Review 845
Authors
E. Peverelli1, E. Giardino1, E. Vitali2, D. Treppiedi1, A. G. Lania3, G. Mantovani1
Affiliations
1
Key words ▶ cytoskeleton ● ▶ pituitary tumors ● ▶ GPCR ●
received 14.03.2014 accepted 18.06.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1384520 Published online: July 28, 2014 Horm Metab Res 2014; 46: 845–853 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence M. Giovanna, MD, PhD Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Unità Operativa di Endocrinologia e Diabetologia University of Milan Padiglione Granelli via F. Sforza 35 20122 Milano Italy Tel.: + 39/02/503 20613 Fax: + 39/02/503 20605 [email protected]
Endocrine Unit, Department of Clinical Sciences and Community Health, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy 2 Laboratory of Cellular and Molecular Endocrinology, Humanitas Research Center, Rozzano, Italy 3 Endocrine Unit, IRCCS Humanitas Clinical Institute, Rozzano, University of Milan, Milan, Italy
Abstract
▼
Molecular mechanisms underlying resistance of pituitary tumors to somatostatin (SS) and dopamine (DA) analogues treatment are not completely understood. Resistance has been associated with defective expression of functional somatostatin and dopamine receptors SSTR2, SSTR5, and DRD2, respectively. Recently, a role of cytoskeleton protein filamin A (FLNA) in DRD2 and SSTR 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. No molecular events underlying the reduction of FLNA levels in resistant tumors have been so far identified.
Introduction
▼
Pituitary tumors are rare neoplasia that derive from excessive proliferation of each subtype of pituitary cells and present with specific endocrine syndromes or mass symptoms or both. The main G protein coupled receptors (GPCRs) target of pharmacological treatment of pituitary tumors are the dopamine (DA) receptor DRD2 and somatostatin (SS) receptors SSTR2 and 5. In particular, DRD2 agonists are used in the treatment of prolactin (PRL)- and, to a lesser extent, ACTH-secreting and nonfunctioning pituitary tumors (NFPA), whereas long acting SS analogues, such as octreotide and lanreotide, are currently used in the treatment of pituitary tumors, particularly GHand TSH-secreting tumors [1, 2]. Prolactinomas are the most frequent pituitary tumors and DA agonists normalize PRL levels and reduce tumor size in the majority of patients by binding DRD2, the DA receptor subtype expressed in pituitary lactotrophs (reviewed in [1]). DRD2
FLNA can be phosphorylated by PKA on Ser2152, with increased FLNA resistance to cleavage by calpain and conformational changes affecting FLNA regions involved in SSTR2 and DRD2 binding and signal transduction. In this respect, the effect of cAMP/PKA pathway in the regulation of FLNA stability and/or function by modulating its phosphorylation status could assume particular importance in pituitary, where cAMP cascade plays a crucial role in pituitary cell functions and tumorigenesis. This review will discuss the role of FLNA in the regulation of the main GPCRs target of pharmacological treatment of pituitary tumors, that is, SSTR2 and DRD2, focusing on the effects of cAMP/PKA-mediated FLNA phosphorylation on FLNA biological functions.
couples to Gi/Go proteins to inhibit adenylyl cyclase activity and reduce intracellular cAMP levels. Somatostatin analogues are currently used to successfully treat acromegalic patients since they inhibit cell proliferation and hormone secretion by binding with high affinity SSTR2 and, to a lesser extent, SSTR5, which are both expressed at high density in most GH-secreting pituitary tumors [3, 4]. By coupling with multiple PTXsensitive G proteins, SSTs inhibit adenylyl cyclase activity and some subtypes reduce calcium entry by modulating L-type Ca2 + and K + channels, all these events being involved in the reduction of hormone secretion. Both SSTR2 and SSTR5 mediate the antiproliferative effects of SS, by tyrosine phosphatase activation and ERK1/2 phosphorylation inhibition, respectively, whereas only SSTR2 and SST3 subtypes mediate apoptotic effects [5–8].
