Progression of genotype-specific oral cancer leads to senescence of cancer-associated fibroblasts and is mediated by oxidative stress and TGF-ß.

ABSTRACT

Keratinocyte senescence acts as a barrier to tumor progression but appears to be lost in late pre-malignancy to yield genetically unstable oral squamous cell carcinomas (GU-OSCC); a subset of OSCC possessing wild-type p53 and are genetically stable (GS-OSCC). In this study, fibroblasts from GU-OSCC were senescent relative to fibroblasts from GS-OSCC, epithelial dysplastic tissues or normal oral mucosa, as demonstrated by increased senescence-associated ß-galactosidase (SA ß-Gal) activity and overexpression of p16(INK4A). Keratinocytes from GU-OSCC produced high levels of reactive oxygen species (ROS) and this was associated with an increase in the production of transforming growth factor-ß1 (TGF-ß1) and TGF-ß2 in stromal fibroblasts. Treatment of normal fibroblasts with keratinocyte conditioned media (CM) from GU-OSCC, but not GS-OSCC or dysplastic keratinocytes with dysfunctional p53, induced fibroblast senescence. This phenomenon was inhibited by antioxidants and anti-TGF-ß antibodies. Fibroblast activation by TGF-ß1 preceded cellular senescence and was associated with increased ROS levels; antioxidants inhibited this reaction. Senescent fibroblasts derived from GU-OSCC or normal fibroblasts treated with CM from GU-OSCC or hydrogen peroxide, but not non-senescent fibroblasts derived from GS-OSCC, promoted invasion of keratinocytes in vitro. Epithelial invasion was stimulated by fibroblast activation and amplified further by fibroblast senescence. The data demonstrate that malignant keratinocytes from GU-OSCC, but not their pre-malignant counterparts, produce high levels of ROS, which, in turn, increase TGF-ß1 expression and induce fibroblast activation and senescence in a p5-independent manner. Fibroblasts from GU-OSCC were particularly susceptible to oxidative DNA damage because of high levels of ROS production, downregulation of antioxidant genes and upregulation of pro-oxidant genes. The results demonstrate the functional diversity of cancer-associated fibroblasts and show that malignant keratinocytes from GU-OSCC reinforce their malignant behavior by inducing fibroblast activation and senescence through ROS and TGF-ß-dependent mechanisms.