Literatura académica sobre el tema "Pathway molecolare"

SommarioLe iperplasie surrenaliche bilaterali sono una delle cause di ipercortisolemia endogena cronica associate alla sindrome di Cushing e, in base alla dimensione della lesione, vengono suddivise in forme micro- (PPNAD e iMAD) e macronodulari (PBMAH). Negli ultimi anni sono stati compiuti numerosi passi in avanti nella comprensione delle cause genetiche e molecolari di queste diverse forme di iperplasia. Fra queste, di primaria importanza sono quelle che coinvolgono le mutazioni e la disregolazione di effettori/modulatori della via cAMP/protein chinasi A (PKA). Il signalling della PKA, infatti, rappresenta il pathway molecolare principale che controlla la secrezione di cortisolo nelle cellule corticosurrenali, mediato dall’ACTH. Sebbene le mutazioni della subunità regolatoria R1$\alpha $ α della PKA rappresentino la causa principale di PPNAD familiare e sporadica, l’inattivazione di due fosfodiesterasi, PDE11A e PDE8B, è stata associata a iMAD, ma anche ad alcuni casi di PPNAD e PBMAH. È interessante notare che la PBMAH osservata in sindromi familiari, quali FAP, MEN1 e HLRCC, è stata inizialmente associata all’espressione aberrante dei recettori accoppiati a proteine G con conseguente attivazione della via cAMP/PKA. La recente scoperta dell’associazione fra mutazioni germinali nel gene KDM1A e ARMC5 ha dato un definitivo slancio all’importanza della componente ereditaria nella PBMAH.

The clinical activity of the BRAF inhibitor PLX4032 (vemurafenib) in patients with BRAFV600E mutant melanoma is limited primarily by the development of resistance leading to tumor progression. Strategies to overcome primary and acquired resistance are required. In a panel of 27 genetically characterized patient-derived melanoma cell lines the sensitivity to PLX4032 was dependent on BRAFV600E and independent from other gene alterations that commonly occur in melanoma, such as CDKN2A, and mutations of TP53, PTEN loss, and BRAF and MITF gene amplification. To investigate the molecular basis underlying acquired resistance to BRAF inhibitor, PLX4032-resistant cells were derived from a high sensitive BRAFV600E melanoma cell line, and used as a model. The resistant variant line showed increased AKT and ERK phosphorylation and enhanced IGF-1R/PI3K signaling. Combined treatment with PLX4032 plus PI3K inhibitors resulted in significant cell growth inhibition by decreasing pAKT and pERK signaling. To explore molecular mechanisms underlying primary resistance two melanoma cell lines lacking sensitivity to PLX4032 were used as models. Resistance to PLX4032 was maintained after CRAF down-regulation by siRNA, indicating that CRAF is not involved in the activation of ERK in the resistant cell lines. Treatment with the MEK inhibitor UO126 inhibited cell growth and decreased ERK phosphorylation indicating alternative activation of MEK-ERK signaling. Genetic characterization by MLPA and analysis of pTyr signaling by MALDI-TOF mass spectrometry revealed the activation of MET and SRC signaling, associated with the amplification of MET and of CTNNB1 and CCND1 genes, respectively. Testing of co-inhibition of the MET, SRC and MAPK signaling pathways by the combined treatment with the MET inhibitor, SU11274 or the SRC inhibitor, BMS-354825 plus PLX4032 resulted in a significant inhibitory effect on melanoma cell proliferation, survival, migration and invasive capacity. These results support combinatorial approaches targeting MAPK pathway at different nodes and intercepting parallel signal transduction pathways as a strategy to override resistance to BRAF inhibitors.

The α7 nicotinic acetylcholine receptor (α7 nAChR) has a key role in the innate immune system’s inflammatory response, as part of “cholinergic anti-inflammatory pathway”: a process by which acetylcholine from the vagus nerve reduces the release of the pro-inflammatory cytokine TNFα, thus allowing for a controlled response to infection. The CHRNA7 gene, in humans, is partially duplicated from exon 5-10 and forms an hybrid with four exons (D-A) of a novel gene, FAM7A. This new gene, CHRFAM7A, which is located in the opposite orientation, at 1.6 Mb, from CHRNA7, is not present on every chromosome 15 and a polymorphic variant, in linkage disequilibrium with a 2bp deletion in exon 6, in the same orientation to the CHRNA7 gene, has been described in a cohort of patients with bipolar disorders and schizophrenia. THP-1 monocytic-like cell line expresses only CHRFAM7A, which was down-regulated on treatment with LPS, by a direct transcriptional mechanism reliant on NF-kB. This effect has been confirmed in primary monocytes and macrophages cell cultures, where CHRFAM7A is expressed 200-1000 times more than CHRNA7. Here, the conventional α7 subunit was up-regulated by LPS treatment, thus suggesting the involvement of CHRFAM7A in the regulation of cell surface α7 receptors’ level (a mechanism unique to humans) and the ability of immune cells to respond to acetylcholine, released from the vagus nerve, during infection. This hypothesis seems to be supported by recent works showing that the duplicated form may have a dominant negative effect on the activity of α7 nAChR. Infact, co-expression of CHRFAM7A with α7 results in a significant reduction of the Ach-evoked currents, suggesting the presence of heteromeric non functional receptors at the plasma membrane. The promoter region that regulates the expression of CHRFAM7A is still unknown. To try to identify and characterize this region, 5'-RACE experiments were carried out to map the CHRFAM7A mRNA 5’UTR. RNA was extracted from three different cell lines: THP-1 cells, primary human macrophages and SHSY5Y neuroblastoma cell line. Multiple transcription start sites were identified, depending on the cell line used, suggesting the