40 results on '"Treppiedi D"'
Search Results
2. Cofilin is a cAMP effector in mediating actin cytoskeleton reorganization and steroidogenesis in mouse and human adrenocortical tumor cells
- Author
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Peverelli, E., Catalano, R., Giardino, E., Treppiedi, D., Morelli, V., Ronchi, C.L., Vaczlavik, A., Fusco, N., Ferrero, S., Bertherat, J., Beuschlein, F., Chiodini, I., Arosio, M., Spada, A., and Mantovani, G.
- Published
- 2017
- Full Text
- View/download PDF
3. Dopamine receptor type 2 (DRD2) inhibits migration and invasion of human tumorous pituitary cells through ROCK-mediated cofilin inactivation
- Author
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Peverelli, E., Giardino, E., Treppiedi, D., Locatelli, M., Vaira, V., Ferrero, S., Bosari, S., Lania, A.G., Spada, A., and Mantovani, G.
- Published
- 2016
- Full Text
- View/download PDF
4. TMPRSS2 Expression and Activity Modulation by Sex-Related Hormones in Lung Calu-3 Cells: Impact on Gender-Specific SARS-CoV-2 Infection
- Author
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Treppiedi, D., Marra, G., Di Muro, G., Catalano, R., Mangili, F., Esposito, E., Barbieri, A.M., Arosio, M., Mantovani, G., and Peverelli, E.
- Subjects
Male ,androgen ,estradiol ,SARS-CoV-2 ,Spike ,TMPRSS2 ,Estradiol ,Serine Endopeptidases ,Settore MED/46 - Scienze Tecniche di Medicina di Laboratorio ,COVID-19 ,Metribolone ,Peptidyl-Dipeptidase A ,Settore MED/13 - Endocrinologia ,Cell Line ,Humans ,Female ,Angiotensin-Converting Enzyme 2 ,Lung - Abstract
Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although males and females are at equivalent risk of infection, males are more prone to develop a higher severity disease, regardless of age. The factors that mediate susceptibility to SARS-CoV-2 and transmission are still under investigation. A potential role has been attributed to differences in the immune systems response to viral antigens between males and females as well as to different regulatory actions played by sex-related hormones on the two crucial molecular effectors for SARS-CoV-2 infection, TMPRSS2 and ACE2. While few and controversial data about TMPRSS2 transcript regulation in lung cells are emerging, no data on protein expression and activity of TMPRSS2 have been reported. Aim of the present study was to search for possible modulatory actions played by sex-related hormones on TMPRSS2 and ACE2 expression in Calu-3 cells, to test the effects of sex-steroids on the expression of the 32kDa C-term fragment derived from autocatalitic cleavage of TMPRSS2 and its impact on priming of transiently transfected spike protein. Cells were stimulated with different concentrations of methyltrienolone (R1881) or estradiol for 30 h. No difference in mRNA and protein expression levels of full length TMPRSS2 was observed. However, the 32 kDa cleaved serine protease domain was increased after 100 nM R1881 (+2.36 ± 1.13 fold-increase vs control untreated cells, p0.05) and 10 nM estradiol (+1.90 ± 0.64, fold-increase vs control untreated cells, p0.05) treatment. Both R1881 and estradiol significantly increased the activating proteolytic cleavage of SARS-CoV-2 Spike (S) transfected in Calu-3 cells (+1.76 ± 0.18 and +1.99±,0.76 increase in S cleavage products at R1881 100nM and 10 nM estradiol treatment, respectively, p0.001 and p0.05 vs control untreated cells, respectively). Finally, no significant differences in ACE2 expression were observed between hormones-stimulated cells and untreated control cells. Altogether, these data suggest that both male and female sex-related hormones are able to induce a proteolityc activation of TMPRSS2, thus promoting viral infection, in agreement with the observation that males and females are equally infected by SARS-CoV-2.
- Published
- 2022
5. Detecting precipitation trend using a multiscale approach based on quantile regression over a Mediterranean area
- Author
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Treppiedi, D., primary, Cipolla, G., additional, Francipane, A., additional, and Noto, L.V., additional
- Published
- 2021
- Full Text
- View/download PDF
6. Filamin A (FLNA) Plays an Essential Role in Somatostatin Receptor 2 (SST2) Signaling and Stabilization After Agonist Stimulation in Human and Rat Somatotroph Tumor Cells
- Author
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Peverelli, E., Giardino, E., Treppiedi, D., Vitali, E., Cambiaghi, V., Locatelli, M., Lasio, G. B., Spada, A., Lania, A. G., and Mantovani, G.
- Published
- 2014
7. The cytoskeleton actin binding protein filamin A impairs both IGF2 mitogenic effects and the efficacy of IGF1R inhibitors in adrenocortical cancer cells
- Author
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Catalano, R., primary, Giardino, E., additional, Treppiedi, D., additional, Mangili, F., additional, Morelli, V., additional, Elli, F.M., additional, Serban, A.L., additional, Luconi, M., additional, Mannelli, M., additional, Spada, A., additional, Arosio, M., additional, Mantovani, G., additional, and Peverelli, E., additional
- Published
- 2021
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8. Octreotide and pasireotide effects on medullary thyroid carcinoma (MTC) cells growth, migration and invasion
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Giardino, E., primary, Catalano, R., additional, Mangili, F., additional, Barbieri, A.M., additional, Treppiedi, D., additional, Elli, F.M., additional, Dolci, A., additional, Contarino, A., additional, Spada, A., additional, Arosio, M., additional, Mantovani, G., additional, and Peverelli, E., additional
- Published
- 2021
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9. Dopamine receptor type 2 (DRD2) and somatostatin receptor type 2 (SSTR2) agonists are effective in inhibiting proliferation of progenitor/stem-like cells isolated from nonfunctioning pituitary tumors
- Author
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Peverelli, E, Giardino, E, Treppiedi, D, Meregalli, M, Belicchi, M, Vaira, V, Corbetta, S, Verdelli, C, Verrua, E, Serban, A, Locatelli, M, Carrabba, G, Gaudenzi, G, Malchiodi, E, Cassinelli, L, Lania, A, Ferrero, S, Bosari, S, Vitale, G, Torrente, Y, Spada, A, Mantovani, G, Peverelli E, Giardino E, Treppiedi D, Meregalli M, Belicchi M, Vaira V, Corbetta S, Verdelli C, Verrua E, Serban AL, Locatelli M, Carrabba G, Gaudenzi G, Malchiodi E, Cassinelli L, Lania AG, Ferrero S, Bosari S, Vitale G, Torrente Y, Spada A, Mantovani G, Peverelli, E, Giardino, E, Treppiedi, D, Meregalli, M, Belicchi, M, Vaira, V, Corbetta, S, Verdelli, C, Verrua, E, Serban, A, Locatelli, M, Carrabba, G, Gaudenzi, G, Malchiodi, E, Cassinelli, L, Lania, A, Ferrero, S, Bosari, S, Vitale, G, Torrente, Y, Spada, A, Mantovani, G, Peverelli E, Giardino E, Treppiedi D, Meregalli M, Belicchi M, Vaira V, Corbetta S, Verdelli C, Verrua E, Serban AL, Locatelli M, Carrabba G, Gaudenzi G, Malchiodi E, Cassinelli L, Lania AG, Ferrero S, Bosari S, Vitale G, Torrente Y, Spada A, and Mantovani G
- Abstract
The role of progenitor/stem cells in pituitary tumorigenesis, resistance to pharmacological treatments and tumor recurrence is still unclear. This study investigated the presence of progenitor/stem cells in non-functioning pituitary tumors (NFPTs) and tested the efficacy of dopamine receptor type 2 (DRD2) and somatostatin receptor type 2 (SSTR2) agonists to inhibit in vitro proliferation. They found that 70% of 46 NFPTs formed spheres co-expressing stem cell markers, transcription factors (DAX1, SF1, ERG1) and gonadotropins. Analysis of tumor behavior showed that spheres formation was associated with tumor invasiveness (OR = 3,96; IC: 1.05-14.88, p = 0.036). The in vitro reduction of cell proliferation by DRD2 and SSTR2 agonists (31 +/- 17% and 35 +/- 13% inhibition, respectively, p < 0.01 vs. basal) occurring in about a half of NFPTs cells was conserved in the corresponding spheres. Accordingly, these drugs increased cyclin-dependent kinase inhibitor p27 and decreased cyclin D3 expression in spheres. In conclusion, they provided further evidence for the existence of cells with a progenitor/stem cells-like phenotype in the majority of NFPTs, particularly in those with invasive behavior, and demonstrated that the antiproliferative effects of dopaminergic and somatostatinergic drugs were maintained in progenitor/stem-like cells.
