Obesity increases the risk of colon cancer as well as the expression of many cancer markers, ostensibly due to the interaction between insulin resistance and adipocyte production of hormones, mitogens and cytokines which collaborate to enhance proliferation signaling and impair the DNA damage response. Cdc7 and PCNA are both proteins involved in the DNA damage response as well as DNA replication. Both have also been shown to be upregulated in human tumours. To assess Cdc7 and PCNA roles during the DNA damage response in obese and lean animals, we administered azoxymethane (AOM), a colon-specific carcinogen, to obese and lean rats. Cdc7 and PCNA levels in colonic mucosal protein extracts from obese Zucker rats were compared with those from their lean counterparts. Significant differences were seen between lean and obese animals 3 hours post-AOM (lean Cdc7 levels > obese Cdc7 levels) and 24 hours post-AOM (lean PCNA levels > obese PCNA levels). This result suggests an impaired checkpoint response in obese animals relative to lean animals and supports a previously reported early role for Cdc7 in the checkpoint signaling cascade relative to a later role of PCNA in DNA damage repair. At the time tumours appeared (32 weeks post-AOM), colonic mucosal Cdc7 levels of obese rats exceeded that of their lean counterparts, suggesting that the obese metabolic environment causes upregulation of Cdc7 in obese rat epithelia. Cdc7 and PCNA levels were then compared between tumours and mucosa in obese and Sprague Dawley rats. Tumour Cdc7 levels were upregulated relative to mucosal levels in more samples than tumour PCNA levels, suggesting Cdc7 may be a more sensitive tumour marker. No significant differences in Cdc7 levels were seen between obese tumours and mucosa, likely due to elevation of obese mucosal Cdc7 levels. However, Sprague Dawley (non-obese) rats showed significantly higher Cdc7 and PCNA levels in tumours than mucosa, consistent with previous studies in human tissues. These results suggest that Cdc7 may be a more sensitive tumour marker than PCNA, but that its utility as a biomarker of colon cancer is dependent on the metabolic state (leanness) of the individual.

No description available.

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.

Lynch syndrome (LS) is the most prevalent cause of hereditary colorectal cancer (CRC) and confers high risks for several other types of cancer. Universal tumor screening (UTS) of all newly diagnosed patients with CRC can improve LS identification and decrease associated morbidity and mortality among patients and family members. However, for UTS to be effective, patients who screen positive must pursue genetic counseling and confirmatory germline testing (i.e., high patient reach). The purposes of this study were to characterize UTS programs, identify barriers and facilitators to implementation, document whether there have been negative outcomes, and determine institutional and implementation conditions that are associated with high and low patient reach. Using two conceptual frameworks, RE-AIM and Consolidated Framework for Implementation Research, a baseline survey was conducted of 25 representatives from different institutions performing UTS. Descriptive statistics were used to illustrate similarities and differences among programs. A multiple-case study was then conducted by extracting data from surveys and interviews of representatives from 15 different institutions where UTS programs had been operational for over 6 months and where aggregated patient outcome data were available. Qualitative comparative analysis was performed to make systematic cross-case comparisons and identify conditions uniquely associated with high or low patient reach. Data were triangulated to create models explaining how UTS implementation and system-level factors influence patient reach. Few patient concerns or negative outcomes were reported. UTS procedures and patient reach were highly variable. All 5 high-reach (H-R) centers have genetics professionals disclose positive screening results and either do not require a referral from another health care provider or have streamlined the referral process. Although 2 of the 5 mid-reach (M-R) centers also share these conditions, they have a less automated follow-up procedure and report difficulty contacting patients as a barrier. Both of the academic institutions with low patient reach (L-R) did not receive patient information that would allow them to follow-up on positive screening results. The three non-academic L-R institutions reported a high proportion of challenges to facilitators during implementation and did not have genetic professionals disclose positive screening results to patients. Implementing a combination of procedures to streamline UTS protocols and procedures, eliminate barriers to patient follow-through after a positive tumor screen, and incorporate a high level of involvement of genetic professionals in contacting patients and disclosing screening results are expected to lead to improvement in patient reach

