Dissertations / Theses on the topic 'Breast cancer, microRNAs, miR-106b'

MicroRNAs are small non-coding RNAs which regulate gene expression by binding to 3&rsquo
UTR of their target mRNAs. Deregulated expression of microRNAs is detected in many pathologies including different types of cancers. miR-106b, is a member of miR-106b-25 cluster and overexpressed in many cancers including breast cancer. Based on miR-106b overexpression, we hypothesized that miR-106b may be an oncogene candidate. To explore miR-106b related phenotypes, we used an already miR-106b transfected model cell line system. Stably transfected MCF10A cells were investigated for alterations in cell growth, motility, migration and invasion. Our results showed that miR-106b overexpression caused increased growth motility and migration. On the other hand, based on matrigel invasion assay miR-106b expression caused a reduction in cell invasion. Further studies are needed to be performed to understand the precise role of miR-106b in breast cancer. Studies are underway to detect possible miR-106b targets that may help to explain these phenotypical alterations.

Le cancer du sein est marqué par une grande hétérogénéité. C´est une maladie complexe, fortement influencée par l´environnement pourtant, elle dépend aussi d´une accumulation de mutations génétiques associées à la dérégulation épigénétique des voies clés. Les altérations présentes dans le profil d´expression génique observées dans la tumeur, peuvent être le résultat de mécanismes de régulation des gènes à différent niveaux, comme des modifications post-transcriptionnelles menées par le mécanisme d´ARN d´interférence sous forme de microARN (miARN). Ce mécanisme peut conduire au début et développement du cancer aussi bien qu’à la résistance aux thérapies. Les miARN font partie d’une classe d´ARN non-codants qui ont émergé ces dernières années comme l'un des principaux régulateurs de l'expression des gènes par sa capacité à réguler négativement l'activité des ARN messagers (ARNm). L´importance de cette régulation a été observée par la présence de ce type de contrôle dans plusieurs processus biologiques, parmi eux, des voies liées à la prolifération, différentiation et apoptose. Afin de mieux comprendre les mécanismes d’initiation et progression tumorale dans le cancer du sein, nous avons fait une analyse globale de l´expression des miARN, par la technique de microarray, dans la série de lignées cellulaires 21T. Cette série est un modèle in vitro de la progression tumorale, comprenant la lignée 16N, obtenue à partir de l’épithélium normal infecté par des virus HPV-16, les lignées 21PT et 21NT, qui correspondent au carcinome in situ et les lignées 21MT1 et 21MT2 obtenues à partir d´une effusion pleurale métastatique à l’endroit de la métastase. Etant donné que les miARN jouent un rôle dans la régulation de l´apoptose et d´autres mécanismes de réponse aux dommages fait à l´ADN et que l´irradiation dans des formes différentes est couramment utilisée comme outil diagnostique, par exemple dans des mammographies, nous avons évalué l´expression de miARN après avoir soumis les cellules à des irradiations de haute et basse énergie, et au traitement avec de la doxorubicine. Les tests ont été faits sur les lignées non tumorales (MCF-10A et HB-2) et sur les lignées tumorales (MCF-7 et T-47D). On a pu observer que le rayon X de basse énergie est capable de causer des cassures double brin à l´ADN et de conduire les cellules à l´apoptose. Une légère altération dans les profils d´expression des miARN impliqués dans cette voie, comme let-7a, miR-34a et miR-29b, a aussi été remarquée. En ce qui concerne la réponse aux dommages fait à l´ADN, une upregulation dans l´expression de miR-29b, qui sous des conditions physiologiques normales est régulée négativement, a été observée après les traitements. Le microARNome de la série 21 montre une importante sous-expression de miR-205, un enrichissement du facteur pro-métastatique ZEB-1 et une réduction conséquente dans les niveaux d’e-cadherine, observée par western blot, seulement dans les lignées métastatiques (21MT). L´ensemble des résultats, suggèrent que miR-29b peut être un bio-marqueur potentiel du stress génotoxique et que miR-205 peut participer du processus de transition épithélium-mésenchyme et, en outre, quand il est sous-exprimé, peut augmenter le potentiel métastatique des cellules de la série 21T
