Since the initial description of cancer stem cells (CSCs) as a self-renewing subpopulation of malignant cells with tumor-initiating capacity, a growing body of evidence has supported the existence of CSCs in virtually every tumor type. Our previous work in colorectal cancer has identified the transcription factor CDX1 as a key regulator of colorectal CSC differentiation. CDX1 expression is frequently lost in colorectal cancer, resulting in more aggressive, poorly differentiated tumors with higher proportions of CSCs. Many miRNAs have been implicated in tumor suppression and carcinogenesis, but the roles of miRNAs in differentiation, particularly in colorectal cancer, remain poorly understood. We began by identifying miRNAs downstream of CDX1 by using high-throughput small-RNA sequencing to profile miRNA expression in two pairs of colorectal cancer cell lines with stable CDX1 overexpression or knockdown. Validation of candidates identified by RNAseq in a larger cell line panel revealed miR-215 to be most significantly correlated with CDX1 expression. ChIP-qPCR and promoter reporter assays confirmed that CDX1 directly transactivates miR-215 transcription. MiR-215 is depleted in FACS-enriched CSCs compared to unsorted samples. Overexpression of miR-215 in poorly-differentiated, highly clonogenic cell lines causes growth arrest and a dramatic decrease in colony formation. miR-215 knockdown using a miRNA sponge causes an increase in clonogenicity and impairs differentiation in CDX1-high cell lines. Indeed, the effects of CDX1 expression on both gene expression and colony morphology can be attenuated by miR-215 inhibition, indicating that miR-215 is a functional mediator of CDX1. Microarray studies following miR-215 overexpression indicate that miR-215 induces terminal differentiation-associated growth arrest, due in part to direct silencing of BMI1 expression and de-repression of BMI1 target genes including CDKN1A. Our work situates miR-215 as a link between CDX1 expression and BMI1 repression that governs differentiation in colorectal cancer. We further characterize another miRNA-transcription factor axis in colorectal cancer, and we identify the novel miR-3189-3p as a potent effector of cell death with potential therapeutic implications.

The cancer stem cell (CSC) model of carcinogenesis and progression posits that within a tumor lies a subpopulation of cells that solely possess the ability to initiate a tumor and to differentiate into tumor cell lineages. Although the behavior of such cells is known, the challenge is to identify factors that characterize the CSC subpopulation. In this thesis, cell lines were identified that, when grown in three-dimensions, gave rise to organized colonies containing lumens originating from differentiating cells (“lumen lines”) and to densely-packed, spherical colonies originating from non-differentiating cells (“dense lines”). A microarray comparison of the pair identified genes upregulated in dense lines, including CD55 and BMI1, and in lumen lines, including CDX1 (Chapter 3). CD55 was used to isolate CD55high CSCs via flow cytometry that are able to self-renew, differentiate, initiate more colonies, proliferate more rapidly and exhibit an increased G2/M cell cycle population as opposed to unfractionated cells. Furthermore, the CD55high cells were able to give rise to more differentiated, lumen colonies in vitro, indicating that CD55 enriches for cells possessing a capacity to differentiate, and were able to enrich the CD24highCD44high putative CSC population further (Chapter 4). CDNA induction of BMI1 and CDX1 expression led to increased clonogenicity/proliferation and decreased clonogenicity/proliferation, respectively, and incorporation of a CDX1 reporter construct into the SW1222 cell line identified CDX1+ cells as a low-expressing population of CD55 (Chapter 5). Finally, co-culture of cell lines in an in vivo-like environment with intestinal myofibroblasts promoted the CSC population by enhancing clonogenicity, proliferation and expression of CD55 (Chapter 6). The results of this thesis implicate CD55 as a potent marker of colorectal cancer stemness, link the expression of BMI1 and CDX1 to cancer stemness and differentiation, respectively, and identify a role for the in vivo stem cell niche in maintaining the CSC population.

