Impact of Glycosyltransferases on the Phenotype, Signaling and Transcriptome of Colorectal Cancer Cell Lines. Focus on the role of glycosyltransferases B4GALNT2 and FUT6 and their cognate Sda and sLex antigens

In colorectal cancer (CRC), two carbohydrate structures are modulated: the Sda antigen, synthesized by B4GALNT2, and sLex antigen, mainly synthesized by FUT6. sLex antigen is often overexpressed and associated with worse prognosis; B4GALNT2/Sda antigen are dramatically downregulated but their role in tumor progression and development is not fully clear. TCGA interrogation revealed a dramatic down-regulation of B4GALNT2 mRNA in CRC, compared with normal samples. Patients with higher B4GALNT2 mRNA in CRC samples displayed longer survival. Yet, methylation and miRNA expression play a relevant role in B4GALNT2 downregulation in CRC. To clarify the mechanisms linking the B4GALNT2/Sda expression level to CRC phenotype, three different CRC cell lines were modified to express B4GALNT2: LS174T cell line, in which the constitutively expressed sLex antigen was partially replaced by Sda; SW480/SW620 pair, both lacking Sda and sLex antigens. In LS174T cells, the expression of B4GALNT2 reduced the ability to grow in poor adherence conditions and the expression of ALDH, a stemness marker. In SW620 cells, B4GALNT2 expression impacted on the main aspects of malignancy. In SW480 cells the expression of B4GALNT2 left unchanged the proliferation rate and the wound healing ability. To clarify the impact of sLex on CRC phenotype, the SW480/SW620 pair were permanently transfected to express FUT6 cDNA. In both cell lines, overexpression of FUT6/sLex boosted the clonogenic ability in standard growth conditions. Conversely, the growth in soft agar and the capacity to close a wound were enhanced only in SW620 cells. Transcriptome analysis of CRC cell lines transfected either with B4GALNT2 or FUT6 showed a relevant impact of both enzymes on gene expression modulation. Overall, current data may help to personalize therapies for CRC patients according to the B4GALNT2 levels and support a causal effect of this glycosyltransferase on reducing malignancy independently of sLex inhibition.

Development and characterisation of a neurogenic model of brain cancer

Primary glioblastoma (GB), the most common and aggressive adult brain tumour, is refractory to conventional therapies and characterised by poor prognosis. GB displays striking cellular heterogeneity, with a sub-population, called Glioblastoma Stem Cells (GSCs), intrinsically resistant to therapy, hence the high rate of recurrence. Alterations of the tumour suppressor gene PTEN are prevalent in primary GBM, resulting in the inhibition of the polarity protein Lgl1 due to aPKC hyperactivation. Dysregulation of this molecular axis is one of the mechanisms involved in GSC maintenance. After demonstrating that the PTEN/aPKC/Lgl axis is conserved in Drosophila, I deregulated it in different cells populations of the nervous system in order to individuate the cells at the root of neurogenic brain cancers. This analysis identified the type II neuroblasts (NBs) as the most sensitive to alterations of this molecular axis. Type II NBs are a sub-population of Drosophila stem cells displaying a lineage similar to that of the mammalian neural stem cells. Following aPKC activation in these stem cells, I obtained an adult brain cancer model in Drosophila that summarises many phenotypic traits of human brain tumours. Fly tumours are indeed characterised by accumulation of highly proliferative immature cells and keep growing in the adult leading the affected animals to premature death. With the aim to understand the role of cell polarity disruption in this tumorigenic process I carried out a molecular characterisation and transcriptome analysis of brain cancers from our fly model. In summary, the model I built and partially characterised in this thesis work may help deepen our knowledge on human brain cancers by investigating many different aspects of this complicate disease.

