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.

New Synthetic Polyamines as Multi-Target-Directed Ligands for the Treatment of Alzheimer's Disease and Cancer: Design, Synthesis and Biological Evaluation

Alzheimer's disease (AD) and cancer represent two of the main causes of death worldwide. They are complex multifactorial diseases and several biochemical targets have been recognized to play a fundamental role in their development. Basing on their complex nature, a promising therapeutical approach could be represented by the so-called "Multi-Target-Directed Ligand" approach. This new strategy is based on the assumption that a single molecule could hit several targets responsible for the onset and/or progression of the pathology. In particular in AD, most currently prescribed drugs aim to increase the level of acetylcholine in the brain by inhibiting the enzyme acetylcholinesterase (AChE). However, clinical experience shows that AChE inhibition is a palliative treatment, and the simple modulation of a single target does not address AD aetiology. Research into newer and more potent anti-AD agents is thus focused on compounds whose properties go beyond AChE inhibition (such as inhibition of the enzyme β-secretase and inhibition of the aggregation of beta-amyloid). Therefore, the MTDL strategy seems a more appropriate approach for addressing the complexity of AD and may provide new drugs for tackling its multifactorial nature. In this thesis, it is described the design of new MTDLs able to tackle the multifactorial nature of AD. Such new MTDLs designed are less flexible analogues of Caproctamine, one of the first MTDL owing biological properties useful for the AD treatment. These new compounds are able to inhibit the enzymes AChE, beta-secretase and to inhibit both AChE-induced and self-induced beta-amyloid aggregation. In particular, the most potent compound of the series is able to inhibit AChE in subnanomolar range, to inhibit β-secretase in micromolar concentration and to inhibit both AChE-induced and self-induced beta-amyloid aggregation in micromolar concentration. Cancer, as AD, is a very complex pathology and many different therapeutical approaches are currently use for the treatment of such pathology. However, due to its multifactorial nature the MTDL approach could be, in principle, apply also to this pathology. Aim of this thesis has been the development of new molecules owing different structural motifs able to simultaneously interact with some of the multitude of targets responsible for the pathology. The designed compounds displayed cytotoxic activity in different cancer cell lines. In particular, the most potent compounds of the series have been further evaluated and they were able to bind DNA resulting 100-fold more potent than the reference compound Mitonafide. Furthermore, these compounds were able to trigger apoptosis through caspases activation and to inhibit PIN1 (preliminary result). This last protein is a very promising target because it is overexpressed in many human cancers, it functions as critical catalyst for multiple oncogenic pathways and in several cancer cell lines depletion of PIN1 determines arrest of mitosis followed by apoptosis induction. In conclusion, this study may represent a promising starting pint for the development of new MTDLs hopefully useful for cancer and AD treatment.

Transcriptional functions of DNA Topoisomerases at a genome-wide scale and a single gene levels

