| Background:Endometrial cancer(EC)is the result of abnormal growth of endometrial cells,a wide variety of which are generally endometrial carcinomas(EC)derived from the glandular epithelium and the most common type is endometrial adenocarcinoma(Endometrial adenocarcinoma,EAC).Endometrial cancer is the most common tumor of female reproductive system in developed countries and regions,also the third leading cause of deathfor female,following ovarian and cervical cancers.The main symptom of endometrial cancer is abnormal vaginal bleeding,however other clinical manifestations such as pelvic pain etc.are not common.For this reason,those endometrial cancers with unconspicuous symptoms could not be diagnosed as early as possible.Environmental and genetic factors are essential in the development and progression of endometrial cancer.It is generally believed that long-term exposure to the environment of excessive estrogen and insufficient progesterone increase the chance of incidence rate of endometrial atypical hyperplasia and endometrial cancer.Therefore,factors that can increase estrogen levels,including obesity,hypertension,diabetes,breast cancer,infertility and postmenopausal delay etc.,are critical for endometrial cancer.At the same time,genetic factor is another main reason for the occurrence of endometrial cancer as well.For example,those patients suffering from Lynch syndrome generally have a high risk of endometrial cancer.In addition,mutations in some oncogenes or tumor suppressor genes can also result in the incidence of endometrial cancer and resistance of chemoradiotherapy.For instance,the over-activated Akt signaling pathway caused by mutations of PTEN/PI3K/Akt molecules in endometrial cancer promotes cell survival,proliferation and treatment resistance(radiotherapy,chemotherapy and progesterone treatment),leading to tumor initiation and progression.Endometrial biopsy combined with transvaginal ultrasound is the standard diagnosis of endometrial cancer.The detection of estrogen receptor(ER),progesterone receptor(PR)and cell proliferating nuclear antigen(Ki-67)levels can help predicting the occurrence and development of endometrial cancer and its prognosis.At present,the main clinical theraputic method of endometrial cancer is radical hysterectomy including the cervix,bilateral fallopian tubes and ovaries,supplemented by radiotherapy,chemotherapy or hormone therapy.However,due to primary or acquired treatment resistance,the 5-year survival rate of endometrial cancer patients has not been significantly improved over the past few decades.Therefore,to enhance the sensitivity of chemo and hormone therapy is critical for improving the prognosis of endometrial cancer.FKBP51 protein(FK506-binding protein 51)is a family member of immunoaffinity proteins whose expression level can be significantly up-regulated by steroid hormones(glucocorticoid,androgen and progesterone).FKBP51 protein is extensively participated in a variety of biological processes with important regulatory roles,including protein folding and transport,the formation of steroid hormone receptor(GR,AR,PR)complex.Also,FKBP51 is involved in regulating the initiation and development of tumors as well as their sensitivity to radiotherapy and chemotherapy through different signaling pathways(mainly Akt,NF-?B).As mentioned obove,endometrial cancer is a neoplastic closely related to steroid hormones(estrogen and progesterone)and Akt signaling pathway.Therefore,whether FKBP51 plays a role in the occurrence and development of endometrial cancer?However,there is no relevant report so far.In addition,can FKBP51 become the target gene and thus affect the sensitivity for chemotherapy in endometrial cancer?This query is also worthy of our in-depth study.Therefore,our research aimed to clarify the role and its mechanism of FKBP51 in the initiation,progression and treatment sensitivity of endometrial cancer,also to provide new insights and directions for the clinical treatment of endometrial cancer.In this paper,we intended to carry out our study from three aspects:Part I.Expression of FKBP51 