Preclinical evaluation of a MYCN-specific inhibitor for the treatment of small cell lung cancer

Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, characterized by rapid growth, early metastasis and acquired drug resistance. SCLC is usually sensitive to initial treatment, however, most patients relapse within few months; thus more effective therapies are urgently needed. Key genetic alterations very frequently observed in SCLC include loss of TP53 and RB1 and mutations in the MYC family genes (MYC, MYCL or MYCN). One of them is amplified and overexpressed in a mutually exclusive manner and represents the most prominent activating oncogene alteration in this malignancy. In particular, MYCN amplification is associated with tumor progression, treatment failure and poor prognosis. Given the role of MYCN in SCLC and its restricted expression profile, MYCN represents a promising therapeutic target; although it is considered undruggable by traditional approaches. An innovative approach to target the oncogene concerns specific MYCN expression inhibition, acting directly at the level of DNA, through an antigene peptide nucleic acid (agPNA) oligonucleotide, called BGA002. This thesis focused on the study of BGA002, as a possible targeted therapeutic strategy for the treatment of MYCN-related SCLC. In this context, BGA002 proved to be a specific and highly effective inhibitor. Furthermore, MYCN silencing induced alterations in many downstream pathways and led to apoptosis, in concomitance with autophagy reactivation. Moreover, systemic administration of BGA002 was effective in vivo as well, significantly increasing survival in MNA mouse models, even in the scenario of multidrug-resistance. In addition, BGA002 treatment successfully reduced N-Myc protein expression and, more importantly, caused a massive diminishment in tumor vascularization in the multidrug-resistant model. Overall, these results proved that MYCN inhibition by BGA002 may represent a new promising precision medicine approach, to treat MYCN-related SCLC.

Study of genes involved in her2-positive breast cancer progression for identification of new therapeutic targets

HER2 overexpression is observed in 20-30% of invasive breast carcinomas and it is correlated with poor prognosis. Although targeted therapies have revolutionized the treatment of HER2-positive breast cancer, a high number of patients presented primary or acquired resistance to monoclonal antibodies and tyrosine kinase inhibitors. Tumor heterogenicity, epithelial to mesenchymal transition (EMT) and cancer stem cells are key factors in target therapy resistance and tumor progression. The aim of this project was to discover alternative therapeutic strategies to over-come tumor resistance by harnessing immune system and looking for new targetable molecules. The results reported introduce a virus-like particles-based vaccine against HER2 as promising therapeutic approach to treat HER2-positive tumors. The high and persistent anti-HER2 antibody titers elicited by the vaccine significantly inhibited tumor growth and metastases onset. Furthermore, the polyclonal response induced by the vaccine also inhibited human HER2-positive breast cancer cells resistant to trastuzumab in vitro, suggesting its efficacy also on trastuzumab resistant tumors. To identify new therapeutic targets to treat progressed breast cancer, we took advantage from a dynamic model of HER2 expression obtained in our laboratory, in which HER2 loss and cancer progression were associated with the acquisition of EMT and stemness features. Targeting EMT-involved molecules, such as PDGFR-β, or the induction of epithelial markers, like E-cadherin, proved to be successful strategy to impair HER2-negative tumor growth. Density alterations, which might be induced by anti-HER2 target therapies, in cell culture condition of a cell line with a labile HER2 expression, caused HER2 loss probably as consequence of more aggressive subpopulations which prevail over the others. These subpopulations showed an increased EMT and stemness profile, confirming that targeting EMT-involved molecules or antigen expressed by cancer stem cells together with anti-HER2 target therapies is a valid strategy to inhibit HER2-positive cells and simultaneously prevent selection of more aggressive clone.

Plasma-activated liquids as promising tools for non-invasive selective treatment of epithelial ovarian cancer