The DNA topology is an important modifier of DNA functions. Torsional stress is generated when right handed DNA is either over- or underwound, producing structural deformations which drive or are driven by processes such as replication, transcription, recombination and repair. DNA topoisomerases are molecular machines that regulate the topological state of the DNA in the cell. These enzymes accomplish this task by either passing one strand of the DNA through a break in the opposing strand or by passing a region of the duplex from the same or a different molecule through a double-stranded cut generated in the DNA. Because of their ability to cut one or two strands of DNA they are also target for some of the most successful anticancer drugs used in standard combination therapies of human cancers. An effective anticancer drug is Camptothecin (CPT) that specifically targets DNA topoisomerase 1 (TOP 1). The research project of the present thesis has been focused on the role of human TOP 1 during transcription and on the transcriptional consequences associated with TOP 1 inhibition by CPT in human cell lines. Previous findings demonstrate that TOP 1 inhibition by CPT perturbs RNA polymerase (RNAP II) density at promoters and along transcribed genes suggesting an involvement of TOP 1 in RNAP II promoter proximal pausing site. Within the transcription cycle, promoter pausing is a fundamental step the importance of which has been well established as a means of coupling elongation to RNA maturation. By measuring nascent RNA transcripts bound to chromatin, we demonstrated that TOP 1 inhibition by CPT can enhance RNAP II escape from promoter proximal pausing site of the human Hypoxia Inducible Factor 1 (HIF-1) and c-MYC genes in a dose dependent manner. This effect is dependent from Cdk7/Cdk9 activities since it can be reversed by the kinases inhibitor DRB. Since CPT affects RNAP II by promoting the hyperphosphorylation of its Rpb1 subunit the findings suggest that TOP 1inhibition by CPT may increase the activity of Cdks which in turn phosphorylate the Rpb1 subunit of RNAP II enhancing its escape from pausing. Interestingly, the transcriptional consequences of CPT induced topological stress are wider than expected. CPT increased co-transcriptional splicing of exon1 and 2 and markedly affected alternative splicing at exon 11. Surprisingly despite its well-established transcription inhibitory activity, CPT can trigger the production of a novel long RNA (5’aHIF-1) antisense to the human HIF-1 mRNA and a known antisense RNA at the 3’ end of the gene, while decreasing mRNA levels. The effects require TOP 1 and are independent from CPT induced DNA damage. Thus, when the supercoiling imbalance promoted by CPT occurs at promoter, it may trigger deregulation of the RNAP II pausing, increased chromatin accessibility and activation/derepression of antisense transcripts in a Cdks dependent manner. A changed balance of antisense transcripts and mRNAs may regulate the activity of HIF-1 and contribute to the control of tumor progression After focusing our TOP 1 investigations at a single gene level, we have extended the study to the whole genome by developing the “Topo-Seq” approach which generates a map of genome-wide distribution of sites of TOP 1 activity sites in human cells. The preliminary data revealed that TOP 1 preferentially localizes at intragenic regions and in particular at 5’ and 3’ ends of genes. Surprisingly upon TOP 1 downregulation, which impairs protein expression by 80%, TOP 1 molecules are mostly localized around 3’ ends of genes, thus suggesting that its activity is essential at these regions and can be compensate at 5’ ends. The developed procedure is a pioneer tool for the detection of TOP 1 cleavage sites across the genome and can open the way to further investigations of the enzyme roles in different nuclear processes.

Farmacogenomica della Clofarabina nel trattamento delle Leucemie Acute pediatriche: identificazione di nuovi bersagli molecolari e del profilo genomico associato all’efficacia terapeutica del farmaco antitumorale

In quest’ultimi decenni si è assistito ad un notevole miglioramento nella terapia delle Leucemie Acute (LA) pediatriche, nonostante tutto si assiste oggi ad una fase di plateau della curva di sopravvivenza e le leucemie continuano a costituire la principale causa di morte pediatrica per malattia. Ulteriori progressi nel trattamento delle LA potrebbero essere ottenuti mediante studi di farmacogenomica che, identificando le componenti genetiche associate alla risposta individuale ai trattamenti farmacologici, consentono il disegno di terapie personalizzate e tumore-specifiche, ad alta efficacia e bassa tossicità per ciascun paziente. Il lavoro svolto è stato, dunque, finalizzato allo studio della farmacogenomica del farmaco antitumorale Clofarabina (CLO) nel trattamento delle LA pediatriche al fine di identificare marcatori genetici predittivi di risposta delle cellule leucemiche al farmaco, delucidare i meccanismi di resistenza cellulare ed individuare nuovi bersagli verso cui indirizzare terapie più mirate ed efficaci. L’analisi in vitro della sensibilità alla CLO di blasti provenienti da pazienti pediatrici affetti da Leucemia Acuta Linfoblastica (LAL) e Mieloide (LAM) ha consentito l’identificazione di due sottopopolazioni di cellule LAL ad immunofenotipo T a diversa sensibilità alla CLO. Mediante DNA-microarrays, si è identificata la “signature” genetica specificamente associata alla diversa risposta delle cellule LAL-T al farmaco. Successivamente, la caratterizzazione funzionale dei geni differenziali e l’analisi dei pathways hanno consentito l’identificazione specifica di potenziali biomarcatori di risposta terapeutica aprendo nuove prospettive per la comprensione dei meccanismi di resistenza cellulare alla CLO e suggerendo un nuovo bersaglio terapeutico per le forme LAL-T a bassa sensibilità al farmaco. In conclusione, nel lavoro svolto si sono identificati set di geni e pathways di rilievo biologico per la risposta delle cellule LAL-T alla CLO suggerendo marcatori genetici in grado di identificare i soggetti eleggibili per il trattamento o verso cui disegnare terapie innovative. Il lavoro è paradigma per l’applicazione della farmacogenomica in altre neoplasie.

