Abstract
Cushing’s disease is a rare but devastating and difficult to manage condition. The somatostatin analogue pasireotide is the only pituitary-targeting pharmaceutical approved for the treatment of Cushing’s disease but is accompanied by varying efficacy and potentially severe side effects. Finding means to predict which patients are more likely to benefit from this treatment may improve their management. More than half of corticotroph tumours harbour mutations in the USP8 gene, and there is evidence of higher somatostatin receptor 5 (SSTR5) expression in the USP8-mutant tumours. Pasireotide has a high affinity for SSTR5, indicating that these tumours may be more sensitive to treatment. To test this hypothesis, we examined the inhibitory action of pasireotide on adrenocorticotrophic hormone synthesis in primary cultures of human corticotroph tumour with assessed USP8 mutational status and in immortalized murine corticotroph tumour cells overexpressing human USP8 mutants frequent in Cushing’s disease. Our in vitro results demonstrate that pasireotide exerts a higher antisecretory response in USP8-mutant corticotroph tumours. Overexpressing USP8 mutants in a murine corticotroph tumour cell model increased endogenous somatostatin receptor 5 (Sstr5) transcription. The murine Sstr5 promoter has two binding sites for the activating protein 1 (AP-1) and USP8 mutants possibly to mediate their action by stimulating AP-1 transcriptional activity. Our data corroborate the USP8 mutational status as a potential marker of pasireotide response and describe a potential mechanism through which USP8 mutants may regulate SSTR5 gene expression.
Introduction
Cushing’s disease is caused by corticotroph tumours and is the most common form of endogenous hypercortisolism (Newell-Price et al. 2006, Lacroix et al. 2015). Chronic exposure to elevated cortisol levels is associated with increased morbidity and overall mortality (Dekkers et al. 2013, Clayton et al. 2016, Valassi et al. 2019). Early diagnosis and successful treatment are important goals to achieve, as the mortality rate in patients with the persistent disease is around ten times higher compared to patients in remission (Fleseriu et al. 2021).
The first-line treatment for Cushing’s disease is the surgical removal of the pituitary tumour with a success rate varying between 79% in microadenomas and 40% in macroadenomas (Dimopoulou et al. 2014, Petersenn et al. 2015). Other therapeutic options in patients with contraindication to surgery or with either persistent or recurrent disease are medical therapy, pituitary radiotherapy and bilateral adrenalectomy (Ritzel et al. 2013, Pivonello et al. 2015, Tritos & Biller 2018, Feelders et al. 2019).
The somatostatin analogue pasireotide is the only pituitary-targeting pharmaceutical that is approved for the treatment of patients with persistent or recurrent Cushing’s disease (Pivonello et al. 2015, Fleseriu et al. 2021). Pasireotide has a high affinity for the somatostatin receptor (SSTR) 5, which is abundantly expressed in corticotroph tumours and whose expression is not affected by the high plasma cortisol levels (Hofland et al. 2005, van der Hoek et al. 2005, Batista et al. 2006). Clinical trials and real-world evidence have demonstrated pasireotide efficacy in normalizing 24-h urinary free cortisol (24hUFC) levels in patients with Cushing’s disease and also higher prevalence and severity of hyperglycaemia (Boscaro et al. 2009, 2014, Colao et al. 2012, Schopohl et al. 2015, Petersenn et al. 2017, Lacroix et al. 2018, Pivonello et al. 2019). Furthermore, a meta-analysis reported biochemical normalization in 41% of patients, clearly demonstrating resistance to pasireotide treatment in more than half of cases (Broersen et al. 2018).
Almost half of corticotroph tumours carry somatic mutations in the ubiquitin-specific protease 8 (USP8) gene (Ma et al. 2015, Perez-Rivas et al. 2015, Reincke et al. 2015, reviewed in Sbiera et al. 2019). Studies reported USP8-mutant tumours to be smaller in size but with worse postoperative outcome in the long-term compared with WT tumours (Ma et al. 2015, Perez-Rivas et al. 2015, Reincke et al. 2015, Hayashi et al. 2016, Faucz et al. 2017, Albani et al. 2018b). There is evidence that USP8-mutant corticotroph tumours have higher SSTR5 immunoreactivity score, suggesting a better response to pasireotide (Hayashi et al. 2016, Castellnou et al. 2020). Based on this, a recent consensus suggested that thse USP8 mutational status could be utilized as a marker of pasireotide response (Fleseriu et al. 2021). The aim of the present study was to determine the impact of USP8 mutations on pasireotide response in human and murine corticotroph tumour in vitro.
Material and methods
Patients and samples
The study included 24 corticotroph tumours freshly obtained after transsphenoidal surgery (used for primary cell culture) and 51 archived formalin-fixed paraffin-embedded (FFPE) tumours (used for immunohistochemistry). A part of the FFPE cohort was described elsewhere (Albani et al. 2018b ) (Table 1). Biochemical diagnosis of Cushing’s syndrome was based on abnormal 24hUFC, 1 mg dexamethasone suppression test overnight (low-dose suppression test LDDST) and late-night salivary cortisol (Dimopoulou et al. 2014). All the patients with suspected central hypercortisolism, based on the baseline adrenocorticotrophic hormone (ACTH) level and on the response to the high-dose dexamethasone suppression test and to the corticotrophin-releasing hormone (CRH) stimulation test (100 μg human CRH i.v.), underwent MRI. When the tumour was not clearly visible in the MRI or in case of inconclusive test results, the inferior petrosal sinus sampling (IPSS) was performed. Once the diagnosis of CD was made, transsphenoidal surgery was performed as first-line therapy. The study was approved by the ethics committee of the LMU Munich (Nr. 643-16), and all subjects gave written informed consent.