- Published
- 2017
10. Cofilin is a mediator of RET-promoted medullary thyroid carcinoma cell migration, invasion and proliferation
- Author
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Giardino, E., primary, Catalano, R., additional, Barbieri, A.M., additional, Treppiedi, D., additional, Mangili, F., additional, Spada, A., additional, Arosio, M., additional, Mantovani, G., additional, and Peverelli, E., additional
- Published
- 2019
- Full Text
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11. Cytoskeleton actin-binding proteins in clinical behavior of pituitary tumors
- Author
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Mantovani, G, primary, Treppiedi, D, additional, Giardino, E, additional, Catalano, R, additional, Mangili, F, additional, Vercesi, P, additional, Arosio, M, additional, Spada, A, additional, and Peverelli, E, additional
- Published
- 2019
- Full Text
- View/download PDF
12. Dopamine receptor type 2 (DRD2) and somatostatin receptor type 2 (SSTR2) agonists are effective in inhibiting proliferation of progenitor/stem-like cells isolated from nonfunctioning pituitary tumors
- Author
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Peverelli E, Giardino E, Treppiedi D, Meregalli M, Belicchi M, Vaira V, Corbetta S, Verdelli C, Verrua E, Serban AL, Locatelli M, Carrabba G, Gaudenzi G, Malchiodi E, Cassinelli L, Lania AG, Ferrero S, Bosari S, Vitale G, Torrente Y, Spada A, Mantovani G, Peverelli, E, Giardino, E, Treppiedi, D, Meregalli, M, Belicchi, M, Vaira, V, Corbetta, S, Verdelli, C, Verrua, E, Serban, A, Locatelli, M, Carrabba, G, Gaudenzi, G, Malchiodi, E, Cassinelli, L, Lania, A, Ferrero, S, Bosari, S, Vitale, G, Torrente, Y, Spada, A, and Mantovani, G
- Subjects
Adult ,Male ,ERG1 Potassium Channel ,Carcinogenesis ,Dopamine Agents ,pituitary adenoma ,tumor stem cells ,somatostatin ,Spheroids, Cellular ,Humans ,Pituitary Neoplasms ,dopamine ,drug resistance ,pituitary adenomas ,Cell Proliferation ,Cyclin D3 ,Cyclin-Dependent Kinase Inhibitor p27 ,DAX-1 Orphan Nuclear Receptor ,Drug Resistance, Neoplasm ,Female ,Gene Expression Regulation, Neoplastic ,Gonadotropins ,Middle Aged ,Neoplasm Recurrence, Local ,Neoplastic Stem Cells ,RNA Splicing Factors ,Receptors, Dopamine D2 ,Receptors, Somatostatin - Abstract
The role of progenitor/stem cells in pituitary tumorigenesis, resistance to pharmacological treatments and tumor recurrence is still unclear. This study investigated the presence of progenitor/stem cells in non-functioning pituitary tumors (NFPTs) and tested the efficacy of dopamine receptor type 2 (DRD2) and somatostatin receptor type 2 (SSTR2) agonists to inhibit in vitro proliferation. They found that 70% of 46 NFPTs formed spheres co-expressing stem cell markers, transcription factors (DAX1, SF1, ERG1) and gonadotropins. Analysis of tumor behavior showed that spheres formation was associated with tumor invasiveness (OR = 3,96; IC: 1.05-14.88, p = 0.036). The in vitro reduction of cell proliferation by DRD2 and SSTR2 agonists (31 +/- 17% and 35 +/- 13% inhibition, respectively, p < 0.01 vs. basal) occurring in about a half of NFPTs cells was conserved in the corresponding spheres. Accordingly, these drugs increased cyclin-dependent kinase inhibitor p27 and decreased cyclin D3 expression in spheres. In conclusion, they provided further evidence for the existence of cells with a progenitor/stem cells-like phenotype in the majority of NFPTs, particularly in those with invasive behavior, and demonstrated that the antiproliferative effects of dopaminergic and somatostatinergic drugs were maintained in progenitor/stem-like cells.
- Published
- 2016
13. A novel pathway activated by somatostatin receptor type 2 (SST2): Inhibition of pituitary tumor cell migration and invasion through cytoskeleton protein recruitment
- Author
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Peverelli, E., Giardino, E., Treppiedi, D., Catalano, R., Mangili, F., Locatelli, M., Lania, A. G., Arosio, M., Spada, A., and Mantovani, G.
- Subjects
Tumor ,Filamins ,GH-secreting pituitary tumors ,cofilin ,invasion ,filamin A ,somatostatin receptor 2 ,Actin Depolymerizing Factors ,Animals ,Cell Line, Tumor ,Cell Movement ,Cytoskeleton ,Humans ,Neoplasm Invasiveness ,Phosphorylation ,Pituitary Neoplasms ,Rats ,Receptors, Somatostatin ,Signal Transduction ,Somatostatin ,rhoA GTP-Binding Protein ,Cell Line ,Receptors - Abstract
The pharmacological therapy of GH-secreting pituitary tumors is based on somatostatin (SS) analogs that reduce GH secretion and cell proliferation by binding mainly SS receptors type 2 (SST2). Antimigratory effects of SS have been demonstrated in different cell models, but no data on pituitary tumors are available. Aims of our study were to evaluate SST2 effects on migration and invasion of human and rat tumoral somatotrophs, and to elucidate the molecular mechanism involved focusing on the role of cofilin and filamin A (FLNA). Our data revealed that SST2 agonist BIM23120 significantly reduced GH3 cells migration (-22% ± 3.6%, p 0.001) and invasion on collagen IV (-31.3% ± 12.2%, p 0.01), both these effects being reproduced by octreotide and pasireotide. Similar results were obtained in primary cultured cells from human GH-secreting tumors. These inhibitory actions were accompanied by a marked increase in RhoA/ROCK-dependent cofilin phosphorylation (about 2.7-fold in GH3 and 2.1-fold in human primary cells). Accordingly, the anti-invasive effect of the SS analog was mimicked by the overexpression in GH3 cells of the S3D phosphomimetic cofilin mutant, and abolished by both phosphodeficient S3A cofilin and a specific ROCK inhibitor that prevented cofilin phosphorylation. Moreover, FLNA silencing and FLNA dominant-negative mutants FLNA19-20 and FLNA21-24 transfection demonstrated that FLNA plays a scaffold function for SST2-mediated cofilin phosphorylation. Accordingly, cofilin recruitment to agonist-activated SST2 was completely lost in FLNA silenced cells. In conclusion, we demonstrated that SST2 inhibits rat and human tumoral somatotrophs migration and invasion through a molecular mechanism that involves FLNA-dependent cofilin recruitment and phosphorylation.
