The Role And Mechanism Of Androgen/Androgen Receptor In Epithelial Ovarian Cancer

Androgens Ovarian cancer is one the most fatal gynaecological cancers and the fifth most common cause of cancer-related deaths in women worldwide.It had caused an estimate of 184,799 deaths in 2018 worldwide.Ovarian cancer is a heteroarenes disease that characterized into different subsets and epithelial ovarian cancer(EOC)is the most common type.Primary cytoreductive surgery followed by platinum-based chemotherapy treatment is well-accepted in clinic and has been the first-line therapy for many years.However,despite many advances have been achieved in surgery and chemotherapy over the last 30 years,there has been no remarkable improvement in the five-year survival of patients with advanced EOC.Epidemiology studies suggested that EOC usually occurs in postmenopausal women.The age of patients that first diagnosed ranged from 50 to 84 years old in Australia and Western countries and less than 65 years old in Asia and Middle East.As we know,ovary is not only an endocrine organ but also a target of and regulated by sex steroid hormones.After menopause,the oestrogen secretion abruptly declines,while androgen levels are gradually reduced and maintained for many years.However,androgen is in a state of relative or absolute excess in postmenopausal women.There are many studies showed that androgen contributes to the pathogenesis of ovarian cancer,however,in the phase ? clinical studies of antiandrogen therapy for EOC,neither flutamide nor bicalutamide showed good antitumor effects.Based on the contradictions,the purpose of this study was to explore the role of androgen receptor(AR)in the androgen pathogenesis of EOC and the possible mechanism.Part one Protein levels of androgen receptor in the cancer tissues and the para-cancerous tissues and its clinical relevanceObjective:To explore the protein level of AR in the cancer tissues and the para-cancerous tissues and its clinical relevance.Methods:Cancer biopsies were obtained from patients with epithelial ovarian cancer(EOC)admitted to the Second Hospital of Hebei Medical University from 2010 to 2013.All patients had complete clinic data and were followed up to 5 years post-surgery.There were 87 tissue cores with a diameter of 1.5 mm in a microarray block.The microarray included tissues from patients with papillary serous carcinoma(PSC,N=48),endometrioid ovarian carcinoma(EMC,N=6),mucinous ovarian carcinoma(MUC,N=12),clear cell ovarian carcinoma(CCC,N=4)and para-cancerous tissues adjacent to tumor(Para-ca,N=17).All samples involved in the study were obtained from perimenopausal or postmenopausal women.We used immunostaining to detect the expression of androgen receptor.The expression levels of the target protein were scored base on the percentage of cells that stained positive by 2 independent pathologists.To better measure the expression of AR,Tissue Quest software(Tissue Gnostics)was used and results were given as the percentage of tissue stained positive per millimeter squared of total specimen area.Results:1.Protein Levels of AR in the TMA.The IHC staining showed that the majority of androgen receptors were expressed in the nucleus.The semiquantitative analysis of the AR expression in the tissue microarray chips was performed by Tissue Quest software(Tissue Gnostics)and the result suggested that the AR protein expression level in the ovarian cancer tissues was significantly higher compared to the para-cancerous tissues,P=0.017.The AR positive rate in ovarian cancer tissues(67.1%)was higher than that in the para-cancerous tissues(41.2%),P=0.048.2.The clinical relevance of AR in EOC.The expression of the AR protein was associated with tumour-differentiated grades.We showed that the AR positive rate in lower grade tumours(76%)was higher than that in the higher gread tumors(50%),P=0.041.Such correlation was not discovered when refers to the different pathological subtypes(P=0.129).Furthermore,our data suggested that there was no association between the AR protein and age,the serum level of ca125,the FIGO stage or clinical stages(P>0.05).3.The role of AR in the prognosis of EOC.The Kaplan-Meier method was applied to analyse whether the expression of AR affects the survival rate of EOC patients.The results showed that the prognosis of the patients in the AR positive group was worse compared to the AR negative group(P=0.009).Conclusions:1.The expression of AR in the cancer tissues was significantly higher than that in the para-cancerous tissues.2.The AR expression was not associated with patients' age,gravidity,parity,serum CA125 level,FIGO stages,the clinical stages or the pathological subtypes.However,our data suggested that AR was highly expressed in tumours with lower grade.3.The prognosis of AR-positive patients were worse than that of AR-negative patients.Part Two The effect of testosterone and AR on the growth of epithelial ovarian cancer cellsObjective:SKOV3 and A2780,two ovarian cancer cell lines,were used to explore the effect of androgens(testosterone)on the proliferation of ovarian cancer cells,and further reveal the role of classical androgen nuclear receptors.Methods:1.The expression of androgen receptor in EOC cell lines were detected by immunofluorescence and western blot.2.To determine whether testosterone was associated with proliferation in AR positive and AR negative ovarian cancer cells and to titrate an appropriate concentration,different amount of testosterone(ranging from1 n M to 100 n M)was added to SKOV3 cells and A2780 cells for 24/48 hours before the proliferation rates were measured.3.SKOV3 