Dipartimento di Farmacia e Scienze della Salute e della Nutrizione - Tesi di Dottorato

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Questa collezione raccoglie le Tesi di Dottorato afferenti al Dipartimento di Farmacia e Scienze della Salute e della Nutrizione dell'Università della Calabria.

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Start date: 2010End date: 2019

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Now showing 1 - 10 of 90
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    GPER mediates the up-regulation of acid synthase (fasn) intuced by 17B-estradiol in cancer cell and in cancer-associated fibroblasts (CAFs)
    (2012-11-26) Santolla, Maria Francesca; Maggiolini, Marcello; Sisci, Diego
    Activation of lipid metabolism is an early event in carcinogenesis and a central hallmark of many tumors. Fatty acid synthase (FASN) is a key lipogenic enzyme catalyzing the terminal steps in the de novo biogenesis of fatty acids. In cancer cells, FASN may act as a metabolic oncogene given that it confers growth and survival advantages to these cells, whereas its inhibition effectively and selectively kills tumor cells. Hormones like estrogens and growth factors contribute to the transcriptional regulation of FASN expression also through the activation of downstream signaling and a crosstalk among diverse transduction pathways. In this study, we demonstrate for the first time that 17β-estradiol (E2) and the selective GPER ligand G-1 regulate FASN expression and activity through the GPER-mediated signaling which involved the EGFR/ERK/c-fos/AP1 transduction pathway, as ascertained by using specific pharmacological inhibitors, performing gene-silencing experiments and ChiP assays in breast SkBr3, colorectal LoVo, hepatocarcinoma HepG2 cancer cells and breast cancerassociated fibroblasts (CAFs). In addition, the proliferative effects induced by E2 and G-1 in these cells involved FASN as the inhibitor of its activity, named cerulenin, abolished the growth response to both ligands. Our data suggest that GPER may be included among the transduction mediators involved by estrogens in regulating FASN expression and activity in cancer cells and CAFs that strongly contribute to cancer progression.
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    FoxO3a reactivation restores the sensitivity to the antiestrogen treatment in tamoxifen resistant breast cancer
    (2017-06-12) Donà, Ada Alice; Andò, Sebastiano; Sisci, Diego
    Resistance to endocrine treatments is a major clinical challenge in the management of estrogen receptor alpha positive (ER+) breast cancers (BC). Although multiple mechanisms leading to endocrine resistance have been proposed, the poor outcome of this subgroup of BC patients demands additional studies. Here we show that the expression of FoxO3a transcription factor is strongly reduced in ER+ BC MCF-7 cells (wtMCF-7) that developed resistance to Tamoxifen (TamR). On the other hand, FoxO3a silencing (siF3a) was able to counteract Tam induced growth inhibition in wtMCF-7, demonstrating that FoxO3a is a mediator of cell response to Tam. To analyze the role of FoxO3a in the acquisition of a Tam resistant phenotype, TamR clones bearing an active FoxO3a (F3aAAA), whose expression can be induced by Doxycycline (Dox) were developed. FoxO3a re-activation was able to re-establish the sensitivity of TamR cells to the antiestrogen, inhibiting proliferation and cell cycle progression, as well as restoring Tam dependent apoptotic response. For a closer look at the molecular mechanisms involved, an unbiased proteomics analysis on F3aAAA-inducible TamR cells was conducted, unveiling novel interesting and potential mediators of the anti-proliferative and pro-apoptotic activity of FoxO3a, all worthy of future investigations. Kaplan-Meier (K-M) survival curves confirmed the relevance of FoxO3a also in a clinical setting, since high levels of the transcription factor strongly correlate to a positive response to tamoxifen therapy. Finally, to assess if FoxO3a reactivation is able to restore the sensitivity to Tam also in vivo, the widely used anti-epileptic drug (AED) Lamotrigine (LTG; Lamictal), which is able to induce FoxO3a expression in TamR cells leading to growth inhibition, was also tested on TamR deriving xenografts tumors, where it showed the same effects observed in vitro. Altogether, our data indicate that FoxO3a could not only be considered a good prognostic factor in ER+ BC, predicting a positive response to endocrine therapy, but also a key target to be exploited in combination therapy. In this context, LTG might represent a valid candidate to be used as an adjuvant to Tam therapy in patients at risk.
