Work in our lab has resulted in the development of a novel approach to creating a more developmentally plastic human dermal fibroblast (hDF) phenotype that allows for the study of molecular mechanisms involved in cell-fate conversion. Culturing hDF under defined culture conditions (5% O2 and supplementation with fibroblast growth factor FGF2) induces induced the regeneration competent (iRC) phenotype that is characterized by stem cell gene expression, and increased life-span in vitro. The work presented in this thesis further characterizes the system, and describes an overall shift in extracellular matrix and adhesion molecules in human dermal fibroblasts (hDF) undergoing the transition to a more developmentally plastic phenotype (iRC). This work suggests that we create the initiation phase of Mesenchymal-to-Epithelial Transition (MET) during conversion to the iRC phenotype. This transition is marked by loss of integrin alpha-11 (α11) and its binding partner Collagen-I (COL-I). Moreover, we describe the mechanism for the down-regulation of α11 that is mediated by FGF2 activation of ERK1/2 through systematic investigation of several potential molecular mechanisms. The body of work presented here shows that the ERK 1/2 mediated down-regulation of α11 is independent of activation of TGF-β1-mediated regulation of α11. In addition to down-regulation of α11, an overall shift in the transcript levels of other adhesion molecules is observed, which demonstrates that iRC are most likely transitioning their attachment to a laminin and fibronectin-based matrix. These results suggest that iRC may be producing a more “pro-regenerative matrixâ€�. We hypothesize that the changes in integrin expression profile and interaction with ECM serve as a feedback loop during the iRC phenotype shift. Our findings suggest that this “pro-regenerativeâ€� shift in attachment of iRC as well as the ERK 1/2 mediated down-regulation of α11 could be exploited in wound healing biology and fibrosis research. Manipulation of the dynamic relationship between TGF-β1 and FGF2 has the potential to reduce scar deposition. Further identification of molecular mechanisms controlling this phenotype conversion will allow development of strategies for in situ manipulation of wound healing outcomes.

The temporomandibular joint (TMJ) helps move the mandible and handle the forces associated with mastication. Much like other joints in the body, the TMJ can be afflicted with disorders that impair its function. Osteoarthritis (OA), one of the most common degenerative joint diseases worldwide, has been implicated as a prevalent temporomandibular disorder with limited treatment options. Fibroblast growth factor 2 (Fgf2), a gene important in bone remodeling, has been shown to lead to murine knee OA phenotypes in its germline ablation studies. In this study, the articular chondrocyte-specific ablation of the gene is studied in the temporomandibular joint condylar cartilage of female mice using a Col2CreERT2 knockout system. Micro-CT imaging suggested phenotypic changes in the condylar head samples of the conditional knockout samples in comparison to the control. Safranin o stains on frozen sections revealed phenotypic changes in cell morphology in the deeper layers of the cKO cartilage tissues in comparison to the control. Immunohistochemistry staining indicated a significant decrease in BMP2 protein expression and increasing trends in proteins responsible for cartilage degradation such as MMP13 in cKO samples in comparison to the control. These results suggest that the conditional ablation of Fgf2 results in phenotypic disruptions in the condylar cartilage of the TMJ. Further studies would be needed to indicate the validity behind these apparent disruptions and to further evaluate the molecular markers responsible for these changes.

