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.

The role of interleuchin 6 (IL-6) in the promotion of an aggressive and stem cell-like phenotype in human breast cancer cells and in stem/progenitor cells expanded in vitro as mammospheres

High serum levels of Interleukin-6 (IL-6) correlate with poor outcome in breast cancer patients. However no data are available on the relationship between IL-6 and stem/progenitor cells which may fuel the genesis of breast cancer in vivo. Herein, we address this issue in mammospheres (MS), multi-cellular structures enriched in stem/progenitor cells of the mammary gland, and also in MCF-7 breast cancer cells. We show that MS from node invasive breast carcinoma tissues express IL-6 mRNA at higher levels than MS from matched non-neoplastic mammary glands. We find that IL-6 mRNA is detectable only in basal-like breast carcinoma tissues, an aggressive variant showing stem cell features. Our results reveal that IL-6 triggers a Notch-3-dependent up-regulation of the Notch ligand Jagged-1, whose interaction with Notch-3 promotes the growth of MS and MCF-7 derived spheroids. Moreover, IL-6 induces a Notch-3-dependent up-regulation of the carbonic anhydrase IX gene, which promotes a hypoxia-resistant/invasive phenotype in MCF-7 cells and MS. Finally, an autocrine IL-6 loop relies upon Notch-3 activity to sustain the aggressive features of MCF-7-derived hypoxia-selected cells. In conclusion, our data support the hypothesis that IL-6 induces malignant features in Notch-3 expressing, stem/progenitor cells from human ductal breast carcinoma and normal mammary gland.

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.

Classificazione su base molecolare dei tumori mammari della cagna mediante metodica immunoistochimica

Background. A new classification system of human breast tumours based on the immunohistochemical characterization has been applied to mammary tumours of the female dog with the aim to verify its association with invasion and grade, and prognostic aid in veterinary medicine. Methods. Forty-five canine mammary carcinomas with a two-year post-mastectomy follow-up were selected from our database, and the following antibodies were applied: anti-cytokeratines 14, 5/6, oestrogen receptor (ER), progesterone receptor (PR), and ERB-B2. . The tumours were grouped for phenotype as: luminal-like (ER+ and/or PR+, CK14-, CK5/6-) type A (ERB-B2-), and B (ERB-B2+); basal-like (ER-, PR-, CK14+ and/or CK5/6+, ERB-B2-); ERB-B2 (ER-, PR-, CK14-, CK5/6-, ERB-B2+). Association with invasion, grade and histotypes were evaluated and Kaplan-Meier survival curves estimated, then compared by survival analysis. Results. Thirty-five cases with luminal pattern (ER+ and PR+) were subgrouped into 13 A type and 22 B type, if ERB-B2 positive or negative . Most luminal-like A and basal-like cases were grade 1 carcinomas, while the percentage of luminal B cases was higher in grade 2 and 3 (Pearson Chi-square P=0.009). No difference in the percentage of molecular subtypes was evidenced between simple and complex/mixed carcinomas (Pearson Chi-square P=0.47). No significant results were obtained by survival analysis, even if basal-like had a more favourable prognosis than luminal-like. Conclusion. The panel of antibodies identified only 3 groups (luminal-like A and B, and basal-like) in the dog. Even though canine mammary tumours may be a model of human breast cancer, the existence of the same types of carcinoma as in the woman need to be confirmed. Canine mammary carcinomas show high molecular heterogeneity, which would benefit from a classification based on molecular differences. However, by multivariate analysis, the molecular classification appears a variable with a dependent value if compared to invasion and grade that are independent, suggesting that, at present, caution should be used in the application of such a classification to the dog, in which invasion and grade supply the most important prognostic information.

Cellular and molecular effects of cross reacting material 197 (CRM197) on HT-29 colon cancer cell line

Cross Reacting Material 197(CRM197) is a Diphteria toxin non toxic mutant that had shown anti-tumor activity in mice and humans. CRM197 is utilized as a specific inhibitor of heparin-binding epidermal growth factor (HB-EGF), that competes for the epidermal growth factor receptor (EGFR), overexpressed in colorectal cancer and implicated in its progression. We evaluated the effects of CRM197 on HT-29 human colon cancer cell line behaviour and, for CRM197 recognized ability to inhibit HB-EGF, its possible effects on EGFR activation. In particular, while HT-29 does not show any reduction of viability after CRM197 treatment, or changes in cell cycle distribution, in EGFR localization or activation, they show a change in gene expression profile analyzed by microarray. This is the first study where the CRM197 treatment on HT-29 show the alteration of a specific and selected number of genes.

