Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

The dynamics of cell signalling are critical to cell fate decisions. Human Epidermal growth factor Receptors (HERs)-mediated Ras/Raf/MEK/ERK and PI3K/Akt signalling cascades relay extracellular signals from the plasma membrane to targets in the nucleus and cytoplasm and play pivotal roles in cell fate determination including proliferation, differentiation and cell survival. Both pathways, once activated, are further regulated by complex feedback loops which may exert either positive or negative effects on cascade components and can result in signalling oscillation. In this study, heregulin (HRG) - and epidermal growth factor (EGF)- stimulated oscillation of both p-ERK1/2 and p-Akt expression in breast cancer cell lines was demonstrated. The oscillation was cell line dependent and was observed in MCF-7 and MCF-7/HER2-18 cells but not in BT474 cells. The oscillation was augmented by cycloheximide implicating transcriptional involvement. Gene expression analysis identified 29 genes as possible candidates involved in the transcriptional feedback regulation. Apart from the feedback regulation, feedforward regulation was also observed. To expedite the analyses In-cell Western and Reverse Phase Protein Array (RPPA) assays were developed. A scheme of transcriptional feedback loops regulating the oscillation in the ERK1/2 pathway is proposed, including negative feedback loops to ERK1/2 from DUSPs, early positive and late negative feedback loops to MEK1/2 and positive feedback loops to HER-3 from AREG, HB-EGF, CYR61 and CTGF. Two HER-2-targeted inhibitory monoclonal antibodies were investigated – trastuzumab and pertuzumab. Trastuzumab not only inhibited the growth of HER-2 over-expressing MCF-7/HER2- 18 cells and BT474 cells but also that of EGF-driven MCF-7 cells which expressed low/moderate HER-2 levels. Pertuzumab blocked the growth of both MCF-7 and MCF-7/HER2-18 driven by either EGF or HRG. When used in combination with trastuzumab, pertuzumab had much more potent activity in inhibiting cell growth and signalling than either single drug. Trastuzumab and pertuzumab had opposing effects on immediate p-ERK1/2 signalling and trastuzumab’s effects on signalling could be mimicked by the PI3K inhibitor LY294002. PTPN13, a non-receptor type tyrosine protein phosphatase, is a proposed tumour suppressor in breast cancer. This was investigated as a candidate regulator of the signalling oscillation and although not observed as a transcriptional modulator of the oscillation, its high expression level was observed to be associated with cell growth inhibition in MCF-7/HER2-18 cells by trastuzumab. Moreover, immunohistochemical analysis of 121 clinical tumours which had received trastuzumab treatment revealed the correlation between the expression level of PTPN13 and the mutation status of PIK3CA. In conclusion, the observed oscillation may contribute to the elucidation of the complex regulation of signalling pathways, which is vital to the different cell fate decision made through the same core pathway. The synergy between trastuzumab and pertuzumab supports the clinical use of the combination treatment and suggested PI3K/Akt pathway as the major pathway in controlling tumour growth.

