Abstract B16: AGR2 is a marker of disease progression and a potential therapeutic target in non-muscle invasive bladder cancer
Authors: Maia Andreia , Martin-Fernandez Javier , Sopena Josep Maria Gaya , Rodriguez-Faba Oscar , Redorta Joan Palou , Castillo-Martin Mireia 
Published: August 2020

Abstract
Introduction: Non-muscle invasive bladder cancer (NMIBC) is a heterogeneous disease that includes tumors with different risks of progression to an aggressive muscle-invasive cancer (MIBC). Patients with T1 bladder cancer have an overall mortality of 33% and a cancer-specific mortality of 14%, three years after diagnosis. Nowadays, clinicopathologic features alone are not sufficient to effectively classify NMIBC patients according to their risk of tumor progression. Thus, the discovery of new biomarkers is essential to help clinicians decide the most appropriate treatment and consequently to increase quality of life of patients, especially for those who undergo radical cystectomy, a high-morbidity approach. DeltaNp63 has previously been reported by our group as a protective factor of high-grade (HG) NMIBC progression: from the 132 patients whose tumors showed nuclear deltaNp63 expression, none suffered disease progression after a median follow-up of 62.1 months.
Methods: Two commercially available HG-NMIBC cell lines (RT112 and BF-TC905) were used to study the role of AGR2 in tumor progression. A stable knockdown of AGR2 (AKO) was performed (using shRNA technology) in cells previously knocked down for deltaNp63 (NKO). In vitro functional studies (cell cycle, proliferation, invasion, and wound healing assays) were performed comparing the parental cells, NKO and AKO cells. A bladder orthotopic mouse model was used to study tumor initiation and metastatic capacity in vivo. Immunohistochemistry (IHC) analyses of deltaNp63 and AGR2 in 163 human HG-NMIBC tissue specimens with a mean follow-up of 100.8 months were performed.
Results: NKO cells showed higher proliferation and invasive capacities in vitro and higher tumor and metastasis formation in vivo than the original parental cells. Interestingly, NKO cells displayed AGR2 gene upregulation and protein overexpression. AKO cells showed a reversion of the aggressive phenotype of NKO cells in vitro, presenting lower cell proliferation (cell cycle and proliferation assays), lower invasion capacities, and slower wound healing abilities. The in vivo results support in vitro studies, showing higher tumor formation in NKO (RT112 cell line), thus confirming the importance of AGR2 in bladder tumor progression. IHC analyses revealed that tumors with an aggressive phenotype (deltaNp63 loss and overexpression of AGR2) showed a significant higher progression rate.
Conclusion: This work identified AGR2 as a progression biomarker in HG-NMIBC and confirmed the role of deltaNp63 as a protective factor. This helped understand better the mechanisms of NMIBC progression, opening the possibility of novel therapies for patients (e.g., AGR2 targeted approaches) and ultimately improving patients' overall survival and quality of life, by deciding the most adequate treatment for each patient, taking into account the molecular characteristics of their HG-NMIBC tumor.
Citation Format: Andreia Maia, Javier Martin-Fernandez, Josep Maria Gaya Sopena, Oscar Rodriguez-Faba, Joan Palou Redorta, Mireia Castillo-Martin. AGR2 is a marker of disease progression and a potential therapeutic target in non-muscle invasive bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr B16.