Integrazione temporale del segnale mediato da recettori accoppiati a proteine G: il potenziale ruolo di G15 nella cancerogenesi

G15 \ue8 una proteina G eterotrimerica della famiglia di Gq/11, specifica del sistema ematopoietico, la cui funzione \ue8 sostanzialmente ignota. In questo studio si descrive la sua eccezionale resistenza al generale processo di desensibilizzazione che virtualmente regola l\u2019attivit\ue0 di ogni recettore accoppiato a proteine G (GPCR). Il segnale di G15 \ue8 infatti risultato refrattario alla desensibilizzazione del recettore \u3b22 adrenergico indotta da sovraespressione di arrestina. Allo stesso modo, il livello d\u2019espressione di arrestina non ha influito sul segnale mediato da G15 in risposta all\u2019attivazione dei recettori V2 per la vasopressina e \u3b4 oppioide. Esperimenti di co-immunoprecipitazione hanno dimostrato che la subunit\ue0 G\u3b115 dell\u2019eterotrimero (a differenza di G\u3b1q and G\u3b1s) viene stabilmente reclutata da un recettore V2 che una mutazione rende costitutivamente desensibilizzato. L\u2019arrestina oltre a rendere meno efficiente il signalling dei GPCR, ne favorisce l\u2019internalizzazione in compartimenti endosomiali. La co-espressione di G\u3b115 ha parzialmente ristabilito la presenza sulla membrana plasmatica del mutante costitutivamente desensibilizzato ristabilendone anche parte delle capacit\ue0 segnalatorie. Tuttavia, confrontando l\u2019effetto dell\u2019agonista sull\u2019attivit\ue0 verso gli effettori di G15, quali PKD1 e PLC, con l\u2019effetto sull\u2019internalizzazione del recettore, sono state osservate alcune peculiarit\ue0 funzionali che suggeriscono che G15 stabilizzi una struttura conformazionale intermedia al normale processo di attivazione. Nel loro insieme, questi risultati suggeriscono un nuovo meccanismo per cui la regolazione dei GPCR pu\uf2 essere aggirata e G15 pu\uf2 trasdurre segnali estremamente prolungati quando le cellule siano \u201ccronicamente\u201d esposte all\u2019effetto di ormoni, neurotrasmettitori etc. Questo aspetto ci ha portato ad ipotizzare che la presenza di G15 possa essere associata a stati fisiologici caratterizzati da fenomeni di deregolazione, in particolare con la formazione di tumori. Uno screening ha dimostrato la presenza di G15 in 10 fra 40 linee cellulari tumorali analizzate, tutte derivate da tessuti che in origine risultano negativi. Il messaggero di G\u3b115 e la corrispondente proteina sono anche risultati presenti in biopsie di cancro prostatico e pancreatico. Particolarmente rilevante appare il fatto che G\u3b115 fosse presente nella frazione proliferante del tumore cos\uec come dimostrato con xenotrapianti di tumore in topi nudi.G15 is a heterotrimeric G protein of the Gq/11 family. In this study, we describe its exceptional poor sensitivity to the general regulatory mechanism of G protein-coupled receptor (GPCR) desensitization. Enhancing \u3b22 adrenergic receptor desensitization by arrestin overexpression, did not affect signalling to G15. Similarly, increased levels of arrestin did not affect G15 signalling triggered by the activation of V2 vasopressin and \u3b4 opioid receptors. Furthermore, co-immunoprecipitation experiments showed that G15 \u3b1 subunit (as opposed to G\u3b1q and G\u3b1s) is recruited to a V2 vasopressin receptor mutant that is constitutively desensitized by \u3b2-arrestin. Interestingly, co-expression of G\uf06115 partially rescued cell surface localization and signalling capabilities of the same mutant and supported its sustained activity on downstream effectors including PKD1. However, differential modulation by the agonist was observed while comparing effectors activity to receptor internalization. Taken together, these findings provide evidence for a novel mechanism whereby GPCR desensitization can be bypassed and G15 can support sustained signalling in cells chronically exposed to hormones or neurotransmitters. This aspect led us to hypothesize that G15 presence could be associated to poorly regulated physiological states, and in particular with tumor formation. A screening demonstrated G\u3b115 presence in 10 out of 40 human cancer cell lines analyzed, all derived from a variety of tissues reported as negative. G\u3b115 mRNA and protein were also found in pancreatic and prostate tumor biopsies. Notably, G\u3b115 was present in the proliferating fraction of the tumor as demonstrated by xenotransplantation of the tumors in nude mice