cells were pretreated with flutamide,a competitive antagonist of AR,for 1 h in indicated groups.An MTS assay was used to detect proliferation rate of the SKOV3 cells.4.sh310-AR was used to knock down the AR expression in SKOV3 cells.The transfection efficiency was determined by RT-PCR and western blot.Testosterone was used to treat cells and the proliferation was measured by MTS assay.5.AB6398-AR was used to enhance the AR expression in SKOV3 cells.The transfection efficiency was determined by RT-PCR and western blot.The cells were treated by testosterone and the proliferation was measured by MTS assay.6.AB6398-AR was used to enhance the AR expression in A2780 cells.The transfection efficiency was determined by RT-PCR and western blot.The cells were treated as indicated above and the proliferation was measured by MTS assay.Results:1.The results suggested that the SKOV3 cells were androgen receptor-positive while the A2780 cells did not express androgen receptor.2.Testosterone enhanced cell proliferation in the SKOV3 cells.After the 24-hour treatment,the cell number increased by 10.45%(T=1 n M),16.92%(T=10 n M),and 21.39%(T=100 n M)in the SKOV3 cells compared to controls and all tested significant by statistical analysis.It is important to point out that there was no difference between the T=10 n M and T=100 n M groups.However,when the testosterone treatment prolonged to 48 h,the cell number increased by 12.15%(T=1 n M),12.45%(T=10 n M),and 6.35%(T=100 n M),which were significantly lower comparing to 24 h treatment.No obvious pro-proliferation effect was found in the testosterone concentration group and time group in the A2780 cells.3.After the pretreatment of flutamine for 1 h,the OD values of the four groups as follows: DMSO(1.73±0.27),T(2.14±0.34),F(1.35±0.04),F+T(1.43±0.07).The difference was statistically significant.Compared to the cells treated with testosterone alone,the enhanced proliferation was almost entirely abolished in the flutamide pretreated cells(F+T group).4.The sh310-AR markedly reduced the AR m RNA and protein expression levels in SKOV3 cells(P<0.05),and the testosterone enhanced proliferation in the sh310-AR/SKOV3 cells(1.71±0.09)was significant lower than that in the sh310-NC/SKOV3 cells(1.87±0.09),P=0.043.5.The AB6398-AR increased the AR m RNA and protein expression levels in SKOV3 cells(P<0.05)and the testosterone enhanced proliferation in the AB6398-AR/SKOV3 cells(2.77±0.12)was significant higher than that in the AB6398-NC/SKOV3 cells(2.34±0.12),P=0.001.6.In the A2780 cells,the AB6398-AR only upregulated the AR m RNA level(P=0.002)but did not affect the protein expression(P=0.317),and no difference of proliferation was observed,P=0.134.Conclusions:1.Testosterone could stimulate cell proliferation only in the AR positive SKOV3 cells but not in the AR negative A2780 cells.2.The stimulation of cell proliferation to the SKOV3 cells by Testosterone could be inhibited by flutamide.3.The testosterone induced proliferation rate changed in consistent of the AR expression levels in SKOV3 cells.Part three The potential mechanism of androgen/androgen receptor in epithelial ovarian cancerObjective: To explore the potential mechanism of androgen/androgen receptor in ovarian cancerMethods:1.Immunohistochemistry(IHC)was used to detect the AKT and p-AKT expression in the tissue.2.Western blot(IHC)was used to detect the AKT and p-AKT expression in the EOC cell lines.3.The sh310-AR were transfected into the SKOV3 cell line and the AKT and p-AKT expression were to detect by Western blot.4.The AB6398-AR transfected into the SKOV3 cell line and the AKT and p-AKT expression were to detect by Western blot.5.The BEZ235 and testosterone were used to treat the SKOV3 cells for 24 h and the MTS assay was used to measure the proliferation.Results:1.The p-AKT protein expression level in ovarian cancer tissues was remarkably higher compare to the para-cancerous tissues(P=0.034)while such difference was not observed in terms of the AKT expression(P=0.815).The p-AKT/AKT ratio in the ovarian cancer tissues was higher than that in the para-cancerous tissues(P=0.005).2.The results indicated that both AKT and p-AKT protein levels in the SKOV3 cells were increased compared to the A2780 cells(AKT:P=0.003?p-AKT:P=0.009).Similarly,the p-AKT/AKT ratio in the SKOV3 cells was increased compared to the A2780 cells(P=0.041).3.The p-AKT expression declined after the sh310-AR knocking down the AR expression(P=0.001)while such difference was not observed in terms of the AKT expression(P=0.953).4.The p-AKT expression increased after the AB6398-AR upregulating the AR expression in SKOV3 cells(P=0.002)while there was no significant change in the AKT expression(P=0.681).5.BEZ235 combined T treated the SKOV3 cell for 24 h,the OD value of the four groups as follows: DMSO(2.09±0.10),T(2.71±0.12),B(1.55±0.12),BEZ235+T(1.50±0.15).The difference was statistically significant.The testosterone induced proliferation was completely abolished when co-culturing with the BEZ235.Conclusions:1.The p-AKT protein expression level and the rate of p-AKT/AKT in ovarian cancer tissues were remarkably higher compare to the para-cancerous tissues while such difference was not observed in terms of the AKT expression.2.Both AKT and p-AKT protein levels in the SKOV3 cells were increased compared to the A2780 cells.3.Knock down/over expression of the androgen receptor in SKOV3 cells resulted in a corresponding decrease/increase in p-AKT levels without significant change in total AKT levels.4.The testosterone induced proliferation was completely abolished when co-culturing with the BEZ235.