existence of alternative promoters. A series of constructs that recapitulate the mapping of the CHRFAM7A regulatory region, according to the transcription start sites identified, was also generated. They were cloned into a reporter vector and their functionality was tested by transient transfection both in THP-1 and SHSY5Y cell models. Through these experiments, an intronic region (-702/-208 bp from ATG codon, in exon B) and an Alu sequence (-1155/-821 bp) were identified as negative regulators of reporter gene transcription. Future experiments will allow us to identify other regulatory sites, important for proper CHRFAM7A gene expression in different tissues. Furthermore, two variants exist for CHRFAM7A gene, due to alternative splicing, that gives rise to two protein products of predicted 36 and 47 KDa, whose function is currently unknown. The N-terminally portion of each variant would lack the majority of the ligand binding domain, but the protein product retains the transmembrane region that forms the ion channel. To clarify the function of CHRFAM7A gene and to study the cellular localization of these two isoforms, CHRFAM7A (both variants) and CHRNA7 tagged proteins were generated, by cloning their cDNAs into pcDNA3.1/myc-His and pcDNA3.1/V5-His vectors. The α7 specific chaperon, RIC3, was also cloned into pcDNA3.0 vector and co-transfected with the tagged proteins, in order to increase their folding and expression. The constructs were transfected into Hela cells and characterized by immunofluorescence and western blotting experiments. The use of nicotine as therapy for chronic inflammatory diseases has often been characterised by excessive side effects due to a lack of specificity for just one receptor type. For this reason, the study of the regulation of α7 and its duplicated isoform subunits in response to pro-inflammatory stimuli, is important to understand their role in the “cholinergic anti-inflammatory pathway”, in order to discover selective nicotinic receptor agonists and to develop novel anti-inflammatory treatments.

1. Abstract The p63 transcription factor, homolog to the p53 tumor suppressor, plays a crucial role in epidermal and limb development. Dominant mutations in the p63 gene give rise to several human congenital syndromes characterized by skin, craniofacial and limb defects. One of the syndromes caused by p63 mutations is the Split-Hand/Foot Malformation-IV (SHFM-IV) syndrome, characterized by the loss of central rays of hands and feet. These developmental defects are due to failure of Apical Ectodermal Ridge (AER) development. The correct limb outgrowth and patterning is guarantee by the expression of key molecules including Fybroblast Growth Factor 8 (FGF8), p63 and the DLX5 and DLX6 transcription factors. In this context, the study of the molecular mechanisms regulating p63 stability and function is fundamental for understanding the molecular bases of the SHFM-IV pathogenesis: indeed p63 as been proposed to be one of the crucial regulators of limb and epidermal development. Little is known on the post-translational modifications and the upstream signalling pathway controlling ΔNp63α functions, one of the most expressed p63 isoform in epithelial tissues and in the AER cells. The projects performed during my PhD thesis achieved to the identification of FGF8 as a crucial regulator of ΔNp63α stability and activity in human osteosarcoma and keratinocyte cell lines. FGF8 determined also ΔNp63α protein stabilization in mice embryonic limb buds put in culture at Embryonic day 10.5 (E10.5). In particular, treatments with FGF8 of human osteosarcoma cell lines (U2OS) and human keratinocytes (HaCat), activate the tyrosine kinase c-Abl, leading to ΔNp63α phosphorylation and consequent acetylation by the p300 acetyl-transferase, promoting ΔNp63α stabilization and transcriptional activation. Moreover, I have found that p300 interacts with ΔNp63α determing its acetylation on lysine K193E, in vitro. Interestingly, this regulatory cascade is not active on the natural ΔNp63αK193E mutant associated to the SHFM-IV syndrome. Indeed, the ΔNp63αK193E mutant displays promoter specific altered DNA binding activity that results in altered expression of ΔNp63α target genes involved in limb development (like Perp, Ikkα and DLX5 gene) (Manuscript in preparation). One of the mechanism by which FGF8 promotes ΔNp63α stability and activation, is inhibiting its interaction with Pin1, a prolyl isomerase known to positively regulate p53 and p73 in response to DNA damage stress. In particular, PIN1 has an opposite effect on ΔNp63α respect to p53 and p73: it promotes ΔNp63α degradation through the proteasome pathway. Moreover, ΔNp63α mutant proteins, associated with SHFM-IV or EEC syndromes, characterized by limb defects, are not degraded by PIN1 overexpression. These data were confirmed also by in vivo experiments on PIN1 Knock-Out (KO) mice, where lack of PIN1 expression caused the accumulation of p63 in the embryonic limbs and ectoderm compared to wild-type littermates. Moreover, I found that FGF8 is a downstream target of the transcription factor Dlx5. Indeed, in the limb buds of both p63 and DLX5;DLX6 KO mice, the AER is poorly stratified and FGF8 expression is severely reduced. All these data suggest that DLX5, ΔNp63α, FGF8 and PIN1 participate in a regulatory loop essential for AER stratification, normal patterning and morphogenesis of the limb buds (1). The work performed during my PhD contributes to a better understanding of the regulatory mechanisms controlling ΔNp63α function and stability. We have identified FGF8 as a crucial upstream signal required for ΔNp63α activation and stabilization during limb development: mutations or altered expression of regulators in this pathway leads to abnormal limb development and onset of pathogenesis.

Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends.

Background. The most frequent adrenocortical tumors (ACT) are benign forms defined as adrenocortical adenomas (ACA) with an estimated incidence of about 7.3%. Malignant forms, adrenocortical carcinomas (ACC) are rare endocrine tumors with poor prognosis and incidence of 1-2 cases per million people in the population. Currenlty ACC treatments are ineffective and substantially are based on the use of mitotane (o,p'DDD) with or without traditional chemotherapeutic agents. Because of dissimilar prognosis of ACA or ACC it is important to differentiate these two forms. Many tumors stimulate the growth of blood vessels by the angiogenesis process. Recently, vascular endothelial growth factor (VEGF) over-expression in ACC and VEGF receptors (VEGFR-1 and VEGFR-2) expression were found on tumor cells; these findings suggest a possible autocrine effect of VEGF for cell growth. Sorafenib, a multikinase inhibitor, inhibits the phosphorylation of VEGFR-2 and induces in vivo tumor growth arrest. Everolimus, a rapamycin derivative, inhibits mTOR, which is involved in cell survival and located downstream toVEGFR-2. It stops production of angiogenic growth factors in several neuroendocrine tumors. To date few genetic alterations were identified in ACT involving adrenocortical tumorigenesis signaling pathways; in particular the Ras/Raf/MEK/ERK and the Wnt/β-catenin signaling pathways seem to be often altered. β-catenin constitutive activation is an alteration frequently found in ACT. It is a multifunctional molecule involved in the processes of cell adhesion together with cadherin (E-cadherin and N-cadherin). Cadherins have been implicated in the development of many cancers, but information regarding their expression in the ACT is very limited. Aim. The objective of this study is: a) to evaluate the expression of VEGF, VEGFR-1 and VEGFR-2 in normal and tumoral adrenocortical tissues; b) to examine the effect of the two drugs, sorafenib and everolimus, in vitro on cellular viability, on apoptosis and on signal pathways of 2 stabilized lines (H295R, SW13) and in ACT primary cultures and in vivo in xenografts mouse models; c) to analyze the presence of genetic alterations in key components of the Ras/Raf/MEK/ERK signaling pathway (BRAF, H-RAS, K-RAS, N-RAS genes) and of Wnt/β-catenin signaling pathway (CTNNB1 and AXIN2 genes) and to investigate the β-catenin expression in relation to the cell adhesion molecules E-cadherin and N-cadherin. Material and Methods. 24 adrenocortical carcinomas (ACC), 37 aldosterone producing adenoma (APA), 28 cortisol producing adenoma (CPA), 6 non-secreting adenomas (NSA) and 8 normal adrenal gland tissues (NA) were collected. The VEGF and its receptors (VEGFR-1 and VEGFR-2) gene expression was evaluated in 63 ACT by real-time PCR. In cell lines (SW13 and H295R) and in primary ACT cultures, cell viability was observed by incubating sorafenib and everolimus in a range of concentrations from 10 µM to 0.1 nM by MTT test. By fluorescence microscopy (TUNEL) and flow cytometric analysis (Annexin-V), apoptosis was evaluated; by western blot, the involvement of PI(3)K/Akt/mTOR and in Ras/Raf/MEK/ERK signaling pathways was analyzed. The effects of drugs, alone or in combination, were tested in vivo in ACC xenografted models. By high resolution melting analysis (HRM), mutation analysis was performed on 95 ACT on BRAF (exons 11 and 15), H-RAS (exons 2 and 3), N-RAS (exons 2 and 3), K-RAS (exons 2 and 3), CTNNB1 (exon 3), AXIN2 (exon 7). Only samples with altered melting curves were sequenced. Finally, by real-time PCR and immunohistochemistry (IHC), β-catenin, N-cadherin and E-cadherin gene expression was evaluated in 68 ACT. Results. VEGF, VEGFR-1, VEGFR-2 expression was found in both adrenocortical normal and tumoral tissues. Compared to normal adrenal glands, a significant VEGF over-expression was observed in 65% (12/18) (P = 0.049) ACC and in 61% (20/33) (P = 0.025) APA. In SW13 and H295R cell viability, sorafenib and everolimus showed a dose-dependent response, while by fluorescence (TUNEL) and by flow cytometry (Annexin V) the compounds revealed an apoptotic effect. By western blot, sorafenib induced a complete decrease in Akt, ERK1/2 and p70S6K phosphorylation, while everolimus totally abolished p70S6K phosphorylation. Out of 8 primary cultures, 3 ACC and 3 ACA significantly responded to sorafenib and everolimus treatments. In vivo experiment a significant reduction of the tumor mass and an increase in median survival (especially in xenograft subjected to combined treatment) were observed. In HRM analisys some alterations in key components of the Ras/Raf/MEK/ERK signaling pathway were found, i.e. 2 BRAF mutations and 4 H-RAS mutations. In Wnt/β-catenin signaling pathway 18 alterations in CTNNB1 gene (5 APA, 6 CPA, 2 NSA, 5 ACC) and a single mutation in AXIN2 gene in H295R cells were observed. In RT-PCR β-catenin is over-expressed in approximately 50% of ACC (12/24) and in 51% of ACA (24/47). By IHC a significant accumulation of cytoplasmic and/or nuclear β-catenin has been observed in 47% of ACC (7/15) and 33% of ACA (11/33). In all ACT expression of E-cadherin was not detected. By RT-PCR N-cadherin down-regulation has been found in 75% of ACC (18/24) and in 60% of ACA (28/47). Similar results were obtained by IHC: N-cadherin down-regulation was observed in 100% (15/15) ACC and in 55% (18/33) ACA. Conclusion. Our data underline the importance of angiogenesis in adrenocortical tumors system. Anti-VEGF strategies, such as new tyrosine kinases and mTOR inhibitors currently used in different tumors, may represent a new therapeutic tool for adrenocortical tumors. The identification of new anti-angiogenic and Wnt/β-catenin signaling targets has contributed to a better understanding of adrenocortical tumorigenesis and to generate the basis for the development of new targeted drugs (targeted therapy).