Breast tumors are characterized by their high heterogeneity. Breast cancer is a complex disease, which has its development strongly influenced by environmental factors, combined with a progressive accumulation of genetic mutations and epigenetic dysregulation of critical pathways. Changes in gene expression patterns may be a result of a deregulation in epigenetic events as well as in post-transcriptional regulation driven by RNA interference endogenously represented by microRNA (miRNA). These mechanisms are capable to promote the initiation, maintenance and progression of carcinogenesis and are also implicated on the development of therapy resistance. miRNAs form a class of non-coding RNAs, which have emerged in recent years as one of the major regulators of gene expression through its capacity to silence messenger RNAs (mRNAs) containing a partially complementary sequence. The importance of regulation mediated by miRNAs was observed on their ability to regulate a wide range of biological processes, including cell proliferation, differentiation and apoptosis.To gain insights into the mechanisms involved in breast cancer initiation and progression we conducted a miRNA global expression on 21T series that are an in vitro model of breast cancer progression, comprising cell lines derived from the same patient, which include a normal epithelia (16N), primary in situ ductal carcinoma (21PT and 21NT) and cells derived from pleural effusion of lung metastasis (21MT-1 and 21MT-2). Considering the importance of miRNAs in the regulation of apoptosis, and that irradiation in different spectra is commonly used in diagnostic procedures, as mammography and on radiotherapy, we evaluated the miRNA expression after cell low and high energy irradiation and doxorubicin treatment to determine whether miRNAs are useful biomarkers to detect cell response after DNA damage. The experiments were done on the non-tumoral cell lines MCF-10A and HB-2 and on the breast carcinoma derived cell lines MCF-7 and T-47D. We observed that low energy X-rays is able to promote DNA strand breaks and apoptosis and to slightly change the expression of miRNAs involved on this pathway, such as let-7a, miR-34a and miR-29b. Regarding DNA stress response pathways, an upregulation on miR-29b expression, that in normal conditions is downregulated in tumor cell lines could be observed after all treatments. The microRNAome of 21T series revealed a significant downregulation of miR-205, an enrichment of the pro-metastatic factor ZEB-1, potential target for miR-205 and the consequent reduction of e-cadherin levels in 21MT cells checked by western blot. Our results indicate that miR-29b is a possible biomarker of genotoxic stress and that miR-205 can participate on the metastatic potential of 21T cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Os tumores de mama são caracterizados pela sua alta heterogeneidade. O câncer de mama é uma doença complexa, que possui o seu desenvolvimento fortemente influenciado por fatores ambientais, combinada a uma progressiva acumulação de mutações genéticas e desregulação epigenética de vias críticas. Alterações nos padrões de expressão gênica podem ser resultado de uma desregulação no controle de eventos epigenéticos, assim como, na regulação pós-transcricional pelo mecanismo de RNA de interferência endógeno via microRNA (miRNA). Estes eventos são capazes de levar à iniciação, à promoção e à manutenção da carcinogênese, como também ter implicações no desenvolvimento da resistência à terapia Os miRNAs formam uma classe de RNAs não codificantes, que durante os últimos anos surgiram como um dos principais reguladores da expressão gênica, através da sua capacidade de regular negativamente a atividade de RNAs mensageiros (RNAms) portadores de uma seqüencia parcialmente complementar. A importância da regulação mediada por miRNAs foi observada pela capacidade destas moléculas em regular uma vasta gama de processos biológicos incluindo a proliferação celular, diferenciação e a apoptose. Para avaliar a expressão de miRNAs durante a progressão tumoral, utilizamos como modelo experimental a série 21T que compreende 5 linhagens celulares originárias da mesma paciente diagnosticada com um tumor primário de mama do tipo ErbB2 e uma posterior metástase pulmonar. Essa série é composta pela linhagem obtida a partir do tecido normal 16N, pelas linhagens correspondentes ao carcinoma primário 21PT e 21NT e pelas linhagens obtidas um ano após o diagnóstico inicial, a partir da efusão pleural no sítio metastatico 21MT1 e 21MT2. O miRNAoma da série 21T revelou uma redução significativa nos níveis de miR-205 e nos níveis da proteina e-caderina e um enriquecimento do fator pró-metastático ZEB-1 nas células 21MT. Considerando a importância dos miRNAs na regulação da apoptose, e que a irradiação em diferentes espectros é comumente usada em procedimentos de diagnóstico como mamografia e na radioterapia, avaliamos a expressão de miRNAs após irradiação de alta e baixa energia e do tratamento doxorrubicina. Para os ensaios foram utilizados as linhagens não tumorais MCF-10A e HB-2 e as linhagens de carcinoma da mama MCF-7 e T-47D. Observou-se que raios-X de baixa energia são capazes de promover quebras na molécula do DNA e apoptose assim como, alterar sensivelmente miRNAs envolvidos nessas vias como o let-7a, miR-34a e miR-29b. No que diz respeito à resposta a danos genotóxicos, uma regulação positiva sobre a expressão de miR-29b, o qual em condições normais é regulado negativamente foi observada uma regulação positiva sobre miR-29b expressão após todos os tratamentos em células tumorais. Nossos resultados indicam que miR-29b é um possível biomarcador de estresse genotóxico e que miR-205 pode participar no potencial metastático das células 21T.
