Abstract
Cushing’s disease (CD) is a rare endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor. Pasireotide is the only pituitary-targeted drug approved for adult patients. Nevertheless, many side effects are encountered and curative therapy is still challenging. Ubiquitin-specific peptidase 8 (USP8) plays a crucial role in the modulation of corticotroph cells growth and ACTH secretion. Here, we explored the anticancer potential of the USP8 inhibitor RA-9 in USP8-WT human tumor corticotroph cells and murine AtT-20 cells. Our results showed that RA-9 causes cell proliferation decrease (−24.3 ± 5.2%, P < 0.01) and cell apoptosis increase (207.4 ± 75.3%, P < 0.05) in AtT-20 cells, as observed with pasireotide. Moreover, RA-9 reduced ACTH secretion in AtT-20 cells (−34.1 ± 19.5%, P < 0.01), as well as in AtT-20 cells transfected with USP8 mutants, and in one out of two primary cultures in vitro responsive to pasireotide (−40.3 ± 6%). An RA-9 mediated decrease of pERK1/2 levels was observed in AtT-20 cells (−52.3 ± 13.4%, P < 0.001), comparable to pasireotide, and in primary cultures, regardless of their in vitro responsiveness to pasireotide. Upregulation of p27 was detected upon RA-9 treatment only, both in AtT-20 cells (167.1 ± 36.7%, P < 0.05) and in one primary culture tested (168.4%), whilst pCREB level was similarly halved in AtT-20 cells by both RA-9 and pasireotide. Altogether, our data demonstrate that RA-9 is efficient in exerting cytotoxic effects and inhibitory actions on cell proliferation and hormone secretion by modulating the expression of pERK1/2, pCREB and p27. Inhibition of USP8 might represent a novel strategy to target both USP8-WT and USP8-mutated tumors in CD patients.
Introduction
Cushing’s disease (CD) is a severe disorder characterized by hypercortisolism in most cases due to adrenocorticotropic hormone (ACTH) oversecretion from a pituitary corticotroph tumor (Newell-Price et al. 2006). Chronic exposure to glucocorticoid excess is responsible for multisystem complications such as hypertension, diabetes mellitus, dyslipidemia, osteoporosis, infections, cardiovascular disease, and mental disorders (Pivonello et al. 2008), contributing to increased mortality and impaired quality of life (Clayton et al. 2011, van Haalen et al. 2015). Surgical excision of the pituitary tumor represents the first-line option treatment for patients with CD, although recurrence occurs in 15 to 66% of patients (Nieman et al. 2015). In relapsed cases or when the surgery fails, the second generation somatostatin analog (SSA) pasireotide is used to pharmacologically target the tumor and lower ACTH and cortisol levels (Bruns et al. 2002, Lewis et al. 2014). Thanks to its high affinity to the most expressed somatostatin receptor on corticotropinomas – somatostatin receptor type 5 (SST5) – (Hofland et al. 2005, Batista et al. 2006, Hofland 2008, Tateno et al. 2009) pasireotide mediates mitogen-activated protein kinase (MAPK) activity inhibition, exerting both antiproliferative and antisecretory actions in vitro (Batista et al. 2006, Treppiedi et al. 2019) and in vivo (Colao et al. 2012, Lu et al. 2013, Simeoli et al. 2016). Moreover, pasireotide showed pro-apoptotic effects in vitro (Treppiedi et al. 2019). However, in clinical practice, many side effects are currently observed during pasireotide administration, such as gall bladder disease, gastrointestinal symptoms and hyperglycemia (Simeoli et al. 2020). Thus, curative therapy for CD is still challenging and novel pharmacological strategies of treatment directly targeting the tumor are needed (Fukuoka et al. 2011, Bertagna & Guignat 2013, Asari et al. 2019).
Ubiquitin-specific proteases (USPs) are a subclass of deubiquitinating enzymes (DUBs) that play key regulatory roles in a multitude of therapeutically relevant processes including cancer (Reyes-Turcu et al. 2009, Farshi et al. 2015) and are rapidly emerging as promising targets for drug design (Coughlin et al. 2014, Mirzapoiazova et al. 2020). The role of USPs in pituitary tumorigenesis has been highlighted by Reincke and colleagues in 2015 with the identification of gain-of-function somatic mutations in the USP8 gene showing a peculiar specificity for corticotropinomas (Reincke et al. 2015). In this context, USP8 mutations lead to enhanced deubiquitination of EGF receptor (EGFR) and consequent unbalanced EGFR signaling, with extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) overactivation, proopiomelanocortin (POMC) transcription, ACTH production and cell growth. Deregulation of the oncosuppressor p27kip1 and cAMP-response element-binding protein (CREB) have been encountered in USP8-mutated corticotropinomas as well (Weigand et al. 2019). Interestingly, Sesta and colleagues have recently found that POMC is subject to ubiquitination, regardless of USP8 sequence status (Sesta et al. 2020). These pieces of evidence have raised the hypothesis of the ubiquitin-proteasome system as a target for CD treatment, independently of USP8 mutations. Indeed, a USP8 inhibitor (9-oxo-9H-indeno[1,2-b]pyrazine-2,3-dicarbonitrile) has been already successfully tested for its effectiveness in reducing EGFR expression, POMC transcription, ACTH secretion and cell proliferation in murine AtT-20 corticotroph tumor cells (Kageyama et al. 2020), however, no data concerning the use of USP8 inhibitors in human ACTH-secreting pituitary tumor cells have been reported so far.
