Thesis (Ph. D.)--University of Texas at Austin, 2006. / Vita. Includes bibliographical references.

Resumo: Com o objetivo de caracterizar pelo método histológico e imunoistoquimíco o câncer de mama de cadelas sem raça definida (SRD) foram estudados 40 animais com distribuição uniforme nos estágios I, II, III e IV da doença. Todos os animais foram submetidos a anamnese, exame físico e complementares (Citopatológico, Raio x, Ultra Som) para o estadiamento clínico do câncer (TNMc), amostra histológica da lesão para caracterização do tipo de tumor e amostra imunoistoquímica para determinação molecular das células em apoptose (caspase 3) e proliferação (PCNA). A amostra foi caracterizada com predominância de idade acima de seis anos (92,5%), obesos (57,5%) com ingestão de dieta mista (80%) caseira-industrial. 80% das cadelas já tiveram cria, apresentando pseudogestação (40%), tumoração das mamas inguinais (70%), em ambas as cadeias, com nódulos multicêntricos (82,5%) de incidência bilateral (60%), prevalecendo nódulos de tamanho inferior a 2cm de diâmetro (37,5%). Prevaleceu o carcinoma simples (70%) em relação ao carcinoma complexo (30%). Os animais com carcinoma simples apresentaram características de porte grande (50%), obesidade (57%), cios regulares (89%) e multíparas (82%) e prevalência idêntica entre os estadiamentos clínico (70%). O estadio mais grave (IV) esteve associado aos animais com idade média de 8 anos, ausência de obesidade (60%). A imunomarcação de PCNA e caspase-3 não apresentou diferença significativa entre os tipos de tumor, suas características clínicas e grau histológico, no entanto a caspase-3 teve associação significativa entre o estadiamento clínico (I<III e II<III) e presença de ulceração tumoral (P<0,049) / Abstract: The severity of breast cancer in mongrel animals is not defined yet in its clinical, histologic and imunoistoquimical associative aspects. With the aim to establish that characterization we studied 40 bitches, 3-15 years old, mixed breed attended at the U. Vet. Hospital ambulatories in compliance with current ethical and pathological rules. The animals were alloted according to their clinical stage in groups stage I, II, III and IV with 10 animals each. After physical examination and lifestyle records. The tumor nodules were evaluated macro-and-microscopically, the later through histopathologicaly and imunoistopathologicaly. From these data the animals were classified as predominantly up to six years old (92,5%), obese (57,5%), presenting simple carcinoma (CS) more frequent (70%) than complex carcinoma type (30%). 80% of the animals wasanþt nuliparus, showing pseudopregnancies (40%), having multicentric nodules (82,5%) sizing less than two cm diameter (37,5%), located inguinally (70%) in both side mammary chains. The immunohistochemistry label for PCNA and caspase-3 were not associated with tumors type, clinical aspects and histological grade, however only caspase-3 had significant association with clinical stage (I<III and II<III) and tumors ulceration (P<0,049) / Orientador: Enio Pedone Bandarra / Coorientador: Renée Laufer Amorim / Banca: Maria Denise Lopes / Banca: Luiz Henrique do Araujo Machado / Banca: Paulo César Maiorka / Banca: Andrigo Barbosa de Nardi / Doutor