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    Activated FXR inhibits leptin signaling and counteracts tumor-promoting activities of cancer-associated fibroblasts in breast malignancy
    (2017-06-12) Vircillo, Valentina; Andò, Sebastiano; Catalano, Stefania
    Cancer-associated fibroblasts (CAFs), the principal components of the tumor stroma, play a central role in cancer development and progression. As an important regulator of the crosstalk between breast cancer cells and CAFs, the cytokine leptin has been associated to breast carcinogenesis. The nuclear Farnesoid X Receptor-(FXR) seems to exert an oncosuppressive role in different tumors, including breast cancer. In this study, we demonstrated, for the first time, that the synthetic FXR agonist GW4064, inhibiting leptin signaling, affects the tumor-promoting activities of CAFs in breast malignancy. GW4064 inhibited growth, motility and invasiveness induced by leptin as well as by CAF-conditioned media in different breast cancer cell lines. These effects rely on the ability of activated FXR to increase the expression of the suppressor of the cytokine signaling 3 (SOCS3) leading to inhibition of leptin-activated signaling and downregulation of leptin-target genes. We further extend our data investigating whether FXR agonist may directly influence CAF phenotype. We demonstrated that FXR is expressed in different CAFs and treatment with GW 4064 is able to induce the transcription of key FXR target genes, including SHP (Small Heterodimer Partner) and BSEP (Bile Salt Export Pump). Interestingly, FXR activation is able to significantly reduce CAF motility, without influencing their proliferation capabilities. Accordingly, IPA (Ingenuity Pathway Analysis) on FXR-modulated genes highlighted cellular movement as the most significantly represented biologic process and evidenced a marked reduction in the activity of Rho signaling and Integrin proteins, with activation z-score of -1, -0,5 respectively. Moreover, our data showed a reduction in stress fibers formation in GW 4064 -treated CAFs. Activated FXR is able to reduce tumor promoting effects of CAFs on breast cancer cells, due to the ability of GW 4064 to reduce CAF secreted soluble factors, including IGF-1 (Insulin Growth Factor-1), FGF-9 (Fibroblast Growth Factor 9), TGF-3 (Transforming Growth Factor Beta 3) and others key mediators involved in the crosstalk tumor-stroma. Indeed, our data demonstrate how ER-breast cancer cell lines, MCF-7 and T47D, cocoltured with conditioned media derived from GW4064-treated CAFs, exhibit a significantly reduced anchorage-independent growth and migration. In vivo xenograft studies, using MCF-7 cells alone or co-injected with CAFs, showed that GW4064 administration markedly reduced tumor growth. Thus, FXR ligands might represent an emerging potential anti-cancer therapy able to block the tumor supportive role of activated fibroblasts within the breast microenvironment
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    Stimulatory actions of IGF-I are mediated by IGF-IR cross-talk with GPER and DDR1 in mesothelioma and lung cancer cells
    (2017-06-12) Avino, Silvia; Andò, Sebastiano; Maggiolini, Marcello
    Insulin-like growth factor-I (IGF-I)/IGF-I receptor (IGF-IR) system has been largely involved in the pathogenesis and development of various tumors. We have previously demonstrated that IGF-IR cooperates with the G-protein estrogen receptor (GPER) and the collagen receptor discoidin domain 1 (DDR1) that are implicated in cancer progression. Here, we provide novel evidence regarding the molecular mechanisms through which IGF-I/IGF-IR signaling triggers a functional cross-talk with GPER and DDR1 in both mesothelioma and lung cancer cells. In particular, we show that IGF-I activates the transduction network mediated by IGF-IR leading to the up-regulation of GPER and its main target genes CTGF and EGR1 as well as the induction of DDR1 target genes like MATN-2, FBN-1, NOTCH 1 and HES-1. Of note, certain DDR1-mediated effects upon IGF-I stimulation required both IGF-IR and GPER as determined knocking-down the expression of these receptors. The aforementioned findings were nicely recapitulated in important biological outcomes like IGFI promoted chemotaxis and migration of both mesothelioma and lung cancer cells. Overall, our data suggest that IGF-I/IGF-IR system triggers stimulatory actions through both GPER and DDR1 in aggressive tumors as mesothelioma and lung tumors. Hence, this novel signaling pathway may represent a further target in setting innovative anticancer strategies
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    Idrogel funzionali per la veicolazione di principi attivi
    (2017-06-12) Spataro, Tania; Andò, Sebastiano; Picci, Nevio
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    Identification and biochemical-molecular characterization of mitochondrial carrier proteins in human andmodel organisms and associated diseases