Breast tumors are characterized by their high heterogeneity. It is a complex disease, which has its development strongly influenced by environmental factors, combined with a progressive accumulation of genetic mutations and epigenetic dysregulation of critical pathways. Changes in gene expression patterns may be a result of a deregulation in epigenetic events as well as in post-transcriptional regulation driven by RNA interference endogenously represented by microRNA (miRNA) these mechanisms are capable to promote the initiation, maintenance and progression of carcinogenesis; they are also implicated on the development of therapy resistance. miRNAs form a class of non-coding RNAs which have emerged in recent years as one of the major regulators of gene expression through its capacity to silence messenger RNAs (mRNAs) containing a partially complementary sequence. The importance of regulation mediated by miRNAs was observed on their ability to regulate a wide range of biological processes including cell proliferation, differentiation and apoptosis.To gain insights into the mechanisms involved in breast cancer initiation and progression conducted a miRNA global expression on 21T series that are an in vitro model of breast cancer progression comprising cell lines derived from the same patient which include a normal epithelia (16N), primary in situ ductal carcinoma (21PT and 21NT) and cells derived from pleural effusion of lung metastasis (21MT-1 and 21MT-2). Considering the importance of miRNAs in the regulation of apoptosis, and that irradiation in different spectra is commonly used in diagnostic procedures as mammography and on radiotherapy, we evaluate the miRNA expression after cell low and high energy irradiation and doxorubicin treatment to determine whether miRNAs are useful biomarkers to detect cell response after DNA damage. The experiments were done on the non-tumoral cell lines MCF-10A and HB-2 and on the breast carcinoma derived cell lines MCF-7 and T-47D. We observed that of low energy X-rays is able to promote DNA strand breaks and apoptosis and to slightly change the expression of miRNAs involved on this pathway such as let-7a, miR-34a and miR-29b. Regarding DNA stress response pathways an upregulation on miR-29b expression, that in normal conditions is downregulated in tumor cell lines could be observed after all treatments. The microRNAome of 21T series revealed a significant downregulation of miR-205, an enrichment of the prometastatic factor ZEB-1, potential target for miR-205 and the consequent reduction of ecadherin levels in 21MT cells checked by western blot. Our results indicate that miR-29b is biomarkers of genotoxic stress and that miR-205can participate on the metastatic potential of 21T cells.

microRNA dependent gene expression regulation has roles in diverse processes such as differentiation, proliferation and apoptosis. Therefore, deregulated miRNA expression has functional importance for various diseases, including cancer. miR-125b is among the commonly downregulated miRNAs in breast cancer cells . Therefore we aimed to characterize the effects of miR-125b expression in MCF7 breast cancer cell line (BCCL) to better understand its roles in tumorigenesis. Here, we investigated mir-125 family members

Esta tesis consiste en el estudio de la regulación por microRNAs del proceso de metástasis de carcinomas ductales invasivos de mama. Hemos encontrado una expresión alterada de varios microRNAs en muestras tumorales, incluyendo las familia miR-200 y miR-181, miR-210, miR-101 y miR-10b, a lo largo de la progresión desde tumores primarios a metástasis regionales y a distancia. miR-7, miR-30, miR-148 y miembros de la familia let-7 se encontraron diferencialmente expresados entre tumores primarios no metastáticos vs primarios metastáticos a ganglios linfáticos. Además, los miembros del cluster 1 de la familia miR-200 se econtraban sobreexpresados en las metástasis ganglionares, las cuales mantenían o tenían una expresión más elevada de E-cadherina respecto de sus correspondientes tumores primarios. Adicionalmente y de acuerdo con la tendencia observada en los tejidos tumorales, observamos niveles elevados de miR-200b y miR-7 en la sangre de pacientes con metástasis regional o a distancia en comparación con pacientes con tumores primarios no metastáticos en el momento del diagnóstico. Centrándonos en la significación biológica de la mayor expresión de los miembros de la familia miR-200 en asociación con la progresión metastática, utilizamos el modelo celular MCF10CA1h para inducir la expresión de miR-200. Observamos que miR-200 promovía una transición mesénquima-epitelio que conllevaba la inducción de un marcado programa epitelial. Éste era acompañado de un fuerte incremento de la actividad aldehido deshidrogenasa (ALDH), mayor capacidad de crecimiento en mamoesferas y la transición de un immunofenotipo CD44+ CD24- a CD44+ CD24+, lo cual era indicativo de la adquisición de características de progenitor luminal por parte de células que expresaban miR-200. Además, las células MCF10CA1h que expresaban miR-200 desarrollaron la capacidad de diferenciarse y organizarse en estructuras tubuloalveolares ramificadas similares a las de la mama normal. Se observó además una mayor capacidad de crecimiento tumoral y de colonización metastática in vivo. Además de la expresión de marcadores epiteliales, la expresión de miR-200 resultó en la inducción de la expresión de marcadores basales y de diferenciación luminal in vitro y de forma más prominente in vivo. Todo ello refueza la idea de que miR-200 induce características de progenitor luminal así como de agresividad en células tumorales mamarias. A nivel mecanístico encontramos que la mayor capacidad de autorrenovación promovida por miR-200 era debida en parte a la inhibición de su ya conocidad diana ZEB2 así como de una activación de la ruta de señalización PI3K-AKT. Finalmente, la morfología de los tumores formados in vivo por células que expresaban miR-200 recordaba a la de los carcinomas metaplásicos mamarios, por lo que decidimos estudiarlos. De hecho, observamos que el componente epitelial de tumores metaplásicos expresaban niveles significativamente superiores de miR-200 que el componente mesenquimal y además, mostraban un perfil de marcadores compatible con el de células luminales progenitoras. En vista de los resultados, proponemos que los microRNAs de la familia miR-200 inducen características de células progenitoras luminales, las cuales asociadas a un fenotipo epitelial, promueven la capacidad metastática de las células tumorales.
