Heterocyclic aromatic amines (HCAs) are formed in meat cooked at high temperatures for a long time or over an open flame. In this context 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), the most abundant HCA in cooked meat, has been suggested to be involved in colon and prostate carcinogenesis. In the latter case it has been reported that: (1) roughly 50% of Fischer F344 male rats treated with PhIP develop carcinomas in the ventral prostate lobe at 1 year of age; (2) inflammation precedes prostatic intraepithelial neoplasia in PhIP-fed rats; (3) inflammation specifically occurs in the ventral prostate lobe of PhIP-fed rats. To test whether PhIP by itself leads to inflammation in the colon and whether a human-relevant concentration of PhIP is able to induce preneoplastic lesions in the colon, male F344 rats were fed 0.1 or 100 ppm PhIP for up to 10 months and thereafter the colon tissue was analyzed histochemically. In none of the experimental groups signs of acute or chronic colonic inflammation were observed. 0.1 ppm PhIP leads to the development of hyperplastic and dysplastic lesions in the colon of single animals, but the incidence of these lesions does not reach a statistical significance. In contrast, in rats fed 100 ppm PhIP for 10 months hyperplastic and dysplastic colonic lesions were induced in a statistically significant number of animals. It is concluded that: (1) the induction of preneoplastic lesions in rat colon by PhIP is not preceded or accompanied by an inflammatory process; (2) a human-relevant concentration of PhIP alone is not sufficient to initiate colon carcinogenesis in rats.

Cannabinoids (CBs) are compounds that activate the CB1 and CB2 receptors. CB receptors mediate many different physiological functions, and cannabinoids have been reported to decrease tumor cell viability, proliferation, migration, as well as to modulate metastasis. In this thesis, the effects of cannabinoids on human colorectal carcinoma Caco-2 cells (Paper I) and mouse P19 embryonal carcinoma (EC) cells (Paper III) were studied.  In both cell lines, the compounds examined produced a concentration- and time-dependent decrease in cell viability. In Caco-2-cells, HU 210 and the pyrimidine antagonist 5-fluorouracil produced synergistic effects upon cell viability. The mechanisms behind the cytocidal effects of cannabinoids appear to be mediated by other than solely the CB receptor, and a common mechanism in Caco-2 and P19 EC cells was oxidative stress. However, in P19 EC cells the CB receptors contribute to the cytocidal effects possibly via ceramide production. In paper II, the association between CB1 receptor immunoreactivity (CB1IR) and different histopathological variables and disease-specific survival of colorectal cancer (CRC) was investigated. In microsatellite stable (MSS) cases there was a significant positive association of the tumor grade with the CB1IR intensity. A high CB1IR is indicative of a poorer prognosis in MSS with stage II CRC patients. Paper IV focused on the cytotoxic effects of cannabinoids during neuronal differentiation. HU 210 affected the cell viability, neurite formation and produced a decreased intracellular AChE activity. The effects of cannabinoids on embryonic development and survival were examined in Paper V, by repeated injection of cannabinoids in fertilized chicken eggs. After 10 days of incubation, HU 210 and cannabidiol (without CB receptor affinity), decreased the viability of chick embryos, in a manner that could be blocked by α-tocopherol (antioxidant) and attenuated by AM251 (CB1 receptor antagonist). In conclusion, based on these studies, the cannabinoid system may provide a new target for the development of drugs to treat cancer such as CRC. However, the CBs also produce seemingly unspecific cytotoxic effects, and may have negative effects on the neuronal differentiation process. This may be responsible for, at least some of, the embryotoxic effects found in ovo, but also for the cognitive and neurotoxic effects of cannabinoids in the developing and adult nervous system.

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.

