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. 2012 Jun;32(11):2160-7.
doi: 10.1128/MCB.00305-12. Epub 2012 Apr 2.

Fbw7 and p53 cooperatively suppress advanced and chromosomally unstable intestinal cancer

Jonathan E Grim  1 , Sue E Knoblaugh, Katherine A Guthrie, Amanda Hagar, Jherek Swanger, Jessica Hespelt, Jeffrey J Delrow, Tom Small, William M Grady, Keiichi I Nakayama, Bruce E Clurman
Affiliations

Fbw7 and p53 cooperatively suppress advanced and chromosomally unstable intestinal cancer

Jonathan E Grim et al. Mol Cell Biol. 2012 Jun.

Abstract

Colorectal cancer (CRC) remains a major cause of cancer mortality worldwide. Murine models have yielded critical insights into CRC pathogenesis, but they often fail to recapitulate advanced-disease phenotypes, notably metastasis and chromosomal instability (CIN). New models are thus needed to understand disease progression and to develop therapies. We sought to model advanced CRC by inactivating two tumor suppressors that are mutated in human CRCs, the Fbw7 ubiquitin ligase and p53. Here we report that Fbw7 deletion alters differentiation and proliferation in the gut epithelium and stabilizes oncogenic Fbw7 substrates, such as cyclin E and Myc. However, Fbw7 deletion does not cause tumorigenesis in the gut. In contrast, codeletion of both Fbw7 and p53 causes highly penetrant, aggressive, and metastatic adenocarcinomas, and allografts derived from these tumors form highly malignant adenocarcinomas. In vitro evidence indicates that Fbw7 ablation promotes genetic instability that is suppressed by p53, and we show that most Fbw7⁻/⁻; p53⁻/⁻ carcinomas exhibit a CIN⁺ phenotype. We conclude that Fbw7 and p53 synergistically suppress adenocarcinomas that mimic advanced human CRC with respect to histopathology, metastasis, and CIN. This model thus represents a novel tool for studies of advanced CRC as well as carcinogenesis associated with ubiquitin pathway mutations.

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Figures

Fig 1
Fig 1
Tumorigenesis in FPV mice. (A) Kaplan-Meier overall survival curves for the indicated genotypes, with the cumulative incidence of colon cancer superimposed (purple curve). *, P < 0.01 for FPV compared to all other groups by a log rank test (FPV versus FP, P < 0.001; FPV versus FV, P = 0.006; FPV versus PV, P = 0.005; FP versus FV, P < 0.001; FP versus PV, P = 0.001). (B) Gross appearance of an FPV tumor (T). st, stomach. The ruler shown is in cm. (C to I) Representative FPV tumor histopathology is shown. All sections are stained with hematoxylin and eosin except those in panels J and K. (C) Low-power image of an infiltrative adenocarcinoma. (D and E) High-power image of the section from panel C, showing penetration of neoplastic glands into the muscularis (D) and lymphatics (E). (F) Lymph node metastasis. The architecture is effaced by neoplastic glands (inset, low-power view). (G and H) Low (G, inset)-, medium (G)-, and high-power (H) views of liver metastasis. (I to K) Representative FPV invasive adenocarcinoma, stained with hematoxylin and eosin (I), anti-β-catenin antibody (J), and anti-Ki-67 antibody (K). Diffuse cytoplasmic and nuclear accumulation of β-catenin is indicative of aberrant Wnt activity, whereas Ki-67 staining shows the high proliferative rate of this tumor. Scale bars are in micrometers.
Fig 2
Fig 2
p53 status impacts Fbw7 substrate regulation, proliferation, and cell fate anomalies in the Fbw7 null gut. (A) Western blot analyses of the indicated proteins in lysates obtained from intestinal epithelial cells of the indicated genotypes. (B to E) Quantitated immunostaining of intestines is shown for Paneth cells (lysozyme) (B), goblet cells (combined periodic acid-Schiff and Alcian blue) (C), and proliferating cells (Ki-67) (D). The statistical significance of these findings is shown in panel E. (F) Real-time PCR analyses of the indicated genes in gut epithelial cells from all four genotypes. Triplicate runs of three biologic replicates were performed for each genotype, and data were normalized with GAPDH and expressed relative to the data for the control genotype (FP).
Fig 3
Fig 3
FPV cell lines grow as malignant tumors in NOD/scid/gamma (NSG) mice. Three cell lines derived from FPV tumors, termed FPV1, FPV2, and FPV3, were implanted subcutaneously in NSG mice (n ≥ 2 per cell line; representative sections are shown). H&E-stained sections of subcutaneous tumors are shown at low power (A, B, and C) and high power (D, E, and F). Bars indicate the scale in micrometers.
Fig 4
Fig 4
Aneuploidy in FPV cell lines and tumors. (A) Table summarizing the features of the three FPV cell lines described below. The DNA index is calculated as aneuploid G1 DNA content/diploid G1 DNA content. Note that FPV2 has two separate aneuploid peaks. Mean chromosome numbers are listed, with the ranges indicated in parentheses. The number of metaphase spreads counted is indicated for each cell line. Micronucleation is expressed as a ratio of the cells with micronuclei to the total cell number. (B) Flow cytometry profiles of FPV1, FPV2, and FPV3. Each sample contained normal diploid nucleus kidney cells as controls (blue peaks) in addition to the established tumor cells (red peaks). (C) Representative FPV cell line metaphase spreads. (D) Table summarizing the ploidy of 10 FPV carcinomas determined by flow cytometry. Nuclei were isolated from fixed and embedded tumor samples and processed for flow cytometry. The percentages of total cells that were aneuploid and the DNA indexes as well as the general phenotypes of the aneuploid cells are indicated. The presence of metastases (Mets) in each animal is indicated. LN, lymph node. Note that tumors 1367 and 1634 have two separate aneuploid peaks. (E) Representative profiles of a near-diploid tumor (left) and a near-tetraploid tumor (right). Aneuploid peaks are shown in red, and normal diploid cells are shown in dark blue. (F) Array comparative genomic hybridization of three FPV tumors (after laser capture microdissection) and three FPV cell lines. DNA was labeled after whole-genome amplification and hybridized to Agilent CGH arrays. Normal tissues were used as control DNAs, and the cell lines were compared with sex-mismatched normal spleen DNA.
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