Molecular analysis of Papillomavirus-induced cutaneous tumors in equids

Papillomavirus associated tumors are well recognized entities in humans as well as in animals. Here is reviewed the current understanding of human papillomavirus (HPV) associated cancers to better understand the oncogenic mechanisms of Equine papillomavirus (EcPV) and Bovine Papillomavirus (BPV) in horses. In the first part of this study the interactions between Equine papillomavirus 2 (EcPV-2) and cell cycle proteins are discussed. EcPV-2 has been recognized as the cause of genital squamous cell carcinomas (SCCs) in horses, but the exact mechanism of carcinogenesis is not fully understood. The aim of the first part of this study is to assess the expression of cell cycle proteins p53, p16, pRB and Cyclin D1 in a series of equine SCCs and papillomas. Results confirm the role of EcPV-2 in the pathogenesis of genital SCCs. Moreover, in a small subset of ocular SCCs, EcPV-2 was detected for the first time. By immunohistochemistry, p53 was mostly expressed in ocular SCCs with a suprabasal localization. Regarding p16, overexpression was associated with increased mitotic index but not with viral infection. Investigation on pRB and Cyclin D1 proteins did not show significant correlation with other variables. The second part of this study is focused on the carcinogenetic mechanisms of BPV in equine sarcoids. The aim of the second part of this study was to characterize the typical histomorphological features of equine sarcoids, assess the expression of cell cycle proteins and Ki-67 proliferation index. Our results confirm that the typical histological features of sarcoids cannot be used to correctly classify the clinical types. Moreover, in a subset of sarcoids low pRB-Cyclin D1 scores were associated with simultaneous high p16 expression. The Ki-67 proliferation index confirm the low proliferative activity of sarcoids, except for tumors displaying a fascicular pattern. Finally, a subset of sarcoids recurred after excision.

Identificazione di bersagli terapeutici e realizzazione di tecnologie innovative in oncologia ortopedica

Controlled delivery of anticancer drugs through osteotropic nanoparticles (NP) is a novel approach for the adjuvant therapy of osteolytic bone metastases. Doxorubicin (DXR) is widely used in chemotherapy, although its activity is restricted by dose-dependent cardiotoxicity and marrow toxicity. However, its efficacy can be improved when specific targeting at the tumor site is obtained. The aim of this study was to obtain osteotropic biodegradable NP by nanoprecipitation of a copolymer between poly(D,L-lactide-co-glycolide) (PLGA) and an osteotropic bisphosphonate, sodium alendronate (ALE). NP were subsequently characterised for their chemical-physical properties, biocompatibility, and the ability to inhibit osteoclast-mediated bone resorption, and then loaded with DXR. The effectiveness of NP-loaded DXR was investigated through in vitro and in vivo experiments, and compared to that of free DXR. For the in vitro analysis, six human cell lines were used as a representative panel of bone tumors, including breast and renal adenocarcinoma, osteosarcoma and neuroblastoma. The in vitro uptake and the inhibition of tumor cell proliferation were verified. To analyse the in vivo activity of NP-loaded DXR, osteolytic bone metastases were induced through the intratibial inoculation in BALB/c-nu/nu mice of a human breast cancer cell line, followed by the intraperitoneal administration of the free or NP-loaded DXR. In vitro, aAll of the cell lines were able to uptake both free and NP-loaded drug, and their proliferation was inhibited up to 80% after incubation either with free or NP-loaded DXR. In addition, in vivo experiments showed that NP-loaded DXR were also able to reduce the incidence of bone metastases, not only in comparison with untreated mice, but also with free DXR-treated mice. In conclusion, this research demonstrated an improvement in the therapeutic effect of the antineoplastic drug DXR, when loaded to bone-targeted NP conjugated with ALE. Osteotropic PLGA-ALE NP are suitable to be loaded with DXR and offer as a valuable tool for a tissue specific treatment of skeletal metastases.