published_or_final_version / abstract / Medicine / Master / Master of Philosophy

published_or_final_version / Pathology / Doctoral / Doctor of Philosophy

Um dos grupos de doenças mais importante que acomete o desenvolvimento da caixa craniana humana é o das craniossinostoses, caracterizado pelo fechamento prematuro de uma ou mais suturas cranianas. Entre as formas mendelianas das craniossinostoses sindrômicas, mutações dominantes em FGFR2 são uma das causas mais frequentes e estão associadas às síndromes de Apert, de Crouzon e de Pfeiffer. A sinalização intracelular subseqüente à ativação de FGFR2, tanto selvagem quanto mutante, é bastante intrincada e pode sofrer inúmeras bifurcações. As porções iniciais destas vias, imediatamente subsequentes à ativação do receptor, são relativamente bem compreendidas. Grande parte, porém, do controle dessas vias, principalmente no que tange a regulação transcricional e sua associação com alterações em comportamentos celulares, não é entendido. Assim sendo, os objetivos gerais deste trabalho foram: 1) estudar o potencial de diferenciação e o perfil diferencial de transcrição gênica de culturas primárias de células fibroblastóides isoladas a partir do periósteo das suturas coronais de pacientes acometidos por síndrome de Apert (heterozigotos para a mutação de ganho de função p.Ser252Trp em FGFR2, a mutação mais comum em pacientes com esta síndrome) e 2) estudar o potencial de diferenciação osteogênico e o perfil transcricional respectivamente de células mesenquimais e de tecido provenientes de sutura coronal de um modelo murino para Síndromes de Crouzon/Pfeiffer (heterozigotos para a mutação p.Cys342Tyr em Fgfr2, a mutação mais comum associada a estas síndromes). Certificamo-nos da expressão gênica e proteica de FGFR2 nas células fibroblastóides humanas e de Fgfr2 nas células mesenquimais murinas. Em seguida, testamos o potencial osteogênico (in vitro e in vivo ) e adipogênico (in vitro ) das células de pacientes com Síndrome de Apert, comparadas a células do mesmo tecido mas de indivíduos sem esta mutação e o potencial osteogênico (in vitro ) das células mesenquimais de camundongos portadores da mutação p.Cys342Tyr em Fgfr2, comparadas a células também das suturas coronais mas de animais selvagens. O potencial de diferenciação das células mutantes, nos dois grupos de experimentos, foi muito aumentado em relação ao potencial das células livres destas mutações. Conduzimos experimentos de microarrays de expressão gênica (sistema CodeLink) com 7 amostras de culturas primárias de células de pacientes com S. de Apert e as comparamos com 7 amostras de culturas primárias controles. Identificamos 263 genes com valores de expressão estatisticamente diferentes (SNR ≥ |0.4|, P ≤ 0,05) nas amostras de pacientes com S. de Apert quando comparadas às controles (118 superexpressos, 145 subexpressos). Categorias funcionais enriquecidas foram regulação de proliferação celular, metabolismo de nucleotídeos, regulação de expressão gênica, adesão celular, organização de matriz extracelular e cascata PI3K MAPK. Para a validação deste experimento constatamos superexpressão, por PCR em tempo real, de genes identificados como superexpressos na assinatura de expressão associada às células mutadas, além de verificarmos o mesmo comportamento destes genes em células controles tratadas com FGF2 exógeno para superativação do receptor. Os experimentos de expressão gênica com os tecidos de suturas coronais do modelo murino foram feitos com 15 amostras de tecidos de animais mutantes em 3 grupos de 5 e comparadas a amostras de mesmo tecido de animais selvagens agrupadas da mesma forma. Identificamos três listas de genes diferencialmente expressos: a primeira contendo 188 transcritos (P ≤0,05, FC ≥ 1,5,sendo 91 superexpressos e 97 subexpressos), e as outras duas filtradas previamente para coeficiente de variação < 50% dentro de cada grupo, contendo 488 transcritos (P ≤0,05, FC ≥ 1,2, sendo 183 superexpressos e 305 subexpressos) e 31 transcritos (P ≤0,05, FC ≥ 1,5, sendo 11 superexpressos e 20 subexpressos). Categorias funcionais mais enriquecidas foram crescimento, proliferação e ciclo celular, diferenciação celular, sinalização célula-célula, resposta imune mediada por células e sinalização por receptor Wnt. Estes resultados nos permitiram: a) demonstrar que células fibroblastóides de periósteo craniano de paciente portadores de S. de Apert (mutação p.Ser252Trp em FGFR2) e células mesenquimais do modelo murino para S. de Crouzon e Pfeiffer, portador da mutação p.Cys342Tyr em Fgfr2, apresentam potencial osteogênico aumentado, agregando evidências que sugerem que esta alteração de comportamento celular tem função fundamental no desencadeamento das craniossinostoses nestas síndromes; b) revelar assinaturas de expressão gênicas associadas a estas mutações nas condições estudadas, que podem reger este comportamento celular anormal; c) identificar um novo