Neoplastic cells of aggressive tumours can differentiate into endothelial-like cells acquiring the expression of endothelial markers (e.g. CD31) and the ability to participate in the establishment of the tumour vasculature. These tumour-mediated vascular structures promote cancer progression and their presence associates with poor outcome. In triple negative breast cancer (TNBC), we identified platelet-derived growth factor receptor beta (PDGFRP) as an important player of this process. Interestingly, PDGFRp is a promising target in breast cancer; however, therapies with multi-target tyrosine-kinase inhibitors against this receptor have been disappointing. Thus, we aimed at investigating the role of microRNAs (miRNAs) as targets or drugs for the modulation of PDGFRp-mediated vasculogenic properties of TNBC, focusing on miR-9 and miR-200 family. We showed that miR-9 and mir-200 promoted and inhibited, respectively, the in vitro formation of vascular-like structures (loops) in MDA-MB-231 and MDA-MB-157 TNBC cell lines. MiR-9 was induced upon PDGFR~ activation and mediated loop formation ability partially through the direct targeting of STARD13. Mir-200, instead, indirectly suppressed PDGFRp by the inhibition of ZEB1. To confirm these effects in vivo, we generated MDA-MB-231 xenografted mice models for either the stable modulation of miRNAs or the peritumoural delivery of miRNA-based drugs. Notably, both miR-9 inhibition and miR-200c restoration strongly decreased the number of tumour-derived vascular lacunae, identified as vascular-like structures lined by CD31-positive tumour cells. Finally, in TNBC specimens, immunohistochemistry and immunofluorescence analyses indicated that PDGFRp identified tumour cells engaged in vascular lacunae. Interestingly, the presence of PDGFRp-positive structures negatively associated with miR-200c expression.

Tumour infiltrating Iymphocytes influence colorectal cancer (CRC) progression. However, Iymphocyte infiltration comes in different flavours and evidence has been provided that the spatial distribution of immune cells within the tumour tissue is an important immunological feature. The aim of this thesis was to investigate how the dual localization of tumour infiltrating Iymphocytes (TILs) can affect their function in the tumour microenvironment. The project started with the analysis of the CD3 compartment, as CD3+ T cell infiltration (C03-TILs) is a recognized positive prognostic factor for CRC patients. Results here presented show that CD3+ tumour- infiltrating Iymphocytes are present both interspersed in the tumour tissue or scattered throughout the stroma (CD3- TILs) and also aggregated in lymphoid structures showing features of tertiary lymphoid tissue (CD3-TLT). Tumour- associated TLT had a peculiar compartmentalization, with CD3+ T cells and CD20+ B lymphocytes holding complementary positions and with distinct types of dendritic cell populations among them. The presence of HEVs (High Endothelial Venules) suggests a role for TLT in T cell recruitment at the tumour site. To test this hypothesis in human cancer, I performed a whole tissue analysis of the CD3+ infiltrate on CRC sections and found a positive correlation between CD3-TIL and CD3- TLT densities. I further confirmed the hypothesis in vivo in a murine model of colitis- associated cancer (AOM/DSS). AOM/DSS treated mice had expanded TLT compared to control mice. Intravenously injected GFP+ splenocytes localised in TLT of tumour- bearing mice more than in control mice. I then investigated the clinical significance of CD3-TLT in relationship with CD3-TILs in a cohort of 351 CRC patients. In patients with node-negative (without lymph node metastasis, stage 11) CRC, a high density of CD3-TLT and CD3-TILs associated to a better prognosis, while in patients with node- positive (presence of lymph node metastasis, stage Ill) CRC, TLT and TIL density were irrelevant in predicting patient prognosis, thus behaving as biomarkers only for early stage CRC patients. In the second part of my thesis, I analysed the distribution of B cells in colorectal cancer and their : possible contribution to disease progression. Despite still controversial, increasing evidence that B cells play a role in cancer progression has been provided, bringing up the hypothesis that also B-cell responses should be considered as targets of immunotherapeutic approaches. Similarly to CD3+ cells, I showed that, both in human and in preclinical models of CRC, B cells display a dual geographical distribution, either within tertiary lymphoid tissue (CD2D-TLT) or dispersed at the tumour invasive margin (CD2D-TILs). Therefore, I evaluated the role of B cells according to their localization in the microenvironment. I found that CD2D- TLT associated to better prognosis, while CD2D-TILs did not. Interestingly, C02D-TLl correlated with CD2D-TILs only among patients who experienced cancer recurrence. This result suggests that, when located within a lymphoid site, B cells might have a protective anti-tumour function, participating in an anti-tumour immune response. Conversely, the distribution of B cells scattered in the microenvironment is likely to reflect a non-specific pro-tumour inflammatory reaction. To confirm the hypothesis in vivo and attempt to dissect the dual function of B cells, I took advantage of two CRC preclinical models in which B cells present a distinct geographical distribution in the tumour microenvironment. In the first model, B cells mainly localise within TLT, while in the second one B cells diffusely infiltrate the mucosa, without forming aggregates. I found that in a model in which B cells localize primarily within TLT, the genetic deficiency of B cells significantly increased tumour formation, suggesting that B cells within TLT might exert an important anti-tumour function. In contrast, in a model in which B cells only localize within the tissue, genetic deficiency of B cells reduces tumour growth, suggesting that infiltrating B-TILs might have a pro-tumour role. Therefore, the occurrence of TLT is associated with Iymphocyte infiltration in CRC, contributing to recruitment of CD3-TILs. TLT and TILs work together to set up an anti- tumour immune response in patients with low-risk early-stage colorectal cancer. Thus, TLT represents a novel prognostic biomarker for CRC. As to the B cell compartment, their differential distribution in the tumour site corresponds to distinct prognostic functions. This evidence suggests that the design of novel immunotherapeutic drugs depleting B cells should take into account their ability to selectively targeting CD20-TILs but not C20-TLT.

