Hypothesis / aims of study
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].
Study design, materials and methods
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).
Interpretation of results
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.