The Wilms' Tumour Suppressor gene 1, WT1, encodes for a complex protein which is essential in mammals throughout life. Its roles vary with the developmental stages: in the embryo, it regulates the epithelial-mesenchymal balance required for a correct organogenesis and acts as a tumour suppressor; in the adult, it is involved in the maintenance of tissue homeostasis and has been controversially considered as an oncogene. Breast cancer is one of the adult tumours in which WT1 oncogenic function was first hypothesised. This malignancy is the most common in women, with more than one million cases being diagnosed worldwide every year, and represents the leading cause of cancer related deaths. Because of its major health burden, this disease has been extensively studied and special attention has also been paid to normal mammary gland biology: several works have shown that breast cancer can be divided into many molecular subtypes, which may reflect the cell of origin of the tumour; moreover, many genes involved in the normal development of the mammary gland have been proven to also play a role in breast tumorigenesis. WT1 expression has been previously reported in both healthy mammary glands and breast cancer samples, however, its function in this context is not well understood and the evidence gathered so far is extremely contradictory. This thesis aimed to investigate the exact role played by WT1 in both mammary gland and breast cancer biology, using a combination of in vivo and in vitro techniques. Following flow cytometry isolation, Wt1 mRNA expression was detected in the myoepithelial and stem cell subpopulations of the healthy gland. To investigate the effects of WT1 loss, Wt1 conditional mice were crossed with two different mammary specific Cre lines: the knockout animals developed, bred and lactated normally, however, they showed a significant increase of ductular branches during pregnancy, suggesting that WT1 may be involved in the regulation of branching morphogenesis. In order to study WT1 role in mammary tumours, the gene was knocked out in a breast cancer mouse model and knocked down in several breast cancer cell lines, using both constitutive and inducible lentivirus-based systems. WT1 loss did not seem to affect cell proliferation, but resulted in a significant increase in cell migration in vitro and in the upregulation of mesenchymal markers. Furthermore, bioinformatics analysis showed that the WT1-positive tumours mainly belong to the luminal/ER-positive subtypes and express lower levels of mesenchymal markers than the WT1-negative tumours. As a whole, the findings of this thesis characterise WT1 expression in the healthy mammary gland and provide the first evidence of its possible function in this organ; moreover, this work seems to rule out an oncogenic role for WT1 in breast cancer, while suggesting that it could be an upstream regulator of cell migration. Additional experiments are required to confirm this result in vivo and verify whether it could lead to any clinical application.

Made available in DSpace on 2014-06-11T19:31:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-02-27Bitstream added on 2014-06-13T19:41:03Z : No. of bitstreams: 1 santos_das_dr_jabo.pdf: 2572223 bytes, checksum: aea5701db88f8bf27923f6c9ed630f4e (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Este estudo utilizou 15 fêmeas bufalinas, provenientes de abatedouros frigoríficos dos estados do Maranhão, São Paulo e Paraná para estudo morfológico, macroscópico, microscópico e morfométrico das papilas mamárias. O material para estudo macroscópico foi pesado, medido e fotografado, e os resultados do peso papilar foram de: cranial direita (PCD) 20, 95g; caudal direita (PCaD) 21,49g; cranial esquerda (PCE) 23,52g e para caudal esquerda (PCaE) 21,35g. Os valores médios para comprimento do ducto papilar, número de pregas e comprimento da cisterna papilar foram de: 0,67cm; 5,5 e 2,17cm para PCD; 0,62cm; 6,0 e 2,26cm para PCaD; 0,71cm; 6,0 e 2,26cm para PCE e 0,74cm; 5,7 e 2,57cm para PCaE, respectivamente. A análise de correlação de Pearson indicou não existir relação entre o peso corpóreo e o peso glandular. Fragmentos das regiões proximal, média e distal foram colhidos e fixados em solução aquosa de formol 10%, cortados com 10, 20 e 40mm e corados em Azan, Picrocírus F3BA e Weigert-Van Gieson, para a análise mesoscópica. Cortes com 5mm corados em HE, tricrômico de Masson foram utilizados para a análise histológica e morfométrica. A estrutura histológica da papila mamária em búfalas mostrou-se similar aquela dos bovinos, a camada muscular é formada por fibras em duas disposições, a interna circular e a externa longitudinal, e com epitélio cúbico biestratificado em quase toda a sua extensão. Na porção média da papila a camada mais desenvolvida é a muscular com 22,90mm de espessura, a qual possui grande quantidade de tecido conjuntivo denso (7,10 mm). / Fifteen Murrah female buffaloes, proceeding from abattoirs of Maranhão, São Paulo and Paraná states, were used in this study and submitted for morphologic, macroscopic, microscopic and morphometric analysis of the mammary papilla. The material for macroscopic study was weighed, measured and photographed, the results for papillary weight had been: cranial right (PCD) 20.95g; caudal right (PCaD) 21.49g; left cranial (PCE) 23.52g and for left caudal (PCaE) 21.35g. The average values for the papillary duct length, number of folds and length of the papillary cistern had been respectively for PCD: 0.67cm, 5.5, 2.17cm; PCaD: 0.62cm, 6.0, 2.26cm; PCE: 0.71cm, 6.0, 2.26cm and PcaE: 0.74cm, 5.7, 2.57cm. The analysis of de Pearson correlation coefficient test between corporeal weight and the glandular weight indicated not relation existence. Proximal, meddle and distal tissue samples had been collected and fixed in 10% formol watery solution and cut with 10-20-40mm and stained in Azan, Picrosirus F3BA and Weigert-Van Gieson, for the mesoscopic analysis. Cuts of 5mm and had stained in HE and Masson's trichrome were submitted to the morphologic and morphometric analysis. Buffalo's' mammary papilla structure revealed to be similar to the described for bovines, the muscular layer was composed by two bundles of smooth muscle fibers, the external circular layer and the longitudinal external one. The mucosa was bi-stratified cuboidal ephitelia in almost all its extension. The papilla meddle portion muscular layer was 22.90mm of thickness which contained large amount of connective tissue (7.10mm).

