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      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

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          Abstract

          The high expression of somatostatin receptor 2 (SST<sub>2</sub>) 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 SST<sub>2</sub> signaling and expression. The aim of this study was to explore FLNA involvement in SST<sub>2</sub> intracellular trafficking in tumor somatotroph cells. By biotinylation assay, we found that FLNA silencing abolished octreotide-mediated SST<sub>2</sub> internalization in rat GH3 cell line (28.0 ± 2.7 vs. 4 ± 4.3% SST<sub>2</sub> 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% SST<sub>2</sub> internalization, control versus FLNA siRNA cells, respectively, p < 0.05). In addition, confocal imaging revealed impaired SST<sub>2</sub> 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 SST<sub>2</sub> receptors both in rat and human tumor somatotroph cells. Although FLNA expression knock down did not prevent the formation of β-arrestin2-SST<sub>2</sub> complex in GH3 cells, it significantly impaired efficient SST<sub>2</sub> 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 SST<sub>2</sub>-Rab5 and SST<sub>2</sub>-Rab4 colocalization, respectively, in control versus FLNA siRNA cells). Altogether these data support an important role for FLNA in the mediation of octreotide-induced SST<sub>2</sub> trafficking in GH-secreting pituitary tumor cells through Rab5 and 4 sorting endosomes.

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          Most cited references 38

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          Filamins as integrators of cell mechanics and signalling.

          Filamins are large actin-binding proteins that stabilize delicate three-dimensional actin webs and link them to cellular membranes. They integrate cellular architectural and signalling functions and are essential for fetal development and cell locomotion. Here, we describe the history, structure and function of this group of proteins.
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            Resistance to somatostatin analogs in acromegaly.

            Somatostatin analogs (SA) are widely used in acromegaly, either as first-line or adjuvant treatment after surgery. First-line treatment with these drugs is generally used in the patients with macroadenomas or in those with clinical conditions so severe as to prevent unsafe reactions during anesthesia. Generally, the response to SA takes into account both control of GH and IGF-I excess, with consequent improvement of clinical symptoms directly related to GH and IGF-I excess, and tumor shrinkage. This latter effect is more prominent in the patients treated first-line and bearing large macroadenomas, but it is also observed in patients with microadenomas, even with little clinical implication. Predictors of response are patients' gender, age, initial GH and IGF-I levels, and tumor mass, as well as adequate expression of somatostatin receptor types 2 and 5, those with the highest affinity for octreotide and lanreotide. Only sporadic cases of somatostatin receptor gene mutation or impaired signaling pathways have been described in GH-secreting tumors so far. The response to SA also depends on treatment duration and dosage of the drug used, so that a definition of resistance based on short-term treatments using low doses of long-acting SA is limited. Current data suggest that response to these drugs is better analyzed taking together biochemical and tumoral effects because only the absence of both responses might be considered as a poor response or resistance. This latter evidence seems to occur in 25% of treated patients after 12 months of currently available long-acting SA.
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              Differential effects of octreotide and pasireotide on somatostatin receptor internalization and trafficking in vitro.

              The clinically used somatostatin analogs, octreotide and lanreotide, act primarily by binding to somatostatin receptor 2 (sst2). In contrast, the novel multireceptor ligand pasireotide (SOM230) binds with high affinity to somatostatin receptor subtypes sst1, sst2, sst3, and sst5. SOM230 is currently under clinical evaluation for treatment of acromegaly, Cushing's disease, and octreotide-resistant carcinoid tumors. However, the effects of SOM230 on internalization and postendosomal sorting of individual human somatostatin receptor subtypes have not been determined so far. Here we show that SOM230 was less potent than octreotide in inducing internalization and signaling of sst2 receptors expressed in human embryonic kidney cells. In contrast, SOM230 was more potent than octreotide in inducing internalization and signaling of sst3 and sst5 receptors. Both SOM230 and octreotide stimulated a rapid down-regulation of sst3 but not of sst2 or sst5 receptors. SOM230 and octreotide profoundly differed in their patterns of sst2-stimulated beta-arrestin mobilization. Whereas octreotide-mediated receptor activation led to the formation of stable complexes facilitating the internalization of sst2 and beta-arrestin-2 into the same endocytic vesicles, SOM230-mediated receptor activation led to the formation of unstable complexes that dissociated at or near the plasma membrane. Consequently, sst2 receptors recycled rapidly to the plasma membrane after endocytosis in SOM230-treated cells, but not in octreotide-treated cells. We show that SOM230 modulates somatostatin receptor trafficking in a manner clearly distinct from octreotide and somatostatin. These findings may provide an explanation for the differential regulation of somatostatin receptor responsiveness during long-term administration of stable somatostatin analogs.
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                Author and article information

                Journal
                NEN
                Neuroendocrinology
                10.1159/issn.0028-3835
                Neuroendocrinology
                S. Karger AG
                0028-3835
                1423-0194
                2020
                July 2020
                01 October 2019
                : 110
                : 7-8
                : 642-652
                Affiliations
                aDepartment of Clinical Sciences and Community Health, University of Milan, Milan, Italy
                bPhD Program in Endocrinological Sciences, Sapienza University of Rome, Rome, Italy
                cDepartment of Pathophysiology and Transplantation, University of Milan, Milan, Italy
                dNeurosurgery Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy
                eEndocrine Unit, IRCCS Humanitas Clinical Institute, Humanitas University, Rozzano, Italy
                fEndocrinology Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy
                gInstitute of Pharmacology and Toxicology and Bio-Imaging Center, University of Würzburg, Würzburg, Germany
                hInstitute of Metabolism and Systems Research, University of Birmingham, Birmingham, United Kingdom
                iCentre of Membrane Proteins and Receptors, University of Birmingham, Birmingham, United Kingdom
                Author notes
                *Giovanna Mantovani, Endocrinology Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Department of Clinical Sciences and Community Health, University of Milan, Via F. Sforza, 35, IT–20122 Milan (Italy), E-Mail giovanna.mantovani@unimi.it
                Article
                503791 Neuroendocrinology 2020;110:642–652
                10.1159/000503791
                31574507
                © 2019 S. Karger AG, Basel

                Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

                Page count
                Figures: 5, Pages: 11
                Categories
                Research Article

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