Clinical, radiological and biochemical presentation of the patients included in the FFPE cohort.
| Variable | Total, n | ||
|---|---|---|---|
| Age at diagnosis (years), mean ± s.d. | 48 | 13 | 51 |
| Female patients, n (%) | 41 | (80) | 51 |
| Disease presentation, n (%) | 51 | ||
| Cushing’s disease | 47 | (92) | |
| CTP-BADX/NS | 4 | (8) | |
| USP8 mutant tumours, n (%) | 21 | (41) | 51 |
| Macroadenomas, n (%) | 17 | (34) | 50 |
| Cavernous sinus invasion, n (%) | 8 | (25) | 32 |
| Postoperative biochemical remission, n (%) | 38 | (81) | 47 |
| Basal plasma ACTH (pg/mL), median (IQR) | 70 | 48 | 33 |
| Basal levels of cortisol (µg/dL), median (IQR) | 24 | 10 | 33 |
| 24-h urinary free cortisol (µg/24 h), median (IQR) | 584 | 1099 | 25 |
| Cortisol after low-dose dexamethasone suppression test (µg/dL), median (IQR) | 25 | 14 | 12 |
Primary cell culture
Primary cultures were derived from the 24 corticotroph tumours freshly obtained after transsphenoidal surgery. Tumours were washed with HDB buffer (15 mmol/L HEPES (pH 7.4), 137 mmol NaCl, 5 mmol/L KCl, 0.7 mmol/L Na2HPO4, 10 mmol/L glucose, 2.5 mg/mL amphotericin B, 105 units/L penicillin/streptomycin). Part of each tumour was snap-frozen and stored at −80°C for DNA extraction, while the rest was enzymatically dispersed in collagenase, as previously described (Stalla et al. 1988). Cell viability was determined by trypan blue staining and was considered acceptable when above 80%. Cells were seeded in 96-well plates (20,000 cells per well) in Dulbecco’s modified Eagle’s medium (DMEM; Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS; Gibco) and 105 IU/L penicillin–streptomycin; Invitrogen and left to recover for 3days, before being treated with 10 nM pasireotide in low serum medium (1% FCS DMEM) for 3 days. For each tumour culture, each condition was in triplicates.
ACTH determination
ACTH was determined in the primary cell culture supernatants using a RIA as previously described (Stalla et al. 1988). ACTH values were normalized with cell viability values obtained with the non-radioactive colorimetric WST-1 assay at 450 nm (Roche Molecular Biochemicals) and are presented as pg/mL/OD 450 nm. A physiologically relevant response was arbitrarily set at ≥20% secretion suppression compared to vehicle control.
USP8 sequencing
DNA extraction from the 24 frozen tumours was performed using a Maxwell Instrument and Maxwell Tissue DNA Kit (Promega). The FFPE DNA mini kit (Qiagen) was used for DNA extraction from the 51 FFPE specimens. Exon 14 of USP8 containing the mutational hotspot was amplified by PCR and sequenced as previously described (Perez-Rivas et al. 2015, 2017). Chromatograms were analysed with the Mutation Surveyor software version v4.09 (Soft Genetics).
Immunohistochemistry
SSTR5 immunoreactivity was assessed on the 51 FFPE corticotroph tumours by peroxidase immunohistochemistry on 4 µm formalin-fixed paraffin-embedded sections using the rabbit monoclonal UMB4 antibody (#ab109495, Abcam). After deparaffinization and consecutive rehydration, antigen retrieval was done by microwaving in citrate buffer of pH 6.0 for 3 × 5 min. Sections were blocked in diluted goat serum for 30 min and incubated with the primary antibody diluted 1:80 in Tris buffer saline (TBS) overnight at 4°C. After washing in TBS, they were incubated in biotinylated goat anti-rabbit IgG (Vector Laboratories) for 30 min, washed and incubated in avidin–biotin complex (Vectastain Elite Kit, Vector Laboratories) for 30 min. Immunoreactivity was visualised with 1 mg/mL diaminobenzidine (DAB, Sigma) as chromogen and 0.01% hydrogen peroxide as substrate, and sections were counter-stained in Toluidine blue, dehydrated and mounted with Entellan (Sigma).
SSTR5 immunoreactivity was mainly cytoplasmic and was scored based on staining intensity as absent (0), weak (1), moderate (2) and strong (3). Immunoreactivity score was calculated as follows: 0 × percentage of cells with absent staining + 1 × percentage of cells with weak staining + 2 × percentage of cells with moderate staining + 3 × percentage of cells with strong staining (H-score; Detre et al. 1995). Each value was divided by 300, which is the hypothetical maximum score of 100% of cells with strong staining, giving an immunoreactivity score range between 0 (no immunoreactivity) and 1 (maximum immunoreactivity). SSTR5 immunoreactive score (IRS) was calculated as previously described and divided into four categories (negative (IRS 0–1), low (2–3), medium, (4–8) and high (9–12)) (Hayashi et al. 2016, Castellnou et al. 2020).
Plasmids
The pME-Flag-USP8 vectors expressing WT and mutant USP8 were previously described (Reincke et al. 2015). The POMC-luc reporter vector has ~800 bp of the human POMC promoter upstream to the luciferase gene (Panomics). The AP1-luc reporter vector has seven repeats of the AP1-responsive sequence upstream to luciferase (Stratagene). pcDNA-3×HA-SSTR5 expresses N-terminal 3× haemagglutinin (HA)-tagged human SSTR5 (cDNA Resource Center, Bloomsburg University). pCI-His-hUbi encodes for histidine-tagged human ubiquitin and was a gift from Astar Winoto (Addgene plasmid 31815; RRID:Addgene_31815) (Young et al. 2011).
Cell culture and luciferase assay
Mouse corticotroph tumour AtT20/D16vF2 (ATCC® CCL-89™) cells were obtained from the American Type Culture Collection and authentized. Cells were cultured in DMEM supplemented with 10% heat-inactivated FCS, 2 nmol/L glutamine and 105 IU/L penicillin–streptomycin at 37°C and 5% CO2. Cell culture materials were from Life Technologies, Nunc and Sigma–Aldrich.