    (2017-06-12) Muto, Luigina; Andò, Sebastiano; Dolce, Vincenza
    The mitochondrial carriers (MCs) are transmembrane proteins found in the mitochondrial inner membrane, which catalyze the translocation of solutes through the membrane. These belong to a family of carrier proteins, the SLC25 or Mitochondrial Carrier Family (MCF). Their function is to create a connection between mitochondria and cytosol, facilitating the flow of a large variety of solutes across the permeability barrier of the inner mitochondrial membrane, which is necessary for many physiological processes. The functional information obtained from the study of mitochondrial carrier was fundamental in correlating MCs physiological and pathological roles in cellular metabolism. It was possible to identify genes, and their possible defects, responsible for the onset of certain diseases such as the Stanley syndrome, Amish microcephaly, HHH syndrome (hyperornithinemia, hyperammonemia and homocitrullinuria) and type II citrullinemia, their molecular basis and their symptoms. Further studies on the functional characterization of the gene family SLC25 will clarify other diseases caused by a mitochondrial carrier deficiency. This work was focused in particular on the study of some carriers belonging to the MCF: - the mitochondrial glycine carrier, important in heme synthesis and congenital sideroblastic anemia; - the mitochondrial dephosphocoenzyme A carrier, important in regulating the compartmentalization of the CoA, the study of which is crucial for a better understanding of some neurodegenerative diseases that depend on the biosynthesis of CoA; - the mitochondrial oxoglutarate carrier, of which the functional and structural rearrangements required for substrate transport were analyzed The studies were focused on the biochemical and molecular characterization of human glycine carrier protein (GlyC) and its yeast homolog (Hem25p) providing evidence that they are mitochondrial carriers for glycine. Glycine carrier is required for the uptake of glycine in the mitochondrial matrix, where this amino acid is condensed with succinyl coenzyme A to yield δ-aminolevulinic acid, necessary for heme biosynthesis. A detailed knowledge of this transporter could be helpful to clearly understand congenital sideroblastic anemia (CSA), caused by defects of heme biosynthesis in developing erythroblasts. In particular, Hem25p was cloned into a bacterial expression system (Escherichia coli BL21), overexpressed at high levels as inclusion bodies, and purified by Ni2+-NTAagarose affinity chromatography. The protein was then reconstituted in liposomes and its transport activity of glycine was observed. The kinetic constants, Km and Vmax, were calculated. Subsequently, other evidences of glycine uptake were obtained carrying out experiments on mitochondrial proteins from the yeast wild-type strain, the hem25Δ strain and the hem25Δ HEM25-pYES2. The protein subcellular localization was found to be mitochondrial. Furthermore, the hem25Δ mutant manifested a defect in the biosynthesis of δ-aminolevulinic acid and displayed reduced levels of downstream heme and mitochondrial cytochromes. The observed defects were rescued by complementation with yeast HEM25 or human SLC25A38 genes. This work may suggest new therapeutic approaches for the treatment of congenital sideroblastic anemia. In human, the transport of CoA across the inner mitochondrial membrane has been attributed to two different genes, SLC25A16 and SLC25A42. Presumed orthologs of both genes are present in many eukaryotic genomes, but not in that of D. melanogaster, which contains only one gene, CG4241, phylogenetically close to SLC25A42. CG4241 encodes a long and a short isoform of the dPCoA carrier, respectively dPCoAC1 and dPCoAC2, which arise from an alternative translational start site. dPCoAC1 and dPCoAC2 were expressed as inclusion bodies in E. coli C0214, and reconstituted in proteoliposomes to observe the transport activity in order to characterize them functionally.The functional characterization of the D. melanogaster dPCoA carrier is of particular interest as it is the first mitochondrial carrier showing a particular substrate specificity for dPCoA and ADP. The expression of both isoforms in a S. cerevisiae strain lacking the endogenous putative mitochondrial CoA carrier restored the growth on respiratory carbon sources and the mitochondrial levels of CoA. The results reported here and the proposed subcellular localization of some of the enzymes of the fruit fly CoA biosynthetic pathway, suggest that dPCoA may be synthesized and phosphorylated to CoA in the matrix, but it can also be transported by dPCoAC to the cytosol, where it may be phosphorylated to CoA by the monofunctional dPCoA kinase. Thus, dPCoAC may connect the cytosolic and mitochondrial reactions of the CoA biosynthetic pathway without allowing the two CoA pools to get in