Anna Maria Barbieri

Objective: An SF3B1 (Somatic splicing factor 3B subunit 1) mutation has been associated with prolactin-secreting pituitary neuroendocrine tumours (PitNET) with increased proliferation, invasion, dopamine agonist resistance and reduced progression-free survival. Design: We screened a multi-centric cohort of 127 patients with prolactin-secreting PitNET for the SF3B1R625H mutation using digital PCR. Methods: A comparative analysis was conducted between wild-type and mutated tumours, assessing clinical parameters, including age at diagnosis, sex distribution, prolactin levels, tumour size, extent of invasion, recurrence, and response to dopamine agonists. Results: Somatic SF3B1R625H mutation was found in 21/127 patients (17%), who were diagnosed at a younger age (p=0.04) and had larger tumour diameter (p=0.03). Patients who needed transcranial surgery had a higher mutation frequency in their tumour samples compared to the transsphenoidal group (p=0.01). The occurrence of mutation was similar in males and females. Preoperative and postoperative prolactin levels were comparable in patients with mutant and wild-type tumours. No associations were observed between mutation and tumour invasiveness, Ki-67 proliferation index, p53 expression, recurrence and dopamine agonist resistance. Hypopituitarism at presentation, visual deficits, number of surgeries and disease-free survival were not different between the groups. Conclusion: SF3B1 somatic mutation status in lactotroph tumours—as assessed by digital PCR technology—is associated with a younger age at diagnosis and larger tumour diameter. However, in our cohort it does not appear to associate with histological features, higher recurrence, treatment resistance, tumour invasiveness, or long-term outcomes in our multi-centric cohort.

Filamin A (FLNA) is poorly expressed in adrenocortical carcinomas (ACC) compared to adenomas (ACA). Its presence is associated to a less aggressive tumour behaviour, potentially due to its role in negatively regulating IGF1R signalling. Upregulation of G2/M Wee1 kinase was shown in FLNA‐deficient mouse neural progenitor cells, and it has been reported in several tumours. This study explored Wee1 expression in ACC and its regulation by FLNA, the effects of Wee1 inhibitor AZD1775, and the impact of FLNA on its efficacy in ACC cell lines and primary cells. Analysis of FLNA and Wee1 proteins revealed elevated Wee1 and reduced FLNA in ACC compared to normal adrenal gland. FLNA knockdown increased Wee1 protein in NCI‐H295R, MUC‐1, and in primary ACC cells. Higher p‐CDK1 and cyclin B1 were shown in FLNA‐silenced MUC‐1, while decreased Wee1, p‐CDK1 and cyclin B1 resulted after FLNA overexpression. Wee1 reduction was reverted by lactacystin treatment and FLNA transfection increased p‐Wee1 (Ser123), suggesting FLNA's role in targeting Wee1 for degradation. AZD1775 dose‐dependently reduced proliferation and viability in ACC cell lines and primary cultures, and it triggered MUC‐1 cell death. Similar effects were induced by Wee1 silencing. FLNA depletion augmented AZD1775's efficacy in reducing proliferation and potentiating apoptosis in MUC‐1 and primary cells. In conclusion, we demonstrated that FLNA regulates Wee1 expression by promoting its degradation, suggesting that low FLNA typical of ACC leads to increased Wee1 with consequent cancer cells growth. It proposes Wee1 inhibition as a new potential therapeutic approach for ACC, particularly for those lacking FLNA.

Somatostatin receptor ligands (SRLs) with high affinity for somatostatin receptors 2 and 5 (SSTR2 and SSTR5) are poorly efficacious in NF-PitNETs, expressing high levels of SSTR3. ITF2984 is a pan-SSTR ligand with high affinity for SSTR3, able to induce SSTR3 activation and to exert antitumoral activity in the MENX rat model. The aim of this study was to test ITF2984’s antiproliferative and proapoptotic effects in NF-PitNET primary cultured cells derived from surgically removed human tumors and to characterize their SSTR expression profile. We treated cells derived from 23 NF-PitNETs with ITF2984, and a subset of them with octreotide, pasireotide (SRLs with high affinity for SSTR2 or 5, respectively), or cabergoline (DRD2 agonist) and we measured cell proliferation and apoptosis. SSTR3, SSTR2, and SSTR5 expression in tumor tissues was analyzed by qRT-PCR and Western blot. We demonstrated that ITF2984 reduced cell proliferation (−40.8 (17.08)%, p < 0.001 vs. basal, n = 19 NF-PitNETs) and increased cell apoptosis (+41.4 (22.1)%, p < 0.001 vs. basal, n = 17 NF-PitNETs) in all tumors tested, whereas the other drugs were only effective in some tumors. In our model, SSTR3 expression levels did not correlate with ITF2984 antiproliferative nor proapoptotic effects. In conclusion, our data support a possible use of ITF2984 in the pharmacological treatment of NF-PitNET.

