Abstract A011: Bladder cancerization is linked to epithelial basalization and is reversed by EGF inhibition
Authors: Kershner Aaron M. , Prado Kris B. , Kiss Bernhard M. , Mrouj Karim , Penland Lolita , Lu Wan-Jin , Choi Jiyun , Diaz Edward , Lachmansingh Rovin , Chen Bertha , Kao Chia-Sui , Liao Joseph C. , Beachy Philip A. 
Published: May 2024

Abstract
Introduction: Epithelial changes can extend beyond the boundaries of an incipient tumor, leading to cancer recurrence even after effective excision of the primary tumor. This ‘field cancerization’ and consequent high recurrence rate is especially pronounced in bladder cancer. The purpose of this study is to characterize the urothelium in the context of ‘field cancerization’ in a mouse model of bladder cancer as well as in patients with bladder cancer and to determine if reversal of the field effect will prevent tumor formation. Results: We investigated field cancerization by single cell transcriptomics (scRNA-Seq) in a murine (male FVB mice) bladder carcinogenesis model using N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN) and found that prior to overt tumor formation urothelial cells throughout the entire bladder take on a strongly basal phenotype, characterized by reduced expression of differentiation markers (i.e. Upk1a, Upk2, and Upk3a) and increased expression of basal markers (i.e. Krt14, Krt6a, Krt5). Using monocle, we applied single cells from a BBN treated mouse bladder library to a pseudotime trajectory generated from a control mouse bladder library in order to unbiasedly characterize urothelial differentiation in the non-tumor urothelium of BBN treated mouse bladders. scRNA-Seq analysis of non-tumor urothelium from bladder cancer patients revealed a similar ‘basalization’ among non-tumor urothelial cells. In order to apply our single cell findings to bulk RNA-seq data, we developed a basal index from differentially expressed genes in basal clusters versus non-basal clusters from healthy control human urothelium single cell libraries, and we applied this basal index to non-tumor bladder samples from the The Cancer Genome Atlas (TCGA). We found that among a small subset of patients with paired tumor and non-tumor urothelium in the bladder TCGA dataset, the basal index, when applied to the non-tumor urothelium, was prognostic of survival. Finally, we found that basalization in BBN treated mice could be reversed pharmacologically by epidermal growth factor (EGF) pathway antagonism using erlotinib and trametinib and that over a 4-month time course this reversal dramatically decreased tumor incidence and improved survival, suggesting a new, more effective approach using mechanism-based cancer drugs for the prevention of bladder cancer recurrence in patients. Conclusions: The non-tumor urothelium in BBN treated mouse bladders, prior to overt tumor formation, demonstrates a basal phenotype which we characterize with scRNA-Seq and pseudotime analysis. Similarly, in patients with bladder cancer we observe that the non-tumor urothelium also takes on a basal phenotype. This ‘basalization’ of the non-tumor urothelium may potentially be prognostic, and pharmacologic reversal of the basal phenotype in the non-tumor urothelium using EGF pathway inhibitors prevents tumors in BBN treated mice.
Citation Format: Aaron M. Kershner, Kris B. Prado, Bernhard M. Kiss, Karim Mrouj, Lolita Penland, Wan-Jin Lu, Jiyun Choi, Edward Diaz, Rovin Lachmansingh, Bertha Chen, Chia-Sui Kao, Joseph C. Liao, Philip A. Beachy. Bladder cancerization is linked to epithelial basalization and is reversed by EGF inhibition [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr A011.