Mutational analyses of cancer genomes have identified novel candidate cancer genes with hitherto unknown function in cancer. To enable phenotyping of mutations in such genes, we have developed a scalable technology for gene knock-in and knock-out in human somatic cells based on recombination-mediated construct generation and a computational tool to design gene targeting constructs. Using this technology, we have generated somatic cell knock-outs of the putative cancer genes ZBED6 and DIP2C in human colorectal cancer cells. In ZBED6-/- cells complete loss of functional ZBED6 was validated and loss of ZBED6 induced the expression of IGF2. Whole transcriptome and ChIP-seq analyses revealed relative enrichment of ZBED6 binding sites at upregulated genes as compared to downregulated genes. The functional annotation of differentially expressed genes revealed enrichment of genes related to cell cycle and cell proliferation and the transcriptional modulator ZBED6 affected the cell growth and cell cycle of human colorectal cancer cells. In DIP2C-/-cells, transcriptome sequencing revealed 780 differentially expressed genes as compared to their parental cells including the tumour suppressor gene CDKN2A. The DIP2C regulated genes belonged to several cancer related processes such as angiogenesis, cell structure and motility. The DIP2C-/-cells were enlarged and grew slower than their parental cells. To be able to directly compare the phenotypes of mutant KRAS and BRAF in colorectal cancers, we have introduced a KRASG13D allele in RKO BRAFV600E/-/-/ cells. The expression of the mutant KRAS allele was confirmed and anchorage independent growth was restored in KRASG13D cells. The differentially expressed genes both in BRAF and KRAS mutant cells included ERBB, TGFB and histone modification pathways. Together, the isogenic model systems presented here can provide insights to known and novel cancer pathways and can be used for drug discovery.