grupo de genes associados à patofisiologia da Síndrome de Apert ou às características fenotípicas do modelo murino investigado, podendo também ser genes candidatos a outras craniossinostoses de causa desconhecida. / Craniosynostosis is one of the most important group of diseases linked to the development of the human skull and is characterized by the premature fusion of one or more cranial sutures. Dominant mutations in FGFR2 are frequent molecular causes amongst the mendelian inherited forms of the syndromic craniosynostosis and are associated to Apert, Crouzon and Pfeiffer syndromes. The intracellular signaling pathways following the activation of wild type or mutant FGFR2 are very complex due to several possible bifurcations. The initial portions of these pathways, immediately following the receptor activation, are relatively well delineated. However the great majority of the events related to the control of these pathways is still not well understood, mainly concerning its transcriptional regulation and its association to other cell behavior anomalies. Therefore the key scopes of this work were: 1) to study the differentiation potential and the differential gene expression profile of primary fibroblastoid cell cultures isolated from the periosteum of the coronal sutures of Apert Syndrome patients (heterozygous for the mutation p.Ser252Trp in FGFR2, the most common cause of the Apert Syndrome condition) and 2) to study the osteogenic differentiation potential and the transcriptional profile of mesenchymal cells and tissue isolated from the coronal sutures of a mouse model for the Crouzon and Pfeiffer Syndromes (heterozygous for the p.Cys342Tyr mutation in Fgfr2, the mutation most commonly associated to these syndromes). We assured the FGFR2 /FGFR2 gene and the protein expression in human fibroblastoid cells and Fgfr2 /Fgfr2 expression in the mesenchymal murine cells. We tested the (in vitro and in vivo ) osteogenic and the (in vitro ) adipogenic potentials of the Apert Syndrome patients cells compared to cells from the same tissue but from subjects without this mutation and the (in vitro ) osteogenic potential of mesenchymal cells from mice bearing the p.Cys342Tyr mutation in Fgfr2 compared to coronal suture cells but from wild type mice. On both experiments the differentiation potential of the mutant cells were very increased when compared to the potential of the wild type cells. We conducted gene expression microarray experiments (CodeLink system) using 7 samples from primary cultures of cells from Apert Syndrome patients compared to 7 samples from primary control cultures. We identified 263 genes with significantly different expression (SNR ≥ |0.4|, P ≤ 0,05) associated to the Apert Syndrome profile (118 upregulated, 145 downregulated). Enriched functional cathegories were regulation of cell proliferation, nucleotide metabolism, gene expression regulation, cell adhesion, extracellular matrix organization and PI3K MAPK cascades. In order to validate this gene expression signature we confirmed through Real-Time PCR the upregulation of genes identified as upregulated in the Apert cell profile in samples from the microarray experiment and in control cells treated with exogenous overactivate the receptor. The gene expression experiments with the coronal suture tissues from the mouse model were performed with 15 samples of mutant animal tissue in 3 groups of 5 and compared to samples from the same tissue of wild type animals, with identical grouping. We identified three sets of differentially expressed genes: the first set containing 188 transcripts (P ≤0,05, FC ≥ 1,5, 91 upregulated e 97 downregulated), and the other two filtered for coeficient of variation < 50% in each group, containing 488 transcripts (P ≤0,05, FC ≥ 1,2, sendo 183 upregulated and 305downregulated) e 31 transcripts (P ≤0,05, FC ≥ 1,5, 11 upregulated and 20 downregulated). The most enriched functional categories were growth, proliferation and cell cycle, cell differentiation, cell-to-cell signaling, cell mediated immune response and Wnt receptor signaling. These results allowed us: a) to demonstrate that fibroblastoid cells from coronal periosteum PF Apert Syndrome patients (p.Ser252Trp mutation in FGFR2) and mesenchymal cells from the coronal tissue of the mouse model for Crouzon and Pfeiffer syndromes (bearing the p.Cys342Tyr in Fgfr2) have enhanced osteogenic potential, summoning evidences suggesting that this cell behavior alteration have a fundamental role to the craniosynostotic process in these syndromes; b) to unravel gene expression signatures linked to these mutations in the studied conditions, that could orchestrate this abnormal cell behavior; c) to identify a ser of genes associated to the pathophysiology of Apert Syndrome and to the phenotypic characteristics of the animal model investigated, which might be candidate genes to other craniosynostosis of unknown cause.

