Investigation Of The Role Of HSF1 In The Pathogenesis Of Cushing's Disease

Part I: Evaluation of the expression of heat shock factor 1 in human corticotroph adenomas and correlation with clinical parametersObjective To investigate the possible correlation between HSF1 expression in human normal corticotroph adenomas and ACTH secretion,plasma cortisol levels and tumor diameter.Methods A series of 47 corticotroph adenomas and 4 normal human pituitaries from Biobank of Clinical Neuroendocrinology in Max Planck institute of Psychiatry were enrolled in this study.Immunohistochemical staining for HSF1 was performed on paraffin-embedded sections.Intensity of immunoreactivity was scored in a semi-quantitative manner.Graph Pad Prism Ver.7.0 was used for statistical analysis with documented clinical data and HSF1 expression level.Results In the cohort of 47 corticotroph adenomas(44 females and 3 males),14 corticotroph adenomas were diagnosed as macroadenomas(mean tumor diamaeter 15.86±4.67 mm),25 cases as microadenomas(mean tumor diameter 7.68±1.91mm).Among these,3 macroadenomas,1 microadenoma,and 1 case whose diameter was unavailable,were classified as invasive.Hormone levels were on average as following: 9:00 serum cortisol 827.2±290.4 nmol/ml,midnight cortisol 653±347.7 nmol/ml,urinary free cortisol(UFC)2488.69±1932.99 nmol/ml and 9:00 ACTH 99.77±52.23 ng/ml.Immunostaining for HSF1 was detected in all normal pituitaries and corticotroph adenomas.Quantification of HSF1 expression in corticotroph adenomas showed a higher score in 55.31%(n=26)of corticotroph adenomas and no difference with normal pituitaries in 44.69%(n=21)of cases.HSF1 score showed no correlation with gender(p=0.8382),tumor invasiveness(p=0.2374),9:00 serum cortisol(p=0.4627),ACTH(p=0.6679),UFC (p=0.9048),and tumor diameter(p=0.5581).Conclusion HSF1 is differentially expressed in human corticotroph adenomas.No significant correlation between HSF1 expression level and analysed clinical parameters was found in our cohort.Part II: Determination of activation status of HSF1 in a cellular model of corticotroph adenoma and effect of HSF1 inactivation on cell proliferation and ACTH synthesisObjective To investigate the acitivity of HSF1 in At T20 cells and the effect of HSF1 inactivation on cell proliferation and the regulation of ACTH synthesis in corticotroph adenomaMethods Western blot and HSE-Luc reporter gene assay were used to determine the response of At T20 to cellular heat shock stress stimulation and HSF1 pharmacological inhibitior KRIBB11.Cell Titer-Glo assay and cell counting were used to evaluate cell viability and cell proliferation after HSF1 inactivation.FACS analysis was used to detect cell cycle distribution.Caspase-Glo 3/7 assay was performed to detect the effect of HSF1 in the activation of apoptosis process.Pomc-Luc and Nur RE-Luc reporter assasy were used to determine the effect of KRIBB11 on Pomc promoter activity both in basal and dexamethasone-stimulated condition in At T20 cells.Quantitative RT-PCR was used to evaluate the effect of KRIBB11 on Pomc expression in At T20 cells.Radioimmunoassay was used to measure ACTH secretion in the supernatant of At T20 cells,primary cultures from mouse pituitary cells and primary cultures from human corticotroph adenoma cells.Transient silencing of HSF1 by si RNA was performed to confirm the effects of HSF1 pharmacological inhibition.Results Heat shock did not affect the expression of HSF1 in At T20 cells.KRIBB11 treatment caused a downshift of the band specific for HSF1 in compassion to untreated cells.In addition,by reporter gene assay,we found a strongly increased HSE promoter activity after heat shock stimulation compared to non-heat shock group(p<0.01).HSP90 N-terminal inhibitor 17-AAG augmented HSE promoter activity under both un-stressed and stressed status(p<0.01 vs vehicle).KRIBB11 treatment exert a suppressive effect on basal and 17-AAG stimulated HSE promoter activity both in absence and in presence of heat shock in At T20 cells(p<0.01 vs vehicle).Furthermore,treatment with KRIBB11 exhibited dose-and time-dependent inhibition of cell viability in At T20 cells,with an IC50 of 