For luciferase assays and RNA extraction, cells were plated and the day after, they were transfected using SuperFect (Qiagen) following the manufacturer’s instructions. For experiments with reporter plasmids, cells were assayed 24 h after transfection. The transfection efficacy was monitored by cotransfection with the RSV-β-gal construct, and results are presented as luciferase: β-galactosidase activity ratio. The empty vector pME-Flag was used as ‘mock’ negative control.
AtT-20 cells were transfected with a Fos siRNA pool against mouse c-Fos (#sc-29222, Santa Cruz Biotechnology) for 48 h. Scrambled unspecific siRNA (# sc-37007, Santa Cruz) was used as control.
RNA extraction and quantitative qRT-PCR
RNA was extracted from AtT-20 cells 48 h after transfection as indicated for each experiment using Trizol (Invitrogen) per manufacturer’s instructions. 1 µg RNA was reverse transcribed using QuantiTect RT kit (Qiagen), and qPCR was done with QuantiFast SYBR Green PCR kit (Qiagen) on LightCycler (Roche). Primers were against mouse Sstr5 (5’-GTGGACAGCCGTGGAGAC-3’ and 5’-TGAGCGCTCTGCTCTTTG-3’) and TfIIb (5’-tggagatttgtccaccatga-3’ and 5’-gaattgccaaactcatca-aaact-3’). Primers against mouse Fos (5’-CATCCTCC-CGCTGCAGTAG-3’ and 5’- GCGCAAAAGTCCT-GTGTGTT-3’) were used to validate Fos knockdown. Gene expression was normalized to TfIIb transcript levels.
SSTR5 Immunoprecipitation
AtT-20 cells overexpressing HA-tagged human SSTR5 (pcDNA-3xHA-SSTR5), ubiquitin (pCI-His-hUbi) and the different USP8 vectors (pME-Flag-USP8) or empty plasmid were pretreated with 25 nM MG132and 200 µM chloroquine (both Sigma–Aldrich) for 3 and 1 h, respectively. Then, cells were treated with the SSTR5 ligand BIM 23,052 (#2842, Tocris) or vehicle for 1 h at a final concentration of 1 µM. Cells were scraped in cold lysis buffer (20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 50 mM NaF, 1% Nonidet P-40, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µg/mL aprotinin, 1 µg/mL leupeptin and1 µg/mL pepstatin A) supplemented with 25 nM MG132, 200 µM chloroquine and 10 mM N-ethylmaleimide (reagents from Sigma–Aldrich). Supernatants were collected after centrifugation, and immunoprecipitation and immunoblotting were performed following standard procedures. HA-tagged human SSTR5 was immunoprecipitated using an HA-Tag antibody (anti-HA.11 Epitope Tag Antibody, #901513, Biolegend) and protein A/G agarose (Sigma–Aldrich). Membranes were immunoblotted with antibodies against His-tag (HRP-conjugated His-Tag (27E8), #9991, Cell Signaling) to detect ubiquitin, USP8 (HPA004869, Sigma) to detect USP8 binding and SSTR5 (UMB4, #ab109495; Abcam) to monitor human SSTR5 overexpression. Human USP8 overexpression was monitored using Flag-Tag (M2, #F3165, Sigma–Aldrich). Signals were detected using ECL Clarity (Biorad).
Statistical analysis
Statistical analysis was performed using the statistical software package SPSS version 24.0 (IBM SPSS Statistics). Normal distribution of the data was examined using the Shapiro–Wilk test. Student’s t-test and Mann–Whitney U-test were used to compare USP8 mutant and WT tumour groups in terms of ACTH suppression after treatment and SSTR5 immunoreactivity, respectively. Data are represented as mean ± standard deviation (s.d.) or median (interquartile range, IQR). Statistical significance was considered when P < 0.05.
Results
Screening 51 archival FFPE corticotroph tumours revealed significantly higher SSTR5 immunoreactivity score in 21 USP8 mutant compared to 30 WT (median (IQR), 0.5 (0.3–0.85) vs 0.2 (0–0.3); P = 0.0007; Fig. 1A) and IRS (median (IQR), 5 (4-9) vs 4 (1.5–4.5); P = 0.0115; Supplementary Fig. 1, see section on supplementary materials given at the end of this article). The most frequent mutation was the substitution c.2159C>G (p.720P>R), which was found in 9/21 (43%) cases. The other variants were the deletion c.2155_2157delTCC in 7/21 (33%) and the substitutions c.2152T>C (p.718S>P) in 4/21 (19%) and c.2159C>A (p.720P>Q) in 1 case (5%). We observed higher SSTR5 immunoreactivity scores in the USP8-mutant tumour groups regardless of mutation type (median (IQR) for p.Ser718P, p.S718del and p.Pro720Arg/Gln: 0.9(0.6), 0.5(0.6) and 0.6(0.7), respectively versus WT 0.2(0.3)). These differences were significant for the p.Ser718P and p.S718del mutant groups (P = 0.013 and 0.041, respectively) but did not reach statistical significance for the p.Pro720Arg/Gln group (P = 0.229). Dividing in four IRS categories, we observed a trend towards higher incidence of USP8 mutations in the tumours with higher SSTR5 IRS (P = 0.065; Supplementary Table 1).
(A) SSTR5 immunoreactivity score in 51 FFPE corticotroph tumour cohort: USP8 WT (n = 30) and mutant (n = 21) tumours. Data are median (IQR). * P = 0.0007. (B) Antisecretory response to in vitro pasireotide treatment in a separate cohort of 24 primary cultures of human WT (n = 11) and mutant (n = 13) corticotroph tumours. For each culture, each condition (control or pasireotide treatment) was given in triplicates. Data are calculated as ACTH (pg/mL) to cell viability (WST-1 colorimetric assay; OD450 nm) and presented as % suppression to individual vehicle controls. * P = 0.044 (t-test).
