Abstract A16: Wnt antagonist SFRP1 functions as secreted mediator of senescence

Abstract The purpose of this study was 1) to identify the mediator(s) of senescence that are secreted from senescent cells and induce senescence in an autocrine and paracrine fashion and 2) to dissect the mechanism of senescence induction by such secreted mediator(s). Cellular senescence has emerged as critical tumor suppressive mechanism in recent years, but relatively little is known about how senescence occurs. We undertook a quantitative proteomic analysis of proteins secreted from human primary fibroblasts induced to senesce by DNA damage, Ras oncogene, or replicative telomere shortening, and determined that a number of previously reported SASP (senescence-associated secretory phenotype) factors are oversecreted in different combinations from three types of senescent cells. This analysis also identified the oversecretion of SFRP1, a secreted antagonist of Wnt signaling, upon DNA damage-induced senescence. SFRP1 is a tumor suppressor whose expression is frequently silenced by promoter methylation in various types of human cancers. SFRP1 oversecretion occurred upon treatment with different DNA damaging agents or by oxidative stress. Interestingly, nonsenescent fibroblasts acquired senescence phenotypes upon co-culture with senescent fibroblasts induced to senesce by DNA damage or by SFRP1 overexpression; this effect was alleviated by the addition of anti-SFRP1 antibodies to the culture medium. This experiment suggested that secreted SFRP1 can mediate the “spreading of senescence.” We went on to show that SFRP1 lentiviral expression or recombinant SFRP1 treatment induces senescence. Conversely, SFRP1 knockdown or addition of anti-SFRP1 antibodies to the culture medium inhibited the DNA damage-induced or oxidative stress-induced senescence in fibroblasts. The role of SFRP1 in senescence induction was also examined in epithelial cells. Upon etoposide-induced DNA damage, human primary retinal pigment epithelial (RPE) cells displayed SFRP1 oversecretion and underwent senescence. Etoposide-induced senescence of RPE cells was alleviated by SFRP1 shRNAs or by anti-SFRP1 antibody. Further, recombinant SFRP1 induced senescence in RPE cells. Recombinant SFRP1 also induced senescence in primary mammary epithelial cells and MCF-7 breast cancer cells, which lack SFRP1 expression due to promoter methylation. These results suggest that SFRP1 functions as secreted mediator of senescence in both fibroblasts and epithelial cells. Regarding the downstream pathways, we demonstrated that SFRP1 induces senescence by silencing Wnt signaling. A pharmacological inhibition of Wnt signaling or knock-down of β-catenin also resulted in senescence. Furthermore, we demonstrated the senescence-inducing activity for all five members of the SFRP family of secreted Wnt antagonists as well as DKK1, which belongs to a distinct class of secreted Wnt antagonists. SFRP1-induced or Wnt downregulation-induced senescence was accompanied by dephosphorylation of Rb and was abolished by Rb knock-down, indicating the critical importance of the Rb pathway in senescence induction. Although the primary mode of SFRP1 inactivation in tumors appears to be promoter methylation, there are also reports of cancer-associated mutations in the coding region of SFRP1. We analyzed two SFRP1 mutants found in colon cancer and glioblastoma and determined that both of these SFRP1 mutants are defective for antagonizing Wnt signaling and inducing senescence. Based on these results, we propose SFRP1 is an extracellular component of stress-induced senescence signaling that responds to potentially carcinogenic stresses such as DNA damage and oxidative insult and induces cellular senescence in an autocrine and paracrine fashion, which may lead to non-cell autonomous tumor suppression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A16.