Presupposti dello studio. I più comuni tumori corticali della ghiandola surrenalica (ACT) sono tumori benigni definiti adenomi corticosurrenalici (ACA) con una incidenza stimata di circa 7,3%. I tumori maligni, i carcinomi corticosurrenalici (ACC), sono invece rare neoplasie endocrine, con prognosi infausta ed incidenza di circa 1-2 casi per milione. I trattamenti per l’ACC sono inefficaci nella maggior parte dei casi ed attualmente si basano sull’uso di mitotane (o,p’DDD) con o senza i tradizionali agenti chemioterapici. Poiché le prognosi di ACA o di ACC sono estremamente differenti è importante differenziare queste due tipologie di tumore. Numerosi tumori attraverso il processo di angiogenesi stimolano la crescita dei vasi ematici dell’ospite. Recentemente è stata osservata una over-espressione del fattore di crescita endoteliale vascolare (VEGF) negli ACC ed i recettori di VEGF (VEGFR-1 e VEGFR-2) trovati anch’essi sulle cellule tumorali, suggeriscono un possibile effetto autocrino di VEGF per la crescita cellulare. Il farmaco sorafenib, un inibitore multichinasico, inibisce la fosforilazione di VEGFR-2 ed induce in vivo l’arresto della crescita di alcuni tumori. Il farmaco everolimus, analogo della rapamicina, inibisce mTOR coinvolto nella sopravvivenza cellulare e situato valle di VEGFR-2; è inoltre in grado bloccare la produzione di fattori pro-angiogenici in molti tumori neuroendocrini. Ad oggi, le alterazioni genetiche note che colpiscono gli ACT sono limitate ed inoltre nella tumorigenesi corticosurrenalica sono coinvolte una grande varietà di vie del segnale, in particolar modo la via del segnale di Ras/Raf/MEK/ERK e la via di Wnt/β-catenina risultano spesso alterate. Un’alterazione frequentemente riscontrata negli ACT è l’attivazione costitutiva della β-catenina, una molecola multifunzionale coinvolta anche nei processi di adesione cellulare assieme alle caderine (E-caderina ed N-caderina). Le caderine sembrano essere coinvolte nello sviluppo di molti carcinomi, ma le informazioni riguardanti la loro espressione negli ACT è molto limitata. Scopo. L’obbiettivo dello studio è stato quello di: a) valutare l’espressione di VEGF, VEGFR-1 e VEGFR-2 nei tessuti surrenalici normali e/o tumorali; b) esaminare l’effetto dei due farmaci, sorafenib ed everolimus, in vitro sulla vitalità cellulare, sull’apoptosi e su alcune pathways cellulari in linee stabilizzate (H295R, SW13), in colture primarie di ACT ed in vivo in modelli murini xenotrapiantati; c) analizzare la presenza di alterazioni geniche nei componenti chiave della via del segnale di Ras/Raf/MEK/ERK (studiando i geni BRAF, H-RAS, K-RAS, N-RAS) e nella via del segnale di Wnt/β-catenina (valutando i geni CTNNB1 e AXIN-2) ed indagare l’espressione della β-catenina in relazione alle molecole di adesione cellulare E-caderina ed N-caderina. Materiali e Metodi. Sono stati raccolti 24 carcinomi corticosurrenalici (adrenocortical carcinoma, ACC), 37 adenomi secernenti aldosterone (aldosterone producing adenoma, APA), 28 adenomi secernenti cortisolo (cortisol producing adenoma, CPA), 6 adenomi non secernenti (non-secreting adenoma, NSA) e 8 campioni di surrene normale (normal adrenal, NA). L’espressione genica di VEGF e dei suoi recettori (VEGFR-1 e VEGFR-2) è stata valutata in 63 ACT tramite real-time PCR. Nelle linee cellulari (SW13 e H295R) e nelle colture primarie corticosurrenaliche, attraverso il test MTT è stata osservata la vitalità cellulare incubando con sorafenib ed everolimus in un range di concentrazioni da 10 μM a 0,1 nM. In microscopia a fluorescenza (TUNEL) e dall’analisi citofluorimetrica (Anessina