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.

Activity and mechanisms of action of novel organosulfur derivatives of the HDAC inhibitor Valproic Acid in human experimental models of non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) represents the leading cause of cancer death worldwide, and 5-year survival is about 16% for patients diagnosed with advanced lung cancer and about 70-90% when the disease is diagnosed and treated at earlier stages. Treatment of NSCLC is changed in the last years with the introduction of targeted agents, such as gefitinib and erlotinib, that have dramatically changed the natural history of NSCLC patients carrying specific mutations in the EGFR gene, or crizotinib, for patients with the EML4-ALK translocation. However, such patients represent only about 15-20% of all NSCLC patients, and for the remaining individuals conventional chemotherapy represents the standard choice yet, but response rate to thise type of treatment is only about 20%. Development of new drugs and new therapeutic approaches are so needed to improve patients outcome. In this project we aimed to analyse the antitumoral activity of two compounds with the ability to inhibit histone deacethylases (ACS 2 and ACS 33), derived from Valproic Acid and conjugated with H2S, in human cancer cell lines derived from NSCLC tissues. We showed that ACS 2 represents the more promising agent. It showed strong antitumoral and pro-apoptotic activities, by inducing membrane depolarization, cytocrome-c release and caspase 3 and 9 activation. It was able to reduce the invasive capacity of cells, through inhibition of metalloproteinases expression, and to induce a reduced chromatin condensation. This last characteristic is probably responsible for the observed high synergistic activity in combination with cisplatin. In conclusion our results highlight the potential role of the ACS 2 compound as new therapeutic option for NSCLC patients, especially in combination with cisplatin. If validated in in vivo models, this compound should be worthy for phase I clinical trials.

6-(Methylsulfonyl) hexyl isothiocyanate as potential chemopreventive agent: molecular and cellular profile in leukaemia cell lines

Despite numerous therapeutic interventions cancer is still today the second leading cause of death. A growing interest has been addressed to isothiocytanates and more recently, the 6- (methylsulfonyl) hexyl isothiocyanate (6-MITC), the main constituent of the rhizome of Wasabia Japonica, has stimulated the interest of researchers. Aim of the research was to study if 6-MITC is able to modulate the main mechanisms underlying chemopreventive process in leukemic cells lines, verify the selectivity of action and the safety of use in terms of mutagenicity. The study was conducted on different cell types. In particular, Jurkat and HL-60 cells were treated with increasing concentrations of 6-MITC and cell viability, induction of apoptosis, cell cycle analysis, autophagy modulation and stimulation of differentiation were evaluated by flow cytometry. PBL, the non-transformed counterparty of leukemia cells, was used to analyse the selectivity of action by studying the same mechanisms previously indicated. Finally, safety of use and antimutagenicity were studied in TK6 cells adopting an automated protocol in flow cytometry. The achieved results have demonstrated that isothiocyanate modulates many signaling pathways involved in chemopreventive mechanism. In fact, 6-MITC induces apoptosis of both transformed cells, limits tumor growth by slowing down the cell cycle of Jurkat cells and blocks HL-60 cell cycle, increases the autophagic flux and induces cytodifferentiation of promyelocytic HL-60 into macrophage and granulocytic phenotypes. Furthermore, the results obtained with 6-MITC on PBL from healthy donors suggest that the isothiocyante is a good selective cytotoxic agent. Essential feature of a good chemopreventive agent is selectivity toward cancer cells and low toxicity towards non-transformed cells. Finally, the analysis of the micronuclei revealed that 6-MITC is not mutagenic, ensuring safe use, and that instead, it is able to counteract the mutagenic activity of the aneuploidogen Vinblastine, demonstrating another important and interesting chemopreventive activity.

