As interações entre as células neoplásicas e o estroma têm um papel central na progressão tumoral Crescimento e invasão das neoplasias depende de modificações controladas dos componentes da matriz extracelular. A fronte de invasão tem sido considerada a região mais agressiva das neoplasias malignas, pois suas células são as que apresentam a maior habilidade de invadir os tecidos adjacentes. Por essa razão, a região da fronte de invasão é a área mais crucial da interface tumorhospedeiro. O objetivo deste trabalho é investigar, através de imunoistoquímica, a expressão e distribuição das proteínas da membrana basal laminina e colágeno tipo IV e da matriz extracelular colágeno tipo III, fibronectina e tenascina, na região de fronte de invasão do carcinoma epidermóide de lábio, a neoplasia maligna mais freqüente na cavidade oral. Os achados foram comparados à graduação histológica de malignidade da fronte de invasão. Vinte casos de carcinoma epidermóide de lábio, todos apresentando elastose solar ? que evidencia a participação da radiação ultravioleta do sol no processo ? foram estudados. A graduação histológica de malignidade da fronte de invasão foi feita de acordo com os critérios propostos por Bryne et al. (1989), usando-se cortes de 5 ?m corados por hematoxilina e eosina. As reações imunoistoquímicas foram realizadas em cortes de 3 ?m obtidas a partir de material fixado em formol e emblocado em parafina e usando-se o método da estreptavidina-bioina e anticorpos apropriados. Dos 20 casos estudados, 10 foram graduados como bem diferenciados (BD), 7 moderadamente diferenciados (MD) e 3 pobremente diferenciados (PD). Tanto laminina quanto colágeno tipo IV tiveram resultados semelhantes, apresentando descontinuidade da marcação com freqüência. Quase todos os casos mostraram algum grau de descontinuidade na fronte de invasão para esses dois antígenos. Alguns poucos casos revelaram, inclusive, a perda total da marcação nessa área. Não foi possível relacionar-se a graduação histológica de malignidade com o grau de descontinuidade da membrana basal. Dezessete casos mostraram perda total do colágeno tipo III na região da fronte de invasão. Dentre os 3 casos que apresentaram marcação para a proteína , dois eram BD e 1 MD. A fibronectina foi muito abundante em todos os casos estudados. Essa proteína estava presente em uma área espessa de marcação iniciando-se na região de membrana basal. A marcação para a tenascina foi muito variável dentre os casos e não estava relacionada com a graduação histológica de malignidade ou com a intensidade da resposta inflamatória. Em conclusão: os resultados para laminina, colágeno IV e III sugerem uma importante atividade invasiva na fronte de invasão da neoplasia. Embora o padrão de expressão das proteínas estudadas estava alterada na região de fronte de invasão em comparação com o epitélio normal da região, não houve correlação com o grau histológico de malignidade. / Interaction between tumor cells and stroma plays a central role in cancer progression. Local growth and invasion of neoplasms depend on the controlled modification of components of the extracellular matrix. The invasive front has been considered the most aggressive part of a tumor whose cells have the greatest ability to invade surrounding tissue. Therefore, this is the most crucial area at the tumourhost interface. The aim of this study was to investigate the immunohistochemical expression and distribution of the basement membrane proteins laminin and collagen type IV, and the extracellular matrix proteins collagen type III, fibronectin and tenascin, in the invasive front region of lip SCC, the most frequent malignancy of the oral cavity, and compare the findings to the histological malignancy grading of the invasive front. Twenty cases of lip SCC, all showing solar elastosis, were studied. Histological grading of the invasive front was carried out according to the criteria proposed by Bryne et al. (1989), using 5?m sections stained with H&E. Immunohistochemistry reactions were performed in 3?m sections obtained from formalin-fixed, paraffin-embedded tissue, using the streptavidin-biotin-peroxidase method and appropriate antibodies. Of the 20 cases studied, 10 were graded welldifferentiated (WD), 7 moderately-differentiated (MD) and 3 poorly-differentiated (PD). Both laminin and collagen IV had similar results presenting frequent discontinuity. Almost every case showed at least some discontinuity in the invasive front. A few cases have showed a total loss of immunostaining in the area. It was not possible to determine a correlation between histological grade and degree of basement membrane discontinuity. Seventeen cases have shown total loss of collagen III expression in the invasive front. The three cases that presented positivity were represented by 2 WD and 1 MD SCC. Fibronectin was very abundant in all cases. It was present in a thick area starting from the basement membrane. Staining for tenascin was very variable among the cases and it was not correlated to the histological grading or intensity of inflammatory response. The results for laminin, collagen IV and III suggest invasive activity in the tumoral invasive front. Although the pattern of the proteins were altered in the invasive front of lip SCC in comparison to normal lining epithelium, these changes were not correlated to histological malignancy grade.