The maintenance of an epithelial gene program is essential for the metastatic growth of epithelial cancers, including breast cancer. However, little is known of the molecular and cellular mechanisms leading to the enhanced proliferative and survival properties of metastatic epithelial cells. We report here that forced expression of miR-200s in MCF10CA1h mammary cells induced not only a strong epithelial program but also aldehyde dehydrogenase (ALDH) activity, mammosphere growth and ability to form branched tubuloalveolar structures while promoting orthotopic tumor growth and lung colonization in vivo. This was accompanied with the expression of luminal differentiation markers in vitro and in vivo, the overall phenotype being compatible with a promotion of luminal progenitor traits. Interestingly, the morphology of tumors formed in vivo by MCF10CA1h cells expressing miR-200s was reminiscent of metaplastic breast cancer (MBC) and the epithelial components of MBC samples expressed significantly higher levels of miR-200s than their mesenchymal components and displayed a marker profile compatible with luminal progenitor cells. Additionally, the miR-200 family showed enhanced expression along progression of invasive ductal breast cancer (IDC) from primary tumors to distant metastases, further reflected in higher blood levels of miR-200b in patients with regional or distant metastases relative to patients with primary node-negative tumors. We propose that the expression of epithelial gene programs through miR-200 family microRNAs promote traits of highly proliferative mammary luminal progenitor cells, thereby exacerbating the growth and metastatic properties of transformed mammary epithelial cells.

Au cours de ma thèse, j’ai étudié la contribution de miR-205 dans le développement du cancer du sein. MiR-205 a été choisi suite à l'analyse comparative de l'expression du miRome entre la lignée « normale » MCF10A et une lignée cancéreuse dérivée MCF10A-CA1a. J’ai démontré que l’expression de miR-205 augmente durant la tumorigenèse tandis que miR-205 est non détectable dans la lignée cellulaire ayant un potentiel métastatique. De plus, j’ai montré que les cellules souches du cancer du sein expriment miR-205, contrairement à la population non souche. En utilisant des cultures de cellules épithéliales 3D, j’ai corrélé la fonction tumorigène de miR-205 à la répression de l'apoptose et non à une prolifération accrue. De plus, le niveau d'expression de la E-Cadhérine dépend de la quantité de miR-205 dans les différentes lignées cellulaires de MCF10A. Les études de perte de fonction suggèrent que la E-Cadhérine est impliquée dans le phénotype acini miR-205-Dépendant, en corrélation avec la transformation de cellules épithéliales du sein. L’ensemble de ces résultats met en lumière la complexité et la duplicité des miRNA durant le processus de cancérisation. Ce type d’étude ouvre des perspectives d’utilisation des miRNA dans le cadre des diagnostics et/ou thérapeutiques
During the time I was working on my thesis, I aimed to understand the role of miR-205 in breast cancer development. MiR-205 was chosen from the comparative analysis of total micro-RNAs expression in non-Transformed and tumorigenic cell lines of the MCF10A breast epithelial cell model. I demonstrated the complexity of miR-205 functions during breast epithelial cell transformation by showing miR-205 overexpression in transformed non-Invasive cell lines and miR-205 down-Regulation in cell line with metastatic potential. Moreover, we demonstrated increased level of miR-205 expression in breast cancer stem cells in comparison with non-Stem cells. Using 3D cultures of breast epithelial cells, I succeeded to correlate the tumorigenic function of miR-205 with its role in modulation of acinar size, and to attribute it to the apoptosis repression but not increased proliferation. Further, I was able to show that miR-205 exercises its oncogenic functions via targeting ZEB1, an inhibitor of E-Cadherin. Indeed, E-Cadherin expression level depends on the amount of miR-205 in different MCF10A cell lines. Downregulating E-Cadherin restored normal acinar morphology in miR-205 expressing cells, consistent with E-Cadherin being involved in the miR-205-Dependent acini phenotype that correlates with tumorigenic breast epithelial cell transformation