The DNA damage response (DDR) is an intricate signalling network established by eukaryotic cells in order to manage the insults to their DNA, arising both from exogenous and from endogenous sources. Thanks to the activation of DDR, cells can block cell cycle progression, to have time to repair the DNA lesions; alternatively, in presence of irreparable damages, cells are committed to apoptosis or senescence, to prevent the replication of damaged genomes, because mutations could be maintained, leading to genomic instability and predisposition to cancer development. One of the proteins recently found to play an important role in the DDR is DBC1 (deleted in breast cancer 1, CCAR2). In particular, DBC1 is a substrate of ATMjATR and a biological inhibitor of the deacetylase SIRT1, the main protein involved in p53 deacetylation. DBC1, through its physical interaction with SIRT1, blocks its deacetylase activity, preserving p53 acetylation and activation and promoting p53-dependent apoptosis. The data presented in this thesis further characterize the mechanism through which DBC1 regulates SIRT1, as I found that it co-operates with Chk2 and the proteasome activator REGy to exert this function. Moreover, these studies unravel a novel role for DBC1 in the repair of heterochromatic DNA lesions through the promotion of Chk2 activity towards KAP1, a Chk2 substrate involved in the modulation of chromatin remodelling. In addition, the data obtained with a genome wide expression analysis of cells lacking DBC1 revealed a crosstalk between DBC1 and TSPYL2, a protein that we found to playa crucial role in the modulation of p53 activation and induction of apoptosis, by regulating the activity of SIRTl and p300. Overall, these studies extend the knowledge of DBCl functions in DDR and DNA repair, and report for the first time a novel role for TSPYL2 in the regulation of p53 activation upon DNA lesions.