Citation: Endocrine-Related Cancer 29, 8; 10.1530/ERC-22-0088
Four USP8 WT cases (three female and one male; all microadenomas) presented with higher SSTR5 levels, but no clinical differences were observed compared to the rest of WT tumours that had low SSTR5 immunoreactivity (age at diagnosis (P = 0.622), sex (P = 1.00), disease presentation (P = 1.00), tumour size (P = 0.216), invasion (P = 1.00), basal ACTH (P = 0.352), basal cortisol P = 0.595), 24hUFC (P = 0.844) or cortisol after dexamethasone suppression test (P = 0.522)).
To examine the impact of USP8 mutational status on the antisecretory response to pasireotide, we studied a separate cohort of 24 corticotroph tumours in primary culture: 11 USP8 WT and 13 mutants (c.2159C>G (p.720P>R; n = 4), c.2152T>C (p.718S>P; n = 5), S718 deletion c.2155_2157delTCC (n = 3) and long deletion c.2154_2172delCTCCCCAGATATAACCCA (n = 1)). Pasireotide treatment in vitro suppressed ACTH secretion to a significantly higher extent in the USP8-mutant corticotroph tumours (% of ACTH suppression USP8 mutant, 36 ± 26 vs WT 17 ± 13; t-test P = 0.044; Fig. 1B). Pasireotide suppressed ACTH secretion by more than 20% in 14 cases: 5 USP8 WT and 9 mutants (1 p.720P>R, 4 p.718S>P and 4 deletions). We observed more responders than non-responders in the USP8 mutant group (9/14, 64%) than in the WT group (5/11, 45.5%), but the difference was not statistically significant (χ-square P = 0.408). Focusing on the pasireotide responders, there was a trend towards higher ACTH suppression in the USP8-mutant tumours compared to WT, which did not reach statistical significance (% of ACTH suppression in USP8 mutant, 49 ± 21 vs WT 29 ± 6; t-test P = 0.067).
To further study the mechanisms responsible for the improved response to pasireotide in USP8-mutant corticotroph tumour cells, we overexpressed the most common USP8 mutants, p.Ser718Pro, p.Pro720Arg and p.Ser718del, in immortalized AtT-20 cells. All USP8 mutants significantly increased human POMC promoter activity (ANOVA P = 0.0145) similar to what we reported using the rat Pomc promoter (Reincke et al. 2015). In accordance with what we observed in primary human corticotroph tumour cultures, pasireotide suppressed human POMC promoter activity more potently in the USP8 mutant overexpressing cells compared to WT (ANOVA P = 0.0007; Fig. 2A).
(A) Suppressive action of pasireotide on human POMC promoter activity in immortalized mouse corticotroph tumour AtT-20 cells overexpressing USP8 WT, p.Ser718Pro, p.Pro720Arg, p.Ser718del or empty pME vector (mock). Cells were transfected for 24 h before being treated with 10 nM pasireotide for 6 h in serum-free medium. Data are calculated as luciferase to β-galactosidase ratio and as means ± s.d. for three experiments with each transfection condition in triplicates presented as percentage of each vehicle treatment control. RLA, relative luciferase activity. * P < 0.05 to mock (t-test). (B and C). Real-time qRT-PCR data showing the impact of USP8 mutants on endogenous Sstr5 (B) and Sstr2 (C) expression. Data of Sstrx/TfIIb obtained in triplicates. (D) Effect of USP8 mutants vs WT (wt) on AP-1 transcriptional activity. Data are calculated as luciferase to β-galactosidase ratio and as means ± s.d. for three experiments with each transfection condition in triplicates and presented as percentage of empty pME vector (mock). * P < 0.005 to mock (t-test). (E) Real-time qRT-PCR data showing the impact of knocking down Fos on the stimulatory action of USP8 mutants on endogenous Sstr5 transcription. RNA was extracted from AtT-20 cells transfected with the various USP8 vectors and siRNA against mouse Fos for 48 h. Data of Sstr5/TfIIb obtained in triplicates and presented as fold change to empty pME (mock)-scramble control. * P < 0.05 to mock-scramble. All error bars are s.d.
Citation: Endocrine-Related Cancer 29, 8; 10.1530/ERC-22-0088
USP8 mutant overexpression in AtT-20 cells resulted in significantly higher endogenous Sstr5 expression (ANOVA P < 0.0001; Fig. 2B). In contrast, no effects were observed on the expression of Sstr2, a somatostatin receptor that is highly expressed in AtT-20 cells (Fig. 2C). The murine Sstr5 promoter has two binding sites for the AP-1 (Gordon et al. 1999). USP8 mutants stimulated AP-1 transcriptional activity significantly more compared to WT (ANOVA P = 0.0325; Fig. 2D) and knocking down the gene encoding for the AP-1 subunit Fos abolished their stimulatory action on Sstr5 transcription (Fig. 2E).
Considering the role of the USP8 deubiquitinase as a rescue protein (Mizuno et al. 2005), we investigated whether it may also regulate SSTR5 at the posttranslational level. Somatostatin-14 treatment was shown to induce SSTR3 ubiquitination in heterologous cell systems (Tulipano et al. 2004). However, in our study, we did not observe ubiquitin binding in human SSTR5 immunoprecipitates after treatment with the SSTR5 agonist BIM 23052, indicating a lack of ubiquitination (Fig. 3A). In addition, co-immunoprecipitation did not detect any physical association between WT or mutant USP8 and overexpressed human SSTR5 (Fig. 3B).