Η ανάγκη εξατομίκευσης της θεραπείας ασθενών που πάσχουν από καρκίνο κρίνεται επιτακτική, λόγω του στενού θεραπευτικού εύρους των αντινεοπλασματικών φαρμάκων και των παρατηρούμενων δια-ατομικών διαφορών στη φαρμακοκινητική και στην κλινική ανταπόκριση. Ένα πολύ χρήσιμο εργαλείο στην εξατομίκευση της θεραπείας θεωρείται ότι είναι η πληθυσμιακή φαρμακοκινητική-φαρμακοδυναμική μοντελοποίηση, καθώς μπορεί να περιγράψει τις σχέσεις δόσης-ανταπόκρισης, να εξηγήσει την παρατηρούμενη μεταβλητότητα στην έκθεση στο φάρμακο ή στην κλινική ανταπόκριση και να καθοδηγήσει την επιλογή της δόσης βάσει της βέλτιστης αναλογίας οφέλους-κινδύνου για τη δεδομένη θεραπεία. Η παρούσα διδακτορική διατριβή επικεντρώθηκε στην μπεβασιζουμάμπη, ένα σχετικά καινούριο φάρμακο στοχευμένης θεραπείας για το οποίο δεν υπάρχουν αρκετά διαθέσιμα στοιχεία που αφορούν στη φαρμακοκινητική και φαρμακοδυναμική συμπεριφορά του. Σκοπός της συγκεκριμένης μελέτης ήταν ο χαρακτηρισμός της αλληλεπίδρασης της μπεβασιζουμάμπης με τον μοριακό της στόχο, VEGF165, σε ενήλικες ασθενείς με μεταστατικό ορθοκολικό καρκίνο που λαμβάνουν το φάρμακο σε συνδυασμό με χημειοθεραπεία (FOLFIRI, FOLFOX ή CAPIRI). Για αυτόν τον λόγο, προσδιορίστηκαν αρχικά οι συγκεντρώσεις της ολικής μπεβασιζουμάμπης και του ελεύθερου VEGF165 σε διάφορους κύκλους θεραπείας και στη συνέχεια, εφαρμόζοντας τη μη γραμμική μικτών επιδράσεων μοντελοποίηση με το υπολογιστικό πρόγραμμα NONMEM 7.3, αναπτύχθηκε ένα φαρμακοκινητικό μοντέλο σύνδεσης της μπεβασιζουμάμπης με τον VEGF165. Επιπλέον, διερευνήθηκε η επίδραση των δημογραφικών δεδομένων και των VEGF μονονουκλεοτιδικών πολυμορφισμών στην αλληλεπίδραση μεταξύ της φαρμακοκινητικής της μπεβασιζουμάμπης και των συγκεντρώσεων του VEGF165. Είναι η πρώτη φορά που χρησιμοποιήθηκε η TMDD προσέγγιση για τον χαρακτηρισμό της in vivo αλληλεπίδρασης μπεβασιζουμάμπης-VEGF165. Σύμφωνα με αυτή την προσέγγιση, η φαρμακοκινητική ενός φαρμάκου επηρεάζεται από την υψηλής συγγένειας δέσμευση με τον μοριακό του στόχο και την επακόλουθη αποικοδόμηση του σχηματιζόμενου συμπλόκου μέσω ενδοκυττάρωσης. Το αναπτυχθέν μοντέλο επέτρεψε την ικανοποιητική περιγραφή της φαρμακοκινητικής της μπεβασιζουμάμπης και των ιδιοτήτων σύνδεσής της με τον VEGF165. Η κάθαρση της μπεβασιζουμάμπης βρέθηκε να είναι 0.18 L/day, η τιμή αναφοράς της συγκέντρωσης του ελεύθερου VEGF165 ήταν 212 ng/L, η σταθερά του ρυθμού απομάκρυνσης του ελεύθερου VEGF165 ήταν 0.401 day-1 και η Kss ήταν 267 nM. Η επίδραση του πραγματικού σωματικού βάρους συνυπολογίστηκε στην εκτίμηση όλων των φαρμακοκινητικών παραμέτρων του μοντέλου. Κάποιες στατιστικά μη σημαντικές συσχετίσεις παρατηρήθηκαν μεταξύ της συγγένειας δέσμευσης του φαρμάκου και των VEGF-2578C/A και VEGF-634G/C πολυμορφισμών. Το αναπτυχθέν μοντέλο θα μπορούσε να αποτελέσει ένα χρήσιμο εργαλείο στην εξατομίκευση της θεραπείας και στην αξιολόγηση της κλινικής ανταπόκρισης ασθενών που λαμβάνουν μπεβασιζουμάμπη σε συνδυασμό με χημειοθεραπεία. / The need for individualized treatment in cancer patients is considered crucial due to the narrow therapeutic range of antineoplastic drugs and the observed inter-individual differences in pharmacokinetics and clinical response. Population pharmacokinetic-pharmacodynamic modeling has been recognized as a beneficial tool for personalizing treatment, as it can describe the dose-response relationships, explain the observed variability in drug exposure or response and guide dose selection based on the optimal benefit-risk ratio for a given treatment. The current doctoral thesis was focused on bevacizumab, a relatively new targeted therapy drug for which no sufficient data are available regarding its pharmacokinetic and pharmacodynamic behavior. The aim of the present study was to characterize the interaction of bevacizumab with its molecular target, VEGF165, in adult patients with metastatic colorectal cancer who receive the drug in combination with chemotherapy (FOLFIRI, FOLFOX or CAPIRI). For this reason, the concentrations of total bevacizumab and free VEGF165 were first determined in different cycles of treatment and then, a pharmacokinetic model for bevacizumab binding to VEGF165 was developed by using nonlinear mixed-effects modeling implemented in NONMEM 7.3 software. Moreover, the effect of demographic data and VEGF single nucleotide polymorphisms on the interplay between bevacizumab pharmacokinetics and VEGF165 concen-trations was investigated. This is the first time the TMDD approach was applied to characterize the in vivo bevacizumab-VEGF165 interaction. According to this approach, the pharmacokinetics of a drug is affected by its high affinity binding to its molecular target and subsequent degradation of the formed complex via endocytosis. The developed model allowed an adequate description of bevacizumab pharmacokinetics and its binding properties to VEGF165. Bevacizumab clearance was found to be 0.18 L/day, the free VEGF165 concentration at baseline was 212 ng/L, the elimination rate constant of free VEGF165 was 0.401 day-1, and Kss was 267 nM. The effect of actual body weight was taken into account in the estimation of all pharmacokinetic model parameters. Correlations, which were not statistically significant, were noticed between the binding affinity of the drug and the VEGF-2578C/A and VEGF-634G/C polymorphisms. The developed model could become a useful tool for individualizing treatment and evaluating clinical response of patients receiving bevacizumab in combination with chemotherapy.