Development of 3D Cancer Cell Models to Test Metabolic Interventions as an Anticancer Strategy

In recent years, it has become evident that the role of mitochondria in the metabolic rewiring is essential for cancer development and progression. The metabolic profile during tumorigenesis has been performed mainly in traditional 2D cell models, including cell lines of various lineages and phenotypes. Although useful in many ways, their relevance can be often debatable, as they lack the interactions between different cells of the tumour microenvironment and/or interaction with the extracellular matrix 1,2. Improved models are now being developed using 3D cell culture technology, contributing with increased physiological relevance 3,4. In this work, we improved a method for the generation of 3D models from healthy and tumour colon tissue, based on organoid technology, and performed their molecular and biochemical characterization and validation. Further, in-plate cryopreservation was applied to these models, and optimal results were obtained in terms of cell viability and functionality of the cryopreserved models. We also cryopreserved colon fibroblasts with the aim to introduce them in a co-culture cryopreserved model with organoids. This technology allows the conversion of cell models into “plug and play” formats. Therefore, cryopreservation in-plate facilitates the accessibility of specialized cell models to cell-based research and application, in cases where otherwise such specialized models would be out of reach. Finally, we briefly explored the field of bioprinting, by testing a new matrix to support the growth of colon tumour organoids, which revealed promising preliminary results. To facilitate the reader, we organized this thesis into chapters, divided by the main points of work which include development, characterization and validation of the model, commercial output, and associated applications. Each chapter has a brief introduction, followed by results and discussion and a final conclusion. The thesis has also a general discussion and conclusion section in the end, which covers the main results obtained during this work.

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.

Mitochondrial dysfunction in a cell model of thyroid oncocytoma

The role of mitochondrial dysfunction in cancer has long been a subject of great interest. In this study, such dysfunction has been examined with regards to thyroid oncocytoma, a rare form of cancer, accounting for less than 5% of all thyroid cancers. A peculiar characteristic of thyroid oncocytic cells is the presence of an abnormally large number of mitochondria in the cytoplasm. Such mitochondrial hyperplasia has also been observed in cells derived from patients suffering from mitochondrial encephalomyopathies, where mutations in the mitochondrial DNA(mtDNA) encoding the respiratory complexes result in oxidative phosphorylation dysfunction. An increase in the number of mitochondria occurs in the latter in order to compensate for the respiratory deficiency. This fact spurred the investigation into the presence of analogous mutations in thyroid oncocytic cells. In this study, the only available cell model of thyroid oncocytoma was utilised, the XTC-1 cell line, established from an oncocytic thyroid metastasis to the breast. In order to assess the energetic efficiency of these cells, they were incubated in a medium lacking glucose and supplemented instead with galactose. When subjected to such conditions, glycolysis is effectively inhibited and the cells are forced to use the mitochondria for energy production. Cell viability experiments revealed that XTC-1 cells were unable to survive in galactose medium. This was in marked contrast to the TPC-1 control cell line, a thyroid tumour cell line which does not display the oncocytic phenotype. In agreement with these findings, subsequent experiments assessing the levels of cellular ATP over incubation time in galactose medium, showed a drastic and continual decrease in ATP levels only in the XTC-1 cell line. Furthermore, experiments on digitonin-permeabilised cells revealed that the respiratory dysfunction in the latter was due to a defect in complex I of the respiratory chain. Subsequent experiments using cybrids demonstrated that this defect could be attributed to the mitochondrially-encoded subunits of complex I as opposed to the nuclearencoded subunits. Confirmation came with mtDNA sequencing, which detected the presence of a novel mutation in the ND1 subunit of complex I. In addition, a mutation in the cytochrome b subunit of complex III of the respiratory chain was detected. The fact that XTC-1 cells are unable to survive when incubated in galactose medium is consistent with the fact that many cancers are largely dependent on glycolysis for energy production. Indeed, numerous studies have shown that glycolytic inhibitors are able to induce apoptosis in various cancer cell lines. Subsequent experiments were therefore performed in order to identify the mode of XTC-1 cell death when subjected to the metabolic stress imposed by the forced use of the mitochondria for energy production. Cell shrinkage and mitochondrial fragmentation were observed in the dying cells, which would indicate an apoptotic type of cell death. Analysis of additional parameters however revealed a lack of both DNA fragmentation and caspase activation, thus excluding a classical apoptotic type of cell death. Interestingly, cleavage of the actin component of the cytoskeleton was observed, implicating the action of proteases in this mode of cell demise. However, experiments employing protease inhibitors failed to identify the specific protease involved. It has been reported in the literature that overexpression of Bcl-2 is able to rescue cells presenting a respiratory deficiency. As the XTC-1 cell line is not only respiration-deficient but also exhibits a marked decrease in Bcl-2 expression, it is a perfect model with which to study the relationship between Bcl-2 and oxidative phosphorylation in respiratory-deficient cells. Contrary to the reported literature studies on various cell lines harbouring defects in the respiratory chain, Bcl-2 overexpression was not shown to increase cell survival or rescue the energetic dysfunction in XTC-1 cells. Interestingly however, it had a noticeable impact on cell adhesion and morphology. Whereas XTC-1 cells shrank and detached from the growth surface under conditions of metabolic stress, Bcl-2-overexpressing XTC-1 cells appeared much healthier and were up to 45% more adherent. The target of Bcl-2 in this setting appeared to be the actin cytoskeleton, as the cleavage observed in XTC-1 cells expressing only endogenous levels of Bcl-2, was inhibited in Bcl-2-overexpressing cells. Thus, although unable to rescue XTC-1 cells in terms of cell viability, Bcl-2 is somehow able to stabilise the cytoskeleton, resulting in modifications in cell morphology and adhesion. The mitochondrial respiratory deficiency observed in cancer cells is thought not only to cause an increased dependency on glycolysis but it is also thought to blunt cellular responses to anticancer agents. The effects of several therapeutic agents were thus assessed for their death-inducing ability in XTC-1 cells. Cell viability experiments clearly showed that the cells were more resistant to stimuli which generate reactive oxygen species (tert-butylhydroperoxide) and to mitochondrial calcium-mediated apoptotic stimuli (C6-ceramide), as opposed to stimuli inflicting DNA damage (cisplatin) and damage to protein kinases(staurosporine). Various studies in the literature have reported that the peroxisome proliferator-activated receptor-coactivator 1(PGC-1α), which plays a fundamental role in mitochondrial biogenesis, is also involved in protecting cells against apoptosis caused by the former two types of stimuli. In accordance with these observations, real-time PCR experiments showed that XTC-1 cells express higher mRNA levels of this coactivator than do the control cells, implicating its importance in drug resistance. In conclusion, this study has revealed that XTC-1 cells, like many cancer cell lines, are characterised by a reduced energetic efficiency due to mitochondrial dysfunction. Said dysfunction has been attributed to mutations in respiratory genes encoded by the mitochondrial genome. Although the mechanism of cell demise in conditions of metabolic stress is unclear, the potential of targeting thyroid oncocytic cancers using glycolytic inhibitors has been illustrated. In addition, the discovery of mtDNA mutations in XTC-1 cells has enabled the use of this cell line as a model with which to study the relationship between Bcl-2 overexpression and oxidative phosphorylation in cells harbouring mtDNA mutations and also to investigate the significance of such mutations in establishing resistance to apoptotic stimuli.