Side population is a minor subpopulation (SP) of some cell lines, exporting staining dye Hoechst 33342 out of their cytoplasm. It is discussed as a possible source of "cancer stem cells", "tumour initiating cells" or "metastasis initiating cells". However, broad literature suggest, that stemness and other privileged properties of SP are very variable between different cell types, cell lines and stage of disease. Cell lines TRAMP are the only widely available murine models for testing of prostate cancer therapy. We noticed in literature a mention about existence of 1-2% of cells constituting side population, but detailed characteristic have not been described until now. In this diploma thesis, we worked on characterisation of SP of the TRAMP-C2 cell line in comparison to other cells (nonSP). In the first part, we compared stem properties of SP and nonSP. We started with checking the existence of SP by its verapamil sensitivity. Using mRNA analysis, we showed that neither SP nor nonSP have increased c-Kit expression and that there are no differences in Bmi-1 expression. We found that SP is heterogenic mixture of CD24-CD44-, CD24-CD44+ and CD24+CD44+ cells, while nonSP is almost solely CD24-CD44+. We documented that SP and nonSP returned back to original SP ratio during cultivation. Then we showed on...

La littérature récente suggère que certaines sous populations cellulaires appelées cellules initiatrices de tumeurs (CIT) seraient particulièrement résistantes aux traitements actuels qui visent préférentiellement les cellules prolifératives et, de ce fait, participeraient activement à l'initiation de récidives tumorales et à la formation des métastases. La caractérisation de ces cellules ainsi que de leurs régulateurs revêt par conséquent un intérêt considérable pour la recherche fondamentale et translationnelle. La progastrine est une pro-hormone sécrétée en quantité anormalement importante par la majorité des tumeurs colorectales et qui joue un rôle important dans la promotion du développement tumoral. Les données publiées du laboratoire montrent en effet que l'inhibition de la PG conduit à la baisse des activités WNT/b-caténine et Notch qui sont des voies de signalisations anormalement activées dans les cancers colorectaux (CCR) et nécessaires au maintien des CIT coliques. Ainsi, le but de ce travail de thèse a été de déterminer si la PG possède un rôle régulateur des CIT coliques. Nos résultats indiquent que la PG est fortement exprimée dans des modèles cellulaires enrichis en CIT tels que le sont les colosphères ou les cellules exprimant fortement l'activité enzymatique des aldéhydes déshydrogénases (ALDH+). Le ciblage de la PG, soit par ARN interférent soit par l'utilisation d'anticorps neutralisant la PG dans des lignées cellulaires ou dans des cellules fraichement isolées de biopsies d'adénocarcinomes coliques humains, altère fortement la capacité d'initiation de colosphères sur plusieurs générations alors que le phénotype est sauvé par l'ajout de PG exogène. Ces résultats reflètent un potentiel d'autorenouvellement moindre des CIT in vitro en absence de PG. Le nombre de cellules ALDH+ diminue aussi significativement en absence de PG. À l'inverse, l'ajout de PG exogène stimule da capacité d'initiation des colosphères et augmente le nombre de cellules ALDH+. In vivo, la diminution de l'expression de la PG dans les cellules ALDH+ permet un délai dans l'initiation tumorale et diminue de 16x la propagation des CIT lors du deuxième passage chez la souris immunodéprimée. Ainsi, la PG est sécrétée par les CIT coliques, et, en retour, cette pro-hormone régule le maintien de ces cellules. Enfin, même si le rôle de la PG dans le tissu intestinal sain n'est pas primordial, nos résultats montrent qu'elle est produite par des cellules situées au fond des cryptes intestinales. Nos résultats viennent conforter l'idée selon laquelle le ciblage de la PG représente une stratégie thérapeutique prometteuse pour le traitement du CCR. / Recent literature strongly suggests that tumor initiating cells (TIC), resistant to chemotherapies, would be responsible for recurrence and metastasis development in colorectal cancer. Discovering new targets to eradicate these cells become urgent. Our team has shown that Progastrin (PG), a hormonal precursor abnormally secreted by most of colorectal tumors, would be a promising therapeutic target for colorectal cancer (CRC). In a mouse model developing spontaneously intestinal tumors, PG expression reduction leads to tumor growth decrease and remaining tumors display an increase of cell differentiation. Interestingly, tumorigenesis diminution is mediated by WNT-bcatenin and Notch transcriptional activity inhibition. These pathways being abnormally activated in CRC and essential for TIC survival and self-renewal, the aim of this work is thought to characterize PG involvement in TIC phenotype. Our results indicate that Progastrin is produced in TIC enriched models such as in colonospheres and in cells that highly expressed Aldehyde Dehydrogenase (ALDH+) activities. Targeting PG by RNAi or using neutralizing antibodies in cell lines or in cells that were freshly isolated from human's colon adenocarcinoma leads to decrease colonosphere number and self-renewal. Moreover, ALDH+ cells number decline after PG downregulation. In contrast, PG supplementation increases colonospheres initiation as well as ALDH+ cell number. In vivo, PG downregulation in ALDH+ cells that usually secretes PG leads to tumor initiation delay even after a second passage of bulk cells in immunodeficient mice. These results suggest also TIC frequency decrease in absence of PG. Thus, we show that PG is secreted by TIC and regulates their activities. Finally, PG role is not primordial for intestinal tissue homeostasis but our results show that this pro-hormonal precursor is preferentially produced at the bottom of intestine crypt cells. To conclude, our results strongly consolidate the idea of targeting Progastrin as a promising therapeutic strategy for colorectal cancer management.