<p>Apoptotic dysregulation is a hallmark of cancer cells. The inability of cancer cells to undergo apoptosis may lead to therapeutic resistance. Inflammatory breast cancer (IBC) is a highly aggressive subtype of breast cancer that is often characterized by ErbB2 overexpression and ErbB2 activation. ErbB-targeting is clinically relevant using trastuzumab (anti-ErbB2 antibody) and lapatinib (small molecule ErbB1/2 inhibitor). However, acquired resistance is a common outcome even in IBC patients who show an initial clinical response, which limits the efficacy of these agents. Little is known about the molecular mechanisms of therapeutic resistance in IBC cells. We hypothesized that apoptotic dysregulation leads to therapeutic resistance of IBC cells to therapeutic agents, including ErbB-targeting agents. To determine whether apoptotic dysregulation and changes in anti-apoptotic proteins leads to resistance of IBC cells to therapeutic agents, we performed a variety of in vitro-based studies using agents that are used in the clinic to treat IBC patients. The sensitivity of both ErbB2 overexpressing and ErbB1 activated IBC cells to various therapeutic agents was evaluated using various cell death and apoptosis assays, and anti-apoptotic protein expression post-treatment was determined using western blot analysis. The overarching theme observed was that x-linked inhibitor of apoptosis protein (XIAP) expression inversely correlated with sensitivity of cells to therapeutic agents with various mechanisms of action, including TNF-related apoptosis inducing ligand (TRAIL), doxorubicin, cisplatin, paclitaxel, and two ErbB-targeting agents: trastuzumab and a lapatinib-analog (GW583340). Moreover, there was a specific and marked overexpression of XIAP in cells with de novo resistance to trastuzumab and with acquired resistance to GW583340. The observed overexpression was identified to be caused by IRES-mediated XIAP translation. Stable XIAP overexpression using a lentiviral system reversed sensitivity to therapeutic agents (TRAIL and GW583340) in parental IBC cells. Moreover, XIAP downregulation in cells resistant to therapeutic agents (TRAIL, trastuzumab, and GW583340) resulted in decreased viability and increased apoptosis, demonstrating that XIAP is required for survival of cells with resistance to these agents. A novel mechanism of GW583340 oxidative stress-induced mediated apoptosis was identified, and resistant cells had increased antioxidant expression and capability. Interesting, inhibition of XIAP function overcame this increase in antioxidant potential, demonstrating a new function for XIAP in oxidative stress-induced apoptosis. These studies establish the feasibility of development of an XIAP inhibitor that potentiates apoptosis for use in IBC patients with resistance to therapeutic agents.</p> / Dissertation

Thesis (Ph. D.)--Cornell University, 2001. / Vita. Includes bibliographical references (leaves 167-194).

published_or_final_version / Pathology / Master / Master of Medical Sciences

Thesis (Ph. D.)--University of Washington, 2005. / Vita. Includes bibliographical references (leaves 154-192).