Methylation Dynamics of RASSF1A and Its Impact on Cancer

5-methyl cytosine (5mC) is a key epigenetic mark entwined with gene expression and the specification of cellular phenotypes. Its distribution around gene promoters sets a barrier for transcriptional enhancers or inhibitor proteins binding to their target sequences. As a result, an additional level of regulation is added to the signals that organize the access to the chromatin and its structural components. The tumor suppressor gene RASSF1A is a microtubule-associated and multitasking scaffold protein communicating with the RAS pathway, estrogen receptor signaling, and Hippo pathway. RASSF1A action stimulates mitotic arrest, DNA repair and apoptosis, and controls the cell cycle and cell migration. De novo methylation of the RASSF1A promoter has received much attention due to its increased frequency in most cancer types. RASSF1A methylation is preceded by histones modifications and could represent an early molecular event in cell transformation. Accordingly, RASSF1A methylation is proposed as an epigenetic candidate marker in many cancer types, even though an inverse correlation of methylation and expression remains to be fully ascertained. Some findings indicate that the epigenetic abrogation of RASSF1A can promote the alternative expression of the putative oncogenic isoform RASSF1C. Understanding the complexity and significance of RASSF1A methylation is instrumental for a more accurate determination of its biological and clinical role. The review covers the molecular events implicated in RASSF1A methylation and gene silencing and provides a deeper view into the significance of the RASSF1A methylation patterns in a number of gastrointestinal cancer types

Pleiotropic effects of sphingosine-1-phosphate signaling to control human chorionic mesenchymal stem cell physiology

Chorionic stem cells represent a promising opportunity for regenerative medicine. A deeper understanding of the stimuli that regulate their physiology, could lead to innovative clinical approaches. We revealed the presence of multiple sphingosine-1-phosphate (S1P) receptor isoforms in chorion-derived mesenchymal stem cells (CMSCs). Their activation simultaneously propagated from the plasma membrane through Gi and other heterotrimeric G proteins and further diverged toward extracellular-signal-regulated kinase 1/2 (ERK1/2), p38 and protein kinase D 1. At a functional level, S1P signaling inhibited CMSC migration, while promoting proliferation. Instead, a reduction of cell density was obtained when S1P was combined to treatments that increased cAMP intracellular concentration. Such surprising reduction of cell viability was relatively specific as it was not observed with stromal stem cells from bone marrow. Neither it was observed by activating analogous G proteins with bradykinin nor by inducing cell death via a cAMP-independent pathway. S1P could thus reveal novel keys to improve CMSC differentiation programs acting on cAMP concentration. Furthermore, S1P receptor agonists/antagonists could become instrumental in favoring CMSC engraftment by controlling cell motility

cAMP Response Element-Binding Protein Controls the Appearance of Neuron-Like Traits in Chorion Mesenchymal Cells

Mesenchymal stromal cells (MSC) from bone marrow have been reported to undergo the initial phases of neural differentiation in response to an increase of intracellular cAMP. We investigated the possibility that a similar effect applies to chorion-derived MSC

