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. 2011 Jan 11;108(2):704-9.
doi: 10.1073/pnas.1015027108. Epub 2010 Dec 27.

E2f Binding-Deficient Rb1 Protein Suppresses Prostate Tumor Progression in Vivo

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Free PMC article

E2f Binding-Deficient Rb1 Protein Suppresses Prostate Tumor Progression in Vivo

Huifang Sun et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Mutational inactivation of the RB1 tumor suppressor gene initiates retinoblastoma and other human cancers. RB1 protein (pRb) restrains cell proliferation by binding E2f transcription factors and repressing the expression of cell cycle target genes. It is presumed that loss of pRb/E2f interaction accounts for tumor initiation, but this has not been directly tested. RB1 mutation is a late event in other human cancers, suggesting a role in tumor progression as well as initiation. It is currently unknown whether RB1 mutation drives tumor progression and, if so, whether loss of pRb/E2f interaction is responsible. We have characterized tumorigenesis in mice expressing a mutant pRb that is specifically deficient in binding E2f. In endocrine tissue, the mutant pRb has no detectable effect on tumorigenesis. In contrast, it significantly delays progression to invasive and lethal prostate cancer. Tumor delay is associated with induction of a senescence response. We conclude that the pRb/E2f interaction is critical for preventing tumor initiation, but that pRb can use additional context-dependent mechanisms to restrain tumor progression.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Pituitary and thyroid tumorigenesis in Rb1-deficient mice. (A) The graph shows cumulative survival for Rb1-deficient mice of the indicated genotypes (sample size = n). Mice dying for reasons unrelated to tumorigenesis are censored from the data and indicated by cross hatches. Differences in cumulative survival between Rb1 or Rb1654 mice, either in the presence or absence of E2f1, are not statistically significant by log rank test (P > 0.05). (B) Western blot analysis of pRb in tissue extracts from mice of the indicated genotypes. The letter B represents nontumor-bearing brain tissue and serves as a positive control. The letters T and P are extracts from thyroid or pituitary tumors, respectively. Hsp70 levels serve as a protein loading control. (C) RNA was extracted from the indicated tissues of Rb1654/+ mice, amplified by RT-PCR, and sequenced. The chromatogram is shown for the sequence surrounding codon 654 (wild type, CGA; Rb1654, TGG).
Fig. 2.
Fig. 2.
Prostate tumorigenesis in Rb1-deficient mice. (A) The graph displays cumulative survival for Rb1 and Rb1654 mice (sample size = n). Mice dying for reasons unrelated to tumorigenesis have been censored from the data and are indicated by cross hatches. Survival of Rb1654 mice is significantly longer than Rb1 mice by log rank test (P < 0.001). (B) Rb1 protein expression was analyzed in tissues from mice of the indicated genotypes by Western blotting. The letter B is normal brain tissue and serves as a positive control. LM, NM, and P represent liver metastasis, lymph node metastasis, and primary prostate tumor, respectively. Hsp70 serves as a protein loading control. (C) RNA was extracted from the indicated tissues of Rb1654 mice, amplified by RT-PCR, and sequenced. The chromatogram is shown for the sequence surrounding codon 654 (wild type, CGA; Rb1654, TGG).
Fig. 3.
Fig. 3.
Senescence in PrE cultured in vitro. (A) PrE rescued from embryos of the indicated genotypes were cultured in vitro under optimal growth conditions, in the absence of serum, or upon treatment with H2O2. Cells were stained for SA-bgal activity and representative images captured. (Scale bar, 25 μM.) (B) The fraction of PrE of the indicated genotypes staining positive for SA-bgal activity was quantitated with or without H2O2 treatment. The results show the mean and SD for three independent experiments. The percentage of SA-bgal positive cells is significantly higher in H2O2-treated Rb1654/− (P < 0.001) or wild-type PrE (P < 0.001) than in treated Rb1−/− PrE by Student's t test. (C) PrE isolated from proximal prostate tissue of Rb1654 or Rb1 adult mice were treated with H2O2 and the fraction of SA-bgal positive cells counted. SA-bgal staining is not observed in untreated cells. The results show the mean and SD of three independent experiments. The percentage of SA-bgal positive cells is significantly higher in Rb1654 PrE than in Rb1 PrE (P = 0.004 by Student's t test). (D) PrE from C with or without H2O2 treatment were pulse labeled with BrdU and the fraction of cells incorporating BrdU counted. The results show the mean and SD of three independent experiments. The percentage of BrdU-labeled cells is significantly lower in H2O2-treated Rb1654 PrE than in similarly treated Rb1 PrE (P = 0.003 by Student's t test).
Fig. 4.
Fig. 4.
Senescence in prostate tissue in vivo. (A) Frozen tissue sections showing proximal prostatic ducts from mice of the indicated genotypes were stained for SA-bgal activity and counterstained with eosin. Representative duct cross-sections are marked with the letter D. (Scale bar, 50 μm.) (B) Frozen sections of prostate tumors from Rb1654 or Rb1 mice were stained for SA-bgal and counterstained with eosin. (Scale bar, 200 μm.) (C) Frozen sections of rescued prostate tissue of the indicated genotype treated with T+E2 were stained for SA-bgal activity. Representative ducts are marked with the letter D. (Scale bar, 50 μm.)
Fig. 5.
Fig. 5.
Cell proliferation and p16 immunostaining in prostate tissue. (A) Proximal prostate tissue sections from an 18-wk-old Rb1 mouse or a 22-wk-old Rb1654 mouse (both selected for the presence of mPIN lesions) as well as a wild-type mouse, were immunostained for p16 and counterstained with hematoxylin. P marks ducts with involved mPIN lesions; D marks a normal duct. (Scale bar, 50 μm.) (B) Consecutive sections from tissue in A were immunostained for pH3 and counterstained with hematoxylin. P marks ducts with involved mPIN lesions. The graph at Right depicts the percentage of pH3 positive cells in mPIN lesions, primary prostate tumors, or metastatic tumors from mice of the indicated genotype. The mean and SD are shown for 29 Rb1 mPIN lesions, 15 Rb1654 lesions, two primary tumors for each genotype, and multiple metastatic tumors from two different mice for each genotype. The asterisk marks a statistically significant difference between genotypes (Student's t test, P < 0.05). (C) Prostate ventral lobe sections from mice of the indicated genotype were immunostained for p16 and counterstained with hematoxylin. The box outlines the area magnified in the Lower panel. The arrow indicates a representative atypical preneoplastic cell positively stained for p16. The arrowhead shows an atypical cell lacking p16 staining. (Scale bars, 50 μm.)
Fig. 6.
Fig. 6.
E2f target gene expression in prostate tissue. RNA was extracted from prostate tissue of the indicated genotype and the expression levels of the indicated genes determined by quantitative RT-PCR. The data represent the fold change in expression relative to a wild-type prostate tissue reference sample, normalized to β-actin RNA levels. The mean and SD of data from two mice for each genotype, each performed in triplicate, are shown. The asterisk marks a statistically significant difference between genotypes (Student's t test, P < 0.05).

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