Les cellules souches cancéreuses (CSCs) représentent une petite sous-population de cellules dans la tumeur qui sont capables de s'autorenouveler. Ce sont des cellules impliquées dans l'initiation et la croissance métastasique des tumeurs, ainsi que dans la résistance aux traitements conventionnels. Ces CSCs expriment les facteurs de pluripotence Oct4, Nanog et Sox2, lorsqu’elles sont fortement indifférenciées. Mon travail de thèse a consisté à analyser les effets anticancéreux de produits phytochimiques sur des CSCs. Nous avons ainsi étudié l’activité anti-carcinogénique sélective du roopérol sur un modèle de CSC tératocarcinomale. Le roopérol entraîne un processus pro-apoptotique et induit l’activation de la p53 et de la caspase 3, via la formation intracellulaire d'espèces réactives d’oxygène (ERO), ce qui conduit à une chute de l'expression d’Oct4 et Nanog. Un tel effet n’est pas observé dans les cellules souches normales (CSNs). Nous avons donc étudié le mécanisme impliqué dans la résistance des CSNs au traitement par le roopérol. Ce dernier n'y provoque aucune formation de ERO, mais active sélectivement les protéines de pro-survie Akt et Bad. Dans une 2eme étude, nous avons examiné le potentiel anticancéreux sélectif de plusieurs dérivés triterpéniques de type avicine, issus de différentes espèces d’Albizzia africaines, sur un modèle de carcinome épidermoïde. Après criblage, nous avons choisi d'étudier plus en profondeur les mécanismes moléculaires mis en jeu par le composé qui présentait la plus grande activité pro-apoptotique. Cette analyse a été effectuée dans un modèle de mélanome métastasique agressif connu pour exprimer Oct4. Nous démontrons que l'agent pharmacologique entraîne une apoptose sélective des cellules de mélanome, via une activation de la p38 MAPK et de la caspase 3, suivie d’une diminution de l’expression d’Oct4. Un tel effet apoptotique n’a pas été observé dans les cellules normales. Ces résultats permettent ainsi de mettre en évidence de nouveaux et puissants composés anticancéreux, capables d’induire sélectivement et in vitro l’apoptose des CSCs et leurs descendants, tout en épargnant les CSNs et leurs descendants. / Cancer stem cells (CSCs) are a small subpopulation of cells in the tumor which are able to self-renew. These cells are involved in the initiation and the metastatic growth of tumors, as well as in the resistance to conventional treatments. In their highly undifferentiated state, CSCs are known to express the stemness factors Oct4 and Nanog. The aim of this work was therefore to analyze the effects of phytochemicals on Oct-4 expressing teratocarcinomal stem-like cells. In a first study, we investigated the selective anti-carcinogenic activity of rooperol, the aglycone of the plant-derived compound hypoxoside. We observed that the rooperol-induced apoptosis in CSCs was associated with an oxidative stress, dependent of the activation of p53 and cleaved caspase 3 expression. These modifications were accompanied by reduced expression of the stemness factors. Such effect was however not observed in normal stem cells (NSCs). We investigated therefore the mechanism involved in the resistance of NSCs to the drug. In contrast to CSCs, rooperol does not cause ROS (reactive oxygen species) formation in NSCs and selectively activates the pro-survival proteins Akt and Bad. In a second study, we examined the selective anti-cancer potential of several acacic acid-type saponins, from different species of African Albizzia, on epidermoid carcinoma cells; in a next step, we investigated the molecular targets of the anti-cancer compound which showed the greatest proapoptotic activity. The study was performed on a model of aggressive metastatic melanoma cell known to express Oct4. We observed that the pharmacological agent had a selective pro-apoptotic effect, via the activation of p38 MAPK and caspase 3, followed by a decrease on the expression of Oct4. Such effect could not be detected in normal cells. These results highlight new and powerful anticancer compounds able to induce selectively apoptosis of CSCs and their descendants, while sparing NSCs and their descendants.