in human endometrial samples and its clinical significance1.Objective:To detect the expression of FKBP51 in human normal endometrial and endometrial adenocarcinoma tissues,and to explore the relationship between FKBP51 expression and clinical pathological features.2.Methods:(1)Sample collection 98 samples of endometrial adenocarcinoma and 97 cases of normal endometrial specimens were obtained from Shaanxi Chaoying Biotech Co.,Ltd and gynecologydepartment of Provincial Hospital of Shandong University.Among the normal specimens,50 cases of which were proliferative endometria,while 47 cases were secretory endometria.None of the patients had received any form of treatment(including surgery,chemoradiation or endocrine therapy)before surgery.This study passed the approval of the Ethical Review Board and collected samples only after aquiring patient informed consent.(2)Immunohistochemistry Immunohistochemical method was used to detect the expression of FKBP51 protein in normal endometrial and endometrial adenocarcinoma specimens.The staining results were independently scored by two pathologists.In addition,we also carried out image analysis by Image-Pro Plus software to obtain the average optical density(AOD)of each picture's stained area.(3)Statistical processing:The result of negative expression rate and AOD of FKBP51 in endometrial tissues were analyzed by statistical software SPSS,and differences were identified statistically significant when p<0.05.3.Results:The occurrance of endometrial adenocarcinoma is largely due to the lack of sufficient progesterone levels;moreover,in normal menstrual cycle,the level of progesterone during the proliferative phase is evidently lower than the secretory phase.In addition,FKBP51 expression can be drastically induced by progesterone.Therefore,FKBP51 expression in tissues of endometrial adenocarcinoma and normal proliferative phase with less level of progesterone should be lower than that in normal secretory phase of endometrium.Remarkably,the results were consistant with our findings:(1)Compared with the normal endometrial tissues,FKBP51 expression in endometrial adenocarcinoma was significantly decreased and the negative rate was increased.(2)There is no evidence showed that FKBP51 expression was related to the pathological grade or clinical stage of endometrial adenocarcinoma.(3)In normal endometrial tissues,FKBP51 expression was significantly lower in the proliferative phase than that in the secretory phase,however the negative rate was increased.(4)FKBP51 expression fluctuated with the age of patients:rose first but then declined which was in accordance with the variation of progesterone level in vivo.It was further confirmed that FKBP51 was a progesterone closely-related protein.4.Conclusion:The result of immunohistochemistry confirmed that FKBP51 expression in endometrial adenocarcinoma tissues<in normal endometrial tissues of proliferative phase<in normal secretory-phase endometrial tissues.It is speculated that FKBP51,which is closely related to progesterone,may be involved in the occurrence and development of endometrial adenocarcinoma.Part II.The role and mechanism of FKBP51 on cell biological behavior in endometrial adenocarcinoma1.Objective:To evaluate the effect of FKBP51 on the malignant behavior of endometrial adenocarcinoma cells,and to investigate its mechanism.2.Methods:(1)Establishment of two stable cell lines Two human endometrial adenocarcinoma cell lines,KLE,Ishikawa,were selected for their relatively high and low FKBP51 expressions.The constructed shRNA-FKBP51 plasmids were transfected into KLE cells,meanwhile puromycin screening method was applied to establish monoclonal cell lines that stably knock-down FKBP51 expression,containing shR-FKBP51 and control group shR-Ctrl.Additionally,the constructed lentiviral vector with wild-type FKBP51 cDNA was uesd to infect Ishikawa cells and to establish two stable cell lines:LV-FKBP51 and the control group LV-Vec.The expression level of FKBP51 in transfected cells were detected by Western Blot,the interference and overexpression efficiency were tested.