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Background: Behavioral interventions and evidence based guidelines along the cancer control continuum can reduce the burden of cancer. Objectives: This dissertation aims to increase our understanding of colorectal cancer screening (CRCS) decisional conflict and breast cancer survivorship care. This project: 1) assesses CRCS decisional conflict in a general population, 2) uses the Theory of Triadic Influence to model and evaluate direct and indirect associations between CRCS decisional conflict and colonoscopy adherence, 3) assesses post-treatment breast cancer care. Methods: Data from a questionnaire administered to randomly selected adults, 50-75 years, living in six MN communities (N=1,268) and the 2010 Behavioral Risk Factor Surveillance System (BRFSS) (N=1,024, women ages 27-99) were used. Multivariable logistic regression was used to identify characteristics associated with high CRCS decisional conflict; then structural equation modelling (SEM) was performed to assess direct and indirect associations of CRCS decisional conflict and colonoscopy adherence. Using BRFSS data, multivariable logistic regression was performed to assess the association between years since diagnosis and the type of clinician providing the majority of care for breast cancer survivors after treatment completion. Results: Greater colonoscopy barriers (OR=1.04; 95% CI: 1.02-1.05) and CRCS-specific confusion (OR=1.12; 95% CI: 1.10-1.15) as well as a healthcare provider not discussing CRCS options (OR=1.67; 95% CI: 1.18-2.37) were associated with increased odds of high CRCS decisional conflict. A similar relationship was found in the SEM analyses: both greater levels of perceived colonoscopy barriers and CRCS confusion were associated with higher decisional conflict (standardized total effects=0.42 and 0.39, respectively, p-values < 0.01). CRCS decisional conflict was associated with increased non-adherence to colonoscopy. This relationship was mediated by CRCS-specific self-efficacy and intention (standardized total effect=0.14, p-value <0.01). Among breast cancer survivors, women 0–1 and 2–3 years since diagnosis were 2.1-2.6 times more likely to have a cancer-related clinician providing the majority of care compared to women 6+ years since diagnosis (95% CIs: 1.0-4.3; 1.4-4.6). Conclusions: Decreasing colonoscopy barriers and CRCS-specific confusion could decrease CRCS decisional conflict and ultimately increase CRCS uptake. National policies to move breast cancer follow-up care to a primary care provider might be well-received by cancer survivors.

Introduction: Shared decision making (SDM) has been advocated as an optimal approach to medical decision-making. Yet, little is known about how patients perceive SDM and whether patient-defined SDM is associated with patient outcomes. Methods: This three-manuscript dissertation used a mixed-methods approach including a systematic literature review and both qualitative and quantitative research methods. The aims were to: (1) systematically review the patient outcomes studied in relation to SDM and identify under what measurement contexts SDM is associated with which types of patient outcomes; (2) use in-depth, qualitative interviews to develop a conceptual model of patient-defined SDM and compare this to recent decisions that patients labeled as shared; and (3) apply the model of patient-defined SDM to the context of colorectal cancer screening. Results: Study 1 found that 39 studies measured SDM and evaluated it with a patient outcome, and only 43% of patient outcomes assessed were significantly associated with SDM. Patient reports of SDM were most likely to be associated with outcomes. Study 2 found that patients’ conceptual definition of SDM included four components: exchange of information, active listening, patient-self advocacy, and a personalized physician recommendation. Patient descriptions of recent decisions labeled as shared ranged from very simple recommendations through complex interactions, with the only commonality among shared decisions being that the patient and physician ultimately agreed. Study 3 found that the most commonly observed component of patient-defined SDM was patient self-advocacy (76%) and least common was a personalized physician recommendation (23%). Only 9% visits contained all four patient-defined SDM components. In adjusted models, physician provision of information around the process and potential side effects of colorectal cancer screening was associated with an increase in screening. There were differences in screening rates by the patient’s initial verbal response to the physician recommendation with those who initially refused being least likely to be screened (40%) and patients who did not verbalize a response to the recommendation being most likely to be screened (70%). Discussion: Findings across the three studies highlight the complexity of studying and measuring SDM and emphasize the importance of the patient’s perspective on SDM.

