Inactivation of p53 and Pten promotes invasive bladder cancer

Abstract

Although bladder cancer represents a serious health problem worldwide, relevant mouse models for investigating disease progression or therapeutic targets have been lacking. We show that combined deletion of p53 and Pten in bladder epithelium leads to invasive cancer in a novel mouse model. Inactivation of p53 and PTEN promotes tumorigenesis in human bladder cells and is correlated with poor survival in human tumors. Furthermore, the synergistic effects of p53 and Pten deletion are mediated by deregulation of mammalian target of rapamycin (mTOR) signaling, consistent with the ability of rapamycin to block bladder tumorigenesis in preclinical studies. Our integrated analyses of mouse and human bladder cancer provide a rationale for investigating mTOR inhibition for treatment of patients with invasive disease.

Figure 1.

Targeted gene deletion in bladder epithelium via Adeno-Cre delivery. (A) Schematic of bladder anatomy showing the layers of the epithelium. Adeno-Cre (1X10¹⁰ PFU) was delivered into the bladder lumen of R26R reporter mice and the location and extent of recombination was analyzed 3 d later by visualization of β-galactosidase staining (B) or by immunofluorescence with colabeling by cytokeratin 7 to mark epithelial cells (C).

Figure 2.

p53 and Pten collaborate in suppression of invasive bladder cancer in mutant mice. (A) Survival curve for mice of the indicated genotypes injected with Adeno-Cre and monitored for the length of time indicated. (B) Gross bladder phenotype following injection of Adeno-Cre into the bladder of p53^+/+; Pten^+/+ or p53^flox/flox; Pten^flox/flox mice. (C–H) Representative hematoxylin and eosin (H&E)-stained sections from bladders of normal (p53^+/+; Pten^+/+; C,D) or mutant (p53^flox/flox; Pten^flox/flox; E–H) mice injected with Adeno-Cre. Arrows and inset in panels F and H show mitotic figures. (I–N) H&E-stained sections of human bladder, showing representative histology for comparison with the mouse bladder tumors. Bars, 100 μm.

Figure 3.

Altered p53 and PTEN are associated with poor survival in human bladder cancer. (A–F) Tissue microarray analyses of p53 and PTEN showing immunohistochemical staining of human bladder tumors. (G,H) Multivariate survival analyses. Kaplan-Meier plot for patients with clinical outcome data for Pten immunostaining in all patient samples (G) and showing Pten-positive and Pten-negative tumors in patients harboring p53 mutations (H). (I–P) Functional analyses of p53 and PTEN inactivation in human bladder cells. Strategy: RT4 human bladder cancer cells were infected with lentiviral RNAi (MOI = 5) for p53 and/or PTEN, recombined with rat embryonic bladder mesenchyme, and grown under the renal capsule of a nude mouse for 3 mo. (I, M) Gross morphology of the renal grafts. (J,N) H&E-stained sections. (K,L,O,P) Immunostained sections. Bars, 100 μm.

Figure 4.

Preclinical analyses in the mutant mouse models. (A–I) Activation of mTORC1 in mouse and human bladder cancer. (A) Western blot analyses showing the expression/activation of the indicated components of the mTORC1 pathway in normal bladder (p53^+/+; Pten^+/+) or bladder tumors (p53^flox/flox; Pten^flox/flox) mice. Shown are representative data from analyses of five or more independent tumors. (B–E) Immunostaining for activated p-Akt and p-S6 kinase in sections from control mice (p53^+/+; Pten^+/+) or bladder tumors from p53^flox/flox; Pten^flox/flox mice. (F–O) Inhibition of mTORC1 inhibits bladder tumor growth in preclinical studies. One week following delivery of Adeno-Cre to the p53^flox/flox; Pten^flox/flox mice, mice were randomly enrolled into the vehicle (Veh) and rapamycin (Rap) groups and delivered rapamycin (or vehicle) via daily i.p. injection for up to 5 mo. (F,G) Gross morphology. (H,I) H&E-stained sections. (J–M) Immunostained sections. (N) Summary of bladder weights. (O) Summary of proliferation rates.