Progression of genotype-specific oral cancer leads to senescence of cancer-associated fibroblasts and is mediated by oxidative stress and TGF-β.
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 β-galactosidase (SA β-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-β1 (TGF-β1) and TGF-β2 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-β antibodies. Fibroblast activation by TGF-β1 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-β1 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-β-dependent mechanisms.
Department of Oral and Dental Sciences, University of Bristol, Bristol BS1 2LY, UK.
Yazan
Y
Hassona
Nicola
N
Cirillo
Kue Peng
KP
Lim
Andrew
A
Herman
Max
M
Mellone
Gareth J
GJ
Thomas
Gayani N
GN
Pitiyage
E Ken
EK
Parkinson
Stephen S
SS
Prime
D000975:Antioxidants / Q000494:pharmacology; D002294:Carcinoma, Squamous Cell / Q000235:genetics; D045744:Cell Line, Tumor; D016922:Cellular Senescence; D017077:Culture Media, Conditioned / Q000494:pharmacology; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000096:biosynthesis; D004249:DNA Damage; D018450:Disease Progression; D005347:Fibroblasts / Q000502:physiology; D005838:Genotype; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015603:Keratinocytes / Q000378:metabolism / Q000502:physiology; D009061:Mouth Mucosa / Q000502:physiology; D009062:Mouth Neoplasms / Q000235:genetics / Q000473:pathology*; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000276:immunology / Q000378:metabolism*; D053781:Transforming Growth Factor beta2 / Q000276:immunology / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000235:genetics; D001616:beta-Galactosidase / Q000378:metabolism
D000975:Antioxidants; D017077:Culture Media, Conditioned; D019941:Cyclin-Dependent Kinase Inhibitor p16; D016877:Oxidants; D017382:Reactive Oxygen Species; C495901:TP53 protein, human; D053773:Transforming Growth Factor beta1; D053781:Transforming Growth Factor beta2; D016159:Tumor Suppressor Protein p53; D006861:Hydrogen Peroxide; D001616:beta-Galactosidase