UC San Diego

Senescent cells are one source of the chronic inflammation associated with the diseases and debilities of aging. Whereas cellular senescence in fibroblasts is well documented, how this process is orchestrated in epithelial cells, the origin of human carcinomas, is much less understood. I used normal primary human oral keratinocytes (NOKs) to elucidate senescence programs in a prototype mucosal epithelial cell that undergoes senescence spontaneously. Widely-accepted assays were used to characterize senescence phenotypes in these cells, and showed that p21WAF1/CIP1 is not a reliable marker of senescence for NOKs, as it is for fibroblasts. Transcriptome analysis by RNA-seq, and analysis by mass spectrometry of secreted proteins, both soluble and those associated with extracellular vesicles (EVs), were performed. Findings regarding NOK senescence were buttressed using other methods, including microscopy and immunoblotting. Identification of top upregulated and downregulated genes at the mRNA level emphasizes the pro-inflammatory and cancer evasion aspects of NOK senescence. Two of the donors displayed an expected repression of DNA repair genes, correlating with downregulation of E2F1 mRNA and protein, but a divergent result was evidenced for the third donor. Analysis of RNA-seq results within the context of the canonical senescence associated secretory phenotype (SASP) and inflammatory pathways (NF-κB, interferon, and p38MAPK) known to operate in senescence was conducted, and secretion of elements of the canonical soluble SASP was measured. RNA-seq results highlighted potential senolytic targets, including immune evasion mechanisms. The secretome analysis identified a number of pro-inflammatory damage associated molecular patterns (DAMPs) and other molecules that likely contribute to the deleterious effects of senescent cells. The data enabled me to propose additions to the senescence associated secretory phenotype, including HSP60, which was found on the surface of EVs. A co-incubation experiment showed that EVs from senescent NOKs can create inflammation by stimulating STING-dependent interferon pathway signaling in THP-1 monocytes. Sequencing of DNA from these EVs showed that they are associated with mitochondrial and nuclear DNA. These results highlight important senescence changes in epithelial cells in terms of how these cells contribute to chronic inflammation and age-related diseases, and how this inflammation might be alleviated.