In the present study, we used RA-9 as a cell-permeable and potent inhibitor of USP8 (Anchoori et al. 2011) in both AtT-20 cells and human primary cultures from ACTH-secreting pituitary tumors. Anticancer properties of RA-9 have been previously documented in multiple cancer cell lines in vitro and in in vivo experiments (Issaenko & Amerik 2012, Coughlin et al. 2014, Mirzapoiazova et al. 2020). Here, we compared RA-9-mediated downstream effects on hormone secretion, cell proliferation and apoptosis with those elicited by pasireotide in tumor corticotrophs.
Materials and methods
Chemicals
USP8 inhibitor RA-9 was purchased from Merck KGaA (Darmstadt, DE). Pasireotide was from Novartis Pharma AG (Basel, CH). In this study, 5 µM was chosen as optimal concentration for experiments based on the preliminary dose-response curve.
ACTH-secreting pituitary cell culture
The study was approved by the Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Ethics Committee, and all patients with ACTH-secreting pituitary tumors who underwent trans-sphenoidal surgery gave their informed consent to the use of their sample. None of the patients was pharmacologically pre-treated with SSAs. Seven different ACTH-secreting pituitary tumor specimens were partially stored at −80°C for subsequent genetic analysis and partially dissociated to obtain primary cell cultures, according to previously published protocols (Peverelli et al. 2014). Briefly, tumors were subjected to enzymatic dissociation in Dulbecco’s modified Eagle’s medium (DMEM) containing 2 mg/mL collagenase (Merck KGaA, Darmstadt, DE) at 37°C for 2 h. The digested tissue was passed on a 100 μm filter (nylon cell strainer, BD Transduction Laboratories, Lexington, UK) to remove undigested material. Dispersed cells were cultured in DMEM (Merck KGaA, Darmstadt, DE) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine and antibiotics (Gibco, Invitrogen, Life Technologies Inc.). In order to exclude fibroblast contamination, ACTH-secreting primary cultures were daily checked by visual inspection with an optical microscope. Murine pituitary tumor corticotroph AtT-20 cells (ATCC CRL-1795™) were cultured in DMEM (Life Technologies) supplemented with 10% FBS and antibiotics. Pituitary cells were kept at 37°C in a humidified atmosphere with 5% CO2.
DNA extraction and Sanger sequencing
Genomic DNA was extracted from frozen ACTH-secreting pituitary tumors and AtT-20 cells with the Puregene Core Kit A according to the manufacturer’s instructions (Qiagen). USP8 exon 14 was PCR amplified with GoTaq G2 DNA Polymerase (Promega) using a C1000 Touch Thermal Cycler (Bio-Rad Laboratories); PCR primers sequences are available on request. Direct sequencing was performed using the BigDye™ Terminator v3.1 Cycle Sequencing Kit and the 3130xl Genetic Analyzer (Applied Biosystems).
Plasmids transfection
pME-FLAG expression vector coding for WT USP8 was kindly provided by Prof Marily Theodoropoulou. S718del, S718P and P720R USP8 mutants were generated from USP8 WT by QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) as described previously (Mangili et al. 2020). AtT-20 cells were transiently transfected with USP8 S718del, USP8 S718P, USP8 P720R expression vectors for 96 h using Lipofectamine 2000 (Invitrogen) as transfection reagent and following the instruction of the manufacturer. Transfection efficiency was monitored by Western blot analysis using an anti-USP8 antibody (Santa Cruz Biotechnology). Empty vector was used in each experiment as negative control (mock).
ACTH levels determination
Human primary cultured cells from three ACTH-secreting pituitary tumors and AtT-20 cells were seeded in 24-well plate at a density of 1.25 × 105/well. The day after (or 96 h after USP8 mutants transfection), the culture medium was replaced with 300 µL of fresh medium containing RA-9 5 µM or pasireotide 10 nM for 4 h. In the case of RA-9 pre-treatment, cells were pre-incubated with RA-9 5 µM for 30 min, and subsequently medium was replaced with fresh medium containing pasireotide for 4 h. For each experiment, a parallel well with cells kept in a fresh medium without stimuli for 4 h was considered as a basal condition. After treatment, culture media were collected to detect ACTH. Human ACTH was measured by specific chemiluminescent immunometric assay (Immulite 2000, Siemens Medical Solutions Diagnostics) with an inter-assay coefficient of variation ranging from 6.1 to 10.0%, an intra-assay coefficient of variation ranging from 6.7 to 9.5% and sensitivity of 5 pg/mL. Murine ACTH levels were determined using a specific Elisa immunoassay kit (Fine Test, Wuhan Fine Biotech Co., Ltd, Wuhan, CN), according to the manufacturer’s instructions and previously published protocol (Mangili et al. 2020). Absorbance was read at 450 nm in a Victor2 multilabel plate reader (Perkin Elmer). Hormone levels were normalized on the protein content measured by the BCA assay. Data were plotted and analyzed with the Curve Expert 1.4 program. Hormone detection was done in triplicate and experiments were replicated three times.