Considerando que o colágeno tipo V (Col V) pode ser exposto em processos de remodelamento tecidual, gerando autoimunidade em diversas patologias, postulamos que a administração oral de Col V possa modular a inflamação e remodelamento da sinovite articular experimental, evitando a destruição articular. Neste estudo a artrite foi induzida em ratos Lewis, machos com aproximadamente 250g, por injeção intra-articular de 500 ug de albumina de soro bovino metilada (mBSA) emulsificada em adjuvante de Freund completo (ACF) (10 ul), seguido por reforços intra-articulares de mBSA (500 ug) em solução salina (50 ul), após 7 e 14 dias da primeira injeção (grupo com artrite induzida; AI). Animais sem indução de artrite receberam solução salina (50 ml) por via intra-articular (grupo controle; CT). Após a primeira injeção de mBSA/ACF, a suplementação de Col V bovino (300ul/500ug) foi administrada por via oral nos animais com AI, durante 15 e 30 (AI-ColV15, n=10; AI-ColV30, n=10 ) dias e nos animais CT por 30 (CT-ColV30, n=10) dias. Nossos dados demonstraram que após 15 e 30 dias, a administração oral de Col V levou à diminuição do edema e infiltrado inflamatório severo, conteúdo total de fibras de colágeno, colágeno tipo I, subpopulação de linfócitos, macrófagos, IL-1beta, IL-10 e metaloproteinase 2 no tecido sinovial, e redução IL-1beta, TNFalfa, IL-17 e IL-10 no soro. Ocorreu maior expressão de caspase 9 no tecido sinovial dos animais que receberam o Col V. Pode-se concluir que a suplementação com Col V bovino reduziu a inflamação sinovial e resposta fibrótica independentemente do tempo, possivelmente por aumento da apoptose das células inflamatórias / Whereas type V collagen (Col V) can be exposed in tissue remodeling process, leading to autoimmunity in various pathologies, we postulate that the oral administration Col V can modulate inflammation and remodeling of joint experimental synovitis, preventing joint destruction. In this study, arthritis was induced in Lewis male rats of approximately 250g, by intra-articular injection of 500 ?g serum albumin methylated from bovine serum (mBSA) (500 ug) emulsified in complete Freund\'s adjuvant (CFA) (10 ul), followed by stiffeners intraarticular of mBSA (500 ug) in saline (50 ul) after 7 and 14 days from the first injection (group induced arthritis; AI). Animals received no arthritis induction brine (50 ul) by intraarticular (control group; CT). After the first injection of mBSA / CFA supplementation Col V bovine (300ul / 500ug) was administered orally in animals with AI for 15 and 30 (AI-ColV15, n = 10, I-ColV30, n = 10) days and animals CT per 30 (CT-ColV30, n = 10) days. Our data showed that after 15 and 30 days, oral administration of Col V led to the reduction of edema and severe inflammatory infiltrate, total content of collagen fibers, collagen type I, subpopulation of lymphocytes, macrophages, IL-1beta, IL-10 and metalloproteinase 2 in synovial tissue and reduced the IL-1beta, TNFalfa, and IL-17 IL-10 serum. There increased expression of caspase-9 in the synovial tissue of animals receiving Col V. It can be concluded that supplementation with bovine Col V reduced synovial inflammation and fibrotic response regardless of time, possibly by increased apoptosis of inflammatory cells

O câncer de mama é no Brasil o segundo maior causador de morte entre mulheres. Os hormônios sexuais estão dentre os vários fatores indutores ou promotores da carcinogênese. ADAMTS (uma desintegrina e metaloproteinase com domínios trombospondina) é uma enzima Zn2+/Ca2+dependente. Avaliamos a influência dos hormônios na expressão e localização da ADAMTS-1 em 3 linhagens de mama. qPCR demonstrou que a progesterona estimulou o aumento de mRNA de ADAMTS-1 na MCF-10A e na MDA-MB-231, o tratamento com estrógeno e progesterona estimulou a diminuição. No Western blot observamos nas linhagens MCF-10A e MCF-7 que os hormônios tendem a aumentar os níveis de ADAMTS-1, e o estrógeno estimulou uma acentuada secreção em MCF-7. Na linhagem MDA-MB-231, os tratamentos demonstraram uma tendência a diminuir os níveis de ADAMTS-1 intracelular. Na imunofluorescência e Western blot observamos a predominância de ADAMTS-1 nuclear. Os resultados sugerem que os hormônios regulam a expressão de ADAMTS-1 nas células normais e tumorais, e que está predominantemente presente no núcleo celular. / Breast cancer is the second largest cause of death among women in Brazil. Sex hormones are one of the several inducers factors or cancer promoters. ADAMTS (a disintegrin and metaloproteinase with thrombospondin motifs) are dependent on Zn2+/Ca2+ enzymes. We evaluated influence of hormones on ADAMTS-1 levels and localization in three different breast cell lines. qPCR showed that progesterone stimulated an increase of ADAMTS-1 mRNA in MCF-10A, however, in MDA-MB-231, the treatment by estrogen and progesterone decreased levels. Western blot analysis showed a tendency to increased ADAMTS-1 levels in MCF-10A and MCF-7 due to the hormone treatment. Treatment by estrogen led to a marked secretion in MCF-7. In MDA-MB-231 the treatment showed a tendency to decreased intracellular ADAMTS-1 protein levels. Immunofluorescence and Western Blot we observed ADAMTS-1 predominant in the nucleus. Results suggest that sex hormones regulate the ADAMTS-1 expression in normal and tumoral breast cells, ADAMTS-1 is predominant in the cellular nucleus.