V) è stato valutato il grado di apoptosi indotto dai farmaci e tramite western blotting è stato analizzato il loro possibile coinvolgimento nelle vie di PI(3)K/Akt/mTOR e di Ras/Raf/MEK/ERK. Gli effetti dei farmaci, da soli o in combinazione, sono stati testati poi in vivo in modelli murini di ACC xenotrapiantati con cellule H295R ed SW13. Attraverso l’analisi di high resolution melting (HRM) abbiamo ricercato in 95 ACT la presenza di mutazioni attivanti nei geni BRAF (esoni 11 e 15), H-RAS (esoni 2 e 3), N-RAS (esoni 2 e 3), K-RAS (esoni 2 e 3), CTNNB1 (esone 3), AXIN2 (esone 7). I campioni aventi curve di melting dubbie o alterate sono stati sottoposti tutti al sequenziamento. Infine, è stata valutata in 68 ACT l’espressione genica di β-catenina, N-caderina ed E-caderina attraverso le tecniche di real-time PCR e di immunoistochimica (IHC). Risultati. Sia i tessuti corticosurrenalici normali che tumorali esprimono VEGF, VEGFR-1, VEGFR-2. Rispetto ai surreni normali, una significativa over-espressione di VEGF è stata osservata in circa il 65% (12/18) (P = 0.049) degli ACC analizzati ed in circa il 61% (20/33) (P = 0.025) degli APA. Nella valutazione della vitalità cellulare le cellule SW13 ed H295R rispondono in modo dose-dipendente ai farmaci sorafenib ed everolimus mentre in fluorescenza ed in citofluorimetria è stato osservato un effetto apoptotico. Mediante western, sorafenib induce una completa diminuzione della fosforilazione di Akt, ERK1/2 e P70S6K mentre everolimus, abolisce totalmente la fosforilazione di p-P70S6K. Delle 8 colture primarie sottoposte ai farmaci, 3 carcinomi e 3 adenomi rispondono in modo significativo sia al trattamento con sorafenib che all’everolimus, mentre 2 casi di ACC sono resistenti ad entrambi i farmaci. In vivo è stata osservata una significativa riduzione della massa tumorale ed un aumento della sopravvivenza mediana soprattutto nei topi sottoposti al trattamento farmacologico combinato. Attraverso le indagini in HRM sono state riscontate alcune alterazioni nei componenti della via del segnale di Ras/Raf/MEK/ERK: 2 ACC con mutazioni nel gene BRAF e 1 APA, 1 CPA e 2 ACC con mutazioni nel gene H-RAS. Per quanto riguarda l’indagine genetica della via del segnale di Wnt/β-catenina sono state osservate 18 alterazioni nel gene CTNNB1 (5 APA, 6 CPA, 2 NSA, 5 ACC) ed una sola mutazione nel gene AXIN2 nelle cellule H295R. In RT-PCR l’espressione di β-catenina è over-espressa in circa 50% degli ACC (12/24) ed in circa il 51% degli ACA (24/47) mentre in IHC circa il 47% degli ACC (7/15) ed il 33% degli ACA (11/33) presenta un notevole accumulo citoplasmatico e/o nucleare di β-catenina. In tutti gli ACT non è stata rilevata l’espressione di E-caderina, mentre per quanto riguarda N-caderina, in RT-PCR circa il 75% degli ACC (18/24) ed il 60% degli ACA (28/47) possiedono una down-regolazione. Risultati simili sono stati ottenuti in IHC in cui il 100% degli ACC (15/15) e il 55% degli ACA (18/33) presentano una down-regolazione di N-caderina. Conclusioni. I nostri dati sottolineano l’importanza del sistema angiogenico nei tumori corticosurrenalici. I nuovi inibitori tirosin-chinasici e di mTOR attualmente usati in alcuni tumori come strategie anti-VEGF potrebbero rappresentare un nuovo strumento terapeutico per i tumori della ghiandola surrenale. L’identificazione quindi di nuovi target molecolari anti-angiogenetici e di target nella via del segnale di Wnt/β-catenina ha contribuito ad una migliore comprensione ed a un approfondimento della tumorigenesi corticosurrenalica generando le basi per lo sviluppo di nuovi farmaci mirati (target therapy).