Ruolo dei recettori tirosinchinasici in oncologia animale

Disregolazioni dei recettori tirosinchinasici (RTK) sono di frequente riscontro nei tumori dell’uomo e in molti casi sono indicatori biologici che permettono di definire in maniera più accurata la prognosi dei pazienti. Possono rappresentare inoltre marker predittivi per la risposta a terapie antitumorali con farmaci a bersaglio molecolare. Numerosi inibitori tirosinchinasici (TKI) sono attualmente in corso di studio o già disponibili per l’utilizzo in oncologia umana, e molti di questi hanno dimostrato una significativa efficacia utilizzati singolarmente o in combinazione a terapie convenzionali. Studi recenti indicano che un quadro analogo di disregolazione dei recettori tirosinchinasici è presente anche nelle neoplasie dei piccoli animali, e ne suggeriscono in molti casi un’implicazione prognostica. Gli inibitori tirosinchinasi sono da poco entrati nell’arena dell’oncologia veterinaria, ma i primi risultati lasciano supporre che siano destinati ad essere integrati definitivamente nei protocolli terapeutici standard. La tesi consiste in una parte introduttiva in cui sono trattate le principali funzioni biologiche dei recettori tirosinchinasici, la loro struttura e il loro ruolo nell’oncogenesi e nella progressione tumorale in medicina umana e veterinaria. Si affrontano inoltre le principali metodiche di laboratorio per l’analisi molecolare in oncologia e i meccanismi d’azione dei farmaci inibitori tirosinchinasici, con un cenno ai prodotti maggiormente utilizzati e alle loro indicazioni. Segue la presentazione e la discussione dei risultati di quattro studi relativi alla valutazione delle disregolazioni del recettore tirosinchinasico Kit (espressione aberrante e mutazioni genomiche) nel mastocitoma cutaneo del gatto e del recettore del fattore di crescita epidermico (EGFR) nel carcinoma squamocellulare cutaneo del gatto e nei tumori polmonari primitivi del cane, con particolare attenzione al loro ruolo prognostico.

Virus oncogeni a dna in tumori umani: studio delle infezioni da papillomavirus e poliomavirus in neoplasie di diversa origine.