Peverelli E et al. FLNA Phosphorylation in Pituitary Tumors … Horm Metab Res 2014; 46: 845–853
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Filamin A in Somatostatin and Dopamine Receptor Regulation in Pituitary and the Role of cAMP/PKA Dependent Phosphorylation
846 Review
▼
To date, the promises raised by the large bulk of preclinical data concerning the effects of long acting SS analogues and DRD2 agonists on hormone secretion and cell proliferation have been only in part fulfilled. Indeed, clinical practice demonstrated a great variability in the frequency and entity of favorable responses during chronic treatment of patients with pituitary tumors with these agents. In particular, 10 % of patients with prolactinoma and 30 % of patients with acromegaly are resistant to cabergoline and octreotide, respectively, while most NFPA and ACTH secreting adenomas are unresponsive to both drugs [9–12], with a major impact on mortality, morbidity, and quality of life. Moreover, the occurrence of resistance to DRD2 agonists has been associated with the shift of a prolactinoma to an invasive tumor or even a carcinoma [12]. Despite intensive research, the molecular events involved in pituitary tumors resistance to pharmacological treatment are still unclear. Concerning resistance to dopaminergic drugs, reduction in DRD2 expression has been reported in PRL-secreting tumors, particularly in DA-resistant with respect to DA-sensitive tumors [13–15], but the molecular determinants of this low expression have not been identified. No mutation of the DRD2 gene has been found in prolactinomas [16], the only finding being a correlation of some DRD2 polymorphisms with DA resistance [17]. More controversial is the association of SS resistance to the reduction of SSTR expression, since some tumors are resistant to therapy despite high SSTR2 expression [18–25]. Also in this case, the molecular events responsible for reduced SST expression are still largely unknown, mutations in their coding sequence and/ or loss of heterozygosity in loci where they are located being very rare events [26–29]. The hypothesis of post-receptor alterations involved in resistance to SS analogues is supported by the dissociation between the antisecretory and antiproliferative effects of SS analogues observed in some acromegalic patients [30]. Alterations in SSTRs signal transduction, involving AIP and ZAC1, have been investigated. Interestingly, acromegalic patients
Scaffold region
Rep. 24 (Dimerization)
Rep. 21 (integrin interaction)
Rod 2
Rep. 19–20 (SSTR2 interaction)
Hinge2
FLNA monomer
Rep. 19 (DRD2 interaction) Hinge1
Ser2152
Rod 1
Rep. 1 ABD
Fig. 1 Schematic representation of a FLNA dimer. The actin-binding domain (ABD) at the N-terminus is shown. The rod-1 domain (repeats 1–15), rod-2 domain (repeats 16–23), and repeat 24 are separated by 2 hinge regions. Regions involved in the interaction with partner proteins are shown. Black circles: repeats involved in DRD2/SST2 binding. Gray circles: scaffold region. The repeat 24 mediates FLNA homodimerization. The serine residue target of PKA-mediated phosphorylation is indicated.
with AIP germline mutations or tumors with low levels of AIP are typically resistant to SS analogues [31]. Growing evidence revealed that GPCR expression, localization, and signaling are regulated by interaction with different cytoskeleton proteins, including filamin A (FLNA) [32], suggesting another possible mechanism underlying drug resistance. This actin-binding protein is involved in the regulation of expression and signaling of DRD2 and SSTR2, with important consequences for pituitary tumor responsiveness to drugs targeting these receptors.
FLNA Role in Pituitary Tumor Responsiveness to DRD2 Agonists and Somatostatin Analogues
▼
FLNA structure and functions
Although originally it was believed that GPCRs mediate signal transduction only through the activation of coupled G proteins, it is now well established that these receptors directly interact through their intracellular loops with cytoplasmic and surface proteins involved in GPCRs stabilization, desensitization, internalization, and signal transduction. Among these, arrestins are well known as both GPCR signal terminators and signal transducers, and more recently also cytoskeleton proteins have been recognized to play a considerable role. Indeed, cytoskeleton not only plays a paramount role in cell morphology maintenance, cell migration and adhesion, cell division and organelles localization and movement within the cytosol but it also participates in extracellular signal transduction and regulates activity of several receptors. The 3 main structural components of the cytoskeleton are microtubules, intermediate filaments and microfilaments, resulting from the polymerization of different monomers, which undergo continual turnover and rearrangement and are specifically associated with a number of partner proteins that determine the structural and functional differences. In particular, microfilaments consist of actin filaments that are polymerized, depolymerized, and cross-linked into bundles and networks with the help of multiple families of cytoskeletal specific actin binding proteins. Filamins belong to the family of the actin cross-linking proteins, characterized by a conserved actin binding domain followed by a rod-like domain that allows to dimerize. Some of these proteins such as α-actinin form parallel actin bundles, whereas filamins form orthogonal networks. The filamin family consists of 3 homologous high-molecular weight proteins, FLNA, FLNB, and FLNC, encoded by genes located on different chromosomes (chr.X, chr.3, and chr.7, respectively). The 3 isoforms of filamin in mammals show a strong homology in the entire sequence and possess highly conserved genomic organization [33]. Human FLNA, mapping to Xq28, is the first actin filament cross-linking protein identified in nonmuscle cells [34] and is the most abundant filamin isoform in adults. The expression of FLNC is restricted to skeletal and cardiac muscle, whereas FLNA and B are ubiquitously expressed. Several studies suggest that filamins are essential for normal human development, and mutations in the respective genes cause a wide range of developmental malformations of the brain, bone, and heart with moderate to lethal consequences [35]. In particular, FLNA mutations cause periventricular nodular heterotropia, a brain malformation due to abnormal neuronal migration, in which a subset of neurons fails to migrate into the developing cerebral cortex [36] or a wide spectrum of con-
Peverelli E et al. FLNA Phosphorylation in Pituitary Tumors … Horm Metab Res 2014; 46: 845–853
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Mechanisms of Resistance of Pituitary Adenomas to Pharmacological Treatment
genital malformations, such as otopalotodigital syndrome, frontometaphyseal dysplasia and Melnick Needles syndrome [37]. FLNA is composed of 2 subunits of 280 kDa each that self-assemble. A schematic representation of a FLNA dimer structure is shown in ● ▶ Fig. 1. Each monomer possesses an actin-binding domain (ABD) at the N-terminus, which consists of 2 calponin homology domains, followed by 24 immunoglobulin (Ig)-like repeats of about 96 amino acid residues. Two calpain-sensitive hinge regions separate the 24 repeats in a rod-1 domain (repeats 1–15), rod-2 domain (repeats 16–23), and repeat 24. A secondary ABD of lower affinity is located in the rod-1 domain, whereas rod-2 is involved in the interaction with partner proteins. The repeat 24 is the self-association domain that mediates FLNA homodimerization, allowing the formation of V-shaped flexible structures, which results in the perpendicular cross-linking of actin filaments. Besides stabilizing actin filaments in a three-dimensional structure, FLNA anchors several transmembrane proteins, such as integrins, ion channels, and several GPCRs with which it directly interacts, to the actin cytoskeleton [32]. In addition, FLNA functions as important signaling scaffold by binding a variety of proteins, including receptors, ion channels, intracellular signaling molecules, kinases, and transcription factors (reviewed in [38]). These interactions are regulated by phosphorylation events, proteolysis, mechanical forces, competition, and multimerization of partners.