TFE-fusion renal cell carcinomas (TFE-fusion RCCs) are caused by chromosomal translocations that lead to the overexpression of the TFE3 and TFEB genes (Kauffman et aI, 2014). The mechanisms causing kidney tumour development starting from TFE3/TFEB gene overexpression, remain largely uncharacterized and effective targeted therapies are yet to be identified, hence the need to model these diseases in an experimental animal system (Kauffman et al, 2014). Here we show that kidney-specific TFEB overexpression, in both constitutive and inducible conditional transgenic mouse lines, resulted in a phenotype characterized by renal clear cells, multi-layered basement membranes, severe cystic pathology, and ultimately papillary carcinomas with hepatic metastases. These features closely recapitulate the phenotype observed in both TFEB- and TFE3-mediated human kidney tumors. Analysis of kidney samples from these mice revealed both transcriptional induction of genes belonging to the WNT pathway and enhanced WNT βcatenin signalling. The use of specific WNT signalling inhibitors normalized the proliferation rate of primary kidney cells derived from transgenic mice and significantly rescued the disease phenotype in the mouse model. These data shed new light on the mechanisms underlying TFE-fusion RCCs and suggest a possible therapeutic strategy based on the inhibition of the WNT pathway.

Identifying disease-related variants is a primary aim of human genetics. In breast cancer, loss of heterozygosity at specific loci was previously demonstrated in paired tumour and circulating plasma cell-free DNA (cfDNA) samples. However, alterations unique to cfDNA were also found in all cases, suggesting disease progression. These results prompted the characterisation of the circulating breast cancer genome in more detail in this thesis, to test the hypothesis that cfDNA acts as a surrogate tumour marker. This was achieved using Affymetrix SNP 6.0 technology and bioinformatics to map SNP and copy number variation (CNV), comparing cfDNA with matched normal lymphocyte and tumour DNA in 65 breast cancer patients and 8 healthy female controls. Results in this thesis show that comparison of cfDNA SNP genotypes can distinguish between primary breast cancer patients and healthy controls (p<0.0001), and between pre-surgical breast cancer patients and those who already had surgery/treatment (p=0.0016). A significant difference (p=0.0006) was also found between cfDNA samples taken an average of 3 years apart in women on follow-up, again suggesting progression. In addition, CNV amplification was observed in matched tumour and cfDNA at numerous loci on different chromosome arms. Many of these tumour-specific CNVs contributed significantly to disease through logistic regression analysis and remained detectable in cfDNA up to 12 years after diagnosis despite no other evidence of disease. This finding strongly infers breast cancer dormancy in the majority of patients on follow-up. In addition, candidate CNVs were validated by real-time qPCR and additional bioinformatics revealed key SNP and CNV signatures of breast cancer patients. If validated in other patient series, the results could alter the diagnostic and prognostic landscape of breast cancer. Future studies will focus on developing high throughput approaches to target common SNPs/CNVs with a view to developing a targeted custom DNA chip for screening and monitoring.

There is substantial systemic and local morbidity associated with complex reconstruction of the maxillofacial (face, mouth, jaws and neck) region and the distant donor site of the free flap used to reconstruct the defect. This morbidity may be alleviated by careful patient preparation, selection of operation, improving wound care and new surgical procedures. Many aspects of morbidity are not readily amenable to investigation because of a multitude of confounding clinical factors. Improvements in the quality of care may be obtained by careful evaluation of outcomes and comparison with the literature. This body of work represents a reflection on my surgical practice, animal experimental studies and computer modelling techniques which have reduced morbidity and improved my ability to counsel patients. Major elements include the techniques of suprafascial dissection of the soft tissue radial flap and prophylactic internal fixation of the osteocutaneous radial donor site which have become increasingly accepted within the United Kingdom and overseas. The impact of differing flap choice, particularly in the medically compromised group, has been considered. The quality of the clinical documentation has been improved. Biomechanical issues raised by the clinical studies were investigated in laboratory work using the sheep tibia model of the human radius. A computer based model of the sheep tibia, created using a finite element analysis technique, was validated against the preceding biomechanical studies. This simulation was used to investigate the most effective design of osteotomy cuts and type of plate for reinforcement of a straight osteotomised bone, such as the radius, or segmental defects of the mandible. Interest in my work is reflected in the high rate of presentations at the Annual Scientific Meetings of the British Association of Oral & Maxillofacial Surgery combined with the joint second highest rate of successful conversion to publication in the United Kingdom.