I virus tumorali inducono oncogenesi nel loro ospite naturale o in sistemi animali sperimentali, manipolando diverse vie cellulari. Ad oggi, sono stati identificati sette virus capaci di causare specifici tumori umani. Inoltre HPV, JCV ed SV40, sono stati associati con un grande numero di tumori umani in sedi corporee non convenzionali, ma, nonostante molti anni di ricerca, nessuna eziologia virale è stata ancora confermata. Lo scopo di questo studio è stato di valutare la presenza ed il significato sia di JCV ed SV40 in tumori ossei umani, e di HPV nel carcinoma della mammella (BC), galattoforectomie (GF), secrezioni mammarie patologiche (ND) e glioblastoma multiforme (GBM). Tecniche di biologia molecolare sono state impiegate per esaminare campioni di tessuto tumorale di 70 tumori ossei (20 osteosarcomi [OS], 20 tumori a cellule giganti [TCG], 30 condrosarcomi [CS]), 168 BCs , 30 GFs, 59 GBM e 30 campioni di ND. Il genoma di SV40 e JCV è stato trovato nel 70% dei CS + 20% degli OS, e nel 13% dei CS +10% dei TCG, rispettivamente. Il DNA di HPV è stato rilevato nel 30% dei pazienti con BC, nel 27% dei campioni GF e nel 13% dei NDs. HPV16 è stato il genotipo maggiormente osservato in tutti questi campioni, seguito da HPV18 e HPV35. Inoltre, il DNA di HPV è stato trovato nel 22% dei pazienti con GBM, in questo tumore HPV6 era il tipo più frequentemente rilevato, seguito da HPV16. L’ ISH ha mostrato che il DNA di HPV è situato all’interno di cellule tumorali mammarie e di GBM. I nostri risultati suggeriscono un possibile ruolo di JCV, SV40 e HPV in questi tumori, se non come induttori come promotori del processo neoplastico, tuttavia diversi criteri devono ancora essere soddisfatti prima di chiarirne il ruolo.

Targeting the p53–MDM2 interaction by the small-molecule MDM2 antagonist Nutlin-3a: a new challenged target therapy in adult Philadelphia positive acute lymphoblastic leukemia patients

The human p53 tumor suppressor, known as the “guardian of the genome”, is one of the most important molecules in human cancers. One mechanism for suppressing p53 uses its negative regulator, MDM2, which modulates p53 by binding directly to and decreasing p53 stability. In testing novel therapeutic approaches activating p53, we investigated the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph-) leukemic cell line models, and primary B-Acute lymphoblastic leukemia (ALL) patient samples. In this study we demonstrated that treatment with Nutlin-3a induced grow arrest and apoptosis mediated by p53 pathway in ALL cells with wild-type p53, in time and dose-dependent manner. Consequently, MDM2 inhibitor caused an increase of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from Ph+ ALL patients with the T315I Bcr-Abl kinase domain mutation. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis in sensitive cell lines was performed. A total of 621 genes were differentially expressed (p < 0.05). We found a strong down-regulation of GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) (fold-change -1.35 and -1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells. Both genes are repressors of INK4/ARF and p21. Given the importance of BMI in the control of apoptosis, we investigated its pattern in treated and untreated cells, confirming a marked decrease after exposure to MDM2 inhibitor in ALL cells. Noteworthy, the BMI-1 levels remained constant in resistant cells. Therefore, BMI-1 may be used as a biomarker of response. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph-ALL.

Study of PI3K/Akt signaling pathway as potential molecular target for T-cell acute lymphoblastic leukemia (T-ALL) treatment: pan-inhibition of PI3K catalitic isoforms as better therapeutic approach

Class I phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases consisting of a regulatory subunit and one of four catalytic subunits (p110α, p110β, p110γ or p110δ). p110γ/p110δ PI3Ks are highly enriched in leukocytes. In general, PI3Ks regulate a variety of cellular processes including cell proliferation, survival and metabolism, by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Their activity is tightly regulated by the phosphatase and tensin homolog (PTEN) lipid phosphatase. PI3Ks are widely implicated in human cancers, and in particular are upregulated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to loss of PTEN function. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. At present different compounds which target single or multiple PI3K isoforms have entered clinical trials. In the present research, it has been analyzed the therapeutic potential of the pan-PI3K inhibitor BKM120, an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T-lymphoblasts. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. BKM120 efficacy was confirmed in in vivo studies to a subcutaneous xenotransplant model of human T-ALL. Because it is still unclear which agents among isoform-specific or pan inhibitors can achieve the greater efficacy, further analyses have been conducted to investigate the effects of PI3K inhibition, in order to elucidate the mechanisms responsible for the proliferative impairment of T-ALL. Overall, these results indicated that BKM120 may be an efficient treatment for T-ALLs that have aberrant up-regulation of the PI3K signaling pathway and strongly support clinical application of pan-class I PI3K rather than single-isoform inhibitors in T-ALL treatment.