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Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG
Introduction: MicroRNAs are small molecules of single-stranded non-coding RNAs that regulate gene expression in the post-transcriptional phase and are being evaluated as prognostic and predictive markers in breast cancer. Objective: The aim of this study was to evaluate the expression of hsa-miR-205ab and hsa-miR-218a microRNAs and BRCA1 protein in breast cancer samples and their associations with the pathological and prognostic clinical aspects of tumors. Method: A group of 92 breast cancer cases was evaluated for clinical-pathological aspects, five-year survival, expression of BRCA1, hsa-miR-205ab and hsa-miR-218a. BRCA1 expression was assessed by immunohistochemistry and the quantification of the miRNAs by quantitative PCR in real time. The clinical-pathological aspects were compared in relation to the expression of BRCA1, hsa-miR- 205ab and hsa-miR-218a using the Mann-Whitney test. Survival curves were generated by the Kaplan-Meyer method and compared by the long-rank method. Results: The group of tumors comprised 56 cases of non-triple-negative breast carcinomas and 36 tumors with triple-negative phenotype. Hypoexpression of hsa-miR-205ab and hsa-miR-218a was associated with larger tumors (> 2 cm), presence of lymph node metastasis and distant metastasis, the highest histological grade, triple negative phenotype, absence of expression of BRCA1 and lower survival. (P = 0.044), more advanced stages (p = 0.005), lymph node involvement (p = 0.038), presence of distant metastasis (p = 0, 0008) and absence of BRCA1 expression (p = 0.039). Conclusion: The hypoexpression of hsa-miR-205ab and hsa-miR-218a was associated with factors of poor prognosis, absence of BRCA1 expression and the lowest survival rate in breast cancer patients evaluated in this study.
Introdução: Os microRNAs são pequenas moléculas de RNAs não-codificantes de cadeia simples, que regulam a expressão gênica na fase pós-transcricional e vem sendo avaliados como marcadores prognósticos e preditivos no câncer de mama. Objetivo: O objetivo deste estudo foi avaliar a expressão dos microRNAs hsa-miR-205ab e hsa-miR-218a e da proteína BRCA1 em amostras de câncer de mama e suas associações com os aspectos clínico patológicos e prognósticos dos tumores. Método: Um grupo de 92 casos de câncer de mama foi avaliado quanto aos aspectos clínico-patológicos, a sobrevida em cinco anos, a expressão de BRCA1, hsa-miR-205ab e hsa-miR-218a. A expressão de BRCA1 foi avaliada por imuno- histoquímica e a quantificação dos miRNAs por PCR quantitativa em tempo real. Os aspectos clínicos-patológicos foram comparados em relação à expressão de BRCA1, hsa-miR-205ab e hsa-miR-218a, utilizando o teste de Mann-Whitney. As curvas de sobrevida foram geradas pelo método de Kaplan-Meyer e comparadas pelo método de long-rank. Resultados: O grupo de tumores compreendeu 56 casos de carcinomas de mama não triplo-negativos e 36 tumores com fenótipo triplo-negativo. A hipoexpressão de hsa-miR-205ab e hsa-miR-218a foi associada aos tumores maiores (> 2 cm), à presença de metástase linfonodal e de metástase a distância, ao grau histológico mais elevado, fenótipo triplo negativo, ausência de expressão de BRCA1 e menor sobrevida. A sobrevida das pacientes em função das características clínico patológicas foi influenciada pelo fenótipo triplo-negativo (p=0,044), estádios mais avançados (p=0,005), acometimento linfonodal (p=0,038), presença de metástase à distância (p=0,0008) e ausência da expressão de BRCA1 (p=0,039). Conclusão: A hipoexpressão de hsa-miR-205ab e hsa-miR-218a foi associada aos fatores de pior prognóstico, ausência de expressão de BRCA1 e à menor sobrevida nas pacientes com câncer de mama avaliadas neste estudo.

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

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references.