(A) SSTR5 immunoprecipitation experiment showing lack of ubiquitination. AtT-20 cells were transfected with overexpressing vectors for N-terminus HA-tagged human SSTR5 and/or different Flag-USP8 constructs plus ubiquitin (6×Hist-Ubiquitin) and left for 48 h in 2% FCS DMEM. The empty vectors pcDNA3.1+(HA) and pME(Flag) were used as mock controls for SSTR5 and Flag-USP8 constructs, respectively. Transfected cells were pretreated with MG132 and chloroquine before being treated with 10 nM BIM 23,052 for 1 h in serum-free medium. Overexpressed human SSTR5 was immunoprecipitated with the anti-HA-tag (C29F4) antibody and immunoblotted with anti-His-tag to detect overexpressed His-tagged ubiquitin. Blots were stripped with 100 mmol/L Tris–HCl pH2.0 and blotted with anti-SSTR5 (UMB4) to monitor human SSTR5 overexpression. SSTR5 is detectable as a broad band migrating between ~55 and 100 kDa due to glycosylation (the size of the unglycosylated SSTR5 is ~40 kDa; Lupp et al. 2011). In the inset, total lysate immunoblots showing overexpressed Flag-USP8 forms (blotted with the FLAG M2 antibody against Flag-tag) and His-tagged ubiquitin (blotted with the His-tag antibody). (B) Immunoprecipitation experiment showing lack of interaction between SSTR5 and USP8 forms. Overexpressed human SSTR5 was immunoprecipitated with the HA antibody and immunoblotted with anti-USP8 or SSTR5 (UMB4) antibodies.
Citation: Endocrine-Related Cancer 29, 8; 10.1530/ERC-22-0088
Discussion
Pasireotide responder patients experience significant clinical benefits, such as improvement of arterial blood pressure, BMI , lipid profile, quality of life and reduced cardiometabolic risk and also side effects like hyperglycaemia (Boscaro et al. 2009, Colao et al. 2012, Henry et al. 2013, Silverstein 2016, Petersenn et al. 2017, Albani et al. 2018a , Samson et al. 2021). Therefore, finding ways to predict whether a patient would benefit from pasireotide treatment could greatly facilitate patient management. To this end, lower baseline UFC was linked to higher rates of UFC normalization under pasireotide treatment (Colao et al. 2012, Witek et al. 2018). Recently, a consensus statement has highlighted the potential of USP8 mutational status as a molecular marker of pasireotide response (Fleseriu et al. 2021).
Our present in vitro study reports a significantly better response to pasireotide treatment in terms of suppressed ACTH secretion in human corticotroph tumours carrying USP8 mutations. We have previously shown that pasireotide suppresses POMC promoter activity in a murine corticotroph tumour cell model (Castillo et al. 2011), and herein, we show that introducing human USP8 mutants improved this suppressive action.
These observations postulate a regulatory role of USP8 mutants specifically on SSTR5. We have previously reported that USP8 mutants rescue cell surface receptors like EGFR by deubiquitinating them (Reincke et al. 2015); therefore, we hypothesized that a similar mechanism may be responsible for the higher SSTR5 immunoreactivity observed in USP8-mutant corticotroph tumours. Receptor ubiquitination after somatostatin binding was previously observed for SSTR3, where it is involved in agonist-induced receptor internalization and endosomal sorting (Tulipano et al. 2004). However, we did not observe human SSTR5 ubiquitination after treatment with an SSTR5-specific ligand or any physical association between USP8 and this receptor. Accordingly, our results stipulate that SSTR5 is unlikely to be ubiquitinated and a direct client of the USP8 deubiquitinase.
Most studies, including ours, assess SSTR5 by immunohistochemistry, but transcriptomic and other analyses showed increased transcript levels in USP8 mutant tumours (Hayashi et al. 2016, Neou et al. 2020), indicating a transcriptional primary mode of action. Indeed, our study reveals an increase in endogenous Sstr5 transcript levels in murine corticotroph tumour cells overexpressing USP8 mutants. This is in accordance with the higher SSTR5 expression and immunoreactivity score observed in human USP8-mutant corticotroph tumours (Hayashi et al. 2016, Castellnou et al. 2020, Neou et al. 2020). In contrast, we did not observe any effect on Sstr2 transcription, reflecting the lack of significant differences in SSTR2 transcript levels between USP8 WT and mutant human corticotroph tumours (Hayashi et al. 2016).
To understand how USP8 mutants may affect endogenous Sstr5 transcription in AtT-20 cells, we reviewed the mouse Sstr5 promoter and observed that it contains AP-1-binding site (Gordon et al. 1999). We have previously shown that USP8 mutants potentiate EGFR-induced AP-1 transcriptional activity (Reincke et al. 2015), and herein, we observed that their stimulatory action also remains in the absence of EGFR overexpression. AP-1 is a collective term that refers to dimers of transcription factors belonging to the JUN, FOS, ATF and MAF families of basic region and leucine zipper (bZIP) domain proteins (Eferl & Wagner 2003). There is evidence that Fos (also known as c-Fos) is regulated by ubiquitination that causes its degradation (Stancovski et al. 1995). A potential direct impact on c-Fos ubiquitination was out of the scope of the present study, nevertheless, the lack of stimulatory action on Sstr5 transcription in Fos knockdown cells led us to conclude that USP8 mutants exert their effect via c-Fos.
A limitation in our study is the lack of clinical data on pasireotide response in patients with Cushing’s disease. In our cohort, five patients were treated with pasireotide: four had USP8 WT corticotroph tumours and did not show improvement after pasireotide treatment. One patient had a USP8 mutant corticotroph tumour (p.718S>P) but showed no improvement after 3 months of treatment (s.c., up to 1200 µg bid), which had to be terminated because of severe hyperglycaemia and gastrointestinal complaints.
In conclusion, our in vitro results on human corticotroph tumours highlight for the first time the role of USP8 mutational status on the antisecretory response to pasireotide and reveal a potential mechanism through which mutant USP8 forms may upregulate SSTR5 transcription. Extensive clinical studies are needed to consolidate the use of a positive USP8 mutational status as a predictor of successful pasireotide treatment response in Cushing’s disease patients for whom surgery is contraindicated, not feasible or not successful.