Made available in DSpace on 2014-06-11T19:27:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-02-28Bitstream added on 2014-06-13T18:56:52Z : No. of bitstreams: 1 paiva_ce_me_botfm.pdf: 1255697 bytes, checksum: 3b134e3a9a4224016105b510cbe1c230 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O câncer de mama (CM) ocupa o primeiro lugar em incidência e o segundo em mortalidade entre as mulheres no mundo. É considerada uma doença heterogênea com alterações em diversas vias de sinalização molecular. Os fatores prognósticos atuais são incapazes de predizer a evolução da totalidade dos pacientes, assim como os marcadores preditivos não conseguem evitar que grande parte das pacientes com CM sejam submetidas à tratamento desnecessário. A busca de outros marcadores tumorais, tanto prognósticos quanto preditivos tem sido alvo de inúmeras pesquisas, que buscam predizer a evolução clínica de um número maior de pacientes. Dois marcadores tumorais com possíveis implicações clínicas são o TGFB1 e o seu receptor TGFBR2. No entanto, o papel do TGFB1 na carcinogênese e na progressão do câncer de mama ainda não é totalmente conhecido. Desta forma, o objetivo deste estudo foi avaliar a expressão do TGFB1 e do TGFBR2 em tumores de mama e correlacionar com os dados clínicos e histopatológicos. Para tanto, foram analisadas 49 amostras de carcinomas ductais invasivos primários e sete amostras de mamas normais como controles pelas técnicas de RT-PCR quantitativa em tempo real (qRTPCR) e imunohistoquímica (IHQ). Os resultados mostraram que baixos níveis de expressão protéica de TGFB1 nas células tumorais estavam associados a menor sobrevida livre de doença. Os tumores apresentaram acentuada diminuição da expressão do transcrito TGFBR2 quando comparados ao controle normal. Além disso, foram observados baixos níveis dos transcritos TGFB1 e TGFBR2 nos tumores com fenótipos mais agressivos (alto índice proliferativo e estadio avançado). Esses dados sugerem que o TGFB1 atua como supressor tumoral em CMs e que a diminuição da expressão TGFB1 e TGFBR2 é um evento importante na carcinogênese mamária. A detecção dos níveis de expressão protéica... / Breast cancer (BC) is the leading female cancer in incidence and the second one in mortality worldwide. BC is considered a heterogeneous disease, that presents abnormalities in several molecular pathways. The known prognostic markers are not capable to predict the outcome of all the patients, as well as the predictive markers are not capable to avoid unnecessary treatments. It is necessary to search for other tumor markers, even prognostic and predictive, regarding the prediction of a higher number of patient’s outcome. Two tumor markers with possible clinical implications are TGFB1 and its receptor TGFBR2. However the role of TGFB1 in carcinogenesis and in the BC progression is not totally understood yet. In this way, the aim of this study was to analyze the expression of TGFB1 and TGFBR2 in breast tumors and to correlate it with clinical and histopathological data. For this, it were analyzed 49 ductal invasive carcinomas and 7 normal breast tissues as controls with quantitative real time PCR (qRT-PCR) and immunohistochemistry (IHC) technologies. The results showed that low expression levels of TGFB1 protein in tumor cells were associated with a lower disease-free survival. Tumors presented highly diminished TGFBR2 transcript expression when compared with normal controls. Moreover, low levels of TGFB1 and TGFBR2 transcripts were observed in tumors with aggressive phenotypes (high proliferation index and advanced stage). These results suggests that TGFB1 acts like a tumor supressor in BCs and that the TGFB1 and TGFBR2 lowering expression is an important event in mammary carcinogenesis. The detection of tumor TGFB1 protein expression levels can be helpful in clinical practice like a prognostic marker in mammary carcinomas.

