Introduction: Van Gogh-Like 2 (VANGL2) is a scaffolding planar cell polarity protein involved in non-canonical Wnt signalling. It has been shown to have crucial roles in regulating epithelial development and homeostasis. Moreover, VANGL2 has been implicated in human cancers, with increased expression and copy number amplification seen in several cancer contexts. Many related components within this pathway have also been linked to cancer development, with VANGL2 expression known to regulate factors involved in cell migration and extracellular matrix (ECM) remodelling in cell lines. These cellular processes tend to be erroneously activated in cancer. VANGL2 is known to inhibit the classical driver pathway of colorectal cancer (CRC), canonical, or β- catenin dependant, Wnt signalling, in CRC cell lines. The aim of this thesis is to determine the expression of VANGL2 in CRC, and to investigate how VANGL2 may act to regulate intestinal homeostasis and disease. Methods: Transcriptional verification of VANGL2 expression in the mouse intestine was carried out by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and transcripts localised within the murine colon using RNA-In Situ Hybridisation (RNAISH). Expression and localisation of the VANGL2 protein and related non-canonical Wnt signalling components was confirmed using immuno-histochemistry (IHC). Furthermore, using a combination of human Tissue Micro-Array (TMA), transcriptional data and genomic data, we determined an association between VANGL2 on tumour grade and disease-free survival. To functionally validate the effects of VANGL2 on colorectal biology, we used a model in which VANGL2 is selectively deleted from the colonic epithelium using Villin-CreERT Vangl2flox mouse lines. Using a combination of molecular biology methods, we identified the ECM as differentially regulated following VANGL2 modulation. To test the role of VANGL2 in colorectal cancer, we used a murine colorectal cancer model in which adenomatous polyposis coli (APC) is deleted from colonic epithelium resulting in the formation of cancer concurrently with deletion of Vangl2. We evaluated survival of these mice as well as tumour number and size. Tumour tissue was analysed using IHC, qRT-PCR and 3-Dimensional organoid culture. Results: Within this thesis I have illustrated that the murine colonic epithelium expresses Vangl2, and other components known to interact with VANGL2 including Vangl1, Wnt5A, and Protein Tyrosine Kinase 7 (Ptk7). I have also shown that VANGL2 is expressed within the human colonic epithelium. I go on to show that 9.2% of human CRC possesses VANGL2 transcriptional alterations which correlates with a worsened disease-free survival (DFS) rate among patients. Using IHC, I also show that higher grade CRC is associated with increased VANGL2 expression. In our murine cancer model, mice with single or dual-copy loss of VANGL2 were found to have a reduced number of colonic tumours, while maintaining similar tumour size. Investigations to identify how VANGL2 may have control of tumour initiation were carried out focussing on the ECM. I found that, contrary to what I have discovered in the healthy murine colon, tumours from VANGL2-deficient mice had increased transcription of the ECM markers Secreted protein acidic and rich in cysteine (Sparc) and Decorin (Dcn), as well as increased expression of the ECM regulators Matrix Metallopeptidase 9 (Mmp9) and Tissue Inhibitor of Metalloproteinases 1 (Timp1). Changes in the ECM was also seen at the protein level, with increases in staining for the ECM components Col1 (Collagen, type I), and Laminin in VANGL2-deficient tissue. The ECM modulator Connective Tissue Growth Factor (Ctgf), is implicated in multiple cancers including CRC and is increased within VANGL2-deficient tumours at both the transcript and protein level, implicating Ctgf in increasing the ECM of these tumours.

