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. 2009 May;48(5):408-19.
doi: 10.1002/mc.20479.

Tumor suppressor gene co-operativity in compound Patched1 and suppressor of fused heterozygous mutant mice

Jessica Svärd  1 , Björn Rozell, Rune Toftgård, Stephan Teglund
Affiliations

Tumor suppressor gene co-operativity in compound Patched1 and suppressor of fused heterozygous mutant mice

Jessica Svärd et al. Mol Carcinog. 2009 May.

Abstract

Dysregulation of the Hedgehog signaling pathway is central to the development of certain tumor types, including medulloblastoma and basal cell carcinoma (BCC). Patched1 (Ptch1) and Suppressor of fused (Sufu) are two essential negative regulators of the pathway with tumor suppressor activity. Ptch1(+/-) mice are predisposed to developing medulloblastoma and rhabdomyosarcoma, while Sufu(+/-) mice develop a skin phenotype characterized by basaloid epidermal proliferations. Here, we have studied tumor development in Sufu(+/-)Ptch1(+/-) mice to determine the effect of compound heterozygosity on the onset, incidence, and spectrum of tumors. We found significantly more (2.3-fold) basaloid proliferations in Sufu(+/-)Ptch1(+/-) compared to Sufu(+/-) female, but not male, mice. For medulloblastoma, the cumulative 1-yr incidence was 1.5-fold higher in Sufu(+/-)Ptch1(+/-) compared to Ptch1(+/-) female mice but this strong trend was not statistically significant. Together this suggests a weak genetic interaction of the two tumor suppressor genes. We noted a few rhabdomyosarcomas and pancreatic cysts in the Sufu(+/-)Ptch1(+/-) mice, but the numbers were not significantly different from the single heterozygous mice. Hydrocephalus developed in approximately 20% of the Ptch1(+/-) and Sufu(+/-)Ptch1(+/-) but not in Sufu(+/-) mice. Interestingly, most of the medulloblastomas from the Sufu(+/-)Ptch1(+/-) mice had lost expression of the remaining Ptch1 wild-type allele but not the Sufu wild-type allele. On the contrary, Sufu as well as Gli1 and Gli2 expression was upregulated in the medulloblastomas compared to adult cerebellum in Ptch1(+/-) and Sufu(+/-)Ptch1(+/-) mice. This suggests that Sufu expression may be regulated by Hedgehog pathway activity and could constitute another negative feedback loop in the pathway.

