A Novel Mechanism Regulating Dopamine Receptor Type 2 Signal Transduction in Pituitary Tumoral Cells: The Role of cAMP/PKA-Induced Filamin A Phosphorylation

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.


INTRODUCTION
Pituitary neuroendocrine tumors (PitNETs) are endocrine tumors that represent the 10-25% of all intracranial neoplasms classified on the base of their secretory activity. PitNETs may give rise to neurological manifestations, visual defects depending on mass spread effect, or endocrine syndromes related to hormonal hypersecretion. Although generally benign, infiltration of tumor tissue into near structures together with medical therapy resistance, are the main obstacles to their treatment (1,2).
Dopamine receptor type 2 (DRD2) represents the principle target for pharmacological therapy with dopamine agonists (DAs) due to its ability to induce tumor mass shrinkage and to regulate excessive hormone secretion in prolactin (PRL)secreting PitNETs (3).
Despite some clinical benefits have been shown (4,8,21), a strong support for the use of DAs in ACTH-secreting PitNETs is lacking.
The molecular mechanisms underlying pharmacological resistance have not been fully understood and no biomarkers predictable of response to medications are yet available.
In the last years, a role for cytoskeleton and its associated proteins in regulating receptors expression and signaling has been highlighted (22).
FLNA is an homodimer of two subunits of 280 kDa each, containing an actin-binding domain at N-terminus, followed by 24 Ig-like repeats. The repeat 24 at the C-terminal acts as dimerization site and confers the protein its own characteristic 'V' shape (26). The main mechanism that regulates FLNA activity is the phosphorylation by protein kinase A (PKA) on Ser2152 residue, at the repeat 20 (27). This post translational modification affects the protein functions as it is involved in cell migration (28,29), integrin binding (30), intracellular localization (29) and calpain cleavage (31).
In particular, the Ig19-20 region, which is involved in the formation of an autoinhibitory structure when FLNA is dephosphorylated (32), is implicated in the direct interaction with DRD2 (33). Therefore, it is eligible to suppose that PKAinduced FLNA phosphorylation (P-FLNA) might relate to its ability to bind DRD2 and its effectors, thus modulating DRD2 signal transduction.
In GH-secreting PitNETs, our group demonstrated that P-FLNA regulates SSTR2 signal transduction. We showed that phosphorylation switches FLNA function from a scaffold for signaling proteins, allowing SSTR2 signal transduction, to a signal termination protein that hampers all SSTR2 antitumoral effects, including inhibition of cell proliferation, migration and increase of cell apoptosis (25).
In the light of these premises, the aims of the present study were to evaluate in PRL-and ACTH-secreting pituitary tumor cell lines the modulation of FLNA phosphorylation by cAMP pathway activation and DRD2 agonist and to study the effects of P-FLNA on DRD2 intracellular signal transduction. Total proteins extracted from cultured cells were quantified by BCA assay, separated on SDS/polyacrylamide gels and transferred to a nitrocellulose filter. Phospho-Filamin A (Ser2152), Filamin A, phospho-ERK1/2, ERK1/2, c-Myc and cyclin D3 antibodies (Cell Signaling Technology, Danvers, MA, USA) were used at 1:1,000. P27 and D2DR antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) were diluted at 1:200. Phospho-AKT antibody was diluted at 1:2,000 and AKT at 1:1,000 (Immunological Science, Rome, IT). Primary antibodies were incubated overnight at 4°C, secondary antibodies anti-rabbit or anti-mouse (Cell Signaling Technology, Danvers, MA, USA) were used at 1:2,000 at room temperature for 1 h. Anti-GAPDH antibody (Ambion, Thermo Fisher Scientific, Waltham, MA, USA) was used at 1:4,000 for 1 h at room temperature. Chemiluminescence was detected trough a ChemiDOC-IT Imaging System (UVP, Upland, CA, USA) and then analysed with NIH ImageJ software.
These vectors were transiently transfected in MMQ and AtT-20 cells for 72 h at 37°C using Lipofectamine 2000 reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), according to the instruction of the manufacturer. Transfection efficiency was monitored by Western blot analysis for each experiment by using an anti-c-Myc antibody (Cell Signaling Technology, Danvers, MA, USA). Empty vectors were used in each experiment as negative controls.

