Clinical significance of filamin A in patients with acromegaly and its association with somatostatin and dopamine receptor profiles

Filamin-A (FLNA) plays a crucial role in somatostatin receptor (sst) subtype-2 signaling in somatotropinomas. Our objective was to investigate the in vivo association between FLNA and sst2 expression, sst5 expression, dopamine receptor subtype-2 (D2) expression, somatostatin receptor ligand (SRL) responsiveness and tumor invasiveness in somatotropinomas. Quantitative real-time PCR was used to evaluate the absolute mRNA copy numbers of FLNA/sst2/sst5/D2 in 96 somatotropinomas. FLNA, sst2 and sst5 protein expression levels were also evaluated using immunohistochemistry. The Knosp-Steiner criteria were used to evaluate tumor invasiveness. Median FLNA, sst2, sst5 and D2 copy numbers were 4,244, 731, 156 and 3,989, respectively. Thirty-one of the 35 available tumors (89%) were immune positive for FLNA in the cytoplasm and membrane but not in the nucleus. FLNA and sst5 expression were positively correlated at the mRNA and protein levels (p < 0.001 and p = 0.033, respectively). FLNA was positively correlated with sst2 mRNA in patients who were responsive to SRL (p = 0.014, R = 0.659). No association was found between FLNA and tumor invasiveness. Our findings show that in somatotropinomas FLNA expression positively correlated with in vivo sst5 and D2 expression. Notably, FLNA was only correlated with sst2 in patients who were controlled with SRL. FLNA was not associated with tumor invasiveness.

Previous studies by the group of Giovana Mantovani have demonstrated the important role of the cytoskeleton protein filamin A (FLNA) in sst2 expression and signaling in somatotropinomas [7][8][9] . FLNA is encoded by a gene located in chromosomal region Xq28, and it is a cytoskeletal protein that organizes actin filaments into stress fibers and networks 10 . This process is important for conformational changes at the cell membrane, where it acts as a key mediator of cytoskeleton reorganization 11 . FLNA binds diverse transmembrane proteins, such as G-protein-coupled receptors (GPCRs), ion channels and integrins, and anchors these proteins to the actin cytoskeleton; moreover, FLNA acts as an interface for protein-protein interactions 10,[12][13][14] . Peverelli and et al. 7 demonstrated an association between FLNA expression and response to pharmacological therapy in somatotropinomas and suggested that a reduction of FLNA expression was another mechanism of resistance to SRLs, at least in vitro. Their study indicated that changes in FLNA expression altered the sst2 signaling pathway in somatotropinomas 7 . The same group recently demonstrated that sst2 inhibited rat and human tumoral somatotrophs migration and invasion in vitro via a molecular mechanism that involved FLNA-dependent cofilin recruitment and phosphorylation 9 . FLNA is also crucial for D2 expression and signaling in prolactinomas 15 . However, these results were demonstrated in in vitro cell models; no in vivo studies have confirmed the results.
Previous studies demonstrated that FLNA was involved in the control of cell mobility and extracellular matrix degradation in some tumoral tissues 16,17 and FLNA knockdown enhanced metalloproteinase activity, which stimulated invasion, cancer cell migration and metastasis formation 16,18 . However, FLNA levels and its clinical relevance in somatotropinoma samples/patients were not examined. Therefore, the aim of this study was to analyze FLNA expression levels and its association with sst2, sst5 and D2 expression in human somatotropinoma samples and to investigate the association of FLNA expression with SRL responsiveness and tumor invasiveness in patients with acromegaly. Median GH level was 18.8 ng/mL (1.1-120) at time of diagnosis, and median IGF-I level was 325% ULNR (101-734). Data of treatment with first-generation SRL prior to surgery were not available in 21 patients. Sixty-two patients were treatment-naïve, and 13 patients were treated prior to surgery (2 of these patients were also treated with cabergoline). Nine patients used cabergoline after surgery. Radiotherapy was not performed in any patient prior to surgery.

Results
Among the 96 patients who were included, data regarding responses to first-generation SRLs were available in 40 of the 96 included patients, and 23 (57.5%) of these 40 patients were controlled. One patient was excluded from the analysis of sst2 and sst5 mRNA levels because qPCR data were not obtainable due to the poor quality of the samples.
FLNA, sst2 and sst5 protein expression. Immunohistochemistry (IHC) revealed that FLNA was expressed in 89% of the formalin-fixed paraffin-embedded (FFPE) somatotropinoma samples that were available for this analysis (n = 31 out of 35 samples). IHC also revealed that FLNA staining was low in 32% of the somatotropinomas (score-1; n = 10) compared with the moderate or intense staining found in 68% of the samples [score-2 (n = 12) and score-3 (n = 9)] (Fig. 1). Notably, FLNA was expressed at the membrane and cytoplasmic levels but not at the nuclear level in our cohort of somatotropinoma samples.
Correlation of FLNA with sst2, sst5 and D2 expression at the mRNA and/or protein levels.