Melanoma is an aggressive disease with high metastatic potential and resistance to cytotoxic agents. Early-stage melanomas can be successfully cured by surgery and, as in all solid tumours, morbidity and mortality of melanoma are a consequence of local invasion and metastatic spread. The molecular mechanisms involved in the progression of the malignancy, the genetic markers associated to metastatic melanoma dissemination and the acquisition of chemoresistance are only beginning to be defined. An understanding of the molecular biology of melanoma provides a necessary basis to enable the generation of more effective therapeutic modalities. RaLP, a new member of the SHC family of adaptor proteins was previously characterized in our laboratory as a determinant in the regulation of migration of melanoma cells in short-term assays in vitro. In this study we further characterized the role of RaLP in the progression of melanoma. We have verified that the expression of RaLP significantly correlates with the most important prognostic markers of melanoma (Breslow thickness, Clark’s level of invasion, ulceration, mitotic index and presence of metastasis in lymph nodes) and that patients with RaLP expressing tumours had reduced disease-free survival and overall survival, suggesting that RaLP can be identified as a novel prognostic molecular marker and an independent prediction factor of melanoma progression. We have shown that permanent RaLP silencing does not interfere with the proliferation of three different metastatic melanoma cell lines, while it significantly decreases their migration and this phenomenon was observed even after extended time in culture. The phenotype could be rescued by the overexpression of RaLP in the silenced cells, suggesting that RaLP is a central molecule that positively regulates migration of melanoma cells. Besides regulating migratory abilities of the melanoma cells, RaLP positively influences their invasive potential, by regulating collagen matrix digestion. We also tested cell – cell and cell – ECM adhesion abilities of melanoma cells after RaLP ablation. We observed that RaLP decreases adhesion of the cells to each other in cell – cell adhesion assays and negatively regulates adhesion of melanoma cells to different matrices in cell – ECM adhesion assay. Analyzing gene expression profiles of RaLP – proficient and – deficient cells we have shown that RaLP is involved in the regulation of the NOTCH molecular pathway. Our in vitro studies suggest that RaLP expression in melanoma might facilitate dissociation of metastatic cells from a tumour mass by loosening cell – cell adhesion, and favour invasion of the surrounding tissues. We still do not know the exact signalling cascade by which RaLP regulates cell motility and adhesion processes and additional studies are necessary to fully understand its role in melanoma progression.

La malattia di Parkinson (MdP) é il più comune disturbo neurodegenerativo del movimento, che colpisce circa 2-3% delle persone sopra i 65 anni di età. L´eziologia della malattia non è ancora definita ma casi familiari hanno fornito indizi sui meccanismi coinvolti nella malattia. In particolare, alterazioni genetiche nei geni codificanti alfa-sinucleina (aSyn) e leucine-rich repeat kinase 2 (LRRK2) sono alla base di forme autosomiche dominanti della malattia, le quali ricalcano le caratteristiche salienti della forma sporadica. Le inclusioni intraneuronali, definite corpi di Lewy e costituite prevalentemente da aSyn, sono uno dei segni distintivi della presentazione neuropatologica nella MP. I meccanismi coinvolti nella formazione, mantenimento e degradazione delle inclusioni sono tutt´ora oggetto di studio approfondito. Si ipotizza che diversi processi cellulari siano alla base di tali meccanismi, con una particolare attenzione rivolta all´autofagia. Inoltre, diversi geni associati ad aumento di rischio di MdP rivestono un ruolo nella biologia lisosomale e nell´autofagia stessa. Linee cellulari ricombinanti di neuroblastoma che sovra esprimono LRRK2 WT o G2019S riportano difetti in diversi stadi della via autofagia-lisosomi, in particolare accumulo di autofagosomi, alterazioni nella morfologia dei lisosomi e nel loro numero. Le cellule LRRK2 G2019S specificamente sono caratterizzate da inclusioni proteiche positive per pS129-aSyn. L´inibizione farmacologica dell´attività chinasica di LRRK2 in queste cellule corregge i deficit nella via autofagia-lisosomi e riduce il numero di inclusioni di pS129-aSyn. RIT2, che codifica per la proteina Rin, è un nuovo fattore di rischio genetico per la MdP che è stato scarsamente studiato finora. Si tratta di una piccola GTPasi che media la crescita neuritica tramite le vie di segnalazione delle MAPK p38 ed ERK1/2. Inoltre, RIT2 è arricchiato nei neuroni della substantia nigra pars compacta nei roditori e la sua espressione è ridotta nel cervello di pazienti di MdP. Tuttavia, non sono disponibili evidenze sperimentali sulle vie di segnalazione dipendenti da Rin e/o sulle eventuali interazioni con proteine legate causalmente alla MdP. Abbiamo appurato che i livelli di mRNA di RIT2 sono ridotti nelle cellule WT e G2019S LRRK2 ed inoltre l´attività del suo promotore è ridotta nelle stesse linee cellulari. La sovra espressione acuta di RIT2 produce una fenocopia del trattamento con l´inibitore chinasico di LRRK2, in riferimento al numero di autofagosomi, alla morfologia e numero di lisosomi e la loro attività proteolitica. In aggiunta, la sovra espressione di RIT2 riduce il carico di inclusioni positive per p S129-aSyn. Questa strategia inoltre riduce la fosforilazione di LRRK2 a carico della S1292, ma non della S935, e questo aspetto la differenzia dal trattamento farmacologico con inibitore di LRRK2. Inoltre, i livelli di espressione proteina totale di LRRK2 non vengono ridotti dalla sovra espressione di RIT2, a differenza di quanto accade in seguito ad inibizione farmacologica. La sovra espressione di RIT2 tramite vettori virali in un modello in vivo di tossicità di aSyn nel topo porta ad un miglioramento dell´attività motoria e riduce la perdita neuronale dopaminergica, indotta dall´espressione virale di aSyn. Inoltre, aSyn induce un aumento dei livelli di pS1292 LRRK2, indicativi di un´aumentata attività di LRRK2 endogena. La co-espressione con RIT2 previene tale aumento, suggerendo un´azione di RIT2 su LRRK2 stessa. Questi dati indicano che aSyn, RIT2 e LRRK2 sono alla base di meccanismi cellulari comuni, specialmente autofagia, e modulano a valle il catabolismo di aSyn. La modulazione dell´espressione di RIT2 potrebbe quindi costituire una nuova strategia terapeutica indirizzata alla patologia da aSyn, applicabile sia alla MdP familiare che idiopatica e, idealmente, alle altre sinucleinopatie.