Cell proliferation assay
Cell proliferation was determined in AtT-20 cells by colorimetric measurement of 5-bromo-20-deoxyuridine (BrdU) incorporation during DNA synthesis in proliferating cells. Cells were seeded at a cell density of 5 × 105 cells/well in a 96-well plate in a starved medium at 37°C. The day after, cells were incubated with a complete medium containing RA-9 5 µM or pasireotide 10 nM for 48 h at 37°C, based on preliminary time-course experiments. In the case of RA-9 and pasireotide co-treatment, RA-9 was added 30 min before and during pasireotide stimulation. A parallel well with a fresh medium was considered as a basal condition. Cells were subsequently exposed to BrdU for 2 h according to the instruction of the manufacturer (GE Healthcare Cell Proliferation Kit). Each determination was done in quintuple, and experiments were repeated three times.
Cell apoptosis assay
AtT-20 cells were seeded at a cell density of 2.5 × 104 cells/well in a 96-well plate. The following day cells were incubated with a complete medium containing RA-9 5 µM or pasireotide 10 nM for 48 h at 37°C, based on preliminary time-course experiments. In the case of RA-9 and pasireotide co-treatment, RA-9 was added 30 min before and during pasireotide stimulation. A Parallel well with a fresh medium was considered as a control basal condition. Cell apoptosis was measured using Apo-ONE homogenous caspase-3/7 assay (Promega). The liberation of Rhodamine 110 substrate was detected by an absorbance plate reader. Each determination was done in quintuple, and experiments were repeated three times.
Western blot analysis
Phospho-ERK1/2 levels were evaluated in AtT-20 cells and in primary cultures from five different ACTH-secreting pituitary tumors. Phospho-CREB levels were evaluated in AtT20 cells only. Cells were seeded in a 6-well plate at a cell density of 3 × 105 cells/well (AtT-20 cells) or in a 24-well plate at a cell density of 1.25 × 105 cells/well (primary cultures) overnight at 37°C in serum-free medium (for phospho-ERK1/2) or complete medium (for phospho-CREB). The day after, the medium was replaced with a complete fresh medium containing or not containing RA-9 5 µM for 30 min. After that, the medium was removed, and cells were incubated with a fresh complete medium containing or not containing pasireotide 10 nM for 10 min. p27 expression levels were measured in AtT-20 cells and in one primary culture from an ACTH-secreting pituitary tumor. The day after seeding, the medium was replaced with fresh medium containing RA-9 5 µM or pasireotide 10 nM for 48 h, based on preliminary time-course experiments. For RA-9 and pasireotide co-treatment, RA-9 was added 30 min before and during pasireotide stimulation. Parallel well with a fresh medium was considered as a basal condition. Total proteins were extracted with lysis buffer (Cell Signaling) and 30 μg of proteins were separated by SDS-polyacrylamide gradient (4–12%) gels and transferred to a nitrocellulose filter. Specific primary antibodies against phosphor-ERK1/2 and phosphor-CREB were from Cell Signaling and diluted 1:1000. Membranes were stripped and reprobed with antibodies against total ERK1/2 or total CREB (Cell Signaling), diluted 1:1000. The primary antibody anti-p27 was from Santa Cruz Biotechnology and diluted 1:200. Secondary anti-rabbit or anti-mouse horseradish peroxidase-linked antibodies were used (1:2000 dilution). For analysis of USP8 transfection efficacy, an antibody against USP8 was used (Santa Cruz Biotechnology, 1:200). GAPDH (Life Technologies) was considered as housekeeping and diluted 1:4000. Chemiluminescence was detected using the ChemiDoc-IT imaging system (UVP) and densitometrical analysis of the resulted bands was performed with NIH ImageJ software.
Statistical analysis
The results are expressed as the mean ± s.d. A paired two-tailed Student’s t-test was used to assess the significance between two series of data. Statistical analysis was performed by GraphPad Prism 7.0 software and P < 0.05 was accepted as statistically significant.
Results
RA-9 exerts antiproliferative and pro-apototic effects in AtT-20 cells
First, our subclone of AtT-20 cells and all human samples used for in vitro experiments were screened for USP8 mutations resulting USP8 WT. Then, we tested whether targeting USP8 with RA-9 might exhibit anticancer effects in corticotroph tumor cells. The same cells were treated with pasireotide as a comparison. As expected, 48 h of cells being exposed to pasireotide slightly but significantly reduced cell proliferation (−13.8 ± 4.6% reduction, P < 0.01 vs basal). A statistically relevant decrease of cell growth was also seen after RA-9 incubation (−24.3 ± 5.2% reduction, P < 0.01 vs basal). Similar anti-proliferative effects were exerted by RA-9 and pasireotide co-incubation (−27.9 ± 17.1% reduction, P < 0.05 vs basal) (Fig. 1A). At the same time, cell apoptosis was induced by pasireotide (+38.4 ± 35.5% increase, P < 0.05 vs basal), RA-9 (+107.4 ± 75.3% increase, P < 0.05 vs basal) and the combination of the two compounds (+109.9 ± 106.0% increase, P < 0.05 vs basal) with no additive or synergistic effects (Fig. 1B).
