Academic literature on the topic 'SCUOLA DI DOTTORATO DI RICERCA IN BIOMEDICINA MOLECOLARE'

2008/2009
Non-syndromic cleft lip with or without cleft palate (NSCLP) is one of the most common birth defect. Genetic studies on human populations have identified numerous predisposing factors, as MYH9 and JARID2, whose role during palatogenesis remains obscure. In order to improve our knowledge on pathogenetic mechanisms, we carried out expression studies during mouse palate development using RNA in situ hybridization. As reference genes (Tgfb3, Irf6, Pvrl1, Foxe1 and Tp63) known to be required for correct palate formation, Jarid2 and Myh9 are expressed in the epithelial cells of the palatine processes before and at the time of contact. Then, this signal decreases and eventually disappears concomitantly with the degradation of the medial epithelial cells. Consistent with these observations, RT-PCR carried out on dissected palatal shelves detected products of Myh9 and Jarid2 from embryonic day E14.0 to E15.0 with a pick at E14.5, the stage when shelves appose in the midline. Taken together, these expression studies strongly support the association studies that designate these genes as predisposing factors for NSCLP. In multifactorial diseases as NSCLP once genetic studies demonstrate association, a major challenge is to identify mutations. In this regard, we started analyzing two in linkage disequilibrium SNPs (rs3752462 of MYH9 and rs2076056 of JARID2) that could be involved in defective splicing mechanisms, as hypothesized on the basis of their localization within splice sites. Using a hybrid minigene assay, however, we demonstrated that none of the allelic variants lead to any aberrant products at least in HeLa cells, the model used for this study. Moreover, we investigated whether alternative splicing events of the Myh9 gene detected in cochlea and brain (Li et al., 2008) could also be found in other tissues and palate. A small insertion of 12 bp between exon 4 and 5 (loop1) due to an alternative splicing was detected in all adult tissues analyzed. The same insertion was not detected in embryonic tissues, such as palatal samples at different stages of development, and brain. These results suggest that the genomic region of loop1 should be investigated in all risk haplotypes to search for pathogenetic variants. In conclusion, further investigations should be planned to explore the role of MYH9 and JARID2 in orofacial development in order to dissect signaling pathways during palate formation and identify the causative variants contributing to NSCLP.
XXII Ciclo
1973

2008/2009
The design of osseous implants, either load bearing or not, with desired mechanical and surface features that promote integration with bone and avoid risks of bone resorption and implant failure due to shear stresses, is still a challenging endeavour. The mechanical stresses which the skeleton undergoes affect bone formation and resorption processes. Bone remodelling is often promoted by adequate stress/strain conditions which are able to prevent bone mass loss. The largely used metallic implants offer several advantages like easy shape casting and modelling but include also several drawbacks like high stiffness if compared with the mechanical properties of native bone. A new generation of bone prosthesis is therefore indispensable to overcome the limitations of the obsolete metallic devices. In the orthopaedic framework, promising results have been achieved in the recent decades by three-dimensional structures named scaffolds. It is mandatory for any optimal scaffold to act as a temporary three-dimensional support for cell adhesion, growth and mineral matrix deposition. Moreover, ideal scaffolds should be able to integrate into surrounding tissue and mimic the structure and morphology of the natural bone tissue. Strict requirements for scaffolds are biocompatibility, a design closely resembling the natural extracellular matrix, an appropriate surface chemistry to promote cellular attachment, differentiation and proliferation and a sufficient mechanical strength to withstand in vivo stresses and physiological loading. Finally, the degradation of the ideal scaffold should proceed in a controlled way, keeping a sufficient structural integrity until the newly grown tissue has replaced the scaffold's supporting functions. Coupling a three-dimensional porous scaffold and a load bearing structure with suitable mechanical properties it is possible to obtain a device where the osteoconductive and osteoinductive properties of the former are synergistically linked with the mechanical ones of the latter. In this work both the aspects – osteointegration and load bearing – of an ideal prosthesis have been investigated. Alginate/Hydroxyapatite composite scaffolds were developed to be used either as scaffolds for sub-critical defects or as coatings for load bearing non-metallic bone prostheses. In both cases the investigation aimed to select suitable components and casting procedures to obtain the best results. The features of the single components and of the final three-dimensional structure were extensively investigated in order to obtain the most clarifying characterization both in terms of physical-chemical properties and in terms of biological responsiveness. The experimental section of this work involved physical-chemical analysis that helped to characterize both the organic and the inorganic components of the scaffold, respectively alginate and hydroxyapatite, before and after composite assembling. This investigation, based on several techniques (NMR, Rheology, XRD, Raman and TEM) allowed to characterize in detail the scaffold’s components and revealed the possibility of using the hydroxyapatite as a source of calcium ions for the gelification of the alginate without loosing the paramount osteoinductive properties of the mineral. Micro Computed Tomography (µ-CT) was employed to understand quantitatively the architectural features of the three-dimensional matrix obtained after alginate gel casting process. Moreover, this tool allowed to assess the influence of different manufacturing protocols (e.g. concentration of the components, casting temperatures) on the scaffold’s final structure. The results obtained by means of µ-CT coupled with the ones of Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) analysis of the scaffolds showed an optimal interconnected porous structure with pore sizes ranging between 100 m and 300 m and over 88% porosity. Proliferation assays and SEM observations demonstrated that human osteosarcoma cell lines were able to proliferate, maintain osteoblast-like phenotype and massively colonize the scaffold structure. Once the in vitro behaviour of the structure was clear, in vivo tests were performed. Cone-like Alg/HAp scaffolds were tested on skeletally mature female New Zealand White rabbits and compared with positive (bioactive glass scaffold) and negative (without any implant) controls. Ex vivo investigations of the dissected samples were based on µ-CT and histological analysis and revealed high level of osteointegration and osteoconduction of the scaffolds. Moreover, efforts have been made to link the porous structure to the non-metallic fibre reinforced composite used as load bearing unit. Overall, these combined results indicate that the structure here developed is promising for being employed in orthopaedic applications.
