No description available.

No / Dystroglycan is a ubiquitously expressed cell adhesion molecule frequently found to be altered or reduced in adenocarcinomas, however the mechanisms or consequences of dystroglycan loss have not been studied extensively. We examined the consequence of overexpression or RNAi depletion of dystroglycan on properties of in vitro growth migration and invasion of LNCaP, PC3, and DU145 prostate cancer cell lines. RESULTS: Using LNCaP cells we observed cell density-dependent changes in beta-dystroglycan with the appearance of several lower molecular weight species ranging in size from 43 to 26 kDa. The bands of 31 and 26 kDa were attributed to proteolysis, whereas bands between 43 and 38 kDa were a consequence of mis-glycosylation. The localization of beta-dystroglycan in LNCaP colonies in culture also varied, cells with a mesenchymal appearance at the periphery of the colony had more pronounced membrane localization of dystroglycan. Whereas some cells demonstrated nuclear dystroglycan. Increased dystroglycan levels were inhibitory to growth in soft agar but promoted Matrigel invasion, whereas reduced dystroglycan levels promoted growth in soft agar but inhibited invasion. Similar results were also obtained for PC3 and DU145 cells. This study suggests that changes in beta-dystroglycan distribution within the cell and/or the loss of dystroglycan during tumorigenesis, through a combination of proteolysis and altered glycosylation, leads to an increased ability to grow in an anchorage independent manner, however dystroglycan may need to be re-expressed for cell invasion and metastasis to occur.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

Fibroblasts are the principal cellular component of the connective tissue. They are a heterogeneous group of cells which contribute to the structure of connective tissue and wound healing by their ability to produce extracellular matrix (ECM). Fibroblasts and cells derived from them are involved in many pathological processes such as formation of malignant tumors and fibrosis. Tumor progression which finally leads to metastasis is a serious biomedical problem. There is a growing body of the recent evidence showing an important role of the tumor stroma and its interaction with cancer cells in cancer progression. Tumor stroma comprises mainly of myofibroblasts and their products, namely ECM, soluble factors, and enzymes. Myofibroblasts contribute more or less to all steps of cancer progression. Furthermore myofibroblasts play a key role in fibrosis, another serious human disease which is not efficiently treatable and which is associated with cancer progression. These facts made us to search for molecular means capable of eliminating the myofibroblastic phenotype. We succeeded to entirely dedifferentiate primary myofibroblasts by concomitant inhibition of TGFβ signaling and perturbation of MAPK signaling in a chick model that we have introduced. Malignant fibroblasts form sarcomas. ECM is the first...

O câncer de mama é a principal causa de morte por câncer entre as mulheres no mundo. Fatores genéticos, comportamentais e ambientais afetam o risco de aparecimento dessa doença e seu desenvolvimento e progressão ocorrem pelo acúmulo de alterações genéticas/epigenéticas que levam a manutenção de sinais proliferativos nas células, fuga dos agentes supressores de crescimento e resistência à morte celular. O gene PHLDA1 (de Pleckstrin Homology-Like Domain, Family A, Member 1) codifica uma proteína de 401 aminoácidos que já foi descrita envolvida em distintos processos biológicos incluindo morte celular e, dessa forma, é frequentemente associada ao câncer. Perda progressiva de PHLDA1 já foi descrita em melanoma primário e metastático enquanto sua superexpressão foi descrita para tumores intestinais e pancreáticos. Em dados prévios de nosso grupo de pesquisa o gene PHLDA1 foi encontrado diferencialmente expresso em tumores de mama onde sua ausência estava relacionada com sobrevida livre de doença e sobrevida global reduzidas nas pacientes. Estudos do gene PHLDA1 em linhagens de mama são escassos e a compreensão de seu papel funcional e de como sua ausência pode estar relacionada com a redução da sobrevida em pacientes com câncer de mama permanecem obscuros. Com o objetivo de compreender a função de PHLDA1 em células epiteliais de mama, nós investigamos os efeitos da supressão do gene PHLDA1 em células MCF10A. A redução da expressão foi alcançada a partir de transfecção das células com vetores plasmidiais contendo shRNAs específicos para o transcrito de PHLDA1 e subsequentemente foram realizados ensaios funcionais. A expressão diminuida de PHLDA1 foi capaz de induzir acentuadas alterações morfológicas e comportamentais nas células MCF10A, incluindo mudança no padrão de ancoragem