Putrescine, spermidine and spermine are the polyamines biosynthesized by human cells via ornithine decarboxylase (ODC) and are also sourced from the diet. Polyamines are required for malignant and normal cell growth and development. Pharmacological suppression of polyamine biosynthesis, by difluoromethylornithine, and inflammation, via sulindac, has demonstrated ~70% efficacy in preventing premalignant colorectal adenomas (CRA) in a clinical trial; however, high polyamine intakes mitigated this preventative action. Further, dietary polyamines increase the dysplasia of CRA in initiated animal models of colorectal cancer (CRC) and are hypothesized to function as tumor promoters. Human research on dietary polyamines was limited until the development of a dietary database in 2007 but, continues to be limited by the lack of a biomarker of exposure. Chapter 1 of this dissertation tests the hypothesis that dietary polyamines increase risk of CRA in polyp-formers (n = 1164) and found evidence to support this hypothesis. However, only women, younger participants and certain genotypes experienced more risk of CRA with high polyamine exposure. Chapter II tests the hypothesis that dietary polyamines increase the risk for CRC in an average risk cohort of post-menopausal women (n = 87,620) and did not find evidence to support this hypothesis in the whole population. Rather, dietary polyamines were non-significantly protective against CRC and significantly protective when paired with aspirin use and against CRC-specific death. There was some evidence to support an increase in risk of CRC in younger participants with high polyamine exposure. Overall, the first two chapters suggest that dietary polyamines protect the colorectum in normal risk individuals but promote carcinogenesis in high risk individuals. Chapter III tests the hypothesis that dietary polyamine intake correlates with urinary polyamine output in a group of overweight/obese, older men (n = 36) and Chapter IV tests the hypothesis that intake of highly ripe sweet cherries will increase urinary polyamine output in a subgroup of 10 men from Chapter III. The findings from these chapters suggest there may be a positive correlation, but that a better measure of dietary polyamine intake is needed to determine if urinary polyamines are biomarkers of exposure to polyamines.

Colorectal cancer (CRC) is the third most common cancer in the United States (U.S.) and is the second leading cause of cancer deaths. Although the incidence of CRC has been decreasing with CRC screenings, disparities of CRC and screening prevalence exist for racial and ethnic groups. The CRC incidence rates have dramatically increased in Korean Americans, however, there is little known about their CRC screening behaviors and the factors that may predict screening behaviors have not been fully investigated. The purposes of this study were to describe CRC screening behaviors and identify the predictors and barriers influencing CRC screening behaviors among Korean Americans. A sample of 254 Korean Americans participated in this study. Correlation, Multiple logistic regression, and Chi-square were used to analyze data. In this study, Korean American had lower rates of CRC screenings compared to the general U.S. population. Only 20% of the sample had ever had a fecal occult blood Test (FOBT), 49% had ever had a colonoscopy, and 19% responded they had ever had a sigmoidoscopy in their lifetime. Korean Americans had low rates of perception of cancer screening (annual physical exam and periodic cancer screening), moderate CRC knowledge, low cancer fatalism, limited CRC literacy, lack of health care access, and a low rate of receiving the physician's recommendation of CRC screenings. The greatest predictors influencing CRC screening were perception of cancer screening for a FOBT, and the physician's recommendation for a colonoscopy and a sigmoidoscopy. There were no significant differences by gender in CRC screening behaviors. However, significant differences were found between the two groups divided by length of U.S. residence. Compared to those who have lived in the U.S.>10 years, new immigrants had lower rates of all three CRC screening, lower perception of cancer screening, higher uninsured, less receiving physician's recommendation, and higher perceived barriers to CRC screening. The findings of this study suggest that improved efforts are needed to increase CRC screenings among Korean Americans. Further research is needed to increase a physician's recommendation for CRC screenings and awareness for the importance of annual checkups and periodic cancer screening among Korean Americans.