A Parede lateral da faringe parece ser a estrutura central envolvida no colapso da faringe nos pacientes com síndrome da apnéia obstrutiva do sono (SAOS). Os pacientes com roncos e apnéia têm uma faringe mais colapsável associada a um espessamento da musculatura da parede lateral da faringe, em grande parte formada pelo músculo constritor superior da faringe. O endomísio da matriz extracelular do músculo esquelético tem uma íntima relação com as células musculares, influenciando suas propriedades mecânicas e biológicas, podendo modificar seu comportamento e função. As fibras colágenas e elásticas formam o arcabouço do tecido conectivo, enquanto os proteoglicanos e as glicoproteínas estruturais (fibronectina) têm importante papel na propriedade de adesão e hidratação dos tecidos e as metaloproteínas são responsáveis pela degradação dos componentes da matriz extracelular. Os fatores determinantes da complacência excessiva da musculatura da parede lateral são desconhecidos. É possível que a MEC tenha um papel relevante neste aspecto. O objetivo deste estudo é descrever e comparar a densidade dos componentes da matriz extracelular do endomísio da musculatura da parede lateral da faringe em controles, roncadores e apnéicos. Neste estudo prospectivo foram avaliados 61 pacientes maiores de 18 anos e não obesos do Departamento de Otorrinolaringologia do Hospital das Clínicas da Faculdade de medicina da Universidade de São Paulo, com indicação de cirurgias faríngeas no período de 2005 e 2006. Os pacientes foram divididos em três grupos: controles (14 pacientes sem roncos ou SAOS), 18 roncadores primários e 29 pacientes com SAOS. Os espécimes foram obtidos da musculatura da parede lateral da faringe (constritor superior da faringe) durante cirurgias faríngeas. Por meio de análise histoquímica e imunohistoquímica foi determinada a área proprocional do colágeno total, fibras elásticas, colágeno tipos I e III, metaloproteínas 1 e 2, versican e fibronectina, no endomísio destas amostras e comparada entre os grupos. Os resultados foram ainda correlacionados com dados antropométricos. O colágeno tipo I foi o componente mais abundante da matriz extracelular no constritor superior da faringe. Observamos uma correlação positiva entre o colágeno I e a idade (r=0,42, p=0,01) e inversa com o colágeno III (r=-0,28, p=0.027). O índice de massa corpórea também mostrou uma correlação inversa com o colágeno III (r=-0,311, p=0,017). Não houve diferenças estatisticamente significantes para os componentes da MEC entre os grupos. Em resume, a análise histológica da musculatura da parede lateral da faringe de um largo grupo de pacientes com distúrbios do sono, revelaram que a composição da matriz extracelular é relacionada à idade e o peso, e não pode ser envolvida nos mecanismos que levam a colapsibilidade e espessamento da parede lateral da faringe. / The lateral muscular pharyngeal wall appears to be the predominant structure involved in the collapse of the pharynx in obstructive sleep apnea (OSA). OSA patients and snorers have a narrower pharynx which is more susceptible to collapse and is associated with muscle thickening in the lateral pharyngeal wall, largely formed by superior pharyngeal constrictor. The endomysial extracellular matrix of skeletal muscle has an intimate relationship with the muscle cells and has an important influence on its mechanical and biological properties and can modify their behavior and function. Collagen and elastic fibers form the scaffolding of the connective tissue and proteoglycans and structural glycoproteins (fibronectin) have important roles in hydration, resiliency and adhesive properties of the tissues and the matrix metalloproteinases are involved in extracellular matrix components degradation. The determining factors in the compliance of lateral muscular pharyngeal wall are also unknown. It is possible that the ECM has a relevant role in this aspect. Our objective was to determine and compare the density of the components of the extracellular matrix in the deep, structural lateral pharyngeal muscular walls in snorers, OSA and control patients and correlate with age and weight. In this prospective study we evaluated 61 patients older than 18 and nonobese with indications for pharyngeal surgery at the department of Otolaryngology of the Hospital das Clínicas of the São Paulo University, between 2005 and 2006. The patients were divided in three groups: controls (14 patients), 18 primary snorers and 29 