Colorectal cancer (CRC) is one of most common cancer diseases worldwide. In Swedenapproximately 5,000 new cases of CRC are generated each year, which makes it the thirdmost common cancer disease among both men and women. The past decades ofimproved treatment strategies have considerably increased the five-year survival for CRCpatients. However more could be achieved in this area, in particular for metastatic CRC,which is the cause of most CRC-related deaths. Therefore it is important to study thebiological response to certain treatments induced in CRC to find valuable predictiveand/or prognostic factors to select patients for better suited treatments. The aim of this thesis was to gain insight into the molecular changes that occurfollowing irradiation or treatment with SN-38 in rectal cancer patients or colon cancercell lines by studying the RNA expression, protein expression, DNA cell cycledistribution and apoptotic response. The expression of phosphatase of regenerating liver(PRL) proteins was investigated in rectal cancers from 125 patients included in arandomized clinical trial of preoperative radiotherapy (RT). Increased expression of PRLswas seen at the invasive margin of primary and metastatic cancers compared with theinner area of the tumors. Moreover, strong PRL staining at the invasive margin correlatedto distant recurrence and worse survival of patients in the RT group but not in non-RTgroup (Paper I). Radiosensitivity was studied by treating KM12C, KM12SM andKM12L4a colon cancer cell lines with radiation. KM12C is of low metastatic naturecompared with the highly metastatic KM12SM and KM12L4a. Upregulation of ΔNp73and PRL-3 might contribute to the radioresistant phenotype in KM12C. In contrast,KM12L4a shows a high frequency of apoptosis and lack of upregulation of ΔNp73, PRL-3 and survivin, which might explain its radiosensitive phenotype (Paper II). KM12C,KM12SM and KM12L4a were treated with SN-38 which inhibits topoisomerase 1 (topo-1). The results show that SN-38 induces G2/S arrest and possess the capacity to triggerapoptosis in the three cell lines (Paper III). To further elucidate SN-38 effect on these celllines, the gene expression profile following SN-38 treatment was studied. Oligonucleotidearrays consisting of ~27,000 spots were hybridized with sample and reference cDNA.Both unsupervised and supervised hierarchical clustering analysis, and functional analysiswere performed. Supervised hierarchical clustering gives a strong signal of 1453discriminated genes, the vast majority being upregulated. Both upregulated anddownregulated genes point toward a favorable impact of SN-38 regarding the apoptoticpathways. For example RhoB and Bax are upregulated together with downregulation ofKras and survivin, which promotes apoptosis (Paper IV). In conclusion, PRLs may be valuable biomarkers for RT resistance, predicting apoor prognosis in rectal cancer patients. Targeting radio-resistance factors, such asΔNp73 and survivin may contribute to an increased sensitivity to RT. SN-38 affects cellproliferation and apoptosis.

Ulcerative colitis (UC) and Crohn’s disease (CD) are the two common forms of inflammatory bowel disease (IBD). They share similar clinical and demographic features as well as harbor key differences in tissue damage and prognosis. Previous studies indicated that they contributed to the increased rick to Colorectal cancer (CRC). However, whether UC and CD share inflammatory signatures still remains controversial. In addition, no inflammatory signatures have been reported on CRC. To answer these questions, a comprehensive study has been conducted on collected microarray datasets. Our analysis suggests that although CD and UC share common inflammatory pathways, they also present difference. Especially, CD patients are likely to have type I response, while UC patients are inclined to undergo type II response. Pathway enrichment analysis on CRC uncovered two potential CRC-specific inflammatory pathways.

Abstract The development of colorectal cancer ( CRC ) is believed to follow series progress of pathological changes and with correspondent genetic changes of many genes. This includes intestinal epithelial crypts, aberrant focus, adenoma and carcinoma, each of that commonly involved genetic and proteomic alterations. And in genetic level, it usually includes mutations of APC, p53, K-ras and microsatellite instability. The somatic mutations of APC gene mostly occur in MCR ( Mutation Cluster Region ) in codon 1286-1513. The p53 mutations is dispersed in whole gene with 3 hot spots: codon 175, 248 273. K-ras codon 12 and 13 mutations is preferentially involved in polyps growth of CRC. And microsatellite instability is found in 15-25% CRC patients. We collect polyps and various stages CRC samples in Taiwan, and design 6 primer pairs of APC and p53 which is widely used in western countries to analyze mutations of the local CRC genetic changes. We also use two-dimensional electrophoresis and mass spectrometry to identify protein expression changes in CRC. We have found 30 proteins that exhibited either a significant decrease or increase between normal colon tissue and carcinoma, and 3 out of ( TSD1, TSD2, and TSD3 ) these were significantly associated with tumor progression. TSD3 is annotated by mass spectrometry and is identified to be a c1q-related protein. Though there are no report on the function of c1q-related protein, a NCBI virtual northern analysis shows its expression is varied in various cancer. On the other hand, there are only about 56 % genetic changes of APC and p53 during carcinogensis, which is much less than the 70-85 % mutational rate in western CRC patients. It indicates different genetic mutational pattern of CRC in Taiwan.

