Thesis (M.S.H.P.) PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / BACKGROUND: Effective colorectal cancer prevention programs should ensure that patients with adenomas receive timely surveillance colonoscopy consistent with guidelines. The aim of our study was to identify patient-, provider-, and system-level predictors of adherence with surveillance colonoscopy in an ethnically diverse safety-net population. METHODS: We performed a retrospective analysis of average-risk patients age 50-75 with adenomas diagnosed at screening colonoscopy at Boston Medical Center and its affiliated community health centers from 1/1/2005 to 12/31/2007. The primary outcome was on-time follow-up defined as attendance at surveillance colonoscopy within 5.5 years of the screening colonoscopy. We compared frequencies of on-time follow-up and performed multivariable analysis, including ethnicity/language and any variable with P value <0.20 in unadjusted analysis. RESULTS: We identified 891 patients, of which 38% were English-speaking Non-Hispanic blacks, 24% Non-Hispanic white, and 10% Spanish-speaking Hispanics. Overall, 38.2% attended on-time surveillance colonoscopy. In unadjusted analyses, having ≥3 PCP visits in year 3-5 after baseline colonoscopy (OR 3.6 [2.6-5.1]), having “adenoma” on the electronic medical record problem list (OR 2.2 [1.6-2.9]), age (OR 0.98 [0.96-1.0]), and Charlson Index ≥1 (OR 1.3 [1.0-1.8]) were positively associated with adherence. In multivariate analysis, having “adenoma” on the problem list remained significant (adjusted OR (aOR) 1.8 [1.3-2.5]). Significant interactions were observed for ethnicity/language and PCP visits (P=0.008). CONCLUSION: A substantial proportion of adenoma-bearing patients fail to attend surveillance colonoscopy even in a safety net setting. Adding “adenomas” to the EMR problem list improved attendance, suggesting that system-level interventions may increase adherence to surveillance colonoscopy. / 2999-01-01

Yes / This report presents a new library of organometallic iridium(III) compounds of the type [Cp*IrCl(L)] (Cp*=pentamethylcyclopentadienyl and L=a functionalized β‐ketoiminato ligand) showing moderate to high cytotoxicity against a range of cancer cell lines. All compounds show increased activity towards colorectal cancer, with preferential activity observed against the immortalized p53‐null colorectal cell line, HCT116 p53‐/‐, with sensitivity factors (SF) up to 26.7. Additionally, the compounds have excellent selectivity for cancerous cells when tested against normal cell types, with selectivity ratios (SR) up to 35.6, contrary to that of cisplatin, which is neither selective nor specific for cancerous cells (SF=0.43 and SR=0.7–2.3). This work provides a preliminary understanding of the cytotoxicity of iridium compounds in the absence of p53 and has potential applications in treatment of cancers for which the p53 gene is absent or mutant.

Yes / Most cancer patients receive chemotherapy at some stage of their treatment which makes improving the efficacy of cytotoxic drugs an ongoing and important goal. Despite large numbers of potent anti-cancer agents being developed, a major obstacle to clinical translation remains the inability to deliver therapeutic doses to a tumor without causing intolerable side effects. To address this problem, there has been intense interest in nanoformulations and targeted delivery to improve cancer outcomes. The aim of this work was to demonstrate how vascular endothelial growth factor receptor 2 (VEGFR2)-targeted, ultrasound-triggered delivery with therapeutic microbubbles (thMBs) could improve the therapeutic range of cytotoxic drugs. Methods: Using a microfluidic microbubble production platform, we generated thMBs comprising VEGFR2-targeted microbubbles with attached liposomal payloads for localised ultrasound-triggered delivery of irinotecan and SN38 in mouse models of colorectal cancer. Intravenous injection into tumor-bearing mice was used to examine targeting efficiency and tumor pharmacodynamics. High-frequency ultrasound and bioluminescent imaging were used to visualise microbubbles in real-time. Tandem mass spectrometry (LC-MS/MS) was used to quantitate intratumoral drug delivery and tissue biodistribution. Finally, 89Zr PET radiotracing was used to compare biodistribution and tumor accumulation of ultrasound-triggered SN38 thMBs with VEGFR2 targeted SN38 liposomes alone. Results: ThMBs specifically bound VEGFR2 in vitro and significantly improved tumor responses to low dose irinotecan and SN38 in human colorectal cancer xenografts. An ultrasound trigger was essential to achieve the selective effects of thMBs as without it, thMBs failed to extend intratumoral drug delivery or demonstrate enhanced tumor responses. Sensitive LC-MS/MS quantification of drugs and their metabolites demonstrated that thMBs extended drug exposure in tumors but limited exposure in healthy tissues, not exposed to ultrasound, by persistent encapsulation of drug prior to elimination. 