- Published
- 2017
14. cAMP/PKA-induced filamin A (FLNA) phosphorylation inhibits SST2 signal transduction in GH-secreting pituitary tumor cells
- Author
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Peverelli, E., primary, Giardino, E., additional, Mangili, F., additional, Treppiedi, D., additional, Catalano, R., additional, Ferrante, E., additional, Sala, E., additional, Locatelli, M., additional, Lania, A.G., additional, Arosio, M., additional, Spada, A., additional, and Mantovani, G., additional
- Published
- 2018
- Full Text
- View/download PDF
15. Dopamine receptor type 2 ( DRD2 ) and somatostatin receptor type 2 ( SSTR2 ) agonists are effective in inhibiting proliferation of progenitor/stem‐like cells isolated from nonfunctioning pituitary tumors
- Author
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Peverelli, E., primary, Giardino, E, additional, Treppiedi, D., additional, Meregalli, M., additional, Belicchi, M., additional, Vaira, V., additional, Corbetta, S., additional, Verdelli, C., additional, Verrua, E., additional, Serban, A. L., additional, Locatelli, M., additional, Carrabba, G., additional, Gaudenzi, G., additional, Malchiodi, E., additional, Cassinelli, L., additional, Lania, A. G., additional, Ferrero, S., additional, Bosari, S., additional, Vitale, G., additional, Torrente, Y., additional, Spada, A., additional, and Mantovani, G., additional
- Published
- 2017
- Full Text
- View/download PDF
16. Somatostatin Receptor Type 2 (SSTR2) Internalization and Intracellular Trafficking in Pituitary GH-Secreting Adenomas: Role of Scaffold Proteins and Implications for Pharmacological Resistance
- Author
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Treppiedi, D., additional, Peverelli, E., additional, Giardino, E., additional, Ferrante, E., additional, Calebiro, D., additional, Spada, A., additional, and Mantovani, G., additional
- Published
- 2016
- Full Text
- View/download PDF
17. A novel pathway activated by somatostatin receptor type 2 (SST2): Inhibition of pituitary tumor cell migration and invasion through cytoskeleton protein recruitment.
- Author
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Peverelli, E., Giardino, E., Treppiedi, D., Catalano, R., Mangili, F., Locatelli, M., Lania, A. G., Arosio, M., Spada, A., and Mantovani, G.
- Abstract
The pharmacological therapy of GH‐secreting pituitary tumors is based on somatostatin (SS) analogs that reduce GH secretion and cell proliferation by binding mainly SS receptors type 2 (SST2). Antimigratory effects of SS have been demonstrated in different cell models, but no data on pituitary tumors are available. Aims of our study were to evaluate SST2 effects on migration and invasion of human and rat tumoral somatotrophs, and to elucidate the molecular mechanism involved focusing on the role of cofilin and filamin A (FLNA). Our data revealed that SST2 agonist BIM23120 significantly reduced GH3 cells migration (−22% ± 3.6%,
p < 0.001) and invasion on collagen IV (−31.3% ± 12.2%,p < 0.01), both these effects being reproduced by octreotide and pasireotide. Similar results were obtained in primary cultured cells from human GH‐secreting tumors. These inhibitory actions were accompanied by a marked increase in RhoA/ROCK‐dependent cofilin phosphorylation (about 2.7‐fold in GH3 and 2.1‐fold in human primary cells). Accordingly, the anti‐invasive effect of the SS analog was mimicked by the overexpression in GH3 cells of the S3D phosphomimetic cofilin mutant, and abolished by both phosphodeficient S3A cofilin and a specific ROCK inhibitor that prevented cofilin phosphorylation. Moreover, FLNA silencing and FLNA dominant‐negative mutants FLNA19‐20 and FLNA21‐24 transfection demonstrated that FLNA plays a scaffold function for SST2‐mediated cofilin phosphorylation. Accordingly, cofilin recruitment to agonist‐activated SST2 was completely lost in FLNA silenced cells. In conclusion, we demonstrated that SST2 inhibits rat and human tumoral somatotrophs migration and invasion through a molecular mechanism that involves FLNA‐dependent cofilin recruitment and phosphorylation. [ABSTRACT FROM AUTHOR]- Published
- 2018
- Full Text
- View/download PDF
18. Somatostatin Receptor Type 2 (SSTR2) Internalization and Intracellular Trafficking in Pituitary GH-Secreting Adenomas: Role of Scaffold Proteins and Implications for Pharmacological Resistance.
- Author
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Treppiedi, D., Peverelli, E., Giardino, E., Ferrante, E., Calebiro, D., Spada, A., and Mantovani, G.
- Subjects
- *
SOMATOSTATIN receptors , *SOMATOTROPIN , *ADENOMA , *SCAFFOLD proteins , *CELL membranes - Abstract
Somatostatin receptor type 2 (SSTR2), together with SSTR5, represents the main target of medical treatment for growth hormone (GH)-secreting pituitary tumors, since it is expressed in most of these tumors and exerts both antiproliferative and cytostatic effects, and reduces hormone secretion, as well. However, clinical practice indicates a great variability in the frequency and entity of favorable responses of acromegalic patients to long-acting somatostatin analogues (SSAs), but the molecular mechanisms regulating this pharmacological resistance are not completely understood. So far, several potentially implied mechanisms have been suggested, including impaired expression of SSTRs, or postreceptor signal transduction alterations. More recently, new studies exploited the molecular factors involved in SSTRs intracellular trafficking regulation, this being a critical point for the modulation of the available active G-coupled receptors (GPCRs) amount at the cell surface. In this respect, the role of the scaffold proteins such as β-arrestins, and the cytoskeleton protein Filamin A (FLNA), have become of relevant importance for GH-secreting pituitary tumors. In fact, β-arrestins are linked to SSTR2 desensitization and internalization, and FLNA is able to regulate SSTR2 trafficking and stability at the plasma membrane. Therefore, the present review will summarize emerging evidence highlighting the role of β-arrestins and FLNA, as possible novel players in the modulation of agonist activated-SSTR2 receptor trafficking and response in GHsecreting pituitary tumors. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
19. Filamin A in Somatostatin and Dopamine Receptor Regulation in Pituitary and the Role of cAMP/PKA Dependent Phosphorylation
- Author
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Peverelli, E., additional, Giardino, E., additional, Vitali, E., additional, Treppiedi, D., additional, Lania, A., additional, and Mantovani, G., additional
- Published
- 2014
- Full Text
- View/download PDF
20. Detecting precipitation trend using a multiscale approach based on quantile regression over a Mediterranean area
- Author
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L. V. Noto, D. Treppiedi, Antonio Francipane, Giuseppe Cipolla, Treppiedi D., Cipolla G., Francipane A., and Noto L.
- Subjects
Atmospheric Science ,precipitation trend ,quantile regression ,Climatology ,Mann–Kendall test ,sub-hourly extremes ,Settore ICAR/02 - Costruzioni Idrauliche E Marittime E Idrologia ,Environmental science ,Mediterranean area ,Precipitation ,Quantile regression - Abstract
One of the most relevant and debated topics related to the effects of the climate change is whether intense rainfall events have become more frequent over the last decades. It is a crucial aspect, since an increase in the magnitude and frequency of occurrence of heavy rainfall events could result in a dramatic growth of floods and, in turn, human lives losses and economic damages. Because of its central position in the Mediterranean area, Sicily has been often screened with the aim to capture some trends in precipitation, potentially related to climate change. While Mann-Kendall test has been largely used for the rainfall trend detection, in this work a different procedure is considered. Precipitation trends are here investigated by processing the whole rainfall time-series, provided by the regional agency SIAS at a 10-min resolution, through the quantile regression method by aggregating precipitation across a wide spectrum of durations and considering different quantiles. Results show that many rain gauges are characterized by an increasing trend in sub-hourly precipitation intensity, especially at the highest quantiles, thus suggesting that, from 2002 to 2019, sub-hourly events have become more intense in most of the island. Moreover, by analysing some spatial patterns, it has been revealed that the south and the east of Sicily are more interested in significant increasing rainfall trends, especially at the 10-min duration. Finally, the comparison between the two procedures revealed a stronger reliability of the quantile regression in the trend analysis detection, mainly due to the possibility of investigating the temporal variation of the tails of precipitation distribution.
