During the last decade, a new trend in medicine is transforming the nature of healthcare from reactive to proactive. This new paradigm is changing into a personalized medicine where the prevention, diagnosis, and treatment of disease is focused on individual patients. This paradigm is known as P4 medicine. Among other key benefits, P4 medicine aspires to detect diseases at an early stage and introduce diagnosis to stratify patients and diseases to select the optimal therapy based on individual observations and taking into account the patient outcomes to empower the physician, the patient, and their communication. This paradigm transformation relies on the availability of complex multi-level biomedical data that are increasingly accurate, since it is possible to find exactly the needed information, but also exponentially noisy, since the access to that information is more and more challenging. In order to take advantage of this information, an important effort is being made in the last decades to digitalize medical records and to develop new mathematical and computational methods for extracting maximum knowledge from patient records, building dynamic and disease-predictive models from massive amounts of integrated clinical and biomedical data. This requirement enables the use of computer-assisted Clinical Decision Support Systems for the management of individual patients. The Clinical Decision Support System (CDSS) are computational systems that provide precise and specific knowledge for the medical decisions to be adopted for diagnosis, prognosis, treatment and management of patients. The CDSS are highly related to the concept of evidence-based medicine since they infer medical knowledge from the biomedical databases and the acquisition protocols that are used for the development of the systems, give computational support based on evidence for the clinical practice, and evaluate the performance and the added value of the solution for each specific medical problem. / Tortajada Velert, S. (2012). Incremental Learning approaches to Biomedical decision problems [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/17195

Yes / Polysialic acid (PolySia) is an important carbohydrate bio-polymer that is commonly over-expressed on tumours of neuroendocrine origin and plays a key role in tumour progression. PolySia exclusively decorates the neural cell adhesion molecule (NCAM) on tumour cell membranes, modulating cell-cell interactions, motility and invasion. In this preliminary study, we examine the nano-mechanical properties of isogenic C6 rat glioma cells - transfected cells engineered to express the enzyme polysialyltransferase ST8SiaII, which synthesises polySia (C6-STX cells) and wild type cells (C6-WT). We demonstrate that polySia expression leads to reduced elastic and adhesive properties but also more visco-elastic compared to non-expressing wild type cells. Whilst differences in cell elasticity between healthy and cancer cells is regularly assigned to changes in the cytoskeleton, we show that in this model system the change in properties at the nano-level is due to the polySia on the transfected cell membrane surface.

No / Various saponins, plant glycosides with favorable anti-tumorigenic properties, have been used to inhibit tumor cell growth by cell cycle arrest and apoptosis with IC50 values of up to 0.2 μM. We describe several groups of saponins (dioscins, saikosaponins, julibrosides, soy saponins, ginseng saponins and avicins) currently investigated for their use in tumor therapy. We focus on cellular and systemic mechanisms of tumor cell growth inhibition both in vitro and in vivo, combinational approaches with saponins and conventional tumor treatment strategies, and successful syntheses of saponins. The increasing interest in saponins for tumor therapy is very promising for the future development of sophisticated anti-cancer drugs.

The N6-methyladenosine, also known as m6A, is the most common post-transcriptional modification in mRNAs and long non-coding RNAs and that profoundly influences mRNA biology, from early processing in the nucleus to final steps of translation and decay in the cytoplasm. Taking into consideration the importance of RNA in shaping cell fate, m6A is widely recognized as an additional layer in the regulation of gene expression, also thanks to its dynamic and reversible nature. Therefore, it is not surprising that any misregulation in m6A content might lead to the loss of cellular homeostasis. This effect is particularly evident when it comes to stem cells differentiation, embryo development and cancer. In a tumorigenic context, the m6A could affect the development, progression, cancer stem cells (CSCs) renewal and drug resistance of solid and liquid tumours. So, the m6A is consistently becoming a new attractive pharmacological target. Neuroblastoma (NB) is a neuroendocrine tumour of early childhood that derives from undifferentiated cells of the sympathoadrenal lineage of the neural crest. About 50% of patients have a very aggressive form of NB, with an overall survival rate of less than 30% despite heavy treatments. Moreover, NB is a challenging druggable tumour due to a low rate of somatic mutations. Somatic mutations at significant frequency have been identified in only five genes that also show detectable expression. Among these, only one is currently a directly validated druggable target. Two m6A regulators (METTL14 and ALKBH5) are aberrantly expressed in high-risk NB patients, and their alteration in NB cell lines affects tumour aggressiveness. Specifically, the overexpression of the methyltransferase METTL14 increases cell proliferation and invasion in vitro and tumour growth in mice acting as an oncogene, while ALKBH5 restoration affects cell proliferation, apoptosis and invasion in an opposite fashion. Importantly, the demethylase ALKBH5 impaired tumour formation in vivo when costitutively expressed and dramatically slows down tumor progression in mice when is induced by causing massive apoptosis. These data suggest that ALKBH5 acts as a potent tumour suppressor in NB. We discovered that METTL14 and ALKBH5 exert their effect on different levels by affecting mRNA stability or translation, respectively. Although the contribution to NB of the altered stability of transcripts related to mRNA processing in METTL14-overexpressing cells is less understand, the increase translation of pro-apoptotic genes in the ALKBH5-overexpression condition leaves little doubts. Our results unveil the m6A and its regulators as potential therapeutic targets for treating NB. Indeed, in collaboration with the Laboratory of Genomic Screening of Professor Alessandro Provenzani, we presented an encouraging proof-of-concept of the reader YTHDF1 as a possible pharmacological target.