89Zr PET radiotracing showed that the percentage injected dose in tumors achieved with thMBs was twice that of VEGFR2-targeted SN38 liposomes alone. Conclusions: thMBs provide a generic platform for the targeted, ultrasound-triggered delivery of cytotoxic drugs by enhancing tumor responses to low dose drug delivery via combined effects on circulation, tumor drug accumulation and exposure and altered metabolism in normal tissues. / EPSRC funding (EP/I000623/1, EP/K023845/1 and EP/P023266/1) and the MRC for a Confidence in Concept award and MR/L01629X. L.E. McVeigh was funded by an EPSRC PhD Studentship (EP/L504993/1).

Yes / Urinary prostaglandin (PG) E metabolite (PGE-M) and 11-dehydro (d)-thromboxane (TX) B2 are biomarkers of cyclooxygenase-dependent prostanoid synthesis. We investigated (1) the effect of aspirin 300 mg daily and eicosapentaenoic acid (EPA) 2000 mg daily, alone and in combination, on urinary biomarker levels and, (2) whether urinary biomarker levels predicted colorectal polyp risk, during participation in the seAFOod polyp prevention trial. Urinary PGE-M and 11-d-TXB2 were measured by liquid chromatography-tandem mass spectrometry. The relationship between urinary biomarker levels and colorectal polyp outcomes was investigated using negative binomial (polyp number) and logistic (% with one or more polyps) regression models. Despite wide temporal variability in PGE-M and 11-d-TXB2 levels within individuals, both aspirin and, to a lesser extent, EPA decreased levels of both biomarkers (74% [P ≤ .001] and 8% [P ≤ .05] reduction in median 11-d-TXB2 values, respectively). In the placebo group, a high (quartile [Q] 2-4) baseline 11-d-TXB2 level predicted increased polyp number (incidence rate ratio [IRR] [95% CI] 2.26 [1.11,4.58]) and risk (odds ratio [95% CI] 3.56 [1.09,11.63]). A low (Q1) on-treatment 11-d-TXB2 level predicted reduced colorectal polyp number compared to placebo (IRR 0.34 [0.12,0.93] for combination aspirin and EPA treatment) compared to high on-treatment 11-d-TXB2 values (0.61 [0.34,1.11]). Aspirin and EPA both inhibit PGE-M and 11-d-TXB2 synthesis in keeping with shared in vivo cyclooxygenase inhibition. Colorectal polyp risk and treatment response prediction by 11-d-TXB2 is consistent with a role for platelet activation during early colorectal carcinogenesis. The use of urinary 11-d-TXB2 measurement for a precision approach to colorectal cancer risk prediction and chemoprevention requires prospective evaluation. / Efficacy and Mechanism Evaluation Programme. Grant Number: NIHR128210. Cancer Research UK. Grant Number: C23434/A24939

Yes / Photodynamic Therapy (PDT) is an emerging method to treat colorectal cancers (CRC). Hypericin (HYP) is an effective mediator of PDT and the ABCG2 inhibitor, Febuxostat (FBX) could augment PDT. HT29 and HEK293 cells showed light dependant cytotoxic response to PDT in both 2D and 3D cell models. FBX co-treatment was not found to improve PDT cytotoxicity. Next, ABCG2 protein expression was observed in HT29 but not in HEK293 cells. However, ABCG2 gene expression analysis did not support protein expression results as ABCG2 gene expression results were found to be higher in HEK293 cells. Although HYP treatment was found to significantly reduce ABCG2 gene expression levels in both cell lines, FBX treatment partially restored ABCG2 gene expression. Our findings indicate that FBX co-treatment may not be suitable for augmenting HYP-mediated PDT in CRC but could potentially be useful for other applications. / Royal Society International Exchanges Award (IEC\R3\203014) - UKRI EPSRC Research Programme Grant (753910/B19R13527) - Bowel Cancer UK/RCS Eng Colorectal Research Chair Award (18SC0001) / The full-text of this article will be released for public view at the end of the publisher embargo on 16 Apr 2025.