- Published
- 2021
21. The Novel SSTR3 Agonist ITF2984 Exerts Antimitotic and Proapoptotic Effects in Human Non-Functioning Pituitary Neuroendocrine Tumor (NF-PitNET) Cells.
- Author
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Di Muro G, Catalano R, Treppiedi D, Barbieri AM, Mangili F, Marra G, Di Bari S, Esposito E, Nozza E, Lania AG, Ferrante E, Locatelli M, Modena D, Steinkuhler C, Peverelli E, and Mantovani G
- Subjects
- Humans, Octreotide pharmacology, Octreotide therapeutic use, Receptors, Somatostatin genetics, Antimitotic Agents, Neuroendocrine Tumors drug therapy, Pituitary Neoplasms drug therapy, Pituitary Neoplasms genetics
- Abstract
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.
- Published
- 2024
- Full Text
- View/download PDF
22. A β-Arrestin 2-Biased Dopamine Receptor Type 2 (DRD2) Agonist Is More Efficacious Than Cabergoline in Reducing Cell Proliferation in PRL-Secreting but Not in Non-Functioning Pituitary Tumor Cells.
- Author
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Di Muro G, Mangili F, Esposito E, Barbieri AM, Catalano R, Treppiedi D, Marra G, Nozza E, Lania AGA, Ferrante E, Locatelli M, Arosio M, Peverelli E, and Mantovani G
- Abstract
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.
- Published
- 2023
- Full Text
- View/download PDF
23. Role of filamin A in the pathogenesis of neuroendocrine tumors and adrenal cancer.
- Author
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Treppiedi D, Catalano R, Mangili F, Mantovani G, and Peverelli E
- Abstract
Cell cytoskeleton proteins are involved in tumor pathogenesis, progression and pharmacological resistance. Filamin A (FLNA) is a large actin-binding protein with both structural and scaffold functions implicated in a variety of cellular processes, including migration, cell adhesion, differentiation, proliferation and transcription. The role of FLNA in cancers has been studied in multiple types of tumors. FLNA plays a dual role in tumors, depending on its subcellular localization, post-translational modification (as phosphorylation at Ser2125) and interaction with binding partners. This review summarizes the experimental evidence showing the critical involvement of FLNA in the complex biology of endocrine tumors. Particularly, the role of FLNA in regulating expression and signaling of the main pharmacological targets in pituitary neuroendocrine tumors, pancreatic neuroendocrine tumors, pulmonary neuroendocrine tumors and adrenocortical carcinomas, with implications on responsiveness to currently used drugs in the treatment of these tumors, will be discussed., Competing Interests: The authors have no conflict of interest to declare., (© The authors.)
- Published
- 2022
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- View/download PDF
24. Dimerization of GPCRs: Novel insight into the role of FLNA and SSAs regulating SST 2 and SST 5 homo- and hetero-dimer formation.
- Author
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Treppiedi D, Marra G, Di Muro G, Catalano R, Mangili F, Esposito E, Calebiro D, Arosio M, Peverelli E, and Mantovani G
- Subjects
- Animals, Dimerization, Filamins metabolism, Humans, Rats, Somatostatin, Octreotide metabolism, Octreotide pharmacology, Pituitary Neoplasms pathology
- Abstract
The process of GPCR dimerization can have profound effects on GPCR activation, signaling, and intracellular trafficking. Somatostatin receptors (SSTs) are class A GPCRs abundantly expressed in pituitary tumors where they represent the main pharmacological targets of somatostatin analogs (SSAs), thanks to their antisecretory and antiproliferative actions. The cytoskeletal protein filamin A (FLNA) directly interacts with both somatostatin receptor type 2 (SST
2 ) and 5 (SST5 ) and regulates their expression and signaling in pituitary tumoral cells. So far, the existence and physiological relevance of SSTs homo- and hetero-dimerization in the pituitary have not been explored. Moreover, whether octreotide or pasireotide may play modulatory effects and whether FLNA may participate to this level of receptor organization have remained elusive. Here, we used a proximity ligation assay (PLA)-based approach for the in situ visualization and quantification of SST2 /SST5 dimerization in rat GH3 as well as in human melanoma cells either expressing (A7) or lacking (M2) FLNA. First, we observed the formation of endogenous SST5 homo-dimers in GH3, A7, and M2 cells. Using the PLA approach combined with epitope tagging, we detected homo-dimers of human SST2 in GH3, A7, and M2 cells transiently co-expressing HA- and SNAP-tagged SST2 . SST2 and SST5 can also form endogenous hetero-dimers in these cells. Interestingly, FLNA absence reduced the basal number of hetero-dimers (-36.8 ± 6.3% reduction of PLA events in M2, P < 0.05 vs . A7), and octreotide but not pasireotide promoted hetero-dimerization in both A7 and M2 (+20.0 ± 11.8% and +44.1 ± 16.3% increase of PLA events in A7 and M2, respectively, P < 0.05 vs . basal). Finally, immunofluorescence data showed that SST2 and SST5 recruitment at the plasma membrane and internalization are similarly induced by octreotide and pasireotide in GH3 and A7 cells. On the contrary, in M2 cells, octreotide failed to internalize both receptors whereas pasireotide promoted robust receptor internalization at shorter times than in A7 cells. In conclusion, we demonstrated that in GH3 cells SST2 and SST5 can form both homo- and hetero-dimers and that FLNA plays a role in the formation of SST2 /SST5 hetero-dimers. Moreover, we showed that FLNA regulates SST2 and SST5 intracellular trafficking induced by octreotide and pasireotide., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Treppiedi, Marra, Di Muro, Catalano, Mangili, Esposito, Calebiro, Arosio, Peverelli and Mantovani.)- Published
- 2022
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25. DRD2 Agonist Cabergoline Abolished the Escape Mechanism Induced by mTOR Inhibitor Everolimus in Tumoral Pituitary Cells.