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.

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.

The inhibition of aerobic glycolysis as a therapeutic approach to improve cancer chemotherapy

The aim of the research project discussed in this thesis was to study the inhibition of aerobic glycolysis, that is the metabolic pathway exploited by cancer cells for the ATP generation. This observation has led to the evaluation of glycolytic inhibitors as potential anticancer agents. Lactate dehydrogenase (LDH) is the only enzyme whose inhibition should allow a blocking of aerobic glycolysis of tumor cells without damaging the normal cells which, in conditions of normal functional activity and sufficient oxygen supply, do not need this enzyme. In preliminar experiments we demonstrated that oxamic acid and tartronic acid, two LDH competitive inhibitors, impaired aerobic glycolysis and replication of cells from human hepatocellular carcinoma. Therefore, we proposed that the depletion of ATP levels in neoplastic cells, could improved the chemotherapeutic index of associated anticancer drugs; in particular, it was studied the association of oxamic acid and multi-targeted kinase inhibitors. A synergistic effect in combination with sorafenib was observed, and we demonstrated that this was related to the capacity of sorafenib to hinder the oxidative phosphorylation, so that cells were more dependent to aerobic glycolysis. These results linked to LDH blockage encouraged us to search for LDH inhibitors more powerful than oxamic acid; thus, in collaboration with the Department of Pharmaceutical Sciences of Bologna University we identified a new molecule, galloflavin, able to inhibit both A and B isoforms of LDH enzyme. The effects of galloflavin were studied on different human cancer cell lines (hepatocellular carcinoma, breast cancer, Burkitt’s lymphoma). Although exhibiting different power on the tested cell lines, galloflavin was constantly found to inhibit lactate and ATP production and to induce cell death, mainly in the form of apoptosis. Finally, as LDH-A is able to bind single stranded DNA, thus stimulating cell transcription, galloflavin effects were also studied on this other LDH function.

