Rap1 Regulates Chemotherapy-induced Senescence-associated Secretory Phenotype In Gastric Cancer Cell Line

Cellular senescence is the state of irreversible cell cycle arrest.Senescent cells secrete inflammatory cytokines,chemokines,growth factors,and proteases known as the senescence-associated secretory phenotype（SASP）involving the production of factors that reinforce the senescence arrest,alter the microenvironment,and trigger immune surveillance of the senescent cells.Therapy-induced SASP（TIS）,although tumor-suppressive by enforcing arrest and promoting immune clearance of damaged cells,also provokes tumor-promoting responses.TIS induces local and systemic inflammation,which contribute to short-and long-term side effects due to chemotherapeutic drugs,including bone marrow suppression,cardiac dysfunction,cancer recurrence,and fatigue.In addition,TIS stimulates proliferation and metastasis of drug-resistant cells and promotes the survival of drug-sensitive cancer cells.Moreover,TIS induces epithelial–mesenchymal transition and increases of cancer cells invasiveness.Mammalian telomere is composed of tandem repeats of the TTAGGG sequence bound by a six-subunit protein complex,which is known as shelterin and composed of TRF1,TRF2,TIN2,POT1,TPP1 and Rap1.Mammalian Rap1 is an unique shelterin subunit that plays a role in transcriptional regulation and NFκB signaling rather than telomere protection and length regulation.Rap1 has been reported to be involved in invasion and therapy-resistance of some cancers by mediating NF-κB signaling.Our previous study showed that etoposide-induced Rap1 upregulation participates in TRF2-mediated DNA damage response（DDR）,which promote the etoposide resistance in gastric cancer cells.Since DDR and NF-κB are regulators of senescence-associated secretory phenotype（SASP）,and therapy-induced SASP promote tumor progression and therapy-resistance,we investigated the role of Rap1in chemotherapy-induced SASP.ObjectivesTo investigated the role of Rap1 in chemotherapy-induced SASP in gastric cancer cell line.Methods5μM etoposide treatment was used to induce senescence in the human gastric cancer cell line SGC7901.Senescence was confirmed by detecting senescence-associatedβ-galactosidase（SA-β-gal）staining and the protein level of p53 and p16^INK4a.mRNAs and protein levels of Rap1 and senescence-associated secretory phenotype（SASP）components were detected by RT-PCR and western blot analysis.The secreted levels of SASP factors in the conditioned medium were detected by ELISA analysis.Examined the effect of Rap1 knockdown on etoposide-induced senescence and SASP in gastric cancer cells using Rap1 siRNA.Results1.5μM etoposide treatment inhibited proliferation of SGC7901 cells with no change in cell density after the etoposide-treatment.Senescence was confirmed by increased senescence-associatedβ-galactosidase（SA-β-gal）staining and accumulation of p53 and p16^INK4a proteins.SASP components,IL1A,IL6,IL8,VEGF-A andIFN-γ,were highly induced in senescent SGC7901 cells and in parallel in the conditioned medium,confirming senescence and SASP of SGC7901 cells in response to etoposide.2.5μM etoposide treatment upregulated Rap1 expression in SGC7901 cells.Rap1 upregulation occurred at an early stage（day 1）of etoposide-treatment and sustained in cells undergoing senescence.3.Rap1 knockdown did not alter etoposide-induced senescence.Etoposide treatment repressed cellular proliferation in both SGC7901-Rap1 KD and SGC7901-NC cells.SGC7901-Rap1 KD cells showed similar SA-β-gal staining,p53and p16^INK4a expression level to those of SGC7901-NC cells under etoposide treatment.4.Rap1 knockdown delayed and attenuated SASP expression.The expression of IL1α,IL6,IL8,VEGF-A and IFN-γin SGC7901-NC cells and in parallel in the conditioned medium started to increase from 5 or 7 days after etoposide treatment,while the increase was observed until day 9 after etoposide treatment in SGC7901-Rap1 KD cells.Furthermore,the overall levels of SASP in SGC7901-Rap1KD cells and in parallel in the conditioned medium were significantly reduced as compared with those of SGC7901-NC cells.ConclusionsRap1 was upregulated and sustained during etoposide-induced senescence in gastric cancer cells.Rap1 knockdown attenuated SASP expression without altering etoposide-induced senescence.