In these studies, the microRNA miR-31 was identified as a potent inhibitor of breast cancer metastasis. miR-31 expression levels were inversely associated with the propensity to develop metastatic disease in human breast cancer patients. Additionally, various functional analysis revealed that miR-31 expression was both necessary and sufficient to impede breast cancer metastasis. These effects did not involve confounding influences on primary tumor development; instead, miR-31 exerted its anti-metastatic activities by impinging upon at least three distinct steps of the invasion-metastasis cascade: local invasion, one or more early post intravasation events, and metastatic colonization. At a mechanistic level, miR-31 impaired metastasis via the pleiotropic suppression of a cohort of target genes that otherwise operate to promote metastasis, including integrin a5, radixin, and RhoA. Significantly, the concomitant re-expression of integrin a5, radixin, and RhoA sufficed to override the full spectrum of miR-31'7s anti-metastatic activities. Moreover, the concurrent short hairpin RNA-conferred knockdown of endogenous integrin a5, radixin, and RhoA levels closely phenocopied the known consequences of ectopic miR-31 expression on metastasis. Integrin a5, radixin, and RhoA were found to act during at least partially unique steps of the invasion-metastasis cascade downstream of miR-31. Notably, the temporally controlled re-activation of miR-31 in already-established metastases elicited metastatic regression. These anti-metastatic therapeutic responses were attributable to the capacity of acutely re-expressed miR-31 to induce both cell cycle arrest and apoptosis; such effects arose specifically within the context of the foreign microenvironment present at a metastatic locus. When taken together, these findings provide mechanistic insights concerning the regulation of breast cancer metastasis and suggest that miR-31 may represent a clinically useful prognostic biomarker and/or therapeutic target in certain aggressive human carcinomas. In addition, a novel experimental system for the unbiased identification of metastasisrelevant genes was described. The utility of this system was demonstrated in an initial proof-of-concept screen, which implicated RhoJ as a previously unappreciated modulator of cell motility. Collectively, these observations imply that the single-cell clone-based screening methodology outlined herein may represent a generally useful means by which to enumerate novel regulators of various metastasis-relevant processes.
by Scott J. Valastyan.
Ph.D.

碩士
中山醫學大學
生化微生物免疫研究所
104
Cancer stem cells (CSCs) undergo self-renewal to maintain tumor growth and transform to malignant state. Increased expression of microRNAs (miRNAs), miR-9 and miR-221, has been reported to be associated with cancer aggressiveness, however, the story reside in these miRNA overexpression that endorse tumor cell stemness in outgrowth and invasiveness of breast cancer remains unknown. miR-9 and miR-221 were used to screen in vitro propagation of tumorigenic side-population (SP) of breast cancer cell lines. Paired laser capture microdissected tumor and non-tumor cells were collected from primary cancer tissues of breast cancer patients who were followed up for more than 10 years. Differential expression levels of miRNA in T (tumor)/N (non-tumor) ratio was quantified by qRT-PCR and analyzed their relationship with the clinicopathologic features and outcome of human breast cancer. Morphological and the tumorsphere assay data allows breast CSC formation to be significantly increased upon miR-9 and miR-221 overexpression. On the contrary, cancer cells that lack stem cell properties have limited sphere-forming potential and decreased invasive capability due to inhibition of miR-9 and miR-221 in advanced-stage breast cancer cells. Further, overexpression of both miRNAs showed clinical relevance in tumor tissues categorized as advanced tumor stages and lymph node metastasis (LNM) (p<0.05). In addition, tumors with greater numbers of overexpression of miR-9 and miR-221 were significantly associated with an elevated risk in patients with advanced tumor stage (p-trend=0.003), LNM (p-trend=0.028) and reduced probability of 10-year disease-free survival and overall survival of the patients (log rank P<0.05).Overexpression of miR-9 and miR-221 endow CSCs and given rise to invasiveness of breast cancer cells, thereby opening their potential in earlier prediction of disease progression and an unfavorable outcome in patients with breast cancer.

碩士
中山醫學大學
生化暨生物科技研究所
101
MicroRNAs (miRNAs) are a small non-coding RNAs (~23bps) regulating gene expression by inhibiting mRNA transcription or binding to the specific sequences of 3’-untanslated region (3’-UTR) of the target mRNAs to eventually suppress protein translation. Recently, several miRNAs have been reported to correlate with progression of tumor cells through affecting on the expression of oncogenes and/or tumor suppressor genes that lead to malignant transformation of tumors in different types of human cancer. Chemokine receptor type 4 (CXCR4), a chemokine receptor in the GPCR gene family, encodes a CXC chemokine receptor specific for stromal cell-derived factor-1 and is expressed by cells in the immune system and the central nervous system. In more recent, it has been reported that CXCR4 is overexpressed on several tumor cells, these CXCR4 positive tumor cells may exert pleiotropic effects in regulating processes essential to tumor metastasis through secretion of SDF-1. In this study, to unravel the molecular mechanism underlying CXCR4 signaling by which miRNAs affect breast cancer progression, a computational algorithm combining both miRNA target searching programs of TargetScan 5.1 (www.microrna.org) and microRNA.org was used to putatively predict the CXCR gene might be one of the downstream targets of the microRNA-139 (miR-139) Stepwise, dual-luciferase reporter assay and western blot, we validated that CXCR4 expression levels were reduced after transfection of the human breast cancer cell lines Hs578T and MDA-MB-231 with the precursors of the miR-139. Breast cancers cells whose miR-139 ecotopically expressed have effects on inhibition of migration and invasion. Furthermore, reduced tumor expression level of miR-139 in breast cancer tissue was associated with an unfavorable outcome, including late tumor stage and lymph node metastasis. In conclusion, these findings suggest a role for the miR-139 in inhibiting breast tumor invasiveness and metastasis, moreover, miR-139 downmodulates CXCR4 expression can serve as a novel therapeutic target for the treatment of breast cancer.