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Figures

Figure 1
Figure 1
Survival, growth, and incidence of tumors and other lesions in Sufu+/−Ptch1+/− mice. (A) Kaplan-Meier survival plot of wt, Sufu+/−, Ptch1+/−, and Sufu+/− Ptch1+/− mice followed until one year of age. (B) Cumulative one-year incidence of medulloblastoma (MB), rhabdomyosarcoma (RMS), pancreatic lesions (PL), and hydrocephalus (HC) in female (F) and male (M) Ptch1+/− [F (n=26); M (n=23)] and Sufu+/−Ptch1+/− [F (n=29); M (n=27)] mice. Numbers above the bars represents number of mice with the lesions. No wt or Sufu+/− mouse suffered any of these lesions. (C) Mean weight of female and male wt, Sufu+/−, Ptch1+/−, and Sufu+/−Ptch1+/− mice between 4 to 20 weeks of age. (D) Age distribution of the MB occurrence in Ptch1+/−and Sufu+/−Ptch1+/− mice. (E) Number of epidermal proliferations scored per hematoxylin-eosin stained sagittal section from the plantar surface of the hind paw from one-year old mice of the indicated genotypes. The data represents the average ± standard deviation of the number of proliferations. One-way ANOVA with Tukey's post-test was used for statistical analysis. P-values * = < 0.05, ** = < 0.01, *** = 0.001, and ns = not significant. Note that zero proliferations were encountered in the wt control mice.
Figure 2
Figure 2
Increased expression of Glil, Gli2, and Sufu, but loss of Ptchl expression in most MBs from Ptch1+/− and Sufu+/−Ptch1+/− mice. (A-F) qRT-PCR on total RNA from cerebella of one-year-old mice and MB from mice of different age for (A) Gli1; (B) Gli2; (C) Gli3; (D) Sufu; (E) Ptch1wt+mut; and (F) Ptch1wt. Wt, wild-type; S, Sufu+/−; P, Ptch1+/−; S/P, Sufu+/−Ptch1+/−. Individual samples are numbered 1 through 34. Results shown are means ± standard deviations of mRNA expressions levels relative to a wt sample arbitrarily set to one. Samples are in duplicate from four to seven individual mice in each category.
Figure 3
Figure 3
Expression of molecular markers in medulloblastomas from Ptch1+/− and Sufu+/−Ptch1+/− mice. (A-Y) Immunohistochemistry on representative paraffin-embedded tissue sections from normal adult cerebellum of wt and Sufu+/− mice and MBs from Ptch1+/− and Sufu+/−Ptch1+/− mice. (A-D) Phospho-histone H3. (E-H) Cleaved Caspase 3. (I-L) Cyclin D1. (M-P) Platelet-derived growth factor receptor α. (Q-T) γ-aminobutyric acid (GABA)A receptor, α6 subunit. (U-Y) Sufu. Brown color indicates positive immunostaining and light blue color is the hematoxylin counterstain. Scale bar = 50 μm (A-H, M-Y) and 100 μm (I-L).
Figure 4
Figure 4
Skin from Sufu+/−, Ptch1+/−, and Sufu+/−Ptch1+/− mice develop similar basaloid epidermal proliferations but differ in frequency depending on genotype. (A-Y) Immunohistochemistry on formalin-fixed paraffin-embedded representative skin tissue sections from paws. (A-D) Keratin 5 immunostaining. (E-H) Keratin 5 immunostaining and ß-galactosidase (ß-gal) activity as visualized by X-gal staining (dark blue). Wt and Sufu+/− mice have no LacZ gene and show only weak unspecific staining in the sebaceous glands. (I-L) Keratin 6 immunostaining. (M-P) Keratin 17 immunostaining. (Q-T) Ki67 immunostaining. (U-Y) Cyclin D1 immunostaining. Brown color indicates positive immunostaining and light blue color is the hematoxylin counter-stain. All tissues are from one-year-old mice. Scale bars = 50 μm.
Figure 5
Figure 5
Characterization of the basaloid epidermal proliferations using immunohistochemistry on skin tissue sections from the paw. (A-D) Hemtoxylin-eosin staining and ß-galactosidase (ß-gal) staining. (E-H) Keratin 10 immunostaining. (I-L) β-catenin immunostaining. (M-P) E-cadherin immunostaining. All images are from one-year-old mice. Scale bars = 100 μm (A-D) and 50 μm (E-T).
Figure 6
Figure 6
Increased expression of Gli1 in skin from Ptch1+/− and Sufu+/−Ptch1+/− mice. (A-F) qRT-PCR on total RNA from ventral skin of one-year-old mice for (A) Gli1; (B) Gli2; (C) Gli3; (D) Sufu; (E) Ptch1wt+mut; and (F) Ptch1wt. Wt, wild-type; S, Sufu+/−; P, Ptch1+/−; S/P, Sufu+/−Ptch1+/−. Individual samples are numbered 1 through 17. Results shown are means ± standard deviations of mRNA expressions levels relative to a wt sample arbitrarily set to one. Samples are in duplicate from three to five individual mice in each category.
Figure 7
Figure 7
Development of pancreatic lesions and RMS in Ptch1+/− and Sufu+/−Ptch1+/−mice. (A) H&E-stained pancreatic cyst. (B) PAS and Alcian blue-stained pancreatic cyst. (C) Hematoxylin-eosin (H&E)-stained RMS. (D) Desmin immunostaining of RMS. Scale bars =200 μm (A) and 100 μm (B-D).
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