Yes / In each generation, the germline is tasked with producing somatic lineages that form the body, and segregating a population of cells for gametogenesis. During animal development, when do cells of the germline irreversibly commit to producing gametes? Integrating findings from diverse species, we conclude that the final commitment of the germline to gametogenesis - the process of germ cell determination - occurs after primordial germ cells (PGCs) colonize the gonads. Combining this understanding with medical findings, we present a model whereby germ cell tumors arise from cells that failed to undertake germ cell determination, regardless of their having colonized the gonads. We propose that the diversity of cell types present in these tumors reflects the broad developmental potential of migratory PGCs. / This work was supported by the Howard Hughes Medical Institute where D.C.P. is an Investigator, and the Frontier Research Program from the Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology. P.K.N. is a recipient of the Hope Funds for Cancer Research Fellowship (HFCR-15-06-06) and an Early Career Fellowship from the National Health and Medical Research Council, Australia (GNT1053776).

Carbonic anhydrase IX (CA9) is strongly induced by hypoxia and its overexpression is associated with poor therapeutic outcome in cancer. The function of CAIX is to catalyze the reversible hydration of CO2 to bicarbonate and a proton. This helps hypoxic tumours to maintain a more neutral intracellular pH (pH_i) promoting survival, but produces a more acidic extracellular (pH_e) which promotes invasion and metastasis. Recent evidence has expanded on the role of hypoxia and CAIX by relating them to stem cell niches. In this study, taking advantage of the transmembrane location of CAIX, we show for the first time, a direct marked heterogeneity in response to hypoxia within each tumour cell population studied, associated with major biological differences. Based on CAIX expression pattern under hypoxic conditions, we identify, isolate and characterize two distinct populations of tumour cells, one that express CAIX and the other that does not. Interestingly, we discover that the CAIX positive population is enriched with cells expressing cancer stem cell markers. These include ALDHA1, IGF1, LIN28 and genes involved in epithelial-mesenchymal transition (EMT) and multi-drug resistance (i.e. WNT2, TWIST1, and ABCC2). Accordingly, CAIX+ve cells show higher self-renewal capacity and form tumours significantly faster compared to the CAIX-ve population. Importantly, functional suppression of CAIX in vitro and in vivo, in two breast cancer cell lines resulted in the downregulation of breast cancer stem cell signatures, suggesting that CAIX is not just a marker of stemness but also a regulator of stemness. The molecular mechanism underlying the differential expression of CAIX in the two populations is not HIF-1α-dependent, but instead driven by hypoxia-induced reorganization of chromatin structure. In line with this, we provide experimental evidence showing that the genomic locus encoding CA9 has a more “open” and transcriptionally active chromatin structure in CAIX+ve cells, and a condense and transcriptionally silent chromatin structure in the CAIX-ve cells. Given that HIF induces the transcription of CA9 by binding to hypoxia response elements (HREs) in its promoter we show a significant reduction in binding of HIF to the CA9 promoter of the negative population. We suggest that the reduce HIF binding is a result of the compact chromatin structure of CA9 promoter of the negative cells. Analysis of the transcriptome of the positive and negative populations suggests a symbiotic relationship between these two subpopulations and their environment, likely required to promote tumour growth. This is based on the following observations: Firstly, we identified that CAIX-ve cells express high levels of cytokines and based on this, we suggest that the cytokines secreted by CAIX-ve cells may transmit paracrine signals that regulate the CAIX+ve cells, thus providing a wider hypoxia tolerant microenvironment to protect the stem cell population. Secondly, we identified a metabolic heterogeneity between the CAIX+ve and CAIX-ve cells. The CAIX+ve cells show an upregulation of genes implicated in oxidative phosphorylation, TCA cycle and fatty acid synthesis. Whereas in CAIX-ve cells there is an upregulation of genes implicated in autophagy and mitophagy. Given the above together with the upregulation of oxidative phosphorylation and TCA cycle in the CAIX+ve cells, we proposed the existence of a metabolic symbiosis between the CAIX+ve and CAIX-ve cells. We postulate that the catabolic process such as autophagy and mitophagy in the CAIX-ve cells may results in the overproduction of high-energy metabolites such as lactate, glutamine and ketone bodies which in turns they are been utilized by CAIX+ve cells to fuel mitochondria respiration. Finally, we also demonstrated that in the CAIX+ve cells mTORC1 signaling is upregulated, and contributes to the regulation of CAIX expression. Given the role of mTORC1 in stem cell maintenance and EMT as well as the interdependence of mTORC1 and CAIX expression in the CAIX+ve cells we suggest that mTORC1 signaling may be the critical factor by which CAIX regulates stemness. Interestingly, the subpopulations show a differential sensitivity to HDAC inhibitors, NaBu and SAHA as treatment of MCF7 breast cancer cell line and HCT116 colon cancer cell line leads to elimination of the CAIX+ve population. This is not driven by the downregulation of HIF-1α, the major transcriptional regulator of CAIX. In contrast, we demonstrate that SAHA causes downregulation mTORC1. This suggests that SAHA-induced downregulation of CAIX expression could be due to its effect on mTORC1 pathway. Of wider significance, our findings show that tumours are not homogenous in their response to hypoxia, and distinct signal transduction networks regulate different populations of cells within the tumour. This highlights the need for the utilization of biomarkers, which reveal distinct functional hypoxia profiles of human cancers, and permit the stratification of tumours. Furthermore, the identification of epigenetic regulation of the histones in response to hypoxia for highly selective gene regulation, provides a connection between the epigenetic mechanisms under environmental stress and cancer progression, and is model for development of novel epigenetic cancer therapeutic drugs.