UGT1A1 est responsable de l’inactivation du SN-38, métabolite actif de l’irinotécan utilisé en première intention dans le traitement du cancer colorectal métastatique. Certains facteurs génétiques du gène UGT1A1 sont en partie responsables des variations dans la réponse à cette thérapie. Parallèlement, nous avons émis l’hypothèse qu’un mécanisme épigénétique module les niveaux intratumoraux d'UGT1A1 ainsi que les taux d’inactivation du SN-38 à son site d’action. Nos données démontrent que la méthylation d’îlots CpG spécifiques dans le promoteur et l’exon 1 d’UGT1A1 est associée à la répression de son expression, à la diminution du taux de protéine et d’inactivation du SN-38 dans les cellules tumorales de côlon in vitro. De plus, l’hyperméthylation de ces régions corrèle avec la faible expression du gène dans les tumeurs primaires du côlon de patients. Ces données soulèvent la possibilité que le profil de méthylation du gène UGT1A1 pourrait déterminer la concentration tumorale du métabolite actif et ainsi aider à prédire l’efficacité de la thérapie. / UGT1A1 is the main enzyme involved in the hepatic and tumoral inactivation of SN-38, an anticancer agent used in first line treatment of metastasic colorectal cancer. UGT1A1 genetic factors determine response to irinotecan therapy. We hypothesised that an epigenetic mechanism, more specifically methylation, is involved in tumoral regulation of UGT1A1 levels. Specific CpG islands in the UGT1A1 gene are hypermethylated and linked to the repression of gene expression and to lower levels of SN-38 glucuronidation in colon tumor cells in vitro. In addition, methylation of specific CpG was linked to lower expression of UGT1A1 gene in primary colon tumors from patients. Our data support that methylation profile of the UGT1A1 gene determine SN-38 tumoral concentration and may help to predict tumoral response to irinotecan.

SOX9 est un facteur de transcription à domaine HMG. Il est impliqué dans de multiples processus biologiques au cours du développement et de la vie adulte. En particulier, SOX9 joue un rôle important dans l'homéostasie de l'épithélium intestinal. Nous avons montré que SOX9, cible positive de la voie de signalisation oncogénique Wnt/(beta)-caténine, réprime l'expression de PKC(alpha). Cette répression implique un nouveau mécanisme d'action qui ne nécessite ni la fixation du domaine HMG à l'ADN ni le domaine de transactivation de SOX9. Nous avons également identifié MiniSOX9, un nouveau variant d'épissage de SOX9, résultant de la rétention du second intron. MiniSOX9 est fortement exprimé dans les tumeurs coliques. Il agit en tant que dominant négatif de SOX9, inhibiteur de l'expression du suppresseur de tumeurs PKC(alpha) et activateur de la voie de signalisation oncogénique Wnt/(beta)-caténine. Nos données suggèrent ainsi un rôle primordial de MiniSOX9 dans la tumorigenèse intestinale. Enfin, notre étude protéomique des partenaires de SOX9 et de MiniSOX9 permet d'ouvrir de nouvelles perspectives quant aux rôles de ces deux protéines dans l'homéostasie et la tumorigenèse intestinale / SOX9 is an HMG transcription factor involved in numerous biological processes during development and adult life. It plays an important role especially in the intestinal epithelium homeostasis. In the present study, we demonstrate that SOX9, a positive target of the oncogenic signaling pathway Wnt/(beta)-catenin, represses PKC(alpha) expression. This repression involves a new mechanism of action requiring neither HMG domain binding to DNA nor the transactivation domain of SOX9. We also report the discovery of MiniSOX9, a new SOX9 splice variant, resulting from the second intron retention. MiniSOX9 is highly expressed in colon tumors. It acts as a SOX9 dominant negative, as a repressor of the expression of the tumor suppressor PKC(alpha), and as an activator of the oncogenic signaling pathway Wnt/(beta)-catenin. Our data suggest a crucial role of MiniSOX9 in intestinal tumorigenesis. Finally, a proteomic analysis allowed us to identify potential new SOX9 and MiniSOX9 partners which will be useful to decipher the roles of these two proteins in intestinal homeostasis and tumorigenesis