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.

Human papillomavirus (HPV) and associated diseases: between applied diagnostic and basic research

Human Papillomavirus (HPV) is the cause of cervical cancers (among these, adenocarcinoma, AdCa) and is associated to a subgroup of oropharyngeal carcinomas (OPSCCs). Even if the risk for cancer development is linked to the infection by some viral genotypes, mainly HPV16 and 18, viral DNA alone seems not to be sufficient for diagnosis. Moreover, the role of the virus in OPSCCs has not been totally clarified yet. In the first part of the thesis, the performances concerning viral genotyping in clinical cervical samples of a new pyrosequencing-based test and a well-known hybridization-based assay have been compared. Similar results between the methods have been obtained. However, the former showed advantages in detecting intratype variants, higher specificity and a broader spectrum of detectable HPV types. The second part deals with the evaluation of virological markers (genotyping, viral oncoproteins expression, viral load, physical state and CpG methylation of HPV16 genome) in the diagnosis/prognosis of cervical AdCa and HPV-associated OPSCCs. HPV16 has been confirmed the most prevalent genotype in both the populations. Interestingly, the mean methylation frequency of viral DNA at the early promoter showed the tendency to be associated to invasion for cervical AdCa and to a worse prognosis for OPSCCs, suggesting a promising role as diagnostic/prognostic biomarker. The experiments of the third part were performed at the DKFZ in Heidelberg (Germany) and dealt with the analysis of the response to IFN-k transfection in HPV16-positive cervical cancer and head&neck carcinoma cell lines to evaluate its potential role as new treatment. After 24h, we observed increased IFN-b expression which lead to the up-regulation of genes involved in the antigens presentation pathway (MHC class I and immunoproteasome) and antiviral response as well, in particular in cervical cancer cell lines. This fact suggested also the presence of different HPV-mediated carcinogenic pathways between the two anatomical districts.

Preclinical development of anti-cancer strategies against human HER-2

HER-2 is a 185 kDa transmembrane receptor tyrosine kinase that belongs to the EGFR family. HER-2 is overexpressed in nearly 25% of human breast cancers and women with this subtype of breast cancer have a worse prognosis and frequently develop metastases. The progressive high number of HER-2-positive breast cancer patients with metastatic spread in the brain (up to half of women) has been attributed to the reduction in mortality, the effectiveness of Trastuzumab in killing metastatic cells in other organs and to its incapability to cross the blood-brain barrier. Apart from full-length-HER-2, a splice variant of HER-2 lacking exon 16 (here referred to as D16) was identified in human HER-2-positive breast cancers. Here, the contribution of HER-2 and D16 to mammary carcinogenesis was investigated in a model transgenic for both genes (F1 model). A dominant role of D16, especially in early stages of tumorigenesis, was suggested and the coexistence of heterogeneous levels of HER-2 and D16 in F1 tumors revealed the undeniable value of F1 strain as preclinical model of HER-2-positive breast cancer, closer resembling the human situation in respect to previous models. The therapeutical efficacy of anti-HER-2 agents, targeting HER-2 receptor (Trastuzumab, Lapatinib, R-LM249) or signaling effectors (Dasatinib, UO126, NVP-BKM120), was investigated in models of local or advanced HER-2-positive breast cancer. In contrast with early studies, data herein collected suggested that the presence of D16 can predict a better response to Trastuzumab and other agents targeting HER-2 receptor or Src activity. Using a multiorgan HER-2-positive metastatic model, the efficacy of NVP-BKM120 (PI3K inhibitor) in blocking the growth of brain metastases and the oncolytic ability of R-LM249 (HER-2-retargeted HSV) to reach and destroy metastatic HER-2-positive cancer cells were shown. Finally, exploiting the definition of “oncoantigen” given to HER-2, the immunopreventive activity of two vaccines on HER-2-positive mammary tumorigenesis was demonstrated.