Fibrotic diseases are a leading cause of morbidity and mortality in the developed world. Despite this, the lack of therapies for fibrotic pathological disease states is severe. A large part of the reason for this lack of viable therapies is due to an incomplete understanding of the early processes driving tissue fibrosis, as well as the dismal results of pharmacologic monotherapies at the clinical trial stage in humans thus far. Therefore, better understanding of the upstream mechanisms driving tissue fibrosis is imperative. One of the common mechanisms underlying all fibroses is the presence and activity of the myofibroblast, a contractile mesenchymal cell that deposits high levels of extracellular matrix. Overpersistence of myofibroblasts in the wound site lead to deposition of an acellular, nonfunctional, mechanically aberrant scar that can result in loss of tissue function and, in severe cases, eventual organ failure. Here we investigate the mechanisms under which fibroblast growth factor 2 (FGF2), one member of the mammalian fibroblast growth factor family, antagonizes activation of fibroblasts to myofibroblasts. We identify a gene and protein expression signature induced by FGF2 that is antagonistic to activated myofibroblasts, and we demonstrate that induction of this antifibrotic gene expression paradigm is antagonized by inhibition of the mitogen-activated protein kinase pathways ERK and JNK, each of which lies canonically downstream of FGF2/FGFR signaling, suggesting that the antifibrotic effects of FGF2 as an antagonist to fibroblast activation are likely dependent at least in part upon activation of these cellular signaling pathways. We further demonstrated that, independent of exogenous FGF2 stimulation, inhibition of ERK or JNK signaling in proliferating human dermal fibroblasts was sufficient to induce fibroblast activation, accompanied by a pro-fibrotic extracellular matrix gene expression paradigm. Inhibition of these pathways also resulted in distinct changes in transforming growth factor beta (TGF-β) gene expression paradigms, modulating the expression of both ligands and receptors involved in this pathway, and we verified that activation of fibroblasts via MAPK inhibition was dependent at least in part on activation of TGF-βR signaling. In contrast, inhibition of p38 MAPK was sufficient to antagonize fibroblast activation and subsequent fibrosis-associated extracellular matrix deposition, both in the presence and absence of exogenous TGF-β, via changes in gene expression antagonistic to pro-fibrotic TGF-β/TGF-βR signaling. Broadly, these data suggest that activation of ERK and JNK pathways broadly antagonize fibroblast activation and fibrosis, while activation of p38 drives fibroblast activation and pro-fibrotic fibroblast phenotypes. It is our hope that this information will lead to a better understanding of the way that cellular signaling pathways interact in order to drive fibroblast activation, and better inform the potential effects of kinase inhibitors or related therapeutics for use as anti-fibrotic therapies.

Endometrial adenocarcinoma, originating from the glandular epithelial cells of the uterine endometrial lining, is one of the most prevalent cancers amongst women in the Western world. The prostaglandin F2α (PGF2α) receptor (FP) is upregulated in endometrial adenocarcinoma. A previous microarray analysis of endometrial adenocarcinoma cells (Ishikawa) identified numerous targets of PGF2α-FP signalling including angiogenic factors, VEGF-A, FGF-2, CXCL1 and CXCL8 and antiangiogenic factors ADAMTS1. The regulation of VEGF-A, FGF-2, CXCL1 and CXCL8 was confirmed by previous studies using an in vitro model system, of Ishikawa cells stably expressing the FP receptor to levels observed in cancer (FPS cells). In this thesis, ADAMTS1 expression was found to be upregulated in endometrial adenocarcinoma samples compared to normal endometrium. Using FPS cells, ADAMTS1 expression was regulated in an extracellular signal regulated kinase 1/2 (ERK1/2) independent manner involving activation of nuclear factor of activated T cells (NFAT). Angiogenic and antiangiogenic proteins secreted by epithelial cells, in response to PGF2α-FP receptor signalling, could therefore regulate vascular function in a paracrine manner. Hence this thesis examines the role of angiogenic factors FGF2, CXCL1 and CXCL8, secreted into PGF2α-treated FPS cell conditioned medium (P CM), in the regulation of endothelial cell function in vitro. Firstly, using an in vitro model system, treatment of human umbilical