XXII Ciclo
1978

2012/2013
The tumor suppressor p53 plays a central role in the protection against DNA damage and other forms of stress, primarily by inducing cell cycle arrest or apoptosis. Missense mutation of p53, which is one of the most frequent genetic alterations detected in human cancers, inactivates these growth regulatory functions; in addition, mutant p53 often acquires tumor-promoting activities (gain-of-function). A complete and thorough understanding of the signaling circuitry that regulates wild-type and mutant p53 functions is therefore a primary objective for basic cancer research, since it may lead to development of important tools for diagnosis and therapy of tumors. One crucial component of such knowledge is the molecular mechanism leading to protein activation. Deregulated ribosome biogenesis is commonly observed in cancers as a result of increased biosynthetic demand due to uncontrolled cell proliferation. Cells actively monitor the fidelity of ribosome biogenesis, activating cellular checkpoints when this process is altered. Indeed, perturbations to many aspects of ribosome biogenesis generate a “nucleolar stress” that triggers a p53 response. Evidence in animal models indicates that the ribosomal-p53 checkpoint is indeed important for tumor suppression in vivo. In the recent past, we identified the nucleolar protein GTPBP4 as a novel p53 interactor, and established a functional link between the two proteins by demonstrating that GTPBP4 depletion promotes p53 accumulation and activation. In this Thesis, I demonstrate that GTPBP4 depletion affects 28S rRNA processing, generating a “nucleolar stress” responsible for p53 stabilization. The molecular mechanism responsible for such accumulation relies on the binding of ribosomal proteins to Mdm2. In parallel, I report that the same molecular mechanism leading to wild-type p53 stabilization upon ribosomal stress, can also promote mutant p53 accumulation in cancer cells. Thus, deregulated ribosome biogenesis may contribute to the stability of mutant p53 in tumors. These results collectively suggest that upon alterations in ribosome biogenesis, the TP53 status may be critical to drive the nucleolar surveillance pathway towards a tumor suppressive or oncogenic outcome.
L’oncosoppressore p53 svolge un ruolo cruciale nella protezione della cellula da danni al DNA e da altre forme di stress, principalmente inducendo arresto del ciclo cellulare e apoptosi. Mutazioni missenso nel gene TP53 sono le alterazioni genetiche più frequenti trovate nei tumori umani ed inattivano queste funzioni regolative; oltre a questo, p53 mutata acquisisce nuove proprietà oncogeniche (gain-of-function). Capire i meccanismi regolativi che sono alla base delle funzioni di p53 wild-type e mutata è quindi un fondamentale obiettivo per la ricerca di base nel cancro, dal momento che può portare allo sviluppo di importanti strumenti sia diagnostici che terapeutici. Un aspetto fondamentale di tale ricerca è la comprensione dei meccanismi molecolari responsabili dell’attivazione della proteina. La sintesi dei ribosomi è frequentemente deregolata nei tumori, in quanto le cellule hanno un aumentato fabbisogno biosintetico a causa dell’incontrollata proliferazione. La cellula monitora attivamente la fedeltà della sintesi dei ribosomi, attivando meccanismi molecolari quando questo processo è compromesso. Infatti, alterazioni ad ogni livello nella sintesi dei ribosomi genera uno “stress nucleolare” che attiva la risposta di p53. Alcune evidenze in modelli animali infatti suggeriscono l’importanza delle funzioni oncosoppressive dell’asse molecolare costituito dalle proteine ribosomali e p53. In un lavoro pubblicato recentemente, abbiamo identificato la proteina nucleolare GTPBP4 come nuovo interattore di p53, inoltre abbiamo dimostrato un collegamento funzionale tra le due proteine, in quanto la deplezione di GTPBP4 promuove l’accumulo e l’attivazione di p53. In questa Tesi ho dimostrato che la deplezione di GTPBP4 altera il processamento del RNA ribosomale 28S, causando uno “stress nucleolare” responsabile per la stabilizzazione di p53. Il meccanismo molecolare necessario per la stabilizzazione di p53 dipende dal legame di proteine ribosomali a Mdm2. In parallelo, ho dimostrato che lo stesso meccanismo molecolare responsabile dell’accumulo di p53 wild-type in seguito allo stress ribosomiale, può indurre la stabilizzazione anche di p53 mutata. Quindi, un’alterata sintesi ribosomale può contribuire alla stabilità di mutant p53. Questi risultati suggeriscono che in seguito ad alterazioni nella sintesi dei ribosomi, lo stato di p53 può essere critico nel determinare se la risposta cellulare sia di natura oncosoppressiva oppure oncogenica.
XXV Ciclo
1980

2009/2010
In infants and in the Crigler-Najjar syndrome type I patients, severe hyperbilirubinemia due to high levels of unconjugated bilirubin (UCB) may cause Kernicterus, leading to an irreversible and selective brain damage. The Gunn rat is the animal model for the study of these pathologies. It has been suggested that different enzymes of the phase I (cytochrome P-450-dependent mixed function oxygenases 1A1, 1A2, 2A3), phase II (glutathione-S-transferases α2, α3, µ3, µ4, π) and phase III transporters (particularly, Mrp1) seems to be involved in UCB detoxification pathways. However, to date, their in vivo brain expression has been evidenced only at the blood-brain interfaces, while remains largely unexplored in brain parenchyma. Particularly for Mrp1, in vitro evidence reported a role of this transporter in protection of neural primary cultures from dissected cortex, by extruding bilirubin out of the cell. The aim of this study is establish the developmental profile of these genes in brain parenchyma, and assess their alteration in hyperbilirubinemic jj animals. Due to the high regional selectivity of UCB-induced neurotoxicity, cerebellum (Cll), striatum (St), hippocampus (Hip) and cerebral cortex (Cx) were chosen for this study. Our results regard the Mrp1 protein in cerebral cortex of normobilirubinemic (JJ) rats showed that its expression varied during the post-natal age, reaching the highest levels at 9 days after birth. No changes were found between JJ and Jj (having a temporary hyperbilirubinemia in the first week of life) rat for all ages analyzed. Similarly, no differences were detected among JJ/Jj and jj (hyperbilirubinemic) rats at P2, P17 and P60, while a significant increase (p < 0.005) was evidenced in P9 jj rats as compared to age-matched JJ animals. Our Mrp1 mRNA analysis in four regions of P9 animals by Real Time-qPCR revealed the absence of differences among Cx, Cll, St and Hip of P9 normobilirubinemic JJ rats. Moreover, no variations between jj and JJ control animals were detected. Regarding the Mrp1 protein expression in the same four regions by Western blot analysis, our results showed that the levels of this transporter in normobilirubinemic JJ rats were lower in Cx, similar in Cll, St and Hip (p < 0.05 vs Cx). Comparing genotypes, a reduction on Mrp1 in jj animals (compared to Mrp1 amount in the same region of JJ pups) was detected in Cll, St, but reached the statistical significance only in Hip (p < 0.05 vs Hip JJ). The analysis of CYPs gene expression in P9 Gunn rats indicate that CYP1A1, 1A2 and 2A3 mRNA were differently expressed among Cx, Cll, St and Hip of JJ rat. Similarly a region-specific modulation of CYPs expression in jj Gunn rats (compared to JJ) was pointed-out. Surprisingly, UCB seems to generate a plateau effect on CYPs mRNA levels among brain regions of jj rats. In P60 JJ Gunn rats the CYPs expression is higher than in P9 animals, with the following pattern among regions: Cx  CllSt  Hip. A down-regulation (p < 0.05) in St of P60 jj compared to normal animals was observed. Analyzing the GSTs expression in P9 animals, higher variability in the GSTs expression among the four brain areas was evidenced. In hyperbilirubinemic (jj) rats (compared to JJ), statistically relevant down-regulations were detected for GSTα2 (in St;p < 0.05), GSTα3 (in Hip;p < 0.05), µ3 (in Cx;p < 0.01), µ4 (in Cx; p < 0.05) and π (in Cll: p < 0.05); while GSTµ4 was up-regulated in St (p < 0.05). From P9 to P60, in JJ animals: GSTα3 expression increased (13-75-fold depending on the region); while GSTα2 (5-fold), µ3 (p < 0.05), µ4 (2-fold) and π (2-fold) mRNA amounts decreased. In P60 jj Gunn rats, compared to controls (JJ):a relevant up-regulation of GSTα3 was observed in Cll (p < 0.005) and Hip (p < 0.05), while GSTµ3 in jj was down-regulated (p < 0.05). The Mrp1 results obtained in the present in vivo study seems not to be in agreement with the in vitro data reported, to date. Thus, the Mrp1 expression is low in brain parenchyma and bilirubin affect (up-regulation) only marginally the protein amounts in cortex of P9 animals, while in other regions Mrp1 is not modulated, indicating a marginal role in vivo in bilirubin clearance. Similarly, while in liver GSTα2 and α3 act together as ligandin, this seems not happens in brain where the two subunit are expressed at very low levels (P9: α2 77000- α3 1500 fold difference; P60: α2 112000-α3 2200 fold difference with respect to age matched livers). For all genes under analysis, a very complex and variable pattern of expression among brain areas was evidenced. Consequently, no general rules concerning bilirubin-induced modulation could be drawn, as both up and down-regulation were observed. Additionally, in Cx of P9 jj animals, a translational control of Mrp1 might be hypothesized due to a significant increase in Mrp1 protein, without changes in mRNA level. Therefore, the genomic screening made in this work provides the first general overview on the mRNA developmental profiles of several CYPs and GSTs genes in brain parenchyma (specifically Cx, Cll, St and Hip) of normal rats, and of animals suffering from hyperbilirubinemia, underlying the necessity to find functional evidence to finally understand the role of these enzymes associated with the kernicterus and Crigler-Najjar type I syndrome pathologies.
XXIII Ciclo
1978

2011/2012
Metastasis formation is the final step in a solid tumour progression and is the most common cause of death in cancer patients. Therefore novel therapeutic strategies to prevent development of metastases have a potential impact on cancer mortality. In the last years, work from our lab has identified that, following oncogenic signalling, the phosphorylation-dependent prolyl-isomerase Pin1 amplifies mutant p53 oncogenic functions. In particular, we have demonstrated the relevance of a Pin1/mutant p53 axis in controlling directly a transcriptional “ten genes signature” program relevant for tumour growth and invasion. This signature includes genes relevant for breast cancer progression, correlated to poor outcome of breast cancer patients. Among these, we have unveiled DEPDC1 as a potent promoter of invasiveness and migration in MDA MB231 triple negative breast cancer (TNBC) cells. This gene is has been found overexpressed in different mouse and human tumours, such as breast carcinoma, colorectal and lung adenocarcinomas. The aim of my PhD work was to investigate the role of DEPDC1 in the cancer progression that is linked to cell-biological traits associated with high-grade malignancy - including motility, invasiveness and loss of polarity. We found that the high DEPDC1 expression level positively correlates with clinical outcome and aggressiveness in breast cancer and we demonstrate that DEPDC1 regulates important phenotypes involved in tumour formation and progression. First, we identified a role of DEPDC1 in promoting aggressive cancer phenotypes in vitro by regulating cell motility, polarity and proliferation. Second, we have evaluated that the establishment of lung metastasis in vivo in mice was reduced upon inhibition of DEPDC1. Third, we demonstrate the tumorigenic potential of DEPDC1-V1 alone or cooperating with oncogenic H-Ras on induction of malignant cell transformation. Finally, a preliminary data shows that DEPDC1 is also able to increase the efficiency of mammosphere formation of breast cancer cells, implicating a role in cancer stemness. Until now, our results support the strong impact of DEPDC1 on tumour progression while molecular pathways perturbed by DEPDC1 and by which drive the cancer progression are still unknown. However, our analysis of RNA-seq data upon silencing of DEPDC1, suggests the mechanism by which DEPDC1 could induce an aggressive phenotype altering the gene expression profile of breast cancer cells. Future studies will address the molecular networks by which DEPDC1 drives the metastatic cancer progression that could be useful for discovering the novel therapeutic targets and diagnostic markers in breast cancer.