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Mangili F, Esposito E, Treppiedi D, Catalano R, Marra G, Di Muro G, Barbieri AM, Locatelli M, Lania AG, Mangone A, Spada A, Arosio M, Peverelli E, and Mantovani G
- Subjects
- Drug Interactions, Humans, Proto-Oncogene Proteins c-akt metabolism, beta-Arrestin 2 metabolism, Cabergoline pharmacology, Everolimus pharmacology, Neuroendocrine Tumors drug therapy, Neuroendocrine Tumors metabolism, Neuroendocrine Tumors pathology, Pituitary Neoplasms drug therapy, Pituitary Neoplasms metabolism, Pituitary Neoplasms pathology, Receptors, Dopamine D2 agonists, Receptors, Dopamine D2 metabolism, TOR Serine-Threonine Kinases antagonists & inhibitors, TOR Serine-Threonine Kinases metabolism
- Abstract
The mammalian target of rapamycin (mTOR) inhibitor everolimus has been shown to display antiproliferative effects on a wide spectrum of tumors. In vitro studies demonstrated that everolimus inhibited pituitary neuroendocrine tumor (PitNET) cell growth in a subset of patients. Sensitivity to everolimus is reduced by an escape mechanism that increases AKT phosphorylation (p-AKT), leading to pro-survival pathway activation. Dopamine receptor type 2 (DRD2) mediates a reduction of p-AKT in a subgroup of non-functioning PitNETs (NF-PitNETs) and in prolactin-secreting tumor cells (MMQ cells) through a β-arrestin 2-dependent mechanism. The aim of this study was to investigate the efficacy of everolimus combined with DRD2 agonist cabergoline in reducing NF-PitNET primary cells and MMQ cell proliferation and to evaluate AKT phosphorylation and a possible role of β-arrestin 2. We found that 9 out of 14 NF-PitNETs were resistant to everolimus, but the combined treatment with cabergoline inhibited cell proliferation in 7 out of 9 tumors (-31.4 ± 9.9%, p < 0.001 vs . basal) and reduced cyclin D3 expression. In the everolimus-unresponsive NF-PitNET group, everolimus determined a significant increase of p-AKT/total-AKT ratio (2.1-fold, p < 0.01, vs . basal) that was reverted by cabergoline cotreatment. To investigate the molecular mechanism involved, we used MMQ cells as a model of everolimus escape mechanism. Indeed everolimus did not affect MMQ cell proliferation and increased the p-AKT/total-AKT ratio (+1.53 ± 0.24-fold, p < 0.001 vs . basal), whereas cabergoline significantly reduced cell proliferation (-22.8 ± 6.8%, p < 0.001 vs . basal) and p-AKT. The combined treatment of everolimus and cabergoline induced a reduction of both cell proliferation (-34.8 ± 18%, p < 0.001 vs . basal and p < 0.05 vs . cabergoline alone) and p-AKT/total-AKT ratio (-34.5 ± 14%, p < 0.001 vs . basal and p < 0.05 vs . cabergoline alone). To test β-arrestin 2 involvement, silencing experiments were performed in MMQ cells. Our data showed that the lack of β-arrestin 2 prevented the everolimus and cabergoline cotreatment inhibitory effects on both p-AKT and cell proliferation. In conclusion, this study revealed that cabergoline might overcome the everolimus escape mechanism in NF-PitNETs and tumoral lactotrophs by inhibiting upstream AKT activation. The co-administration of cabergoline might improve mTOR inhibitor antitumoral activity, paving the way for a potential combined therapy in β-arrestin 2-expressing NF-PitNETs or other PitNETs resistant to conventional treatments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mangili, Esposito, Treppiedi, Catalano, Marra, Di Muro, Barbieri, Locatelli, Lania, Mangone, Spada, Arosio, Peverelli and Mantovani.)
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- 2022
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26. P720R USP8 Mutation Is Associated with a Better Responsiveness to Pasireotide in ACTH-Secreting PitNETs.
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Treppiedi D, Marra G, Di Muro G, Esposito E, Barbieri AM, Catalano R, Mangili F, Bravi F, Locatelli M, Lania AG, Ferrante E, Indirli R, Nozza E, Arlati F, Spada A, Arosio M, Mantovani G, and Peverelli E
- Abstract
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.
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- 2022
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27. Molecular mechanisms involved in somatostatin receptor regulation in corticotroph tumors: the role of cytoskeleton and USP8 mutations.
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Peverelli E, Treppiedi D, and Mantovani G
- Abstract
Adrenocorticotropic hormone (ACTH)-secreting pituitary tumors mainly express somatostatin receptor 5 (SSTR5) since SSTR2 is downregulated by the elevated levels of glucocorticoids that characterize patients with Cushing's disease (CD). SSTR5 is the molecular target of pasireotide, the only approved pituitary tumor-targeted drug for the treatment of CD. However, the molecular mechanisms that regulate SSTR5 are still poorly investigated. This review summarizes the experimental evidence supporting the role of the cytoskeleton actin-binding protein filamin A (FLNA) in the regulation of SSTR5 expression and signal transduction in corticotroph tumors. Moreover, the correlations between the presence of somatic USP8 mutations and the expression of SSTR5 will be reviewed. An involvement of glucocorticoid-mediated β-arrestins modulation in regulating SSTRs expression and function in ACTH-secreting tumors will also be discussed., Competing Interests: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (© The authors.)
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- 2022
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28. Pituitary Tumors: Genetic and Molecular Factors Underlying Pathogenesis and Clinical Behavior.
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Spada A, Mantovani G, Lania AG, Treppiedi D, Mangili F, Catalano R, Carosi G, Sala E, and Peverelli E
- Subjects
- Humans, Neuroendocrine Tumors diagnosis, Neuroendocrine Tumors genetics, Neuroendocrine Tumors metabolism, Neuroendocrine Tumors pathology, Pituitary Neoplasms diagnosis, Pituitary Neoplasms genetics, Pituitary Neoplasms metabolism, Pituitary Neoplasms pathology
- Abstract
Pituitary neuroendocrine tumors (PitNETs) are the most common intracranial neoplasms. Although generally benign, they can show a clinically aggressive course, with local invasion, recurrences, and resistance to medical treatment. No universally accepted biomarkers of aggressiveness are available yet, and predicting clinical behavior of PitNETs remains a challenge. In rare cases, the presence of germline mutations in specific genes predisposes to PitNET formation, as part of syndromic diseases or familial isolated pituitary adenomas, and associates to more aggressive, invasive, and drug-resistant tumors. The vast majority of cases is represented by sporadic PitNETs. Somatic mutations in the α subunit of the stimulatory G protein gene (gsp) and in the ubiquitin-specific protease 8 (USP8) gene have been recognized as pathogenetic factors in sporadic GH- and ACTH-secreting PitNETs, respectively, without an association with a worse clinical phenotype. Other molecular factors have been found to significantly affect PitNET drug responsiveness and invasive behavior. These molecules are cytoskeleton and/or scaffold proteins whose alterations prevent proper functioning of the somatostatin and dopamine receptors, targets of medical therapy, or promote the ability of tumor cells to invade surrounding tissues. The aim of the present review is to provide an overview of the genetic and molecular alterations that can contribute to determine PitNET clinical behavior. Understanding subcellular mechanisms underlying pituitary tumorigenesis and PitNET clinical phenotype will hopefully lead to identification of new potential therapeutic targets and new markers predicting the behavior and the response to therapeutic treatments of PitNETs., (© 2021 S. Karger AG, Basel.)
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- 2022
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29. Drug resistance in pituitary tumours: from cell membrane to intracellular signalling.
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Peverelli E, Treppiedi D, Mangili F, Catalano R, Spada A, and Mantovani G
- Subjects
- Adenoma pathology, Animals, Cell Membrane drug effects, Cell Membrane physiology, Humans, Intracellular Signaling Peptides and Proteins physiology, Pituitary Neoplasms pathology, Signal Transduction drug effects, Adenoma drug therapy, Drug Resistance, Neoplasm physiology, Pituitary Neoplasms drug therapy
- Abstract
The pharmacological treatment of pituitary tumours is based on the use of stable analogues of somatostatin and dopamine. The analogues bind to somatostatin receptor types 2 and 5 (SST2 and SST5) and dopamine receptor type 2 (DRD2), respectively, and generate signal transduction cascades in cancerous pituitary cells that culminate in the inhibition of hormone secretion, cell growth and invasion. Drug resistance occurs in a subset of patients and can involve different steps at different stages, such as following receptor activation by the agonist or during the final biological responses. Although the expression of somatostatin and dopamine receptors in cancer cells is a prerequisite for these drugs to reach a biological effect, their presence does not guarantee the success of the therapy. Successful therapy also requires the proper functioning of the machinery of signal transduction and the finely tuned regulation of receptor desensitization, internalization and intracellular trafficking. The present Review provides an updated overview of the molecular factors underlying the pharmacological resistance of pituitary tumours. The Review discusses the experimental evidence that supports a role for receptors and intracellular proteins in the function of SSTs and DRD2 and their clinical importance., (© 2021. Springer Nature Limited.)
