with aberrant activation of the Hedgehog (HH) signalling pathway, which leads to increased expression and activation of glioma (GLI; GLI1 and GLI2) transcription factors, the principal. Received 07.8.13; revised 31.10.13; accepted 04.11.13; Edited by RA Knight effectors of the HH pathway.[16] Targeted expression of an active mutant of GLI2 (GLI2DN – a constitutively active form of GLI2 isoform b, lacking the N-terminal repressor domain) is sufficient to induce the formation of BCC-like lesions in the epidermis of transgenic mice[17,18,19] and is required for their maintenance.[20] GLI2 is consistently upregulated in tumors that display complex genomic alterations.[21,22,23,24,25,26,27,28,29,30] the role of GLI2 in GIN remains elusive. We found that ectopic expression of GLI2DN profoundly affected the genomic stability of human epidermal keratinocytes, partly by suppressing cell cycle checkpoint proteins and disabling the intrinsic apoptotic mechanisms responsible for the elimination of cells with genomic alterations, identifying a novel role for GLI2 in tumorigenesis
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