XXIV Ciclo
1983

2013/2014
Nei vertebrati i telomeri sono strutture specializzate localizzate all’estremità dei cromosomi costituito da sequenze ripetute TTAGGG. L’ accorciamento progressivo del telomero ad ogni divisione cellulare porta ad una disfunzione telomerica e all’attivazione della risposta di danno al DNA alle estremità cromosomiche. L’attivazione del segnale di danno al DNA provoca senescenza o apoptosi, fenomeni legati all’invecchiamento. Per mantenere la funzione del telomero il complesso della telomerasi, costituito dalla componente ad RNA (hTERC) e dalla subunità catalitica (hTERT), aggiunge ripetizioni telomeriche in cellule con alto potenziale replicativo. Due complessi principali sono coinvolti nella regolazione dei telomeri: il complesso shelterin e il complesso della telomerasi. Shelterin è costituito da sei principali proteine: TRF1, TRF2, POT1, TPP1, TIN2 e RAP1, e controlla vari aspetti della funzione telomerica come la lunghezza telomerica, la ricombinazione e la protezione da fattori di risposta al danno al DNA. l’evasione dalla senescenza replicativa raggiunta con la riattivazione della telomerasi, è un passaggio chiave nel processo di tumorigenesi, come osservato nel 90% dei tumori umani. Inoltre, aumentano anche le dimostrazioni che indicano il ruolo centrale di shelterin nella formazione e nella progressione del cancro. L’espressione del complesso shelterin o della telomerasi è strettamente regolata a livello trascrizionale e post-traduzionale, anche se il ruolo dei miRNAs nella regolazione dei telomeri non è ancora stato studiato. Lo scopo del mio progetto di tesi era quello di identificare miRNAs che controllano l’espressione dei componenti di shelterin o del complesso della telomerasi e valutare la rilevanza clinica di questi miRNAs nel contesto del tumore al seno. Per raggiungere questo obiettivo abbiamo eseguito un high-throughput luciferase reporter screening in cellule HeLa identificando un panello di miRNAs che hanno come bersaglio il 3’UTR di componenti del complesso shelterin (TRF1, TRF2, POT1) o del complesso della telomerasi (TERT, DKC1). Con questo screening abbiamo identificato che l’onco-miRNA miR-155 è un efficiente regolatore di TRF1. Il miR-155 è up-regolato in tutti i tipi di tumore al seno ed alti livelli del miR-155 correlano con una bassa espressione dei livelli proteici di TRF1. Inoltre, abbiamo validato con ulteriori saggi luciferasi il targeting di TRF1 da parte del miR-155 ed abbiamo anche scoperto che il miR-155 controlla l’espressione di TRF1 a livello traduzionale. Soprattutto, la bassa espressione dei livelli di mRNA di TRF1 e la bassa espressione dei geni bersaglio del miR-155 correlano con una ridotta sopravvivenza libera da metastasi distanti (DMFS) e con una ridotta sopravvivenza libera da recidive (RFS) in pazienti con cancro al seno luminale estrogeno-positivo (ER+). Questo indica che il targeting di TRF1 da parte del miR-155 è parte di una signature del miR-155 che determina una scarsa sopravvivenza nel cancro al seno di tipo luminale ER+. Soprattutto abbiamo scoperto che il targeting di TRF1 da parte del miR-155 porta ad un aumento della fragilità telomerica, a fusioni telomeriche dei cromatidi fratelli e all’allungamento dei telomeri. Il nostro lavoro dimostra per la prima volta che la regolazione post-trascrizionale di TRF1, mediata dal miR-155, è un efficiente meccanismo per controllare la fragilità telomerica e l’instabilità genomica nel cancro al seno. Invece, per quanto riguarda i miRNAs che regolano la telomerasi nel cancro al seno, abbiamo studiato il miR-296-5p e il miR-512-5p, i quali bersagliano efficientemente hTERT come mostrato dal high-throughput luciferase reporter screening . Entrambi i miRNAs bersagliano specifiche regioni nel 3’UTR di hTERT portando alla degradazione dell’mRNA di hTERT e alla riduzione dell’attività telomerasica. Dati clinici rivelano che il miR-296-5p e il miR-512-5p sono down-regolati nei tessuti tumorali del seno. Inoltre, abbiamo scoperto che l’aumento dell’espressione di hTERT e l’aumentata espressione dei geni bersaglio del miR-296-5p e del miR-512-5p correlano con una scarsa sopravvivenza in pazienti con cancro al seno di tipo basale. Questo evidenzia l’importanza clinica del miR-296-5p e del miR-512-5p nel cancro al seno (basale). A livello molecolare abbiamo mostrato che il miR-296-5p e il miR-512-5p rallentano la proliferazione cellulare e provocano l’ accorciamento della lunghezza dei telomeri in cellule di tumore al seno basale. Soprattutto, abbiamo scoperto che la ri-espressione epigenetica dei geni del miR-296-5p e del miR-512-5p in cellule di tumore al seno basale, determina l’aumento dell’espressione del miR-296-5p e del miR-512-5p e la conseguente riduzione dell’espressione di hTERT. I nostri dati suggeriscono che l’utilizzo di composti che inducono la ri-espressione del miR-296-5p e del miR-512-5p potrebbe essere una strategia promettente per compromettere i meccanismi di mantenimento dei telomeri e la funzione di hTERT nel cancro al seno. I risultati di questo lavoro dimostrano che i miRNAs rappresentano dei nuovi regolatori della funzione telomerica e hanno un impatto sull’omeostasi dei telomeri nel cancro umano. Questo lavoro identifica i miRNAs come nuovi bersagli per modulare la funzione dei telomeri nelle malattie ad essi correlate come il cancro e l’invecchiamento.