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- 2021
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30. Genetic Profiling of a Cohort of Italian Patients with ACTH-Secreting Pituitary Tumors and Characterization of a Novel USP8 Gene Variant.
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Treppiedi D, Barbieri AM, Di Muro G, Marra G, Mangili F, Catalano R, Esposito E, Ferrante E, Serban AL, Locatelli M, Lania AG, Spada A, Arosio M, Peverelli E, and Mantovani G
- Abstract
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.
- Published
- 2021
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31. USP8 inhibitor RA-9 reduces ACTH release and cell growth in tumor corticotrophs.
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Treppiedi D, Di Muro G, Marra G, Barbieri AM, Mangili F, Catalano R, Serban A, Ferrante E, Locatelli M, Lania AG, Arosio M, Spada A, Peverelli E, and Mantovani G
- Subjects
- Adrenocorticotropic Hormone metabolism, Adult, Animals, Cell Proliferation, Corticotrophs metabolism, Corticotrophs pathology, Endopeptidases metabolism, Endosomal Sorting Complexes Required for Transport metabolism, Humans, Mice, Ubiquitin Thiolesterase metabolism, Pituitary ACTH Hypersecretion drug therapy, Pituitary Neoplasms metabolism
- 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.
- Published
- 2021
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32. Filamin A is required for somatostatin receptor type 5 expression and pasireotide-mediated signaling in pituitary corticotroph tumor cells.
- Author
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Treppiedi D, Di Muro G, Mangili F, Catalano R, Giardino E, Barbieri AM, Locatelli M, Arosio M, Spada A, Peverelli E, and Mantovani G
- Subjects
- Animals, Apoptosis, Cell Line, Tumor, Cell Proliferation, Gene Silencing, Hormones metabolism, Humans, Mice, Protein Binding, Somatostatin metabolism, Corticotrophs metabolism, Filamins metabolism, Pituitary Neoplasms metabolism, Receptors, Somatostatin metabolism, Signal Transduction, Somatostatin analogs & derivatives
- Abstract
Somatostatin receptor type 5 (SST5) represents the main pharmacological target in the treatment of adrenocorticotroph hormone (ACTH)-secreting tumors. However, molecular predictors of responsiveness to pasireotide require further investigation. The cytoskeleton protein filamin A (FLNA) modulates the responsiveness to somatostatin analogs (SSA) treatment in other types of pituitary tumors by regulating somatostatin receptor type 2 (SST2)/dopamine receptor type 2 (DRD2) expression and activity. Here, we aimed to test the involvement of FLNA in the modulation of SST5 response to SSA in human and murine tumor corticotrophs. Western blot analysis of human corticotropinomas showed that FLNA and SST5 correlate. Both in human primary cultures and AtT-20 cells, FLNA genetic silencing caused a decrease of receptor expression level. Moreover, pasireotide-mediated SST5 downregulation observed in AtT-20 control cells was no further detected in FLNA silenced cells. In AtT-20 cells, in situ PLA experiments revealed an increased number of SST5-FLNA complexes following pasireotide incubation. Finally, FLNA knock down abolished pasireotide-induced SST5 actions on hormone secretion, cell proliferation and apoptosis. In conclusion, FLNA is implicated in SST5 expression modulation and signaling., (Copyright © 2021 Elsevier B.V. All rights reserved.)
- Published
- 2021
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33. A Novel Mechanism Regulating Dopamine Receptor Type 2 Signal Transduction in Pituitary Tumoral Cells: The Role of cAMP/PKA-Induced Filamin A Phosphorylation.
- Author
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Mangili F, Treppiedi D, Catalano R, Marra G, Di Muro G, Spada A, Arosio M, Peverelli E, and Mantovani G
- Subjects
- Adenoma pathology, Animals, Cell Line, Tumor, Dopamine Agonists pharmacology, Mice, Phosphorylation physiology, Pituitary Neoplasms pathology, Rats, Receptors, Dopamine D2 agonists, Adenoma metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, Filamins metabolism, Pituitary Neoplasms metabolism, Receptors, Dopamine D2 metabolism
- Abstract
The actin binding protein filamin A (FLNA) is required for somatostatin receptor 2 (SSTR2) and dopamine receptor 2 (DRD2) expression and signaling in GH- and PRL-secreting PitNETs, respectively, playing a role in tumor responsiveness to somatostatin receptors ligands and dopaminergic drugs. FLNA functions are regulated by several mechanisms, including phosphorylation. It has been shown that in GH-secreting PitNETs FLNA phosphorylation on Ser2152 (P-FLNA) switches FLNA function from a scaffold that allows SSTR2 signal transduction, to a signal termination protein that hampers SSTR2 antitumoral effects. Aims of the present study were to evaluate in PRL- and ACTH-secreting PitNETs cell lines MMQ and AtT-20 the effects of cAMP pathway activation and DRD2 agonist on P-FLNA and the impact of P-FLNA on DRD2 signal transduction. We found that forskolin increased (+2.2 ± 0.8-fold, p < 0.01 in MMQ; +1.9 ± 0.58-fold, p < 0.05 in AtT-20), and DRD2 agonist BIM53097 reduced (-49.4 ± 25%, p < 0.001 in MMQ; -45.8 ± 28%, p < 0.05 in AtT-20), P-FLNA on Ser2152. The overexpression of a phosphomimetic (S2152D) FLNA mutant in both cell lines prevented DRD2 antiproliferative effects, that were comparable in cells transfected with empty vector, wild-type FLNA as well as phosphodeficient FLNA mutant (S2152A) (-20.6 ± 5% cell proliferation, p < 0.001 in MMQ; -36.6 ± 12%, p < 0.01 in AtT-20). Accordingly, S2152D FLNA expression abolished the expected ability of BIM53097 to increase or decrease, in MMQ and in AtT20 respectively, ERK phosphorylation, an effect that was maintained in S2152A FLNA expressing cells (+1.8 ± 0.65-fold, p < 0.05 in MMQ; -55 ± 13%, p < 0.01 in AtT-20). In addition, the inhibitory effects of DRD2 on hormone secretion (-34.3 ± 6% PRL, p < 0.05 in MMQ; -42.8 ± 22% ACTH, p < 0.05 in AtT-20, in cells expressing S2152A FLNA) were completely lost in S2152D FLNA transfected cells. In conclusion, our data demonstrated that cAMP pathway and DRD2 agonist regulated FLNA activity by increasing or decreasing, respectively, its phosphorylation. Moreover, we found that P-FLNA prevented DRD2 signaling in PRL- and ACTH-secreting tumoral pituitary cell lines, suggesting that this FLNA modification might represent a new regulatory mechanism shared by different GPCRs. In PitNETs expressing DRD2, modulation of P-FLNA might suggest new pharmacological strategies to overcome drug resistance, and P-FLNA might represent a new biomarker for tumor responsiveness to dopaminergic agents., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mangili, Treppiedi, Catalano, Marra, Di Muro, Spada, Arosio, Peverelli and Mantovani.)
- Published
- 2021
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34. Beta-Arrestin 2 Is Required for Dopamine Receptor Type 2 Inhibitory Effects on AKT Phosphorylation and Cell Proliferation in Pituitary Tumors.