vein endothelial cells (HUVECs) with P CM increased endothelial network formation and proliferation, compared to control CM. Immunoneutralisation of FGF2, CXCL1 and CXCL8 from the P CM reduced endothelial cell network formation and proliferation (P<0.05). In addition, inhibition of their receptors (FGFR1 and CXCR2) with chemical antagonists decreased endothelial cell network formation and proliferation (P<0.05) in response to treatment with P CM. This indicates that FGF2, CXCL1 and CXCL8 are paracrine effectors of FP-mediated endothelial cell network formation and proliferation. Next, the mechanisms by which FGF2 regulates P CM-induced endothelial cell network formation and proliferation were investigated. Using specific inhibitors of cell signalling, FGF2-FGFR1 was found to regulate endothelial cell proliferation via the mTOR pathway. In contrast, FGF2-FGFR1 signalling mediated endothelial cell network formation via the regulation of COX-2 expression and PGF2α synthesis in endothelial cells. Endometrial adenocarcinoma, originating from the glandular epithelial cells of the uterine endometrial lining, is one of the most prevalent cancers amongst women in the Western world. The prostaglandin F2α (PGF2α) receptor (FP) is upregulated in endometrial adenocarcinoma. A previous microarray analysis of endometrial adenocarcinoma cells (Ishikawa) identified numerous targets of PGF2α-FP signalling including angiogenic factors, VEGF-A, FGF-2, CXCL1 and CXCL8 and antiangiogenic factors ADAMTS1. The regulation of VEGF-A, FGF-2, CXCL1 and CXCL8 was confirmed by previous studies using an in vitro model system, of Ishikawa cells stably expressing the FP receptor to levels observed in cancer (FPS cells). In this thesis, ADAMTS1 expression was found to be upregulated in endometrial adenocarcinoma samples compared to normal endometrium. Using FPS cells, ADAMTS1 expression was regulated in an extracellular signal regulated kinase 1/2 (ERK1/2) independent manner involving activation of nuclear factor of activated T cells (NFAT). Angiogenic and antiangiogenic proteins secreted by epithelial cells, in response to PGF2α-FP receptor signalling, could therefore regulate vascular function in a paracrine manner. Hence this thesis examines the role of angiogenic factors FGF2, CXCL1 and CXCL8, secreted into PGF2α-treated FPS cell conditioned medium (P CM), in the regulation of endothelial cell function in vitro. Firstly, using an in vitro model system, treatment of human umbilical vein endothelial cells (HUVECs) with P CM increased endothelial network formation and proliferation, compared to control CM. Immunoneutralisation of FGF2, CXCL1 and CXCL8 from the P CM reduced endothelial cell network formation and proliferation (P<0.05). In addition, inhibition of their receptors (FGFR1 and CXCR2) with chemical antagonists decreased endothelial cell network formation and proliferation (P<0.05) in response to treatment with P CM. This indicates that FGF2, CXCL1 and CXCL8 are paracrine effectors of FP-mediated endothelial cell network formation and proliferation. Next, the mechanisms by which FGF2 regulates P CM-induced endothelial cell network formation and proliferation were investigated. Using specific inhibitors of cell signalling, FGF2-FGFR1 was found to regulate endothelial cell proliferation via the mTOR pathway. In contrast, FGF2-FGFR1 signalling mediated endothelial cell network formation via the regulation of COX-2 expression and PGF2α synthesis in endothelial cells. Angiogenesis is maintained by a balance of pro-and antiangiogenic factors. Hence, concomitantly with the upregulation of proangiogenic factors, antiangiogenic proteins ADAMTS1 and regulator of calcineurin 1 (RCAN1) were upregulated by P CM treatment of HUVECs. They were subsequently shown to limit endothelial cell network formation and proliferation in response to P CM. Finally, the role of PGF2α in angiogenesis was investigated using two in vivo models. PGF2α treatment did not increase angiogenesis in a sponge matrigel mouse model. In a xenograft mouse model, PGF2α-FP signalling increased expression of angiogenic factors in human epithelial cells and mouse stroma but this did not enhance microvessel density. Taken together, this thesis had highlighted that PGF2α-FP receptor signalling stimulates expression of pro-and antiangiogenic factors that in turn regulate endothelial cell function. However, in vivo studies demonstrate that PGF2α-FP receptor interaction does not impact on the level of angiogenesis but may control other aspects of vascular function.