The mammary gland contains a subset of cells with regenerative capacity that is able to generate both luminal and myoepithelial mammary epithelial lineages. Those cells are described as mammary epithelial stem cells. The fate of stem cells is tightly controlled by their microenvironment and adhesion receptors on the stem cells play a vital role in the microenvironment–stem cell communication. They facilitate the interaction of stem cells with the extracellular matrix as well as adjacent cells, and they regulate stem cell homing to their niches, as well as stem cell proliferation, self-renewal, and differentiation. Stem cells express high levels of ECM binding adhesion receptors such as β1 and α6-integrins. Those integrins were used to isolate stem cells from the rest of the differentiated epithelial cells within the mammary gland. However, little is known about the role of those integrins in stem cell self-renewal and differentiation. This project aimed to understand how β1-integrin receptors contribute to stem cell behavior. To achieve this, FACS sorting method of stem cells, the organoid assay, and lentivirus knockdown of β1-integrin using shRNA were optimised. The organoid assay was used as an in-vitro test to assess for the frequency of bi-lineage and luminal progenitor cells in a given mammary epithelial population. It is known that bi-lineage cells produce solid organoids in culture while luminal progenitors produce hollow organoids. The frequency of solid and hollow organoids might therefore be an indication of the stem cells and luminal progenitor frequency respectively. My results showed that cells with the highest solid organoid forming ability were within the basal population, which is high for β1- and α6-integrin. The β1-integrin signaling pathway was shown to be important for maintaining the organoid-forming population in basal and luminal populations. Knocking out β1-integrin in MECs resulted in abolishing their solid and hollow organoid-forming activity. Downstream of β1-integrin, I found that Rac1 but not ILK is important in β1-integrin maintenance of solid organoid-forming cells. Active Rac1 was able to rescue solid organoid formation but was not able to rescue hollow organoids in the β1-integrin knockdown cells. β1-integrin and Rac1 deletion resulted in the down regulation of Wnt/β-catenin signaling, which is important for stem cells. This down regulation was rescued using active Rac1. Activating Wnt/ β1-catenin signaling in primary cells (using Wnt3a ligand or GSK3β inhibitor) resulted in an increase in solid organoid and a decrease in hollow organoid formation. When activating Wnt signaling using GSK3I in β1-integrin knockdown cells, the solid organoid activity was rescued. However, Wnt3a did not rescue solid organoid formation in the β1-integrin knockdown cells. When active Rac1 was overexpressed in β1-integrin null cells, Wnt3a was able to activate solid organoid formation. When inhibiting Rac1 in primary MECs, solid but not hollow organoid activity was significantly decreased. Wnt3a or GSK3I addition did not rescue this reduction. Taken these results together, it can be concluded that β1integrin-Rac1 signaling play a role in controlling stem cells and this is might be achieved through controlling Wnt/β-catenin signaling. These studies are important in understanding the role of integrins in mammary stem cells. They will also provide new insight on how integrins might be controlling breast cancer and thereby, help in providing new targets for cancer therapy.

No description available.