célula-célula e reorganização nos filamentos de actina, além de maior taxa de proliferação, migração e invasão das células. Além disso, em condições de baixa ancoragem, as células com expressão reduzida de PHLDA1 apresentaram mamosferas de formato irregular em comparação às células controle. Em conjunto, nossos resultados mostram que a diminuição da expressão de PHLDA1 em células MCF10A está relacionada a um comportamento agressivo e acentuadas alterações morfológicas. Estes dados são consistentes com atividade supressora tumoral de PHLDA1 em células epiteliais de mama / Breast cancer is the leading cause of cancer death among women worldwide. Genetic, behavioral and environmental factors affect the risk of onset of the disease. Breast cancer development and progression involves the accumulation of genetic/epigenetic changes that lead to maintenance of proliferative signals, evasion of growth suppressors and resistance to cell death. The PHLDA1 gene (Pleckstrin Homology-like domain, Family A, member 1) encodes a 401 amino acids protein that has been described involved in different biological processes including cell death and thus, is often associated with cancer. Progressive loss of PHLDA1 has been described in primary and metastatic melanoma while their overexpression has been reported for intestinal and pancreatic tumors. In previous data from our research group the PHLDA1 gene was found differentially expressed in breast tumors where its downregulation was related to shorter disease-free survival and overall survival of the patients. Literature regarding PHLDA1 in mammary epithelial cell lines is scarce and the understanding of their functional role and how its downregulation can be related to poor prognosis in breast cancer patients remain unclear. In order to understand the PHLDA1 function in breast epithelial cells, we investigated the effects of downregulation of PHLDA1 in MCF10A cells. The reduced expression was achieved from transfection of cells with plasmid vectors containing shRNAs for the specific transcript of PHLDA1 followed by functional assays. The decreased expression of PHLDA1 was sufficient to induce marked morphological and behavioral changes in MCF10A cells, including changes in cell-to-cell attachment pattern and actin reorganization, increased proliferation, migration and invasion rate of cells. Furthermore, in independent of attachment condition, cells with reduced expression of PHLDA1 formed mammospheras whit irregular shape compared to control cells. Taken together, our results showed that the decreased expression of PHLDA1 in MCF10A cells is related to aggressive behavior and marked morphological changes. These data are consistent with tumor suppressor activity for PHLDA1 in breast epithelial cells

Invasions of non-native species cause problems in ecosystems worldwide, and despite the extensive effort that has been put into research about invasions, we still lack a good understanding for why some, but not other, communities resist these invasions. In this doctoral thesis I test hypotheses on biotic resistance using a large dataset of more than 1000 both failed and successful introductions of freshwater fish into Swedish lakes. We have found that the classic species richness hypothesis is a poor descriptor of introduction success because it fails to acknowledge that resident species contribute to the resistance in different ways. We developed a new measure of biotic resistance, the weighted species richness, which takes into account that the resident species contributes to the resistance with different strength and sign. Further, we correlated performance traits of species in their role as an invader and as a resident species to predict how the biotic resistance of these communities would develop over time. We found a positive correlation between performance traits: Some species have high introduction success, they make a large contribution to the resistance, and they cause extinctions when introduced but do not go extinct themselves when other species establishes, whereas other species are weak performers in these respects. Thus, the biotic resistance of these communities should grow stronger as non-native species accumulates. These results give us clues about what type of communities that should be most sensitive to further invasions, i.e., communities harboring species weak performers.  My results show that the biotic resistance of communities is an important factor in determining invasibility of a community. They also show that methods for quantifying resistance must take into account how interactions are structured in nature. What determine the biotic resistance of a community is the type of interactions that the resident species have with the invader and not the species richness of the community.