Plasma medicine is a branch of plasma-promising biomedical applications that uses cold atmospheric plasma (CAP) as a therapeutic agent in treating a wide range of medical conditions including cancer. Epithelial ovarian cancer (EOC) is a highly malignant and aggressive form of ovarian cancer, and most patients are diagnosed at advanced stages which significantly reduces the chances of successful treatment. Treatment resistance is also common, highlighting the need for novel therapies to be developed to treat EOC. Research in Plasma Medicine has revealed that plasma has unique properties suitable for biomedical applications and medical therapies, including responses to hormetic stimuli. However, the exact mechanisms by which CAP works at the molecular level are not yet fully understood. In this regard, the main goal of this thesis is to identify a possible adjuvant therapy for cancer, which could exert a cytotoxic effect, without damaging the surrounding healthy cells. An examination of different plasma-activated liquids (PALs) revealed their potential as effective tools for significantly inhibiting the growth of EOC. The dose-response profile between PALs and their targeted cytotoxic effects on EOC cells without affecting healthy cells was established. Additionally, it was validated that PALs exert distinct effects on different subtypes of EOC, possibly linked to the cells' metabolism. This suggests the potential for developing new, personalized anticancer strategies. Furthermore, it was observed that CAP treatment can alter the chemistry of a biomolecule present in PAL, impacting its cytotoxic activity. The effectiveness of the treatment was also preliminarily evaluated in 3D cultures, opening the door for further investigation of a possible correlation between the tumor microenvironment and PALs' resistance. These findings shed light on the intricate interplay between CAP and the liquid substrate and cell behaviour, providing valuable insights for the development of a novel and promising CAP-based cancer treatment for clinical application.

Cancer omics strategies for personalized treatments

The Consensus Molecular Subtypes (CMSs) classification stratifies colorectal cancer (CRC) into four well-defined molecular subgroups, providing incredible support to personalized medicine. Indeed, the huge inter-patient heterogeneity observed in CRC makes it difficult to define a therapeutic strategy from which every patient can benefit. Unfortunately, so far really few targetable biomarkers are known in the CRC setting, leading to an urgent need for new targeted therapies. Here we performed a bioinformatic meta-analysis over a cohort of 1700 CMS-stratified CRC patients, identifying a negative correlation between high levels of anaplastic lymphoma kinase (ALK) expression and relapse-free survival, exclusively in the CMS1 subtype. No correlation with ALK expression was pointed out in the other three subgroups. The association of ALK with CMS1 led to generate the hypothesis that ALK pharmacological inhibition may elicit therapeutic potential in this subgroup. Thus, we tested ALK inhibitors and an ALK-directed ADC on several CRC in vitro models, stratified according to the CMS classification as well as on CRC patient-derived organoids and mice. ALK interception strongly inhibited CMS1-cells, organoids, and tumor proliferation and was responsible for the dampening of ALK activation along with the downstream. Mechanistically, we found that CMS1 cells display several mRNA copies of both ALK and ALKAL2 ligand, suggesting a role for ALK abundance in the differential response to its inhibition. Collectively, these findings support the hypothesis that ALK may represent an attractive target for CMS1 colorectal cancer therapy.

Combining molecular alterations and functional imaging in metastatic castration resistant prostate cancer treated with taxanes

The treatment of metastatic castration-resistant prostate cancer (mCRPC) is currently characterized by several drugs with different mechanisms of action, such as new generation hormonal agents (abiraterone, enzalutamide), chemotherapy (docetaxel, cabazitaxel), PARP inhibitors (olaparib) and radiometabolic therapies (radium-223, LuPSMA). There is an urgent need to identify biomarkers to guide personalized therapy in mCRPC. In recent years, the status of androgen receptor (AR) gene detected in liquid biopsy has been associated with outcomes in patients treated with abiraterone or enzalutamide. More recently, plasma tumor DNA (ptDNA) and its changes during treatment have been identified as early indicators of response to anticancer treatments. Recent works also suggested a potential role of tumor-related metabolic parameters of 18Fluoro-Choline Positron Emission Tomography (F18CH-PET)-computed tomography (CT) as a prognostic tool in mCRCP. Other clinical features, such as the presence of visceral metastases, have been correlated with outcome in mCRPC patients. Recent studies conducted by our research group have designed and validated a prognostic model based on the combination of molecular characteristics (ptDNA levels), metabolic features found in basal FCH PET scans (metabolic tumor volume values, MTV), clinical parameters (absence or presence of visceral metastases), and laboratory tests (serum lactate dehydrogenase levels, LDH). Within this PhD project, 30 patients affected by mCRPC, pre-treated with abiraterone or enzalutamide, candidate for taxane-based treatments (docetaxel or cabazitaxel), have been prospectively evaluated. The prognostic model previously described was applied to this population, to interrogate its prognostic power in a more advanced cohort of patients, resulting in a further external validation of the tool.