Colorectal cancer remains the third largest cause of mortality from cancer related death, with approximately 90% of these deaths attributed to metastatic spread of the disease. There is a need to improve chemotherapy, with the dietary polyphenol curcumin, derived from turmeric, representing a potential candidate as it possess very few side effects and it has shown efficacy in mouse models. Recent advances in our understanding of tumour development have highlighted the existence of tumour initiating cells (TIC), which possess clonogenic potential, are essential for tumour growth, and represent an important therapeutic target. This study sought to determine whether curcumin in combination with oxaliplatin+5-Fluorouracil (OX+5-FU) represented a better combination for targeting TICs, using a variety of models consisting of cells derived directly from colorectal liver metastasis (CRLM). ALDHHigh activity, CD133 and CD26 were all found to mark a spheroid forming population, a method that tests for clonogenicity and selects for TICs. Curcumin significantly reduced the number of spheroids compared to DMSO, and enhanced efficacy of OX+5-FU. The only marker to decrease after treatment was ALDHHigh activity, which was also positively associated with spheroid growth. CD26 expressing cells were identified as a possible chemo-resistant population, however, this population remained unaffected by curcumin. Ex vivo analysis using explant cultures demonstrated that curcumin could significantly decrease ki67 and increase cleaved capase-3 expression, notably enhancing the effects of oxaliplatin in a proportion of patients. A pilot study was undertaken using non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice to reflect a clinical regimen, and assess in vivo whether curcumin could enhance efficacy of OX+5-FU, but the results advocate the use of higher doses as little effect was seen. Overall this body of work contributes to knowledge on propagating CRLM TICs, their expression of known TIC markers, and response to curcumin.

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Suppressors of cytokine signalling (SOCS) are important negative regulators of several signalling pathways and their role in cancer development has been recently investigated. In this thesis; I first examined SOCS 1-7 genes expression in normal and breast cancer tissue using real-time PCR, and correlated this with several clinico-pathological prognostic factors including TNM stage, tumour grade and clinical outcome over lO-year follow-up. SOCS1, SOCS4-7 expression decreased with higher TNM stage, SOCS2-3 expression decreased with higher Nottingham Prognostic Index (NPI), and SOCS7 expression decreased with higher tumour grade. Higher SOCS 1-2, SOCS7 expression levels were found among patients who remained disease-free compared to those who developed local recurrence, and higher SOCS2, SOCS4, and SOCS7 as well as SOCSl-2 levels were found in patients who remained disease-free versus those who developed distant recurrence or died from breast cancer respectively. In survival analysis, higher levels of SOCSl, SOCS3 and SOCS7 were significant predictors of better disease-free survival (DFS) and overall survival (as), while SOCS4 was a significant predictor of better as. Secondly; I studied SOCS7 role. MCF7 and MDA-MB-23l breast cancer cells were transfected with anti-SOCS7 ribozymal transgene, creating sublines with SOCS7 knockdown that was verified by RT-PCR. Data suggest a SOCS7 tumour suppressor role in breast cancer, both in vitro and in vivo, as SOCS7 knockdown has enhanced cellular in vitro growth and migration, and in vivo MCF7- tumour growth in the athymic nude mice. As previous evidence suggested an anti-phospholipase Cy-l (PLCy-l) role for SOCS7, the in vitro cellular functions were evaluated with and without Insulin-like Growth Factor-I (IGF- I), Hepatocyte Growth Factor (HGF), and the PLCy-l inhibitor (U73122) treatment. SOCS7 loss resulted in increased in vitro cellular growth and migration leading to a synergistic effect on their response to IGF-I and HGF. This role could be attributed to SOCS7 downstream interaction with PLCy-l. These data support a tumour suppressing role for several SOCS family members in breast cancer. The study of SOCS7 showed a precise anti PLCy-l signalling role. Further research is required to explore SOCSs diagnostic and therapeutic applications in breast cancer.