Three novel, synthetic biarylheterocycles bearing imidazole terminal groups had previously been discovered with high cytotoxicity (IC₅₀ 16-640 nM) against a number of human tumour cell lines. Notably, this biological activity was independent of duplex DNA binding affinity. The compounds were tested in the NCI 60-cell line panel and COMPARE analysis suggests they have a novel mechanism of action, targeting the product of a 'gene-like sequence' of unidentified function. The identity of likely protein targets was explored using a chemical proteomic strategy. Bespoke affinity matrices for chromatography were prepared in which test compounds were attached to a solid support through a biotin tag. A synthetic route to hit compounds containing a biotin moiety in place of one of the imidazole sidechains was developed. Chemosensitivity studies confirmed that the biotinylated compounds retained their activity showing IC₅₀ = 6.25 μM in a susceptible cell line, compared with > 100 μM for an insensitive cell line. The biotinylated ligands were complexed to a streptavidin-activated affinity column and exposed to cell lysates from the susceptible cell lines. Bound proteins were eluted from the column and separated using SDS-PAGE. Proteins were characterised by MALDI MS and MS/MS and identified using Mascot database searches. Heterogeneous nuclear ribonuclear protein A2/B1 was found to selectively bind to the affinity probes.

Tumours are not autonomous transformed cell populations, but rather a society composed of both malignant and normal, including immune, cells that together foster tumour growth and development. Tumour-associated macrophages have been reported to enhance tumour growth, progression and metastasis. In high-grade non-Hodgkin’s lymphomas, prototypically the B-cell neoplasm, Burkitt’s lymphoma (BL), infiltrating macrophages engulf large numbers of apoptotic tumour cells. Evidence suggests that apoptotic BL cells can condition the tumour microenvironment to promote lymphoma development by selectively attracting macrophages while inhibiting neutrophil infiltration and by stimulating macrophages to produce the B-cell growth and survival factor. Tumour cells grow in a hypoxic and nutrient-deficient environment and the resultant cellular stress can induce apoptosis. It is therefore possible that hostile environmental conditions in the tumour also contribute to the generation of a pro-tumour microenvironment. This thesis describes investigations which examined this hypothesis. BL cells were cultured at high density to mimic conditions of metabolic stress existing in the tumour environment. Cell-free supernatants from such stressed BL cells demonstrated potent chemoattractive activity for mononuclear phagocytes. Supernatants from BL cells that were protected from apoptosis by over-expression of bcl-2 had similar ability, confirming that chemoattractant release was apoptosis-independent. The observation that apyrase and suramin could inhibit the chemotactic activity of these supernatants suggested that nucleotides might be the apoptosis-independent chemoattractant. Detection of ATP in stress supernatants by bioluminescence assay was consistent with this proposal. Significantly, supernatants from BL cells and those transfected with bcl-2 were both found to inhibit neutrophil migration, suggesting the occurrence of a neutrophil migration inhibitory factor whose release was apoptosis-independent. Furthermore, stress supernatants could promote BL cell proliferation in vitro, which was apoptosis and cell line-independent. In order to study the role of TAM in the tumour microenvironment, a novel macrophage model was devised using mouse embryonic stem cells (ES cells). Cells derived from ES cells generated in vitro expressed macrophage-specific markers and were free of dendritic cells and undifferentiated ES cells. ES cell-derived macrophages (ESDM) could