Ce travail de thèse s’inscrit dans la caractérisation des cellules radiorésistantes des cancers du sein, à l’origine des rechutes après traitement par radiothérapie. La théorie des « Cellules Souches Cancéreuses » (CSC) place une sous-population cellulaire présentant une capacité accrue à induire des tumeurs et à proliférer au centre de la résistance à l’irradiation. Au cours de ce travail, nous avons montré que seul le marqueur de CSC CD24-/low permettait de définir une sous population radiorésistante, capable de transmettre une « mémoire » de l’irradiation se traduisant par une instabilité génétique persistante dans la descendance des cellules irradiées. En outre, nous avons montré que CD24 n’est pas uniquement un marqueur, mais bien un acteur de la réponse à l’irradiation. Ainsi, CD24 contrôle la prolifération cellulaire in vitro et in vivo, ainsi que les niveaux de ROS avant et après irradiation. L’ensemble de ces propriétés aboutit à une sensibilité réduite des cellules CD24-/low à l’irradiation γ, ainsi qu’à une baisse de l’instabilité génétique. Ces résultats permettent pour la première fois d’attribuer un rôle aux CSC CD24-/low dans la transmission de l’instabilité chromosomique. De plus, en apportant des informations pour évaluer la radiorésistance intrinsèque des tumeurs mammaires, le marquage CD24 pourrait contribuer à l’amélioration des protocoles de radiothérapie. / This work focuses on the characterization of radiation-resistant breast cancer cells, responsible for relapse after radiotherapy. The “Cancer Stem Cells” (CSC) theory describes a radiation-resistant cellular sub-population, with enhanced capacity to induce tumors and proliferate. In this work, we show that only the CSC marker CD24-/low defines a radiation resistant cell population, able to transmit the “memory” of irradiation, expressed as long term genomic instability in the progeny of irradiated cells. We show that CD24 is not only a marker, but is an actor of radiation-response. So, CD24 expression controls cell proliferation in vitro and in vivo, and ROS level before and after irradiation. As a result, CD24-/low cells display enhanced radiation-resistance and genomic stability. For the first time, our results attribute a role to CD24-/low CSCs in the transmission of genomic instability. Moreover, by providing informations on tumor intrinsic radiation-sensitivity, CD24- marker could help to design new radiotherapy protocols.

Une sous population de cellules au sein des tumeurs mammaires présente une capacité accrue de se renouveler et de reproduire l’hétérogénéité du cancer du sein. Maintenant il est bien connu que les cellules souches présomptives du cancer du sein possèdent des programmes d’expression des gènes qui correspondent à leurs caractéristiques biologiques uniques. Notre groupe a été impliqué dans la caractérisation épigénétique des cellules souches présomptives du cancer du sein et l’importance de la dérégulation des mécanismes épigénétiques comme la méthylation de l’ADN et le microARN au cours de la carcinogenèse. Plus spécifiquement cette étude détaille l’idée que la survie des cellules souches du cancer du sein peut être due à une signalisation via des circuits spécifiques de régulation, y compris la voie d’inflammation IL6-JAK-STAT. Ces cellules présentent une activation constitutive de cette voie associée à une configuration particulière de la chromatine. Une autre part de cette étude est d’explorer l’idée que des changements dans l’expression des microARN sont fondamentaux pour la maintenance des principales caractéristiques de ces cellules, et leur ciblage peut représenter une nouvelle approche de thérapie contre le cancer du sein. De plus, en testant directement les conséquences in vivo de la régulation de miR30a nous ouvrons la voie pour la recherche et la validation de l’utilisation potentielle des microARN comme thérapie anti cancéreuse. Ensemble, nos résultats apportent une nouvelle compréhension du rôle des modifications épigénétiques dans la maintenance des cellules souches du cancer du sein. De façon importante ces découvertes intègrent l’idée que des mécanismes de régulations différents mais coordonnés ont un rôle dans la survie des cellules souches du cancer du sien et donnent une perspective élargie pour la découverte de nouvelles cibles thérapeutiques / A subpopulation of cells within breast tumors is known to display an increased ability to self-renew and reproduce breast cancer cell heterogeneity. It is now known that putative breast cancer stem cells (CSCs) display distinct programs of gene expression that