RA-9 effects on cell proliferation and cell apoptosis. (A) Results of BrdU assay to determine cell proliferation in AtT-20. Cells were cultured in the absence or presence of 5 µM RA-9 and/or 10 nM pasireotide for 48 h. Experiment were repeated three times, and each determination was done in quintuple. Cell proliferation is expressed as percent respect to control untreated cells (basal). Values represent mean ± s.d. **P < 0.01, ***P < 0.001 vs control. (B) Results of cell apoptosis in AtT-20 cells. Cells were incubated with or without RA-9 and/or pasireotide for 48 h. Caspase-3/7 activity was measured in the culture medium. Cell apoptosis is expressed as percent respect to control untreated cells (basal). Data are plotted as mean ± s.d. from three independent experiments. Each determination was done in quintuplicate. *P < 0.05 vs control.
Citation: Endocrine-Related Cancer 28, 8; 10.1530/ERC-21-0093
RA-9 effects on cell proliferation and cell apoptosis. (A) Results of BrdU assay to determine cell proliferation in AtT-20. Cells were cultured in the absence or presence of 5 µM RA-9 and/or 10 nM pasireotide for 48 h. Experiment were repeated three times, and each determination was done in quintuple. Cell proliferation is expressed as percent respect to control untreated cells (basal). Values represent mean ± s.d. **P < 0.01, ***P < 0.001 vs control. (B) Results of cell apoptosis in AtT-20 cells. Cells were incubated with or without RA-9 and/or pasireotide for 48 h. Caspase-3/7 activity was measured in the culture medium. Cell apoptosis is expressed as percent respect to control untreated cells (basal). Data are plotted as mean ± s.d. from three independent experiments. Each determination was done in quintuplicate. *P < 0.05 vs control.
Citation: Endocrine-Related Cancer 28, 8; 10.1530/ERC-21-0093
RA-9 effects on cell proliferation and cell apoptosis. (A) Results of BrdU assay to determine cell proliferation in AtT-20. Cells were cultured in the absence or presence of 5 µM RA-9 and/or 10 nM pasireotide for 48 h. Experiment were repeated three times, and each determination was done in quintuple. Cell proliferation is expressed as percent respect to control untreated cells (basal). Values represent mean ± s.d. **P < 0.01, ***P < 0.001 vs control. (B) Results of cell apoptosis in AtT-20 cells. Cells were incubated with or without RA-9 and/or pasireotide for 48 h. Caspase-3/7 activity was measured in the culture medium. Cell apoptosis is expressed as percent respect to control untreated cells (basal). Data are plotted as mean ± s.d. from three independent experiments. Each determination was done in quintuplicate. *P < 0.05 vs control.
Citation: Endocrine-Related Cancer 28, 8; 10.1530/ERC-21-0093
RA-9 is able to mediate an anti-secretory action in both AtT-20 cells and primary cultures from ACTH-secreting pituitary tumors
Next, we explored a possible RA-9-mediated anti-secretory effect both in AtT-20 cells and human primary cultured cells from corticotropinomas and investigated whether a combined strategy comprising RA-9 and pasireotide might be more effective in lowering ACTH release. Similar to pasireotide, RA-9 significantly decreased ACTH secretion in AtT-20 cells (−42.9 ± 15.2% reduction at pasireotide 10 nM, P < 0.001 vs basal; −34.1 ± 19.5% reduction at RA-9 5 µM, P < 0.01 vs basal). A reduction of ACTH release was achieved in AtT-20 cells pre-treated with RA-9 and then incubated with pasireotide (−37.7 ± 35.4% reduction, P < 0.05 vs basal), although no synergic effect was detected (Fig. 2A). As concerns, the primary cultures used for this experiment, two out of three were considered in vitro responders to pasireotide since a reduction of ACTH levels was observed upon 4 h cells incubation with pasireotide (Fig. 2B). The ability of RA-9 to decrease ACTH secretion was achieved in one out of two of these primary cultures responsive to pasireotide. In this specific primary culture, the combined treatment with RA-9 and pasireotide resulted more efficient in inhibiting ACTH secretion compared with RA-9 or pasireotide alone (−52.4 ± 5.1% reduction at pasireotide 10 nM; −40.3 ± 6% reduction at RA-9 5 µM; −60.4 ± 3.5% reduction at RA-9 5 µM and pasireotide 10 nM). The only primary culture that did not respond to pasireotide in vitro in terms of hormone inhibitiondid not respond to RA-9 either.
RA-9 effects on ACTH secretion. (A) AtT-20 cells were pre-incubated with 5 µM RA-9 for 30 min and then stimulated or not stimulated with 10 nM pasireotide for 4 h. ACTH was measured in culture medium. Experiments were repeated at least three times and each determination was done in triplicate. Values represent mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001 vs untreated cells (basal). (B) Primary cultured cells from three independent ACTHomas were pre-incubated with 5 µM RA-9 for 30 min and then stimulated or not stimulated with 10 nM pasireotide for 4 h. ACTH was measured in culture medium. Each determination was done in triplicate. ACTHomas were divided in two subgroups as in vitro responder or resistant to pasireotide according with in vitro responsiveness to pasireotide and analyzed separately. Values represent mean ± s.d. and are expressed as percent of untreated cells (basal). *P < 0.05, vs untreated cells (basal).