OSA patients. We obtained specimens from the lateral pharyngeal muscular wall (superior constrictor muscle) during pharyngeal surgeries. Using histochemical and immunohistochemical analyses, we determined the fractional area of total collagen, elastic fibers, collagen I and III, matrix metalloproteinase 1 and 2, versican and fibronectin in the endomysium of these samples, and compared among groups. Results were further correlated with anthropometric data. Collagen type I was the most abundant component of the ECM within the pharyngeal muscle. There was a positive correlation between collagen I and age (r=0.419, p=0.01) and an inverse one with type III collagen (r=-0.284, p=0.027). Body mass index inversely correlated with collagen type III (r=-0,311, p=0,017). There were no statistically significant differences for any of the components of the ECM among groups. In summary, histological analysis of the lateral muscular pharyngeal wall of a large group of patients with sleep disturbances revealed that its extracellular matrix composition is age and weight related but may not be involved in the mechanisms leading to the collapsibility of the lateral wall of the pharynx.

Exon shuffling foi primeiramente proposto por Walter Gilbert em 1978 como um mecanismo em que exons de diferentes genes podem ser combinados, levando à formação de novos genes. O mecanismo de exon shuffling é favorecido por recombinações intrônicas e está correlacionado com a simetria de exons. Evidências deste mecanismo provém de análises de combinações de fases de introns, correlações entre bordas de exons e de domínios protéicos e da recorrência de domínios em diversas proteínas. Dessa forma, a evolução de proteínas formadas por exon shuffling pode ser inferida considerando a organização exon-intron dos genes, o padrão de combinações de fases de introns e a organização de domínios nas proteínas. Neste sentido, regiões protéicas que possivelmente foram originadas por eventos de exon shuffling foram identificadas através de análises em larga escala em diferentes espécies eucarióticas. A estratégia foi baseada no alinhamento entre todas as proteínas anotadas de uma determinada espécie e a verificação da presença de introns e suas respectivas fases em torno das regiões alinhadas. Nós verificamos que eventos de exon shuffling em eucariotos antigos, de origem anterior aos Metazoa, são predominantemente simétricos 0-0, enquanto nos metazoários a predominância é de unidades simétricas 1-1. Esses dados confirmam idéias anteriores de que a transição para a multicelularidade animal foi marcada pelo embaralhamento extensivo de exons e domínios 1-1. O metazoário basal Trichoplax adhaerens pode ser considerado um representante desta transição, evidenciada pelas freqüências balanceadas de regiões simétricas 0-0 e 1-1. O sinal de flanqueamento por introns em torno das bordas de domínios protéicos confirmou os resultados obtidos através dos alinhamentos, com a prevalência de domínios 0-0 em não metazoários e 1-1 em metazaoários. Um agrupamento hierárquico de domínios flanqueados por introns foi construído, permitindo identificar domínios ou grupos de domínios com evidência de expansões em períodos específicos, como nos vertebrados. Por fim, os genes envolvidos em eventos de exon shuffling foram analisados quanto ao enriquecimento em termos do Gene Ontology. Os resultados indicaram que este mecanismo contribuiu significativamente para a formação de genes relacionados com uma grande diversidade de termos, alguns dos quais envolvidos diretamente com características de metazoários e vertebrados, tais como matriz extracelular, adesão, coagulação sangüínea, processos do sistema imune e sistema nervoso / Exon shuffling was first proposed by Walter Gilbert in 1979 as a mechanism in which exons from different genes could be combined to lead the creation of new genes. The mechanism of exon shuffling is favored by intronic recombinations and it is correlated with symmetry of exons. Evidence of this mechanism come from analyses of intron phase combinations, correlations between the borders of exons and domains and domain recurrence in several proteins. Taking this into account, the evolution of proteins formed by exon shuffling can be inferred regarding the exonintron organization of the genes, the pattern of intron phase combinations and the protein domain organization. In this sense, protein regions that were probably arose by exon shuffling events were identified through a