correlate with their unique biological characteristics. Our group has been involved in the epigenetic characterization of putative breast CSCs and the importance of the deregulation of epigenetic mechanisms such as DNA methylation and microRNA during carcinogenesis. More specifically, this study is detailing the idea that the survival of breast CSC may be dependent on signaling through specific regulatory circuits, including the well known inflammatory IL6-JAK-STAT pathway. These cells display a constitutive activation of this pathway associated with a distinct chromatin configuration. Another part of the study is exploring the idea that changes in microRNA expression are fundamental in sustaining the main attributes of these cells, and their targeting may represent a novel approach for breast cancer therapy. In addition, by directly testing the in vivo consequences of miR30a regulation, we open a window of opportunity for testing and validating the potential use of microRNAs in anti-neoplastic therapy. Together our results bring a new understanding of the role of epigenetic modifications in the maintenance of breast CSC. Importantly, these findings integrate the idea that different but coordinated regulation mechanisms play a role in the survival of CSC and give a larger perspective for finding novel therapeutic targets

O estudo das células-tronco cancerígenas (CTCs) durante o processo de malignização e no carcinoma epidermóide intra-bucal já instalado é essencial para um melhor entendimento de como essas células participam da formação e manutenção de uma neoplasia. Atualmente, a identificação de células com características de células tronco se dá principalmente através da expressão de marcadores celulares como o ALDH1 e o CD44. A proteína ALDH1 é responsável pela oxidação de aldeídos intracelulares e vem sendo utilizada para o isolamento de CTCs em inúmeros canceres incluindo casos de cabeça e pescoço. A proteína CD44 é uma glicoproteína envolvida na adesão e migração celular, também participa do processo de metástase e já foi associada às CTCs. Nesse trabalho, a expressão dessas proteínas foi analisada em 45 casos de displasias epiteliais e 13 casos de carcinomas epidermóide intra-bucais. As lesões displásicas foram classificadas em casos leves (19), moderados (18) e intensos (8) e foram também divididas em casos de baixo risco (22) e alto risco de transformação maligna (23). A expressão imunohistoquímica para a ALDH1 foi encontrada predominantemente na camada basal em 16 casos de displasias epiteliais e em 7 carcinomas epidermóides, com a marcação difusa pela epitélio neoplásico. A expressão imunohistoquímica de CD44 foi encontrada em 42 displasias epiteliais e em 12 carcinomas epidermóides, sendo que nas displasias, a expressão ocorreu predominantemente na camada basal do epitélio e no carcinoma epidermóide a expressão foi disseminada. Ambos marcadores exibiram aumento de expressão com a evolução do grau das displasias. / The study of cancer stem cells (CTCs) in the process of malignant transformation and intra-oral squamous cell carcinoma already installed is essential for a better understanding of how these cells participate in the formation and maintenance of a neoplasm. Currently, identification of cells with characteristics of stem cells is primarily through the expression of cell markers such as CD44 and ALDH1. The ALDH1 protein is responsible for the oxidation of intracellular aldehydes and has been used for the isolation of CTCs in numerous cancers including head and neck cases. The CD44 protein is a glycoprotein involved in cell adhesion and migration, also participates in the process of metastasis and has been associated with CTCs. In this work, the expression of these proteins was analyzed in 45 cases of epithelial dysplasia and 13 cases of intraoral squamous cell carcinomas. The dysplastic lesions were classified as mild (19), moderate (18) and intense (8) cases and were also divided into low-risk cases (22) and high risk of malignant transformation (23). The immunohistochemical expression for ALDH1 was found predominantly in the basal layer in 16 cases of epithelial dysplasia and squamous cell carcinoma in 7, with diffuse labeling by neoplastic epithelium. Immunohistochemical expression of CD44 was found in 42 epithelial dysplasias and 12 squamous cell carcinomas, and in dysplasias, the expression occurred predominantly in the basal layer of the epithelium and in squamous cell carcinoma expression was widespread. Both markers showed increased expression with the evolution of the degree of dysplasia.