Cell Proliferation Assay
Cell proliferation was assessed by colorimetric measurement of 5bromo-2-deoxyuridine (BrdU) incorporation during DNA synthesis in proliferating cells according to the instruction of the manufacturer (GE Healthcare, Life Science, Buckinghamshire, UK), as previously described (34). MMQ and AtT-20 cells were seeded in starved medium in 96-well polylysine-coated plate at a density of 2 × 10 4 cells/well. 24 h later, cells were transfected with plasmids for 72 h at 37°C, then incubated with or without

Hormone Levels Detection
To detect prolactin (PRL) and adrenocorticotrophic (ACTH) hormone levels specific Elisa immunoassay kits (Fine Test, Wuhan Fine Biotech Co., Ltd, Wuhan, CN) were used.
MMQ and AtT-20 cells were seeded in six-well plate, at a density of 3,3 × 10 5 cells/well. After 24 h cells were transfected with wildtype, S2152A and S2152D FLNA plasmids for 72 h at 37°C. Then cells were counted and re-seeded in a 24-well plate at a density of 12,5 × 10 4 cells/well, in 300 ml of complete medium for AtT-20, while MMQ were re-seeded in a six-well plate at a density of 3,6 × 10 5 cells/well, in 1 ml of complete medium. Cells were than incubated with or without BIM53097 100 nM for 4 h (AtT-20) or 24 h (MMQ), based on preliminary time course experiments. After treatment, culture media were collected to perform the assay, according to the manufacturer's instructions. Absorbance was read at 450 nm in a Victor2 multilabel plate reader (Perkin Elmer, Whaltam, MA, USA). Data were plotted and analysed with the specific Curve Expert 1.4 program. Hormone detection were done in triplicate and experiments were replicated three times for each cell line. Hormone levels were normalized on the protein content, measured by BCA assay. Western blot analysis was carried out before each experiment to test transfection efficiency

Statistical Analysis
The results are expressed as the mean ± S.D. A paired two-tailed Student's t-test was used to detect the significance between two series of data. p < 0.05 was accepted as statistically significant.
No significant differences were found in basal proliferation between empty vector, wild-type and FLNA mutants transfected cells (data not shown).
Conversely, DRD2 agonist incubation did not induce a reduction of AKT phosphorylation levels in AtT-20 cells (data not shown).