Vertebrate telomeres are specialized structures localized at the end of chromosomes that are composed of TTAGGG tandem repeats. Progressive telomere shortening with every cell division, finally leads to telomere dysfunction and the induction of a DNA damage response at chromosome ends. DNA damage signaling provokes senescence or apoptosis, phenomena linked to organismal aging. In order to maintain telomere function the telomerase reverse transcriptase complex, composed by the RNA component (hTERC) and the catalytic subunit (hTERT) replenishes telomere repeats in cells with high replicative potential. Two main complexes are involved in the regulation of telomeres: shelterin and the telomerase complex. Shelterin is composed of six main proteins: TRF1, TRF2, POT1, TPP1, TIN2, and RAP1 and controls various aspects of telomere function such as telomere length, recombination and protection from the DNA damage response factors. The escape from replicative senescence is a key step in tumorigenesis and it is achieved by the reactivation of telomerase activity, as observed in 90% of human cancers. However, increasing body of evidence also indicates a central role of shelterin in cancer formation or progression. The expression of telomerase and shelterin complex is tightly regulated at transcriptional and post-translational level, however the role of miRNAs in telomere regulation is not understood. The aim of my PhD thesis project was to identify miRNAs that control the expression of components of the shelterin or telomerase complex and to evaluate the clinical relevance of these miRNAs in the context of human breast cancer. To achieve this goal we performed a high-throughput luciferase reporter screening in HeLa cells and identified a panel of miRNAs that target the 3’UTR of shelterin (TRF1, TRF2, POT1) or telomerase complex components (TERT, DKC1). In this screening we identified the onco-miRNA miR-155 as efficient regulator of TRF1 expression. miR-155 is efficiently upregulated in across all types of human breast cancer and high miR-155 levels correlate with low TRF1 expression levels. We validated targeting of TRF1 by miR-155 in additional luciferase reporter assays and found that miR-155 controls TRF1 expression on the translational level. Importantly, low TRF1 mRNA expression but also low expression of miR-155 target genes is linked to reduced distant metastasis-free survival and relapse-free survival of estrogen receptor positive luminal breast cancer patients. This indicates that targeting of TRF1 by miR-155 is part of a miR-155 signature that mediates poor survival in ER+ luminal breast cancer. Importantly, we found that targeting of TRF1 expression by miR-155 leads to increased telomere fragility, telomere sister chromatid fusions and telomere elongation. Our work demonstrates, for the first time, that post-transcriptional regulation of TRF1 by miR-155 is an efficient mechanism to control telomere fragility and genomic stability in the context of human breast cancer. Concentrating on miRNA dependent mechanisms of telomerase regulation in human breast cancer we studied miR-296-5p and miR-512-5p that were found to efficiently target hTERT in the high throughput luciferase reporter assay. Both miRNAs target specific motifs in the hTERT 3’UTR, leading to degradation of the hTERT mRNA and reduction of telomerase activity. Clinical data reveal that miR-296-5p and miR-512-5p are downregulated in breast cancer tissues. Importantly, we found that increased hTERT expression but also high expression of miR-296-5p or miR-512-5p target genes is linked to poor survival in basal breast cancer patients. This highlights the clinical relevance of miR-512-5p and miR-296-5p in basal breast cancer. On the molecular levels we showed that miR-296-5p and miR-512-5p impair cell proliferation and provoke progressive telomere shortening in basal breast cancer cell lines. We further found that epigentic de-repression of the miR-296 and miR-512 genes in a basal breast cancer cell line increase the expression of miR-296-5p and miR-512-5p levels to reduce hTERT expression. Our data suggest that the use of drugs that release miR-296-5p or miR-512-5p expression might be a promising strategy to impair telomere maintenance mechanisms and hTERT function in human breast cancer. Altogether, my work demonstrated that miRNAs represent new regulators of telomere function that impact on telomere homeostasis in human cancer. This identifies miRNAs as novel targets to modulate telomere function in telomere related maladies such as cancer and aging.
XXVII Ciclo
1986

2012/2013
HMGA1 is an oncogene encoding for an architectural transcription factor that affects fundamental cell processes, leading to neoplastic transformation. The two main mechanisms by which HMGA1 protein is known to be involved in cancer concern the regulation of gene expression by altering DNA structure and interacting with a conspicuous number of transcription factors. Here we provide evidence of an additional level of gene expression regulation exploited by HMGA1 to exert its oncogenic activity. Starting from protein-protein interaction data showing that HMGA1 interacts with histones, we show that HMGA1 regulates gene expression by affecting the epigenetic status of cancer cells. In particular, it modulates the signalling cascade mediated by the RAS/RAF/MEKK/ERK/RSK2 pathway regulating the levels of histone H3 phosphorylation at Serine 10 and Serine 28. We demonstrate that the down-regulation of these two H3 post-translational modifications by HMGA1 silencing and by inhibitors of the RAS/RAF/MEKK/ERK pathway is linked to cell migration decrease and morphological changes resembling the mesenchymal to epithelial transition.
HMGA1 è un oncogene codificante per un fattore trascrizionale architetturale che influenza fondamentali processi cellulari, portando alla trasformazione neoplastica. I due principali meccanismi tramite cui la proteina HMGA1 è nota essere coinvolta nel cancro riguardano la regolazione dell’espressione genica tramite l’alterazione della struttura del DNA e l’interazione con un cospicuo numero di fattori di trascrizione. Qui forniamo la prova di un addizionale livello di regolazione dell’espressione genica sfruttato da HMGA1 per esercitare la sua attività oncogenica. Partendo da dati d’interazione proteina-proteina che mostrano che HMGA1 interagisce con gli istoni, mostriamo che HMGA1 regola l’espressione genica influenzando lo stato epigenetico delle cellule cancerose. In particolare, essa modula la cascata di segnalazione mediata dalla via di RAS/RAF/MEKK/ERK/RSK2 regolando i livelli di fosforilazione dell’istone H3 sulla Serina 10 e sulla Serina 28. Noi dimostriamo che la down-regolazione di queste due modificazioni post-traduzionali di H3 tramite il silenziamento di HMGA1 e l’utilizzo di inibitori della via di RAS/RAF/MEKK/ERK/RSK2 è correlata alla diminuzione della migrazione cellulare e a cambiamenti morfologici che ricordano la transizione mesenchimo-epiteliale.