L’ilot génomique pks de Escherichia coli et d’autres Enterobacteriaceae code des synthases depolycétides et de peptides non ribosomaux qui permettent l’assemblage d’un composé hybride polycétidepeptidenon ribosomal putative. Ce composé nommé colibactine induit des cassures double-brin de l’ADN descellules eucaryotes.La machinerie enzymatique codée par l’ilot pks comporte une protéine essentielle ClbP, atypique dansce type d’ilot. Nous avons montré que ClbP possède une partie N-terminale catalytique et périplasmique, et unepartie C-terminale associée à la membrane cytoplasmique. La structure cristalline de ClbP et des expériences demutagenèse ont révélé un site actif à sérine et des caractéristiques structurales originales, qui sont compatiblesavec une activité peptidase, confirmée par des analyses biochimiques. Dix homologues de ClbP ont été identifiésin silico dans des ilots génomiques de synthases de peptides non ribosomaux d’espèces bactériennes proches etéloignées. Tous les homologues testés ont présenté une promiscuité fonctionnelle avec ClbP. ClbP est donc leprototype d’une nouvelle sous-famille de peptidases, qui sont probablement impliquées dans la maturation decomposés peptidiques non ribosomaux.Par ailleurs, nous avons réalisé deux études épidémiologiques sur la prévalence de l’ilot pks dansl’espèce E. coli dans deux contextes physiopathologiques, l’urosepsis et les cancers coliques et rectaux. L’ilotpks était significativement associé aux souches issues d’urosepsis comparé à des souches commensales, et auxsouches issues de biopsies de tumeurs coliques comparé à des souches commensales ou issues de biopsies detumeurs rectales, de diverticuloses et de lésions iléales de maladie de Crohn. / The pks genomic island of Escherichia coli and other Enterobacteriaceae encodes polyketide andnonribosomal peptide synthases that build a putative hybrid PK-NRP compound. This compound designatedColibactin induces DNA double-strand breaks in eukaryotic cells.The pks-encoded enzymatic machinery comprises an essential protein ClbP, atypical for this type ofgenomic islands. We report that ClbP harbors a catalytic and periplasmic N-terminal part, and a C-terminal partassociated to the cytoplasmic membrane. ClbP crystal structure and mutagenesis experiments revealed a serineactivesite and original structural features, which are compatible with peptidase activity confirmed bybiochemical assays. Ten ClbP homologs were identified in silico in NRPS-encoding genomic islands of closeand distant-related bacterial species. All tested ClbP homologs showed functional promiscuity with ClbP. ClbPis therefore a prototype of a new subfamily of peptidases, which are probably involved for the maturation ofNRP compounds.Furthermore, we undertook two epidemiological studies on the prevalence of pks island in E. coli in twopathophysiology contexts; urosepsis and colorectal cancers. The pks island was significantly associated withurosepsis strains compared to commensal strains, and strains isolated from biopsies of colon tumors comparedwith commensal strains or strains isolated from biopsies of rectal tumors, diverticulosis and ileal lesions ofCrohn disease.