contact. This work will be useful in the near future to better understand the deficiency of enzymes involved in the CoA biosynthesis associated with a neurodegenerative disorder known as neurodegeneration with brain iron accumulation (NBIA). The oxoglutarate carrier (OGC) plays a key role in important metabolic pathways. Its transport activity has been extensively studied, and, to investigate new structural rearrangements required for substrate translocation, site-directed mutagenesis was used to conservatively replace lysine 122 by arginine. K122R mutant was kinetically characterized, exhibiting a significant Vmax reduction with respect to the wild-type (WT) OGC, whereas Km value was unaffected, implying that this substitution does not interfere with 2-oxoglutarate binding site. Moreover, K122R mutant was more inhibited by several sulfhydryl reagents with respect to the WT OGC, suggesting that the reactivity of some cysteine residues towards these Cys-specific reagents is increased in this mutant. Different sulfhydryl reagents were employed in transport assays to test the effect of the cysteine modifications on single-cysteine OGC mutants named C184, C221, C224 (constructed in the WT background) and K122R/C184, K122R/C221, K122R/C224 (constructed in the K122R background). Cysteines 221 and 224 were more deeply influenced by some sulfhydryl reagents in the K122R background. Furthermore, the presence of 2- oxoglutarate significantly enhanced the degree of inhibition of K122R/C221, K122R/C224 and C224 activity by the sulfhydryl reagent 2-Aminoethyl methanethiosulfonate hydrobromide (MTSEA), suggesting that cysteines 221 and 224, together with K122, take part to structural rearrangements required for the transition from the c- to the m-state during substrate translocation
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    Study of antimicrobial and anticancer activity of new synthetic and natural tools
    (2017-06-12) Dhanyalayam, Dhanya; Andò, Sebastiano; Cappello, Anna Rita;
    Infectious diseases and cancer are the two disease groups that representing the major cause of death worldwide. Unfortunately, antibiotic resistance is the biggest threat in the first case; in fact, new resistance mechanisms continuously are emerging and spreading globally, threatening the ability to treat common infectious diseases. A growing list of infections caused by bacteria, viruses, parasites etc. are becoming harder and harder to treat, and sometimes impossible, as antibiotics become less effective. Without urgent action, we are heading for a post-antibiotic era, in which common infections and minor injuries can once again kill the human population. Concerning cancer, Resistance to chemotherapy and molecularly targeted therapies is a major problem in current research. Drugs side effects and toxicity to normal body cells is also an important threat in cancer treatments. In this regard, these problems are at the forefront of scientific research and technological innovation and are leading to the development of new therapeutic approaches against cancer and infectious disease with fewer side effects and lesser resistance problems. The aim of the present study was to investigate on the new compounds in order to find new possible therapeutic agents against bacteria, parasites and cancer. Infectious diseases are caused by microorganisms such as bacteria, parasites, viruses etc.; in particular, bacterial infectious diseases are caused by either Gram +ve or Gram -ve bacteria. Certainly, antibiotics are the main weapon against infectious bacterial diseases; however, the uncontrolled use of antibiotics to control infections in humans, animals and in agriculture caused the development of drug resistance by bacterial populations. Besides this, infections caused by Gram -ve bacteria are difficult to treat due to the presence of a protective outer membrane consisting of lipopolysaccharides. Therefore, it is clear that there is a need to develop novel classes of antibacterial agents capable of killing bacteria through mechanisms unlike those of the known classes of antibiotics. Then, scientists are currently searching for new approaches to treat infectious diseases, particularly those caused by Gram -ve bacteria, focusing on exactly how the pathogens change and how drug resistance evolves. Since ancient times, metal complexes have been used as antibacterial compounds, metallic silver and silver salts are good examples of this. Silver compounds are particularly interesting since their antibacterial activity can be altered by changing the ligand associated with the silver complex. To date, among silver derivatives, silver sulfadiazine remains one of the most commonly-used antibacterial drugs. Therefore, metal N-heterocyclic carbene (MNHC) complexes appeared as an emerging field of research in medicinal chemistry where NHC complexes of coinage metals (Cu, Au, and Ag) proved to be better antimicrobial