Supplementary materials
This is linked to the online version of the paper at https://doi.org/10.1530/ERC-22-0088.
Declaration of interest
G K S received consultancy and speaker fees from Recordati. J Sc received grants and fees from Novartis, Ipsen, Pfizer and Recordati. The other authors have nothing to disclose.
Funding
This study was supported by the Deutsche Forschungsgemeinschaft (DFG) (project number: 314061271-TRR 205) to M R and M T. A A was supported by the Munich Excellence Training Initiative for Physician Scientists (Metiphys). M R was supported by the Else Kröner-Fresenius Stiftung.
Acknowledgement
The authors thank J Stalla and J L Monteserin-Garcia for their excellent technical assistance.
References
Albani A, Ferraù F, Ciresi A, Pivonello R, Scaroni C, Iacuaniello D, Zilio M, Guarnotta V, Alibrandi A, Messina E, et al.2018a Pasireotide treatment reduces cardiometabolic risk in Cushing’s disease patients: an Italian, multicenter study. Endocrine 61 118–124. (https://doi.org/10.1007/s12020-018-1524-5)
Albani A, Pérez-Rivas LG, Dimopoulou C, Zopp S, Colón-Bolea P, Roeber S, Honegger J, Flitsch J, Rachinger W, Buchfelder M, et al.2018b The USP8 mutational status may predict long-term remission in patients with Cushing’s disease. Clinical Endocrinology 89 454–458. (https://doi.org/10.1111/cen.13802)
Batista DL, Zhang X, Gejman R, Ansell PJ, Zhou Y, Johnson SA, Swearingen B, Hedley-Whyte ET, Stratakis CA & Klibanski A 2006 The effects of SOM230 on cell proliferation and adrenocorticotropin secretion in human corticotroph pituitary adenomas. Journal of Clinical Endocrinology and Metabolism 91 4482–4488. (https://doi.org/10.1210/jc.2006-1245)
Boscaro M, Ludlam WH, Atkinson B, Glusman JE, Petersenn S, Reincke M, Snyder P, Tabarin A, Biller BM, Findling J, et al.2009 Treatment of pituitary-dependent Cushing’s disease with the multireceptor ligand somatostatin analog pasireotide (SOM230): a multicenter, phase II trial. Journal of Clinical Endocrinology and Metabolism 94 115–122. (https://doi.org/10.1210/jc.2008-1008)
Boscaro M, Bertherat J, Findling J, Fleseriu M, Atkinson AB, Petersenn S, Schopohl J, Snyder P, Hughes G, Trovato A, et al.2014 Extended treatment of Cushing’s disease with pasireotide: results from a 2-year, phase II study. Pituitary 17 320–326. (https://doi.org/10.1007/s11102-013-0503-3)
Broersen LHA, Jha M, Biermasz NR, Pereira AM & Dekkers OM 2018 Effectiveness of medical treatment for Cushing’s syndrome: a systematic review and meta-analysis. Pituitary 21 631–641. (https://doi.org/10.1007/s11102-018-0897-z)
Castellnou S, Vasiljevic A, Lapras V, Raverot V, Alix E, Borson-Chazot F, Jouanneau E, Raverot G & Lasolle H 2020 SST5 expression and USP8 mutation in functioning and silent corticotroph pituitary tumors. Endocrine Connections 9 243–253. (https://doi.org/10.1530/EC-20-0035)
Castillo V, Theodoropoulou M, Stalla J, Gallelli MF, Cabrera-Blatter MF, Haedo MR, Labeur M, Schmid HA, Stalla GK & Arzt E 2011 Effect of SOM230 (pasireotide) on corticotropic cells: action in dogs with Cushing’s disease. Neuroendocrinology 94 124–136. (https://doi.org/10.1159/000327429)
Clayton RN, Jones PW, Reulen RC, Stewart PM, Hassan-Smith ZK, Ntali G, Karavitaki N, Dekkers OM, Pereira AM, Bolland M, et al.2016 Mortality in patients with Cushing’s disease more than 10 years after remission: a multicentre, multinational, retrospective cohort study. Lancet: Diabetes and Endocrinology 4 569–576. (https://doi.org/10.1016/S2213-8587(1630005-5)
Colao A, Petersenn S, Newell-Price J, Findling JW, Gu F, Maldonado M, Schoenherr U, Mills D, Salgado LR, Biller BM, et al.2012 A 12-month phase 3 study of pasireotide in Cushing’s disease. New England Journal of Medicine 366 914–924. (https://doi.org/10.1056/NEJMoa1105743)
Dekkers OM, Horváth-Puhó E, Jørgensen JO, Cannegieter SC, Ehrenstein V, Vandenbroucke JP, Pereira AM & Sørensen HT 2013 Multisystem morbidity and mortality in Cushing’s syndrome: a cohort study. Journal of Clinical Endocrinology and Metabolism 98 2277–2284. (https://doi.org/10.1210/jc.2012-3582)
Detre S, Saclani Jotti G & Dowsett M 1995 A ‘quickscore’ method for immunohistochemical semiquantitation: validation for oestrogen receptor in breast carcinomas. Journal of Clinical Pathology 48 876–878. (https://doi.org/10.1136/jcp.48.9.876)
Dimopoulou C, Schopohl J, Rachinger W, Buchfelder M, Honegger J, Reincke M & Stalla GK 2014 Long-term remission and recurrence rates after first and second transsphenoidal surgery for Cushing’s disease: care reality in the Munich Metropolitan Region. European Journal of Endocrinology 170 283–292. (https://doi.org/10.1530/EJE-13-0634)