(2)Detection of cell proliferative ability The effect of FKBP51 on cell proliferation was detected by real-time cellular analysis(RTCA).(3)Detection of Cell cycle and apoptosis Flow cytometry was used to examine the effect of FKBP51 on cell cycle and apoptosis.(4)Cell invasion assay The effect of FKBP51 on cell invasiveness was tested by transwell method.(5)Detection of Akt pathway molecules The level of phosphorylation of Akt(Ser473,Thr3 08)and the level of downstream molecules of the Akt signaling pathway were measured by Western Blot,including the phosphorylation GSK3?(Ser9),FOXO1,p21,p27 and p53 levels,to explore whether FKBP51 regulates the level of Akt signaling pathway.(6)Akt activity assay The effect of FKBP51 on Akt kinase activity was verified by using the Akt kinase assay kit.(7)Detection of cell proliferation after the inhibition of Akt pathway Transfected KLE cells,shR-Ctrl and shR-FKBP51,were treated with DMSO or various concentrations of Akt specific inhibitor MK-2206.After incubating for 0,24,48 and72 hours,the effect of FKBP51 on the proliferation of the two groups of cells were examined by CCK-8 method.(8)Detection of NF-?B pathway molecules The level of I?B and the phosphorylation of p65(Ser536)in transfected cells were detected by Western Blot to reflect the activation of NF-?B signaling pathway.(9)Detection of ER DMSO or estradiol was added to the transfected Ishikawa cells,and changes in FKBP51 and ER levels were detected by Western Blot.3.Results:(1)Obtain two stably transfected cell lines The cell lines KLE shR-FKBP51,Ishikawa LV-FKBP51,were confirmed to stably down regulated or up regulated the expression of FKBP51,compared to their respective control cell lines,KLE shR-Ctrl and Ishikawa LV-Vec.(2)FKBP51 inhibited cell proliferation Compared to the respective control cell lines,the proliferation of KLE shR-FKBP51 was significantly enhanced(p<0.05)by using real-time cellular analysis(RTCA),whereas the proliferation of Ishikawa LV-FKBP51 was obviously decreased(P<0.01),suggesting that FKBP51 can inhibit the proliferation of endometrial adenocarcinoma cells.(3)FKBP51 did not affect cell apoptosis or invasiveness Flow cytometry analysis and transwell assay confirmed that there was no statistical difference in cell apoptosis rate or invasiveness between the experimental groups of KLE shR-FKBP51,Ishikawa LV-FKBP51 and the control groups of KLE shR-FKBP51,Ishikawa LV-FKBP51(p>0.05,p>0.05).(4)FKBP51 inhibited cell cycle progression The results of cell cycle assay showed that in comparison with the respective control cell lines KLE shR-Ctrl and Ishikawa LV-Vec,the number of cells in G0/G1 phase of KLE shR-FKBP51 was significantly decreased(p<0.001),in G2/M phase was increased(p<0.01)and the cell proliferative index(G2/M+S)was also markedly increased(P<0.001).On the contrary,The number of cells in G0/G1 phase of Ishikawa LV-FKBP51 was increased(p<0.05),in G2/M phase was decreased significantly(p<0.01)and the G2/M+S phase was also decreased(p<0.01).These results confirmed that FKBP51 exerted its inhibitory effect on the proliferation of endometrial adenocarcinoma cells by inducing cell cycle arrest in G0/G1 phase.(5)FKBP51 reduced Akt phosphorylation Western blot results showed that in comparison with the respective control cell lines,the phosphorylation level of Akt(Ser473)/Akt was increased(p<0.01)in KLE shR-FKBP51,while decreased in Ishikawa LV-FKBP51(P<0.01).However,there was no statistical difference in the phosphorylation level of Akt(Thr308)(p>0.05,p>0.05).These results indicated that FKBP51 reduced the phosphorylation level of Akt(Ser473)specifically.(6)FKBP51 attenuates the activity of Akt kinase.Western blot results showed that in comparison with the respective control cell lines,the phosphorylation level of GSK-3a mediated by Akt kinase was enhanced in in KLE shR-FKBP5(p<0.01)1,while decreased in Ishikawa LV-FKBP51(P<0.01),confirming the inhibitory effect of FKBP51 on Akt kinase activity in endometrial adenocarcinoma cells.