agents. Herein, it was investigated the, in vitro, antibacterial activity of the newly synthesized silver (Ag) complexes, Iodide[N-methyl-N-(2-hydoxy-cyclopentyl-imidazole-2ylidine]silver(I), Iodide[N-methyl-N-(2-hydoxy-cyclohexyl)-imidazole-2-ylidine]silver(I) and Iodide[N-methyl- N-(2-hydoxy-2-phenyl)ethyl-imidazole-2-ylidine]silver(I), namely AgL6, AgL18 and AgL20, against two Gram +ve (Staphylococcus aureus, Streptococcus pyogenes) and three Gram -ve (Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa) bacteria. Among these, AgL6 showed good antibacterial activity against both Gram +ve and Gram -ve bacteria. However, the minimum inhibitory concentration (MIC) value was 32 μg/mL for Gram +ve and 16 μg/mL for Gram -ve bacteria, which was higher than that displayed by commercial drug, used as control (Silver Sulfadiazine, AgSD). We therefore hypothesized that the poor activity is due to the poor intake of the compound. In order to enhance its antibacterial activity, we have developed “a pharmaceutically-oriented device”, a nanocarrier as a tool for targeted drug delivery. Here it was described, for the first time, the production of a polymer nanostructure in which dextran, a biopolymer, and oleate residues represent the hydrophilic and hydrophobic parts, respectively. This nanoparticle was loaded with AgL6 and the antibacterial activity has been investigated. The results were very interesting, with MIC values being reduced four-fold for both Gram +ve and Gram -ve bacteria. Surprisingly, these values were two-fold lower than for silver sulfadiazine. Briefly, our results showed that K. pneumoniae and E. coli are the most susceptible bacteria to AgL6, followed by P. aeruginosa. In conclusion, the investigated compound AgL6 showed excellent potentiality against bacterial infections. According to the World Health Organization (WHO), 17 diseases caused by bacteria and parasites have been classified as neglected tropical diseases (NTDs). NTDs are endemic in 149 tropical and subtropical countries and affect more than 1 billion people, including 875 million children. These diseases are responsible for over 500,000 deaths per year and are characterized by severe pain and long term disability. Human African Trypanosomiasis (sleeping sickness) is an important disease among them and is caused by two parasites of the genus Trypanosome: Trypanosome brucei rhodesiense and Trypanosome brucei gambiense. Trypanosomiasis is a disease with a devastating socio-economic impact in sub-Saharan Africa through direct infection of humans and livestock. This disease is fatal if left untreated. Current therapy relies on five drugs that have many limitations among which acute toxicity, problems with oral absorption and emergence of trypanosomal resistance, this latter is a major concern owing to the absence of vaccines and therapeutic alternatives. Therefore pharmaceutical research is aimed at the discovery of new drugs, although the investment in this therapeutic area is not attractive owing to the prospect of poor financial returns. Many pharmaceutical industries have already utilized an opportunistic approach by utilizing drugs long since used for other diseases, a process known as “repurposing” of the drug. It is estimated that over half of the drugs used today are derived from natural sources. In the present study, in a search for molecules with trypanocida activity, it was screened 2000 natural extracts from Fungi and Actinomycetes. The extracts showing activity were selected, and the active compound was identified by liquid chromatography and mass spectroscopy. Chaetocin is one of the molecules identified which showed good trypanocidal activity when tested in vitro. Chaetocin is already used as an antibacterial and anticancer drug, here it was repurposed as drug against trypanosomiasis. The results were very surprising because the trypanocidal activity was in the nanomolar range; the IC50 value was found to be 8.3 nM. Next, it was investigated on its mechanism of action. In chaetocin treated cells, morphological changes and chromatin degradation were identified, by fluorescence microscopy and cell-cycle arrest during the G2 phase was proven by cytometry analysis. Finally, it was hypothesized that the enzyme histone methyl transferase, an important enzyme acting in the G2 phase, could be the target for this drug.This study displayed that chaetocin could have great potentiality in the fight against the deadly trypanosomiasis. However, further studies will be needed to reveal whether this compound can cross the blood-brain barrier. In the third part of this thesis it was evaluated the synthesis and anticancer activity of some phosphonium salts. Phosphonium salts are a class of lipophilic cationic molecules that accumulate preferentially in mitochondria and inhibit the growth of human cancer cell lines The aim of the present study was to investigate the effects of a lipophilic phosphonium