large scale analysis in several eukaryotic species. The strategy was based on alignments between all annotated proteins from a given species. Then, the aligned regions were verified in respect with intron phase combinations surrounding them. We have found that exon shuffling events in early eukaryotes are preferentially symmetric of phase 0, while in metazoans, the preference is for 1-1 symmetric units. These data confirms previous ideas that the transition to animal multicellularity was marked by extensive 1-1 exon shuffling. The basal metazoan Trichoplax adhaerens is a representative of this transition, evidenced by the balanced frequencies of 0-0 and 1-1 symmetric regions. The signal of intron flanking around the borders of protein domains corroborated previous analyses, showing that non metazoans have higher frequencies of 0-0 domains and metazoans have higher frequencies of 1-1 domains. A hierarchical clustering of domains flanked by introns was built, allowing us to identify domains or groups of domains with evidence of expansions during specific periods, such as in vertebrates. Finally, genes involved in exon shuffling events were analyzed regarding the Gene Ontology enriched terms. The results indicated that this mechanism significantly contributed to the creation of genes related with a large diversity of terms, some of them are directly involved with features of metazoans and vertebrates, such as extracellular matrix, cell adhesion, blood coagulation and immune and nervous system processes

Heart failure is one of the leading causes of death in the United States. Every year in the United States, more than 800,000 people are diagnosed with heart failure and more than 375,000 people die from heart disease. Current therapies such as heart transplants and bioartificial hearts are helpful, but not optimal. Decellularization of porcine whole hearts followed by recellularization with patient-specific human cells may provide the ultimate solution for patients with heart failure. Great progress has been made in the development of efficient processes for decellularization, and the design of automated bioreactors. In this study, the decellularization of porcine hearts was accomplished in 24 h with only 6 h of sodium dodecyl sulfate (SDS) exposure and 98% DNA removal. Automatically controlling the pressure during decellularization reduced the detergent exposure time while still completely removing immunogenic cell debris. Stimulation of macrophages was greatly reduced when comparing native tissue samples to the processed ECM. Complete cell removal was confirmed by analysis of DNA content. General collagen and elastin preservation was demonstrated by SEM and histology. The compression elastic modulus of the ECM after decellularization was lower than native at low strains but there was no significant difference at high strains. Polyurethane casts of the vasculature of native and decellularized hearts demonstrated that the microvasculature network was preserved after decellularization. A static blood thrombosis assay using bovine blood was also developed. A perfusion bioreactor was designed and right ventricle of the decellularized hearts were recellularized with human endothelial cells and cardiac fibroblasts. An effective, reliable, and relatively inexpensive assay based on human blood hemolysis was developed for determining the remaining cytotoxicity of the cECM and the results were consistent with a standard live/dead assay using MS1 endothelial cells incubated with the cECM. Samples from the left ventricle of the hearts were prepared with 300 µm thickness, mounted on 10 mm round glass coverslips. Human induced pluripotent stem cells were differentiated into cardiomyocytes (CMs) and 4 days after differentiation, cardiac progenitors were seeded onto the decellularized cardiac slices. After 10 days, the tissues started to beat spontaneously. Immunofluorescence images showed confluent coverage of CMs on the decellularized slices and the effect of the scaffold was evident in the arrangement of the CMs in the direction of fibers. This study demonstrated the biocompatibility of decellularized porcine hearts with human CMs and the potential of these scaffolds for cardiac tissue engineering. Further studies can be directed toward 3D perfusion recellularization of the hearts and improving repopulation of the scaffolds with various cell types as well as adding mechanical and electrical stimulations to obtain more mature CMs.