Evaluation of Antitumoral activity of bone targeted drugs/conventional chemotherapies and identification of biomarkers for the selection of patients with breast cancer for the bone targeted therapy in adjuvant setting

Bone metastases are responsible for different clinical complications defined as skeletal-related events (SREs) such as pathologic fractures, spinal cord compression, hypercalcaemia, bone marrow infiltration and severe bone pain requiring palliative radiotherapy. The general aim of these three years research period was to improve the management of patients with bone metastases through two different approaches of translational research. Firstly in vitro preclinical tests were conducted on breast cancer cells and on indirect co-colture of cancer cells and osteoclasts to evaluate bone targeted therapy singly and in combination with conventional chemotherapy. The study suggests that zoledronic acid has an antitumor activity in breast cancer cell lines. Its mechanism of action involves the decrease of RAS and RHO, as in osteoclasts. Repeated treatment enhances antitumor activity compared to non-repeated treatment. Furthermore the combination Zoledronic Acid + Cisplatin induced a high antitumoral activity in the two triple-negative lines MDA-MB-231 and BRC-230. The p21, pMAPK and m-TOR pathways were regulated by this combined treatment, particularly at lower Cisplatin doses. A co-colture system to test the activity of bone-targeted molecules on monocytes-breast conditioned by breast cancer cells was also developed. Another important criticism of the treatment of breast cancer patients, is the selection of patients who will benefit of bone targeted therapy in the adjuvant setting. A retrospective case-control study on breast cancer patients to find new predictive markers of bone metastases in the primary tumors was performed. Eight markers were evaluated and TFF1 and CXCR4 were found to discriminate between patients with relapse to bone respect to patients with no evidence of disease. In particular TFF1 was the most accurate marker reaching a sensitivity of 63% and a specificity of 79%. This marker could be a useful tool for clinicians to select patients who could benefit for bone targeted therapy in adjuvant setting.

Interactions of G-quadruplex binders and Topoisomerase I inhibitors in cancer cells

Top1-DNA cleavage complexes (Top1ccs) trigger an accumulation of antisense RNAPII transcripts specifically at active divergent CpG-island promoters in a replication independent and Top1 dependent manner, leading to transcription-dependent genome instability and altered transcription regulation. Using different cancer cell lines of colon and osteo origins, we show that they display different sensitivity to CPT and G4 binder that is independent from Top1 level. To look at the interactions between Top1 and G4, we show that co-treatment with G4 binders potentiate the cell cytotoxicity of CPT regardless of the treatment sequences. Potentiation is indicated by a reduced inhibition concentration (IC50) with a more profound cytotoxicity in CPT-resistant cell lines, HCT15 and U2OS, hence, indicating an interaction between Top1inhibitor and G4 binders. Moreover, computational analysis confirmed the present of G4 motifs in genes with CPT-induced antisense transcription. G4 motifs are present mostly 5000 bp upstream from transcription start site and notably lower in genes. Comparisons between genes with no antisense transcription and genes with antisense transcription show that G4 motifs in this region are notably lower in the genes with antisense transcripts. Since CPT increases negative supercoils at promoters of intermediate activity, the formation of G4 is also increased in CPT-treated cells. Suprisingly, formation of G4 is regulated in parallel to the transient stabilization of R-loops, indicating a role in response to CPT-induced stress. G4 formation is highly elevated in Pyridostatin treated cells, which previous study shows increased formation of γH2Ax foci. This effect is also seen in the CPT-resistant cell lines, HCT15, indicating that the formation is a general event in response to CPT. We also show that R-loop formation is greatly increased in Pyridostatin treated cells. In order to study the role of R-loops and G4 structures in Top1cc-dependant repair pathway, we inhibited tyrosyl-phosphodiestrase 1 (TDP-1) using a TDP-1 inhibitor.