Dysregulation of cellular metabolism is associated with multiple diseases including cancer. Polyamines are organic cations shown to control gene expression at the transcriptional, post-transcriptional, and translational level. The activity of ornithine decarboxylase (ODC), the first enzyme in polyamine synthesis, is associated with normal and neoplastic growth. A single nucleotide polymorphism (SNP, rs2302615, SNP +316 nucleotides 3' of the transcriptional start site) in the ODC1 gene has been found to be both functional and prognostic for risk of colorectal carcinogenesis. A comprehensive investigation of genetic variability in ODC1 gene was performed. We confirmed frequencies of 12 SNPs occurring in participants of a clinical cancer prevention trial. We identified haplotypes accounting for over 90% of the genetic diversity in the ODC1 gene. Mechanistically, we addressed two of them, which account for more than half of the participants in the clinical trial. Two ODC1 intron 1 SNPs, rs2302616 (SNP +263 nucleotides 3' of the transcriptional start site) and rs2302615, were found to be associated with disease processes. Both of them predicted metachronous adenoma and response to agents targeting the polyamine pathway in participants of the clinical trial. The rs2302616 functionally modulate a DNA G-quadruplex structure and predicted the ODC1 rate-limiting product putrescine by genotype. Both SNPs cooperate to modulate ODC1 transcriptional activity involving both a G-quadruplex structure and Sp1 binding site at rs2302616, and rs2302615 flanked MYC-binding E-boxes. Haplotype analysis, using both these SNPs, might provide better discrimination of both disease prognosis and treatment prediction in cancer chemoprevention clinical trials.

Colorectal cancer, among other tumors, is characterized by elevated levels of prostaglandins due to the up-regulation of cyclooxygenase -2 (COX-2), a key enzyme in the eicosanoid biosynthesis pathway. Prostaglandin E2 (PGE2) is an important prostaglandin that exerts its biological function via four transmembrane G protein coupled receptors (EP1-4), among which the EP4 receptor is the most important. The relevance of EP4 receptor to the carcinogenic process and the consequences of its interaction with PGE2 were explored in this dissertation.Despite the importance of the EP4 receptor in colon carcinogenesis, studies looking at the receptor expression during cancer progression have not been extensive. One study showed that the protein levels of EP4 receptor were elevated in colon cancer whereas another study indicated that mRNA levels were decreased in tumor compared to normal. We expanded these observations and now report that the elevated protein levels of EP4 receptor in cancer are due to increased translation of proteins.In addition, we identified S100P as a novel downstream target of the PGE2/EP4 receptor signaling pathway. S100P has been previously implicated in a number of gastro-intestinal cancers such as pancreatic, gastric and colon cancers. However, its regulation via the PGE2/EP4 receptor signaling pathway has never been investigated. Here, we show that PGE2 via the EP4 receptor signaling leads to the transcriptional activation of S100P and that this activation happens exclusively in the presence of CREB. In summary, this dissertation brings to light novel therapeutic targets which could be used as potential markers to stratify colon cancer patients as well as avenues for clinical intervention for the management of colon carcinogenesis.