XXV Ciclo
1983

2012/2013
Il peptide 2A è un peptide virale di 18-23 aminoacidi presente in alcuni picornavirus, dei virus a RNA che codificano per una poliproteina che viene poi processata per produrre tutte le proteine virali. Il peptide 2A, in particolare, permette la divisione tra le proteine capsidiche e quelle replicative. Durante la traduzione il peptide 2A agisce come elemento autonomo, in grado di mediare il suo processamento al C-terminale: per farlo modifica l'attività del ribosoma, promuovendo l'idrolisi del legame estere tra il peptide nascente e il tRNA anziché che la formazione del legame peptidico, con il conseguente rilascio prematuro della proteina a monte e la continuazione della traduzione dalla Pro a valle. Abbiamo chiamato questa proprietà "terminazione non convenzionale della traduzione", per distinguerla dalla terminazione convenzionale che si verifica in presenza di un codone di STOP e che viene mediata dai fattori di rilascio 1 e 3. Per questa sua attività, sia degli aminoacidi Asn-Pro-Gly al C-terminale del peptide 2A che la Pro al N-terminale della proteina a valle sono essenziali. Grazie alla sua capacità di autoprocessamento, il peptide 2A può essere utilizzato per produrre due proteine da un unico open reading frame (ORF) . Nella prima parte del lavoro abbiamo testato due differenti peptidi 2A (uno derivante dal Foot-and-Mouth Disease Virus (F2A), e uno dal Porcine Teschovirus (T2A) per trovare eventuali differenze tra la loro attività in termini di terminazione non convenzionale e di reinizio della traduzione dalla Pro a valle. Entrambi questi peptidi si sono mostrati molto efficienti nella terminazione non convenzionale, come si nota dalla quasi totale assenza di proteina di fusione. Tuttavia, il reinizio della traduzione dalla Pro valle 2A non è completo, come si evince dal fatto che la proteina a valle viene prodotta in una quantità inferiore rispetto a quella a monte. Abbiamo determinato che F2A funziona meglio T2A, in quanto consentee la sintesi della proteina a valle con maggiore efficienza. Questi risultati indicano che il peptide F2A è un ottimo sistema per la co-espressione di proteine in vivo. Abbiamo sfruttato questa tecnologia per la produzione di anticorpi ricombinante, riuscendo a produrre sia in vitro che in vivo la catena leggera e pesante dell’anticorpo, che inoltre si assembla correttamente. Nella seconda parte del lavoro abbiamo studiato una caratteristica particolare del peptide 2A: quando viene posizionato immediatamente a monte di un codone di stop, l'espressione della proteina a monte è fortemente compromessa. Questa caratteristica è dovuta alla sequenza amminoacidica, e non a quella nucleotidica. La causa di questa compromissione dell’espressione risiede in un forte stalling del ribosoma al sito Gly-STOP, totalmente dipendente dagli ultimi tredici residui del peptide 2A e dalla presenza degli amminoacidi C-terminali Asn-Pro-Gly terminali. Come conseguenza del blocco, viene attivato un controllo di qualità a livello del ribosoma, che induce l’ubiquitinazione e la degradazione da parte del proteasoma della piccola quantità di proteina prodotta. Questa attività di controllo da parte del peptide 2A sulla traduzione è efficace sia su ribosomi legati alla membrana dell’ER, che su quelli liberi nel citosol. Per i ribosomi legati alla membrana, abbiamo identificato il coinvolgimento di alcuni membri del pathway di retrotraslocazione come l’AAA ATPasi p97 e la deubiquitinase YOD1, suggerendo un meccanismo di controllo di qualità che rileva la presenza di ribosomi in stallo sul lato citosolico della membrana dell’ER e la segnala ad effettori presenti nel lato luminale per indurre la degradazione associata all’ER (ERAD) dei peptidi nascenti. E’ noto che uno dei principali mediatori dell’ERAD è l'AAA ATPasi p97/VCP; si che p97 sia necessaria per la retrotraslocazione e la degradazione da parte del proteasoma dei substrati ERAD, e che la sua perdita provoca la loro ritenzione nel lume dell’ER. Di conseguenza, l’attività ATPasica di p97 è ritenuta essenziale per l'estrazione delle proteine dalla membrana dell’ER . Per studiare il ruolo di p97 in ERAD, abbiamo usato un metodo recentemente sviluppato nel nostro laboratorio (Petris et al., 2011) che consiste nella mono-biotinilazione in vivo, da parte della biotin ligasi citosolica Bira, di substrati proteici taggati con il Biotin Acceptor Peptide BAP (GLNDIFEAQKIEWH). Questo metodo consente una discriminazione precisa tra le proteine che si trovano nel lume dell’ER (non biotinilate) e nel citoplasma (biotinilate). Abbiamo analizzato l'effetto di: 1) una forma dominante negativa di p97 (p97QQ), 2) un siRNA specifico per p97, e 3) l’inibitore chimico di p97 DBeQ, in tre diversi substrati modello di ERAD: NS1, il mutante Null Hong Kong dell’α1-antitripsina (NHK-α1AT), e Tetherin. I nostri risultati sono in contrasto con la visione predominante di p97 come fornitore di energia per l'estrazione delle proteine dall’ER in maniera ATP-dipendente. Abbiamo trovato che l'attività di p97 non è coinvolta nella retrotraslocazione di queste proteine, ma piuttosto nella separazione delle proteine, già nel lato citosolico, e nel reclutamento della PNGase. Risultati simili sono stati ottenuti analizzando l'effetto di una forma dominante negativa della deubiquitinase p97-associata YOD1 (YOD1 C160S).