O câncer de mama é a principal causa de morte por câncer entre as mulheres no mundo. Fatores genéticos, comportamentais e ambientais afetam o risco de aparecimento dessa doença e seu desenvolvimento e progressão ocorrem pelo acúmulo de alterações genéticas/epigenéticas que levam a manutenção de sinais proliferativos nas células, fuga dos agentes supressores de crescimento e resistência à morte celular. O gene PHLDA1 (de Pleckstrin Homology-Like Domain, Family A, Member 1) codifica uma proteína de 401 aminoácidos que já foi descrita envolvida em distintos processos biológicos incluindo morte celular e, dessa forma, é frequentemente associada ao câncer. Perda progressiva de PHLDA1 já foi descrita em melanoma primário e metastático enquanto sua superexpressão foi descrita para tumores intestinais e pancreáticos. Em dados prévios de nosso grupo de pesquisa o gene PHLDA1 foi encontrado diferencialmente expresso em tumores de mama onde sua ausência estava relacionada com sobrevida livre de doença e sobrevida global reduzidas nas pacientes. Estudos do gene PHLDA1 em linhagens de mama são escassos e a compreensão de seu papel funcional e de como sua ausência pode estar relacionada com a redução da sobrevida em pacientes com câncer de mama permanecem obscuros. Com o objetivo de compreender a função de PHLDA1 em células epiteliais de mama, nós investigamos os efeitos da supressão do gene PHLDA1 em células MCF10A. A redução da expressão foi alcançada a partir de transfecção das células com vetores plasmidiais contendo shRNAs específicos para o transcrito de PHLDA1 e subsequentemente foram realizados ensaios funcionais. A expressão diminuida de PHLDA1 foi capaz de induzir acentuadas alterações morfológicas e comportamentais nas células MCF10A, incluindo mudança no padrão de ancoragem célula-célula e reorganização nos filamentos de actina, além de maior taxa de proliferação, migração e invasão das células. Além disso, em condições de baixa ancoragem, as células com expressão reduzida de PHLDA1 apresentaram mamosferas de formato irregular em comparação às células controle. Em conjunto, nossos resultados mostram que a diminuição da expressão de PHLDA1 em células MCF10A está relacionada a um comportamento agressivo e acentuadas alterações morfológicas. Estes dados são consistentes com atividade supressora tumoral de PHLDA1 em células epiteliais de mama / Breast cancer is the leading cause of cancer death among women worldwide. Genetic, behavioral and environmental factors affect the risk of onset of the disease. Breast cancer development and progression involves the accumulation of genetic/epigenetic changes that lead to maintenance of proliferative signals, evasion of growth suppressors and resistance to cell death. The PHLDA1 gene (Pleckstrin Homology-like domain, Family A, member 1) encodes a 401 amino acids protein that has been described involved in different biological processes including cell death and thus, is often associated with cancer. Progressive loss of PHLDA1 has been described in primary and metastatic melanoma while their overexpression has been reported for intestinal and pancreatic tumors. In previous data from our research group the PHLDA1 gene was found differentially expressed in breast tumors where its downregulation was related to shorter disease-free survival and overall survival of the patients. Literature regarding PHLDA1 in mammary epithelial cell lines is scarce and the understanding of their functional role and how its downregulation can be related to poor prognosis in breast cancer patients remain unclear. In order to understand the PHLDA1 function in breast epithelial cells, we investigated the effects of downregulation of PHLDA1 in MCF10A cells. The reduced expression was achieved from transfection of cells with plasmid vectors containing shRNAs for the specific transcript of PHLDA1 followed by functional assays. The decreased expression of PHLDA1 was sufficient to induce marked morphological and behavioral changes in MCF10A cells, including changes in cell-to-cell attachment pattern and actin reorganization, increased proliferation, migration and invasion rate of cells. Furthermore, in independent of attachment condition, cells with reduced expression of PHLDA1 formed mammospheras whit irregular shape compared to control cells. Taken together, our results showed that the decreased expression of PHLDA1 in MCF10A cells is related to aggressive behavior and marked morphological changes. These data are consistent with tumor suppressor activity for PHLDA1 in breast epithelial cells