Colorectal cancer (CRC), which includes cancer of the large bowel and rectum is the third most common cancer in men and the second in women and there is a poorer survival rate in less developed regions of the world such as West Africa mainly due to the ‘out of reach’ costs of chemotherapy. Evidence suggests that aspirin, a non-steroidal anti-inflammatory drug (NSAID) has the potential to decrease incidence of, or mortality from, a number of cancers including CRC through several mechanisms of action. However, this evidence is dampened by aspirin’s gastrointestinal (GI) toxicity, which have been found to be mostly age-dependent. The search for potential aspirin-related compounds with the same or better cytotoxic effects against cancer cells accompanied by a safer toxicity profile has been ongoing over the years and led to us to synthesise a number of novel aspirin analogues. One of the mechanisms of action suggested for the anticancer property of aspirin is the COX-dependent pathway. In this thesis SW480 cell line, a CRC cell line that is COX-2 negative and mismatch repair (MMR) proficient was used to study the possible COX-independent mechanism of action for aspirin, its analogues and diflunisal at 0.5 mM. Diflunisal was included in this study because it is also a salicylate with reports of having cytotoxic effects. OE33 and FLO1 oesophageal cancer cells were also employed in the epidermal growth factor receptor (EGFR) and synergy experiments to show effects were not just specific to SW480 cells alone. These aspirin analogues were synthesised, identified using nuclear magnetic resonance (NMR) and infra-red (IR) spectroscopy, and tested for purity using thin layer chromatography (TLC) and melting point. The findings of this study suggest that these compounds breakdown into salicylates and perturb epidermal growth factor (EGF) internalization with PN517 (fumaryldiaspirin) and PN590 (ortho-thioaspirin) also driving EGF co-localization with early-endosome antigen-1 (EEA1). The perturbation of the internalization of EGF by aspirin and PN517 was also observed by a time-lapse assay using live confocal imaging. These compounds also had specific effects on different tyrosine phosphorylation sites of the EGFR, with none but PN590 inhibiting 4 phosphorylation at Y1068, and all but PN502 (ortho-aspirin), PN548 (meta-aspirin) and PN549 (para-aspirin) inhibiting phosphorylation at Y1045 and Y1173. Given that the EGF internalization assay involved the cells being treated with compounds for 2 h, cells were also treated for this same time period and probed with pEGFR 1045, which resulted in the compounds having no significant effect on phosphorylation at that site which is responsible for the ubiquitination of the EGFR. Most of these compounds were apoptotic with some showing a combination of apoptosis and necrosis. Aspirin and its isomers drove apoptotic cell death in SW480 cells via the BCL2-BAX pathway while the thioaspirins appear to follow the p21 pathway by decreasing the expression of the protein. In addition, it was shown that PN502 (aspirin), PN517 and PN590 had synergistic effects when used in combination with oxaliplatin at ED50, ED75 and ED90 in SW480 CRC cells. The cytotoxicity of these compounds individually or in combination was determined using MTT assay followed by the use of the CompuSyn and CalcuSyn software to calculate combination index (CI), which indicated whether a drug combination was synergistic, antagonistic or additive. PN517 and PN524 were synergistic when used in combination with cisplatin in OE33 oesophageal cancer cells. Effect of these compounds on the EGFR indicates a delay or disruption of the signalling pathway involved in the proliferation of cancer cells, thus, translating into protection against tumour formation or progression while the synergistic effects of these compounds when used in combination with platinum compounds can provide patients with less toxic chemotherapeutic regimen especially in patients with CRC tumours that harbour mutant TP53 gene and normally resistant to oxaliplatin. It is therefore proposed that the perturbation of EGF internalization is a novel mechanism of action for aspirin and its analogues in cancer therapy. These positive findings shed light on the understanding of the possible mechanism of action for aspirins and gives hope for a more affordable, less toxic therapy for the prevention, treatment and management of cancer.

