Micromanaging fibroblast senescence: The role of small non-coding RNAs in senescence associated secretory phenotype (SASP)

Introduction: The molecular pathways that dictate the acquisition of senescence-associated secretory phenotype (SASP) by stromal fibroblast during ageing or in response to genotoxic insult have yet to be elucidated. In this thesis the ability of cisplatin-induced SASP of oral fibroblasts to stimulate pro-tumourigenic stromal tumour cross-talk in oral cancer has been studied. Moreover the miRNA expression profile associated with development of SASP in stress-induced premature senescent oral fibroblasts and senescent-CAFs of genetically unstable oral squamous cell carcinoma (GU OSCC) had been thoroughly investigated. Materials and methods: Oral fibroblasts were treated with sub-cyotoxic doses of H2O2 and cisplatin or allowed to undergo replicative exhaustion to induce senescence. Senescence was confirmed by measurement of SA-β-gal activity, cyclin dependent kinase inhibitors (CDKI): p21 and p16, and secreted cytokines and MMP-2 by human cytokine array, ELISA and zymography. MiRNA expression profile was determined by TaqMan miRNA TLDA and candidate miRNAs were validated by qRT-PCR. Transfection was used to study the functional effects of candidate miRNAs. Paracrine effects were assessed by proliferation, migration and invasion assays. pmiR-Reporter vector was used to identify novel functional gene target of miRNAs. Results: Senescent oral fibroblasts showed increased SA-β-Gal activity and higher levels of p21 and p16. They secreted more MMP-2, IL-6, MCP-1, Endothelin-1, PGE2. Inhibiting COX-2 activity and blockade of secreted MCP-1 diminished the stimulatory effect of senescent fibroblasts on oral cancer cells. Both senescent-normal oral fibroblasts and senescent-CAFs demonstrated differential expression of miRNAs. miR-335 and miR-148b were significantly elevated in senescent fibroblasts and was found to target PTEN. A COX-2 mediated inflammatory loop was identified in senescent oral fibroblasts that could be rescued by celecoxib treatment (selective COX-2 inhibitor) via up-regulation of PTEN. Conclusion: In this thesis a SASP-associated miRNA signature had been identified in oral fibroblasts, which may provide insight into the molecular pathways associated with chemotherapy resistance and cancer recurrences in elderly patients