Background: The current adherence to colorectal cancer screening (CRCS) guidelines is suboptimal. How neighborhood characteristics, e.g., racial residential segregation (RRS), area-level socioeconomic status (SES) and physician composition, affect CRCS adherence are not fully understood. We assessed associations between facility proximity to RRS areas, area-level SES, physician composition, and CRCS adherence. Methods: Data sources included 2013 Minnesota Community Measurement, 2009-2013 American Community Survey, 2012 U.S. and 2012-2013 Washington State Behavioral Risk Factor Surveillance System data, and 2013-2014 Area Health Resource File. Logistic regressions and weighted multilevel logistic regressions were used to assess the association between facility proximity to RRS areas and CRCS adherence, and association between area-level SES, physician composition and CRCS adherence, respectively. Results: Facility proximity to RRS areas was positively associated with low CRCS performance, e.g., facilities located < 2 miles away from Hispanic-segregated areas were 3 times more likely to have low CRCS performance than those at ≥5 miles away (odds ratio (OR): 2.83, 95% confidence interval (CI): 1.29, 6.24). Most area-level SES measures showed negative bivariate associations between deprivation and colonoscopy/overall adherence, and measures such as education had relatively strong associations, although few of fully-adjusted associations remained statistically significant. Further, a one-unit increase in the percentage of gastroenterologists among physicians was associated with 3% increase in the odds of colonoscopy (OR: 1.03, 95% CI: 1.01-1.04) and overall adherence (OR: 1.03, 95% CI: 1.01-1.04) in the rural-metropolitan areas. Conclusions: Developing culturally tailored CRCS programs, increasing percentage of gastroenterologists, and targeting deprived communities may improve CRCS adherence.

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.

This work will first introduce the problems related to the colorectal cancer - its epidemiology and screening possibilities. Next the main topic is addressed - i.e. to ascertain the influence of national screening programmes for colorectal cancer on really observed data of lethality and incidence of this disease. Group of so-called APC models was selected as a useful tool for this purpose. Applying these methods on data of The National Oncological Registry of the Czech Republic for the period between 1980 till 2009 this work aims to prove expected reducing effect of area-wide screening programme on incidence and lethality related to colorectal cancer. Using the AP model and data of previous period before the screening introduction (i.e. 1980-1999) the values of incidence and lethality were predicted for the period in question (i.e. 2000-2009). Mere comparison of this predicted values with really observed data showed that real lethality and incidence was significantly lower in both sexes as compared to the model without the screening intervention. Difference between predicted and real data corroborates positive influence of colorectal cancer screening.