The molecular events underlying the variable effectiveness of dopamine receptor type 2 (DRD2) agonists in pituitary neuroendocrine tumors (PitNETs) are not known. Besides the canonical pathway induced by DRD2 coupling with Gi proteins, the β-arrestin 2 pathway contributes to DRD2′s antimitotic effects in PRL- and NF-PitNETs. A promising pharmacological strategy is the use of DRD2-biased agonists that selectively activate only one of these two pathways. The aim of the present study was to compare the effects of two biased DRD2 ligands, selectively activating the G protein (MLS1547) or β-arrestin 2 (UNC9994) pathway, with unbiased DRD2 agonist cabergoline in PRL- and NF-PitNET cells. In rat tumoral pituitary PRL-secreting MMQ cells, UNC9994 reduced cell proliferation with a greater efficacy compared to cabergoline (−40.2 ± 20.4% vs. −21 ± 10.9%, p < 0.05), whereas the G-protein-biased agonist induced only a slight reduction. β-arrestin 2 silencing, but not pertussis toxin treatment, reverted UNC9994 and cabergoline’s antiproliferative effects. In a cabergoline-resistant PRL-PitNET primary culture, UNC9994 inhibited cell proliferation and PRL release. In contrast, in NF-PitNET primary cultures (n = 23), biased agonists did not show better antiproliferative effects than cabergoline. In conclusion, the preferential activation of the β-arrestin 2 pathway by UNC9994 improves DRD2-mediated antiproliferative effects in PRL-PitNETs, suggesting a new pharmacological approach for resistant or poorly responsive tumors.

Somatic mutations in the ubiquitin specific peptidase 8 (USP8) gene have been associated with higher levels of somatostatin (SS) receptor subtype 5 (SSTR5) in adrenocorticotroph hormone (ACTH)-secreting pituitary neuroendocrine tumors (PitNETs). However, a correlation between the USP8 mutational status and favourable responses to pasireotide, the somatostatin multi-receptor ligand acting especially on SSTR5, has not been investigated yet. Here, we studied the impact of USP8 mutations on pasireotide responsiveness in human and murine corticotroph tumor cells. SSTR5 upregulation was observed in USP8 wild-type primary tumor cells transfected with S718del USP8 mutant. However, cell transfection with S718del USP8 and C40-USP8 mutants in in vitro sensitive cultures from USP8 wild-type tumors abolished their ability to respond to pasireotide and did not confer pasireotide responsiveness to the in vitro resistant culture. Pasireotide failed to reduce ACTH secretion in primary cells from one S718P USP8-mutated tumor but exerted a strong antisecretory effect in primary cells from one P720R USP8-mutated tumor. In agreement, AtT-20 cells transfection with USP8 mutants led to SSTR5 expression increase but pasireotide could reduce ACTH production and cyclin E expression in P720R USP8 overexpressing cells, only. In situ Proximity Ligation Assay and immunoflurescence experiments revealed that P720R USP8 mutant is still able to bind 14-3-3 proteins in AtT-20 cells, without affecting SSTR5 localization. In conclusion, P720R USP8 mutation might be considered as a molecular predictor of favourable response to pasireotide in corticotroph tumor cells.

Cushing’s Disease (CD) is a rare condition characterized by an overproduction of ACTH by an ACTH-secreting pituitary tumor, resulting in an excess of cortisol release by the adrenal glands. Somatic mutations in the deubiquitinases USP8 and USP48, and in BRAF genes, have been reported in a subset of patients affected by CD. The aim of this study was to characterize the genetic profile of a cohort of 60 patients with ACTH-secreting tumors, searching for somatic mutations in USP8, USP48, and BRAF hotspot regions. Seven patients were found to carry USP8 somatic mutations in the well-characterized 14-3-3 protein binding motif (n = 5 P720R, n = 1 P720Q, n = 1 S718del); 2 patients were mutated in USP48 (M415I); no mutation was identified in BRAF. In addition, a novel USP8 variant, G664R, located in exon 14, upstream of the 14-3-3 protein binding motif, was identified in 1 patient. Functional characterization of USP8 G664R variant was performed in murine corticotroph tumor AtT-20 cells. Transient transfection with the USP8 G664R variant resulted in a significant increase of ACTH release and cell proliferation (+114.5 ± 53.6% and +28.3 ± 2.6% vs. empty vector transfected cells, p < 0.05, respectively). Notably, USP8 proteolytic cleavage was enhanced in AtT-20 cells transfected with G664R USP8 (1.86 ± 0.58–fold increase of N-terminal USP8 fragment, vs. WT USP8, p < 0.05). Surprisingly, in situ Proximity Ligation Assay (PLA) experiments showed a significant reduction of PLA positive spots, indicating USP8/14-3-3 proteins colocalization, in G664R USP8 transfected cells with respect to WT USP8 transfected cells (−47.9 ± 6.6%, vs. WT USP8, p < 0.001). No significant difference in terms of ACTH secretion, cell proliferation and USP8 proteolytic cleavage, and 14-3-3 proteins interaction was observed between G664R USP8 and S718del USP8 transfected cells. Immunofluorescence experiments showed that, contrary to S718del USP8 but similarly to WT USP8 and other USP8 mutants, G664R USP8 displays an exclusive cytoplasmic localization. In conclusion, somatic mutations were found in USP8 (13.3% vs. 36.5% incidence of all published mutations) and USP48 (3.3% vs. 13.3% incidence) hotspot regions. A novel USP8 variant was identified in a CD patient, and in vitro functional studies in AtT-20 cells suggested that this somatic variant might be clinically relevant in ACTH-secreting tumor pathogenesis, expanding the characterization of USP8 functional domains.