Durant l’exécution de l’apoptose, plus de 2 000 protéines sont protéolysées par les caspases, une famille de protéases à cystéine. Le clivage de plusieurs d’entre elles a pour effet d’interrompre les processus régulant le trafic intracellulaire. Durant mes études, je me suis intéressée à deux substrats potentiels des caspases, soit les sorting nexins SNX1 et SNX2. Leur clivage en N-terminal avait auparavant été identifié par protéomie dans des extraits cellulaires apoptotiques, respectivement aux sites LFAD91[flèche vers le bas]A et VSLD84[flèche vers le bas]S. Conjointement avec le complexe rétromère, SNX1 et SNX2 jouent un rôle essentiel dans le transport rétrograde de cargos, tel le récepteur lysosomal CI-MPR, des endosomes vers le TGN, évitant ainsi leur dégradation aux lysosomes. Notamment, l’association entre SNX1 et SNX2 et le complexe rétromère, via la sous-unité Vps35, requerrait leur domaine N-terminal, or, celui-ci est clivé durant l’apoptose. Dans le but de déterminer l’impact de la protéolyse de SNX1 et SNX2 sur la fonction du complexe rétromère et le transport rétrograde, nous avons étudié leur clivage par les caspases. Nos résultats indiquent qu’in vitro, les caspases initiatrices 8, 9 et 10 protéolysent SNX1 et SNX2 tandis que seule la caspase-6 exécutrice clive SNX2. Plusieurs fragments de SNX1 sont générés par le clivage à 16 sites, dont le site LFAD91[flèche vers le bas]A en N-terminal ainsi que plusieurs suivant un résidu glutamate. Durant l’apoptose, SNX2 est entre autres clivée par la caspase-6, et ce au site VSLD84[flèche vers le bas]S en N-terminal. Nous avons par la suite étudié l’effet de la protéolyse de SNX1 et SNX2 sur la fonction du complexe rétromère. Nos résultats démontrent que SNX2 tronquée, imitant le clivage au site VSLD84[flèche vers le bas]S, n’interagit plus avec Vps35, la sous-unité centrale du complexe rétromère. De plus, la déplétion de SNX1 et SNX2, récapitulant potentiellement leur protéolyse, a pour effet de délocaliser Vps26, une autre sous-unité du complexe rétromère. Par ailleurs, nous avons évalué l’effet de la protéolyse de SNX2 sur la régulation du récepteur Met, lequel serait régulé entre autres par SNX1 et SNX2. La déplétion de SNX2 induit une augmentation de la phosphorylation du récepteur Met et de ERK1/2 suivant sa stimulation. De plus, l’ARNm de SNX1 et SNX2 sont tous deux réduits dans les tissus de tumeurs de patients atteints du cancer colorectal (CCR) et une diminution des niveaux de SNX2 corrèle avec une hausse de mortalité chez ces patients. Pour conclure, notre étude démontre un effet direct de la protéolyse de SNX2 sur le complexe rétromère durant l’apoptose et suggère un lien entre SNX2 et la pathogenèse du CCR. / Abstract: During the execution of apoptosis, more than 2,000 proteins are proteolysed by caspases, a family of cysteine proteases. The cleavage of several of them results in the interruption of intracellular trafficking processes. During my studies, I investigated two potential caspases substrates, namely the sorting nexin SNX1 and SNX2. Their cleavage at their N-terminus has previously been identified in apoptotic cell lysates by proteomics, respectively at LFAD91[down arrow]A and VSLD84[down arrow]S sites. Together with the retromer complex, SNX1 and SNX2 play an essential role in the retrograde transport of cargos, such as the lysosomal receptor CI-MPR, from endosomes to TGN, thus avoiding their degradation by lysosomes. In particular, the association between SNX1 and SNX2 and the retromer complex, via the Vps35 subunit, seems to require their N-terminal domain, which is thought to be cleaved during apoptosis. To determine the impact of SNX1 and SNX2 proteolysis on the function of the retromer complex and retrograde transport, we have first studied their cleavage by caspases. Our results indicate that in vitro, initiator caspases 8, 9, and 10 proteolyze SNX1 and SNX2 while only executioner caspase-6 cleaves SNX2. Several fragments of SNX1 are generated by the cleavage of up to 16 sites, including at the N-terminus LFAD91[down arrow]A site and following glutamate residues. During apoptosis, SNX2 is directly cleaved by caspase-6 at the site VSLD84[down arrow]S in its N-terminus. We next investigated the effect of SNX1 and SNX2 proteolysis on the function of retromer complex. Our results demonstrate that truncated SNX2, mimicking cleavage at the VSLD84[down arrow]S site, no longer interacts with Vps35, the central subunit of retromer complex. Furthermore, depletion of SNX1 and SNX2, potentially recapitulating their proteolysis, redistributes Vps26, another retromer subunit. In addition, we evaluated the effect of SNX2 proteolysis on the regulation of Met receptor, which has been shown to be regulated by SNX1 and SNX2. SNX2 depletion induces an increase in Met and ERK1/2 phosphorylation after stimulation. In addition, both SNX1 and SNX2 mRNAs are reduced in tumor tissues of colorectal cancer patients and decreased expression levels of SNX2 correlates with increased mortality. In conclusion, our study demonstrates a direct effect of SNX2 proteolysis on retromer complex association during apoptosis and suggests a link between SNX2 and the pathogenesis of colorectal cancer.