Infiammazione e cancro: l'interazione COX-2/CA-IX promuove il potenziale invasivo e la sopravvivenza in ipossia delle cellule del cancro colon-rettale

Cyclooxygenase-2/Carbonic anhydrase-IX up-regulation promotes invasive potential and hypoxia survival in colorectal cancer cells Purpose: Cyclooxygenase-2 (COX-2) is a major mediator of inflammation, playing a pivotal role in colorectal carcinogenesis. Hypoxia is an universal hallmark of solid tumour in vivo. This investigation was prompted by the observation that in colorectal cancer cells the expression of COX-2 protein is positively correlated with that of the hypoxia survival gene Carbonic Anhydrase-IX (CA-IX). Experimental Design: Since COX-2 gene expression and activity is increased in hypoxia, and that CA-IX is expressed also in normoxia in colorectal cancer cells, we tested the hypothesis that COX-2 activity in normoxia, as well as in hypoxia may be functionally linked to that of CA-IX gene. We investigated the role of COX-2 and CA-IX in colorectal cancer cell lines. In this regard, we performed RNA interference to knockdown COX-2 gene in vitro and immunohistochemistry to evaluate the protein expression of COX-2 and CA-IX in human colon cancer tissue specimens ex vivo. Results: We found that COX-2, by PGE2 production, controls CA-IX gene expression in an ERK dependent manner. In line with this finding, we also showed that the COX-2 inhibition by a specific short harpin COX-2 RNA (shCOX-2) or by a specific drug (SC-236), down-regulated CA-IX expression in colon cancer cells. We then exposed colon cancer cells to hypoxia stimuli and found that COX-2/CA-IX interplay promoted hypoxia survival. Moreover, we also report that COX-2/CA-IX interplay triggers Matrix Metalloproteinase 2/9 (MMP-2/9) activation and enhances the invasiveness of colorectal cancer cells. Thus given our above observations, we found that CA-IX and COX-2 protein expressions correlate with more aggressive stage colorectal cancer tissues ex vivo. Conclusions: Taken together these data indicate that COX-2/CA-IX interplay promotes an aggressive phenotype (hypoxia survival and invasiveness) which can be modulated in vitro by COX-2 selective inhibition and which may play a role in determining the biological aggressiveness of colorectal tumours. Moreover, in vitro and ex vivo data also suggest that the signatures of inflammation (COX-2) and hypoxia (CA-IX) may be difficult to be disentangled in colon cancer, being both responsible for the up-regulation of the same pathways.

Role of caveolin-1 in the proliferation of solid tumours in vitro

Caveolin-1 (Cav-1), the essential structural constituent of caveolae, which are flask-shaped invaginations of the plasma membrane, has been found to play a key role in the modulation of cell proliferation and cancer development. It seems to act as an oncosuppressor or a promoter of growth, depending on the histotype, stage and grade of each tumour. The aim of this study was to analyze the effects of Caveolin-1 gene silencing on the proliferation of human lung cancer and osteosarcoma in vitro. Our data show that Cav-1 silencing blocks the growth in both metastatic lung cancer cell lines analyzed, suggesting a proliferation promoting action of the protein in these cells. A marked decrease of phospho-Akt, phospho-ERK, STAT3, cyclin D1, CDK4 and consequently of phospho-Rb expression was evident in the cells treated with Cav-1 siRNA. With regards to osteosarcoma, we demonstrated that the suppression of Cav-1 results in the blocking of MG-63 and in the slowing down of HOS proliferation, suggesting a role for Cav-1 as a promoter of tumour growth in these cell lines. A marked decrease of phospho-Akt, cyclin E, CDK2 and phospho-Rb and an increase of p21 expression levels were evident in the cells treated with Cav-1 siRNA. Our results suggest two new cell cycle inhibiting pathways, mediated by Cav-1 knock-down, and provide new insights into the molecular mechanisms underlying the tumour-promoting role of Cav-1 in lung cancer and osteosarcoma. In this work we also investigated the role of estrogens in lung cancer and the functional cross-talk between Cav-1 and estrogens/estrogen receptors in it. Our results show that 17β-estradiol induces proliferation either in RAL or in SCLC-R1 cells and that both cell lines are sensitive to 4-OHT antiproliferative effect. The sensitivity to estrogen stimulation seems to be gender- and/or histological type-independent in metastatic lung cancer in vitro.