RNA Interference and cyclooxygenase-2 (COX-2) regulation in colon cancer cells

Despite new methods and combined strategies, conventional cancer chemotherapy still lacks specificity and induces drug resistance. Gene therapy can offer the potential to obtain the success in the clinical treatment of cancer and this can be achieved by replacing mutated tumour suppressor genes, inhibiting gene transcription, introducing new genes encoding for therapeutic products, or specifically silencing any given target gene. Concerning gene silencing, attention has recently shifted onto the RNA interference (RNAi) phenomenon. Gene silencing mediated by RNAi machinery is based on short RNA molecules, small interfering RNAs (siRNAs) and microRNAs (miRNAs), that are fully o partially homologous to the mRNA of the genes being silenced, respectively. On one hand, synthetic siRNAs appear as an important research tool to understand the function of a gene and the prospect of using siRNAs as potent and specific inhibitors of any target gene provides a new therapeutical approach for many untreatable diseases, particularly cancer. On the other hand, the discovery of the gene regulatory pathways mediated by miRNAs, offered to the research community new important perspectives for the comprehension of the physiological and, above all, the pathological mechanisms underlying the gene regulation. Indeed, changes in miRNAs expression have been identified in several types of neoplasia and it has also been proposed that the overexpression of genes in cancer cells may be due to the disruption of a control network in which relevant miRNA are implicated. For these reasons, I focused my research on a possible link between RNAi and the enzyme cyclooxygenase-2 (COX-2) in the field of colorectal cancer (CRC), since it has been established that the transition adenoma-adenocarcinoma and the progression of CRC depend on aberrant constitutive expression of COX-2 gene. In fact, overexpressed COX-2 is involved in the block of apoptosis, the stimulation of tumor-angiogenesis and promotes cell invasion, tumour growth and metastatization. On the basis of data reported in the literature, the first aim of my research was to develop an innovative and effective tool, based on the RNAi mechanism, able to silence strongly and specifically COX-2 expression in human colorectal cancer cell lines. In this study, I firstly show that an siRNA sequence directed against COX-2 mRNA (siCOX-2), potently downregulated COX-2 gene expression in human umbilical vein endothelial cells (HUVEC) and inhibited PMA-induced angiogenesis in vitro in a specific, non-toxic manner. Moreover, I found that the insertion of a specific cassette carrying anti-COX-2 shRNA sequence (shCOX-2, the precursor of siCOX-2 previously tested) into a viral vector (pSUPER.retro) greatly increased silencing potency in a colon cancer cell line (HT-29) without activating any interferon response. Phenotypically, COX-2 deficient HT-29 cells showed a significant impairment of their in vitro malignant behaviour. Thus, results reported here indicate an easy-to-use, powerful and high selective virus-based method to knockdown COX-2 gene in a stable and long-lasting manner, in colon cancer cells. Furthermore, they open up the possibility of an in vivo application of this anti-COX-2 retroviral vector, as therapeutic agent for human cancers overexpressing COX-2. In order to improve the tumour selectivity, pSUPER.retro vector was modified for the shCOX-2 expression cassette. The aim was to obtain a strong, specific transcription of shCOX-2 followed by COX-2 silencing mediated by siCOX-2 only in cancer cells. For this reason, H1 promoter in basic pSUPER.retro vector [pS(H1)] was substituted with the human Cox-2 promoter [pS(COX2)] and with a promoter containing repeated copies of the TCF binding element (TBE) [pS(TBE)]. These promoters were choosen because they are partculary activated in colon cancer cells. COX-2 was effectively silenced in HT-29 and HCA-7 colon cancer cells by using enhanced pS(COX2) and pS(TBE) vectors. In particular, an higher siCOX-2 production followed by a stronger inhibition of Cox-2 gene were achieved by using pS(TBE) vector, that represents not only the most effective, but also the most specific system to downregulate COX-2 in colon cancer cells. Because of the many limits that a retroviral therapy could have in a possible in vivo treatment of CRC, the next goal was to render the enhanced RNAi-mediate COX-2 silencing more suitable for this kind of application. Xiang and et al. (2006) demonstrated that it is possible to induce RNAi in mammalian cells after infection with engineered E. Coli strains expressing Inv and HlyA genes, which encode for two bacterial factors needed for successful transfer of shRNA in mammalian cells. This system, called “trans-kingdom” RNAi (tkRNAi) could represent an optimal approach for the treatment of colorectal cancer, since E. Coli in normally resident in human intestinal flora and could easily vehicled to the tumor tissue. For this reason, I tested the improved COX-2 silencing mediated by pS(COX2) and pS(TBE) vectors by using tkRNAi system. Results obtained in HT-29 and HCA-7 cell lines were in high agreement with data previously collected after the transfection of pS(COX2) and pS(TBE) vectors in the same cell lines. These findings suggest that tkRNAi system for COX-2 silencing, in particular mediated by pS(TBE) vector, could represent a promising tool for the treatment of colorectal cancer. Flanking the studies addressed to the setting-up of a RNAi-mediated therapeutical strategy, I proposed to get ahead with the comprehension of new molecular basis of human colorectal cancer. In particular, it is known that components of the miRNA/RNAi pathway may be altered during the progressive development of colorectal cancer (CRC), and it has been already demonstrated that some miRNAs work as tumor suppressors or oncomiRs in colon cancer. Thus, my hypothesis was that overexpressed COX-2 protein in colon cancer could be the result of decreased levels of one or more tumor suppressor miRNAs. In this thesis, I clearly show an inverse correlation between COX-2 expression and the human miR- 101(1) levels in colon cancer cell lines, tissues and metastases. I also demonstrate that the in vitro modulating of miR-101(1) expression in colon cancer cell lines leads to significant variations in COX-2 expression, and this phenomenon is based on a direct interaction between miR-101(1) and COX-2 mRNA. Moreover, I started to investigate miR-101(1) regulation in the hypoxic environment since adaptation to hypoxia is critical for tumor cell growth and survival and it is known that COX-2 can be induced directly by hypoxia-inducible factor 1 (HIF-1). Surprisingly, I observed that COX-2 overexpression induced by hypoxia is always coupled to a significant decrease of miR-101(1) levels in colon cancer cell lines, suggesting that miR-101(1) regulation could be involved in the adaption of cancer cells to the hypoxic environment that strongly characterize CRC tissues.