Epithelial to Mesenchymal Transition (EMT) is a dynamic process by which a distinct change in the phenotype and function of epithelial cells render them as mesenchymal cells. Characteristics of mesenchymal cells include the ability to invade, increased migratory kinetics and heightened resistance to apoptosis. Therefore, there is a strong need to fully understand the mechanism for the induction of EMT in pathological conditions such as carcinoma progression. Recent advances highlight two pivotal contributors, soluble growth factor (gf) signals, and mechanical signals, in the process. However, to date, no clear mechanism exists linking the two in epithelial transdifferentiation. Transforming Growth Factor-β1 (TGF-β1), a gf known to induce EMT in breast cancer formation, induces EMT on rigid surfaces and apoptosis on compliant surfaces. It is our belief that a combination of mechanical signals, gf signals, and the type of extracellular matrix (ECM) proteins assembled by cells together drive the process of EMT. Here we investigated the role of the ECM protein fibronectin (FN) in EMT. Upon assembly into elastic, insoluble fibrils through cell-generated forces which become larger on stiffer surfaces, FN is able to serve as a gf delivery system. We examined the following hypothesis: Increased tissue stiffness drives FN assembly, which exposes cryptic binding sites for various gfs, such as TGF-β1, and creates a high concentration of these gfs at the cell surface, which in turn drives EMT. In this project we investigated three aims: (1) evaluate the effect of inhibiting FN fibrillogenesis and gf localization on TGF-β1-induced EMT, (2) assess the effect of TGF-β1 concentration on spatial patterning of ECM dynamics, cell phenotype and adherens junctional force, and (3) probe the role of the FN matrix in TGF-β1-induced spatial patterning of EMT. Results showed that both inhibition of FN fibril assembly and blocking the gf binding site on fibrils significantly attenuated the downstream effects of EMT. In microcontact patterns of epithelial colonies, increasing gf concentration led to spatial patterning of FN fibrils, cell phenotype and cell-cell junctional force. Elimination of FN fibrils effectively attenuated TGF-β1-induced spatial patterning. The knowledge acquired through these studies serves as an addition to an increasingly important body of work aimed at elucidating how physical changes within the microenvironment regulate physiology and pathology. By establishing a novel mechanism by which gf signaling induces EMT through interaction with the extracellular matrix, this research serves to combat the development and initiation of pathological phenomena, such as metastasis.

Lung cancer is the number one cause of cancer related death worldwide, with more than 1.6 million fatalities each year. Non-small cell lung cancer (NSCLC) accounts for 80-85% of all lung cancers, with KRAS being one of the most prevalent oncogenic driver mutations. Therapeutic approaches for KRAS-mutated NSCLC have been extensively explored due to the US National Cancer Institute RAS Initiative, but methods of directly targeting KRAS or downstream effectors, such as MEK, still have poor results. Previous reports have shown that KRAS-mutated NSCLC activate distinct receptor tyrosine kinases (RTKs) depending on the epithelial or mesenchymal state. Epithelial-to-mesenchymal transition (EMT) is known to play a role in the metastasis and poor prognosis of cancer, and is induced by extracellular matrix (ECM) stiffness. Hallmarks of EMT include loss of E-Cadherin and increase in Vimentin. This research investigates the role of KRAS in EMT transition due to increased ECM stiffness in KRAS mutant NSCLC, and how this affects the efficacy of KRAS and MEK inhibition. To understand how KRAS mutations in NSCLC play a role in this stiffness induced EMT, experiments were performed to detect the gene and protein expression of EMT markers, as well as possible sources of mechanosensing, including primary cilia and receptor tyrosine kinases. We hypothesized that KRAS plays a role in activation of mechanosensors and directly correlates to EMT induced by increased mechanical forces. Results show when KRAS was inhibited and there was increased mechanical forces, either from stretch or substrate stiffness, there was a decreased activation of mechanosensors. KRAS inhibition also prevented the cells from undergoing stiffness-induced EMT. This supports our hypothesis that KRAS plays a key role in ECM stiffness induced EMT. Future studies include examining the mechanism behind this phenomenon and in vivo studies.