Antiangiogenic And Finasteride Therapies: Responses Of The Prostate Microenvironment In Elderly Mice.

The aim of this study was to evaluate the structural and molecular effects of antiangiogenic therapies and finasteride on the ventral prostate of senile mice. 90 male FVB mice were divided into: Young (18 weeks old) and senile (52 weeks old) groups; finasteride group: finasteride (20mg/kg); SU5416 group: SU5416 (6 mg/kg); TNP-470 group: TNP-470 (15 mg/kg,) and SU5416+TNP-470 group: similar to the SU5416 and TNP-470 groups. After 21 days, prostate ventral lobes were collected for morphological, immunohistochemical and Western blotting analyses. The results demonstrated atrophy, occasional proliferative lesions and inflammatory cells in the prostate during senescence, which were interrupted and/or blocked by treatment with antiangiogenic drugs and finasteride. Decreased AR and endostatin reactivities, and an increase for ER-α, ER-β and VEGF, were seen in the senile group. Decreased VEGF and ER-α reactivities and increased ER-β reactivity were verified in the finasteride, SU5416 groups and especially in SU5416+TNP-470 group. The TNP-470 group showed reduced AR and ER-β protein levels. The senescence favored the occurrence of structural and/or molecular alterations suggesting the onset of malignant lesions, due to the imbalance in the signaling between the epithelium and stroma. The SU5416+TNP-470 treatment was more effective in maintaining the structural, hormonal and angiogenic factor balance in the prostate during senescence, highlighting the signaling of antiproliferation via ER-β.

Egfr Activating Mutations And Their Association With Response To Platinum-doublet Chemotherapy In Brazilian Non-small Cell Lung Cancer Patients.

The aim of the study was to analyze the frequency of epidermal growth factor receptor (EGFR) mutations in Brazilian non-small cell lung cancer patients and to correlate these mutations with response to benefit of platinum-based chemotherapy in non-small cell lung cancer (NSCLC). Our cohort consisted of prospective patients with NSCLCs who received chemotherapy (platinum derivates plus paclitaxel) at the [UNICAMP], Brazil. EGFR exons 18-21 were analyzed in tumor-derived DNA. Fifty patients were included in the study (25 with adenocarcinoma). EGFR mutations were identified in 6/50 (12 %) NSCLCs and in 6/25 (24 %) adenocarcinomas; representing the frequency of EGFR mutations in a mostly self-reported White (82.0 %) southeastern Brazilian population of NSCLCs. Patients with NSCLCs harboring EGFR exon 19 deletions or the exon 21 L858R mutation were found to have a higher chance of response to platinum-paclitaxel (OR 9.67 [95 % CI 1.03-90.41], p = 0.047). We report the frequency of EGFR activating mutations in a typical southeastern Brazilian population with NSCLC, which are similar to that of other countries with Western European ethnicity. EGFR mutations seem to be predictive of a response to platinum-paclitaxel, and additional studies are needed to confirm or refute this relationship.

Low-density Lipoprotein Cholesterol And Radiotherapy-induced Carotid Atherosclerosis In Subjects With Head And Neck Cancer.

Radiotherapy (RT) is a risk factor for accelerated carotid artery atherosclerotic disease in subjects with head and neck cancer. However, the risk factors of RT-induced carotid artery remodeling are not established. This study aimed to investigate the effects of RT on carotid and popliteal arteries in subjects with head and neck cancer and to evaluate the relationship between baseline clinical and laboratory features and the progression of RT-induced atherosclerosis. Eleven men (age = 57.9 ± 6.2years) with head and neck cancer who underwent cervical bilateral irradiation were prospectively examined by clinical and laboratory analysis and by carotid and popliteal ultrasound before and after treatment (mean interval between the end of RT and the post-RT assessment = 181 ± 47 days). No studied subject used hypocholesterolemic medications. Significant increases in carotid intima-media thickness (IMT) (0.95 ± 0.08 vs. 0.87 ± 0.05 mm; p < 0.0001) and carotid IMT/diameter ratio (0.138 ± 0.013 vs. 0.129 ± 0.014; p = 0.001) were observed after RT, while no changes in popliteal structural features were detected. In addition, baseline low-density lipoprotein cholesterol levels showed a direct correlation with RT-induced carotid IMT change (r = 0.66; p = 0.027), while no other studied variable exhibited a significant relationship with carotid IMT change. These results indicate that RT-induced atherosclerosis is limited to the irradiated area and also suggest that it may be predicted by low-density lipoprotein cholesterol levels in subjects with head and neck cancer.