DNA methylation is a major form of epigenetic modification and involves the addition of a methyl group covalently to the 5-position of the cytosine pyrimidine ring, mostly within the context of CpG dinucleotides in vertebrate somatic cells. Methylation of CpG dinucleotides at promoter regions is generally associated with transcriptional repression. In this context, the methyl-CpG binding proteins (MeCPs) that are capable of recognition of methylated CpG dinucleotides are proposed to play a central role in DNA methylation associated transcriptional repression. Methyl-CpG binding domain protein 4 (MBD4) is an MeCP that possesses a glycosylase domain at its C-terminal, which can excise and repair both G:T and G:U mutations derived from DNA deamination at CpG dinucleotides, in addition to its Nterminal MBD binding domain. MBD4 has been associated with a number of pathways including DNA repair, apoptosis, transcriptional repression, and possibly DNA demethylation processes. However, the precise contribution of MBD4 to these processes remains unclear. To explore the functional repertoire of MBD4 I decided to undertake multiple protein interaction studies to identify potential partner proteins. I performed yeast 2-hybrid screens with an 11.5 day mouse embryonic cDNA library and multiple mass spectrometry of immunoprecipitates of tagged versions of MBD4 that were over-expressed in human cell lines. I detected ~380 potential interacting candidates with these assays. A significant number of candidates were detected in both assay systems. Chosen candidates were further validated by reciprocal co-IP of expressed partners and by immunofluorescence (IF) microscopy to determine their potential co-localisation in mouse and human cell lines. Subsequently, I identified the intervening domain of MBD4 as a novel protein interaction region for tested candidates. My analysis suggests that MBD4 can have a role in regulation of post-replication methyl-error repair/methylation machinery through its direct interaction with DNMT1 (previously shown), UHRF1 (novel) and USP7 (novel), as well as possible cross-talk to histone modification and chromatin remodelling pathways, through partners such as PRMT5 and ACF1. Interestingly the transcription regulatory components KAP1 and CFP1 not only interact with but also dramatically influence the stability of exogenously expressed MBD4 in human cells. In general positive validation by IP and IF demonstrates the robustness of the initial screens, and implies that MBD4 may impact upon several transcriptional and epigenetic networks along with a number of nuclear pathways that include transcriptional repression, DNA repair and RNA processing. To test for transcriptional aberration in the absence of Mbd4 function I profiled two independent mouse cell lines that lack MBD4 activity using Illumina MouseWG-6 v2.0 Expression BeadChip arrays. A number of genes were identified that are significantly up- or down- regulated in both Mbd4-/- MEFs. This included mis-expression of insulin-like growth factor-binding proteins and two paternally imprinted genes Dio3 and H19. The cohort of genes that were mis-expressed in the Mbd4-/- MEFs overlap with genes that responsed to tamoxifen exposure in an ER-positive ZR-75-1 xenograft model. In response to this observation I identified a potential interaction between MBD4 and estrogen receptor α (ERα) by co-IP and IF co-localisation. This suggests that MBD4 might potentiate transcription of estrogen regulated genes via a direct interaction with ERα, supporting a possible link between replication repair remodelling and steroid/thyroid hormone receptor transcriptional regulation. Additionally I performed a pathway analysis by which several developmental genes including Sox9, Klf2 and Klf4, were prioritised as possible MBD4 targets. On this basis I propose a role for MBD4 in acquired diseases such as cancers and autoimmune diseases via transcriptional regulation. I also performed a comparison of MBD4 DNA binding activity with MBD4 homologues from the Medaka fish (Oryzias latipes) and the amphibian, Xenopus laevis. I could show that DNA binding specificity to a series of methylated and mismatched probes is conserved regardless of the poor sequence conservation of the MBD domain of MBD4 between the species. I conclude that MBD4 is integrated in multiple pathways in the nucleus that includes DNA repair, chromatin remodelling, transcriptional regulation and genome stability.