RESUMO: Os microRNAs (miRNAs) são pequenos RNAs não codificantes com função reguladora que regulam a expressão génica ao ligar-se a sequências específicas na região 3’ UTR dos mRNAs. Diversos estudos mostraram que os miRNAs regulam mecanismos fundamentais para o normal funcionamento celular, como crescimento celular, proliferação, diferenciação e apoptose. A expressão de alguns miRNAs é alterada em diversos tipos de cancro, nomeadamente em cancro da mama. Estudos de análise funcional em linhas celulares mostraram que os miRNAs podem funcionar como supressores de tumor ou ter actividade oncogénica. Assim, o valor clínico dos miRNAs como potenciais marcadores para cancro da mama está a ser amplamente estudado de momento. No entanto, apenas se conhecem efeitos de alguns miRNAs. A maior dificuldade, neste âmbito, depreende-se com a identificação de possíveis alvos com relevância biológica para cancro da mama. Visto que os programas bioinformáticos predizem um elevado número de falsos positivos e falsos negativos, é de extrema importância identificar experimentalmente alvos relevantes. Nesta tese procuramos explorar diferentes abordagens da influência de miRNAs em cancro da mama. Começamos por estudar os mecanismos que estão por trás da regulação dos próprios miRNAs. Colocámos a hipótese de serem mecanismos epigenéticos, tais como a metilação de citosinas no DNA, que estão a influenciar os níveis de expressão dos miRNAs. Para tal, tratámos linhas celulares de mama com um agente capaz de desmetilar o DNA e verificámos que os níveis de miRNAs são alterados. Contudo, não conseguimos encontrar uma associação entre a metilação de ilhas CpG nas regiões promotoras dos genes que codificam para os miRNAs. No entanto, não podemos excluir a possibilidade de os níveis de expressão de miRNAs estarem a ser regulados por metilação das suas zonas promotoras, dado que não estudámos todas as regiões promotoras existentes. De seguida, abordámos a influência de dois miRNAs, miR-200c e miR-203, na resistência para fármacos dirigidos a cancro da mama, nomeadamente, Paclitaxel e 5-fluoruacil. Para tal fizemos expressar ambos os miRNAs na linha celular MDA-MB-231 e inibir os mesmos na linha celular MCF-7. Infelizmente não fomos capazes de encontrar significado estatístico nos resultados obtidos. Contudo pudemos concluir que o miR-200c parece ter um efeito contrário nas linhas MCF-7 e MDA-MB-231 no que diz respeito ao tratamento com Paclitaxel e o miR-203 parece aumentar a resistência para o mesmo comporto na linha celular MDA-MB-231. O tratamento com 5-fluoruacil não mostrou qualquer diferença em ambas as linhas. Dado que os estudos in vitro, nesta área, devem ser transpostos para humanos e/ou tecidos humanos, seguidamente procurámos estudar os níveis de expressão do miR-200c e do miR-203 em tecido tumoral mamário, bem como a expressão de dois alvos hipotéticos encontrados utilizando ferramentas bioinformáticas, SIX1 e SOX2. Relativamente ao miR-200c, não encontrámos quaisquer diferenças entre tecido normal e tecido tumoral de mama, nem conseguimos relacionar este miRNA com características clinicopatológicas. Comparativamente detectaram-se diferenças para o miR-203 e conseguimos relacionar este com os estadios iniciais de desenvolvimento tumoral. Conseguimos também demonstrar que o miR-203 pode ser um potencial marcador para discriminar os tumores lobulares invasivos. No que diz respeito à expressão do SOX1 e SOX2, observámos que ambos possuem uma incidência baixa na nossa população e que não se associam com a expressão dos miRNAs em estudo. Por último, procurámos validar alguns alvos do miR-200c e do miR-203. Para tal, efectuámos um estudo comparativo de proteómica, onde fizemos expressar o miR-200c e o miR-203 na linha celular MDA-MB-231 e inibimos os mesmos miRNAs na linha celular MCF-10A. Este estudo exploratório, ainda por terminar, revelou aproximadamente 3000 proteínas diferentemente expressas nas linhas celulares. No entanto, até ao momento conseguimos reduzir esta vasta lista para uma menor com aproximadamente 10 proteínas para cada linha celular. De futuro, serão seguidas outras abordagens para validar estes alvos hipotéticos. Devido ao facto da população recolhida ser recente, o seguimento clínico nos próximos anos permitirá tirar algumas conclusões relativas à resistência à terapia utilizada e a sua relação com a expressão dos miRNAs em estudo.