Role of FLNA in the regulation of DRD2: relevance for PRL-secreting pituitary tumors
Li and colleagues first demonstrated FLNA interaction with the third cytoplasmic loop of DRD2 [39]. The specificity of this interaction was identified in a yeast 2-hybrid screen and confirmed by protein binding. They also showed that this association enhances coupling efficiency of DRD2 to adenylate cyclase and plays a role in cell surface receptor clustering by using as cell model 2 human melanoma cell lines, M2, that does not express FLNA and A7, stably transfected with FLNA. Almost simultaneously, another study using the third intracellular loop of the DRD2 as bait in a yeast 2-hybrid approach to screen a human brain cDNA library, confirmed a specific DRD2/FLNA association, better defining the specific regions of FLNA (repeat 19) and DRD2 (amino acids 211–241 in the N-terminal region of the third intracellular loop) involved [40]. FLNA and DRD2 co-localized in cell cultures of rat striatum. The authors found that DRD2 was predominantly intracellular in M2 cells, whereas it localized at the plasma membrane in A7 cells, suggesting that FLNA is required for the cell surface localization of DRD2. Further confirmation to this hypothesis came from the observation that the overexpression of a dominant negative truncated form of FLNA (repeats 18–19, containing the DRD2, but not the actin, binding domain) caused a marked reduction in both the number and half-life of cell surface DRD2 receptors [41]. The finding that human prolactinomas, and in particular those removed from patients in whom DA analogues treatment did not achieve PRL normalization and tumor shrinkage, showed low levels of both FLNA and DRD2 [42], is the first observation suggesting a role for FLNA in DRD2 regulation in this type of tumor. Alterations of FLNA levels in primary cultured prolactinoma cells by gene silencing or overexpression resulted in corresponding modifications of DRD2 levels, demonstrating that ▶ Fig. 2a). Moreover, these 2 events are causally related [42] ( ● DRD2 signal transduction, including reduction of PRL release and ERK1/2 phosphorylation, was impaired after FLNA silencing,
whereas DA-resistant prolactinomas lacking FLNA recovered PRL responsiveness when transfected with FLNA expression vector [42]. The authors further investigated the mechanisms by which FLNA regulates DRD2 in a cell model of prolactinoma endogenously expressing functional DRD2 and FLNA, that is, MMQ cells. Data show that FLNA is not only required for DRD2 targeting to the cell membrane, but also protects DRD2 against lysosomal degradation, suggesting a role for FLNA in the control of DRD2 fate towards recycling processes or lysosomal degradation ▶ Fig. 2b, c). Since FLNA directly interacts with beta [42] ( ● arrestins [43] that are involved in DRD2 trafficking [44], it is possible to hypothesize the formation of a complex receptor-FLNAarrestin involved in the regulation of DRD2 stability. This protective effect of FLNA from instability has been demonstrated for other receptors, such as calcium-sensing receptor, calcitonin receptor, cystic fibrosis transmembrane conductance regulator, and the high-affinity IgG receptor FcgammaRI [45–48]. These data strongly demonstrate a structural role of FLNA in anchoring DRD2 to actin cytoskeleton and regulating receptor localization and stability, but also suggest an additional functional role of FLNA as scaffold for signaling molecules involved in DRD2 signal transduction. Indeed, since the pituitary has a substantial DRD2 reserve for PRL inhibition, and PRL response reaches the plateaux at about 40 % receptor occupancy in rat pituitary cells [49], the 60 % reduction of DRD2 levels measured in prolactinomas and MMQ cells after FLNA knockdown might not entirely account for the loss of D2R effects on PRL release and cell proliferation. Overall, FLNA is crucial for DRD2 expression and signaling in lactotrophs and the loss of FLNA expression may be one of the mechanisms involved in loss of DA responsiveness in human prolactinomas. Up to now, the molecular events underlying FLNA reduced expression are unknown, the only observation being the absence of alterations in the CpG island with the highest probability to have regulatory functions, excluding epigenetic silencing [42].
