Radiation cystitis (RC) is a painful chronic bladder condition that results from Radiation Therapy (RT) for pelvic cancers and for which no safe and effective therapy exists. It has a long latency, that can take years before becoming chronic and symptomatic. RC is characterized by inflammation and fibrosis in the bladder [1], which can cause symptoms such as pain, urgency, frequency, and hematuria. The molecular mechanisms underlying the development of RC are not fully understood, thus we sought to use our previously developed pre-clinical model to identify and validate proteins that are altered after radiation therapy and that may drive RC. One hypothesis is that radiation may increase the expression of pro-fibrotic cytokines and growth factors, such as Amphiregulin (AREG). This protein is a pro-fibrotic growth factor that belongs to the epidermal growth factor (EGF) family. AREG may play an important role in mediating fibrosis in RC as it has been implicated in the pathogenesis of various fibrotic diseases, including pulmonary fibrosis [2] and liver cirrhosis [3].

Female C57Bl/6 mice received a single dose of 40Gy irradiation using the Small Animal Radiation Research Platform (SARRP). Control and irradiated bladders were harvested at 1 week, 4 weeks, 3 months, and 6 months. RNA was harvested, mRNA sequencing was performed at paired-end 150bp on the Illumina NovaSeq6000 with a target of 30 million reads per sample, and bioinformatics analysis was performed. SV-HUC-1 urothelial cells line from ATCC (CRL-9520) were irradiated using Faxitron X-ray cabinet in a single dose of 1, 3 and 6 Gy and qPCR analysis performed over-time. The levels of the protein of interest were assessed in cell lysate and urine samples from prostate cancer survivors with history of external beam radiation therapy using ELISA (R&D, cat # DAR00).

Differential gene expression study of irradiated bladders over time revealed increased levels of proteins involved in inflammation, immune response, oxidative stress, and DNA repair pathways. mRNA sequencing analysis identified AREG to be increased in mouse bladder tissues in response to irradiation (Fig 1A). This finding was validated using qPCR analysis in bladder tissues samples and human urothelial cells. In addition, we assessed the levels of amphiregulin in the urine of patients with RC diagnosis (Fig 1B).

In this study we used our RC pre-clinical model to identify and validate potential proteins involved in RC progression. AREG expression was significantly increased in the irradiated bladder, starting at 1-week post-RT and continuing up to 6 months post-RT. Exposure of human urothelial cells to radiation also resulted in increased expression levels of AREG, in a dose dependent manner, at different time points. Moreover, AREG was detectable in the urine of prostate cancer survivors with history of pelvic radiation therapy and was significantly higher in the subset of patients with diagnosis of RC.

RC is a debilitating condition for many pelvic-cancer survivors. Current treatment options are limited and largely ineffective. The identification and validation of potential protein targets for RC will be essential for the development of safe and effective therapies for this condition. The amplified production of pro-fibrotic amphiregulin appears to be an important mechanism underlying the development of radiation cystitis and targeting this factor may therefore be a promising approach for the prevention and treatment of this condition. Further studies are needed to elucidate the exact mechanisms by which amphiregulin promotes fibrosis in the bladder and to explore the efficacy of targeting this pathway in the treatment of radiation cystitis.

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Perugorria MJ, Latasa MU, Nicou A, Cartagena-Lirola H, Castillo J, Goñi S, Vespasiani-Gentilucci U, Zagami MG, Lotersztajn S, Prieto J, Berasain C, Avila MA. The epidermal growth factor receptor ligand amphiregulin participates in the development of mouse liver fibrosis. Hepatology. 2008 Oct;48(4):1251-61. doi: 10.1002/hep.22437. PMID: 18634036.

Continence 7S1 (2023) 100801
DOI: 10.1016/j.cont.2023.100801