Abstract IA001: Cellular and molecular underpinnings of field cancerization and progression to invasive carcinoma in bladder cancer

Abstract Two major concerns in clinical management of bladder cancer are: (i) the high frequency of recurrence; and (ii) the morbidity and mortality associated with progression to invasive disease (invasive urothelial carcinoma, IUC). To gain a better understanding of these issues we have used the murine BBN (N-butyl-N-(4-hydroxylbutyl) nitrosamine) experimental model of bladder cancer, as well as tissue samples from normal human bladders and from bladders of patients at various stages of bladder cancer formation and progression. We have documented in the BBN model a CIS-like stage, prior to invasion, in which clonal expansion from a single cell occupies a significant fraction of the urothelium. With this experimental murine model alongside observations from patient samples and data from The Cancer Genome Atlas (TCGA), we have ascertained that a key event in progression of pre-invasive to invasive carcinoma is the silencing of the Sonic hedgehog (Shh) member of the Hedgehog gene family. Silencing-associated invasion appears due to the loss of SHH-induced differentiation signals, which when present slow growth and block invasion. Despite the consistent loss of SHH expression in invasive murine and human bladder cancers, however, TCGA data show few if any mutations in the SHH gene of IUC cells. Instead, our studies of patient samples and cancer-derived cell lines reveal that the loss of SHH expression is due to tighter chromosomal packaging and reduced accessibility of a bladder-specific enhancer located ~200 kb upstream of the SHH transcription start site. We have found that inhibitors of certain chromatin-modifying enzymes, including an FDA-approved drug, are capable of re-activating SHH expression, and are testing the possibility that these drugs may block or reverse invasion of urothelial carcinoma. The high frequency of bladder cancer recurrence is thought to result from field cancerization, that is, an increased probability of new cancer formation in apparent normal urothelium, even after successful resection of the primary tumor. To understand the basis for this field effect, we compared apparent normal urothelium from cancer patients to truly normal urothelium from bladders never affected by malignancy. We found that apparent normal samples display a dramatically increased basal character in transcriptomic assays, using as a metric of basalization an index constructed from single cell RNA-seq analysis of normal urothelium. We and others have demonstrated previously by lineage tracing and cell ablation studies in the BBN model that IUC arises from basal stem cells of the urothelium. The basalization of urothelium distant from the primary tumor that we observe thus may account for the increased probability of cancer formation in the apparent normal urothelium of bladder cancer patients. We have found that several FDA-approved antagonists of EGF signaling can reverse this basalization in the mouse BBN model as well as in organoid culture, and that these drugs dramatically reduce the formation of tumors following BBN exposure. Citation Format: Philip A. Beachy, Kris B. Prado, Aaron M. Kershner, Karim Mrouj, Bernard M. Kiss, Hua Dong, Ozgu Aydogdu, Ye Tian, Joseph C. Liao. Cellular and molecular underpinnings of field cancerization and progression to invasive carcinoma in bladder cancer [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 IA001.