Role of FLNA in the regulation of SSTR2: relevance for GH-secreting pituitary tumors
Recently, it has been shown by surface plasmon resonance (SPR) that FLNA directly interacts with SSTR2 first intracellular loop [50]. Searching for the FLNA region responsible of this binding, the authors found that only FLNA repeats 19–20 bound SSTR2 in a dose-dependent manner and with a high affinity. By co-immunoprecipitation experiments, they also showed that FLNA/SSTR2 interaction occurs in cellulo in different cell lines, such as neuroendocrine pancreatic BON, keratinocyte HaCaT cells, or CHO transfected with SSTR2. FLNA is required for SSTR2-mediated inhibition of cell survival, and the molecular mechanism involves a competition of FLNA with p85, the regulatory subunit of PI3K, for direct binding to SSTR2. Ligand-stimulated FLNA binding results in the disruption of the SSTR2-p85 complex and the subsequent inhibition of PI3K [50]. The authors also found enhanced ligand-induced SSTR2 internalization rate in FLNA-deficient M2 cells with respect to A7 cells, suggesting FLNA requirement for SSTR2 stabilization at the cell membrane. Interestingly, SSTR2 was correctly targeted to the plasma membrane in M2 cells in the absence of agonist, contrary to DRD2, that remained mainly cytoplasmatic in M2 cells [40], implying different mechanisms involved.
Peverelli E et al. FLNA Phosphorylation in Pituitary Tumors … Horm Metab Res 2014; 46: 845–853
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Review 847
Fig. 2 DRD2 expression and localization in prolactinomas depends on FLNA levels. a Representative immunoblots of FLNA and DRD2 in DA-sensitive or resistant prolactinoma cells transiently transfected (72 h) with negative control or FLNA siRNA or pREP4-FLNA. FLNA silenced cells showed a strong decrease in FLNA protein expression that was associated with a reduction of DRD2 expression, whereas FLNA overexpression in DA-resistant tumors was associated with a significant increase of DRD2 expression. The equal amount of protein was confirmed by stripping and reprobing with an anti-GAPDH antibody. b Representative immunoblot of DRD2 in MMQ cells transiently transfected with negative control or FLNA siRNA. FLNA siRNA treated cells showed a strong reduction of DRD2. The lysosome inhibitor chloroquine induced an increase in DRD2 expression in FLNA silenced cells with respect to control cells. The equal amount of protein was confirmed by stripping and reprobing with an anti-GAPDH
Accordingly, recently published data from our lab showed that FLNA expression in human GH-secreting tumors did not correlate with SSTR2 levels, and FLNA silencing in human somatotroph tumoral cells did not affect SSTR2 expression and membrane localization [51]. However, overexpression of a FLNA dominant negative truncated mutant that specifically prevents SSTR2-FLNA binding reduced SSTR2 expression after prolonged agonist exposure in rat GHsecreting cell line GH3, suggesting that FLNA is involved in SSTR2 stabilization. Moreover, our silencing experiments showing that FLNA is required for SSTR2-induced reduction of cyclin D1 and caspase3/7 activation in tumoral somatotrophs [51] suggest a crucial role for FLNA in mediating antiproliferative and proapoptotic signaling of SSTR2, consistent with the view that FLNA participates to signal transduction as scaffold protein for signaling molecules. All these data support a new role for FLNA in the responsiveness of patients with GH-secreting pituitary tumors to pharmacological treatment with SS analogues. Low levels of FLNA, causing loss of coupling of SSTR2 with downstream signal transduction molecules, might explain the resistance to SS analogues in SSTR2 expressing tumors.