Le cancer colorectal (CCR) demeure l'une des principales causes de décès par cancer dans le monde. Récemment, une nouvelle immunothérapie basée sur le ciblage du récepteur à activité tyrosine kinase (RTK) EGFR via l'anticorps cetuximab a montré une amélioration significative de la survie des patients atteints de CCR métastatique (CCRm). Cependant, une grande partie de ces patients présente une résistance qui est associée à la présence de mutations gains de fonction dans la voie de signalisation Ras. Dans ce contexte, l'identification de cibles thérapeutiques Ras-indépendantes pourrait avoir un intérêt particulier. Une analyse pharmacologique effectuée au laboratoire démontre que le nilotinib, un inhibiteur de la TK oncogénique BCR-ABL utilisée en clinique pour le traitement des leucémies myéloïdes chroniques, affecte également les propriétés invasives et métastatiques de cellules de CCR. Nous avons identifié la cible majoritaire de cet inhibiteur dans cette réponse tumorale : il s'agit de DDR1, un récepteur au collagène ayant une activité TK intrinsèque. Mon travail de thèse a mis en évidence que DDR1 est fréquemment et fortement activé dans les métastases de patients atteints de CCR. J'ai ensuite analysé la signalisation DDR1-dépendante dans ces cellules tumorales par phosphoprotéomique quantitative. Cette analyse montre la nature Ras-indépendante de cette signalisation et révèle la protéine de signalisation BCR comme un nouveau substrat essentiel de DDR1 pour induire l'invasion cellulaire. Enfin, je montre que l'inhibition de DDR1 par le nilotinib améliore la réponse au cetuximab y compris pour les cellules tumorales ayant une mutation oncogénique de K-Ras ou B-Raf. En conclusion, ces résultats suggèrent que le ciblage de DDR1 par le nilotinib pourrait avoir un intérêt thérapeutique dans le traitement des CCRm. / Colorectal cancer (CRC) is one of the leading causes of cancer-related death in the Western world. Cancer that metastasizes to distant sites is usually not curable, although chemotherapy can extend survival. Recently, a novel immunotherapy approach based on targeting the EGFR tyrosine kinase (TK) via the antibody cetuximab was shown to significantly improve the survival of patients with metastatic CRC (mCRC). However, only patients with wild-type KRAS may hope to gain from EGFR inhibition. The majority of patients expresses oncogenic K-Ras alleles and thus have hyperactive Ras cascade operating independently from EGFR. Therefore, there is an urgent need for discovery of a new Ras-independent target that regulates tumor cell invasion and metastasis in mCRC. A pharmacological approach in the lab revealed that the small inhibitor of the leukemic TK BCR-Abl also inhibits invasive and metastatic abilities of CRC cells. This effect was also observed in cells expressing deregulated KRAS signalling. We next identified the receptor for collagen and atypical TK DDR1 as that the main target of nilotinib in these cells. Accordingly, DDR1 was found frequently and strongly activated in metastatic nodules of CRC patients. I also characterized DDR1 signalling by quantitative phosphoproteomic. This analysis revealed the Ras-independent nature of DDR1 signalling but also the Rho GTPase regulator BCR as an essential DDR1 substrate that leads to cell invasion. Finally, I demonstrate that nilotinib potentiates cetuximab response in CRC cells expressing oncogenic K-Ras signaling. Overall, these results suggest that the targeting of DDR1 by nilotinib may be of therapeutic value in mCRC.

Le récepteur à neurotrophine TrkC a été identifié comme étant un récepteur à dépendance : en l'absence de son ligand NT-3, il déclenche l'apoptose. En effet, la survie des cellules qui expriment ces récepteurs dépend de la disponibilité en ligand, un mécanisme qui inhibe la prolifération incontrôlée et la migration des cellules tumorales. TrkC, en tant que récepteur à tyrosine kinase, est généralement considéré comme un proto-oncogène. Or nous montrons que l'expression TrkC est diminuée dans une grande fraction des cancers colorectaux humains, principalement par méthylation du promoteur de TrkC. En outre, ce mécanisme confère un avantage sélectif aux lignées cellulaires colorectales pour inhiber la mort des cellules tumorales. De plus, la réexpression de TrkC dans les lignées tumorales colorectales est associée à la mort des cellules tumorales et à l'inhibition in vitro des caractéristiques de transformation cellulaire, et in vivo de la croissance tumorale. Ensemble, ces données permettent de conclure que TrkC est un gène suppresseur de tumeur dans le cancer colorectal. Le mécanisme moléculaire par lequel TrkC déclenche l'apoptose implique le clivage de son domaine intracellulaire, ce qui libère un fragment pro-apoptotique (TrkC