Eferl R & Wagner EF 2003 AP-1: a double-edged sword in tumorigenesis. Nature Reviews: Cancer 3 859–868. (https://doi.org/10.1038/nrc1209)
Faucz FR, Tirosh A, Tatsi C, Berthon A, Hernández-Ramírez LC, Settas N, Angelousi A, Correa R, Papadakis GZ, Chittiboina P, et al.2017 Somatic USP8 gene mutations are a common cause of pediatric Cushing disease. Journal of Clinical Endocrinology and Metabolism 102 2836–2843. (https://doi.org/10.1210/jc.2017-00161)
Feelders RA, Newell-Price J, Pivonello R, Nieman LK, Hofland LJ & Lacroix A 2019 Advances in the medical treatment of Cushing’s syndrome. Lancet: Diabetes and Endocrinology 7 300–312. (https://doi.org/10.1016/S2213-8587(1830155-4)
Fleseriu M, Auchus R, Bancos I, Ben-Shlomo A, Bertherat J, Biermasz NR, Boguszewski CL, Bronstein MD, Buchfelder M, Carmichael JD, et al.2021 Consensus on diagnosis and management of Cushing’s disease: a guideline update. Lancet: Diabetes and Endocrinology 9 847–875. (https://doi.org/10.1016/S2213-8587(2100235-7)
Gordon DF, Woodmansee WW, Lewis SR, James RA, Wood WM & Ridgway EC 1999 Cloning of the mouse somatostatin receptor subtype 5 gene: promoter structure and function. Endocrinology 140 5598–5608. (https://doi.org/10.1210/endo.140.12.7165)
Hayashi K, Inoshita N, Kawaguchi K, Ibrahim Ardisasmita A, Suzuki H, Fukuhara N, Okada M, Nishioka H, Takeuchi Y, Komada M, et al.2016 The USP8 mutational status may predict drug susceptibility in corticotroph adenomas of Cushing’s disease. European Journal of Endocrinology 174 213–226. (https://doi.org/10.1530/EJE-15-0689)
Henry RR, Ciaraldi TP, Armstrong D, Burke P, Ligueros-Saylan M & Mudaliar S 2013 Hyperglycemia associated with pasireotide: results from a mechanistic study in healthy volunteers. Journal of Clinical Endocrinology and Metabolism 98 3446–3453. (https://doi.org/10.1210/jc.2013-1771)
Hofland LJ, van der Hoek J, Feelders R, Van Aken MO, Van Koetsveld PM, Waaijers M, Sprij-Mooij D, Bruns C, Weckbecker G, De Herder WW, et al.2005 The multi-ligand somatostatin analogue SOM230 inhibits ACTH secretion by cultured human corticotroph adenomas via somatostatin receptor type 5. European Journal of Endocrinology 152 645–654. (https://doi.org/10.1530/eje.1.01876)
Lacroix A, Feelders RA, Stratakis CA & Nieman LK 2015 Cushing’s syndrome. Lancet 386 913–927. (https://doi.org/10.1016/S0140-6736(1461375-1)
Lacroix A, Gu F, Gallardo W, Pivonello R, Yu Y, Witek P, Boscaro M, Salvatori R, Yamada M, Tauchmanova L, et al.2018 Efficacy and safety of once-monthly pasireotide in Cushing’s disease: a 12 month clinical trial. Lancet: Diabetes and Endocrinology 6 17–26. (https://doi.org/10.1016/S2213-8587(1730326-1)
Ma ZY, Song ZJ, Chen JH, Wang YF, Li SQ, Zhou LF, Mao Y, Li YM, Hu RG, Zhang ZY, et al.2015 Recurrent gain-of-function USP8 mutations in Cushing’s disease. Cell Research 25 306–317. (https://doi.org/10.1038/cr.2015.20)
Mizuno E, Iura T, Mukai A, Yoshimori T, Kitamura N & Komada M 2005 Regulation of epidermal growth factor receptor down-regulation by UBPY-mediated deubiquitination at endosomes. Molecular Biology of the Cell 16 5163–5174. (https://doi.org/10.1091/mbc.e05-06-0560)
Neou M, Villa C, Armignacco R, Jouinot A, Raffin-Sanson ML, Septier A, Letourneur F, Diry S, Diedisheim M, Izac B, et al.2020 Pangenomic classification of pituitary neuroendocrine tumors. Cancer Cell 37 123 .e5–134.e5. (https://doi.org/10.1016/j.ccell.2019.11.002)
Newell-Price J, Bertagna X, Grossman AB & Nieman LK 2006 Cushing’s syndrome. Lancet 367 1605–1617. (https://doi.org/10.1016/S0140-6736(0668699-6)
Perez-Rivas LG, Theodoropoulou M, Ferraù F, Nusser C, Kawaguchi K, Stratakis CA, Faucz FR, Wildemberg LE, Assié G, Beschorner R, et al.2015 The gene of the ubiquitin-specific protease 8 is frequently mutated in adenomas causing Cushing’s disease. Journal of Clinical Endocrinology and Metabolism 100 E997–E1 004. (https://doi.org/10.1210/jc.2015-1453)
Perez-Rivas LG, Oßwald A, Knösel T, Lucia K, Schaaf C, Hristov M, Fazel J, Kirchner T, Beuschlein F, Reincke M, et al.2017 Expression and mutational status of USP8 in tumors causing ectopic ACTH secretion syndrome. Endocrine-Related Cancer 24 L73–L77. (https://doi.org/10.1530/ERC-17-0054)
Petersenn S, Beckers A, Ferone D, van der Lely A, Bollerslev J, Boscaro M, Brue T, Bruzzi P, Casanueva FF, Chanson P, et al.2015 Therapy of endocrine disease: outcomes in patients with Cushing’s disease undergoing transsphenoidal surgery: systematic review assessing criteria used to define remission and recurrence. European Journal of Endocrinology 172 R227–R239. (https://doi.org/10.1530/EJE-14-0883)