antibody. c Left panel. Representative confocal microscopy images of MMQ cells transfected with negative control siRNA or FLNA siRNA cells for 72 h stained for DRD2. In control cells DRD2 was mainly localized at the plasma membrane, with frequent clustering, whereas in FLNA siRNA cells, DRD2 was redistributed to cytoplasmic vescicles. Right panel. Biochemical analysis of membrane expression of DRD2. Seventy-two hours after siRNA transfection, biotinylated cell surface proteins and total cellular proteins were immunoprecipitated by DRD2 antibody and cell surface DRD2 detected by an antibiotin antibody. Biotinylation assay showed reduced DRD2 expression at the cell membrane in cells transfected with FLNA siRNA for 72 h. The graph shows the quantification of cell surface expression of DRD2. *p
Authors
E. Peverelli1, E. Giardino1, E. Vitali2, D. Treppiedi1, A. G. Lania3, G. Mantovani1
Affiliations
1
Key words ▶ cytoskeleton ● ▶ pituitary tumors ● ▶ GPCR ●
received 14.03.2014 accepted 18.06.2014 Bibliography DOI http://dx.doi.org/ 10.1055/s-0034-1384520 Published online: July 28, 2014 Horm Metab Res 2014; 46: 845–853 © Georg Thieme Verlag KG Stuttgart · New York ISSN 0018-5043 Correspondence M. Giovanna, MD, PhD Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico Unità Operativa di Endocrinologia e Diabetologia University of Milan Padiglione Granelli via F. Sforza 35 20122 Milano Italy Tel.: + 39/02/503 20613 Fax: + 39/02/503 20605 [email protected]
Endocrine Unit, Department of Clinical Sciences and Community Health, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, University of Milan, Milan, Italy 2 Laboratory of Cellular and Molecular Endocrinology, Humanitas Research Center, Rozzano, Italy 3 Endocrine Unit, IRCCS Humanitas Clinical Institute, Rozzano, University of Milan, Milan, Italy
Abstract
▼
Molecular mechanisms underlying resistance of pituitary tumors to somatostatin (SS) and dopamine (DA) analogues treatment are not completely understood. Resistance has been associated with defective expression of functional somatostatin and dopamine receptors SSTR2, SSTR5, and DRD2, respectively. Recently, a role of cytoskeleton protein filamin A (FLNA) in DRD2 and SSTR 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. No molecular events underlying the reduction of FLNA levels in resistant tumors have been so far identified.
Introduction
▼
Pituitary tumors are rare neoplasia that derive from excessive proliferation of each subtype of pituitary cells and present with specific endocrine syndromes or mass symptoms or both. The main G protein coupled receptors (GPCRs) target of pharmacological treatment of pituitary tumors are the dopamine (DA) receptor DRD2 and somatostatin (SS) receptors SSTR2 and 5. In particular, DRD2 agonists are used in the treatment of prolactin (PRL)- and, to a lesser extent, ACTH-secreting and nonfunctioning pituitary tumors (NFPA), whereas long acting SS analogues, such as octreotide and lanreotide, are currently used in the treatment of pituitary tumors, particularly GHand TSH-secreting tumors [1, 2]. Prolactinomas are the most frequent pituitary tumors and DA agonists normalize PRL levels and reduce tumor size in the majority of patients by binding DRD2, the DA receptor subtype expressed in pituitary lactotrophs (reviewed in [1]). DRD2
FLNA can be phosphorylated by PKA on Ser2152, with increased FLNA resistance to cleavage by calpain and conformational changes affecting FLNA regions involved in SSTR2 and DRD2 binding and signal transduction. In this respect, the effect of cAMP/PKA pathway in the regulation of FLNA stability and/or function by modulating its phosphorylation status could assume particular importance in pituitary, where cAMP cascade plays a crucial role in pituitary cell functions and tumorigenesis. This review will discuss the role of FLNA in the regulation of the main GPCRs target of pharmacological treatment of pituitary tumors, that is, SSTR2 and DRD2, focusing on the effects of cAMP/PKA-mediated FLNA phosphorylation on FLNA biological functions.
couples to Gi/Go proteins to inhibit adenylyl cyclase activity and reduce intracellular cAMP levels. Somatostatin analogues are currently used to successfully treat acromegalic patients since they inhibit cell proliferation and hormone secretion by binding with high affinity SSTR2 and, to a lesser extent, SSTR5, which are both expressed at high density in most GH-secreting pituitary tumors [3, 4]. By coupling with multiple PTXsensitive G proteins, SSTs inhibit adenylyl cyclase activity and some subtypes reduce calcium entry by modulating L-type Ca2 + and K + channels, all these events being involved in the reduction of hormone secretion. Both SSTR2 and SSTR5 mediate the antiproliferative effects of SS, by tyrosine phosphatase activation and ERK1/2 phosphorylation inhibition, respectively, whereas only SSTR2 and SST3 subtypes mediate apoptotic effects [5–8].