ABSTRACT: MicroRNAs (miRNAs) are small non-coding regulatory RNAs that modulate gene expression by binding to their target mRNAs. By these means, miRNAs control normal rates of all major cellular pathways. A subset of miRNAs, which are differentially detected between normal and tumour tissue samples, has been identified in breast cancer, and functional analysis in cell line systems has revealed tumour suppressive and oncogenic functions of some of these miRNAs. Hence, the clinical value of these as novel biomarkers for cancer is being actively investigated. However, the function of only a few of these miRNAs in breast cancer has been investigated. One major difficulty is the identification of target mRNAs and proteins with biological significance in breast cancer and consequently the identifications of the pathways they influence. Given the relatively high rates of both false-positives and false-negatives from current miRNAs targets prediction programs, it is critically important to experimentally identify relevant miRNAs targets and the pathways involved in carcinogenesis. In this thesis our main goal was to study the role of miRNAs in breast cancer. Thus, we started by addressing the mechanisms behind regulation of miRNAs expression levels. We hypothesized if that epigenetic mechanisms, such as DNA methylation, could influence miRNAs expression levels. Therefore, we treated breast cell lines with demethylating agents and observed that miRNAs expression levels were altered. However, we failed to prove a direct correlation between the methylation of CpG islands in promoter regions of the miRNAs studied and their expression. Nevertheless, we cannot exclude the possible regulation of miRNAs levels by methylation since we did not study all possible promoter regions of miRNAs genes as their promoter regions have not been fully identified. Next, we addressed the possible effect of miRNAs in breast cancer therapy resistance. For that, we treated breast tumour cell lines with Paclitaxel and 5-fluoruacil, two known chemotherapeutics used in breast cancer, with the ectopic expression or inhibition of miR-200c and miR-203. Unfortunately, we did not find any statistical difference between untreated and treated cells. However, miR-200c seems to have contrary effects in MCF-7 and MDA-MB-231 cells regarding treatment with Paclitaxel and miR-203 seem to augment resistance to Paclitaxel in MDA-MB-231 cells. Both miRNAs did not show any effect in cells treated with 5-Fluoruacil. Since in vitro studies, always lack studies using human tissues. We subsequently studied the expression levels of miR-200c and miR-203 in breast tumour tissues and two putative targets found by bioinformatics tools, SIX1 and SOX2. Concerning miR-200c, we did not detect significant differences between normal breast and tumour tissues in our population. Additionally, we failed to correlate miR-200c with clinicopathological features. Regarding miR-203, we detect statistically differences between normal and tumour tissue and it seems that miR-203 is involved in breast cancer development, mainly in early stages of development. We also show that miR-203 might be a potential marker to discriminate stages in invasive lobular carcinoma. About the expression levels of SIX1 and SOX2, we observe relatively low levels of both proteins through immunohistochemistry and do not found any statistically difference between their expression and their regulators, miR-200c and miR-203. Finally, we address the validation of putative targets of the miR-200c and miR-203. Thus, we conducted a comparative proteomic study to find differently expressed proteins when miR-200c and miR-203 were ectopically expressed or inhibited in breast cell lines. This exploratory study, until now, revealed a small list out of approximately 3000 proteins that are putative targets of both miRNAs and are differently expressed. Further studies will be conducted in order to validate these putative targets. With this thesis, we believe we provide new insight into the involvement of miRNAs in breast cancer and also important knowledge of how miRNAs levels are being regulated and also in the discovery of new targets. We also gathered a considerable study population during this thesis, which allow future correlations on therapy outcomes and survival with the biomarkers studied.

碩士
中山醫學大學
生化暨生物科技研究所
101
MicroRNAs (miRNAs), a family of small molecular of RNAs transcripts, are important gene regulators that play a crucial role in cell tumorigenesis. Recently, it has been reported that miRNA can affect epithelial-mesenchymal transition (EMT) during tumor cell progression. Our previous studies showed that miR-30a downmodulate Vimentin expression and inhibit breast cancer cell migration and invasion. It has been reporter that the Resveratrol, one of the polyphenol chemicals, can may inhibit cancer cell growth, invasion and metastasis. For this reason, the present study was aim to explore the synergistic effect of miR-30a and RES in inhibiting invasion and migration in breast cancer cells, Besides, the molecular mechanism underlying EMT signaling mediated by miR-30a to affect invasion and migration of breast cancer was examined. Initially, we observed that RES inhibits migration and invasion of breast cancer cells Hs578T. Further, increased expression of miR-30a can enhance the inhibitory effect of invasion and migration on the RES treated Hs578T cells. In the presence of miR-30a, mesenchymal related markers (Slug, Vimentin and N-cadherin) were downregulated. Whereas, expression levels of epithelial proteins of the claudin family were increased in RES-treated tumor cells whose miR-30a was ecotopically expressed. In conclusion, our data provide the evidence to support the preferential role of miR-30a in association with RES inhibiting migration and invasion of breast cancer.