The LRIG1 gene (leucine-rich repeats and immunoglobulin like domains-1) at chromosome 3p14 is a proposed tumour suppressor gene whose gene product negatively regulates various receptor tyrosine kinases. This function has been the basis for classifying LRIG1 as a potential tumour suppressor gene (TSG). The ERBB receptor family is important in malignant cellular functions such as proliferation, survival, adhesion, migration and differentiation. In breast cancer, amplification of the ERBB2 proto-oncogene is an important negative prognostic factor. The epidermal growth factor receptor (EGFR/ERBB1), is expressed in colorectal cancer and has been correlated to a worse prognosis. Until recently, immunohistochemical analysis of EGFR expression was used to select patients suitable for treatment with EGFR targeted antibodies. This thesis characterizes LRIG1 in breast and colorectal cancer to gain further knowledge of the gene and its expression. Also, the EGFR expression in metastases and the invasive margin of colorectal cancers was investigated to correlate changes to clinical factors. Breast cancer samples and matched normal tissues were evaluated for LRIG1 and the ERBB receptors at gene, RNA and protein levels. An increase in copy number of the LRIG1 gene was evident. Also, increased LRIG1 copy number was associated with high levels of ERBB2 mRNA. Another set of breast cancer tumours were analysed for LRIG1 by FISH analysis. The results were coherent with the previous results. To further analyze the correlation to ERBB2, tumours with LRIG1 increased copy number were analysed for ERBB2. The data showed that 89% of tumours with increased LRIG1 copy number were either ERBB2 amplified or had an increased copy number of ERBB2. To investigate LRIG1 and the EGFR in colorectal cancer, the gene and protein expression was analysed by several methods in tumours and corresponding normal tissues. There were no significant changes at gene level found, but at the protein level, both over- and under expression were seen. No evident correlation between LRIG1 and EGFR expression was detected. The ERBB receptor family expression in colorectal cancer tumours and corresponding metastases was investigated to explore if the expression was altered in the metastatic lesion. The results showed that the EGFR expression was lost in the corresponding metastases in 33% of the tumours and that the same percentage of tumours gained expression in the metastases. Co-expression of the ERBB family members was also analysed; there was a significant increase of ERBB3/ERBB4 co-expression in late stage tumours. EGFR expression at the invasive margin of colorectal cancers was analysed to clarify whether expression correlated to the patient’s prognosis. Significant correlation to survival and the presence of budding was seen. In conclusion, 34% of the breast cancer tumours studied had an increased copy number of LRIG1 with a significant co-incidental increase in ERBB2 copy number. This raises the question of a functional correlation between LRIG1 and ERBB2, a finding that might be of clinical importance. The studies of EGFR and the ERBB receptors in colorectal cancer reflect the heterogeneity of EGFR expression in tumours. In addition, these findings suggest that survival of the patients correlates to an increasing EGFR expression at the invasive margin.

Colorectal cancer is the third most diagnosed cancer and second leading cause of cancer related deaths in Canada. As Ontario has the largest population in Canada, it also has great disparities in colorectal cancer incidence. The region of Timiskaming has the highest incidence for colorectal cancer, while the region of Peel has the lowest incidence for colorectal cancer in Ontario. The purpose of this study is to identify the dominant non-nutritional modifiable environmental risk factors in the region of Timiskaming compared to the region of Peel that may be associated with diverging colorectal cancer incidence rates. The three objectives of the study included performing a systematic review on available published literature, creating an assessment questionnaire tool regarding environmental exposures, and utilizing the questionnaire assessment tool within a pilot study group while expanding it into the communities of interest. Findings indicate that there are dominant non-nutritional modifiable environmental risk factors in the regions of Timiskaming and Peel that may be associated with colorectal cancer. The dominant factors identified are tobacco/smoking, alcohol use, pesticides/organochlorines, and metal toxins. Following this study, it is imperative that recommendations are directed at a community level and relate to the assessment of potential non-nutritional modifiable environmental risk factors. Future research should accompany a larger sample size, multiple participant communities, and catering of the questionnaire tool towards the communities of interest. / UOIT