Class of 2017 Abstract / Objectives: To determine the efficacy and toxicity of fluorouracil/leucovorin/oxaliplatin (FOLFOX) versus capecitabine/oxaliplatin (XELOX) in the treatment of colorectal cancer (CRC) in the adjuvant (aCRC) and metastatic (mCRC) setting in Veterans at the Southern Arizona Veteran Affairs Health Care System (SAVAHCS). Methods: A retrospective chart review was conducted to collect efficacy and toxicity data. Subjects were included based on age, treatment setting and regimen in the preset 5-year period, and appropriate diagnosis via International Classification of Diseases-Revision 9 (ICD-9) codes. Efficacy was measured via 1-year disease-free survival (DFS) for aCRC, progression-free survival (PFS) for mCRC, and overall survival (OS) for both settings. Results: A total of 79 subjects were initially enrolled with 51 and 54 all-male subjects included in the efficacy and toxicity analysis, respectively. Mean range of age was 63-72 years old. Subjects were divided into four groups: FOLFOX aCRC (17) and mCRC (19), XELOX aCRC (10) and mCRC (8). No difference was found in 1-year DFS and OS between aCRC groups, and PFS between mCRC groups; a higher incidence of 1-year OS with FOLFOX in the mCRC setting was noted (p = 0.03). No difference was found in toxicity between FOLFOX and XELOX, except a higher incidence of hand-foot syndrome in XELOX (p = 0.0007). Conclusions: Efficacy between FOLFOX and XELOX in aCRC and mCRC is similar, while toxicity is slightly more prevalent in XELOX due to increased hand-foot syndrome incidence. These findings agreed with the results reported by prospective clinical trials.

Petroleum Technology Development Fund (PTDF) Nigeria / The full text will be available at the end of the embargo period: 21st March 2026

Colorectal cancer (CRC) is the second most common cause of cancer-related death in the UK and Western world. More than 90% of sporadic CRCs harbour mutations in the multi-functional tumour suppressor gene Adenomatous polyposis coli (<i>Apc</i>). The most commonly studied function of APC is its role as a scaffold for the β-catenin destruction complex involved in Wnt signalling. However, APC binds many other proteins. For example, it directly binds to and stabilises microtubules and actin. These non-Wnt related functions of APC are poorly understood. My PhD examines non-Wnt functions of APC. To this end, I created degron-tagged APC in DT40 cells that allowed for the rapid, conditional degradation of endogenous APC. The aim was to identify the immediate effects on cellular processes. Then, to identify the contribution of different APC domains by measuring the ability to rescue any defects when reintroducing fragments of APC. However, creation of these degron-tagged <i>Apc </i>knock-in cell lines resulted in hypomorphic phenotypes and auxin-associated off-target effects. Nonetheless, I compared the response of APC^high, APC^low, and APC^minimal cells to DNA damaging agents and Taxol® but found no significant differences. Subsequently, I focused on the relationship between APC and apoptosis. Previous observations suggested that deficiency in <i>Apc </i>rendered cells less sensitive to low doses of Taxol®. However, <i>Apc </i>deficient cells were more readily killed when Taxol® was combined with the Bcl-2 inhibitor, ABT-737. One possible explanation is the increase in Bcl-2 protein upon <i>Apc </i>depletion. However, I found that ABT-737, Taxol® and <i>Apc </i>depletion each cause activation of the unfolded protein response. This suggests that these treatments elicit a stress response that can stimulate apoptosis. Moreover, the same treatments also cause changes in mitochondria. Importantly, all of these effects do not require an increase in the β-catenin protein. Together, my data reveal novel links between APC and apoptosis that could be exploited clinically.