Nat2, Xrcc1 And Hogg1 Polymorphisms, Cigarette Smoking, Alcohol Consumption And Risk Of Upper Aerodigestive Tract Cancer.

To evaluate associations between polymorphisms of the N-acetyltransferase 2 (NAT2), human 8-oxoguanine glycosylase 1 (hOGG1) and X-ray repair cross-complementing protein 1 (XRCC1) genes and risk of upper aerodigestive tract (UADT) cancer. A case-control study involving 117 cases and 224 controls was undertaken. The NAT2 gene polymorphisms were genotyped by automated sequencing and XRCC1 Arg399Gln and hOGG1 Ser326Cys polymorphisms were determined by Polymerase Chain Reaction followed by Restriction Fragment Length Polymorphism (PCR-RFLP) methods. Slow metabolization phenotype was significantly associated as a risk factor for the development of UADT cancer (p=0.038). Furthermore, haplotype of slow metabolization was also associated with UADT cancer (p=0.014). The hOGG1 Ser326Cys polymorphism (CG or GG vs. CC genotypes) was shown as a protective factor against UADT cancer in moderate smokers (p=0.031). The XRCC1 Arg399Gln polymorphism (GA or AA vs. GG genotypes), in turn, was a protective factor against UADT cancer only among never-drinkers (p=0.048). Interactions involving NAT2, XRCC1 Arg399Gln and hOGG1 Ser326Cys polymorphisms may modulate the risk of UADT cancer in this population.

A Putative Otu Domain-containing Protein 1 Deubiquitinating Enzyme Is Differentially Expressed In Thyroid Cancer And Identifies Less-aggressive Tumours.

This study aimed to identify novel biomarkers for thyroid carcinoma diagnosis and prognosis. We have constructed a human single-chain variable fragment (scFv) antibody library that was selected against tumour thyroid cells using the BRASIL method (biopanning and rapid analysis of selective interactive ligands) and phage display technology. One highly reactive clone, scFv-C1, with specific binding to papillary thyroid tumour proteins was confirmed by ELISA, which was further tested against a tissue microarray that comprised of 229 thyroid tissues, including: 110 carcinomas (38 papillary thyroid carcinomas (PTCs), 42 follicular carcinomas, 30 follicular variants of PTC), 18 normal thyroid tissues, 49 nodular goitres (NG) and 52 follicular adenomas. The scFv-C1 was able to distinguish carcinomas from benign lesions (P=0.0001) and reacted preferentially against T1 and T2 tumour stages (P=0.0108). We have further identified an OTU domain-containing protein 1, DUBA-7 deubiquitinating enzyme as the scFv-binding antigen using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. The strategy of screening and identifying a cell-surface-binding antibody against thyroid tissues was highly effective and resulted in a useful biomarker that recognises malignancy among thyroid nodules and may help identify lower-risk cases that can benefit from less-aggressive management.

Primary Relapse Site Pattern In Women With Triple-negative Breast Cancer.