Tenofovir disoproxil fumarate (TDF) is an antiretroviral drug commonly used for the management of Human Immunodeficiency Virus (HIV) in highly active antiretroviral therapy (HAART) and of chronic Hepatitis B Virus (HBV) infections. Long‐term TDF‐treated subjects present decrease of bone mineral density and rarely severe osteomalacia. Although these adverse effects have been attributed to the impaired proximal tubule function, a possible direct involvement of TDF on osteoblasts should be taken into account. The aim of this study was to evaluate whether sodium phosphate transporters NPT2A (sodium‐dependent phosphate transport protein 2A), NPT2C (sodium‐dependent phosphate transport protein 2C), PIT1 (sodium‐dependent phosphate transporter 1), and PIT2 (sodium‐dependent phosphate transporter 2) were expressed in primary human osteoblasts (HOBs), whether their expression was related to HOBs differentiation and whether TDF could affect mineralization and gene expression. PIT1 and PIT2 were expressed under proliferating conditions and increased after induction of mineralization, while NPT2A and NPT2C were almost undetectable. In HOBs TDF exposure induced a significant dose‐dependent decrease in mineralization. Moreover, TDF caused a reduction of COL1A1 and of ATF4 expression in differentiated HOBs. In summary, HOBs do not express NPT2A and NPT2C and do express PIT1 and PIT2, suggesting a role of these two latter in human osteoblast mineralization. TDF impairs osteoblast mineralization, confirming a direct negative effect on bone. Therefore, in clinical practice, bone damage must be suspected and evaluated also in patients receiving TDF without kidney function alterations.

Objective: The diagnosis of central hypoadrenalism (HPAI) is a major clinical challenge. The gold standard procedure remains insuline tolerance test (ITT). Liquid chromatography tandem mass spectrometry (LC-MS/MS) is considered the best procedure for the evaluation of cortisol levels. This study aimed to evaluate cut-offs of serum (TM-SeC) and salivary cortisol (SaC) by LC-MS/MS and serum cortisol by ECLIA assay (E-SeC) after Standard dose ACTH test (SDCT) in diagnosing HPAI. Design: In this study we performed SDCT in 52 consecutive patients (F/M, 33/19, age 42.9±13 yrs) referred to our Center to evaluate at specific time points E-SeC, TM-SeC and SaC. In the same group of patients we also evaluated E-SeC after ITT, to diagnose HPAI (using a cut off348 nmol/l at 0-min, >671 nmol/l at 30-min or >756 nmol/l at 60-min excluded HPAI, whereas an E-SeC 1012 nmol/l at 30-min or >1021 nmol/l at 60-min excluded HPAI, whereas a TM-SeC 15.8 nmol/l at 30-min or >23.3 nmol/l at 60 min excluded HPAI, whereas a SaC<1.7 nmol/l at 0-min, < 4.7;nmol/l at 30-min or <7.3 nmol/l at 60-min confirmed HPAI. Conclusions: We can conclude that even evaluating TM-SeC and SaC, after SDCT there is large gray area of indeterminate results.

A highly polymorphic Cytosine-Adenosine (CA) repeat sequence microsatellite has been identified in the promoter region of IGF1 gene. Several studies investigated the relationship between IGF1-(CA)n polymorphism and IGF1 levels, with conflicting results. Aim of this study was to investigate the influence of this polymorphism on clinical and biochemical characteristics of acromegalic patients. Eighty-eight acromegalic patients and 104 normal subjects were included in the study. Blood DNA was extracted and analysed by microsatellite technique using capillary electrophoresis. Patients and controls were subdivided in 19/19 [homozygous for the (CA)19 allele], 19/X [heterozygous for the (CA)19 allele] and X/X (any other genotype). The genotype frequency was significantly different between patients and controls, the proportion of 19/19 being lower (28·4% vs. 50·0%) and 19/X and X/X higher in acromegalic patients than in controls (P = 0·004). There were no significant differences in age, gender, basal and nadir GH, IGF1-SDS, tumour size, metabolic parameters, outcome and treatment among the three groups. The different frequency of genotypes in acromegalic patients vs. controls, as well as the lack of relationship between IGF1-(CA)n polymorphism and clinical and biochemical data in acromegalic patients, was confirmed using an additional alternative genotyping considering (CA)19 and (CA)20 homozygotes and heterozygotes vs. alleles with more than 19 of 20 repeats or less. Our results do not support the hypothesis that IGF-(CA)n alleles may have a significant role in determining clinical, biochemical and outcome of patients with acromegaly. The possible role of IGF1 polymorphism on susceptibility to acromegaly remains to be investigated.