Discussion
Filamin A is a cytoskeletal protein that plays important roles in adhesion, conservation of cell shape, migration and intracellular signaling 10 . Our study evaluated the associations between the expression levels of FLNA (at the mRNA and protein level) and sst2, sst5 and D2 expression levels, the responsiveness to first-generation SRLs, and the presence of cavernous sinus invasion for the first time in patients with acromegaly.
No significant correlation between FLNA and sst2 expression was observed in the entire cohort, which is consistent with a previous report from Peverelli et al. 7 . However, we found a positive correlation between FLNA and sst2 in patients who were not pretreated and controlled with SRL therapy. Therefore, our current and previous data suggest that FLNA participates in the process of sst2 regulation and signaling in somatotropinomas. FLNA is involved in sst2 stabilization and signaling in tumoral somatotrophs, where it plays structural and functional roles 7,9 . The cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) pathway may be involved in this process and control FLNA stability via its phosphorylation status 19 . PKA phosphorylation of FLNA may produce conformational changes in regions involved in sst2 signaling and signal transduction pathways 19 . Therefore, one limitation of the present study is that we only assessed the association of FLNA expression with clinical features of the tumors independently of phosphorylation status.  FLNA scaffold function is necessary in somatotropinomas for sst2 to induce apoptosis and inhibit cell proliferation in vitro 7,9 . Peverelli et al. 7 demonstrated that silencing of FLNA inhibited the ability of sst2 to activate caspase and reduce cyclin D1 levels in somatotropinoma primary cell cultures. FLNA stabilized sst2 expression via lysosomal degradation processes after prolonged agonist exposure in vitro 7 . Therefore, a correlation between FLNA and sst2 expression in patients treated with SRL prior to surgery was expected. However, the importance of this mechanism in vivo is not certain. Therefore, we analyzed expression levels of FLNA and sst2 in patients treated with SRLs prior to surgery, but no correlation between FLNA and sst2 was found. Notably, reduced expression levels of sst2 were found in patients treated with first-generation SRLs prior to surgery compared to patients who were not pre-treated, which is consistent with previous studies 20, 21 . It is important to highlight a potential bias in our study because only patients who were not controlled with SRLs underwent surgery, and tumors with lower sst2 expression may have been selected for these analyses.
Notably, patient response to first-generation SRLs did not alter FLNA expression levels. However, sst2 mRNA levels were higher in acromegaly patients who achieved biochemical response and disease control after SRL treatment, which is consistent with previous reports from our group 5,22 .
A partial association was found between FLNA and sst2 in our study, i.e., it was only observed in patients who were not pretreated and controlled with SRL. However, an association between FLNA and sst5 levels was found regardless of SRL pretreatment or responsiveness. These results may be clinically relevant because sst5 is also involved in the inhibitory effects of somatostatin and SRL on GH release and cell proliferation 23 . The exact mechanism of sst5 activation and contribution to the response to first-generation SRL treatment of somatotropinomas is not certain. The data found in this study and previous studies of an association between FLNA and sst2 7,14,19 suggest that FLNA is involved in the transcriptional and signaling regulation of sst5. However, further studies are required to elucidate the molecular mechanisms underlying this putative association. FLNA is required for the membrane localization of several G-protein coupled receptors via anchoring these proteins to the actin cytoskeleton 11 . FLNA gene silencing in parathyroid tumors reduced mRNA and protein expression of a calcium-sensing receptor 24 , which is a key signaling pathway involved in somatostatin and SRL actions via the activation of ssts, including sst5 25,26 . Higher sst5 expression is associated with a worse response to first-generation SRLs and a better response to pasireotide 22,24,27,28 . Gatto et al. 29 demonstrated that a lower sst2/sst5 ratio was associated with a better response to pasireotide in vitro in GH secretion compared to octreotide. Further work is required to complete our understanding of this complex process and fully elucidate the molecular mechanisms underlying the putative association between FLNA and sst5 or sst2 in human somatotropinomas.
We found novel data of a positive correlation between FLNA and D2 mRNA expression in somatotropinomas. Our results are partially consistent with a previous report that suggested an association of FLNA with D2 expression in a prolactinoma cell line (MMQ cells that expressed D2) but not in the somatotropinoma GH3 cell line (no D2 expression) 15 . This same study found that FLNA silencing in cultured prolactinoma cells decreased D2 protein expression and its migration to the plasma membrane, but it did not reduce its transcription 15 . FLNA also interacted with the N-terminal region of D2 (amino acids 211-241) in vitro in human melanoma cell lines, which increased the efficiency of D2 binding to the adenylate cyclase 30 . Li et al. 31 demonstrated that FLNA was required for D2 cell surface localization in primary rat striatal cultures. Therefore, FLNA may command lysosomal degradation of D2 or its relocation to the membrane, and this process may be necessary for resensitization of desensitized D2. D2 silencing impaired extracellular signal-regulated kinase (ERK)1/2 phosphorylation and reduced prolactin release 15,32 . The decrease in FLNA expression in prolactinomas may be one mechanism that leads to DA resistance. We also demonstrated a positive correlation between FLNA and D2 expression in somatotropinomas. Therefore, we speculate that a decrease in FLNA may also be a mechanism of resistance to DA treatment in acromegaly. However, we could not evaluate this hypothesis in our study because only eleven patients were treated with cabergoline. Therefore, further studies with an ample cohort of patients may help clarify this relevant clinical question.
Finally, the involvement of FLNA in cancer progression via regulation of cell proliferation and migration was described previously in other tumors [33][34][35] . The absence of FLNA expression in prolactinomas impaired the inhibition of cell proliferation 15 . Despite the limited subset of cases included in our study, our data indicate that FLNA mRNA levels were not associated with the invasiveness features in our cohort of patients with somatotropinomas.

