Identification of SLC14A1 (UT-B) gene rearrangement in urothelial carcinoma of the bladder

Background: Bladder cancer (BCa) is a common and deadly disease. Over the past decade, a number of genetic alterations have been reported in BCa. Bladder urothelium expresses abundant urea transporter UT-B encoded by Slc14a1 gene at 18q12.3 locus, which plays an important role in preventing high concentrated urea-caused cell injury. Early genome-wide association studies (GWAS) showed that UT-B gene mutations are genetically linked to the urothelial bladder cancer. Methods: In this study, using a fluorescence in situ hybridization (FISH), we examined whether Slc14a1 gene has been changed in BCa with Slc14a1 break-apart DNA probes. We also performed immunohistochemistry evaluating bladder cancer markers. Results: We found Slc14a1 gene rearrangement in one case of BCa from 14 patients. The patient is a 59-year-old male with superficial BCa. Histology showed non-muscle invasive bladder transitional-cell carcinoma. Immunostaining was negative for Syn, CK7, CK20, Villin, and positive for HER2, BRCA1, GATA3. Conclusions: We for the first time report a patient diagnosed with urothelial carcinoma accompanied with split Slc14a1 gene abnormality, a crucial gene in bladder.


Background
Bladder cancer (BCa) is the sixth most common cancer and occurs more often in men than in women. In 2004, the World Health Organization divided bladder tumors into muscleinvasive urothelial carcinoma and non-muscle invasive urothelial neoplasia (1). More than 70% of BCa patients are superficial disease at initial diagnosis, whereas 30% are in muscle-invasive stage (1). An important clinical feature of BCa is an unusually high propensity for recurring than in any other solid cancer. More than 70% of patients with 3 BCa will have a recurrence during the first two years after diagnosis (2,3). The recurrences are often accompanied by grade and/or stage progression with a very poor prognosis (4,5).
In human and some other animals, the urinary bladder is a special organ that is in constant contact with a high concentration of urea, 20-100 times higher than in blood.
Urea is the major end-product of nitrogen metabolism and is excreted by the kidneys.
Urea is a small molecule (~60 Da) and water solubility, however urea permeability across lipid bilayers is very low. Movement of urea across the cell membrane is mediated by urea transporter (UT) proteins (6)(7)(8)(9). In mammals, there are two subfamilies of UT, UT-A and UT-B, encoded by the Slc14a2 and Slc14a1 genes, respectively. UT-A urea transporters are mainly expressed in renal medulla and contribute to the kidney's ability to concentrate urine. UT-B has broadly tissue expression including bladder. Bladder urothelium only expresses UT-B (10)(11)(12). This was confirmed by an RNA-sequencing analysis showing high expression of Slc14a1 (UT-B) and the absence of Slc14a2 (UT-A) in bladder tissues (13).
Evidences have shown that an excess of intracellular urea accumulation damages cells and cell functions (DNA damage, abnormal cell phenotype transformation, genetic pathway activation, etc.) (11,(14)(15)(16). Since the bladder stores urine, its urothelium is inevitably and constantly exposed to a higher concentration of urea than any other tissues. Therefore, the urothelial UT-B stands in a key position to prevent urea-induced insults by lowering intracellular urea levels. To support it, bladder urothelium expresses the most abundant urea transporter UT-B in vivo (10). Disfunction of UT-B will cause intracellular urea accumulation and profoundly affect cells. The impaired urothelial cells are then vulnerable to be attacked by urea and/or by carcinogen in urine, a potential important mechanism of tumorigenesis in BCa (17).
We previously reported no or lower level of UT-B expression both at mRNA and protein levels in BCa. We also found that, in some BCa patients, the BCa expresses a mutant UT-B with a 24-nt in-frame deletion (del24) in exon 4. Or a low amount of UT-B is expressed but the UT-B protein glycosylation is unsialylated (17). All these changes of UT-B gene and/or protein contribute to the reduced urea transport activity in BCa.
When examining UT-B mRNA expression by RT-PCR, we noticed that UT-B mRNA was undetectable in some BCa patients (17). To figure out whether it could be due to UT-B genomic gene (Slc14a1) alteration, we collected 14 cases of BCa and performed fluorescence in situ hybridization (FISH). We found UT-B gene rearrangement occurred in one patient. This is the first report of genomic abnormalities of split SLC14A1 gene identified in BCa. The break-apart Slc14a1 probe set including human Cot-1 DNA was applied to individual slides. Human Cot-1 DNA (Fisher, 15279011) was used to block non-specific sequences.

Patients and tissue samples
The slides were immediately covered with a coverslip and sealed with rubber cement.
After denatured at 86°C for 8 min, the sections were incubated for 16 hours at 38°C using Automated ThermoBrite FISH System (Leica Biosystems). Post-hybridization washes were carried out with 2 ×SSC for 3min at room temperature followed by 2X SSC/0.1% NP-40 solution at 72℃ for 5 min and gradient dehydration in 70%, 85% and 100% ethanol. The

Identification of split UT-B gene (Slc14a1) in BCa
Gene split has been demonstrated in several cancers (18)(19)(20). In this study, we examined

Discussion
BCa is a complex and heterogeneous disease caused by both genetic and environmental factors. The "urogenous contact hypothesis" proposed by Braver (22) in 1987 is still prevalent in the etiology of BCa. However, one important factor that we may have ignored in bladder carcinogenesis is urea. The urea concentration in urine is 20-100 times higher than that in blood in humans (23,24). Interestingly, the risk of BCa for people with spinal cord injuries is 16-28 times higher than that for the general population (25,26), underscoring the important role of endogenous factors, including urea, in the bladder tumorigenesis.
Considering that the bladder urothelium is bathed in fluid with a high urea concentration and the toxicity of intracellular urea accumulation, the urea transporter is extremely important for bladder urothelium by lowering intracellular urea levels. Experimental studies have shown that deletion of UT-B gene causes urea accumulation in urothelium ~9 times higher in UT-B knockout mice (11,27). This is accompanied by increased cell DNA damage, apoptosis and diminished arginine metabolism (11,27). Therefore, loss of UT-B protection will render urothelial cells at risk under constantly high urea insult, then either directly activates genetic pathways that induce BCa or synergistically increases BCa risk by facilitating other carcinogen-induced mechanisms of BCa. In 2011, genome-wide 8 association studies (GWAS) from two independent groups (28,29)  Although the Slc14a1 as a new urinary BCa susceptibility gene was appreciated (28,29), the role and the molecular mechanism of the UT-B in bladder oncogenesis have not been 9 well explored to date. In this study, we for the first time report a case of

Consent for publication
Written consent was obtained from the patient to publish this case report.

Availability of data and material
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
This work was supported by grants from Huai'an Promotion Project for science and technology international cooperation (HAC201708) to S.S. Natural Science Foundation of Huai'an (HAB201801) to X. L. and National Natural Science Foundation of China 81620108029 to B.Y.
The funds provided were used to finance laboratory expenses (materials and reagents). The funder had no role in the design of the study, the collection of data, the analysis and interpretation of results, or in writing the manuscript.