Peverelli E et al. FLNA Phosphorylation in Pituitary Tumors … Horm Metab Res 2014; 46: 845–853
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Filamin A in Somatostatin and Dopamine Receptor Regulation in Pituitary and the Role of cAMP/PKA Dependent Phosphorylation
846 Review
▼
To date, the promises raised by the large bulk of preclinical data concerning the effects of long acting SS analogues and DRD2 agonists on hormone secretion and cell proliferation have been only in part fulfilled. Indeed, clinical practice demonstrated a great variability in the frequency and entity of favorable responses during chronic treatment of patients with pituitary tumors with these agents. In particular, 10 % of patients with prolactinoma and 30 % of patients with acromegaly are resistant to cabergoline and octreotide, respectively, while most NFPA and ACTH secreting adenomas are unresponsive to both drugs [9–12], with a major impact on mortality, morbidity, and quality of life. Moreover, the occurrence of resistance to DRD2 agonists has been associated with the shift of a prolactinoma to an invasive tumor or even a carcinoma [12]. Despite intensive research, the molecular events involved in pituitary tumors resistance to pharmacological treatment are still unclear. Concerning resistance to dopaminergic drugs, reduction in DRD2 expression has been reported in PRL-secreting tumors, particularly in DA-resistant with respect to DA-sensitive tumors [13–15], but the molecular determinants of this low expression have not been identified. No mutation of the DRD2 gene has been found in prolactinomas [16], the only finding being a correlation of some DRD2 polymorphisms with DA resistance [17]. More controversial is the association of SS resistance to the reduction of SSTR expression, since some tumors are resistant to therapy despite high SSTR2 expression [18–25]. Also in this case, the molecular events responsible for reduced SST expression are still largely unknown, mutations in their coding sequence and/ or loss of heterozygosity in loci where they are located being very rare events [26–29]. The hypothesis of post-receptor alterations involved in resistance to SS analogues is supported by the dissociation between the antisecretory and antiproliferative effects of SS analogues observed in some acromegalic patients [30]. Alterations in SSTRs signal transduction, involving AIP and ZAC1, have been investigated. Interestingly, acromegalic patients
Scaffold region
Rep. 24 (Dimerization)
Rep. 21 (integrin interaction)
Rod 2
Rep. 19–20 (SSTR2 interaction)
Hinge2
FLNA monomer
Rep. 19 (DRD2 interaction) Hinge1
Ser2152
Rod 1
Rep. 1 ABD
Fig. 1 Schematic representation of a FLNA dimer. The actin-binding domain (ABD) at the N-terminus is shown. The rod-1 domain (repeats 1–15), rod-2 domain (repeats 16–23), and repeat 24 are separated by 2 hinge regions. Regions involved in the interaction with partner proteins are shown. Black circles: repeats involved in DRD2/SST2 binding. Gray circles: scaffold region. The repeat 24 mediates FLNA homodimerization. The serine residue target of PKA-mediated phosphorylation is indicated.
with AIP germline mutations or tumors with low levels of AIP are typically resistant to SS analogues [31]. Growing evidence revealed that GPCR expression, localization, and signaling are regulated by interaction with different cytoskeleton proteins, including filamin A (FLNA) [32], suggesting another possible mechanism underlying drug resistance. This actin-binding protein is involved in the regulation of expression and signaling of DRD2 and SSTR2, with important consequences for pituitary tumor responsiveness to drugs targeting these receptors.
FLNA Role in Pituitary Tumor Responsiveness to DRD2 Agonists and Somatostatin Analogues
▼
FLNA structure and functions
Although originally it was believed that GPCRs mediate signal transduction only through the activation of coupled G proteins, it is now well established that these receptors directly interact through their intracellular loops with cytoplasmic and surface proteins involved in GPCRs stabilization, desensitization, internalization, and signal transduction. Among these, arrestins are well known as both GPCR signal terminators and signal transducers, and more recently also cytoskeleton proteins have been recognized to play a considerable role. Indeed, cytoskeleton not only plays a paramount role in cell morphology maintenance, cell migration and adhesion, cell division and organelles localization and movement within the cytosol but it also participates in extracellular signal transduction and regulates activity of several receptors. The 3 main structural components of the cytoskeleton are microtubules, intermediate filaments and microfilaments, resulting from the polymerization of different monomers, which undergo continual turnover and rearrangement and are specifically associated with a number of partner proteins that determine the structural and functional differences. In particular, microfilaments consist of actin filaments that are polymerized, depolymerized, and cross-linked into bundles and networks with the help of multiple families of cytoskeletal specific actin binding proteins. Filamins belong to the family of the actin cross-linking proteins, characterized by a conserved actin binding domain followed by a rod-like domain that allows to dimerize. Some of these proteins such as α-actinin form parallel actin bundles, whereas filamins form orthogonal networks. The filamin family consists of 3 homologous high-molecular weight proteins, FLNA, FLNB, and FLNC, encoded by genes located on different chromosomes (chr.X, chr.3, and chr.7, respectively). The 3 isoforms of filamin in mammals show a strong homology in the entire sequence and possess highly conserved genomic organization [33]. Human FLNA, mapping to Xq28, is the first actin filament cross-linking protein identified in nonmuscle cells [34] and is the most abundant filamin isoform in adults. The expression of FLNC is restricted to skeletal and cardiac muscle, whereas FLNA and B are ubiquitously expressed. Several studies suggest that filamins are essential for normal human development, and mutations in the respective genes cause a wide range of developmental malformations of the brain, bone, and heart with moderate to lethal consequences [35]. In particular, FLNA mutations cause periventricular nodular heterotropia, a brain malformation due to abnormal neuronal migration, in which a subset of neurons fails to migrate into the developing cerebral cortex [36] or a wide spectrum of con-
Peverelli E et al. FLNA Phosphorylation in Pituitary Tumors … Horm Metab Res 2014; 46: 845–853
This document was downloaded for personal use only. Unauthorized distribution is strictly prohibited.