Fast method for skeletal tissue gene expression analysis

open9Several chronic diseases have been associated with bone alteration in the last few years. Despite the wealth of information provided by the analysis of the transcriptome in affected tissues, only a limited number of studies evaluated gene expression in bone tissue due to the difficulty to obtain high quality RNA. Therefore, skeletal pathologies have been often associated to a defective maturation process that occurs during recruitment of progenitor stem cells. In order to explore the possibility of analysing the gene expression during osteogenic differentiation in skeletal tissue, a single-step method to extract well-preserved RNA from bone specimens was performed. A comparison between this technique and a traditional method was made by analysing the quality and yield of RNA obtained. In addition, RNAs were assayed by reverse transcription-quantitative polymerase chain reaction to analyse the expression levels of the bone genes associated with the differentiation process in a mouse model. The present data showed that good quality RNA can be obtained from bone tissue by a simple single-step method allowing the expression analysis of the genes encoded by skeletal tissue. In conclusion, the present study allows the possibility to easily obtain good quality RNA from bone tissue that is suitable for gene expression studies of bone diseases.openDalle Carbonare, Luca; Vilei, Maria Teresa; Stranieri, Chiara; Innamorati, Giulio; Rosato, Antonio; Boldrin, Elisa; Sella, Stefania; Giannini, Sandro; Valenti, Maria TeresaDALLE CARBONARE, LUCA GIUSEPPE; Vilei, MARIA TERESA; Stranieri, Chiara; Innamorati, Giulio; Rosato, Antonio; Boldrin, Elisa; Sella, Stefania; Giannini, Sandro; Valenti, Maria Teres

New Insights into the Runt Domain of RUNX2 in Melanoma Cell Proliferation and Migration

The mortality rate for malignant melanoma (MM) is very high, since it is highly invasive and resistant to chemotherapeutic treatments. The modulation of some transcription factors affects cellular processes in MM. In particular, a higher expression of the osteogenic master gene RUNX2 has been reported in melanoma cells, compared to normal melanocytes. By analyzing public databases for recurrent RUNX2 genetic and epigenetic modifications in melanoma, we found that the most common RUNX2 genetic alteration that exists in transcription upregulation is, followed by genomic amplification, nucleotide substitution and multiple changes. Additionally, altered RUNX2 is involved in unchecked pathways promoting tumor progression, Epithelial Mesenchymal Transition (EMT), and metastasis. In order to investigate further the role of RUNX2 in melanoma development and to identify a therapeutic target, we applied the CRISPR/Cas9 technique to explore the role of the RUNT domain of RUNX2 in a melanoma cell line. RUNT-deleted cells showed reduced proliferation, increased apoptosis, and reduced EMT features, suggesting the involvement of the RUNT domain in different pathways. In addition, del-RUNT cells showed a downregulation of genes involved in migration ability. In an in vivo zebrafish model, we observed that wild-type melanoma cells migrated in 81% of transplanted fishes, while del-RUNT cells migrated in 58%. All these findings strongly suggest the involvement of the RUNT domain in melanoma metastasis and cell migration and indicate RUNX2 as a prospective target in MM therapy

Physical activity modulates miRNAs levels and enhances MYOD expression in myoblasts

Stem cells functions are regulated by different factors and non-conding RNAs, such as microRNA. MiRNAsplay an important role in modulating the expression of genes involved in the commitment and differentiation of progenitor cells. MiRNAs are post transcriptional regulators which may be modulated by physical exercise. MiRNAs, by regulating different signaling pathways, play an important role in myogenesis as well as in muscle activity. MiRNAs quantification may be considered for evaluating physical performance or muscle recovery. With the aim to identify specific miRNAs potentially involved in myogenesis and modulated by physical activity, we investigated miRNAs expression following physical performance in Peripheral Blood Mononuclear Cells (PBMCs) and in sera of half marathon (HM) runnners. The effect of runners sera on Myogenesis in in vitro cellular models was also explored. Therefore, we performed Microarray Analysis and Real Time PCR assays, as well as in vitro cell cultures analysis to investigate myogenic differentiation. Our data demonstrated gender-specific expression patterns of PBMC miRNAs before physical performance. In particular, miR223-3p, miR26b-5p, miR150-5p and miR15-5p expression was higher, while miR7a-5p and miR7i-5p expression was lower in females compared to males. After HM, miR152-3p, miR143-3p, miR27a-3p levels increased while miR30b-3p decreased in both females and males: circulating miRNAs mirrored these modulations. Furthermore, we also observed that the addition of post-HM participants sera to cell cultures exerted a positive effect in stimulating myogenesis. In conclusion, our data suggest that physical activity induces the modulation of myogenesis-associated miRNAs in bothfemales and males, despite the gender-associated different expression of certain miRNAs, Noteworthy, these findings might be useful for evaluating potential targets for microRNA based-therapies in diseases affecting the myogenic stem cells population