TAZ role in lung cancer: resistance to BET inhibitors and functional interaction with lncRNA

Despite extensive research and introduction of innovative therapy, lung cancer prognosis remains poor, with a five years survival of only 17%. The success of pharmacological treatment is often impaired by drug resistance. Thus, the characterization of response mechanisms to anti-cancer compounds and of the molecular mechanisms supporting lung cancer aggressiveness are crucial for patient’s management. In the first part of this thesis, we characterized the molecular mechanism behind resistance of lung cancer cells to the Inhibitors of the Bromodomain and Extraterminal domain containing Proteins (BETi). Through a CRISPR/Cas9 screening we identified three Hippo Pathway members, LATS2, TAOK1 and NF2 as genes implicated in susceptibility to BETi. These genes confer sensitivity to BETi inhibiting TAZ activity. Conversely, TAZ overexpression increases resistance to BETi. We also displayed that BETi downregulate both YAP, TAZ and TEADs expression in several cancer cell lines, implying a novel BETi-dependent cytotoxic mechanism. In the second part of this work, we attempted to characterize the crosstalk between the TAZ gene and its cognate antisense long-non coding RNA (lncRNA) TAZ-AS202 in lung tumorigenesis. As for TAZ downregulation, TAZ-AS202 silencing impairs NSCLC cells proliferation, migration and invasion, suggesting a pro-tumorigenic function for this lncRNA during lung tumorigenesis. TAZ-AS202 regulates TAZ target genes without altering TAZ expression or localization. This finding implies an uncovered functional cooperation between TAZ and TAZ-AS202. Moreover, we found that the EPH-ephrin signaling receptor EPHB2 is a downstream effector affected by both TAZ and TAZ-AS202 silencing. EPHB2 downregulation significantly attenuates cells proliferation, migration and invasion, suggesting that, at least in part, TAZ-AS202 and TAZ pro-oncogenic activity depends on EPH-ephrin signaling final deregulation. Finally, we started to dissect the mechanism underlying the TAZ-AS202 regulatory activity on EPHB2 in lung cancer, which may involve the existence of an intermediate transcription factor and is the object of our ongoing research.

Inhibition and characterization of two-metal ion enzymes to treat cancer: dna polymerase Ƞ and topoisomerase II α

Chemotherapeutic drugs can in many ways disrupt the replication machinery triggering apoptosis in cancer cells: some act directly on DNA and others block the enzymes involved in preparing DNA for replication. Cisplatin-based drugs are common as first-line cancer chemotherapics. Another example is etoposide, a molecule that blocks topoisomerase II α leading to the inhibition of dsDNA replication. Despite their efficacy, cancer cells can respond to these treatments over time by overtaking their effects, leading to drug resistance. Chemoresistance events can be triggered by the action of enzymes like DNA polymerase ƞ (Pol η). This polymerase helps also to bypass drug-induced damage in cancer cells, allowing DNA replication and cancer cells proliferation even when cisplatin-based chemotherapeutic drugs are in use. Pol ƞ is a promising drug discovery target, whose inhibition would help in overcoming of drug resistance. This study aims to identify a potent and selective Pol ƞ inhibitor able to improve the efficacy of platinum-based chemotherapeutic drugs. We report the discovery of compound 64 (ARN24964), after an extensive SAR reporting 35 analogs. We evaluated compound 64 on four different cell lines. Interestingly, the molecule is a Pol η inhibitor able to act synergistically with cisplatin. Moreover, we also synthesized a prodrug form that allowed us to improve its stability and the bioavailability. This compound represents an advanced scaffold featuring good potency and DMPK properties. In addition to this central theme, this thesis also describes our efforts in developing and characterize a novel hybrid inhibitor/poison for the human topoisomerase II α enzyme. In particular, we performed specific assays to study the inhibiton of Topoisomesare II α and we evaluated compounds effect on three cancer cell lines. These studies allowed us to identify a compound that is able to inhibit the enzyme with a good pK and a good potency.

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.