Mechanisms of Resistance of Pituitary Adenomas to Pharmacological Treatment
genital malformations, such as otopalotodigital syndrome, frontometaphyseal dysplasia and Melnick Needles syndrome [37]. FLNA is composed of 2 subunits of 280 kDa each that self-assemble. A schematic representation of a FLNA dimer structure is shown in ● ▶ Fig. 1. Each monomer possesses an actin-binding domain (ABD) at the N-terminus, which consists of 2 calponin homology domains, followed by 24 immunoglobulin (Ig)-like repeats of about 96 amino acid residues. Two calpain-sensitive hinge regions separate the 24 repeats in a rod-1 domain (repeats 1–15), rod-2 domain (repeats 16–23), and repeat 24. A secondary ABD of lower affinity is located in the rod-1 domain, whereas rod-2 is involved in the interaction with partner proteins. The repeat 24 is the self-association domain that mediates FLNA homodimerization, allowing the formation of V-shaped flexible structures, which results in the perpendicular cross-linking of actin filaments. Besides stabilizing actin filaments in a three-dimensional structure, FLNA anchors several transmembrane proteins, such as integrins, ion channels, and several GPCRs with which it directly interacts, to the actin cytoskeleton [32]. In addition, FLNA functions as important signaling scaffold by binding a variety of proteins, including receptors, ion channels, intracellular signaling molecules, kinases, and transcription factors (reviewed in [38]). These interactions are regulated by phosphorylation events, proteolysis, mechanical forces, competition, and multimerization of partners.
Role of FLNA in the regulation of DRD2: relevance for PRL-secreting pituitary tumors
Li and colleagues first demonstrated FLNA interaction with the third cytoplasmic loop of DRD2 [39]. The specificity of this interaction was identified in a yeast 2-hybrid screen and confirmed by protein binding. They also showed that this association enhances coupling efficiency of DRD2 to adenylate cyclase and plays a role in cell surface receptor clustering by using as cell model 2 human melanoma cell lines, M2, that does not express FLNA and A7, stably transfected with FLNA. Almost simultaneously, another study using the third intracellular loop of the DRD2 as bait in a yeast 2-hybrid approach to screen a human brain cDNA library, confirmed a specific DRD2/FLNA association, better defining the specific regions of FLNA (repeat 19) and DRD2 (amino acids 211–241 in the N-terminal region of the third intracellular loop) involved [40]. FLNA and DRD2 co-localized in cell cultures of rat striatum. The authors found that DRD2 was predominantly intracellular in M2 cells, whereas it localized at the plasma membrane in A7 cells, suggesting that FLNA is required for the cell surface localization of DRD2. Further confirmation to this hypothesis came from the observation that the overexpression of a dominant negative truncated form of FLNA (repeats 18–19, containing the DRD2, but not the actin, binding domain) caused a marked reduction in both the number and half-life of cell surface DRD2 receptors [41]. The finding that human prolactinomas, and in particular those removed from patients in whom DA analogues treatment did not achieve PRL normalization and tumor shrinkage, showed low levels of both FLNA and DRD2 [42], is the first observation suggesting a role for FLNA in DRD2 regulation in this type of tumor. Alterations of FLNA levels in primary cultured prolactinoma cells by gene silencing or overexpression resulted in corresponding modifications of DRD2 levels, demonstrating that ▶ Fig. 2a). Moreover, these 2 events are causally related [42] ( ● DRD2 signal transduction, including reduction of PRL release and ERK1/2 phosphorylation, was impaired after FLNA silencing,
whereas DA-resistant prolactinomas lacking FLNA recovered PRL responsiveness when transfected with FLNA expression vector [42]. The authors further investigated the mechanisms by which FLNA regulates DRD2 in a cell model of prolactinoma endogenously expressing functional DRD2 and FLNA, that is, MMQ cells. Data show that FLNA is not only required for DRD2 targeting to the cell membrane, but also protects DRD2 against lysosomal degradation, suggesting a role for FLNA in the control of DRD2 fate towards recycling processes or lysosomal degradation ▶ Fig. 2b, c). Since FLNA directly interacts with beta [42] ( ● arrestins [43] that are involved in DRD2 trafficking [44], it is possible to hypothesize the formation of a complex receptor-FLNAarrestin involved in the regulation of DRD2 stability. This protective effect of FLNA from instability has been demonstrated for other receptors, such as calcium-sensing receptor, calcitonin receptor, cystic fibrosis transmembrane conductance regulator, and the high-affinity IgG receptor FcgammaRI [45–48]. These data strongly demonstrate a structural role of FLNA in anchoring DRD2 to actin cytoskeleton and regulating receptor localization and stability, but also suggest an additional functional role of FLNA as scaffold for signaling molecules involved in DRD2 signal transduction. Indeed, since the pituitary has a substantial DRD2 reserve for PRL inhibition, and PRL response reaches the plateaux at about 40 % receptor occupancy in rat pituitary cells [49], the 60 % reduction of DRD2 levels measured in prolactinomas and MMQ cells after FLNA knockdown might not entirely account for the loss of D2R effects on PRL release and cell proliferation. Overall, FLNA is crucial for DRD2 expression and signaling in lactotrophs and the loss of FLNA expression may be one of the mechanisms involved in loss of DA responsiveness in human prolactinomas. Up to now, the molecular events underlying FLNA reduced expression are unknown, the only observation being the absence of alterations in the CpG island with the highest probability to have regulatory functions, excluding epigenetic silencing [42].
