Transforming Growth Factor-alpha reduces carcinogen-induced DNA damage in mini-organ cultures from head-and-neck cancer patients

Abstract

Epidermal Growth Factor Receptor (EGFR) signalling is particularly important in the biology of the vast majority of solid human malignancies. EGFR is over-expressed in up to 90-100% of head-and-neck squamous cell carcinomas (HNSCC), and increased expression of EGFR and its ligand Transforming Growth Factor-alpha (TGF-alpha) is not limited to malignant cells, but also detected in histologically normal mucosa of HNSCC patients, supporting the hypothesis of field carcinogenesis. Permanent EGFR activation via an autocrine stimulatory pathway is thought to be a major factor forcing pre-neoplastic tissue towards malignancy. Our study evaluates the impact of stimulation by TGF-alpha on carcinogen-induced and oxidative DNA damage in mucosa tissue cultures of macroscopically normal biopsies from tumour patients and controls. Effects of TGF-alpha on DNA-repair capacity were investigated. To assess DNA fragmentation, alkaline single-cell gel electrophoresis (comet assay) was used. Stimulation of cultures during 24 h with TGF-alpha decreased benzo(a)pyrene diolepoxide (BPDE)-induced DNA damage by 36% in the tumour group (p < 0.001) and by 7% in controls (n = 30). No statistically significant impact on oxidatively induced DNA fragmentation in both groups (n = 15 and 20, respectively), or DNA repair could be shown n (n = 6). The exact mechanism by which TGF-alpha stimulation reduces BPDE-induced DNA fragmentation remains unclear. It was shown in clinical studies, that EGFR targeting has synergistic effects with chemotherapy in HNSCC and reverses chemo-resistance of epithelial tumours, which was shown to be the consequence of altered expression of multidrug resistance (mdr) efflux pumps. EGFR downstream signalling regulates the transcription of MDR1 (p-glycoproteine) and glutathione homeostasis. BPDE is a substrate of mdr1 and is detoxified by glutathione conjugation. However, our results show a strong DNA-stabilizing effect of stimulation by TGF-alpha in mucosa tissue cultures of tumour patients and may therefore be seen as a physiological response to continued carcinogenic impact on the epithelium of the upper aerodigestive tract.