Carcinogenesis is a progressive process involving multiple genetic alterations in tumor cells and complex interactions in the tumor-host microenvironment. To better understand the contribution of molecular alterations in tumor cells and stromal variables to the development of colorectal cancer (CRC) and identify prognostic factors, in this study we examined the clinicopathological and biological significance of stromal variables, including particularly interesting new cysteine-histidine rich protein (PINCH), inflammatory infiltration, angiogenesis and lymphangiogenesis, as well as hRAD50/hMRE11/hNBS1 proteins and hRAD50 mutation in tumor cell in CRC. PINCH protein expression in the stroma was increased from normal mucosa to primary tumors and further to lymph node metastases. In particular, PINCH expression was most intense at the tumor invasive margin, which was related to low inflammatory infiltration and independently related to an unfavorable prognosis. Low inflammatory infiltration at the tumor invasive margin was related to advanced tumor stage, worse differentiation and microsatellite instability (MSI). Further, it was independently related to an unfavorable prognosis. Increased blood and lymphatic vessel density was observed in the primary tumors compared with the corresponding normal mucosa. However, neither angiogenesis nor lymphangiogenesis was associated with tumor stage and patients’ survival. Moreover, PINCH was present in a proportion of endothelial cells of the tumor vasculature, and PINCH expression in tumor-associated stroma was positively related to blood vessel density. In primary tumor cells of CRC, strong expression of hRAD50, hMRE11 or hNBS1 was related to microsatellite stability (MSS). A high percentage of hMRE11 expression was associated with less local recurrence and high apoptotic activity. Further, we observed that the expression of hRAD50, hMRE11 or hNBS1 among normal mucosa, primary tumors and metastases in MSS CRC differed from that in MSI CRC. In MSS CRC, the expression intensity of hRAD50, hMRE11 and hNBS1 was consistently increased with respect to normal mucosa, but there was no difference between the primary tumors and metastases. In the primary MSS tumors, the expression of individual or combination of hRAD50/hMRE11/hNBS1 was associated with a favorable prognosis in the same series of the CRCs. Moreover, strong/high hRAD50 in MSS primary tumors was related to earlier tumor stage, better differentiation and high inflammatory infiltration, whereas strong hNBS1 expression tended to be independently related to a favorable prognosis in MSS CRC with earlier tumor stage. However, in MSI CRC, there were neither differences in the expression of hRAD50/hMRE11/hNBS1 among normal mucosa, primary tumors and metastases, nor any association of the protein expressions with clinicopathological variables. On the other hand, frameshift mutations of (A)9 at coding region of hRAD50 were only found in MSI CRC. Our study indicates that 1) PINCH is likely a regulator of angiogenesis, and PINCH expression at the tumor invasive margin is an independent prognostic indicator in CRC. 2) Inflammatory infiltration at the tumor invasive margin is also an independent prognostic indicator in CRC. The lack of association between high inflammatory infiltration and MSI may help to explain the non-association of MSI with survival in CRC patients. 3) Angiogenesis and lymphangiogenesis occur in the early stage of CRC development, but do not associate with CRC progression and patients’ prognosis. 4) hRAD50/hMRE11/hNBS1 may act dependently and independently, playing different roles in MSS and MSI CRC development. In MSS CRC, the strong expression of the three proteins, associated with a favorable prognosis, may present the cellular response against tumor progression. Expression of hNBS1 may be a prognostic indicator for MSS CRC patients in the earlier tumor stage. In MSI CRC, the frameshift mutations at the coding region of hRAD50 may contribute to tumor development.

Philosophiae Doctor - PhD / Tumour-specific protein markers are usually present at elevated concentrations in patient biopsy tissue; therefore tumour tissue is an ideal biological material for studying cancer proteomics and biomarker discovery studies. To understand and elucidate cancer pathogenesis and its mechanisms at the molecular level, the collection and characterisation of a large number of individual patient tissue cohorts are required. Since most pathology institutes routinely preserve biopsy tissues by standardised methods of formalin fixation and paraffin embedment, these archived, FFPE tissues are important collections of pathology material, often accompanied by important metadata, such as patient medical history and treatments. FFPE tissue blocks are conveniently stored under ambient conditions for decades, while retaining cellular morphology due to the modifications induced by formalin. / 2022