Smurf2 regulates the senescence response and suppresses tumorigenesis in mice

Abstract

The E3 ubiquitin ligase Smurf2 mediates ubiquitination and degradation of several protein targets involved in tumorigenesis and induces senescence in human cells. However, the functional role of Smurf2 in tumorigenesis has not been fully evaluated. In this study, we generated a mouse model of Smurf2 deficiency to characterize the function of this E3 ligase in tumorigenesis. Smurf2 deficiency attenuated p16 expression and impaired the senescence response of primary mouse embryonic fibroblasts. In support of a functional role in controlling cancer, Smurf2 deficiency increased the susceptibility of mice to spontaneous tumorigenesis, most notably B-cell lymphoma. At a premalignant stage of tumorigenesis, we documented a defective senescence response in the spleens of Smurf2-deficient mice, consistent with a mechanistic link between impaired senescence regulation and increased tumorigenesis. Taken together, our findings offer the genetic evidence of an important tumor suppressor function for Smurf2.

Figure 1. Characterization of a mouse model of Smurf2 deficiency

(A) Schematics of the trapped Smurf2 allele. En2-1: mouse En2 intron 1; SA: splicing acceptor; pA: SV40 polyadenylation signal. (B) Southern analysis of genomic DNA of RRA098 cells or Smurf2^+/T mouse tail using Neo as a probe. Specific bands are indicated by arrows at left with DNA ladder at right. (C) Elevated Smurf2 expression in Cre-expressing ES clones, which were identified by genomic PCR with primers P1 and P2 shown in (A). (D) Smurf2 expression in wild-type (+/+), Smurf2^+/T (+/T) and Smurf2^T/T (T/T) MEFs analyzed by Western and quantitative RT-PCR. Smurf2 expression in wild-type was set as 100% after normalization with β-actin. Error bars were standard deviations of three independent experiments. (E) Smurf2 expression in tissues of 2-month-old wild-type and Smurf2^T/T littermates.

Figure 2. Impaired senescence response in Smurf2-deficient MEFs

(A) Cell proliferation and (B) Senescence analyses in early passage (P2) wild-type (+/+), Smurf2^+/T (+/T) and Smurf2^T/T (T/T) MEFs. Student t-test was used in pair-wise comparison with statistical significance indicated as: * (P<0.05), ** (P<0.01), and *** (P<0.001). (C) Immortalization assay of MEFs starting from passage 2 with population doubling (PD) set as 0. (D) Western analysis of p53 and p21 in MEFs following doxorubicin treatment. (E) Western analysis of senescence regulators in MEFs passaged in (B).

Figure 3. Increased spontaneous tumorigenesis in Smurf2-deficient mice

(A) Kaplan-Meier curves of tumor-free survival of wild-type (+/+), Smurf2^+/T (+/T) and Smurf2^T/T (T/T) mice. Statistical significance was analyzed by the log-rank test. (B) Representative spleens of a moribund Smurf2^T/T mouse and an age-matched wild-type mouse. (C) Representative H&E and B220 staining of lymphomas in spleen, liver and kidney of Smurf2^T/T mice, in comparison to sections of wild-type mice. (D) Representative H&E staining of other types of tumors found in Smurf2-deficient mice. Scale bars for 10× and 50× images are 200 µm and 100 µm, respectively.

Figure 4. Impaired senescence response in spleens of aged Smurf2-deficient mice

(A) Representative staining for SA-β-gal activity and quantitation (10 randomly selected fields) in spleens of 12-month-old premalignant wild-type (+/+) and Smurf2^T/T (T/T) mice. (B) Western and quantitative RT-PCR analyses of p16 expression in spleens of 12-month-old mice. The relative expression of p16 in wild-type was set to be 1 after normalization with β-actin. Error bars were standard deviations of three independent experiments. Student t-test was used in statistical analysis (*:P<0.05, **:P<0.01 and ***:P<0.001).