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. 2020 Sep 7;9:e54542. doi: 10.7554/eLife.54542

Figure 4. BMP inhibition reduces Pten-deficient propagation potential and inhibits skin and prostate tumor initiation.

(A) The aberrant organoid-forming capacity of Pten-deficient basal cells is abrogated by BMP inhibitor (NOGGIN) treatment. Cre activity was induced in vitro by treatment with hydroxy-tamoxifen for the first passage of organoid derived from basal cells of Krt5CreERT2Ptenf/f mice. From passage 5, organoids were cultured in presence or absence of NOGGIN. Organoid-forming potential was assessed as in Figure 1A. (B) Krt5CreERT2Ptenf/f tamoxifen-treated mice were injected with either K02288 or PBS for 4 weeks. (C–D) Representative histological sections of prostate tissue (C) and skin tissue (D) stained with H&E showing an absence of PIN and skin hyperplasia in K02288-treated as compared to mock-treated mice. (E) Kaplan-Meier skin tumor free survival curves of tamoxifen-induced Krt5CreERT2Ptenf/f treated or not with K02288 (log-rank (Mantel-Cox) test; p<0.0001, n = 7 and n = 14, respectively). Tick-marks represent sacrificed animals. (F) Bmp5 loss rescues the aberrant organoid-forming capacity of Pten-deficient basal cells. Cre activity was induced in vivo by tamoxifen injection in adult mice 4 weeks prior to organoid propagation potential assessment. (G–L) Eight-week-old mice were treated with tamoxifen and sacrificed 4 weeks later. (H–I) Bmp5 loss and Gata3 overexpression rescues Pten-deficient prostate and skin hyperplasia. Shown are representative H&E pictures of prostate (H) and skin (I) tissues. (J) Kaplan-Meier skin tumor free survival curves of tamoxifen-induced Krt5CreERT2Ptenf/f, Krt5CreERT2Ptenf/fBmp5SE/SE and Krt5CreERT2Ptenf/fRosa26G3/+ mice (log-rank (Mantel-Cox) test; p<0.0001, n = 22, n = 17 and n = 9, respectively). Tick-marks represent sacrificed animals. (K–L) Representative FACS phenotype (K) and percentage of SCA1hi cells (L) in the luminal compartment as defined by Lin(CD45, CD31, TER119)-EpCAM+CD49fMed of total prostate (one-way ANOVA; ***p<0.0001). (M) Representative H&E pictures of prostate tissues of Krt5CreERT2Ptenf/f and Krt5CreERT2Ptenf/fBmp5SE/SE mice sacrificed 9 weeks after tamoxifen treatment. See also Figure 4—figure supplements 1,2.

Figure 4—source data 1. Statistical analysis for Figure 4A–F and Figure 4—figure supplement 2A; Figure 4—figure supplement 2B.
Figure 4—source data 2. Statistical analysis for Figure 4E–J.
Figure 4—source data 3. Statistical analysis for Figure 4L.

Figure 4.

Figure 4—figure supplement 1. BMP inhibition corrects Pten-deficient tumor phenotypes.

Figure 4—figure supplement 1.

(A–B) Growth rate of cells upon organoid passage for the indicated genotypes and treatment calculated from nonlinear regression curve fit of data from Figure 4A and B, respectively (one-way ANOVA; *p<0.05, **p<0.005, ***p<0.0001). (C–D) Higher magnification H&E staining pictures of skin tissue from Figure 4D and I.
Figure 4—figure supplement 2. K02288 acts downstream of AKT pathway.

Figure 4—figure supplement 2.

(A–B) BMPR inhibition affect the growth of Pten-deficient cell lines. Shown is the growth curve (A) and protein and phosphorylation levels of AKT (B) in CaP2 and CaP8 culture treated or not with K02288 at the indicated concentration (one-way ANOVA; p<0.0001).
Figure 4—figure supplement 2—source data 1. Statistical analysis for Figure 4—figure supplement 2A.
Figure 4—figure supplement 2—source data 2. Full unedited gels for Figure 4—figure supplement 2B.