Type II diabetes and hyperinsulinemia are associated with increased risks of developing colorectal cancer (CRC). Detailed mechanisms underlying this correlation, however, are yet to be explored. The present study demonstrates that insulin increases the expression of proto-oncogenes c-Myc and cyclin D1 via both translational and transcriptional mechanisms. We show here that insulin stimulates c-Myc gene translation via an Akt/PKB-dependent mechanism involving the mTOR signaling pathway. More importantly, we show for the first time that transcriptional stimulation of c-Myc and cyclin D1 expression by insulin involves a novel Akt/PKB-independent signal crosstalk between insulin and canonical Wnt signaling pathways. We then identified p21-activated protein kianse 1 (PAK-1) as a novel mediator for insulin and Wnt/beta-catenin (b-cat) molecular crosstalk, involving MEK/ERK signaling. Furthermore, we found that insulin treatment leads to increased b-cat phosphorylation at Ser675, and this is associated with increased b-cat nuclear content and increased b-cat interaction with Tcf/Lef-binding elements (TBEs) of the human c-Myc gene promoter. Lastly, we demonstrated that insulin signaling directly alters the expression levels of components of the Wnt signaling pathway, including fizzled homology 4 (Fdz-4) and TCF7L2 (=TCF-4). This study not only demonstrated the existence of signaling crosstalk between insulin and canonical Wnt signaling pathways at multiple levels, it reveals molecular mechanisms for observed correlation between CRC and hyperinsulinemia. The growing evidence implicating PAK-1 in various human tumorigenesis has emerged PAK-1 as a potential therapeutic target. Our discovery of PAK-1 functioning as a novel central mediator for insulin and Wnt signaling crosstalk in intestinal cells suggests that PAK-1 may potentially be a good target candidate for treating patients with CRC, especially those who have Type II diabetes or experience hyperinsulinemia.

It has been hypothesized that tumours are caricatures of normal tissue organization, where a minority cell population, the ‘stem cell’ of cancer, holds the exclusive ability for tumour propagation. These cancer stem cells (CSCs), or tumour-initiating cells, possess extensive self-renewal ability, through which they ensure maintenance of the tumourigenic clone. Such cells have been identified in various cancers, including colorectal, and have been proposed to be the source of tumour re-initiation following therapy. An important and currently unanswered question in solid tumours is whether all CSCs are equal or whether there is a gradient of potency within the CSC compartment. Using primary human colon tumour cells and sensitive in vivo functional assays, we have determined that colon CSCs are not uniform; rather, they vary with respect to their proliferative capacity, which is also linked to their response to chemotherapy. These findings hold therapeutic implications for colorectal cancer treatment since all types of CSCs must be eradicated to remove the risk of tumour relapse. While the CSC model may provide attractive answers to some challenging questions, it remains controversial. Ascertaining the importance of CSCs will come from targeted CSC therapies. Here we demonstrate that human colorectal CSC function is dependent on the self-renewal regulator BMI-1. Down-regulation of BMI-1 inhibits the ability of colorectal tumour-initiating cells to self-renew resulting in the abrogation of their tumourigenic potential. Treatment of primary colorectal cancer xenografts with small molecule BMI-1 inhibitors resulted in colorectal tumour-initiating cell loss with long-term and irreversible impairment of tumour growth. Targeting the BMI-1 related self-renewal machinery provides the basis for a new therapeutic approach in the treatment of colorectal cancer. Collectively, we have advanced the CSC field in two areas of importance. We show for the first time that the CSC pool encompasses a gradient of proliferative potential linked to chemotherapeutic response. Second, we provide critical proof for the clinical relevance of CSCs by inhibiting tumour growth through targeting of the self-renewal machinery. This body of work significantly advances our understanding of colorectal tumour-initiating cells.

It has been hypothesized that tumours are caricatures of normal tissue organization, where a minority cell population, the ‘stem cell’ of cancer, holds the exclusive ability for tumour propagation. These cancer stem cells (CSCs), or tumour-initiating cells, possess extensive self-renewal ability, through which they ensure maintenance of the tumourigenic clone. Such cells have been identified in various cancers, including colorectal, and have been proposed to be the source of tumour re-initiation following therapy. An important and currently unanswered question in solid tumours is whether all CSCs are equal or whether there is a gradient of potency within the CSC compartment. Using primary human colon tumour cells and sensitive in vivo functional assays, we have determined that colon CSCs are not uniform; rather, they vary with respect to their proliferative capacity, which is also linked to their response to chemotherapy. These findings hold therapeutic implications for colorectal cancer treatment since all types of CSCs must be eradicated to remove the risk of tumour relapse. While the CSC model may provide attractive answers to some challenging questions, it remains controversial. Ascertaining the importance of CSCs will come from targeted CSC therapies. Here we demonstrate that human colorectal CSC function is dependent on the self-renewal regulator BMI-1. Down-regulation of BMI-1 inhibits the ability of colorectal tumour-initiating cells to self-renew resulting in the abrogation of their tumourigenic potential. Treatment of primary colorectal cancer xenografts with small molecule BMI-1 inhibitors resulted in colorectal tumour-initiating cell loss with long-term and irreversible impairment of tumour growth. Targeting the BMI-1 related self-renewal machinery provides the basis for a new therapeutic approach in the treatment of colorectal cancer. Collectively, we have advanced the CSC field in two areas of importance. We show for the first time that the CSC pool encompasses a gradient of proliferative potential linked to chemotherapeutic response. Second, we provide critical proof for the clinical relevance of CSCs by inhibiting tumour growth through targeting of the self-renewal machinery. This body of work significantly advances our understanding of colorectal tumour-initiating cells.