Um dos grupos de doenças mais importante que acomete o desenvolvimento da caixa craniana humana é o das craniossinostoses, caracterizado pelo fechamento prematuro de uma ou mais suturas cranianas. Entre as formas mendelianas das craniossinostoses sindrômicas, mutações dominantes em FGFR2 são uma das causas mais frequentes e estão associadas às síndromes de Apert, de Crouzon e de Pfeiffer. A sinalização intracelular subseqüente à ativação de FGFR2, tanto selvagem quanto mutante, é bastante intrincada e pode sofrer inúmeras bifurcações. As porções iniciais destas vias, imediatamente subsequentes à ativação do receptor, são relativamente bem compreendidas. Grande parte, porém, do controle dessas vias, principalmente no que tange a regulação transcricional e sua associação com alterações em comportamentos celulares, não é entendido. Assim sendo, os objetivos gerais deste trabalho foram: 1) estudar o potencial de diferenciação e o perfil diferencial de transcrição gênica de culturas primárias de células fibroblastóides isoladas a partir do periósteo das suturas coronais de pacientes acometidos por síndrome de Apert (heterozigotos para a mutação de ganho de função p.Ser252Trp em FGFR2, a mutação mais comum em pacientes com esta síndrome) e 2) estudar o potencial de diferenciação osteogênico e o perfil transcricional respectivamente de células mesenquimais e de tecido provenientes de sutura coronal de um modelo murino para Síndromes de Crouzon/Pfeiffer (heterozigotos para a mutação p.Cys342Tyr em Fgfr2, a mutação mais comum associada a estas síndromes). Certificamo-nos da expressão gênica e proteica de FGFR2 nas células fibroblastóides humanas e de Fgfr2 nas células mesenquimais murinas. Em seguida, testamos o potencial osteogênico (in vitro e in vivo ) e adipogênico (in vitro ) das células de pacientes com Síndrome de Apert, comparadas a células do mesmo tecido mas de indivíduos sem esta mutação e o potencial osteogênico (in vitro ) das células mesenquimais de camundongos portadores da mutação p.Cys342Tyr em Fgfr2, comparadas a células também das suturas coronais mas de animais selvagens. O potencial de diferenciação das células mutantes, nos dois grupos de experimentos, foi muito aumentado em relação ao potencial das células livres destas mutações. Conduzimos experimentos de microarrays de expressão gênica (sistema CodeLink) com 7 amostras de culturas primárias de células de pacientes com S. de Apert e as comparamos com 7 amostras de culturas primárias controles. Identificamos 263 genes com valores de expressão estatisticamente diferentes (SNR &#8805; |0.4|, P &#8804; 0,05) nas amostras de pacientes com S. de Apert quando comparadas às controles (118 superexpressos, 145 subexpressos). Categorias funcionais enriquecidas foram regulação de proliferação celular, metabolismo de nucleotídeos, regulação de expressão gênica, adesão celular, organização de matriz extracelular e cascata PI3K MAPK. Para a validação deste experimento constatamos superexpressão, por PCR em tempo real, de genes identificados como superexpressos na assinatura de expressão associada às células mutadas, além de verificarmos o mesmo comportamento destes genes em células controles tratadas com FGF2 exógeno para superativação do receptor. Os experimentos de expressão gênica com os tecidos de suturas coronais do modelo murino foram feitos com 15 amostras de tecidos de animais mutantes em 3 grupos de 5 e comparadas a amostras de mesmo tecido de animais selvagens agrupadas da mesma forma. Identificamos três listas de genes diferencialmente expressos: a primeira contendo 188 transcritos (P &#8804;0,05, FC &#8805; 1,5,sendo 91 superexpressos e 97 subexpressos), e as outras duas filtradas previamente para coeficiente de variação &lt; 50% dentro de cada grupo, contendo 488 transcritos (P &#8804;0,05, FC &#8805; 1,2, sendo 183 superexpressos e 305 subexpressos) e 31 transcritos (P &#8804;0,05, FC &#8805; 1,5, sendo 11 superexpressos e 20 subexpressos). Categorias funcionais mais enriquecidas foram crescimento, proliferação e ciclo celular, diferenciação celular, sinalização célula-célula, resposta imune mediada por células e sinalização por receptor Wnt. Estes resultados nos permitiram: a) demonstrar que células fibroblastóides de periósteo craniano de paciente portadores de S. de Apert (mutação p.Ser252Trp em FGFR2) e células mesenquimais do modelo murino para S. de Crouzon e Pfeiffer, portador da mutação p.Cys342Tyr em Fgfr2, apresentam potencial osteogênico aumentado, agregando evidências que sugerem que esta alteração de comportamento celular tem função fundamental no desencadeamento das craniossinostoses nestas síndromes; b) revelar assinaturas de expressão gênicas associadas a estas mutações nas condições estudadas, que podem reger este comportamento celular anormal; c) identificar um novo grupo de genes associados à patofisiologia da Síndrome de Apert ou às