The 2A peptide is a short viral peptide of 18-23 amino acids, present in some picornaviruses. These RNA viruses encode a polyprotein that is late-processed to produce all viral proteins. 2A allows the primary cleavage between the capsidic and the replicative proteins. It works as an autonomous element during translation, capable of mediating self-processing at its C-terminus. It modifies the ribosome activity, promoting hydrolysis of the ester bond between the nascent peptide and the tRNA rather than the formation of the peptide linkage, resulting in a premature release of the upstream protein and continuation of translation from the in-frame downstream Pro codon. We call this property “non-conventional translation termination”, in contrast to the “conventional translation termination” occurring at a STOP codon site and mediated by the Release Factors 1 and 3. For its activity both the amino acids Asn-Pro-Gly at the C-terminus of the 2A peptide, and the Pro at the N-terminus of the downstream protein are essential. Due to its property of self-processing at the C-terminus, in mammalian cells it can be used to produce two proteins from a single open reading frame (ORF). In the first part of the work we tested two different 2A peptides (one derived from the Foot-and-Mouth Disease Virus (F2A) and one from the Porcine Teschovirus (T2A)) in order to see possible differences between their activity in term of non-conventional translation termination and re-initiation of translation from the 2A downstream Pro. We found that both these peptides were very efficient in terminating translation, in fact we observed only a very little amount of fusion protein. However, re-initiation from the 2A downstream Pro was not complete, in fact we observed that the downstream protein was produced in a lower amount than the upstream one. We also determined that F2A worked better than T2A, allowing synthesis of the downstream protein with higher efficiency. These results indicate that F2A peptide is a powerful system for the co-expression of proteins in vivo. We exploited this technology for the production of recombinant antibodies. We found that both in vitro and in vivo antibody’s light and heavy chains were produced, and the complete antibody was properly folded. In the second part of the work we investigated a particular feature of the 2A peptide: when it was positioned immediately upstream of a STOP-codon, expression of the upstream protein was heavily impaired. We found that the compromised expression was due to the amino acidic and not to the nucleotidic sequence of the peptide. We found also that it was a consequence of a strong ribosome stalling at the Gly-STOP-codon boundary, totally dependent on the last thirteen 2A residues and on the presence of Asn-Pro-Gly as C-terminal amino acids. As a consequence of the stalling, a ribosome-mediated quality control is activated, inducing ubiquitination and proteasome degradation of the small amount of protein produced. This 2A regulatory activity on translation was effective on both membrane-bound and free ribosomes. We found that, for membrane-bound ribosomes, members of the retrotranslocation pathway such as the AAA-ATPase p97 and the deubiquitinase YOD1 were involved, thus suggesting a quality control mechanism that senses the presence of stalled ribosomes on the ER membrane cytosolic side and signals to effectors present in the luminal side to induce ERAD (Endoplasmic Reticulum Associated Degradation). It is widely known that one of the main players in ER Associated Degradation is the AAA-ATPase p97/VCP. It is generally thought that p97 ATPase activity is required for retro-translocation and proteasomal degradation, and that its loss causes retention of ERAD substrates in the ER lumen. As a consequence, p97 ATPase activity is considered essential for extraction of proteins from the ER membranes. To study the role of p97 in ERAD, we used a method recently developed in our laboratory (Petris et al., 2011), which is based in the specific in vivo mono-biotinylation by the cytosolic biotin ligase BirA of protein substrates tagged with the 15 amino acids-long Biotin Acceptor Peptide BAP (GLNDIFEAQKIEWH). This method allows a precise discrimination between the proteins located in the Endoplasmic Reticulum (non biotinylated) or in the cytoplasm (biotinylated). We analysed the effect of: 1) a dominant negative form of p97 (p97QQ), 2) a p97 specific siRNA, and 3) the p97 chemical inhibitor DBeQ, on the three different ERAD model substrates NS1, Null Hong Kong mutant of α1 anti-trypsin (NHK-α1AT), and Tetherin. Our results clearly challenge the predominant view of p97 as energy provider for the extraction of unfolded proteins from a putative retrotranslocon in an ATP-dependent manner. We found that p97 activity is not involved in the retrotranslocation of these proteins from ER to cytosol, but rather in the segregation of proteins already in the cytosolic side and in the recruitment of PNGase. Similar results were obtained analysing the effect of a dominant negative form of the p97-associated deubiquitinase YOD1 (YOD1 C160S).
XXVI Ciclo
1985

2011/2012
Mesenchymal Stem/Stromal Cells (MSCs) are the precursors of various cell types that compose both normal and cancer tissue microenvironments. In order to support the widely diversified parenchymal cells and tissue organization, MSCs are characterized by a large degree of heterogeneity, although available analyses of molecular and transcriptional data do not provide clear evidence. Moreover a wealth of studies has demonstrated a significant role of the microenvironment and MSCs in tumor growth. In the course of the years MSCs were isolated by different groups from different tissues, both healthy and cancerous. The laboratory in which I conducted my PhD project was able to purify MSCs from different healthy tissues (N-MSCs) and, using an adapted protocol, from High-Grade Serous Ovarian Carcinomas (HG-SOC-MSCs). It was possible to shown that these cells do not possess gross chromosomal aberrations and are not tumorigenic in vivo. To better characterize these cells, an integrative bioinformatics analysis was conducted using the deep-CAGE-derived expression profiles obtained from HG-SOC-MSCs, N-MSCs and the FANTOM5 large comprehensive primary cells and tissues dataset. When compared to the other cells and tissues, HG-SOC-MSCs showed a correlation with mesothelial cells and cells hypothesized to have a mesothelial origin, such as smooth muscle cells and fibroblasts. It is known that mesothelial cells in culture can alternate between epithelioid and fibroblastoid morphologies and express high levels of either keratin or vimentin or both depending on their state of growth and the presence of EGF. When cultivated in the absence of EGF, which is known to induce a morphological switch in mesothelial cells, HG-SOC-MSCs switch from a fibroblast-like to an epithelial-like shape. N-MSCs in the same culture conditions, instead, do not change morphology. Moreover, in absence of EGF, HG-SOC-MSCs but not N-MSCs raise the levels of Keratin 7 both in protein and in mRNA. Starting from the list of up-regulated genes in HG-SOC-MSCs compared to N-MSCs a list of mesothelial-related genes was generated. This mesothelial-related gene list was compared to high-throughput gene expression datasets of MSCs derived from other tissues. Such analysis revealed that the mesothelial-related signature is specific to HG-SOC-MSCs. Moreover, Kaplan-Meier survival analysis conducted on a comprehensive SOC microarray dataset showed that patients with higher levels of the mesothelial-related gene signature displayed shorter progression-free survival time. Such correlation was rather specific for HG-SOC given that its performance was either statistically non-significant in the case of lung cancer or correlated with good prognosis in the case of breast cancer. Altogether, the study allowed us to assign a specific identity for MSCs derived from high-grade serous ovarian cancer. We demonstrated a cell-type specific transcriptional activity associated with HG-SOC-MSCs, which identifies them compared to N-MSCs from other districts and position them close to primary mesothelial and mesothelail-derived cells within the FANTOM5 dataset.