Type II diabetes and hyperinsulinemia are associated with increased risks of developing colorectal cancer (CRC). Detailed mechanisms underlying this correlation, however, are yet to be explored. The present study demonstrates that insulin increases the expression of proto-oncogenes c-Myc and cyclin D1 via both translational and transcriptional mechanisms. We show here that insulin stimulates c-Myc gene translation via an Akt/PKB-dependent mechanism involving the mTOR signaling pathway. More importantly, we show for the first time that transcriptional stimulation of c-Myc and cyclin D1 expression by insulin involves a novel Akt/PKB-independent signal crosstalk between insulin and canonical Wnt signaling pathways. We then identified p21-activated protein kianse 1 (PAK-1) as a novel mediator for insulin and Wnt/beta-catenin (b-cat) molecular crosstalk, involving MEK/ERK signaling. Furthermore, we found that insulin treatment leads to increased b-cat phosphorylation at Ser675, and this is associated with increased b-cat nuclear content and increased b-cat interaction with Tcf/Lef-binding elements (TBEs) of the human c-Myc gene promoter. Lastly, we demonstrated that insulin signaling directly alters the expression levels of components of the Wnt signaling pathway, including fizzled homology 4 (Fdz-4) and TCF7L2 (=TCF-4). This study not only demonstrated the existence of signaling crosstalk between insulin and canonical Wnt signaling pathways at multiple levels, it reveals molecular mechanisms for observed correlation between CRC and hyperinsulinemia. The growing evidence implicating PAK-1 in various human tumorigenesis has emerged PAK-1 as a potential therapeutic target. Our discovery of PAK-1 functioning as a novel central mediator for insulin and Wnt signaling crosstalk in intestinal cells suggests that PAK-1 may potentially be a good target candidate for treating patients with CRC, especially those who have Type II diabetes or experience hyperinsulinemia.

Colorectal Cancer is the main type of cancer in Spain. Up to 90% of the cases are sporadic in nature and its aetiology is still unclear. It is supposed to be a multi-factorial disease, where factors play an important role in the tumor onset and development, like microbiota. The main goal of this study was to describe and quantify the bacterial community of the intestinal mucosa associated to colorectal cancer patients. This work has revealed the existence of a bacterial dysbiosis in colorectal cancer patients, which is in agreement with previous research. Specific phylotypes previously descrived using stool samples and also new phylotypes were associated with this disease.Furthermore, streptococcal populations have been studied and also a case report from a patient who present an infection caused by E. faecalis at the same time of CRC diagnosed. Future research should focus on specific aspects of intestinal microbiota such as its interaction with the host, together with the mechanisms by which bacteria can affect on the onset of tumor in the colon. / El càncer colorectal és el tipus de càncer més abundant a Espanya. Fins el 90% dels casos són d'origen espontani i la seva etiologia és desconeguda malgrat existeixen diversos factors que poden afectar en el desenvolupament tumoral, com la microbiota. L'objectiu d'aquesta tesi ha estat analitzar la composició de la comunitat microbiana en mostres de mucosa intestinal quantitativa i qualitativament. Els resultats mostren una disbiosi en els malalts de càncer colorectal i una associació amb l'augment o disminució d'espècies bacterianes, així com l'augment de determinats filotips/gèneres. També s'ha analitzat les poblacions d'estreptococs i l'exposició d'un cas clínic d'un pacient amb càncer colorectal amb una infecció causada per E. faecalis. Estudis focalitzats en aspectes més específics de la relació hoste-microbiota, així com explorar nous mecanismes induïts per bacteris són necessaris per comprendre alguns aspectes de la carcinogènesis colorectal.