HIGH-RESOLUTION SPATIOTEMPORAL ANALYSIS OF SOMATOSTATIN RECEPTOR TYPE 2 (SSTR2) - FILAMIN A (FLNA) INTERACTION BY SINGLE-MOLECULE IMAGING

DOTTORATO DI RICERCA IN MEDICINA CLINICA E SPERIMENTALE (XXIX ciclo) Abstract ENG Cognome: TREPPIEDI Nome: DONATELLA Matricola n.: R10592 Title: "High-resolution spatiotemporal analysis of Somatostatin Receptor Type 2 (SSTR2) \u2013 Filamin A (FLNA) interaction by single-molecule imaging" Background: SSTR2 is the main pharmacological target to treat acromegalic patients harboring GH-secreting pituitary adenomas. However, about 30% of patients displays resistance to somatostatin analogues (SSAs). Recent published data from our group demonstrated that the cytoskeletal protein FLNA plays an essential role in tumor responsiveness by regulating SSTR2 signaling and stability after prolonged stimulation. To date, there are no evidence in the literature describing the dynamic of SSTR2-FLNA interaction at the plasma membrane in vivo. Aim: Aim of my PhD project was to follow the spatiotemporal behavior of SSTR2-FLNA complexes in real time by high resolution strategy. In particular I wanted to investigate the presence of a spatial distribution of SSTR2-FLNA complexes at the plasma membrane, estimate SSTR2 lateral mobility and elucidate a possible involvement of FLNA in regulating this biological phenomenon. A further goal was to evaluate the impact of FLNA-SSTR2 binding on ligand-induced SSTR2 clusters organization and internalization. Materials and Methods: To characterize the dynamics of SSTR2-FLNA complexes in living CHO cells we used the single-molecule imaging approach, a method that combines the labelling of SNAP/CLIP-tagged proteins (SNAP-tagged SSTR2 and CLIP-tagged FLNA) with small organic fluorophores and the use of a total internal reflection fluoresence (TIRF) microscope. To calculate SSTR2 lateral mobility, mean square displacement (MSD) analysis were performed using the u-track algorithm implemented in Matlab. Confocal microscopy was used to evaluate the receptor surface distribution, agonist-mediated clusterization and alignment with actin filaments. Immunofluoresce experiments were assessed to evaluate the colocalization between SSTR2 clusters and AP-2, one of the early endocytosis marker, whereas the overall SSTR2 internalization process was analyzed by both imaging (confocal microscopy) and biochemical (biotinylation assay) strategies. All these SSTR2 aspects were evaluated in the presence or the absence of FLNA interaction. In particular, the overexpression of the dominant negative mutant FLNA 19-20 was used to abolish SSTR2-FLNA binding. Results: First, the motion of freely diffusing SSTR2 particles was observed to slow down in CHO cells treated with 100nM BIM23120 for 5-10min (mean diffusion coefficients from 0,123\ub5m2*s-1 to 0,101\ub5m2*s-1). MSD analysis showed a significant increase in the SSTR2 fraction with diffusion coefficient values 64 0.05\u3bcm2*s-1 with respect to unstimulated cells (28,1% vs 14,4%, 100nM BIM23120 vs control, respectively, expressed as fraction of total particles, P < 0.05). The presence of the FLNA truncated mutant, that selectively prevents SSTR2-FLNA binding (FLNA 19-20), did not influence the SSTR2 agonist effect on receptor mobility. Such data was further confirmed in melanoma cell lines A7 (FLNA-expressing cells), M2 (FLNA-lacking cells). Then, we described the nature of the interactions between SSTR2 particles and FLNA fibers as extremely dynamic and transient under resting condition, whereas they resulted long-lasting and more stable after100nM BIM23120 exposure. Interestingly, when both FLNA and SSTR2 were expressed at single molecule level, SSTR2-FLNA complexes formation was seen to occur preferentially along actin filaments, in stimulated cells only. Furthermore, when overexpressed and activated by the agonist, SSTR2 was observed to undergo clusters formation, and FLNA-SSTR2 binding was required to preserve SSTR2 clusters alignment on actin structures and colocalization with the clathrin coated pits marker AP-2. In addition, quantitative analysis of the agonist-triggered SSTR2 internalization demonstrated a significant reduction of the internalization rate in the presence of FLNA 19-20 compared to negative control (FLNA 17-18), at all the tested time points (eg. 45,3%\ub11,4% internalization vs 71,4%\ub13,1% in FLNA 19-20 vs FLNA 17-18 transfected cells after 30min stimulation with 100nM BIM23120, respectively, P < 0,001), accordingly with biotinylation results. Discussion: The behavior of SSTR2-FLNA interactions were characterized for the first time by means high spatio-temporal resolution strategy. In particular we observed that the SSTR2 agonist is able to modulate the dynamic properties of receptor particles, as demonstrated by a significant increase in the immobile receptor fraction observed upon stimulation, compared to the basal state. Even though FLNA does not contribute to the enrichment of the static receptor population, it seems to act as scaffold platform where ligand-activated receptors preferentially stop, likely to initiate their signaling transduction, then followed by internalization. Moreover, we highlighted a key role of FLNA in anchoring SSTR2 complexes to the cortical actin cytoskeleton. In fact, the abolished SSTR2-FLNA interaction resulted in a remarkably reduced alignment of agonist-induced SSTR2 clusters along actin filaments, revealing that SSTR2 clusters cannot properly associate with the actin cytoskeleton without binding to FLNA. Furthermore, we demonstrated that like for other GPCRs and transmembrane proteins, FLNA interaction is required to initiate and sustain ligand triggered-SSTR2 endocytosis. Indeed, a decrease colocalization between SSTR2 clusters and AP2-defined pits, and a strong impairment of the internalization rate were both associated to the overexpression of FLNA-19-20. Altogether these results let us to speculate that, by tethering SSTR2 to actin filaments, FLNA may facilitate the formation of ligand-inducible complexes with interacting proteins that are necessary for the efficient endocytosis of the receptor into clathrin-coated vesicles. Conclusion: In conclusion, the present work provides evidence for the involvement of FLNA and the cortical actin cytoskeleton in the formation of compartmentalized domains at the plasma membrane, where SSTR2 are first assembled into functional units, and then subjected to endocytosis. Indeed, the deep understanding of these aspects may be useful to clarify the molecular mechanisms by which the cells can modulate the amount of active receptors at their surface, thus determining a variable responsiveness to SSAs, with possible implications in the pharmacological resistance to the clinical management of acromegaly