Abstract Collagen XVII (BP180) is a transmembrane component of hemidesmosomes, which connect basal keratinocytes to the basement membrane. The extracellular domain of collagen XVII is proteolytically shed from the cell surface and released to the extracellular matrix. Apart from its function in epithelial cell adhesion, collagen XVII has been suggested to participate in keratinocyte motility. The collagen XVII expression pattern was studied in wounds of oral mucosa and in epithelial tumors. During re-epithelialization, collagen XVII was expressed in the keratinocytes distal to the wound edge, but not in the leading cells of the epithelial tip. Collagen XVII upregulation was observed in moderate/severe dysplasias of oral mucosa. In follicular ameloblastomas and basal cell carcinomas, collagen XVII expression was reduced in peripheral cells, whereas cytoplasmic staining was detected in central tumor cells. Tongue squamous cell carcinomas showed increased collagen XVII expression in grade II/III tumors, particularly in areas of invasive growth. The results suggest a correlation between overexpression of collagen XVII and the invasive potential of the tumor. For the first time, the role of collagen XVII in the regulation of malignant migration was explored. The presence of COL15, the cell adhesion domain of collagen XVII, induced migration of tongue squamous cell carcinoma cells in transmigration assays. Experiments with specific function-blocking integrin antibodies revealed that the promigratory function of COL15 is mediated by αv and α5 integrins. The role of the matrix metalloproteinase (MMP) family of proteolytic enzymes in wound re-epithelialization was studied in a transgenic mouse model. In these mice, a specific inhibitor of MMPs, TIMP-1, was overexpressed in cells that normally produce MMP-9. The healing of cutaneous wounds was found to be significantly delayed, but not prevented, due to the impaired ability of keratinocytes to migrate to the wound area. These results suggest that collagen XVII may participate in epithelial tumor progression and invasion by promoting migration of tumor cells. Based on the present study, epithelial cell-derived MMPs play a significant role in the migration of wound keratinocytes during re-epithelialization.

Activin-A is a member of transforming growth factor-β (TGF-β) superfamily of cytokines which includes TGF-βs, Activins, Nodal, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs) and anti-Mullerian hormone (AMH). TGF-β, Activin and Nodal are known to activate SMAD2/3, while BMPs and GDFs are known to activate SMAD1/5/8 signaling pathways. Activin-A binds to type II transmembrane serine threonine kinase receptor (ActRIIA or ActRIIB), which in turn activates type I receptor (ActRIB) leading to phosphorylation of SMAD2/SMAD3. Upon phosphorylation, SMAD2/3 forms a complex with SMAD4, which then translocates to nucleus. In the nucleus, SMAD2/3/4 complex, along with other co-factors regulates expression of a large number of genes. Unlike TGF-β, role of Activin in cancer is not well understood. Activin has been shown to be overexpressed in several cancers including metastatic prostate cancer, colorectal cancer, lung cancer, hepatocellular carcinoma and pancreatic cancer. Activin signaling has been shown to promote aggressiveness of esophageal squamous cell carcinoma and enhancing skin tumorigenesis and progression. Nodal, which binds to the same set of receptors, has also been shown to be overexpressed in several cancers. However, role of Activins in breast cancer progression is not well studied. Activin is expressed by normal breast epithelium and is known to play a role in mammary gland development. Earlier, a study had reported downregulation of Activin signaling in breast tumors. On the contrary, increased serum level of Activin has been reported in women with metastatic breast cancers. It is pertinent to mention here that TGF-β, which has been implicated in the progression and metastatic spread of breast cancers, also functions through the same set of downstream effectors- SMAD2 and SMAD3. Hence we wanted to evaluate the status of Activin signaling pathway in breast tumors and investigate its functional role in cancer progression. Gene expression profiling of 80 breast tumors and 20 normal samples was earlier performed in our laboratory revealed overexpression of INHBA in tumors compared to normal tissue samples. An independent set of 30 tumor and 15 normal samples were used to verify these results. Real-time PCR analysis revealed around 11.31 fold upregulation (p<0.001) of INHBA in breast tumors in comparison to normals. While no change in expression of INHA was observed, INHBB was found to be significantly