Parkinson’s disease (PD) is the most common neurodegenerative movement disorder and affects about 2-3% of the population over the age of 65. The etiology of PD is still undefined and familial cases provided hints for mechanisms involved in the disease. In particular, alpha-synuclein (aSyn) and leucine-rich repeat kinase 2 (LRRK2) gene alterations lead to autosomal dominant PD with many commonalities to the idiopathic disorder. aSyn containing inclusions, termed Lewy bodies, are one of the neuropathological hallmarks of PD, but the mechanisms involved in formation, maintenance and degradation of these inclusions are still under intense investigation. Several cellular processes are hypothesized to mediate these mechanisms, with a prominent attention directed towards autophagy. Additionally, several PD risk genes have a role in lysosomal biology and autophagy itself. Recombinant neuroblastoma cell lines overexpressing WT and G2019S LRRK2 display defects at different stages of the autophagy-lysosome pathway (ALP), specifically accumulation of autophagosomes and abnormalities in lysosomal morphology and number. Interestingly, G2019S LRRK2 cells display inclusions staining positive for pS129-aSyn. Pharmacological inhibition of LRRK2 kinase activity in G2019S LRRK2 cells reverts ALP defects and most importantly reduces the number of pS129-aSyn inclusions. RIT2, coding for the protein Rin, is a novel PD gene risk factor and has been scarcely investigated. It is a small GTPase mediating neurite outgrowth through p38 and ERK1/2 MAPK signaling pathways. Moreover, Rin is enriched in rodent substantia nigra pars compacta neurons and is reduced in human PD brains. However, the relevance of Rin-dependent signaling pathways and/or its interactions with PD-causing proteins is currently lacking. We observed that RIT2 mRNA levels are reduced in WT and G2019S LRRK2 cells and also RIT2 promoter activity is reduced in these cell lines. Acute overexpression of RIT2 phenocopies the effects of LRRK2 kinase inhibitor treatment, specifically on the number of autphagosomes, morphology and number of lysosomes and their proteolytic function. Moreover, RIT2 overexpression strongly reduces the burden of pS129-aSyn inclusions. Interestingly, acute RIT2 overexpression reduces phosphorylation levels of S1292, but not S935 LRRK2, an aspect distinct from pharmacological LRRK2 kinase inhibition. Furthermore, total LRRK2 levels are not decreased with RIT2 overexpression, unlike after LRRK2 kinase inhibitor treatment. Viral overexpression of RIT2 in an in vivo aSyn mouse model ameliorates motor deficits and reduces dopaminergic neuron loss, induced by viral overexpression of aSyn. Viral aSyn overexpression induces an increase in pS1292 LRRK2 levels, indicating an enhanced activity of endogenous LRRK2. The co-expression of aSyn with RIT2 prevents the increase of LRRK2 kinase activity, indicating that RIT2 is acting on LRRK2. This data suggest that aSyn, LRRK2 and RIT2 might share common cellular mechanisms, in particular autophagy, and ultimately impact the degradation of aSyn. The modulation of RIT2 expression might constitute a new strategy to combat aSyn pathology, applicable to both familial and idiopathic PD and, possibly, other synucleinopathies.

Rett syndrome (RTT) is a rare and progressive neurodevelopmental disorder that occurs in 1:10,000-15,000 females. RTT is characterized by a normal early growth followed by, above all, motor and cognitive regression. RTT is caused by mutations of MECP2 gene, located on X-chromosome and subjected to random inactivation, that generate a very variable phenotype. Methyl-CpG-binding protein-2 (MeCP2) is a transcriptional factor involved in brain connectivity, neural circuits and importantly, in synaptic plasticity and deficits. However, the molecular mechanisms related with these defects are largely unknown. In previous works, we showed that c-Jun N-terminal protein kinase (JNK), a stress-activated kinase, was strictly involved in synaptic dysfunction related to neurodegenerative disease (Alzheimer’s disease and ischemic stroke) and that its specific inhibition, using the cell permeable D-JNKI1 peptide, led to a recovery of dendritic spines structure and to restoration of functionality supported by a rescue of cognitive deficits. We here proved for the fist time that JNK signalling is powerfully activated in RTT mice and acts as a key modulator of synaptic dysfunctions. The MeCP2 tm 1.1 Bird male mice (referred as Mecp2 y/-) were chosen for our evaluation because, despite not presenting mosaicism, they show an early onset and a most severe phenotype within a homogeneity of genetic background. D-JNKI1 treatment (22mg/kg) was administrated for the first time at 3rd week of age with an intraperitoneal injection and repeated after 3 weeks. Well-being and behavioural studies were effectuated with a weekly examination of food and water intake, weight and locomotor abilities. At 7 weeks, mice were sacrificed and tissues were processed for biochemical evaluations. In brain, we found a strong activation of JNK’s preferential target, c-Jun, a nuclear transcription factor. D-JNKI1 chronic treatment improved general mice wellbeing. Treated mice showed a rescue of motor deficits and an improvement of motor coordination, parameters evaluated with Rotarod and Open field behavioural tests. Since RTT is characterized by locomotor impairment, we checked in cerebellum the synaptic dysfunction evaluating AMPA and NMDA receptors levels. Isolating the post-synaptic region, we found that D-JNKI1 treatment rescued receptor levels. Moreover, PSD95 and Shank3 analysis revealed that the decrement of Mecp2 -/y mice was reported to control level thanks to D-JNKI1 treatment. Moreover, our studies were focused on inflammatory pathway triggered by JNK and we found an astrogliosis and a microgliosis activation, completely rescue by D-JNKI1 treatment. We then move to translational medicine to strengthen the JNK pivotal role in RTT by using Human RTT iPSCs. The mutant neuronal-IPSc presented JNK activation while isogenic control neuronal-IPSc did not; furthermore, we found that D-JNKI1 inhibited JNK activity. The results on iPSCs added value to the clinical relevance of the proposed treatment. RTT is a rare and incurable progressive postnatal female neurodegenerative disorder and the manipulation of JNK pathway may represent the development of an innovative strategy to tackle Rett Syndrome. JNK plays had shown a key role in both mice and human mutated neuronal-IPSc and consequently its relevance in clinical study. We now need to better characterize D-JNKI1 effect in female mosaicist model, closer to the human phenotype.