características fenotípicas do modelo murino investigado, podendo também ser genes candidatos a outras craniossinostoses de causa desconhecida. / Craniosynostosis is one of the most important group of diseases linked to the development of the human skull and is characterized by the premature fusion of one or more cranial sutures. Dominant mutations in FGFR2 are frequent molecular causes amongst the mendelian inherited forms of the syndromic craniosynostosis and are associated to Apert, Crouzon and Pfeiffer syndromes. The intracellular signaling pathways following the activation of wild type or mutant FGFR2 are very complex due to several possible bifurcations. The initial portions of these pathways, immediately following the receptor activation, are relatively well delineated. However the great majority of the events related to the control of these pathways is still not well understood, mainly concerning its transcriptional regulation and its association to other cell behavior anomalies. Therefore the key scopes of this work were: 1) to study the differentiation potential and the differential gene expression profile of primary fibroblastoid cell cultures isolated from the periosteum of the coronal sutures of Apert Syndrome patients (heterozygous for the mutation p.Ser252Trp in FGFR2, the most common cause of the Apert Syndrome condition) and 2) to study the osteogenic differentiation potential and the transcriptional profile of mesenchymal cells and tissue isolated from the coronal sutures of a mouse model for the Crouzon and Pfeiffer Syndromes (heterozygous for the p.Cys342Tyr mutation in Fgfr2, the mutation most commonly associated to these syndromes). We assured the FGFR2 /FGFR2 gene and the protein expression in human fibroblastoid cells and Fgfr2 /Fgfr2 expression in the mesenchymal murine cells. We tested the (in vitro and in vivo ) osteogenic and the (in vitro ) adipogenic potentials of the Apert Syndrome patients cells compared to cells from the same tissue but from subjects without this mutation and the (in vitro ) osteogenic potential of mesenchymal cells from mice bearing the p.Cys342Tyr mutation in Fgfr2 compared to coronal suture cells but from wild type mice. On both experiments the differentiation potential of the mutant cells were very increased when compared to the potential of the wild type cells. We conducted gene expression microarray experiments (CodeLink system) using 7 samples from primary cultures of cells from Apert Syndrome patients compared to 7 samples from primary control cultures. We identified 263 genes with significantly different expression (SNR &#8805; |0.4|, P &#8804; 0,05) associated to the Apert Syndrome profile (118 upregulated, 145 downregulated). Enriched functional cathegories were regulation of cell proliferation, nucleotide metabolism, gene expression regulation, cell adhesion, extracellular matrix organization and PI3K MAPK cascades. In order to validate this gene expression signature we confirmed through Real-Time PCR the upregulation of genes identified as upregulated in the Apert cell profile in samples from the microarray experiment and in control cells treated with exogenous overactivate the receptor. The gene expression experiments with the coronal suture tissues from the mouse model were performed with 15 samples of mutant animal tissue in 3 groups of 5 and compared to samples from the same tissue of wild type animals, with identical grouping. We identified three sets of differentially expressed genes: the first set containing 188 transcripts (P &#8804;0,05, FC &#8805; 1,5, 91 upregulated e 97 downregulated), and the other two filtered for coeficient of variation &lt; 50% in each group, containing 488 transcripts (P &#8804;0,05, FC &#8805; 1,2, sendo 183 upregulated and 305downregulated) e 31 transcripts (P &#8804;0,05, FC &#8805; 1,5, 11 upregulated and 20 downregulated). The most enriched functional categories were growth, proliferation and cell cycle, cell differentiation, cell-to-cell signaling, cell mediated immune response and Wnt receptor signaling. These results allowed us: a) to demonstrate that fibroblastoid cells from coronal periosteum PF Apert Syndrome patients (p.Ser252Trp mutation in FGFR2) and mesenchymal cells from the coronal tissue of the mouse model for Crouzon and Pfeiffer syndromes (bearing the p.Cys342Tyr in Fgfr2) have enhanced osteogenic potential, summoning evidences suggesting that this cell behavior alteration have a fundamental role to the craniosynostotic process in these syndromes; b) to unravel gene expression signatures linked to these mutations in the studied conditions, that could orchestrate this abnormal cell behavior; c) to identify a ser of genes associated to the pathophysiology of Apert Syndrome and to the phenotypic characteristics of the animal model investigated, which might be candidate genes to other craniosynostosis of unknown cause.