10?M.The result was confirmed by analogue inhibitory effect on cell proliferation by typan blue exclusion test.KRIBB11 induced cell cycle arrest in G2/M phase.Caspase-Glo 3/7 assay revealed that KRIBB11 did not influence apoptotic process in At T20 cells under non-cytotoxic concentrations.HSF1 inhibition decreased POMC promoter activity both in basal and dexamethasone-stimulated conditions,and a similar tendency was displayed on Nur RE element promoter activity.The effect on Pomc promoter activity was further confirmed by real-time PCR after 6 and 24 h of treatment and by silencing with two different si RNA sequences targeting HSF1.Moreover,treatment for 24 h with Kribb11 reduced ACTH secretion in the supernatant of At T-20 cells in a dose-dependent manner in basal conditions with enhancing the suppressive effect of low concentrations of dexamethasone.The same inhibitory effect on ACTH secretion was observed by silencing HSF1.In addition,48 h treatment with the compound showed an inhibitory effect on ACTH secretion in three out of four primary cultures from corticotroph adenomas,which was not potentiated by dexamethasone co-treatment,thus confirming the results obtained with At T-20 cells.Interestingly,HSF1 inhibition didn't affect neither the basal nor the dexamethasone-induced suppression of ACTH secretion in mouse normal pituitary cells.Conclusions HSF1 is constitutively activated in basal conditions in At T20 cells,and its activity can be either positively or negatively modulated.HSF1 specific inhibitor KRIBB11 exerts its HSF1 inhibitory effect by reducing its activation in At T20 cells.HSF1 inactivation has a suppressive effect on cell viability and proliferation without inducing apoptosis,accompanied with cell cycle arrest in G2/M phase.Pharmacological inhibition and silencing of HSF1 is decreasing ACTH production in corticotroph adenomas in vitro.HSF1 could be a potent therapeutic target for corticotraph adenomas.Part III: HSF1 inhibition regulate glucocorticoid receptor activity and activate its transrepression effect on POMC promoterObjective To explore the effect of HSF1 inhibition on glucocorticoid receptor activity in At T20 cellsMethods MMTV-Luc reporter assasy and quantitative RT-PCR were used to determine the effect of GR agonist dexamethasone or HSF1 pharmacological inhibition on GR transcriptional activity and expression in At T20 cells.Western blot for total cellular protein extract was used to evaluate GR and HSP70 expression after HSF1 inactivation.Cytosolic or nuclear protein extract were analyzed by western blot to test the effect of dexamethasone or HSF1 inhibition on GR nuclear translocation.HSF1 si RNA were used to confirm the effects of HSF1 pharmacological inhibition.Results Both basal and dexamethasone-stimulated MMTV-reporter activities were increased by KRIBB11 in comparison either to vehicle or dexamethasone alone,with maximal effect at 10 ?M in absence of dexamethasone and a 3.2-fold increase in promoter activity with KRIBB11 at the tested concentrations(2.5,5,10?M)with dexamethasone stimulation.KRIBB11 and dexamethasone caused a downregulation of the transcriptional rate of glucocorticoid receptor.This effect was reverted after a prolonged treatment with dexamethasone or KRIBB11 after 24 h.As expected,treatment with KRIBB11 decreased Hsp70 and Hsf1 gene transcription.By western blot,a progressive increase in GR expression was shown at increasing concentrations of the HSF1 inhibitor,but no obvious change of HSP70 was observed.KRIBB11 inhibited the proteasome inhibitor MG132 induced expression of HSP70,but no obvious alteration was observed in presence of the protein translation inhibitor cycloheximide.In addition,by detecting nuclear and cytoplasmic protein extracts,KRIBB11 was proved to not have an additive effect on GR translocation compared to dexamethasone alone.By transiently silencing HSF1,similar effect on GR activity and GR expression was obtained from two different sequences.Conclusion HSF1 inhibition increases glucocorticoid receptor activity and activates the feedback regulation mechanism of its transcription,exert its transrepression effect on POMC promoter.