La tyrosine kinase cytoplasmique Src est un régulateur clé de la signalisation induite par les facteurs de croissance et les intégrines. Src présente des propriétés oncogéniques lorsqu'elle est dérégulée, une situation fréquemment retrouvée dans les cancers colorectaux (CCR). Sous sa forme activée, Src participe à la croissance tumorale et à la formation de métastases. Cependant, les mécanismes de dérégulation impliqués sont mal connus. En effet, SRC est rarement muté dans ces cancers. Mon travail de thèse a mis en évidence un nouveau mécanisme de dérégulation de Src via l'inactivation de SLAP. SLAP est une protéine de signalisation de type adaptateur qui régule négativement la signalisation lymphocytaire. De part son association avec l'E3-ligase Cbl, il induit la dégradation de substrats importants de la tyrosine kinase Lck nécessaires à l'activation lymphocytaire. Je montre que SLAP est également exprimé dans le tissu épithélial colique et que son expression est fréquemment perdue au cours de la progression tumorale colorectale. SLAP inhibe la tumorigénicité et le potentiel métastasant des cellules de CCR. Sur le plan moléculaire, SLAP définit une boucle de rétrocontrôle d'une voie oncogénique Src/EphA2/Akt initiée par Src via la dégradation protéasome-dépendante du récepteur EphA2 impliquant l'ubiquitine E4-ligase UBE4A. Ces résultats révèlent un nouveau mécanisme d'induction oncogénique de Src, une fonction insoupçonnée de suppresseur de tumeurs de SLAP et montrent l'importance d'une E4-ligase et des protéines adaptatrices dans la régulation négative de la signalisation initiée par les tyrosine kinases dans la progression tumorale. / The cytoplasmic tyrosine kinase Src mediates intracellular signaling induced by growth factors and integrins. When deregulated, Src acquires oncogenic properties. Src deregulation largely occurs in the absence of mutation of the corresponding gene but the underlying molecular mechanisms involved in this process are still unclear. Here I uncovered a novel mechanism of Src oncogenic induction in colorectal cancer (CRC) via SLAP silencing. SLAP is an adaptor protein and signaling molecule that controls lymphocytes activation. By association with E3-ligase Cbl, SLAP induces proteasomal degradation of important components of T cell receptor signaling, which impedes lymphocytes activation. I show that SLAP is also expressed in the epithelial tissue of the colon, but its expression is frequently lost during tumorigenesis. I also show that SLAP controls tumorigenicity and invasiveness of CRC cells. At the molecular level, SLAP specifies a feedback loop of a Src/EphA2/Akt oncogenic signaling that is initiated by Src itself. Precisely, phosphorylation of EphA2 on Tyr594 by Src creates a binding site for SLAP-SH2 to elicit receptor degradation. This novel SLAP function is independent of Cbl but requires its interaction with the E4-ligase UBE4A. SLAP down-regulation observed in cancer cells dramatically increases EphA2 levels and amplifies a Src/EphA2/Akt signaling required for cell tumorigenicity. Thus, SLAP inactivation defines a novel mechanism of Src oncogenic induction in human cancer.