- Author
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Mangili F, Giardino E, Treppiedi D, Barbieri AM, Catalano R, Locatelli M, Lania AG, Spada A, Arosio M, Mantovani G, and Peverelli E
- Subjects
- Animals, Cell Line, Tumor, Cell Proliferation physiology, Cells, Cultured, Dopamine Agonists pharmacology, Humans, Phosphorylation physiology, Rats, Receptors, Dopamine D2 agonists, Pituitary Neoplasms metabolism, Proto-Oncogene Proteins c-akt metabolism, Receptors, Dopamine D2 metabolism, beta-Arrestin 2 metabolism
- Abstract
Dopamine receptor type 2 (DRD2) agonists are the first-choice treatment for prolactin-secreting pituitary tumors but are poorly effective in nonfunctioning pituitary neuroendocrine tumors (NF-PitNETs). DRD2 reduces AKT phosphorylation in lactotrophs, but no data are available in NF-PitNETs. DRD2 effects on AKT are mediated by a β-arrestin 2-dependent mechanism in mouse striatum. The aim of this study was to investigate DRD2 effects on AKT phosphorylation and cell proliferation in human primary cultured NF-PitNET cells and in rat tumoral lactotroph cells MMQ, and to test β-arrestin 2 involvement. We found that the DRD2 agonist BIM53097 induced a reduction of the p-AKT/total-AKT ratio in MMQ (-32.8 ± 17.6%, p < 0.001 vs. basal) and in a subset (n = 15/41, 36.6%) of NF-PitNETs (subgroup 1). In the remaining NF-PitNETs (subgroup 2), BIM53097 induced an increase in p-AKT. The ability of BIM53097 to reduce p-AKT correlated with its antimitotic effect, since the majority of subgroup 1 NF-PitNETs was responsive to BIM53097, and nearly all subgroup 2 NF-PitNETs were resistant. β-Arrestin 2 was expressed in MMQ and in 80% of subgroup 1 NF-PitNETs, whereas it was undetectable in 77% of subgroup 2 NF-PitNETs. In MMQ, β-arrestin 2 silencing prevented DRD2 inhibitory effects on p-AKT and cell proliferation. Accordingly, β-arrestin 2 transfection in subgroup 2 NF-PitNETs conferred to BIM53097 the ability to inhibit both p-AKT and cell growth. In conclusion, we demonstrated that β-arrestin 2 is required for DRD2 inhibitory effects on AKT phosphorylation and cell proliferation in MMQ and NF-PitNETs, paving the way for a potential role of β-arrestin 2 as a biomarker predicting NF-PitNETs' responsiveness to treatment with dopamine agonists., (© 2020 S. Karger AG, Basel.)
- Published
- 2021
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35. Cytoskeleton Protein Filamin A Is Required for Efficient Somatostatin Receptor Type 2 Internalization and Recycling through Rab5 and Rab4 Sorting Endosomes in Tumor Somatotroph Cells.
- Author
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Treppiedi D, Mangili F, Giardino E, Catalano R, Locatelli M, Lania AG, Spada A, Arosio M, Calebiro D, Mantovani G, and Peverelli E
- Subjects
- Adenoma pathology, Animals, Cells, Cultured, Endosomes drug effects, Endosomes metabolism, Growth Hormone-Secreting Pituitary Adenoma pathology, Humans, Octreotide pharmacology, Protein Transport drug effects, Proteolysis drug effects, Rats, Signal Transduction drug effects, Somatotrophs drug effects, Somatotrophs metabolism, Somatotrophs pathology, rab4 GTP-Binding Proteins metabolism, rab5 GTP-Binding Proteins metabolism, Adenoma metabolism, Endosomes physiology, Filamins physiology, Growth Hormone-Secreting Pituitary Adenoma metabolism, Receptors, Somatostatin metabolism
- Abstract
The high expression of somatostatin receptor 2 (SST2) in growth hormone (GH)-secreting tumors represents the rationale for the clinical use of somatostatin analogs (SSAs) in acromegaly. Recently, the cytoskeletal protein Filamin A (FLNA) has emerged as key modulator of the responsiveness of GH-secreting pituitary tumors to SSAs by regulating SST2 signaling and expression. The aim of this study was to explore FLNA involvement in SST2 intracellular trafficking in tumor somatotroph cells. By biotinylation assay, we found that FLNA silencing abolished octreotide-mediated SST2 internalization in rat GH3 cell line (28.0 ± 2.7 vs. 4 ± 4.3% SST2 internalization, control versus FLNA small interfering RNAs (siRNA) cells, respectively, p < 0.001) and human GH-secreting primary cultured cells (70.3 ± 21.1 vs. 24 ± 19.2% SST2 internalization, control versus FLNA siRNA cells, respectively, p < 0.05). In addition, confocal imaging revealed impaired SST2 recycling to the plasma membrane in FLNA silenced GH3 cells. Coimmunoprecipitation and immunofluorescence experiments showed that FLNA, as well as β-arrestin2, is timely dependent recruited to octreotide-stimulated SST2 receptors both in rat and human tumor somatotroph cells. Although FLNA expression knock down did not prevent the formation of β-arrestin2-SST2 complex in GH3 cells, it significantly impaired efficient SST2 loading into cytosolic vesicles positive for the early endocytic and recycling markers Rab5 and 4, respectively (33.7 ± 8.9% down to 25.9 ± 6.9%, p < 0.05, and 28.4 ± 7.4% down to 17.6 ± 5.7%, p < 0.01, for SST2-Rab5 and SST2-Rab4 colocalization, respectively, in control versus FLNA siRNA cells). Altogether these data support an important role for FLNA in the mediation of octreotide-induced SST2 trafficking in GH-secreting pituitary tumor cells through Rab5 and 4 sorting endosomes., (© 2019 S. Karger AG, Basel.)
- Published
- 2020
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36. Stem Cells in Pituitary Tumors: Experimental Evidence Supporting Their Existence and Their Role in Tumor Clinical Behavior.
- Author
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Mantovani G, Giardino E, Treppiedi D, Catalano R, Mangili F, Spada A, Arosio M, and Peverelli E
- Abstract
Although generally benign, pituitary tumors frequently show local invasiveness and resistance to pharmacological therapy. After the demonstration of the existence of pituitary gland stem cells, over the past decade, the presence of a stem cell subpopulation in pituitary tumors has been investigated, analogous to the cancer stem cell model developed for malignant tumors. This review recapitulates the experimental evidence supporting the existence of a population of stem-like cells in pituitary tumors, focusing on their potential role in tumor initiation, progression, recurrence and resistance to pharmacological therapy., (Copyright © 2019 Mantovani, Giardino, Treppiedi, Catalano, Mangili, Spada, Arosio and Peverelli.)
- Published
- 2019
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37. Somatostatin analogs regulate tumor corticotrophs growth by reducing ERK1/2 activity.