MicroRNAs expression analysis in high grade gliomas

Several biomarkers had been proposed as useful parameters to better define the prognosis or to delineate new target therapy strategies for glioblastoma (GBM) patients. MicroRNAs could represent interesting molecules, for their role in tumorigenesis and cancer progression and for their specific tissue expression. Although many studies have tried to identify a specific microRNAs signature for glioblastoma, by now an exhaustive GBM microRNAs profile is far to be well defined. In this work we set up a real-time qPCR, based on LNA primers, to investigate the expression of 19 microRNAs in brain tumors, focusing our attention on GBMs. MiRNAs expression in 30 GBM paired FFPE-Fresh/Frozen samples was firstly analyzed. The good correlation obtained comparing miRNAs results confirmed the feasibility of performing miRNAs analysis starting from FFPE tissues. This leads to many advantages, as a good disposal of archival tumor and normal brain specimens and the possibility to verify the percentage of tumor cells in the analyzed sample. In the second part we compared 3 non-neoplastic brain references to use as control in miRNAs analysis. Normal adjacent the tumor, epileptic specimens and a commercial total RNA were analyzed for miRNAs expression and results showed that different non-neoplastic controls could lead to important discrepancies in GBM miRNAs profiles. Analyzing 50 FFPE GBMs using all 3 non-neoplastic references, we defined a putative GBM miRNAs signature: mir-10b, miR-21 and miR-27a resulted upregulated, while miR-7, miR-9, miR-26a, miR-31, miR-101, miR-137, miR-222 and miR-330 were downregulated. Comparing miRNAs expression among GBM group and gliomas of grade I, II and III, we obtained 3 miRNAs (miR-10b, mir-34a and miR-101) showing a different regulation status between high grade and low grade gliomas. Intriguingly, miR-10b was upregulated in high grade and significantly downregulated in low grade gliomas, suggesting that could be a candidate for a GBM target therapy.

