Role of Intracellular IL-1RA in Oral Keratinocyte Senescence and Oral Cancer Development

Introduction: Head and neck cancer still causes significant mortality, with a survival rate of 50% during the first 5 years. There is a need for identification of the molecules involved in the oral malignant transformation process that could be targeted in an attempt to reduce malignant transformation rates of precursor lesions. Recently, the IL-1 receptor antagonist (IL-1RA) has been reported to be downregulated in head and neck squamous cell carcinoma, but it is not known how its downregulation affects oral cancer development. IL-1RA seems to be important for the regulation of cellular senescence (which includes the senescence-associated secretory phenotype or SASP), an anti-tumour response that can promote cancer development if not well regulated. Thus, the aim of this project was to explore the function of IL-1RA in oral carcinogenesis, keratinocyte senescence, the development of the SASP and its relationship with OSCC development. Methods: The expression of IL-1RN, icIL-1RA, sIL-1RA and IL-1R1 was analysed in a panel of different cell lines, including normal (NOK), dysplastic (OD) and cancerous oral keratinocytes (OSCC) using qPCR, western blot, and confocal microscopy. OSCC and OD cell lines were transiently transfected with a plasmid encoding for icIL-1RA1. Cell migration, cell proliferation and secretion of IL-6 and IL-8 were assessed. Senescence in normal (NOK) and dysplastic oral (OD) keratinocytes was achieved by replicative exhaustions and assessed by evaluating γH2AX by immunofluorescence, p16 and p21 expression by qPCR and immunoblotting, and β-galactosidase activity. SASP factors were analysed using qPCR, western blot or ELISA. The CRISPR/Cas9 system was used to knock out icIL-1RA type 1 (icIL-1RA1) in primary normal and dysplastic oral keratinocytes. Results: icIL-1RA type 1 (icIL-1RA1) is endogenously expressed in NOKs and is downregulated in OD and OSCC. Transient re-expression of icIL-1RA1 in immortal OD and OSCC cell lines showed limited effects on proliferation, migration and cytokine secretion During senescence of normal and dysplastic oral keratinocytes, IL-1RA expression decreases, which is accompanied with an increase in the expression of established components of the SASP. Knock out of icIL-1RA1 in NOK and OD cells (KO OD) caused a significant increase of IL-6, IL-8 and phospo-P65 and induced premature senescence of OD cells compared to wild-type (WT). No differences in abundance of IL-6, IL-8 or phospho-P65 between senescent WT and senescent KO OD cells were observed, which corresponded with the endogenous decrease of IL-1RA in WT senescent OD cells. Conclusions: Intracellular IL-1RA1 is endogenously expressed in oral keratinocytes and is downregulated in OD and OSCC. icIL-1RA downregulation is more likely to have pro-oncogenic effect before the cells become immortal or during immortalization (by enabling an increase in IL-6 and IL-8 secretion), rather than when the cells have already achieved immortality. We have also shown that icIL-1RA1 is an important factor in the regulation of senescence and the SASP in oral keratinocytes. Downregulation of icIL-1RA1 in dysplastic cells can facilitate the development of a premature and de-regulated SASP, which may contribute to malignant transformation.