Introduction:

SCBC is an aggressive subtype of bladder cancer with high metastatic potential and few effective therapies. The defining mutations of neuroendocrine cancers include loss-of-function mutations in p53 and Rb and gain-of-function mutations in driver oncogenes (e.g., MYC). We engineered an orthotopic mouse model of SCBC leveraging these defining mutations to investigate the genetic and epigenetic drivers of SCBC.

Methods:

Lentiviral particles carrying Cre recombinase were produced using Lenti-sgNeo#2/Cre. The bladders of genetically engineered Rb1^fl/fl Trp53^fl/fl Myc^LSL/LSL (RPM) mice were transduced with Cre recombinase-expressing lentivirus via transurethral catheterization. Mice were monitored by micro-ultrasound (mUS) and bioluminescent imaging and detected tumors were verified by histology. SCBC morphology was confirmed by hematoxylin and eosin (H&E) staining and synaptophysin immunohistochemistry (IHC) by a genitourinary pathologist. Whole transcriptome (RNAseq) analysis was performed to correlate transcriptomic profile of neuroendocrine mouse tumors to a cohort of human SCBC tumors. Candidate driver genes were prioritized and association with prognosis in human tumors was evaluated (TCGA BLCA, n=417). The function of Forkhead box protein family members coordinately up and down regulated in human and murine SCBC was investigated in human urothelial carcinoma and murine syngeneic cell lines.

Results:

Transurethral catheterization successfully transduced the bladder urothelium without evidence of ectopic (non-urothelial) exposure. RPM mice developed SCBC visible on mUS and bioluminescent imaging within 8-10 weeks. As expected in neuroendocrine tumors, mice developed spontaneous visceral and lung metastases. High grade neuroendocrine morphology and NE markers were confirmed on H&E staining and IHC, respectively, by our genitourinary pathologist. Western blot analysis confirmed Myc expression and suppression of TP53 and RB1. Transcriptomic profiling of both mouse and human SCBC demonstrated concordant gene expression. Gene expression profiling of mouse neuroendocrine bladder tumors and normal bladder showed increased FOXA2 expression in the tumor and high intratumoral FOXA2 protein expression was confirmed on IHC. TCGA-BLCA analysis showed significantly worse overall survival for patients whose bladder tumors overexpressed FOXA2 (p=0.01). Expression of FOXA2 in human urothelial cancer lines harboring p53 and RB mutations led to increased expression of neuroendocrine markers by immunocytochemistry.

Conclusion:

We developed a novel genetically engineered orthotopic murine model of SCBC which is histologically and morphologically similar to human SCBC. Gene expression profiling of neuroendocrine bladder tumors from these mice suggest FOXA2 may play a role in the initiation and development of the neuroendocrine phenotype. Ongoing work seeks to further characterize the role of FOXA2 in neuroendocrine differentiation and identify other genetic and epigenetic markers that may cooperatively contribute to the development of this aggressive variant of bladder cancer.

Funding: VeloSano Grant