Involvement of microRNAs in Androgen Receptor-dependent Breast Cancers

Triple negative breast cancer (TNBC) is a very aggressive tumor subtype characterized by the lack of expression of estrogen receptor 1 (ESR1), due in the most of cases to an increased expression of DNA methyltransferases (DNMTs) and hypermethylation in CpG islands, resulting in gene silencing. Furthermore, in ESR1- negative breast cancers, androgen receptor (AR) is highly expressed and some studies suggest that it can drive tumor progression and might represent a therapeutic target. A correlation between microRNAs, small non-coding RNAs that regulate gene expression, and DNMTs was investigated in a TNBC cell line to restore a normal methylation pattern of ESR1, leading to its re-expression and conferring again sensitivity to selective estrogen receptor modulators (SERMs). miR-148A and miR-29B were found to be involved in the reduction of the expression of DNMT1 and DNMT3A and in a slight increase of ESR1 expression, but not at protein level. Then, we found a down-regulation of AR by miRs-7, -9, -27a, -27b, -29a, -29b, -29c, -127-3p, -127-5p and -376 at 48h post transfection and an up-regulation by miR-15a and miR-16 at every time considered. We concomitantly investigated a possible increase of Tamoxifen, Herceptin and Metformin sensitivity after AR silencing in MDA-MB 453 and T-47D cell lines. Cells seemed more sensitive when silenced for AR only in MDA-MB-453 at 24h post Tamoxifen treatment. Studies on Metformin have basically confirmed an increase of drug sensitivity due to AR silencing in both cell lines. Analysis of Herceptin showed how MDA-MB 453 samples silenced for AR have a slight decrease in the percentage of proliferating cells, demonstrating a possible increase in the response to treatment. These preliminary data provide the basis for further study of the modulation of the expression of AR by microRNAs and it will be interesting to understand the molecular mechanisms underlying these interactions.

Studio di marcatori immunoistochimici, analisi FISH e ruolo dei microRNA nelle neoplasie gliali di basso e di alto grado

Gliomas are the most common primary brain tumours. Despite advances in surgical techniques, postoperative supportive care, radiation and adjuvant systemic therapy, the life expectancy of patients with high grade glioma has remained essentially poor. Furthermore differential diagnosis among astrocytomas, oligodendrogliomas and oligoastrocytomas is very challenging and subject to inter-observer variability. The purpose of the research was: 1) to investigate a series of high grade and low grade gliomas at gene and protein (immunohistochemistry) levels to disclose possible genetic portraits of malignancy; 2) to verify the utility of Nogo-A, Olig-2 and synaptophysin in providing a correct histological diagnosis of oligodendroglioma and to investigate a possible complementary role in selecting the best areas suitable for detecting 1p/19q codeletion using FISH analysis; 3) to study the role of microRNA in high grade gliomas. In order to obtain these goals large series of brain tumors were studied with DNA microarrays, immunohistochemistry and RT-PCR The results demonstrated that: - Overexpression of IGFBP-2 and CDC20 is highly related to glioblastomas and their immunopositivity can be useful for the identification of glioblastoma in small biopsies. - Nogo-A is the most useful and specific marker in differentiating oigodendrogliomas from other gliomas. Furthermore, using a Nogo-A driven FISH analysis, it is possible to identify a larger number of 1p19q codeletions in gliomas. - microRNAs can be studied in paraffin embedded tissues better than in fresh tissues. A series of six microRNA, significatively deregulated in glioblastomas, may represent a genetic signature with prognostic and predictive value and could constitute candidates for novel anti-cancer therapeutics.

Influence of genetic variants and post transcriptional factors on imatinib transporters as determinants of the pharmacological response

Introduction – Although imatinib (IM) is a recognized gold standard in chronic myeloid leukemia (CML) therapy, resistance has emerged in a significant proportion of patients. Aim – The aim of this study was: (1) to investigate the role of genetic variants in genes encoding for IM transporters, as candidate of IM responsiveness and (2) to test the influence of miRNAs on IM response, focusing on efflux transporters. Methods – As a first step, a panel of polymorphisms (SNPs) was genotyped in a subgroup population of 189 patients enrolled in the Tyrosine Kinase Inhibitor Optimization and Selectivity (TOPS) trial. The association with cytogenetic response and molecular response (MR) was assessed for each SNP. As a second step, an in vitro IM-resistant model (K-562 CML cell line) was established. miRNAs profiles were analyzed using Taqman arrays and in silico search was performed for miRNAs deregulated after IM treatment. mRNA and protein expression were quantified using TaqMan realtime PCR and Western blotting, respectively. Results – (1) Among Caucasian patients, ABCB1 rs60023214 significantly correlated with complete MR (P = 0.005). Concerning SNPs combination in IM uptake transporters, the associations with treatment outcomes were statistically significant for both major and complete MR (P = 0.005 and P = 0.01, respectively). (2) ABCB1 protein was not expressed under any conditions of treatment, differently from ABCG2. Two deregulated miRNAs, namely miR-212 and miR-328, were identified to be inversely correlated with ABCG2 (r2= 0.57; p=0.03 and r2=0.47; p=0.06, respectively). Experiments of loss and gain of function confirmed the functional influence of these miRNAs on ABCG2. Conclusion – The multiple candidate gene approach identified single and combination of SNPs that can be proposed as predictor of IM response. The in vitro study suggested that IM resistance could be mediated by miRNA-dependent mechanism. Further studies are needed to validate these preliminary findings.