migrate towards apoptotic BL cells and engulf them. However, ESDM migrated to stress supernatants with decreasing efficiency as they matured. Preliminary data indicated that the phagocytic ability of ESDM to engulf apoptotic cells increased as they matured, consistent with distinct roles for circulating monocytes and tissue macrophages with regard to this function. Considering the high yields and purities of ESDM described here, together with their non-malignant nature and genetic versatility these cells should provide a superior source of undifferentiated mononuclear phagocytes with which to elucidate the molecular mechanisms underlying tumour infiltration and microenvironmental conditioning by TAM. In conclusion, this work suggests that under conditions of pre-apoptotic stress, BL cells have the capacity to regulate their micro-environment upstream of their apoptosis programme to promote net tumour growth through paracrine signals that attract supportive macrophages and inhibit destructive neutrophils and through release of autocrine/juxtacrine tumour growth factors.

Tumour tissue is characterised by fluctuating oxygen concentrations, decreased nutrient supply, and acidic pH. Angiogenic signalling pathways that drive a certain metabolic 'configuration' may give endothelial cells a selective advantage in the tumour environment. Previously it has been shown that glycolysis drives proliferation, migration and tip cell formation during sprouting of endothelial cells (De Bock, Georgiadou et al. 2013), and is increased by VEGFA. DLL4-NOTCH has been shown to limit angiogenesis and slow down proliferation of endothelial cells, and promote stalk cell formation during angiogenic sprouting, leading to sprout elongation. DLL4-NOTCH is implicated in tumour angiogenesis, and its overexpression is a potential mechanism of resistance to anti-VEGFA therapy (Li, Sainson et al. 2011). This thesis aimed at investigating the effect of the DLL4-NOTCH signalling pathway on endothelial metabolism and its implications in angiogenesis. Firstly, it was found that DLL4-NOTCH decreases the glycolytic rate and mitochondrial respiratory parameters in endothelial cells. When given exogenous fatty acids, DLL4-NOTCH activation caused increased fatty acid uptake, storage and oxidation. This shows that the induction of DLL4-NOTCH signalling results in increased fatty acid utilisation. Secondly, this research identified fatty acid oxidation as a target metabolism pathway for angiogenic therapy. More specifically, inhibition of fatty acid oxidation decreased proliferation of endothelial cells, decreased sprout elongation in the sprouting assay, and decreased sprouting from the axial vein in the zebrafish model. ATP production was not affected. Therefore, it was hypothesised that DLL4-NOTCH activation promotes and maintains the stalk cell phenotype through an increase of fatty acid oxidation, thereby promoting biomass production for endothelial cell proliferation and growth during angiogenic sprout elongation. Thirdly, a key fatty acid metabolism gene, fatty acid binding protein 4 (FABP4), was identified, that is positively regulated by NOTCH at its promoter region. FABP4 is a candidate for mediating increased fatty acid flux in endothelial cells in response to DLL4-NOTCH. This study shows that FABP4 is induced by VEGFA in a manner dependent on DLL4-NOTCH, and the insulin-responsive transcription factor FOXO1 was required for FABP4 expression in response to DLL4-NOTCH. FABP4 is pro-angiogenic and implicated in tumour angiogenesis in ovarian cancer omental metastasis. Taken together, this study shows for the first time that DLL4-NOTCH signalling increases FABP4 induction, contributing to a key pro-angiogenic pathway, and also fatty acid utilisation in endothelial cells, and thereby contributes to the formation of blood vessels.

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