Luminal epithelial cells in the mammary gland require two types of signals to proliferate: soluble signals (growth factor signals) and signals from the extracellular matrix (ECM). The composition of the ECM is sensed by adhesion receptors such as integrins. Integrins modulate cell behaviour and play a key role in cell cycle entry. Altered integrin expression and signalling has been associated with breast cancer and studies using mouse mammary epithelial cells (MECs) have shown that the absence of β1-integrin induces growth arrest. However, it is not completely understood how integrins transduce the signals from the plasma membrane to the nucleus to induce cell cycle entry. Thus, the first aim of this project was to determine how β1-integrin controls proliferation in MECs. I established a model to study the effects of depleting β1-integrin using the FSK7 mammary epithelial cell line. The proliferation defect observed in this β1-integrin knockdown model was rescued by expressing a constitutively active Rac1 or Pak. Moreover, inhibiting Rac1 or Pak prevented normal proliferation in MECs in a similar fashion as β1-integrin depletion. Furthermore, in this thesis I have identified the complex comprised of Src, paxillin and p130Cas as a potential link between β1-integrin and Rac1. These results provide an insight into the mechanism that regulates proliferation downstream of β1-integrin. During breast cancer initiation, β1-integrin signals are disrupted. This indicates that additional signals must be driving proliferation during tumorigenesis. Therefore, the second aim of this project was to test whether expression of breast oncogenes can overcome the proliferation defect present in β1-integrin null cells. In order to do so, an oncogenic ErbB2, a constitutively active form of Akt (myrAkt) and the Notch1 intracellular domain (NICD) were transfected in the β1-integrin knockdown MECs. The results showed that ErbB2 overcomes the need for β1-integrin by signalling to Pak. NICD does not require β1-integrin to drive proliferation by an unknown mechanism. Expression of myrAkt did not restore normal levels of proliferation in β1-integrin depleted MECs. This finding suggests that Akt is not sufficient to induce cell cycle entry by itself and instead, both Akt and Erk signalling are needed to exert this function. This work has further delineated the specific signals controlling proliferation downstream of β1-integrin, and has provided a model to test the dependence of oncogenes for β1-integrin to drive proliferation in MECs. These studies are important to understand the role of β1-integrin in breast cancer formation and to define the types of breast cancer where β1-integrin can be used as an effective therapeutic target.

Continuing the study of the relationships between fibrin investment of the tumor, vascularity, and tumor growth, we decided to investigate the relationship of warfarin sodium anticoagulation with tumor growth and vascularization. It was reasoned that if the previously observed altered tumor growth was due the heparin’s anticoagulant effect rather than a direct effect upon the tumor, another anticoagulant with a different mechanism of action would have the same tumor growth reducing capabilities. Warfarin sodium produces reduced fibrin polymer formation by a mechanism entirely different from that of heparin. Heparins’ immediate anticoagulant activity results from a blockade of thrombin’s activity results from a blockade of thrombin’s activity on fibrinogen, prevention of prothrombin conversion to thrombin, and a reduction in platelet adhesiveness. Warfarin’s delayed activity, however, is through an inhibition of vitamin K activity leading to reduced synthesis of several clotting factors. With the decision to use warfarin sodium, experiments were designed to test the hypothesis that a reduction of prevention of fibrin formation and thus tumor encasement with this polymer would alter tumor growth. It was also hypothesized that, accompanying the altered tumor growth, several macroscopic factors including tumor vascularization, extent of tumor attachment, vasodilation of host blood vessels in the locale of the implanted tumor, and local edema fluid would be altered. Experiments were conducted to determine the relationship between the dose of warfarin sodium administered and the degree of alteration of tumor growth and the related factors. An inverse dose-response relationship between dose of warfarin and tumor growth and the related parameters was hypothesized.

The mammary gland of lactating transgenic animals has several advantages for production of heterologous proteins including a high cell density that results in high concentrations of secreted protein and the ability to perform several types of post-translational modifications. Transgenes were constructed from the 4.1 kbp murine Whey Acidic Protein promoter (mWAP) and the three cDNAs coding for the Aα, Bβ and γ fibrinogen chains to evaluate the requirements of the transgenic murine mammary gland for high level secretion of fully assembled human fibrinogen. After introducing the constructs into the murine zygotes by microinjection, secretion of fully assembled fibrinogen into milk was measured at concentrations between 10 ug/ml to 200 ug/ml. In one line of mice the total secretion of fibrinogen and unassembled subunits approached 700 ug/ml in milk. The level of assembled fibrinogen was proportional to the lowest amount of subunit produced where both the Bβ and γ chains were rate limiting. Also, the subunit complexes γ₂, Aαγ₂ and the individual subunits Aα, Bβ and γ were found as secretion products. This is the first time that secretion of individual Bβ-subunits by any cell type has been reported and suggests the organization of the secretion pathway in mammary epithelia is different from that in liver. Glycosylated forms of individual Bβ-chain contained a complex saccharide with low mannose. Glycosylation of the γ-chain was also observed. These results suggest the 4.1 mWAP promoter can drive expression of fibrinogen cDNAs to high levels and that the amount of fully assembled fibrinogen secreted is equal to the level of the lowest expressing chain. / Master of Science