Ricerca di marcatori molecolari nella prevenzione dei tumori di testa e collo

Oral cavity cancers (OSCC) are among the most malignances worldwide. OSCC tipically affects men in their IV or V dedade of life, and the most relevant risk factors are tobacco and alcohol consumption. OSCCs generally exhibit poor prognosis, and late stage identification correlates with higher mortality rates. Basic prognostic factors, are tumor size and presence of lymph node and/or distance metastases (T classification, N, M). However, tumors with the same TNM grade and similar morphology may have completely different evolution, because of their intrinsic biological characteristics. For these reasons, the identification of new molecular markers with a predictive value, could represent useful tools in OSCC prevention, prognosis and treatment. In the first part of my PhD project I evaluated the loss of heterozygosity as a possible cause of deregulation of well-known tumor suppressors genes. Obtained data put on light the importance of this rearrangement and genes PDCD4, CTNB1, CASP4 and HSP23, in the onset and progression of OSCC. Subsequently, the analysis of the expression profile of miRNAs, led to the identification of some miRNAs that seems to be involved in cancer development and metastatic progression. In both cases, we need further investigations to understand whether these molecules may be used ideal markers in OSCC diagnosis and treatment.

Analisi molecolare delle fasi evolutive della micosi fungoide

Lo scopo del progetto triennale del dottorato di ricerca è lo studio delle alterazioni genetiche in un gruppo di pazienti affetti da micosi fungoide ed un gruppo di pazienti affetti da sindrome di Sezary. Dalle biopsie cutanee è stato estratto il DNA e analizzato, comparandolo con DNA sano di riferimento, utilizzando la tecnica array-CGH, allo scopo di identificare la presenza di geni potenzialmente implicati nel processo di oncogenesi. Questa analisi è stata eseguita, per ogni paziente, su biopsie effettuate ad una fase iniziale di malattia e ad una fase di progressione della stessa. Sugli stessi pazienti è stata inoltre eseguita un’analisi miRNA. Si ipotizza che il profilo d’espressione dei miRNA possa infatti dare informazioni utili per predire lo stato di malattia, il decorso clinico, la progressione tumorale e la riposta terapeutica. Questo lavoro è stato poi eseguito su biopsie effettuate in pazienti affetti da sindrome di Sezary che, quando non insorge primitivamente come tale, si può considerare una fase evolutiva della micosi fungoide. La valutazione delle alterazioni genetiche, ed in particolare la correlazione esistente tra duplicazione e delezione genetica e sovra/sottoespressione genetica, è stata possibile attraverso l’interpretazione e la comparazione dei dati ottenuti attraverso le tecniche array-CGH e miRNA. Sono stati comparati i risultati ottenuti per valutare quali fossero le alterazioni cromosomiche riscontrate nei diversi stadi di malattia. L’applicazione dell’array-CGH e della metodica di analisi mi-RNA si sono rivelate molto utili per l’identificazione delle diverse aberrazioni cromosomiche presenti nel genoma dei pazienti affetti da micosi fungoide e sindrome di Sezary, per valutare la prognosi del paziente e per cercare di migliorare o trovare nuove linee terapeutiche per il trattamento delle due patologie. Lo studio di questi profili può rappresentare quindi uno strumento di grande importanza nella classificazione e nella diagnosi dei tumori.

Meccanismi replicativi e di regolazione dell'espressione genica di Parvovirus B19

Il Parvovirus B19, virus patogeno umano della famiglia Parvoviridae, mostra uno specifico tropismo per i precursori eritroidi e una limitata replicazione in alcune linee cellulari megacarioblastoidi. Allo scopo di sviluppare sistemi utili allo studio delle caratteristiche biologiche del virus, diversi laboratori si sono occupati della costruzione di cloni genomici di B19 dotati di competenza funzionale e capaci di generare virus infettante. Parte del presente lavoro ha riguardato l’analisi funzionale di diversi cloni genomici di B19 e ha permesso di caratterizzare le regioni terminali del virus e di identificare requisiti essenziali per la loro funzionalità. Nel contesto intracellulare, esistono differenti livelli di restrizione in relazione alla capacità della cellula di supportare la replicazione virale, non ancora del tutto caratterizzati. Inoltre si sono accumulate evidenze circa la capacità del B19 di instaurare persistenza in numerosi tessuti. Non sono ancora note le caratteristiche funzionali del genoma virale in questo stato, è possibile che il virus persista in forma silente e meccanismi epigenetici possano regolare tale silenziamento. In questo studio è stato analizzato lo stato di metilazione del genoma di B19 e il suo possibile effetto sul ciclo replicativo virale ed è stata investigata la possibile associazione del DNA virale agli istoni cellulari nel corso di infezione in vitro. I risultati ottenuti confermano la presenza di questi meccanismi epigenetici, potendo ipotizzare che giochino un importante ruolo nella regolazione della funzionalità virale e nell’interazione B19-cellula e siano un elemento critico per l’adattamento del virus nell’ambiente in cui si trova. Inoltre l’ipotesi che anche i microRNA possano assumere un importante significato nell’interazione B19-cellula è stata proposta da diversi lavori e nel presente studio è stata valutata la produzione di queste piccole molecole durante l'infezione in vitro, ricercando microRNA (cellulari e/o virali) con omologia di sequenza per il genoma di B19 e quindi specifici per il virus.

