Novel transgenic knockout model of basal-squamous bladder cancer.

Abstract

459 Background: The recent description of the highly aggressive Basal-Squamous molecular subtype of bladder cancer (BLCa) requires the development of new in vivo models for the study of this clinical entity. Although loss of the transcription factor Forkhead box A1 ( FOXA1) is significantly associated with the Basal-Squamous subtype, cooperating genetic alterations are unknown. Herein, we identify genetic alterations that potentially cooperate with FOXA1 loss to drive tumorigenesis and/or the Basal-Squamous phenotype and develop a novel transgenic model for the study of Basal-Squamous BLCa. Methods: We interrogated The Cancer Genome Atlas (TCGA) BLCa study to identify common genetic alterations associated with FOXA1 copy number (CN) loss. Based on our results, we utilized a bladder-specific Cre-LoxP ( Uroplakin II-Cre) to constitutively knock-out (KO) Foxa1 and/or Pten in mouse urothelium. Two cohorts of mice were aged for 6 and 12 months, and a third cohort was exposed to the bladder-specific carcinogen N-butyl-(4-hydroxybutyl)-nitrosamine (BBN; 0.05%) for 12 weeks. Following all experiments, the bladder tissue was isolated and characterized. Results: The TCGA data revealed that combined CN loss of both FOXA1 and PTEN occurs in 17% of MI BLCa patients. At 6 months of age, our mice developed urothelial hyperplasia, whereas at 12 months of age, double KO ( Foxa1-/-/Pten-/-) mice developed carcinoma in situ (CIS) with SqD, enriched for basal (Krt5/6 and 14) and reduced in luminal (Gata3 and Pparγ) markers. Increased tumor stage was significantly associated with combined KO of at least one allele of Foxa1 and Pten following BBN exposure. Moreover, upregulation of apoptosis related genes was observed by RNA-sequencing data. Conclusions: In advanced BLCa, CN loss of FOXA1 and PTEN is common. Genetic ablation of Foxa1 and Pten results in CIS with SqD and a pathologic profile consistent with the Basal-Squamous subtype and accelerated tumor onset following carcinogen exposure. Therefore, we describe the development of a novel model for the study of Basal-Squamous BLCa potentially useful for preclinical studies.