O câncer de mama é o tipo mais freqüente e o segundo mais letal no mundo. Embora ass taxas de sobrevida dos pacientes tenham aumentado consideravelmente nas últimas décadas, indicadores prognósticos desfavoráveis são associados a pacientes com diagnóstico em fase avançada e presença de metástases, frequentemente associadas à existência de células-tronco tumorais (CTT). As CTT são indiferenciadas e capazes de autorrenovação e diferenciação, o que as torna fundamentais para a manutenção da heterogeneidade celular intratumoral. As CTTs são altamente invasivas, tumorigênicas e resistentes a tratamentos convencionais, sendo frequentemente associadas ao surgimento de metástase e recidiva após tratamento. O microambiente tumoral modula as CTT por meio de células e da matriz extracelular (MEC), uma estrutura biologicamente dinâmica, complexa e que regula processos celulares como migração, invasão e diferenciação. A MEC é composta por uma grande variedade de moléculas, peptídeos e macromoléculas, sendo o colágeno seu componente mais abundante. A alta densidade mamográfica é frequentemente associada a elevada rigidez da MEC e deposição aumentada de colágeno fibrilar, principalmente colágeno tipo I (Col I), e é um dos maiores fatores de risco independentes para o desenvolvimento do câncer de mama. A alta densidade de Col I e rigidez da MEC também está associada à maior agressividade tumoral e metástase. Col I também induz o fenótipo tronco tumoral em diversos tipos celulares tumorais, embora o papel da densidade sobre este efeito seja pouco esclarecido. Nosso estudo avaliou a hipótese de a alta densidade de Col I induzir o fenótipo tronco tumoral. Cultivamos linhagens normais (MCF-10A) e tumorais (MDA-MB-231 e MCF-7) de mama em géis de baixa, média e alta densidade de Col I. Também cultivamos células em superfície bidimensional (2D) e em suspensão para geração de mamoesferas (ME), representando o cultivo tradicional e de enriquecimento de CTTs, respectivamente. Avaliamos os níveis do imunofenótipo tronco (CD44+CD24-), expressão gênica e proteica de marcadores de CTTs e de resposta mecânica ao substrato (mecanotransdução), bem como potencial clonogênico, autorrenovação celular e alinhamento fibrilar de géis de Col I. Alta densidade de Col I elevou os níveis da subpopulação CD44+CD24- e inibiu o alongamento celular da linhagem MDA-MB-231, porém não modulou a expressão de marcadores de CTT, bem como potencial clonogênico, autorrenovação celular e alinhamento fibrilar de géis de Col I. A alta densidade de Col I induziu aumento dos níveis totais da isoforma variante da glicoproteína CD44 (CD44v), receptor de estrógeno (RE &#945) e do fator de pluripotência Sox2 em linhagem MCF-7 derivada de ME. Entretanto, os níveis nucleares dos fatores de transcrição (RE &#945 e Sox2) permaneceram inalterados. Em comum, a alta densidade de Col I não elevou os níveis nucleares do mecanotransdutor YAP em linhagens MDAMB-231 e MCF-7 derivada de ME. Concluímos que a alta densidade de Col I induz parcialmente o fenótipo molecular, mas não o funcional, de células tumorais mamárias. / Breast cancer is the most frequent and second deadliest cancer type worldwide. Although patient survival rates have increased considerably in recent decades, unfavorable prognostic indicators are associated with patients with advanced disease stage at diagnosis and presence of metastases, frequently associated with the existence of cancer stem cells (CSC). CSC are undifferentiated and capable of self renewal and differentiation, making them fundamental for the maintenance of intratumoral cellular heterogeneity. CTTs are highly invasive, tumorigenic and resistant to conventional treatments, and are frequently associated with the onset of metastasis and relapse after treatment. The tumor microenvironment modulates CTT by means of cells and the extracellular matrix (ECM), a biologically complex and dynamic structure that regulates cell processes such as migration, invasion and differentiation. ECM is composed of a large variety of molecules, peptides and macromolecules, with collagen being its most abundant component. High mammographic density is often associated with high MEC stiffness and increased deposition of fibrillar collagen, mainly type I collagen (Coll I), and is one of the main independent risk factors for breast câncer development. High Coll I density and ECM stifness are also associated with increased tumor aggressiveness and metastasis. Coll I also induces tumor stemness in several tumor cell types, although the role of its density on this effect is unclear. Our study evaluated the hypothesis that high Coll I density induces the tumor stemness. We cultured normal-like- (MCF-10A) and tumoral (MDA-MB-231 and MCF-7) breast cell lines in low-, medium- and high-density Coll I gels. We also cultured cells in twodimensional (2D) surface and in suspension for the generation of mammospheres (MS), representing the traditional cell culture and CSC enrichment, respectively. We evaluated the levels of the CSC immunophenotype (CD44+CD24), gene/protein expression of CSC markers and mechanical response to the substrate (mechanotransduction), as well as the clonogenic potential, cell self-renewal and fibrillar alignment of Col I gels. High Coll I density increased the levels of the CD44+CD24- subpopulation and inhibited cell elongation of the MDA-MB-231 cell line, but did not modulate the expression of CSC markers as well as clonogenic potential, cell self-renewal and fibrillar alignment of Col I gels. High Coll I density increased total levels of the variant CD44 glycoprotein (CD44v), estrogen receptor (ER) and the pluripotency factor Sox2 in MS-derived MCF-7. However, the nuclear levels of the transcription factors (ER &#945 and Sox2) remained unchanged. In common, high Coll I density did not increase nuclear levels of the mechanotransducer YAP in MDA-MB- 231 and MS-derived cell lines. We conclude that high Coll I density partially induces the molecular stemness, but not the functional, phenotype of mammary tumor cells.