downregulated in tumor samples. These results indicated upregulation of Activin-A in breast tumors. Further, a significant upregulation of ACVR2A and SMAD2 which act as signal transducers of Activin signaling pathway, was observed in breast tumors. Interestingly, while an increase in the expression of TGF-β1 was observed, TGFBR2 was found to be significantly downregulated in breast tumors. In addition, PCR analysis revealed significant downregulation of FST, β-glycan, IGSF1 and IGSF10, which act as negative regulators of Activin signaling pathway. Functional antagonism between TGF-β/Activin and BMP signaling pathway has been shown in both development and disease. Further analysis revealed that various BMPs including BMP2, BMP4 and BMP6 are downregulated in breast tumors compared to normal tissue samples. Various components and regulators of BMP signaling pathway were also found to be deregulated, indicating suppression of BMP signaling in breast tumors. To evaluate whether Activin signaling is active in breast tumor cells, immunohistochemistry with another set of 13 normal and 29 tumor samples was performed. Immunohistochemistry analysis revealed that most of the tumors have higher levels of Activin-A compared to normals tissues. Interestingly, no significant changes in expression of Activin-A was observed between normals and low grade tumors, suggesting that Activin-A may play an important role towards the late stages of the disease. In good correlation, breast tumors showed increased phospho SMAD2 and phospho SMAD3 levels compared to normal tissues. Also, in the same set of tumors, BMP2 staining showed a reduced expression pattern compared to normal tissues. Expression of inhibin in some normal and breast tumor samples revealed that most of the tumor samples have lower levels of inhibin compared to normal tissues. In order to understand the role of Activin-A in cancer progression, a panel of cell lines was selected. Treatment of cells with Activin-A resulted in activation of canonical SMAD as well as non-canonical Erk1/2 and PI3K signaling pathways. However, Activin-A treatment did not lead to activation of TAK1/p38 MAPK pathway. To begin with, it was important to evaluate effect of Activin-A on proliferation of various cell lines. Primarily, SMAD2/3 signaling pathway inhibits proliferation of normal epithelial cells, and hence, it is considered to have a tumor suppressive role. owever, this signaling pathway remains intact in most ( 98%) of the breast cancers. BrdU incorporation assay showed that Activin-A does not promote proliferation of cells under monolayer culture conditions. However, soft agar assay results showed that Activin signaling promotes anchorage independent growth of cancer cells. TGF-β is widely known as an inducer of epithelial mesenchymal transition (EMT). Also, EMT is considered to be a prerequisite for epithelial cells to undergo migration and invasion. During EMT, cells loose epithelial characteristics and acquire mesenchymal features along with cytoskeletal rearrangement. Treatment of cells with Activin-A resulted in downregulation of E-cadherin and upregulation of various mesenchymal markers. In addition, confocal microscopy imaging revealed a mesenchymal morphology of cells treated with Activin-A. Also, collagen gel contraction assay results indicated that Activin-A enhances the contractile property of HaCaT cells significantly. Cells undergone EMT are believed to acquire migratory and Invasive behaviour. In agreement with this, both scratch assay and trans-well migration assay showed that Activin-A enhances the migration of various cell lines. Further, Trans-well matrigel invasion assays were performed to assess how Activin affects invasion of various cancer cells. Matrigel invasion assay results showed that Activin-A enhances invasion of various cancer cell lines significantly. Also, RT-PCR, zymography and Luciferase assay results showed that Activin-A induces MMP2 expression. As described earlier, Activin-A activates both canonical as well as non canonical signaling pathways. In this direction, it was interesting to investigate the contribution of SMAD signaling pathway in pro-tumorigenic actions of Activin-A. Inhibiting SMAD3 activity either by its stable knockdown or by using a SMAD3 specific small molecule inhibitor revealed that Activin-A regulation of EMT markers is SMAD3 dependent. Further, it was observed that SMAD3 contributes significantly in mediating Activin-A induced migration and invasion. Hence, it is likely that SMADs may play an important role in breast tumor progression. Next, stable overexpression of Activin-A in MCF-7 or its knockdown