Preclinical modeling of plasmacytoid urothelial carcinoma to study cancer mechanisms and therapeutic response

Plasmacytoid urothelial carcinoma (PUC) is an aggressive histologic subtype of bladder cancer with poor patient outcomes and worse response to standard chemotherapies than conventional histology urothelial carcinoma (CUC). The hallmark molecular alteration in PUC is loss of E-cadherin, which can occur through mutations or promoter hypermethylation of CDH1 , the gene encoding E-cadherin. Aside from E-cadherin loss, the mutational spectrum of PUC is similar to that of CUC, although with higher rates of mutations in RB1 and p53 in PUC. E-cadherin, a transmembrane glycoprotein essential for epithelial adhesion and integrity, is a central regulator of EMT. Decreased expression of E-cadherin promotes EMT through transcriptional repressors (Snail, Slug, Twist, ZEB1/2), a cadherin switch, and epigenetic remodeling, driving enhanced invasiveness and metastatic potential. Despite our knowledge regarding the function of E-cadherin, the precise mechanism by which E-cadherin loss promotes disease progression and therapeutic resistance within urothelial carcinoma is not understood. Additionally, due to the lack of a preclinical PUC model and the limited ability to perform randomized controlled trials in this rare tumor type, clinicians face uncertainty regarding which therapeutic agents are most effective, reinforcing the need for innovative preclinical studies. Therefore, we aim to interrogate the impact of CDH1 loss in PUC on cancer phenotypes, elucidate mechanistic insights, and identify novel therapeutic strategies for PUC through preclinical modeling. To model PUC biology, we utilized CRISPR/Cas9 mediated knockout to generate CDH1 loss in two luminal (UPPL1541, UPPL1595) and one basal (BBN963) previously established murine bladder cancer cell lines using three independent gRNAs. Resulting CDH1 mutations at the CRISPR-Cas9 target site were confirmed by Sanger sequencing and E-cadherin protein loss confirmed by western blot. Compared to non-targeted, CDH1-intact control cells, CDH1-knockout cells demonstrate increased N-cadherin protein expression and increased expression of the mesenchymal markers Snail, Slug, ZEB1, and Vimentin by western blot. CDH1 knockout cells did not demonstrate a difference in cell proliferation compared to non-targeting controls, as determined by cell-titer glo assay. On transwell invasion assay, CDH1 knockout cells demonstrate significantly increased cell invasion compared to nontargeted, CDH1-intact control cells. These findings demonstrate that CDH1 loss is associated with upregulation of the epithelial-to-mesenchymal transition and enhanced in vitro cell invasion in urothelial carcinoma. We will continue to advance this work by dissecting tumor-microenvironment interactions and therapeutic responses in syngeneic models, positioning this platform as a novel preclinical resource for the clinically-challenging PUC variant.