Conclusion
Our data revealed that FLNA expression levels positively correlated with sst5 and D2, but not sst2, expression in somatotropinomas. However, a positive correlation between FLNA and sst2 was found in patients who were controlled with SRLs, which suggests that FLNA is important for sst2 signaling. We did not find any association of FLNA with tumor invasiveness. Therefore, the exact role of FLNA in somatotropinomas is not certain, and further studies are needed to better understand its connection to sst2, sst5 and D2 and its association with pharmacological treatment using drugs targeting these receptors, such as pasireotide and cabergoline.

Subjects and methods. The Ethics Committee of Hospital Universitário Clementino Fraga Filho and
Medical School/Universidade Federal do Rio de Janeiro approved this study. All participants and/or their legal guardians provided informed consent prior to entering the study. All methods and experimental protocols were performed in accordance with the approved guidelines and regulations of our institutions following the principles of the Declaration of Helsinki. and stored at −80 °C for molecular analyses. Immunohistochemistry was performed in cases with available paraffin-embedding tumor samples. Biochemical diagnosis of acromegaly was based on current criteria 36 . An expert neuroradiologist with experience in sellar MRI interpretation analyzed images. Tumor maximum diameter and the presence or absence of cavernous sinus invasion were analyzed according to modified Knosp-Steiner criteria 37 .

Patients and tumors.
Criteria for cure and response to somatostatin receptor ligands. Patients were considered not cured based on nadir GH levels higher than 1.0 ng/mL after an oral glucose tolerance test (OGTT) or with plasma IGF-I levels higher than age-matched normal levels three months after surgery. Biochemical response to medical treatment was assessed using GH and IGF-I levels after 6 months of treatment with octreotide LAR at a maximum dose of 30 mg or lanreotide autogel at a maximum dose of 120 mg. Patients with GH levels > 1.0 μg/L and/or IGF-I levels higher than age-matched normal levels were considered uncontrolled.

Methods
Hormonal assessment. Plasma GH levels were measured using a chemiluminescence assay kit (IMMULITE 2000; DPC -Diagnostic Products Corp., Inc., Los Angeles, CA, USA). The coefficients of variation (CV) inter-and intra-assay were 6.0 and 5.8%, respectively. The International Reference Preparation (IRP) for GH was 98/574.
Plasma IGF-I levels were measured using a chemiluminescence assay kit (IMMULITE 2000; DPC). The interand intra-assay CVs were 6.6 and 3.6%, respectively. The IRP for IGF-I was 87/518. IGF-I levels are expressed as a percentage of the upper limit of normal range (%ULNR).
Immunohistochemistry. IHC staining was performed in FFPE samples using the Dako Envision system.
The optimum antibody concentration for FLNA IHC analyses (1:150) using a commercially available human filamin antibody (Anti-Filamin A, Abcam, Cambridge, UK ab76289) was selected following antibody dilution tests (1:100; 1:150, 1:200) in uterus samples. A negative control image was taken from a uterus sample without the addition of primary antibody. IHC was performed in FFPE from somatotropinomas using standard procedures, as previously reported 38 .
Quantitative PCR. Total RNA was extracted from tumor samples using the Allprep ® Universal kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Absolute mRNA copy number levels of FLNA, sst2, sst5 and D2 were analyzed using quantitative real-time RT-PCR (qPCR) and the Sybergreen ® method, as previously described by our group 39 . Supplemental Table 2 lists the primers. The expression level (copy-number) of each of the transcripts analyzed was adjusted to a normalization factor of the expression levels of three housekeeping genes using the GeNorm 3.3 visual basic application to control for variations in the amount of RNA used and the efficiency of the reverse-transcription reaction 27 . The results are reported as gene copy number/NF. Statistical analysis. Statistical tests were performed using SPSS version 23.0 for Mac (IBM, Chicago, IL, USA). The results are reported as median values (minimum-maximum). The Mann-Whitney U non-parametric test was used to compare numeric variables between groups, and correlation coefficients were calculated using Spearman rank order R. Fisher's exact test or the χ 2 test was used to compare frequencies between groups according to sample size. A p value < 0.05 was considered significant.