in MDA-MB-231 and H460 cells was performed to assess the effect of Activin-A on the behaviour of these cells. BrdU assay indicated no change in proliferation of cells upon overexpression or knockdown of Activin-A. However, soft agar assay results showed that Activin-A expression affects anchorage independent growth of these cells. MCF-7 cells are generally considered to be less aggressive in their tumor forming ability. Activin-A overexpressing MCF7 cells and control cells were respectively injected into right and left flank of immunocompromised mice and followed till the tumors reached to a prominent size. Our results show that Activin-A overexpressing MCF-7 cells have better tumor forming ability in comparison to control cells. In contrast to MCF-7 cells, MDA-MB-231 cells are known to be aggressive in their tumorigenic potential. In order to understand the effect of Activin-A knockdown on the tumor forming ability in MDA-MB-231 cells, 0.5 million cells (optimal cell number generally used is 1-2 million) were injected subcutaneously in immunocompromised mice. The results showed that while control cells gave rise to a tumor in 7 out of 10 animals, Activin-A knockdown cells could form a tumor in only 3 out of 10 animals. Also, the tumors formed by control cells were significantly larger by weight as compared to tumors formed by knockdown cells. Further, immunohistochemistry showed that tumors formed by MCF-7 cells overexpressing Activin-A have higher Ki-67 percentage as compared to control tumors. One of the factors known to be important for tumor growth is VEGF, which leads to recruitment of blood vessels and hence providing nourishment to the tumor cells. Hence Activin-A regulation of VEGF expression was evaluated next. Activin-A treatment or its stable overexpression in MCF-7 cells resulted in increased VEGF expression in these cells. This was also confirmed by VEGF promoter activity assay. To assess if Activin-A can play a role in metastatic spread of cancer cells, tail vein injection of Activin-A overexpressing MCF-7 cells was performed in immunocompromised mice. Even though no significant difference was found in the number of nodules formed by control or Activin-A overexpressing cells, it was observed that Activin-A overexpressing cells formed much bigger nodules as compared to the control cells. This suggests that Activin-A may play an important part in the establishment of metastases from the disseminated cancer cells. Tumor forming ability of cancer cells and aggressiveness of various cancers has been associated with the presence of cells having stem-like phenotype. In this direction, CD44high and CD24low expression status was analysed upon overexpression and knockdown of Activin-A in MCF-7 and MDA-MB-231 cells respectively. FACS analysis of Activin-A overexpressing MCF-7 cells and Activin-A knockdown MDA-MB-231 cells shows that Activin-A expression leads to enrichment of breast cancer stem-like cells. In conclusion, this study highlights the importance of Activin-A signaling pathway in the progression of breast tumors. It is also important to note the role of SMAD signalling in the progression of breast cancers since these effectors are common between TGF-β, Activin and nodal factors, which have been shown to be involved in cancer progression in a context dependent manner.

Src kinase plays a crucial role in a multitude of fundamental cellular processes. Src is an essential component of signalling pathways controlling cellular proliferation, motility or differentiation, and is often found deregulated in tumours. Src activity is therefore maintained under stringent and complex regulation mediated by SH3 and SH2 domains and the phosphorylation state of tyrosines 416 and 527. Active Src adopts an open conformation whereas inactive state of the kinase is characterised by a compact structure stabilised by inhibitory intramolecular interactions. We identified phosphorylation of tyrosine 90 within binding surface of SH3 domain as a new regulatory switch controlling Src kinase activation. Using substitutions mimicking phosphorylation state of the residue we demonstrated that tyrosine 90 phosphorylation controls Src catalytic activity, conformation and interactions mediated by the SH3 domain, representing a positive regulatory mechanism leading to elevated activation of mitogenic pathways and increased invasive potential of cells. Based on correlation between compactness of Src structure and its catalytic activity, we constructed a FRET-based sensor of Src conformation enabling to measure the dynamics of Src activation in cells with spatio-temporal resolution. We found that...