salt, (11-methoxy, 11-oxoundecyl)triphenylphosphonium bromide (MUTP) along with two other newly synthesized phosphine oxide salts, 3,3’-(methylphosphoryl)dibenzenaminium chloride and 3,3’-(phenylphosphoryl)dibenzenaminium chloride (SBAMPO and SBAPPO) on proliferation, in two human cancer cell lines: human breast cancer cells (MCF-7) and human uterine cervix adenocarcinoma cells (HeLa) and to elucidate their mechanism. The cancer cell mitochondrial membrane potential is relatively high when compared to normal cells, this force the phosphonium salts to accumulate, preferencially, in the mitochondria and inhibit their function. The results showed that only MUTP exhibits anti-proliferative effects on both cell lines, without affecting normal breast epithelial cell proliferation. More specifically, it was demonstrated that MUTP treatment of breast cancer cells is associated with impaired cell cycle progression, as determined by cytometry analysis. The G1/S cell cycle arrest was confirmed by an increased expression level of two proteins involved in cell cycle regulation, p21 and p53. Recently, there has been a surge of interest in developing compounds selectively targeting mitochondria for the treatment of neoplasms. The critical role of mitochondria in cellular metabolism and respiration supports this therapeutic rationale. Dysfunction in the processes of energy production and metabolism contributes to attenuation of response to pro-apoptotic stimuli and increased ROS production both of which are implicated in the initiation and progression of most human cancers. Therefore, in order to characterize the mitochondrial function in MCF7 cells, after MUTP treatment, the cells were stained with specific metabolic probes and analyzed by FACS. The outcomes displayed that MUTP treatment decreased mitochondrial mass and mitochondrial membrane potential and increased the ROS production. In agreement with these findings, the reduction in the expression of the mitochondrial oxidative pathway (OXPHOS) enzymes revealed a bioenergetics failure, induced by MUTP, in treated cells. TUNEL assay, DNA Laddering and Western blot analysis of caspase-3, caspase-9 and Bax confirmed the apoptotic effect of MUTP treatment. Taken together, all these data suggest that MUTP may be capable of selectively targeting neoplastic cell growth and therefore has potential applications as an anticancer agent.
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    Role of ERalpha/NOTCH4 axis in sustaining stemness in breast cancer cells
    (2018-02-27) Elena Spina, Elena Spina; Andò, Sebastiano
    Early detection and new therapeutic strategies have improved breast cancer patient outcome and survival rates in the last years. However, breast cancer still remains the second leading cause of cancer-related deaths among women worldwide, and approximately 30% of patients eventually experience a tumor relapse. Treatment failure is mainly due to metastatic process and resistance to conventional therapy. Over the past decade it has been established the existence of a subpopulation of cancer stem cell (CSC) within breast cancers that is responsible for tumor initiation, progression and resistance to endocrine therapies. It is well known the “driving role” of oestrogens and its receptor alpha (ERα), in development and progression of breast cancer disease, but still unknown their role in regulating breast CSCs (BCSCs). In the past few years, several studies revealed the presence of gain-of-function mutation in ESR1, gene encoding for ERα, in metastatic breast cancer patients after long-term endocrine therapies treatment. Particularly, Y537N, Y537S and D538G are the most frequent “hot spot” mutations within ERα hormone-binding domain (HBD) that lead to ligand-independent ERα activity and consequently, resistance to endocrine therapy. Here, we studied how HBD-ESR1 mutations might account for a mechanism of metastatic process and endocrine resistance, sustaining stem cell-like phenotype. As experimental model, we used breast cancer cell lines expressing wild-type and HBDESR1 mutations. Our results, using in vivo and in vitro experiment (mammosphereforming assay and CD44+/CD24- phenotype analysis) have suggested an enrichment of BCSCs activity by HBD-ESR1 mutations, that seems to be sustained by Notch4 signaling through constitutive hyper phosphorylation of Serine 118 residue of ERα that has been demonstrated related to stem cell phenotype and tumor initiation, in mutant-expressing cells. Experiments conducted using CRISPR-Cas9 knock-in of Y537S-ERα mutation confirmed the role of this mutation in tumor initiation and progression as obtained using HBD-ESR1 stable clones. We propose a potential novel role of HBD-ESR1 mutations in sustaining BCSCs activity, that could have clinical relevance, suggesting new molecular biomarker and target to aim better therapeutic strategies for ERα-positive breast cancer metastatic patients.
