Given the frequent SFRP1 gene silencing in a wide variety of tumor types as well as SFRP1-mediated inhibition of tumor cell proliferation, SFRP1 has long been considered a tumor suppressor, but its biological function(s) relevant to tumor suppression remained poorly defined. This study revealed a novel senescence-inducing activity of SFRP1 and raised a possibility that SFRP1 suppresses tumors by acting as a secreted mediator of cellular senescence.
We have found that different stresses such as DNA or oxidative damage induce SFRP1 secretion from human primary fibroblasts ( and ) and that secreted SFRP1 in turn mediates senescence phenotypes in stressed fibroblasts ( to and ). Lentiviral expression of SFRP1 caused proliferation arrest and senescence phenotypes in primary fibroblasts ( to ). Furthermore, addition of purified recombinant SFRP1 to the culture medium induced senescence in primary fibroblasts (), which suggests that extracellular SFRP1 can directly induce senescence. SFRP1 also mediated senescence in epithelial cells. Upon etoposide treatment, retinal pigment epithelial cells oversecreted SFRP1 (), and SFRP1 shRNAs or anti-SFRP1 antibody treatment attenuated etoposide-induced senescence in these cells ( and ). Further, recombinant SFRP1 induced a senescence phenotype in retinal pigment epithelial cells (), mammary epithelial cells (), and MCF-7 breast cancer cells (). MCF-7 cells are one of many cancer cell lines whose SFRP1 loci are silenced by promoter methylation (38
), and senescence induction by reexposing MCF-7 to SFRP1 is consistent with the notion that the evasion of SFRP1-induced senescence contributes to tumorigenesis. Lending further support to this notion is the finding that the senescence-inducing activity of SFRP1 is impaired in SFRP1 mutants found in glioblastoma and colon cancer (). Inactivation of SFRP1 by promoter methylation or by mutation of the coding region may allow preneoplastic cells to escape stress-induced senescence and accumulate further mutations to develop into a full-blown tumor. We note, however, that SFRP1 is reported to sensitize cells to apoptosis (27
), which might also contribute to tumor suppression.
We also provided several lines of evidence suggesting that SFRP1 induces senescence by antagonizing Wnt signaling. SFRP1 expression in IMR-90 fibroblasts, which induced senescence phenotypes, also resulted in reduced soluble β-catenin levels () and repression of Wnt/β-catenin-dependent transcription (). SFRP1-induced senescence was inhibited by coexpression of Wnt3 () or by lithium chloride-mediated stimulation of Wnt signaling (). Furthermore, cancer-associated SFRP1 mutants that display compromised senescence-inducing activity were also defective for antagonizing Wnt signaling (). We also demonstrated senescence-inducing activity for all five SFRP family members () as well as DKK1 (), which belongs to a distinct class of secreted Wnt antagonists. The role of Wnt inhibition in cellular senescence was also supported by senescence induction upon pharmacological inhibition of Wnt signaling () and upon knockdown of β-catenin (). Previous work by Ye et al. indicated that Wnt2 expression and Wnt signaling are repressed in human fibroblasts undergoing replicative senescence or Ras-induced senescence and that Wnt2 RNAi induces senescence phenotypes in human fibroblasts (44
). Our study extended these observations by revealing the role of secreted Wnt antagonists in senescence induction and further established the link between Wnt downregulation and cellular senescence.
In terms of the downstream target of SFRP1, we demonstrated that SFRP1-induced senescence is accompanied by dephosphorylation of Rb () and that Rb knockdown abolishes SFRP1-induced senescence (), indicating the critical importance of the Rb pathway in SFRP1-induced senescence. p53 knockdown also abolished SFRP1-induced senescence (); however, we believe this is secondary to the compromised Rb dephosphorylation by SFRP1 in p53 knockdown cells () since SFRP1 expression in primary fibroblasts did not induce p53 or a p53 transcriptional target, p21 (). β-Catenin RNAi-induced senescence was also accompanied by Rb dephosphorylation (), and Rb knockdown abolished β-catenin RNAi-induced senescence (), further supporting the role of the Rb pathway in Wnt downregulation-induced senescence.
One possible mechanism for Rb activation by Wnt downregulation is through repression of Wnt/β-catenin target genes such as CDC25A, c-Myc, and cyclin D, which play critical roles in the G1
/S transition and regulate Rb phosphorylation. However, we did not observe significant changes in the expression of these Wnt target genes upon SFRP1-induced senescence (data not shown). Alternatively, a nontranscriptional function(s) of Wnt/β-catenin pathway may modulate Rb phosphorylation and senescence induction. Recent work demonstrated that Wnt signaling inactivates GSK3 by sequestering the enzyme in multivesicular bodies (MVBs), which affects the half-life of 20% of all cellular proteins (39
). Interestingly, β-catenin also localizes in MVBs and is required for MVB formation (39
). Altered half-lives of critical signaling proteins upon Wnt downregulation might contribute to Rb dephosphorylation and senescence induction.
While a large body of evidence supports the role of SFRP1 and other SFRP family members as human tumor suppressors, inactivation of SFRP family members in mice, thus far, has not been shown to result in tumorigenesis. SFRP1 knockout mice displayed abnormal bone formation but did not develop tumors up to 2 years of age (41
). We tested the effect of SFRP1 expression in mouse embryonic fibroblasts and did not observe proliferation arrest or senescence phenotypes. There are a number of important differences between human and mouse cells including a critical difference in sensitivity to oxidative stress (33
). In fact, mouse embryonic fibroblasts were reported to undergo senescence upon Wnt signaling due to increased mitochondrial biogenesis and concomitant generation of reactive oxygen species (45
). Although the lack of tumors in SFRP knockout mice could be due to compensation by remaining SFRP family members, it is also possible that there are species differences in senescence induction and tumor suppression by SFRPs.
Wnt signaling is deregulated in a large number of human tumor types, and together with Notch signaling, it was classified as one of 12 commonly altered, core signaling pathways in human pancreatic cancers (19
). Recently, several chemical inhibitors of Wnt signaling were developed and were reported to display antitumor activity (7
). A chemical screen for compounds that both stabilize Axin and promote β-catenin turnover identified an FDA-approved drug, pyrvinium pamoate, as a potent inhibitor of Wnt signaling (40
). Pyrvinium selectively activates casein kinase 1α, which results in stabilization of Axin and degradation of β-catenin and Pygopus, a nuclear cofactor of β-catenin. Pyrvinium treatment of colon cancer cells with deregulated Wnt signaling efficiently inhibited proliferation (40
). Consistent with a link between Wnt downregulation and cellular senescence, we found that pyrvinium displays senescence-inducing activity (). Compounds that mimic or enhance the action of SFRP1 and other secreted Wnt antagonists may become a new class of anticancer agents that suppress cancer proliferation by inducing senescence.
Based on the results presented here, we propose that 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. As noted above, the other four SFRP family members as well as DKK1 are also able to induce senescence although the stimuli that induce the secretion of these Wnt antagonists are not well understood. Future work should clarify the precise roles of secreted Wnt antagonists in cellular senescence and tumor suppression.