(7)FKBP51 regulated downstream molecules of the Akt signaling pathway.Western blot results showed that in comparison with the respective control cell lines,the phosphorylation of GSK3?(Ser9)was increased(p<0.01),while p21,p27 and p53 were decreased in KLE shR-FKBP51(p<0.01).On the contrary,the phosphorylation of GSK3?(Ser9)was declined(p<0.01),while p21,p27 and p53 were enhanced in Ishikawa LV-FKBP51(p<0.01).These results confirmed that FKBP51 attenuated the activation of Akt signaling pathway,leading to increased level of cycle-inhibitory molecules,thereby induced cell cycle arrest and inhibition of cell proliferation.(8)Akt inhibitor can reverse the induction of cell proliferation caused by the interference of FKBP51 expression The addition of different concentrations of Akt inhibitor MK-2206 to the transfected KLE cell lines showed that with the increasing concentration of MK-2206,the difference in cell proliferative ability between KLE shR-FKBP51 and KLE shR-Ctrl was gradually narrowed down until there was no difference.These results comfirmed that FKBP51 regulated cell proliferation through the Akt signaling pathway.(9)FKBP51 did not affect the activation of NF-?B signaling pathway Western Blot results showed that that there was no statistical difference in intracellular I?B degradation and the phosphorylation level of p65(Ser536)between KLE shR-FKBP51,Ishikawa LV-FKBP51 and their control groups KLE shR-FKBP51,Ishikawa LV-FKBP51(p>0.05,p>0.05).These results indicated that neither knock-down nor overexpress FKBP51 could interfer the activation of NF-?B signaling pathway.(10)FKBP51 did not affect the level of ER Western Blot results showed that FKBP51 did not affect the expression of ER,while estrogen did not affect the expression of FKBP51 neither.It suggested that FKBP51 might not inhibit the proliferation of endometrial adenocarcinoma cells through the ER pathway.4.Conclusion:FKBP51 decreased Akt(Ser473)phosphorylation,weakened the activity of Akt kinase,induced cell cycle arrest,and inhibited the proliferation of endometrial adenocarcinoma cells.Part ?.The role and mechanism of FKBP51 on sensitivity to chemotherapy and endocrine-therapy in endometrial adenocarcinoma1.Objective:To investigate the effect of FKBP51 on chemotherapy and hormone therapy sensitivity in endometrial adenocarcinoma cells,and to explore the mechanism.2.Methods:(1)The effect of FKBP51 on cell chemosensitivity.Different concentrations of cisplatin and paclitaxel were added to the two transfected cell lines KLE and Ishikawa respectively,the cell viability was tested by CCK-8 assay.(2)Establishment of two progesterone receptor(PR)positive cell lines For lacking of PR in KLE cells,another FKBP51 interfered cell line was needed beside the well-established Ishikawa cell line.The constructed shRNA-FKBP51 plasmids were transfected into RL95-2 cells,meanwhile puromycin screening method was applied to establish monoclonal cell lines that stably knock-down FKBP51 expression,containing shR-FKBP51 and control group shR-Ctrl.(3).The effect of FKBP51 on cell progestin sensitivity Different concentrations of progesterone preparations(medroxyprogesterone acetate,MPA)were added to the two transfected cell lines RL95-2 and Ishikawa respectively,cell viability was tested by CCK-8 assay.(4)The effect of FKBP51 on progesterone-regulated molecules The level of MPA induced proteins,including ER,estradiol dehydrogenase(17?-HSD type 2),p21,p27 and p53,were detected by Western Blot.(5)The role of Akt pathway in the enhancement of MPA sensitivity by FKBP51:a.Detection of Akt phosphorylation Phosphorylated Akt levels were detected by Western Blot with the treatment of DMSO or MPA.b Detection of cell sensitivity to progesterone after Akt pathway was inhibited Under the treatment of MPA,different concentrations of MK-2206 were added to the transfected RL95-2 cells lines,and cell viability was measured by CCK-8 assay.c.Detection of progesterone-related molecules after Akt pathway was inhibited In the presence of MPA,MK-2206 was added to the transfected RL95-2 cells lines,and the levels of PR and p21 were detected by Western Blot.