Donatella Treppiedi

Dopamine receptor type 2 (DRD2) and somatostatin receptor type 2 (SSTR2) agonists are effective in inhibiting proliferation of progenitor/stem-like cells isolated from non functioning pituitary tumors

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.

Dopamine and Somatostatin Analogues Resistance of Pituitary Tumors: Focus on Cytoskeleton Involvement.

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.

Filamin A in somatostatin and dopamine receptor regulation in pituitary and the role of cAMP/PKA dependent phosphorylation.

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. 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.

Somatostatin Receptor Type 2 (SSTR2) Internalization and Intracellular Trafficking in Pituitary GH-Secreting Adenomas: Role of Scaffold Proteins and Implications for Pharmacological Resistance

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 post-receptor 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 GH-secreting pituitary tumors.

A novel pathway activated by somatostatin receptor type 2 (SST2): Inhibition of pituitary tumor cell migration and invasion through cytoskeleton protein recruitment

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.

Expression of protein kinase A regulatory subunits in benign and malignant human thyroid tissues: A systematic review

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.

cAMP/PKA-induced filamin A (FLNA) phosphorylation inhibits SST2 signal transduction in GH-secreting pituitary tumor cells

An efficient intracellular response to somatostatin analogs (SSA) in pituitary tumors requires filamin A (FLNA). Since cAMP pathway plays an important role in GH-secreting pituitary tumors pathogenesis and FLNA is phosphorylated by PKA on S2152, aim of this study was to investigate in tumoral somatotrophs the impact of cAMP pathway activation and SSA stimulation on FLNA phosphorylation and the consequences on SST2 function. We found a PKA-mediated increase (2-fold) and SST2 agonist-induced decrease (-50%) of FLNA phosphorylation in GH3, GH4C1 and primary somatotroph tumor cells. This modification regulates FLNA function. Indeed, phosphomimetic S2152D FLNA mutant, but not phosphodeficient S2152A, abolished the known SSA antitumoral effects, namely: 1) inhibition of cell proliferation, reduction of cyclin D3 and increase of p27; 2) increase of cell apoptosis; 3) inhibition of cell migration via RhoA activation and cofilin phosphorylation. Coimmunoprecipitation and immunofluorescence assays showed that S2152A FLNA was recruited to activated SST2, whereas S2152D FLNA constitutively bound SST2 on the plasma membrane, but prevented Gαi proteins recruitment to SST2. In conclusion, we demonstrated that FLNA phosphorylation, promoted by cAMP pathway activation and inhibited by SSA, prevented SST2 signaling in GH-secreting tumoral pituitary cells.