Caratterizzazione del soppressore tumorale miR-101 nel colon carcinoma

I microRNA sono una classe di piccole molecole di RNA non codificante che controllano la stabilità di numerosi RNA messaggeri, perciò sono considerati come “master regulator” dell’espressione genica. Ogni tumore è caratterizzato da un profilo di espressione alterato dei microRNA. Il miR-101 è un oncosoppressore represso nei tessuti tumorali ed è candidato come biomarcatore del cancro colon-rettale. È regolato da numerosi eventi fisiologici e patologici, come angiogenesi e carcinogenesi. Gli eventi molecolari coinvolti nella regolazione dell’espressione del miR-101 sono scarsamente conosciuti, poiché è trascritto da due loci genici non caratterizzati. L’obiettivo di questo lavoro è di caratterizzare i geni del miR-101 ed individuarne i regolatori molecolari coinvolti nella cancerogenesi colon-rettale.

Analisi di espressione di microRNA in Tumore a Cellule Giganti: identificazione dei target con ruolo di potenziali biomarcatori

Il Tumore a Cellule Giganti dell’osso (TCG) è una rara neoplasia che rappresenta il 5% dei tumori di natura ossea; sebbene venga considerato un tumore a decorso benigno può manifestare caratteri di aggressività locale dando origine a recidive locali nel 10-25% dei casi, e nel 2-4% dei casi metastatizza a livello polmonare. In questo studio è stata valutata l’espressione dei miRNA mediante miRNA microarray in 10 pazienti affetti da TCG, 5 con metastasi e 5 liberi da malattia; sono stati riscontrati miRNA differenzialmente espressi tra i 2 gruppi di pazienti e la successiva validazione mediante Real Time PCR ha confermato una differenza significativa per il miR-136 (p=0.04). Mediante analisi bioinformatica con il software TargetScan abbiamo identificato RANK e NF1B come target del miR-136 e ne abbiamo studiato l’espressione mediante Real Time PCR su una più ampia casistica di pazienti affetti da TCG, metastatico e non, evidenziando una maggior espressione di NF1B nel gruppo di pazienti metastatici, mentre RANK non ha dimostrato una differenza significativa. L’analisi di Western Blot ha rilevato una maggiore espressione di entrambe le proteine nei pazienti metastatici rispetto ai non metastatici. Successivamente è stato condotto uno studio di immunoistochimica su TMA di 163 campioni di pazienti affetti da TCG a diverso decorso clinico che ha dimostrato una maggiore e significativa espressione di entrambe i target nei pazienti con metastasi rispetto ai non metastatici; le analisi di popolazione mediante Kaplan-Meier hanno confermato la correlazione tra over-espressione di RANK, NF1B e ricaduta con metastasi (p=0.001 e p<0.0005 rispettivamente). Lo studio di immunoistochimica è stato ampliato alle proteine maggiormente coinvolte nell’osteolisi che risultano avere un significato prognostico; tuttavia mediante analisi di ROC, la co-over-espressione di RANK, RANKL e NF1B rappresenta il migliore modello per predire la comparsa di metastasi (AUC=0.782, p<0.0005).

Role of Non-coding RNAs in chemotherapeutic treatments

The transcribed ultraconserved regions (T-UCRs) are a group of long non-coding RNAs involved in human carcinogenesis. The factors regulating the expression of T-UCRs and their mechanism of action in human cancers are unknown. In this work it was shown that high expression of uc.339 associates with lower survival in 204 non-small cell lung cancer (NSCLC) patients. Moreover, it was shown that uc.339 found up-regulated in archival NSCLC samples, acts as a decoy RNA for miR-339-3p, -663-3p and -95-5p. So, Cyclin E2, a direct target of three microRNAs is up-regulated, inducing cancer growth and migration. Evidence of this mechanism was provided from cell lines and primary samples confirming that TP53 directly regulates uc.339. These results support a key role for uc.339 in lung cancer.