The Role Of TR4 Nuclear Receptor In Clear Cell Renal Cell Carcinoma Metastasis

Background and objective:Clear cell renal cell carcinoma （ccRCC） is the most lethal urological tumor, which accounts for 2%-3% of adult malignancies in the world. RCC is heterogeneous and comprises several histologic subtypes according to the differences in genetics, biology, and behavior. The most common and aggressive RCC subtype is clear cell RCC （ccRCC） with the highest rates of local invasion, metastasis, mortality, and refractory to current treatments. Recently, advancements in understanding of the VHL gene pathway in ccRCC have produced pharmaceutic outcomes based on specific molecular targets that have changed the treatment landscape for patients with metastatic RCC. Unfortunately, the vast majority of treated patients with mRCC eventually develop progressive disease because of acquired resistance or other reasons. Hence, a better understanding of the mechanisms involved in the pathogenesis of ccRCC and more effective therapeutic approaches are urgently required.While testicular nuclear receptor 4 （TR4） might promote prostate cancer metastasis through CCL2/CCR or miR-373-3p signals, our understanding of its role in clear cell renal cell carcinoma （ccRCC） is limited. Here we sought to identify the role of TR4 in ccRCC metastasis and some detailed mechanism.Methods and results:1. To confirm the role of TR4 in clear cell renal cell carcinoma metastasis in clinical and in vitroWe first applied the IHC staining to examine the TR4 expression in clinical ccRCC tissue samples and found TR4 expressions in ccRCC tumors from patients with distant metastases are higher than in tumors from metastasis-free patients. Futher we identified that patients with higher TR4 expression had significantly lower recurrence-free survival than patients with lower TR4 expression. Lastly the in vitro results are consistent with the human clinical data showing up-regulation of TR4 expression might be correlated with ccRCC metastasis.2. TR4 promote ccRCC metastasis via miR-32-5p/TR4/HGF/Met signalsInvasion and migration assays, after manipulation of the TR4 expression in ccRCC cells, confirmed TR4’s positive roles in promoting ccRCC invasion/migration through modulating the HGF/Met signals. Mechanism dissection further found that the TR4 might alter the HGF/Met signals at the transcriptional level via direct binding to the TR4-response-element of the HGF promoter. We also identified a TR4 upstream regulator, the microRNA （miR-32-5p）, as the ccRCC metastasis suppressor that could down-regulate TR4 signals via directly targeting the 3’UTR of TR4 to decrease TR4 protein expression. Finally, the orthotopic xenografts model also confirmed the in vitro cell lines data.3. TR4 promote ccRCC metastasis via circ 000002/miR-19s-3p/miR-29s-3p/IGF-1 signalsMechanism dissection further found that the TR4 might alter the circ₀00002 signals at the transcriptional level via direct binding to the TR4-response-element of the ZRANB1 promoter. Circ₀00002 might sponge the miR-19s-3p and miR-29s-3p to influence the distribution of these miRNA and then promote the up-regulate the expression of target gene IGF-1 to promote the ccRCC metastasis.Conclusion:In summary, TR4 can promote ccRCC metastasis in vitro and in vivo. The detailed mechanism dissection showed that TR4 might up-regulate HGF/Met/MMP2/MMP9 signals which might be suppressed by th miR-32-5p. TR4 also might up-regulate circ₀00002/miR-19s-3p/miR-29s-3p/IGF-1 signals to promote ccRCC metastasis.