Detecting precipitation trend using a multiscale approach based on quantile regression over a Mediterranean area

One of the most relevant and debated topics related to the effects of the climate change is whether intense rainfall events have become more frequent over the last decades. It is a crucial aspect, since an increase in the magnitude and frequency of occurrence of heavy rainfall events could result in a dramatic growth of floods and, in turn, human lives losses and economic damages. Because of its central position in the Mediterranean area, Sicily has been often screened with the aim to capture some trends in precipitation, potentially related to climate change. While Mann-Kendall test has been largely used for the rainfall trend detection, in this work a different procedure is considered. Precipitation trends are here investigated by processing the whole rainfall time-series, provided by the regional agency SIAS at a 10-min resolution, through the quantile regression method by aggregating precipitation across a wide spectrum of durations and considering different quantiles. Results show that many rain gauges are characterized by an increasing trend in sub-hourly precipitation intensity, especially at the highest quantiles, thus suggesting that, from 2002 to 2019, sub-hourly events have become more intense in most of the island. Moreover, by analysing some spatial patterns, it has been revealed that the south and the east of Sicily are more interested in significant increasing rainfall trends, especially at the 10-min duration. Finally, the comparison between the two procedures revealed a stronger reliability of the quantile regression in the trend analysis detection, mainly due to the possibility of investigating the temporal variation of the tails of precipitation distribution

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

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

Cofilin is a mediator of RET-promoted medullary thyroid carcinoma cell migration, invasion and proliferation.

Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor that originates from parafollicular thyroid C cells and accounts for 5% of thyroid cancers. In inherited cases of MTC, and in about 40% of sporadic cases, activating mutations of the receptor tyrosine kinase proto-oncogene RET are found. Constitutively active RET triggers signaling pathways involved in cell proliferation, survival and motility, but the mechanisms underlying malignant transformation of C-cells have been only partially elucidated. Cofilin is a key regulator of actin cytoskeleton dynamics. A crucial role of cofilin in tumor development, progression, invasion and metastasis has been demonstrated in different human cancers, but no data are available in MTC. Interestingly, RET activation upregulates cofilin gene expression. The aim of this study was to investigate cofilin contribution in invasiveness and growth of MTC cells, and its relevance in the context of mutant RET signaling. We found that cofilin transfection in human MTC cell line TT significantly increased migration (178 +/- 44%, p &lt; 0.001), invasion (165 +/- 28%, p &lt; 0.01) and proliferation (146 +/- 18%, p &lt; 0.001), accompanied by an increase of ERK1/2 phosphorylation (2.23-fold) and cyclin D1 levels (1.43-fold). Accordingly, all these responses were significantly reduced after genetic silencing of cofilin (-55 +/- 10% migration, p &lt; 0.001, -41 +/- 8% invasion, p &lt; 0.001, -17 +/- 3% proliferation, p &lt; 0.001). These results have been confirmed in primary cells cultures obtained from human MTCs. The inhibition of constitutively active RET in TT cells by both the RET pharmacological inhibitor RPI-1 and the transfection of dominant negative RET mutant (RET.TK) resulted in a reduction of cofilin expression (-37 +/- 8%, p &lt; 0.001 and -31 +/- 16%, p &lt; 0.01, respectively). Furthermore, RPI-1 inhibitory effects on TT cell migration (-57 +/- 13%, p &lt; 0.01), but not on cell proliferation, were completely abolished in cells transfected with cofilin. In conclusion, these data indicate that an unbalanced cofilin expression, induced by oncogenic RET, contributes to promote MTC invasiveness and growth, suggesting the possibility of targeting cofilin pathway for more effective treatment of MTC

Stem Cells in Pituitary Tumors: Experimental Evidence Supporting Their Existence and Their Role in Tumor Clinical Behavior

Although generally benign, pituitary tumors frequently show local invasiveness and resistance to pharmacological therapy. After the demonstration of the existence of pituitary gland stem cells, over the past decade, the presence of a stem cell subpopulation in pituitary tumors has been investigated, analogous to the cancer stem cell model developed for malignant tumors. This review recapitulates the experimental evidence supporting the existence of a population of stem-like cells in pituitary tumors, focusing on their potential role in tumor initiation, progression, recurrence and resistance to pharmacological therapy