O câncer de mama representa um importante problema de saúde pública. O microambiente onde as células neoplásicas se encontram desempenha importante papel na tumorigênese e progressão tumoral. Nosso Laboratório estuda o efeito da laminina, importante glicoproteína da matriz extracelular (MEC) e seus peptídeos dentro da biologia tumoral. Nesse projeto detectamos, in vitro e em amostras de câncer de mama humano, peptídeos derivados da clivagem proteolítica da laminina no câncer de mama. Inicialmente analisamos a distribuição das cadeias da laminina-111 no câncer de mama humano. Análise imuno histoquímica demonstrou aumento da marcação para as cadeias alfa1, beta1 e gama1 da laminina em tumores de mama. Células derivadas de câncer de mama (MDA-MB-231 e MCF-7) foram crescidas sobre Matrigel (substrato enriquecido em laminina). Células de mama normal MCF-10A serviram como grupo controle. Amostras controles e tratadas foram submetidas a eletroforese, immunoblot e espectrometria de massas (LC-MS/MS). Immunoblot mostrou a clivagem das cadeias da laminina in vitro e no câncer de mama humano, com a geração de fragmentos de menor massa molecular. Exploramos a liberação de peptídeos gerados pela clivagem da laminina em amostras de células tumorais em cultura e no câncer de mama humano. Análise proteômica mostrou que as bandas derivadas da laminina exibiram a presença de 31 peptídeos, entre eles C16 (KAFDITYVRL) e C28 (RVTLNRL). Previamente já demonstramos que o peptídeo C16 possui alta relevância na regulação tumoral. Diferentes peptídeos foram sintetizados e suas atividades biológicas avaliadas. Entre eles, C16 e C28 aumentaram significantemente a adesão, proliferação, secreção e ativação de MMPs além de invasão de células derivadas do câncer de mama (MDA-MB-231) e também de células derivadas de fibrossarcoma (HT1080). Investigamos também a ação do peptídeo C16 sobre as células tumorais MDA-MB-231 injetadas em zebrafish para avaliação do desenvolvimento tumoral in vivo. C16 promoveu aumento da área tumoral in vivo em comparação aos grupos controles. Concluímos que as cadeias alfa1, beta1 e gama1 da laminina estão aumentadas no câncer de mama. A clivagem da laminina possivelmente ocorre através da atividade de MMPs. As cadeias da laminina que são clivadas in vitro e no câncer de mama humano, geram fragmentos que contém peptídeos com funções biológicas evidentes. Entre os peptídeos observados, detectamos o peptídeo C16 in vitro e in vivo<i/>, também detectamos o peptídeo C28 in vitro. O peptídeo C16 é um grande regulador da invasão de células MDA-MB-231 e células HT1080, além de promover o desenvolvimento tumoral in vivo. Já o peptídeo C28 promove a proliferação de células MDA-MB-231 e HT1080. / Breast cancer represents an important public health problem. The microenvironment plays an important role in tumorigenesis and tumor progression. Our laboratory studies the effect of laminin, an important extracellular matrix glycoprotein (ECM) and its peptides in tumor biology. In this project we detected, in vitro and in human breast cancer samples, peptides derived from proteolytic cleavage of laminin. We initially analyzed the distribution of laminin-111 chains in human breast cancer. Immunohistochemistry demonstrated increased laminin alpha1, beta1 and gamma1 chains in breast tumors. Cells derived from breast cancer (MDA-MB-231 and MCF-7) were grown on Matrigel (laminin-enriched substrate). Normal breast cells MCF-10A served as a control group. Control and treated samples were subjected to electrophoresis, immunoblot and mass spectrometry (LC-MS/MS). Immunoblot showed cleavage of the laminin chains in vitro and in human breast cancer, with generation of lower molecular mass fragments. We explored the release of peptides, generated by laminin cleavage in tumor cell samples in culture and in human breast cancer. Proteomic analysis showed that the bands derived from laminin exhibited the presence of 31 peptides, among them C16 (KAFDITYVRL) and C28 (RVTLNRL). We have previously demonstrated that the C16 peptide has high relevance in tumor regulation. Different peptides were synthesized, and their biological activities were analyzed. Among them, C16 and C28 peptides, significantly increased adhesion, proliferation, secretion and activation of MMPs and cell invasion in breast cancer cells (MDA-MB-231) as well as fibrosarcoma-derived cells (HT1080). In addition, we investigated the action of the C16 peptide on zebrafish-injected MDA-MB-231 tumor cells for evaluation of tumor development in vivo. C16 promoted increase in tumor area in vivo compared to control groups. We conclude that, laminin alpha1, beta1 and gamma1 chains are increased in breast cancer. Laminin chains that are cleaved in vitro and in human breast cancer, generate fragments containing peptides with obvious biological functions. Among the peptides observed, we detected the C16 peptide in vitro and in vivo, we also detected the C28 peptide in vitro. C16 peptide is a major regulator of the invasion of MDA-MB-231 cells and HT1080 cells, in addition C16 promotes tumor development in vivo. C28 peptide promotes the proliferation of MDA-MB-231 and HT1080 cells.