The central theme of this thesis is TAZ, a transcriptional co-activator that works - together with its homologue YAP - as transducer of the mammalian Hippo pathway. Since the beginning of my PhD training, I have been involved in the investigation of TAZ function and regulation in breast cancer stem cells. Our findings established TAZ as a molecular determinant of self-renewal and tumorigenic potential in breast cancer cells. Indeed, the vast majority of high grade breast tumors (known to be enriched with cancer stem cells) display a strong positive staining for TAZ, compared with normal mammary tissue and low grade tumors. Experiments described in the first part of this thesis show that TAZ protein is stabilized in cells that have undergone an epithelial-to-mesenchymal transition, or simply lost the apico-basal polarization proper to epithelial cells. Indeed, in polarized epithelial cells the basolateral determinant Scribble activates the Hippo kinases to inhibit TAZ; in depolarized cells (including cells in malignant carcinomas) this inhibitory mechanism is disabled and TAZ can accumulate. How TAZ overexpression modifies the cell to turn it into a cancer stem cell is the object of ongoing work. Part of these results was published in (Cordenonsi et al., 2011). During the last year, I have collaborated on a second project, dealing with TAZ regulation by Wnt signaling (Azzolin et al., 2012). We have found that Wnt signaling activates TAZ along with β-catenin. This happens because, in the absence of Wnt signals, phosphorylated β-catenin drives TAZ degradation; Wnt stimulation thus leads to the parallel stabilization of β-catenin and TAZ. Experiments presented here show that β-catenin effectively inhibits TAZ activity in the absence of Wnt signaling, and that TAZ stabilization is instrumental in both physiological and pathological Wnt biological responses.
Il tema centrale di questa tesi è TAZ, un co-attivatore trascrizionale che – insieme al suo omologo YAP – funge da trasduttore della Hippo pathway nei mammiferi. Dal primo anno di dottorato, ho partecipato allo studio della funzione e la regolazione di TAZ nelle cellule staminali del tumore al seno. I risultati ottenuti dimostrano che TAZ è un determinante molecolare della capacità di auto- rinnovamento (self-renewal) e delle proprietà tumorigeniche di cellule di tumore mammario. La maggioranza dei tumori di alto grado istologico (che contengono un numero elevato di cellule staminali tumorali) esprimono livelli di TAZ più alti rispetto al tessuto sano della ghiandola mammaria e ai tumori di basso grado istologico. Gli esperimenti descritti nella prima parte di questa tesi dimostrano che TAZ è stabilizzato – a livello di proteina – in cellule epiteliali che hanno subito una trasformazione mesenchimale, o hanno perso la polarità apico-basale tipica delle cellule epiteliali. Infatti, in cellule epiteliali polarizzate Scribble, un determinante basolaterale, attiva le chinasi della Hippo pathway ed inibisce TAZ; in cellule depolarizzate (come molte cellule nei carcinomi maligni) questo meccanismo è inattivo e TAZ si accumula. Come la stabilizzazione di TAZ modifichi le cellule per trasformarle in cellule staminali tumorali è oggetto di studi attualmente in corso. Parte di questi risultati è stata pubblicata in (Cordenonsi et al., 2011). Durante l’ultimo anno, ho collaborato ad un secondo progetto, dedicato alla regolazione di TAZ da parte di Wnt/β-catenina (Azzolin et al., 2012). I segnali Wnt attivano TAZ insieme a β-catenina. Ciò è dovuto al fatto che, in assenza di Wnt, β- catenina fosforilata porta TAZ al proteasoma; la stimolazione con Wnt determina quindi la stabilizzazione parallela di β-catenina e TAZ. Gli esperimenti qui presentati dimostrano che β-catenina inibisce l’attività biologica di TAZ in assenza di Wnt, e che la stabilizzazione di TAZ ha un ruolo funzionale nelle risposte biologiche (sia fisiologiche che patologiche) ai segnali Wnt.