(6)The effect of FKBP51 on PR level DMSO or MPA was added to the transfected RL95-2 cells lines,and levels of FKBP51 and PR were detected by Western Blot.3.Results:(1)FKBP51 did not affect the chemosensitivity of cells.Drug sensitive test showed that there was no statistical difference in cell chemosensitivity between the experimental groups of KLE shR-FKBP51,Ishikawa LV-FKBP51 and the control groups of KLE shR-FKBP51,Ishikawa LV-FKBP51 respectively(p>0.05,p>0.05).(2)Obtain two PR positive cell lines The PR positive cell lines RL95-2 shR-FKBP51,Ishikawa LV-FKBP51,and their respective control cell lines,RL95-2 shR-Ctrl and Ishikawa LV-Vec,were confirmed to stably down regulate and up regulate the expression of FKBP51 by Western Blot.(3)FKBP51 enhanced cell sensitivity to progestin Drug sensitive test showed that compared to the respective control cell lines,cell survival rate and IC50 were significantly enhanced in RL95-2 shR-FKBP51(p<0.01),whereas decreased in Ishikawa LV-FKBP51(p<0.01),suggesting that FKBP51 can enhance the sensitivity of endometrial adenocarcinoma cells to MPA.(4)FKBP51 promoted the expression of progesterone indeuced cycle-inhibitory molecules After treating the cells with MPA,Western Blot results showed that there was no statistical difference in levels of ER and HSD-17?2 between the experimental groups of RL95-2 shR-FKBP51,Ishikawa LV-FKBP51 and the control groups of RL95-2 shR-Ctrl,Ishikawa LV-Vec respectively(p>0.05,p>0.05).However,the levels of p21,p27 and p53 in RL95-2 shR-FKBP51 were decreased(p<0.01),while p21,p27 and p53 in Ishikawa LV-FKBP51 were increased(p<0.01).These results confirmed that FKBP51 promoted the expression of progesterone indeuced cycle-inhibitory molecules.(5)FKBP51 enhanced cell sensitivity to MPA by reducing Akt phosphorylation:a.FKBP51 decreased the level of Akt phosphorylation Western Blot results showed that compared to the respective control cell lines,the phosphorylation of Akt(Ser473)was increased in RL95-2 shR-FKBP51(p<0.01),while decreased in Ishikawa LV-FKBP51(p<0.01),regardless of whether cells were treated with MPA.These results indicated that MPA did not affect the inhibitory effect of FKBP51 on Akt phosphorylation.b.Akt inhibitor can reverse the enhancement of cell sensitivity to MPA caused by the interference of FKBP51 expression Adding different concentrations of Akt inhibitor MK-2206 to the two transfected RL95-2 cell lines showed that with the increasing concentrations of MK-2206,the difference in cell sensitivity to MPA between RL95-2 shR-FKBP51 and RL95-2 shR-Ctrl was gradually narrowed down until there was no difference.These results comfirmed that FKBP51 improved endometrial adenocarcinoma cell sensitivity to MPA through theAkt signaling pathway.c.Akt inhibitor can reverse the inhibition of cycle-suppressors caused by the interference of FKBP51 expression Under the treatment of MPA,Western Blot results showed that with the increase concentration of MK-2206,levels of p21 in RL95-2 shR-FKBP51 and RL95-2 shR-Ctrl were increased gradually,and the difference of p21 expression between RL95-2 shR-FKBP51 and RL95-2 shR-Ctrl were gradually narrowed down until there was no difference.These results comfirmed that FKBP51 improved endometrial adenocarcinoma cell sensitivity to MPA through the Akt signaling pathway.(6)FKBP51 did not affect the expression of PR:a.MPA promoted the expression of FKBP51 In MPA-treated RL95-2 and Ishikawa cell lines,FKBP51 levels were higher than those without MPA.This confirmed the induction of FKBP51 expression by progesterone.b FKBP51 did not affect the level of PR In MPA-treated RL95-2 and Ishikawa cell lines,PR levels were higher than those without MPA.This confirmed the effect of progesterone on the expression of PR,but there was no difference between the experimental group and the empty-vector group,indicating that FKBP51 did not affect the level of PR.4.Conclusion:FKBP51 enhanced the sensitivity of endometrial adenocarcinoma cells to MPA by decreasing the phosphorylation of Akt and increasing the expression of the cycle-inhibitory molecules. |
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