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    Targeting systems vulnerabilities in uveal melanoma by CRISPR Cas/9 focal adhesion kinase (FAK) genome editing and therapeutic inhibition
    (2018-02-27) Rigiracciolo, Damiano Cosimo; Andò, Sebastiano; Maggiolini, Marcello
    Il Melanoma Uveale rappresenta la neoplasia intraoculare più frequente nell’età adulta. Colpisce circa 2,500 individui ogni anno negli USA ed il 50% dei pazienti affetti da tale neoplasia sviluppa metastasi entro 5 anni dalla diagnosi. Non essendo state ancora identificate terapie efficaci, la sopravvivenza in presenza di metastasi è di circa 6 mesi. Il Melanoma Uveale è geneticamente caratterizzato dalla presenza di mutazioni somatiche attivanti a carico degli oncogeni GNAQ e GNA11, che codificano per due diverse subunità α delle proteine G. Tali mutazioni sono state identificate rispettivamente in circa il 94% dei casi di Melanoma Cutaneo ed il 4% dei casi di Melanoma Uveale. Sulla base di tali osservazioni, nel presente lavoro di tesi è stato valutato il ruolo esercitato da una proteina citoplasmatica ad attività tirosin-chinasica associata ai recettori per le integrine denominata FAK (focal adhesion kinase), nella progressione del Melanoma Uveale, sia in vitro che in vivo. In particolare, mediante analisi bioinformatica (www.cbioportal.com) delle alterazioni genomiche di campioni estratti da pazienti affetti da melanoma uveale (n=80), è stato inizialmente determinato che il gene codificante per FAK (PTK2) risulta over-espresso nel 56% dei casi. Inoltre, il presente studio condotto in cellule di Melanoma Uveale OMM1.3 (GNAQ/11 mutate) e in cellule ingegnerizzate per l’espressione di un recettore di membrana accoppiato a proteine-G (Gαq) attivato esclusivamente da ligandi sintetici denominate HEK293 DREADD/Gq, ha dimostrato il coinvolgimento di segnali mediati da GNAQ nell’attivazione di FAK attraverso il reclutamento del fattore coinvolto nello scambio di nucleotidi guaninici denominato TRIO e la proteina appartenente alla super-famiglia di Ras denominata Rho-A. A riprova, saggi biologici hanno dimostrato l’efficacia di specifici inibitori di FAK nei processi di proliferazione cellulare sia in cellule di Melanoma Uveale derivanti da lesioni primarie che da metastasi epatiche. Attraverso l’innovativo approccio genetico denominato CRISPR/Cas 9 genome editing (Clustered Regularly Interspaced Short Palindromic Repeats), il silenziamento dell’espressione di FAK ha ridotto significativamente la crescita del melanoma uveale in modelli sperimentali utilizzati in vivo. Collettivamente, i risultati ottenuti indicano che FAK può essere considerato un potenziale target terapeutico per il trattamento del Melanoma Uveale e di altre neoplasie caratterizzate da mutazioni oncogeniche a carico delle subunità αq/α11 dei recettori di membrana accoppiati a proteine G.