A potential role of RUNX2- RUNT domain in modulating the expression of genes involved in bone metastases: an in vitro study with melanoma cells

Ectopic expression of RUNX2 has been reported in several tumors. In melanoma cells, the RUNT domain of RUNX2 increases cell proliferation and migration. Due to the strong link between RUNX2 and skeletal development, we hypothesized that the RUNT domain may be involved in the modulation of mechanisms associated with melanoma bone metastasis. Therefore, we evaluated the expression of metastatic targets in wild type (WT) and RUNT KO melanoma cells by array and real-time PCR analyses. Western blot, ELISA, immunofluorescence, migration and invasion ability assays were also performed. Our findings showed that the expression levels of bone sialoprotein (BSP) and osteopontin (SPP1) genes, which are involved in malignancy-induced hypercalcemia, were reduced in RUNT KO cells. In addition, released PTHrP levels were lower in RUNT KO cells than in WT cells. The RUNT domain also contributes to increased osteotropism and bone invasion in melanoma cells. Importantly, we found that the ERK/p-ERK and AKT/p-AKT pathways are involved in RUNT-promoted bone metastases. On the basis of our findings, we concluded that the RUNX2 RUNT domain is involved in the mechanisms promoting bone metastasis of melanoma cells via complex interactions between multiple players involved in bone remodeling

Runx2 downregulation, migration and proliferation inhibition in melanoma cells treated with BEL \u3b2-trefoil

Malignant melanoma is a lethal form of skin cancer and highly metastatic tumor has a poor prognosis; BEL \u3b2-trefoil, a lectin, obtained by our group, possesses the ability to act specifically on malignant cells. Therefore, the aim of our study was to investigate the effects of BEL \u3b2-trefoil in melanoma cells in an attempt to evaluate its potential usage as anticancer agent. BEL \u3b2-trefoil was purified by chromatography and A375 and MeWo melanoma cells were treated. Viability and proliferation were evaluated as well as apoptosis, RUNX2 gene expression and migration ability. The treated tumor cells decreased viability as well as proliferative ability. Flow cytometry analysis showed a lessen effect of the treatment on apoptosis. The gene expression analysis showed a reduction of RUNX2 expression in a manner dose depend and migration ability was reduced significantly in both treated cell lines. Our findings suggest that BEL \u3b2-trefoil can be considered a useful tool against malignancy thank to its effect based on the simultaneous proliferation ability reduction as well as the inhibition of migration capacity on melanoma tumor cells

Runx2 stimulates neoangiogenesis through the Runt domain in melanoma

Runx2 is a transcription factor involved in melanoma cell migration and proliferation. Here, we extended the analysis of Runt domain of Runx2 in melanoma cells to deepen understanding of the underlying mechanisms. By the CRISPR/Cas9 system we generated the Runt KO melanoma cells 3G8. Interestingly, the proteome analysis showed a specific protein signature of 3G8 cells related to apoptosis and migration, and pointed out the involvement of Runt domain in the neoangiogenesis process. Among the proteins implicated in angiogenesis we identified fatty acid synthase, chloride intracellular channel protein-4, heat shock protein beta-1, Rho guanine nucleotide exchange factor 1, D-3-phosphoglycerate dehydrogenase, myosin-1c and caveolin-1. Upon querying the TCGA provisional database for melanoma, the genes related to these proteins were found altered in 51.36% of total patients. In addition, VEGF gene expression was reduced in 3G8 as compared to A375 cells; and HUVEC co-cultured with 3G8 cells expressed lower levels of CD105 and CD31 neoangiogenetic markers. Furthermore, the tube formation assay revealed down-regulation of capillary-like structures in HUVEC co-cultured with 3G8 in comparison to those with A375 cells. These findings provide new insight into Runx2 molecular details which can be crucial to possibly propose it as an oncotarget of melanoma