Citation: Endocrine-Related Cancer 28, 8; 10.1530/ERC-21-0093
RA-9 effects on ACTH secretion. (A) AtT-20 cells were pre-incubated with 5 µM RA-9 for 30 min and then stimulated or not stimulated with 10 nM pasireotide for 4 h. ACTH was measured in culture medium. Experiments were repeated at least three times and each determination was done in triplicate. Values represent mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001 vs untreated cells (basal). (B) Primary cultured cells from three independent ACTHomas were pre-incubated with 5 µM RA-9 for 30 min and then stimulated or not stimulated with 10 nM pasireotide for 4 h. ACTH was measured in culture medium. Each determination was done in triplicate. ACTHomas were divided in two subgroups as in vitro responder or resistant to pasireotide according with in vitro responsiveness to pasireotide and analyzed separately. Values represent mean ± s.d. and are expressed as percent of untreated cells (basal). *P < 0.05, vs untreated cells (basal).
Citation: Endocrine-Related Cancer 28, 8; 10.1530/ERC-21-0093
RA-9 effects on ACTH secretion. (A) AtT-20 cells were pre-incubated with 5 µM RA-9 for 30 min and then stimulated or not stimulated with 10 nM pasireotide for 4 h. ACTH was measured in culture medium. Experiments were repeated at least three times and each determination was done in triplicate. Values represent mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001 vs untreated cells (basal). (B) Primary cultured cells from three independent ACTHomas were pre-incubated with 5 µM RA-9 for 30 min and then stimulated or not stimulated with 10 nM pasireotide for 4 h. ACTH was measured in culture medium. Each determination was done in triplicate. ACTHomas were divided in two subgroups as in vitro responder or resistant to pasireotide according with in vitro responsiveness to pasireotide and analyzed separately. Values represent mean ± s.d. and are expressed as percent of untreated cells (basal). *P < 0.05, vs untreated cells (basal).
Citation: Endocrine-Related Cancer 28, 8; 10.1530/ERC-21-0093
RA-9 reduces phospho-ERK1/2 in AtT-20 cells and primary cultures from ACTH-secreting pituitary tumors regardless of their in vitro responsiveness to pasireotide
To investigate the molecular effectors involved in RA-9-induced cellular responses, we focused on the MAPK pathway, whose members are known to regulate cell proliferation and hormone production in the corticotrophs. Specifically, we examined whether any change in phospho-ERK1/2 levels would be observed upon RA-9 incubation in both AtT-20 cells and primary cultured cells from five corticotropinomas, and compared the results with those elicited by pasireotide. As for AtT-20 cells, Western blot experiments revealed that, similar to pasireotide, RA-9 was able to significantly reduce phospho-ERK1/2 levels (−52.0 ± 36.5% reduction at pasireotide 10 nM, P < 0.05 vs basal; −52.3 ± 13.4% reduction at RA-9 5µM, P < 0.001 vs basal). The same extent of phospho-ERK1/2 reduction was reported for cells co-treated with RA-9 and pasireotide (−51.7 ± 20.0% reduction, P < 0.01 vs basal) (Fig. 3A
Modulation of ERK1/2 phosphorylation by RA-9. (A) AtT-20 cells were pre-incubated for 30 min with 5 µM RA-9 and then stimulated or not stimulated with 10 nM pasireotide. A representative immunoblot of phospho-ERK1/2 and total ERK1/2 is shown. The graph shows the densitometrical analysis of phospho-ERK1/2 normalized on total ERK1/2 resulting from three independent experiments. Data are plotted as mean ± s.d., and results are expressed as percent of untreated cells (basal). *P < 0.05, **P < 0.01, ***P < 0.001 vs untreated cells (basal). (B) Human ACTH-secreting tumor cells from five independent ACTHomas were treated for 10 min with 1 µM of pasireotide. ACTHomas were divided in two subgroups as responder (left) or resistant (right) to pasireotide according with in vitro responsiveness to pasireotide and analyzed separately. Representative immunoblots showing phospho-ERK1/2 and total ERK1/2. Graphs show the densitometrical analysis of phospho-ERK1/2 normalized on total ERK1/2 from three independent responsive samples (left) and two independent resistant samples (right). Data are plotted as mean ± s.d. and results are expressed as percent of untreated cells (basal). *P < 0.05, ***P < 0.001, vs untreated cells (basal).
Citation: Endocrine-Related Cancer 28, 8; 10.1530/ERC-21-0093
Modulation of ERK1/2 phosphorylation by RA-9. (A) AtT-20 cells were pre-incubated for 30 min with 5 µM RA-9 and then stimulated or not stimulated with 10 nM pasireotide. A representative immunoblot of phospho-ERK1/2 and total ERK1/2 is shown. The graph shows the densitometrical analysis of phospho-ERK1/2 normalized on total ERK1/2 resulting from three independent experiments. Data are plotted as mean ± s.d., and results are expressed as percent of untreated cells (basal). *P < 0.05, **P < 0.01, ***P < 0.001 vs untreated cells (basal). (B) Human ACTH-secreting tumor cells from five independent ACTHomas were treated for 10 min with 1 µM of pasireotide. ACTHomas were divided in two subgroups as responder (left) or resistant (right) to pasireotide according with in vitro responsiveness to pasireotide and analyzed separately. Representative immunoblots showing phospho-ERK1/2 and total ERK1/2. Graphs show the densitometrical analysis of phospho-ERK1/2 normalized on total ERK1/2 from three independent responsive samples (left) and two independent resistant samples (right). Data are plotted as mean ± s.d. and results are expressed as percent of untreated cells (basal). *P < 0.05, ***P < 0.001, vs untreated cells (basal).