KF). Nous montrons que TrkC KF interagit avec Cobra1, un cofacteur de BRCA1, et que Cobra1 est nécessaire à l'apoptose induite par TrkC. Cobra1 conduit TrkC KF à la mitochondrie, où il favorise l'apoptose apoptosome-dépendante. Ainsi, nous proposons qu'en l'absence de NT-3, le clivage protéolytique de TrkC conduit à la libération d'un fragment tueur qui déclenche l'apoptose mitochondriale, via le recrutement de Cobra1 / The neurotrophin receptor TrkC was recently identified as a dependence receptor, and, as such, it triggers apoptosis in the absence of its ligand, NT-3. Indeed cells that express these receptors are thought to be dependent on ligand availability for their survival, a mechanism that inhibits uncontrolled tumor cell proliferation and migration. TrkC, as a classic tyrosine kinase receptor, is generally considered to be a proto-oncogene. We show that TrkC expression is down-regulated in a large fraction of human colorectal cancers, mainly through promoter methylation. Moreover, we show that TrkC silencing by promoter methylation is a selective advantage for colorectal cell lines to limit tumor cell death. Furthermore, reestablished TrkC expression in colorectal cancer cell lines is associated with tumor cell death and inhibition of in vitro characteristics of cell transformation, as well as in vivo tumor growth. Together, these data support the conclusion that TrkC is a colorectal cancer tumor suppressor. TrkC triggers apoptosis in the absence of its ligand NT-3 : the molecular mechanism for apoptotic engagement involves the double cleavage of the receptor's intracellular domain, leading to the formation of a proapoptotic fragment (TrkC KF). We show that TrkC KF interfacts with Cobra1, a putative cofactor of BRCA1, and that Cobra1 is required for TrkC-induced apoptosis. Cobra1 shuttles TrkC KF to the mitochondria, where it promotes apoptosome-dependent apoptosis. Thus, we propose that, in the absence of NT-3, the proteolytic cleavage of TrkC leads to the release of a killer fragment that triggers mitochondria-dependent apoptosis via the recruitment of Cobra1

L'expression intracellulaire des anticorps (intracorps) est une approche qui permet l'étude et le ciblage des antigènes dans les compartiments intracellulaires. Néanmoins, l'expression d'anticorps entiers fonctionnels dans les cellules reste une tâche difficile en raison de leur grande taille et de leur structure, l'environnement réducteur du milieu intracellulaire étant défavorable à la formation des ponts disulfure. Notre groupe a une forte expertise dans le domaine de l'immunisation intracellulaire et son application pour l'identification de nouvelles cibles thérapeutiques. Pour cela, notre équipe a élaboré des banques de fragments d'anticorps scFv optimisés pour une meilleure expression intracellulaire. Nos travaux antérieurs ont démontré que ces intracorps peuvent cibler spécifiquement des domaines ou des modifications post-traductionnelles de protéines dans des cellules vivantes. Ceci est particulièrement important car il démontre l'un des avantages principaux des intracorps par rapport à l'approche basée sur l'ARNi. Cet avantage a été démontré par un criblage phénotypique dans un modèle d'allergie. En appliquant cette approche à l'étude de l'activation des mastocytes, nous avons pu identifier un nouvel acteur moléculaire impliqué dans la voie de signalisation mise en jeu. Ce travail a été protégé par un brevet européen en 2013 et est publié récemment. Dans le cadre de mon projet de thèse, j'ai construit une nouvelle banque synthétique (HUSCIv) optimisée pour la stabilité, la diversité et l'affinité des scFvs. Pour cela, le scFv 13R4 isolé dans notre équipe a servi de charpente pour le greffage des différentes boucles hypervariables, tout en respectant la diversité des régions CDR observée dans les anticorps naturels humains. Nous avons utilisé la protéine GFP en tant que rapporteur pour étudier le repliement et la solubilité des intracorps. Nos résultats ont clairement démontré que la plupart des intracorps issus de la banque HUSCIv sont soluble dans le cytoplasme des cellules mammifères. Mon projet de thèse décrit ici rapporte l'utilisation de la banque HUSCIv pour un criblage phénotypique dans des cellules de cancer colorectal portant une mutation du gène K-RAS et résistantes au traitement par l'anticorps chimérique Cetuximab. Le projet cherche à sélectionner des scFv capables de restaurer la sensibilité au Cetuximab, avec comme objectif l'identification des cibles intracellulaires impliquées.Pour ce criblage fonctionnel, la banque HUSCIv a été exprimée dans les cellules HCT116 