Despite the remarkable improvements in breast cancer (BC) characterization, accurate prediction of BC clinical behavior is often still difficult to achieve. Some studies have investigated the association between the molecular subtype, namely the basal-like BC and the pattern of relapse, however only few investigated the association between relapse pattern and immunohistochemical defined triple-negative breast cancers (TNBCs). The aim of this study was to evaluate the pattern of relapse in patients with TNBC, namely the primary distant relapse site. One-hundred twenty nine (129) invasive breast carcinomas with follow-up information were classified according to the molecular subtype using immunohistochemistry for ER, PgR and Her2. The association between TNBC and distant relapse primary site was analyzed by logistic regression. Using multivariate logistic regression analysis patients with TNBC displayed only 0.09 (95% CI: 0.00-0.74; p=0.02) the odds of the non-TNBC patients of developing bone primary relapse. Regarding visceral and lymph-node relapse, no differences between in this cohort were found. Though classically regarded as aggressive tumors, TNBCs rarely development primary relapse in bone when compared to non-TNBC, a clinical relevant fact when investigating a metastasis of an occult or non-sampled primary BC.

[why Does The Prevalence Of Cytopathological Results Of Cervical Cancer Screening Can Vary Significantly Between Two Regions Of Brazil?].

To analyze the prevalence of cervical cytopathological results for the screening of cervical cancer with regard to women's age and time since the last examination in Maceió and Rio de Janeiro, Brazil, among those assisted by the Brazilian Unified Health System. Cervical cytopathological results available in the Information System of Cervical Cancer Screening for the year 2011 were analyzed, corresponding to 206,550 for Rio de Janeiro and 45,243 for Maceió. In Rio de Janeiro, examination at one and two year intervals predominated, while in Maceió examination at one and three year intervals had a higher predominance. Women who underwent cervical smear screening in Maceió were older than those in Rio de Janeiro. The prevalence of invasive squamous cell carcinoma was similar for the two cities, but all the other results presented a higher prevalence in Rio de Janeiro: ASCUS (PR=5.32; 95%CI 4.66-6.07); ASCH (PR=4.27; 95%CI 3.15-5.78); atypical glandular cells (PR=10.02; 95%CI 5.66-17.76); low-grade squamous intraepithelial lesions (PR=6.10; 95%CI 5.27-7.07); high-grade squamous intraepithelial lesions (PR=8.90; 95%CI 6.50-12.18) and adenocarcinoma (PR=3.00; 95%CI 1.21-7.44). The rate of unsatisfactory cervical samples was two times higher in Maceió and that of rejected samples for analysis was five times higher in Maceió when compared to Rio de Janeiro. The prevalence rates of altered cervical cytopathological results was significantly higher in Rio de Janeiro than in Maceió. There is no objective information that may justify this difference. One hypothesis is that there may be a difference in the diagnostic performance of the cervical cancer screening, which could be related to the quality of the Pap smear. Thus, these findings suggest that it would be necessary to perform this evaluation at national level, with emphasis on the performance of cervical cancer screening in order to improve the effectiveness of cervical cancer control.

Predictive Value Of Phase I Trials For Safety In Later Trials And Final Approved Dose: Analysis Of 61 Approved Cancer Drugs.

Phase I trials use a small number of patients to define a maximum tolerated dose (MTD) and the safety of new agents. We compared data from phase I and registration trials to determine whether early trials predicted later safety and final dose. We searched the U.S. Food and Drug Administration (FDA) website for drugs approved in nonpediatric cancers (January 1990-October 2012). The recommended phase II dose (R2PD) and toxicities from phase I were compared with doses and safety in later trials. In 62 of 85 (73%) matched trials, the dose from the later trial was within 20% of the RP2D. In a multivariable analysis, phase I trials of targeted agents were less predictive of the final approved dose (OR, 0.2 for adopting ± 20% of the RP2D for targeted vs. other classes; P = 0.025). Of the 530 clinically relevant toxicities in later trials, 70% (n = 374) were described in phase I. A significant relationship (P = 0.0032) between increasing the number of patients in phase I (up to 60) and the ability to describe future clinically relevant toxicities was observed. Among 28,505 patients in later trials, the death rate that was related to drug was 1.41%. In conclusion, dosing based on phase I trials was associated with a low toxicity-related death rate in later trials. The ability to predict relevant toxicities correlates with the number of patients on the initial phase I trial. The final dose approved was within 20% of the RP2D in 73% of assessed trials.