Citation: Endocrine-Related Cancer 28, 8; 10.1530/ERC-21-0093
Modulation of ERK1/2 phosphorylation by RA-9. (A) AtT-20 cells were pre-incubated for 30 min with 5 µM RA-9 and then stimulated or not stimulated with 10 nM pasireotide. A representative immunoblot of phospho-ERK1/2 and total ERK1/2 is shown. The graph shows the densitometrical analysis of phospho-ERK1/2 normalized on total ERK1/2 resulting from three independent experiments. Data are plotted as mean ± s.d., and results are expressed as percent of untreated cells (basal). *P < 0.05, **P < 0.01, ***P < 0.001 vs untreated cells (basal). (B) Human ACTH-secreting tumor cells from five independent ACTHomas were treated for 10 min with 1 µM of pasireotide. ACTHomas were divided in two subgroups as responder (left) or resistant (right) to pasireotide according with in vitro responsiveness to pasireotide and analyzed separately. Representative immunoblots showing phospho-ERK1/2 and total ERK1/2. Graphs show the densitometrical analysis of phospho-ERK1/2 normalized on total ERK1/2 from three independent responsive samples (left) and two independent resistant samples (right). Data are plotted as mean ± s.d. and results are expressed as percent of untreated cells (basal). *P < 0.05, ***P < 0.001, vs untreated cells (basal).
Citation: Endocrine-Related Cancer 28, 8; 10.1530/ERC-21-0093
Pasireotide reduced phospho-ERK1/2 levels in three out of five primary cultures tested (−24.9 ± 4.1% reduction vs basal, P < 0.001; Fig. 3B, left), whereas RA-9 was effective in decreasing phospho-ERK1/2 levels in all primary cultures regardless of their in vitro responsiveness to pasireotide (−32.9 ± 19.8% reduction at RA-9 5 µM vs basal in the sensitive group, P < 0.001; −33.1 ± 11.8% reduction vs basal at RA-9 5 µM in the resistant group, P < 0.05) (Fig. 3B). No significant difference was observed in cells co-treated with RA-9 and pasireotide compared with cells subjected to single treatments.
RA-9 modulates p27kip1 and phospho-CREB expression levels
Then, p27kip1 expression levels were evaluated in murine and human tumoral corticotroph cells after prolonged exposure with RA-9, pasireotide or their combination. Indeed, p27kip1 represents a putative USP8 client with a crucial role in cell-cycle regulation (Weigand et al. 2019). Western blot analysis carried out in AtT-20 cells showed upregulation of p27kip1 at 48 h of treatment with RA-9 (+67.1 ± 36.7% increase, P < 0.05 vs basal), but not pasireotide (Fig. 4A). An increase in p27kip1 expression level was observed in AtT-20 cells co-treated with RA-9 and pasireotide (+72.3 ± 37.7% increase, P < 0.05 vs basal). Similarly, when tested in one primary culture from an ACTH-secreting pituitary tumor, both RA-9 alone and RA-9 in combination with pasireotide were effective in increasing p27kip1 expression (+68.4% increase at 5µM RA-9 vs basal; +49.4% increase at 5 µM RA-9 and 10 nM pasireotide vs basal), whereas no effect was reported with pasireotide alone (Fig. 4B).
RA-9 effects on p27 expression and CREB phosphorylation. AtT-20 cells (A) and primary cultured cells from one ACTHoma (B) were incubated with 5 µM RA-9 and/or 10 nM pasireotide for 48 h. Representative immunoblots of p27 and GAPDH are shown. The graph shows the quantification of p27 normalized to GAPDH, expressed as percent vs untreated cells (basal). For AtT-20 cells experiments were replicated three times. *P < 0.05 vs basal. (C) AtT-20 cells were pre-incubated for 30 min with 5 µM RA-9 and then stimulated or not stimulated with 10 nM pasireotide. A representative immunoblot of phospho-CREB and total CREB is shown. The graph shows the densitometrical analysis and data are plotted as mean ± s.d. from three independent experiments. *P < 0.05 vs untreated cells (basal).
Citation: Endocrine-Related Cancer 28, 8; 10.1530/ERC-21-0093
RA-9 effects on p27 expression and CREB phosphorylation. AtT-20 cells (A) and primary cultured cells from one ACTHoma (B) were incubated with 5 µM RA-9 and/or 10 nM pasireotide for 48 h. Representative immunoblots of p27 and GAPDH are shown. The graph shows the quantification of p27 normalized to GAPDH, expressed as percent vs untreated cells (basal). For AtT-20 cells experiments were replicated three times. *P < 0.05 vs basal. (C) AtT-20 cells were pre-incubated for 30 min with 5 µM RA-9 and then stimulated or not stimulated with 10 nM pasireotide. A representative immunoblot of phospho-CREB and total CREB is shown. The graph shows the densitometrical analysis and data are plotted as mean ± s.d. from three independent experiments. *P < 0.05 vs untreated cells (basal).