DISCUSSION
To date, the molecular mechanisms underlying drug resistance of PitNETs have not been fully understood and no biomarkers predictable of response to pharmacological treatment are yet available. In the recent past a relevant role for cytoskeleton in regulating receptor dynamics was highlighted. The actin binding protein FLNA emerged as important player in the regulation of the complex intracellular processes that dictate pituitary tumors drug responsiveness and invasiveness. It participates in actin filaments crosslinking and in mediating somatostatin and dopamine receptors intracellular effects.
In particular, our group previously demonstrated FLNA essential tasks in mediating SSTR2 and DRD2 expression, intracellular localization and signaling in GH-and PRLsecreting PitNETs, respectively, playing a role in responsiveness to somatostatin analogs and dopaminergic drugs (18,25). Moreover, a latest study positively correlated FLNA and DRD2 expression in somatotropinomas (35), in line with our previous report in MMQ cell line (18). On the other hand, DRD2 expression was not associated with FLNA expression nor to the response to cabergoline treatment in corticotrophinomas and FLNA expression, in turn, did not correlate with dopamine agonist effect (36). However, the evaluation of FLNA protein expression does not provide information about its activity. This last is regulated by several mechanisms, including phosphorylation, mechanical force, intramolecular inhibition, competition with other molecules, alternative splicing and proteolysis (26). FLNA is substrate of phosphorylation of different kinases, among which PKA, that phosphorylates it on the residue S2152, located in Ig20, with important effects on its conformation and activity (25).
DRD2 is the main target for pharmacological therapy with DAs in prolactinomas and different studies indicated it as a potential target also for other DRD2-expressing tumors, such as ACTH-secreting PitNETs, tough there is still a lack in clinical evidences. For these reasons in this study we investigated FLNA phosphorylation (P-FLNA) effects on DRD2 signal transduction in PRL-and ACTH-secreting PitNETs. Our experiments were conducted in two tumoral pituitary cell lines both endogenously expressing DRD2: rat lactotrophs, MMQ, and murine adrenocorticotrophs, AtT-20.
First of all, we showed that FLNA phosphorylation status is regulated by cAMP/PKA. In agreement with previous data obtained in vitro on pituitary tumoral somatotrophs (25) we showed that cAMP/PKA pathway activation induced FLNA phosphorylation at Ser2152 also in pituitary tumoral lactotrophs and adrenocorticotrophs. Indeed, forskolin increased P-FLNA/FLNA ratio and the DRD2 selective agonist, BIM53097, significantly reduced P-FLNA in both cell lines.
To better understand the effects of Ser2152 FLNA phosphorylation on DRD2 signal transduction we transfected phosphodeficient S2152A or phosphomimetic S2152D FLNA mutant in MMQ and AtT-20 cells.
The main pathways involved in cell growth and proliferation regulation are MAPK and PI3K/AKT ones. According to literature, we confirmed a DA-induced increased phosphorylation of ERK1/2 in tumoral lactotrophs, that leads to cell proliferation inhibition (18,37,38). On the other side, BIM53097 treatment determined a reduction of ERK1/2 phosphorylation levels. This result is in line with literature where a reduction of ERK1/2 activity has been associated yet with a reduction of cell proliferation in AtT-20 (39).
FLNA phosphorylation abolished its endogenous functions, in fact, from our results emerged that DRD2 agonist antiproliferative effects were abrogated when S2152D FLNA was overexpressed in both cell models.
DRD2 is able to reduce AKT phosphorylation levels in tumoral lactotrophs (38,40), and we recently demonstrated that BIM53097 determined a significant reduction of AKT activation in MMQ cells and in a subset of NF-PitNETs through a b-arrestin 2-mediated mechanism (41).
In the present study, we showed that phosphomimetic FLNA mutant reverted the ability of BIM53097 to reduce AKT phosphorylation levels in MMQ cells, suggesting that P-FLNA might also influence DRD2 binding to partner molecules, but additional investigations are certainly required in order to elucidate molecular mechanism involved. Moreover, the well-established capability of DAs of reducing PRL levels in tumoral lactotrophs (42,43) was maintained in wild-type and phosphodeficient FLNA mutant transfected cells, but was abrogated by phosphomimetic one.
Accordingly, as pointed out in different studies, even if in variable way (5,44,45), DAs induced an inhibition of ACTH secretion in tumoral corticotrophs via DRD2 activation, effect that we showed to be impaired by S2152D FLNA mutant overexpression.
We must put in evidence that wild-type and S2152A FLNA overexpression did not affect DRD2 effects induced by DRD2agonist incubation. This point is in line with FLNA scaffold role, since it has been demonstrated that the overexpression of a scaffold protein does not necessarily intensify signaling (46). Furthermore, a key role of phosphorylation in regulating FLNA binding properties has been already shown, in particular that phosphorylation on Ser2152 induces integrin binding to Ig21 leading to the separation of the autoinhibitory structure that involves Ig19, Ig20 and Ig21 (30,47). It has also been demonstrated that PKA-dependent FLNA phosphorylation could be allowed by disruption of Ig21 induced by FLNA rearrangement due to ligands recruitment (32).
In our previous study (25) we partially clarified the molecular mechanism by which P-FLNA disrupted SSTR2 signaling. Indeed, trough co-immunoprecipitation experiments we showed a reduction of G i1-2-3 binding to SSTR2 in S2152D FLNA transfected cells with respect to those overexpressing S2152A FLNA. In light of these data, we can hypothesize that a similar mechanism occurred with DRD2. As previous demonstrated FLNA plays a key role in regulating DRD2 expression and signaling in PRL-secreting PitNETs. Indeed, it is required for DRD2-mediated ERK1/2 activation, inhibition of PRL release and cell proliferation (18). Further studies are needed to identify which molecular mediators involved in the DRD2 signaling cascade are affected by FLNA phosphorylation.
To the best of our knowledge, currently there are no other evidences of P-FLNA as key modulator of GPCRs signaling in PitNETs in addition to our previous on somatotrophs (25), or in other tumors as well. We can speculate that phosphorylation might represent a novel regulatory mechanism shared by different GPCRs that switch FLNA function from a scaffold enabling receptor signal transduction to a signal termination protein.
Admittedly, a limitation of our study is the absence of primary cultures of human PRL-or ACTH-secreting PitNETs. Further studies are needed to confirm and strengthen these results in human cells.
In conclusion, our data showed that cAMP pathway and DRD2 agonist regulated FLNA activity by increasing or decreasing, respectively, its phosphorylation on Ser2152. This study additionally turned out that FLNA phosphorylation prevented DRD2 signaling in PRL-and ACTH-secreting tumoral pituitary cell lines. In PitNETs expressing DRD2, modulation of P-FLNA might suggest new pharmacological strategies to overcome drug resistance and P-FLNA might represent a new molecular biomarker for tumor responsiveness to DAs.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.