Figure 1.
Lkb1 and Pten loss in the epithelium of mouse bladder after AhERCre recombinase induction.
A, Cre-mediated recombination is marked by beta-galactosidase reporter gene expression in mouse bladder. AhERCre-positive bladder tissue (Pten -deficient) shows extensive recombination on day 14 after a regime of beta-naphtoflavone injections compared to no recombination in Cre-negative bladder tissue. B, Western blot detection of Lkb1, Pten, phospho-AMPK (Thr172), phospho-mTOR (S2448), phospho-S6 ribosomal protein (S240/244), IRS-1 and phospho-IRS-1 (S307) expression in induced Cre-negative Lkb1fl/fl Pten fl/fl (1, 2) and induced Cre-positive: Pten fl/fl bladder epithelial tissue (3,4), Lkb1fl/fl bladder epithelial tissue (5,6), Lkb1/Pten fl/fl bladder epithelial tissue (7, 8) at day 50. C, Hematoxylin and eosin staining of bladder sections of Cre-negative (WT) and recombined Cre+ Pten fl/fl, Cre+Lkb1f/fl, Cre+ Pten fl/flLkb1fl/fl mice (both treated and non-treated with rapamycin) at day 100 following a combined injection with beta-naphthoflavone and tamoxifen. Lkb1/Pten-deficient bladders show abnormal growth of urothelium which is suppressed by rapamycin. Scale bars correspond to 400 µm (left column), 100 µm (middle column) and 20 µm (right column).
Figure 2.
Bladder tumorigenesis after combined Lkb1 and Pten deletion.
A-C, Hematoxylin and eosin staining of bladder sections of Cre-negative (A) and bladder tumour in recombined Cre+ Pten fl/flLkb1fl/fl mice at day 100 (B) and day 125 (C) following combined injection with beta-naphthoflavone and tamoxifen. Borderlines show different areas of the tumour: more proliferative section is to the left and less proliferative (containing vesicles) is inside the marked area. Scale bars correspond to 100 µm.
Figure 3.
The combined loss of PTEN and Lkb1 induces bladder epithelium hyperproliferation and affects longevity.
A, Immunohistochemical staining with anti-Ki67 antibody showing proliferative cells in the bladder epithelium of Cre-negative and recombined Cre+Ptenfl/fl, Cre+Lkb1f/fl, Cre+ Pten fl/flLkb1fl/fl mice (day 50 for all four genotypes and both days 50 and 125 for double-negatives) B, A graph showing the increase in proliferative cells in the bladder epithelium of Cre+ Pten fl/flLkb1fl/fl mice, C, Kaplan-Meier plot of WT (Cre− Ptenfl/flLkb1fl/fl) cohort is represented in black stars (n = 25), Cre+ Pten fl/fl cohort is represented in green (n = 25), Cre+Lkb1f/flcohort is represented in blue (n = 25), Cre+ Ptenfl/flLkb1fl/flcohort represented in red (n = 25. Cre+Ptenfl/flLkb1fl/fl mice show decreased longevity, where 100% of the cohort did not survive past day 179, χ2 tests confirmed that Cre+Ptenfl/flLkb1fl/fl mice exhibit a significantly reduced average survival compared with Cre-negative cohort (P<0.0001, χ2 = 44.6), while other two cohorts do not show the same tendency: Cre+Ptenfl/fl (P = 0.556, χ2 = 0.347), and Cre+Lkb1fl/fl (P = 0.083506 χ2 = 3.00). Scale bars correspond to 20 µm.
Figure 4.
mTOR/S6K pathway deregulation in Lkb1/Pten-deficient mouse urothelium.
The combined deletion of Lkb1 and Pten in the mouse urothelium led to drastic changes in mTOR/S6K pathway by day 100 after the induction and this is prevented by rapamycin treatment. A, B- Phospho- mTORSer2448 (A) and Phospho- ribosomal S6 proteinSer240/244 (B) immunostaining displaying elevated levels of mTOR and phosho-S6 ribosomal protein phosphorylation, Scale bars correspond to 50 µm.
Figure 5.
Increased Akt phosphorylation and hypoxia in Lkb1/Pten-deficient mouse urothelium.
The drastic changes in AKT/mTOR/S6K pathway in Lkb1/Pten-double deficient urothelium corresponded to hypoxia marker GLUT1 upregulation by day 100 after the induction and this is prevented by rapamycin treatment. A, B- Immunostaining showing a strong increase in Phospho-AKTSer473 (A) and GLUT1 (B) levels. Scale bars correspond to 50 µm.
Figure 6.
Lkb1/Pten-deficient mouse urothelium undergoes EMT.
Mouse bladder epithelium deficient for both Lkb1 and Pten exhibits loss in epithelial markers and acquire mesenchymal properties by day 100. Rapamycin inhibition of mTOR suppresses the observed effect. A, E-cadherin and ZO-1 immunofluorescent co-localisation showing disappearance of these proteins from cell-cell walls in Lkb1/Pten-deficient bladder whereas rapamycin treatment prevents this loss, B, Immunostaining showing a strong increase in Vimentin levels and Snail nuclear localisation after the combined deletion of Lkb1 and Pten and this is mediated via mTOR evidenced from their suppressed levels in rapamycin-treated animals. Scale bars correspond to 20 µm.