High Dose Androgen Suppresses Metastasis In Endometrial Cancer Via β-catenin Signaling Pathway

Objectives: To clarify androgen role in the development and progression of endometrial cancer.the endometrial cancer cells(KLE and HEC-1B)were stimulated with high concentrations of androgen(100n M DHT),and then we observed its effects on the biological behavior of endometrial cancer cells.The mechanisms of androgen action in endometrial cancer cells were also further explored.Materials and methods: This project was divided into two parts: 1.The effects of androgen treatment in the endometrial cancer cells in vitro experiments(1)The KLE and HEC-1B cell lines were treated by 100 n M DHT and cell invasion was calculated at 16 hours by Transwell test.(2)100n M DHT was used to treat KLE and HEC-1B cells and protein expressions of EMT biomarkers were tested by Western Blot to elucidate the relationship between androgen and endometrial cancer cell line EMT.(3)E-cadherin and β-catenin were immunoprecipitated followed by Western Blot with β-catenin and E-cadherin respectively,in response to DHT treatment.Immunofluorescence assay was also performed to further co-localize the E-cadherin and β-catenin after the DHT treatment.(4)Meanwhile,we also investigated the association between androgen and canonical WNT/β-catenin pathway.Cytoplasmic,nuclear and membrane proteins were extracted.The key protein molecules such as GSK3β and β-catenin were examined by Western Blot in response to androgen.2.The effects of androgen treatment in the endometrial cancer cells in vivo experiments The lung metastasis of xenograft tumor models was established.Then the mice were randomly grouped and treated by intraperitoneal injection 3 times per week for 4 weeks with DHT(7mg/kg)or same dose of saline for comparison.Bioluminescence imaging was used to detect tumor growth.Results: 1.The results of Transwell experiments showed that androgen(100n M DHT)inhibited endometrial cancer cells migration(DHT vs DMSO:KLE 18.00±4.506 vs 52.00±3.362;HEC-1B 58.13±2.524 vs 85.63±4.660,P<0.05)and invasion(DHT vs DMSO:KLE 19.80±2.672 vs 43.20±2.939;HEC-1B 39.60±3.219 vs 65.40±5.845,P<0.05).2.After the treatment of androgen(100n M DHT)for 16 hours,the expression of the epithelial marker E-cadherin increased dramatically,while the mesenchymal markers,such as vimentin and N-cadherin decreased significantly in KLE cells.In HEC-1B cells,although E-cadherin expression was not markedly increased after the treatment with androgen,the expression of the mesenchymal markers(N-cadherin and Vimentin)was significantly downregulated.Consistent with the above,we observed an obvious reduction in lamellipodia compared with nontreated cells(KLE and HEC-1B)via F-actin staining.3.Co-Immunoprecipitation assay showed the level of E-cadherin-associated β-catenin was increased after androgen(100n M DHT)treatment.Also,immunofluorescence experiments showed that the co-localization of E-cadherin and β-catenin at the cell membrane was increased after androgen(100n M DHT)treatment.4.In endometrial cancer cells(KLE and HEC-1B),the total protein levels of β-catenin and p-β-catenin(Ser675)were decreased in response to androgen(100n M DHT).And androgen(100n M DHT)triggered a reduction of nuclear β-catenin and p-β-catenin(Ser675).However,β-catenin at the membrane increased following androgen(100n M DHT)treatment.GSK3β participates in a negative feedback loop that could serve to limit the duration of the WNT/β-catenin signal.The androgen group showed that the protein levels of GSK3β were increased.5.The results of bioluminescence imaging revealed that the growth rate of endometrial carcinoma metastases in DHT group was less than that in the control group although not significant.Conclusions:1 Androgen(100n M DHT)inhibited endometrial cancer cells motility,EMT and the formation of the lamellipodia.2 Androgen(100n M DHT)inhibited the β-catenin signaling by upregulating GSK-3β expression and recruited the β-catenin to E-cadherin to stabilize the adhesion junctions at the cell membrane.3 Androgen(100n M DHT)had an inhibitory trend in inhibiting endometrial cancer metastasis in vivo.