- Author
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Treppiedi D, Giardino E, Catalano R, Mangili F, Vercesi P, Sala E, Locatelli M, Arosio M, Spada A, Mantovani G, and Peverelli E
- Subjects
- Animals, Cell Proliferation drug effects, Cell Survival, Corticotrophs drug effects, Corticotrophs metabolism, Humans, Mice, Phosphorylation drug effects, Somatostatin pharmacology, Tumor Cells, Cultured, Corticotrophs cytology, MAP Kinase Signaling System drug effects, Pituitary Neoplasms metabolism, Receptors, Somatostatin metabolism, Somatostatin analogs & derivatives
- Abstract
Pasireotide has been associated with tumor shrinkage in patients with Cushing's disease subjected to long term treatment. However, to date the implicated molecular mechanisms are poorly elucidated. Here, we tested pasireotide-mediated cytostatic and cytotoxic effects in ACTH-secreting primary tumor cultures and murine corticotroph tumor cell line, AtT-20 cells. We found somatostatin receptor type 5 (SST5) expressed in 17 different ACTH-secreting tumors and SST2 detectable in 15 out of the 17 tissues. Pasireotide caused a slight but significant in vitro inhibition of cell growth in 3 out of 6 ACTH-secreting primary cultures (-12.1 ± 4.3%, P < 0.01 at 10 nM), remarkably reduced phospho-ERK1/2 levels in 5 out of 8 samples (-36.4 ± 20.5%, P < 0.01 at 1 μM) and triggered an increase of caspase 3/7 activity in 2 of 4 tumors (17 ± 3.6%, P < 0.05 at 1 μM). Accordingly, in AtT-20 cells, pasireotide significantly inhibited cell proliferation (-10.5 ± 7.7% at 10 nM, P < 0.05; -13.9 ± 10.9% at 100 nM, P < 0.05; -26.8 ± 8.9% at 1 μM, P < 0.01). Similar antiproliferative actions were exerted by BIM23206 and BIM23120 (SST5&2 selective ligands, respectively), whereas octreotide was effective when used at 1 μM (-13.3 ± 9.1%, P < 0.05). Moreover, a reduction of phospho-ERK1/2 was observed upon pasireotide and BIM23206 treatment (-8.4 ± 28.6%, P < 0.01 and -51.4 ± 15.9%, P < 0.001 at 10 nM, respectively) but not after octreotide and BIM23120 incubation. Finally, pasireotide was able to induce cell apoptosis in AtT-20 cells at lower concentration than octreotide. Altogether these data indicate a downstream implication of SST5-mediated phospho-ERK1/2 inhibition by pasireotide resulting in ACTH-secreting tumor cells proliferation reduction. Moreover, we describe for the first time a pro-apoptotic effect of pasireotide in corticotrophs., (Copyright © 2019 Elsevier B.V. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
38. Single-Molecule Microscopy Reveals Dynamic FLNA Interactions Governing SSTR2 Clustering and Internalization.
- Author
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Treppiedi D, Jobin ML, Peverelli E, Giardino E, Sungkaworn T, Zabel U, Arosio M, Spada A, Mantovani G, and Calebiro D
- Subjects
- Actin Cytoskeleton metabolism, Adenoma drug therapy, Adenoma genetics, Adenoma metabolism, Animals, CHO Cells, Coated Pits, Cell-Membrane metabolism, Cricetulus, Filamins ultrastructure, Growth Hormone-Secreting Pituitary Adenoma drug therapy, Growth Hormone-Secreting Pituitary Adenoma genetics, Growth Hormone-Secreting Pituitary Adenoma metabolism, HEK293 Cells, Humans, Protein Binding, Protein Transport, Receptors, Somatostatin agonists, Receptors, Somatostatin ultrastructure, Single Molecule Imaging, Filamins metabolism, Gene Expression Regulation, Neoplastic, Receptors, Somatostatin metabolism, Somatostatin analogs & derivatives
- Abstract
The cytoskeletal protein filamin A (FLNA) has been suggested to play an important role in the responsiveness of GH-secreting pituitary tumors to somatostatin receptor subtype 2 (SSTR2) agonists by regulating SSTR2 expression and signaling. However, the underlying mechanisms are unknown. In this study, we use fast multicolor single-molecule microscopy to image individual SSTR2 and FLNA molecules at the surface of living cells with unprecedented spatiotemporal resolution. We find that SSTR2 and FLNA undergo transient interactions, which occur preferentially along actin fibers and contribute to restraining SSTR2 diffusion. Agonist stimulation increases the localization of SSTR2 along actin fibers and, subsequently, SSTR2 clustering and recruitment to clathrin-coated pits (CCPs). Interfering with FLNA-SSTR2 binding with a dominant-negative FLNA fragment increases SSTR2 mobility, hampers the formation and alignment of SSTR2 clusters along actin fibers, and impairs both SSTR2 recruitment to CCPs and SSTR2 internalization. These findings indicate that dynamic SSTR2-FLNA interactions critically control the nanoscale localization of SSTR2 at the plasma membrane and are required for coupling SSTR2 clustering to internalization. These mechanisms explain the critical role of FLNA in the control of SSTR2 expression and signaling and suggest the possibility of targeting SSTR2-FLNA interactions for the therapy of pharmacologically resistant GH-secreting pituitary tumors.
- Published
- 2018
- Full Text
- View/download PDF
39. Expression of protein kinase A regulatory subunits in benign and malignant human thyroid tissues: A systematic review.
- Author
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Del Gobbo A, Peverelli E, Treppiedi D, Lania A, Mantovani G, and Ferrero S
- Subjects
- Adult, Aged, Aged, 80 and over, Cell Differentiation, Female, Humans, Hyperplasia, Male, Middle Aged, Prognosis, Thyroid Gland pathology, Cyclic AMP-Dependent Protein Kinases metabolism, Protein Subunits metabolism, Thyroid Gland enzymology, Thyroid Neoplasms enzymology, Thyroid Neoplasms pathology
- Abstract
In this review, we discuss the molecular mechanisms and prognostic implications of the protein kinase A (PKA) signaling pathway in human tumors, with special emphasis on the malignant thyroid. The PKA signaling pathway is differentially activated by the expression of regulatory subunits 1 (R1) and 2 (R2), whose levels change during development, differentiation, and neoplastic transformation. Following the identification of gene mutations within the PKA regulatory subunit R1A (PRKAR1A) that cause Carney complex-associated neoplasms, several investigators have studied PRKAR1A expression in sporadic thyroid tumors. The PKA regulatory subunit R2B (PRKAR2B) is highly expressed in benign, as well as in malignant differentiated and undifferentiated lesions. PRKAR1A is highly expressed in follicular adenomas and malignant lesions with a statistically significant gradient between benign and malignant tumors; however, it is not expressed in hyperplastic nodules. Although the importance of PKA in human malignancy outcomes is not completely understood, PRKAR1A expression correlates with tumor dimension in malignant lesions. Additional studies are needed to determine whether a relationship exists between PKA subunit expression and clinical outcomes, particularly in undifferentiated tumors. In conclusion, the R1A subunit might be a good molecular candidate for the targeted treatment of malignant thyroid tumors., (Copyright © 2016. Published by Elsevier Inc.)
- Published
- 2016
- Full Text
- View/download PDF
40. Dopamine and Somatostatin Analogues Resistance of Pituitary Tumors: Focus on Cytoskeleton Involvement.
- Author
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Peverelli E, Treppiedi D, Giardino E, Vitali E, Lania AG, and Mantovani G
- Abstract
Pituitary tumors, that origin from excessive proliferation of a specific subtype of pituitary cell, are mostly benign tumors, but may cause significant morbidity in affected patients, including visual and neurologic manifestations from mass-effect, or endocrine syndromes caused by hormone hypersecretion. Dopamine (DA) receptor DRD2 and somatostatin (SS) receptors (SSTRs) represent the main targets of pharmacological treatment of pituitary tumors since they mediate inhibitory effects on both hormone secretion and cell proliferation, and their expression is retained by most of these tumors. Although long-acting DA and SS analogs are currently used in the treatment of prolactin (PRL)- and growth hormone (GH)-secreting pituitary tumors, respectively, clinical practice indicates a great variability in the frequency and entity of favorable responses. The molecular basis of the pharmacological resistance are still poorly understood, and several potential molecular mechanisms have been proposed, including defective expression or genetic alterations of DRD2 and SSTRs, or an impaired signal transduction. Recently, a role for cytoskeleton protein filamin A (FLNA) in DRD2 and SSTRs receptors expression and signaling in PRL- and GH-secreting tumors, respectively, has been demonstrated, first revealing a link between FLNA expression and responsiveness of pituitary tumors to pharmacological therapy. This review provides an overview of the known molecular events involved in SS and DA resistance, focusing on the role played by FLNA.
- Published
- 2015
- Full Text
- View/download PDF
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