O estudo das células-tronco cancerígenas (CTCs) durante o processo de malignização e no carcinoma epidermóide intra-bucal já instalado é essencial para um melhor entendimento de como essas células participam da formação e manutenção de uma neoplasia. Atualmente, a identificação de células com características de células tronco se dá principalmente através da expressão de marcadores celulares como o ALDH1 e o CD44. A proteína ALDH1 é responsável pela oxidação de aldeídos intracelulares e vem sendo utilizada para o isolamento de CTCs em inúmeros canceres incluindo casos de cabeça e pescoço. A proteína CD44 é uma glicoproteína envolvida na adesão e migração celular, também participa do processo de metástase e já foi associada às CTCs. Nesse trabalho, a expressão dessas proteínas foi analisada em 45 casos de displasias epiteliais e 13 casos de carcinomas epidermóide intra-bucais. As lesões displásicas foram classificadas em casos leves (19), moderados (18) e intensos (8) e foram também divididas em casos de baixo risco (22) e alto risco de transformação maligna (23). A expressão imunohistoquímica para a ALDH1 foi encontrada predominantemente na camada basal em 16 casos de displasias epiteliais e em 7 carcinomas epidermóides, com a marcação difusa pela epitélio neoplásico. A expressão imunohistoquímica de CD44 foi encontrada em 42 displasias epiteliais e em 12 carcinomas epidermóides, sendo que nas displasias, a expressão ocorreu predominantemente na camada basal do epitélio e no carcinoma epidermóide a expressão foi disseminada. Ambos marcadores exibiram aumento de expressão com a evolução do grau das displasias. / The study of cancer stem cells (CTCs) in the process of malignant transformation and intra-oral squamous cell carcinoma already installed is essential for a better understanding of how these cells participate in the formation and maintenance of a neoplasm. Currently, identification of cells with characteristics of stem cells is primarily through the expression of cell markers such as CD44 and ALDH1. The ALDH1 protein is responsible for the oxidation of intracellular aldehydes and has been used for the isolation of CTCs in numerous cancers including head and neck cases. The CD44 protein is a glycoprotein involved in cell adhesion and migration, also participates in the process of metastasis and has been associated with CTCs. In this work, the expression of these proteins was analyzed in 45 cases of epithelial dysplasia and 13 cases of intraoral squamous cell carcinomas. The dysplastic lesions were classified as mild (19), moderate (18) and intense (8) cases and were also divided into low-risk cases (22) and high risk of malignant transformation (23). The immunohistochemical expression for ALDH1 was found predominantly in the basal layer in 16 cases of epithelial dysplasia and squamous cell carcinoma in 7, with diffuse labeling by neoplastic epithelium. Immunohistochemical expression of CD44 was found in 42 epithelial dysplasias and 12 squamous cell carcinomas, and in dysplasias, the expression occurred predominantly in the basal layer of the epithelium and in squamous cell carcinoma expression was widespread. Both markers showed increased expression with the evolution of the degree of dysplasia.