Role of FLNA in the regulation of SSTR2: relevance for GH-secreting pituitary tumors
Recently, it has been shown by surface plasmon resonance (SPR) that FLNA directly interacts with SSTR2 first intracellular loop [50]. Searching for the FLNA region responsible of this binding, the authors found that only FLNA repeats 19–20 bound SSTR2 in a dose-dependent manner and with a high affinity. By co-immunoprecipitation experiments, they also showed that FLNA/SSTR2 interaction occurs in cellulo in different cell lines, such as neuroendocrine pancreatic BON, keratinocyte HaCaT cells, or CHO transfected with SSTR2. FLNA is required for SSTR2-mediated inhibition of cell survival, and the molecular mechanism involves a competition of FLNA with p85, the regulatory subunit of PI3K, for direct binding to SSTR2. Ligand-stimulated FLNA binding results in the disruption of the SSTR2-p85 complex and the subsequent inhibition of PI3K [50]. The authors also found enhanced ligand-induced SSTR2 internalization rate in FLNA-deficient M2 cells with respect to A7 cells, suggesting FLNA requirement for SSTR2 stabilization at the cell membrane. Interestingly, SSTR2 was correctly targeted to the plasma membrane in M2 cells in the absence of agonist, contrary to DRD2, that remained mainly cytoplasmatic in M2 cells [40], implying different mechanisms involved.
Peverelli E et al. FLNA Phosphorylation in Pituitary Tumors … Horm Metab Res 2014; 46: 845–853
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Fig. 2 DRD2 expression and localization in prolactinomas depends on FLNA levels. a Representative immunoblots of FLNA and DRD2 in DA-sensitive or resistant prolactinoma cells transiently transfected (72 h) with negative control or FLNA siRNA or pREP4-FLNA. FLNA silenced cells showed a strong decrease in FLNA protein expression that was associated with a reduction of DRD2 expression, whereas FLNA overexpression in DA-resistant tumors was associated with a significant increase of DRD2 expression. The equal amount of protein was confirmed by stripping and reprobing with an anti-GAPDH antibody. b Representative immunoblot of DRD2 in MMQ cells transiently transfected with negative control or FLNA siRNA. FLNA siRNA treated cells showed a strong reduction of DRD2. The lysosome inhibitor chloroquine induced an increase in DRD2 expression in FLNA silenced cells with respect to control cells. The equal amount of protein was confirmed by stripping and reprobing with an anti-GAPDH
Accordingly, recently published data from our lab showed that FLNA expression in human GH-secreting tumors did not correlate with SSTR2 levels, and FLNA silencing in human somatotroph tumoral cells did not affect SSTR2 expression and membrane localization [51]. However, overexpression of a FLNA dominant negative truncated mutant that specifically prevents SSTR2-FLNA binding reduced SSTR2 expression after prolonged agonist exposure in rat GHsecreting cell line GH3, suggesting that FLNA is involved in SSTR2 stabilization. Moreover, our silencing experiments showing that FLNA is required for SSTR2-induced reduction of cyclin D1 and caspase3/7 activation in tumoral somatotrophs [51] suggest a crucial role for FLNA in mediating antiproliferative and proapoptotic signaling of SSTR2, consistent with the view that FLNA participates to signal transduction as scaffold protein for signaling molecules. All these data support a new role for FLNA in the responsiveness of patients with GH-secreting pituitary tumors to pharmacological treatment with SS analogues. Low levels of FLNA, causing loss of coupling of SSTR2 with downstream signal transduction molecules, might explain the resistance to SS analogues in SSTR2 expressing tumors.
antibody. c Left panel. Representative confocal microscopy images of MMQ cells transfected with negative control siRNA or FLNA siRNA cells for 72 h stained for DRD2. In control cells DRD2 was mainly localized at the plasma membrane, with frequent clustering, whereas in FLNA siRNA cells, DRD2 was redistributed to cytoplasmic vescicles. Right panel. Biochemical analysis of membrane expression of DRD2. Seventy-two hours after siRNA transfection, biotinylated cell surface proteins and total cellular proteins were immunoprecipitated by DRD2 antibody and cell surface DRD2 detected by an antibiotin antibody. Biotinylation assay showed reduced DRD2 expression at the cell membrane in cells transfected with FLNA siRNA for 72 h. The graph shows the quantification of cell surface expression of DRD2. *p