par l'intermédiaire d'un système d'expression rétroviral. Le processus de sélection est basé sur la sélection directe de la prolifération des cellules en utilisant un colorant fluorescent (CMRA). Les cellules dont la prolifération est bloquée sont isolées et un séquençage à haut débit permet de suivre l'évolution des populations de scFv tout au long de l'expérience. Ainsi, ce projet a nécessité un séquençage profond d'un grand nombre de scFv afin de réaliser une analyse statistique. Nous avons réalisé à ce jour deux tours de sélection. Les tests de cytotoxicité réalisés sur les populations sélectionnées ont montré une inhibition significative de la prolifération en présence du Cetuximab d'environ 10%. Ces résultats indiquent l'évolution du phénotype qui tend vers une sélection de scFv inhibiteurs et suggèrent que nous devons réaliser au moins un ou plusieurs tours plus sélectifs avant de formuler des conclusions.L'approche introduite ici est différente de toutes les études existantes en ce qu'elle utilise des banques « naïves », et permet non seulement de répondre à la diversité du protéome, mais aussi d'étudier les messagers secondaires et le métabolisme des cellules. En tant que tel, et par rapport à d'autres approches à grande échelle, celle-ci représente une voie simple pour la découverte de molécules thérapeutiques potentielles. / Intracellular expression of antibodies (intrabodies) permitted the study and targeting of antigens in cellular compartments. However, the expression of functional intrabodies remains a difficult task due to their large size, structure, and the reducing intracellular environment. Our group has a strong expertise in the field of intracellular immunization and the identification of new therapeutic targets. For this purpose, we have developed an scFv library optimized for intracellular expression of scFv antibody fragments. Our previous works have shown the successful use of intrabodies for targeting specific domains or post-translational modifications in living cells. This is particularly important because it demonstrates one of the main advantages of intrabodies compared to the approaches using RNAi. This benefit was demonstrated by a phenotypic screen in a model of allergy. Applying this approach to the study of mast cell activation, we identified a new molecular player involved in the signaling pathway implemented. This work was protected by a European patent in 2013 and was recently published. As part of my thesis project, I designed a new synthetic library (HUSCIv) optimized for scFv stability, diversity and affinity. For this, a highly soluble and hyper-stable framework, scFv13R4 isolated in our group, was used as a scaffold for grafting different hypervariable loops, while respecting the diversity of CDRs observed in human natural antibodies. We used protein GFP as a reporter to study the folding and solubility of intrabodies. Our findings clearly demonstrated that most of the intrabodies from HUSCIv library are soluble in the cytoplasm of mammalian cells. My thesis project described here reports the use of HUSCIv in a phenotypic screen of colorectal cancer cells carrying a mutation in the K-RAS gene and resistant to the treatment with the chimeric antibody Cetuximab. The project seeks to select scFv fragments able to restore the sensitivity to Cetuximab, with the objective to identify the intracellular targets involved. For this functional screen, the HUSCIv library was expressed in HCT116 cells via a retroviral expression system. The selection process is based on the direct selection of cell proliferation using a fluorescent dye (CMRA). The cells whose proliferation is blocked are isolated and the evolution of scFv populations throughout the experiment are tracked via high-throughput sequencing. This sequencing requires a large number of scFvs to perform a statistical analysis. So far, we have achieved two rounds of selection. The cytotoxicity tests carried out on the selected populations showed a significant inhibition of proliferation (10%) in the presence of Cetuximab. These results indicate that the evolving phenotypes are tending towards a selection of scFv inhibitors and suggest that we need to perform at least one or more selective rounds before making conclusions. The approach introduced here is different from all existing studies in that it uses "naive" libraries not only to respond to the diversity of the proteome, but also to study secondary messengers and metabolism in cells. As such, and in comparison to other large-scale approaches, it is a simple way for the discovery of potential therapeutic molecules.