INTRODUÇÃO: Os múltiplos passos da carcinogênese do câncer de cólon envolvem a existência de subpopulações de células tronco tumorais (CSC), responsáveis pela transformação, crescimento e proliferação das células tumorais. As proteínas CD44 e CD166 são marcadores de CSC associados a sinalização celular, adesão, migração, metástase e resposta linfocitária. Alguns fatores podem modular a expressão CSC como a mutação KRAS. OBJETIVO: correlacionar a expressão dos marcadores CD44 e CD166 em carcinoma de cólon metastático e status do oncogene KRAS (selvagem/mutado) com as características clínico-patológicas e desfecho do paciente ao final do seguimento. MATERIAL E MÉTODOS: Foram coletadas 58 amostras de tecido tumoral de pacientes com neoplasia de cólon metastático, tratados com CapeOx no Serviço de Oncologia Clínica do HCFMRPUSP de 2003 a 2012. Foram coletadas informações do prontuário sobre status do gene KRAS, características clínico-patológicas e desfecho clínico, sendo também realizada imunohistoquímica para marcação CD44 e CD166 através da técnica de TMA. Utilizado software SPSS 17 para análise estatística e considerado valor de p<0,050 para significância dos dados. RESULTADOS: A expressão de CD44 e CD166 foi positiva em 41,4% e 43%, respectivamente, e o status KRAS mutado em 48,3%. No subgrupo kAs selvagem e nos idosos (>65 anos), houve associação entre CD44 e CD166, p=0,042 e p=0,001, respectivamente. Pacientes CD166 negativo tiveram 3 vezes mais chances de progressão de doença (p=0,02) do que CD166 positivo. Pacientes Kras mutado e CD166 negativo tiveram 8 vezes risco de progressão (p<0,01). Pacientes CD44 positivo tiveram 4 e 5 vezes mais chances de evoluir com metástases hepática e pulmonar (p<0,01) em relação aos CD44 negativo. Pacientes com a combinação KRAS mutado e CD44 positivo tiveram 7 vezes mais chance de evoluir com metástase pulmonar (p=0,02) em relação a pacientes KRAS selvagem e CD44 negativo. DISCUSSÃO: Na amostra estudada observamos a influência das expressões dos marcadores de CSC e suas combinações com o status de mutação do gene KRAS, de modo que pacientes com CD166 negativo no tumor primário apresentam um desfecho de maior recorrência e o CD44 positivo favorece a evolução para metástases pulmonar e hepática. A mutação do gene KRAS atua modulando a via do EGF influenciando o comportamento biologico do tumor e os desfechos (recidiva e metastases) diretamente relacionados com a expressão dos marcadores de CSC no cancer de colon metastatico. CONCLUSÃO: Este estudo demonstrou interação entre a expressão imuno-histoquímica dos marcadores CSC de cólon (CD166 e CD44) e o status KRAS, podendo carcterizar subgrupos de pacientes com maiores chances de evolução desfavorável e assim propor um modelo de tratamento e seguimento mais individualizado. / BACKGROUND: Colon cancer carcinogenesis has been recently correlated with specific cancer stem cell (CSC) subpopulations which are associated with transformation, growth and spread process of tumor cells. CD44 and CD166 are CSC markers correlated with cell signalization, adhesion, migration, metastasis, and lymphocyte response. Some factors as KRAS mutation could modulate CSC. OBJECTIVE: Analyze CD44 and CD166 expressions in metastatic colon carcinoma and its correlation with KRAS status, clinicopathological features, disease recurrence and patient survival. MATERIAL AND METHODS: Tissues were obtained from 58 patients with confirmed metastatic colon cancer, treated with CapeOx at FMRP-USP from 2003 to 2012. Clinical and outcomes informations and KRAS gene status were obtained from medical records. KRAS status was analyzed with RT-PCR. CD44 and CD166 were analyzed with TMA immunohistochemistry. Statistical analyses were performed using SPSS 17.0. A p-value <0,050 was considered to be statistically significant. RESULTS: CD44 and CD166 expressions were positive in 41,4% and 43%, respectively, and KRAS status was mutate in 48,3%. Wild-type KRAS in elderly patients had statistical association between CD44 and CD166, p=0,042 and p=0,001, respectively. Patients with CD166 negative had 3 fold increase in progression disease (p<0,01). Patients with CD44 positive had 4 and 5 fold increase in liver and lung metastasis (p<0,01), respectively. Patients with combined mutated KRAS and CD44 positive had 7 fold increase in lung metastasis (p=0,02) compared with wildtype KRAS and CD44 negative. DISCUSSION: In this study, the influence of markers expression of colon CSC (CD44 and CD166) and its combinations with status KRAS were proven. Patients with CD166 negative in primary colon tumor are more likely to present higher recurrence and, CD44 positive have a higher chance to develop lung and liver metastasis. KRAS mutation contributed, associated with studied CSC expressions, to cancer biological behavior and agressivness. CONCLUSIONS: This study demonstrated interaction between imunohistochemical expression of colonic CSC markers (CD166 and CD44) and KRAS gene status. Subgroups of patients with worse outcomes could be identified and this biological information contributed to personalized treatments and follow ups that should be proposed for these patients.