Citation: Endocrine-Related Cancer 28, 8; 10.1530/ERC-21-0093
RA-9 effects on p27 expression and CREB phosphorylation. AtT-20 cells (A) and primary cultured cells from one ACTHoma (B) were incubated with 5 µM RA-9 and/or 10 nM pasireotide for 48 h. Representative immunoblots of p27 and GAPDH are shown. The graph shows the quantification of p27 normalized to GAPDH, expressed as percent vs untreated cells (basal). For AtT-20 cells experiments were replicated three times. *P < 0.05 vs basal. (C) AtT-20 cells were pre-incubated for 30 min with 5 µM RA-9 and then stimulated or not stimulated with 10 nM pasireotide. A representative immunoblot of phospho-CREB and total CREB is shown. The graph shows the densitometrical analysis and data are plotted as mean ± s.d. from three independent experiments. *P < 0.05 vs untreated cells (basal).
Citation: Endocrine-Related Cancer 28, 8; 10.1530/ERC-21-0093
Furthermore, we analyzed the effects of RA-9 on the phosphorylated status of CREB, which is a key molecular effector involved in ACTH production and whose altered expression was recently correlated to USP8 unbalanced activity (Weigand et al. 2019). Western blot experiments performed in AtT-20 cells and reported in Fig. 4C demonstrated that phospho-CREB levels were significantly reduced by RA-9 (−46.3 ± 33.1% reduction, P < 0.05 vs basal) and pasireotide (−44.6 ± 31.1% reduction, P < 0.05 vs basal) and their combination (−46.6 ± 27.2% reduction, P < 0.05 vs basal).
RA-9 reduces ACTH release by USP8-mutant cells
As a final step, we asked whether RA-9 could decrease ACTH release in corticotroph tumor cells bearing USP8 mutations. To this purpose, AtT-20 cells were transiently transfected with constructs encoding the most representative USP8 mutants (S718del, S718R and P720R). RA-9 decreased ACTH secretion by cells transfected with USP8 mutants (−29.7 ± 17.4% reduction at USP8 S718del, P < 0.05 vs basal; −31.1 ± 21.2% reduction at USP8 S718P, P < 0.05 vs basal; −35.0 ± 18.0% reduction at USP8 P720R, P < 0.05 vs basal) similarly to what observed in empty vector-transfected cells (−21.1 ± 1.7% reduction, P < 0.001 vs basal) (Fig. 5).
RA-9 decreases ACTH secretion in USP8-mutated AtT-20 cells. AtT-20 cells were transiently transfected with S718del, S718P, P720R USP8 mutants and incubated with 5 µM RA-9. ACTH was measured in culture medium. Experiments were repeated at least three times and each determination was done in triplicate. Values represent mean ± s.d. *P < 0.05; ***P < 0.001 vs untreated cells (basal). For each experiment transfection efficacy was monitored by Western blot.
Citation: Endocrine-Related Cancer 28, 8; 10.1530/ERC-21-0093
RA-9 decreases ACTH secretion in USP8-mutated AtT-20 cells. AtT-20 cells were transiently transfected with S718del, S718P, P720R USP8 mutants and incubated with 5 µM RA-9. ACTH was measured in culture medium. Experiments were repeated at least three times and each determination was done in triplicate. Values represent mean ± s.d. *P < 0.05; ***P < 0.001 vs untreated cells (basal). For each experiment transfection efficacy was monitored by Western blot.
Citation: Endocrine-Related Cancer 28, 8; 10.1530/ERC-21-0093
RA-9 decreases ACTH secretion in USP8-mutated AtT-20 cells. AtT-20 cells were transiently transfected with S718del, S718P, P720R USP8 mutants and incubated with 5 µM RA-9. ACTH was measured in culture medium. Experiments were repeated at least three times and each determination was done in triplicate. Values represent mean ± s.d. *P < 0.05; ***P < 0.001 vs untreated cells (basal). For each experiment transfection efficacy was monitored by Western blot.
Citation: Endocrine-Related Cancer 28, 8; 10.1530/ERC-21-0093
Discussion
To date, pasireotide represents the only pharmacological option approved to directly target the pituitary tumor in patients with CD. However, biochemical remission is observed in only 30% of cases, with about 70% of patients exhibiting hyperglycemic-related events following its administration (Munir & Newell-Price 2010, Mazziotti et al. 2011, Pivonello et al. 2020). Therefore, novel pituitary-directed pharmacological strategies are required.
The recent findings that a consistent subset of patients (35 to 60%) with ACTH-secreting pituitary tumors presents somatic mutations in the USP8 gene have represented an exciting advance in our understanding of CD and shed new light on the ubiquitination/deubiquitination system in the pathophysiology of corticotropinomas (Reincke et al. 2015, Sbiera et al. 2019). The intracellular balance between ubiquitination and deubiquitination determines which proteins are degraded and which are rescued and recycled. USPs and DUBs are indeed key modulators of the steady levels of a variety of proteins involved in cell-cycle progression, apoptosis and DNA damage repair (Li et al. 2002, 2004) whose dysregulation is expected to affect cell proliferation and viability. In this context, small molecules that inhibit ubiquitin-mediated protein degradation independently of the 20S catalytic activity, thus working as DUBs inhibitors (Anchoori et al. 2011), may represent a promising approach for the treatment of cancer (Nicholson et al. 2007, Farshi et al. 2015). The aspect of DUB activity that makes them an attractive therapeutic target for different diseases is their ability to modulate protein fate in a specific or selective manner and the possibility to overcome the onset of chemoresistance (Joo et al. 2011, Selvaraju et al. 2015), while avoiding side effects often reported with proteasomal inhibitors such as Bortezomib (Parma et al. 2012).