XXV Ciclo
1985

2008/2009
Distant metastases of solid tumours are the major cause of cancer death. To improve the effectiveness of chemotherapy, only marginally active on secondary tumours, anti-metastatic agents are needed, i.e. compounds that display the capacity to selectively interfere with metastatic formation and growth. Among metal compounds, NAMI-A, HIm[Ru(III)Cl4Imdmso], has repeatedly shown a peculiar and selective anti-metastatic activity being able to prevent the formation, and to inhibit the growth of established secondary tumours. On these bases, the first phase of the project was aimed to investigate how variations on the NAMI-A chemical structure can influence the anti-metastatic activity. For this purpose, some representative complexes have been chosen: two heterocyclic compounds KP418, HIm[Ru(III)Cl4(Im)2], and KP1019, HInd[Ru(III)Cl4(Ind)2], presenting a different N-donor ligand, and three organometallic compounds, RM175, [(η6-biphenyl)Ru(II)Cl-(ethylendiamine)]PF6, its osmium congener AFAP51, [(η6-biphenyl)Os(II)Cl(ethylene-diamine)]BF4, and RAPTA-T, [RuCl2(η6-toluene)PTA], carrying a PTA ligand instead of ethylendiamine. The effects of the compounds on the interference with some steps of the metastatic progression are evaluated with appropriate in vitro tests, comparing the behaviour of the human MDA-MB-231 highly invasive breast cancer cells to that of human HBL-100 non tumorigenic mammary epithelial cells. To validate the model, the in vitro effects are compared with the in vivo anti-metastatic activity studied in the MCa mammary carcinoma of the CBA mouse. The results obtained highlight the selective activity of the organometallic compound RAPTA-T towards the highly invasive cell line in vitro, accompanied by a selective inhibition of metastasis development in vivo. RAPTA-T seems to act through the modulation of tumour cells-extracellular matrix interactions, and of cell motility. In particular RAPTA-T induces a cytoskeleton remodelling, mainly through the formation of stress fibres, that causes a stiffening of the cell body, particularly evident on MDA-MB-231 cells grown on ECM components. These effects, selectively identified in the highly invasive MDA-MB-231 cells, can be related to the higher ruthenium uptake, detected in this cell line. Cell adhesion, migration and invasion are directly related to actin assembly and disassembly, phenomena regulated by the RhoGTPases. RAPTA-T completely counteracts the increase of trypsin mediated cell detachment induced by the RhoGTPases inhibitor C3 transferase, in MDA-MB-231 cells grown on fibronectin and collagen IV, and on HBL-100 cells grown on collagen IV. These molecular events might stem from the cell surface and involve integrin adhesion molecules, as suggested by the role of ECM components in the functional tests and by the preference of RAPTA-T to bind them, above all, collagen IV, with which RAPTA-T interacts chemically as confirmed by an NMR analysis. The effect of RAPTA-T on cells immediately after the adhesion on the substrate, i.e. mainly adherent with integrinic receptors, suggests that RAPTA-T interacts mainly with integrins in the form already bound with the substrate. Le metastasi dei tumori solidi sono una delle principali cause di morte. Per migliorare l’effetto della chemioterapia, che è attiva solo marginalmente sui tumori secondari, sono necessari agenti anti-metastatici, cioè composti che si mostrino capaci di interferire selettivamente con la formazione e la crescita delle metastasi. Tra i vari composti di sintesi il NAMI-A, HIm[Ru(III)Cl4Imdmso], ha ripetutamente mostrato una selettiva azione anti-metastatica dimostrandosi capace di prevenire la formazione e di inibire la crescita di metastasi già formate. Su queste basi, la prima fase di questo progetto ha avuto lo scopo di indagare come variazioni nella struttura chimica del NAMI-A potessono influenzare l’azione anti-metastatica. A questo scopo sono stati scelti alcuni composti rappresentativi: due composti eterociclici il KP418, HIm[Ru(III)Cl4(Im)2], e il KP1019, HInd[Ru(III)Cl4(Ind)2], e tre composti organometallici, RM175, [(η6-biphenyl)Ru(II)Cl-(ethylendiamine)]PF6, il suo congenere a base di osmio AFAP51, [(η6-biphenyl)Os(II)Cl(ethylene-diamine)]BF4, e RAPTA-T, [RuCl2(η6-toluene)PTA], che al posto dell’etilendiamina presenta il ligando PTA. Gli effetti dei composti sull’interferenza con alcuni step della progressione metastatica sono stati valutati con alcuni test in vitro, paragonando il comportamento della linea cellulare altamente invasiva MDA-MB-231 proveniente da carcinoma mammario, con quello delle HBL-100 linea cellulare dell’epitelio mammario non tumorigenica. Per validare il modello proposto in vitro gli effetti dei composti sono stati paragonati con la loro azione in vivo studiata sul carcinoma mammario (MCa) dei topi CBA. I risultati ottenuti mettono in luce la selettiva azione del composto organometallico RAPTA-T verso la linea cellulare altamente invasiva in vitro, accompagnata dalla inibizione selettiva dello sviluppo delle metastasi in vivo. Il RAPTA-T sembra agire attraverso la modulazione delle interazioni cellula-matrice extracellulare e della motilità cellulare. In particolare il RAPTA-T induce il rimodellamento del citoscheletro, principalmente attraverso la formazione di fibre da stress, questo genera irrigidimento del corpo cellulare, particolarmente evidente sulle MDA-MB-231 cresciute sui componenti della matrice extracellulare. Questi effetti, selettivamente identificati sulla linea altamente invasiva, MDA-MB-231, possono essere correlati ad un più alto assorbimento di rutenio che è stato rilevato in questa linea cellulare. L’adesione cellulare, la migrazione e l’invasione sono direttamente correlate al rimodellamento del citoscheletro actinico, e questi fenomeni sono regolati dalle RhoGTPasi. Il RAPTA-T reverte completamente l’aumentato distacco conseguente al trattamento con la C3 transferasi, agente che inibisce le RhoGTPasi, nelle MDA-MB-231 cresciute su fibronectina e collagene IV e sulle HBL-100 cresciute sul collagene IV. Questi eventi potrebbero coninvolgere l’adesione integrinica ai substrati, come suggerito dal ruolo dei componenti della matrice extracellulare nei test funzionali presi in considerazione, dalla preferenza del RAPTA-T per il legame con questi, e soprattutto il collagene IV con cui il RAPTA-T interagisce chimicamente, come confermato dall’analisi NMR. L’effetto del RAPTA-T su cellule appena dopo l’adesione al substrato, quindi prevalentemente legate attraverso i recettori integrinici, suggerisce che il RAPTA-T interagisca principalmente con le integrine nella forma già legata i substrati.
XXII Ciclo
1978