Petersenn S, Salgado LR, Schopohl J, Portocarrero-Ortiz L, Arnaldi G, Lacroix A, Scaroni C, Ravichandran S, Kandra A & Biller BMK 2017 Long-term treatment of Cushing’s disease with pasireotide: 5-year results from an open-label extension study of a phase III trial. Endocrine 57 156–165. (https://doi.org/10.1007/s12020-017-1316-3)
Pivonello R, De Leo M, Cozzolino A & Colao A 2015 The treatment of Cushing’s disease. Endocrine Reviews 36 385–486. (https://doi.org/10.1210/er.2013-1048)
Pivonello R, Arnaldi G, Scaroni C, Giordano C, Cannavò S, Iacuaniello D, Trementino L, Zilio M, Guarnotta V, Albani A, et al.2019 The medical treatment with pasireotide in Cushing’s disease: an Italian multicentre experience based on ‘real-world evidence’. Endocrine 64 657–672. (https://doi.org/10.1007/s12020-018-1818-7)
Reincke M, Sbiera S, Hayakawa A, Theodoropoulou M, Osswald A, Beuschlein F, Meitinger T, Mizuno-Yamasaki E, Kawaguchi K, Saeki Y, et al.2015 Mutations in the deubiquitinase gene USP8 cause Cushing’s disease. Nature Genetics 47 31–38. (https://doi.org/10.1038/ng.3166)
Ritzel K, Beuschlein F, Mickisch A, Osswald A, Schneider HJ, Schopohl J & Reincke M 2013 Clinical review: Outcome of bilateral adrenalectomy in Cushing’s syndrome: a systematic review. Journal of Clinical Endocrinology and Metabolism 98 3939–3948. (https://doi.org/10.1210/jc.2013-1470)
Samson SL, Gu F, Feldt-Rasmussen U, Zhang S, Yu Y, Witek P, Kalra P, Pedroncelli AM, Pultar P, Jabbour N, et al.2021 Managing pasireotide-associated hyperglycemia: a randomized, open-label, phase IV study. Pituitary 24 887–903. (https://doi.org/10.1007/s11102-021-01161-4)
Sbiera S, Kunz M, Weigand I, Deutschbein T, Dandekar T & Fassnacht M 2019 The new genetic landscape of Cushing’s disease: deubiquitinases in the spotlight. Cancers 11 1761. (https://doi.org/10.3390/cancers11111761)
Schopohl J, Gu F, Rubens R, Van Gaal L, Bertherat J, Ligueros-Saylan M, Trovato A, Hughes G, Salgado LR, Boscaro M, et al.2015 Pasireotide can induce sustained decreases in urinary cortisol and provide clinical benefit in patients with Cushing’s disease: results from an open-ended, open-label extension trial. Pituitary 18 604–612. (https://doi.org/10.1007/s11102-014-0618-1)
Silverstein JM 2016 Hyperglycemia induced by pasireotide in patients with Cushing’s disease or acromegaly. Pituitary 19 536–543. (https://doi.org/10.1007/s11102-016-0734-1)
Stalla GK, Stalla J, Huber M, Loeffler JP, Höllt V, Von Werder K & Müller OA 1988 Ketoconazole inhibits corticotropic cell function in vitro. Endocrinology 122 618–623. (https://doi.org/10.1210/endo-122-2-618)
Stancovski I, Gonen H, Orian A, Schwartz AL & Ciechanover A 1995 Degradation of the proto-oncogene product c-Fos by the ubiquitin proteolytic system in vivo and in vitro: identification and characterization of the conjugating enzymes. Molecular and Cellular Biology 15 7106–7116. (https://doi.org/10.1128/MCB.15.12.7106)
Tritos NA & Biller BMK 2018 Medical therapy for Cushing’s syndrome in the twenty-first century. Endocrinology and Metabolism Clinics of North America 47 427–440. (https://doi.org/10.1016/j.ecl.2018.01.006)
Tulipano G, Stumm R, Pfeiffer M, Kreienkamp HJ, Höllt V & Schulz S 2004 Differential beta-arrestin trafficking and endosomal sorting of somatostatin receptor subtypes. Journal of Biological Chemistry 279 21374–21382. (https://doi.org/10.1074/jbc.M313522200)
Valassi E, Tabarin A, Brue T, Feelders RA, Reincke M, Netea-Maier R, Tóth M, Zacharieva S, Webb SM, Tsagarakis S, et al.2019 High mortality within 90 days of diagnosis in patients with Cushing’s syndrome: results from the ERCUSYN registry. European Journal of Endocrinology 181 461–472. (https://doi.org/10.1530/EJE-19-0464)
van der Hoek J, Waaijers M, Van Koetsveld PM, Sprij-Mooij D, Feelders RA, Schmid HA, Schoeffter P, Hoyer D, Cervia D, Taylor JE, et al.2005 Distinct functional properties of native somatostatin receptor subtype 5 compared with subtype 2 in the regulation of ACTH release by corticotroph tumor cells. American Journal of Physiology: Endocrinology and Metabolism 289 E278–E287. (https://doi.org/10.1152/ajpendo.00004.2005)
Witek P, Biller BMK, Lacroix A, Feelders R, Li Y, Geer EB, Brue T, Ravichandran S, Tauchmanova L, Roughton M, et al.2018 Predictors of response to long-acting pasireotide in patients with Cushing’s disease during a Phase III study. Endocrine Abstracts 56 GP205. (https://doi.org/10.1530/endoabs.56.GP205)
Young JA, Sermwittayawong D, Kim HJ, Nandu S, An N, Erdjument-Bromage H, Tempst P, Coscoy L & Winoto A 2011 Fas-associated death domain (FADD) and the E3 ubiquitin-protein ligase TRIM21 interact to negatively regulate virus-induced interferon production. Journal of Biological Chemistry 286 6521–653 1. (https://doi.org/10.1074/jbc.M110.172288)