The present study provides in vitro evidence for the potential therapeutic role played by the small molecular inhibitor of USP8, RA-9, in both murine and human tumor corticotrophs. It is first worth mentioning that AtT-20 cells and primary cultures used for the experiments were USP8-WT. In this non-mutated context, we showed that BrdU incorporation was attenuated by both RA-9 and pasireotide, reflecting inhibitory effects on tumor cell proliferation in AtT-20 cells. A major determinant of cell fate is regulation of cell-cycle progression (D’Andrea & Pellman 1998, Wilkinson 2009) and the CDK inhibitor p27 is often downregulated in corticotropinomas (Lloyd et al. 1997, Bamberger et al. 1999, Lidhar et al. 1999). Although there is no direct indication in the literature that p27 is regulated through ubiquitination, here we demonstrated that AtT-20 cells exposure to RA-9 but not pasireotide leads to p27 upregulation. Such effect has been also observed in cells co-treated with RA-9 and pasireotide, presumably due to the action mediated by RA-9 only. The same changes in p27 levels have been seen in one primary culture tested. Accordingly, RA-9 has been shown to inhibit proliferation of breast and cervical cancer cell lines as well as of ovarian cancer models, both in vitro and in vivo (Issaenko & Amerik 2012, Coughlin et al. 2014), and induce p27 levels to increase in ovarian and cervical cancers (Issaneko & Amerik 2012). Moreover, we demonstrated that RA-9 is able to induce cell apoptosis in AtT-20 cells. Although we could not observe any statistical difference between the apoptotic responses triggered by pasireotide and RA-9, the cytotoxic effect exerted by RA-9 seemed remarkably higher. It is worth mentioning that cell apoptosis has been previously reported upon both knockdown of USP8 and the use of a small-molecule USP8 inhibitor in NSCLCs (Byun et al. 2013, Jeong 2015). Moreover, the cytotoxic effect of RA-9 has been attributed to the rapid accumulation of poly-ubiquitinated protein causing ‘proteotoxic stress’ (Brnjic et al. 2014, Coughlin et al. 2014). In addition, our data revealed that, in a manner comparable to pasireotide, RA-9 is able to reduce ACTH secretion in AtT-20 cells. A similar result was already observed by Kageyama and co-workers upon AtT-20 cells incubation with a different USP8 inhibitor (Kageyama et al. 2020). As for the anti-secretory property of RA-9 tested in corticotropinomas, our results obtained in one tumor sample in vitro responding to both pasireotide and RA-9 are promising. We are aware of the limitation of our study due to the small number of primary cultures tested for this purpose, but unfortunately, the few amounts of cells obtained after tumor dispersion did not allow us to perform the entire set of experiments in each sample. Nevertheless, in line with our findings, Sesta and colleagues have recently shown that the proteasome system is able to modulate ACTH turnover both in USP8 mutant and WT tumors (Sesta et al. 2020). Because POMC and its precursor prePOMC are direct targets of ubiquitination, they could demonstrate that inhibition of the ubiquitin-proteasome pathway with MG132 leads to increased ACTH secretion and cell content in normal rat pituitary primary cultures and human corticotroph tumors (Sesta et al. 2020). Here, we provide evidence of an anti-secretory role played by RA-9 in the presence of mutated USP8 as well. Similarly, a previous study showed that USP8 knockdown significantly reduced ACTH secretion in primary USP8-mutated corticotroph tumor cells (Ma et al. 2015). Further studies are required to test the efficacy of RA-9 in tumoral corticotroph cells with other rare genetic defects (e.g. USP48, p53, CDKN1B).
USP8 has a specific mode of action through the ERK1/2 pathway, whose signaling cascade is involved in cell growth and hormone secretion (Reincke et al. 2015). RA-9 caused a strong reduction of phosphorylated ERK1/2 levels both in AtT-20 cells and primary cultures regardless of the in vitro responsiveness to pasireotide. Although additional experiments are needed to provide insights into the molecular mechanisms correlating ERK action to the anti-secretory effects of RA-9, our data suggest that inhibition of USP8 might be a powerful approach to reduce both cell proliferation and ACTH release. Finally, we evaluated the effect of USP8 inhibition on CREB phosphorylation. CREB is involved in POMC promoter activation, and its phosphorylation levels have been found to be altered in relation to USP8 mutational status both in AtT-20 cells and in corticotropinomas (Weigand et al. 2019). According to published data reporting CREB as a target of ubiquitination, we observed a decrease of phospho-CREB levels in AtT-20 cells incubated with RA-9. This result was confirmed by pasireotide treatment, although no synergic effect was observed when the two compounds were simultaneously administrated.
In conclusion, the present study shows that, although acting on different targets, RA-9 and pasireotide elicited a comparable spectrum of biological responses in tumor corticotrophs, and inhibition of USP8 may represent a novel approach to control cell proliferation and ACTH secretion of both USP8-WT and USP8-mutated tumors in patients with CD.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This work was supported by AIRC (Associazione Italiana Ricerca Cancro) grant to G M (IG 2017-20594), Italian Ministry of Health grant to G M (PE-2016-02361797), Ricerca Corrente Funds from the Italian Ministry of Health and Progetti di Ricerca di Interesse Nazionale (PRIN) grant to E P (2017N8CK4K).
Acknowledgements
The authors thank Prof Marily Theodoropoulou for kindly providing the pME-FLAG expression vector coding for WT USP8.
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