The extracellular matrix (ECM), as the native cellular substrate, provides necessary mechanical and biological signals to cells. Cells exert forces in the nanonewton range, which when applied over time can strain extracellular matrix fibers until breakage. Cells and tissues inherently interact mechanically with their surrounding matrix, so tissue engineering materials would benefit from the ability to fully exploit mechanical-biochemical interactions to enhance integration with the human body. In this work, I developed an increased understanding of ECM fiber mechanical and mechano-biochemical properties. First, I generated novel composite ECM fibers that can be used to study combinations of ECM proteins in a controlled way. I determined how protein composition impacts mechanical properties of novel single ECM fibers in a hydrated state and showed how mechanical properties can be tuned through composition. Next, I assayed for strain and heparin-sensitive allosteric binding of ligands to fibronectin and fibrin fibers, and determined that the binding of two key growth factors is impacted by strain and heparin. Finally, I investigated the impact of fiber strain, heparin-pretreatment, and growth factor interactions on endothelial cell migration. The novel contributions of this project are the generation of new composite extracellular matrix fiber types with tunable mechanical properties, as well as the identification of extracellular matrix protein mechanosensitive and heparin-sensitive interactions with growth factors and their impact on endothelial cell migration, which could be used to aid in the design of protein-based biomaterials for cardiovascular applications. / 2024-05-23T00:00:00Z

O FGF2 (Fibroblast Growth Factor 2) é um clássico fator peptídico de crescimento que ativa vias intracelulares de sinalização molecular promovendo a transição G0 &#8594; G1 e o comprometimento com o ciclo celular. Não surpreendentemente, seus papéis pró-tumoral e angiogênico estão bem caracterizados e estabelecidos na literatura. No entanto, um crescente corpo de evidências tem indicado que o FGF2 também pode exercer efeitos anti-tumorais in vitro e in vivo, em modelos murinos e também humanos. Neste contexto, nosso grupo publicou em 2008 que o FGF2 exerce um efeito antiproliferativo seletivo em células murinas malignas dependentes de alta atividade de K-Ras e H-Ras. Os genes ras compõem a família de oncogenes mais frequentemente mutada em tumores malignos humanos, alcançando aproximadamente 30% de todos os casos. O desenvolvimento de terapias contra tumores dependentes de Ras fracassou, apesar dos intensos esforços e investimentos desde a descoberta em 1982 de suas mutações ativadoras em múltiplos cânceres. O objetivo deste trabalho foi desvendar os mecanismos moleculares pelo quais o FGF2 inibe irreversivelmente a proliferação de células malignas dependentes da atividade de Ras, empregando como modelos experimentais a linhagem murina Y1 de células adrenocorticais, e 4 linhagens humanas derivadas de sarcomas de Ewing. Identificamos que o efeito citotóxico do FGF2 não se processa por um mecanismo novo e independente das viasproliferativas classicamente ativadas por fatores peptídicos de crescimento. Ao contrário, seu efeito tóxico é resultado de sinalização mitogênica exagerada decorrente de estimulação sustentada por FGF2. A ativação da via de MAPK, principal sinalização mitogênica intracelular, a níveis elevados e sustentados provoca estresse mitogênico, que se propaga para a fase S na forma de estresse replicativo. Nesta situação, a célula passa a depender exageradamente da sinalização protetora de ATR, de modo que a combinação de estimulação com FGF2 e inibição de ATR foi altamente letal para as células malignas dependentes de Ras empregadas neste trabalho. Também analisamos as bases moleculares de resistência a FGF2 exibida por células Y1 anteriormente selecionadas para resistir ao efeito tóxico do FGF2 (Y1FRs). Descobrimos que a pressão seletiva do FGF2 não teve efeito na expressão de seus receptores, mas provocou a eliminação de um dos dois cromossomos que portam a amplificação gênica de ras nesta linhagem, enquanto o segundo cromossomo foi mantido por ser a única fonte de genes ribossomais ativos. Suas cópias de ras, no entanto, mostraram-se transcricionalmente silenciadas. Além disso, as sublinhagens Y1FRs não expressam o principal RasGEF, GRP4, encontrado nas células parentais Y1, o que pode ter influenciado o surgimento do fenótipo resistente ao FGF2. As linhagens resistentes mostraram grande redução no número de cromossomos e aumento da frequência de fusões entre cromossomos não homólogos em relação às células parentais. / FGF2 (Fibroblast Growth Factor 2) is a classic peptide growth factor that activates intracellular molecular signaling pathways promoting the G0 &#8594; G1 transition and cell cycle commitment. Not surprisingly, its pro-tumor and angiogenic roles are well characterized and established in the literature. However, a growing body of evidence has indicated that FGF2 may also exert anti-tumor effects in vitro and in vivo in murine and human models. In this context, our group reported in 2008 that FGF2 exerts a selective antiproliferative effect in murine cells dependent on high activity of K-Ras and H-Ras. Ras genes make up the most frequently mutated oncogene family in human malignant tumors, reaching approximately 30% of all cases. The development of therapies against Ras-dependent tumors has failed despite intense efforts and investments since the discovery in 1982 of its activating mutations in multiple cancers. The objective of this work was to uncover the molecular mechanisms by which FGF2 irreversibly inhibits the proliferation of malignant cells dependent on Ras activity, using as experimental models the Y1 murine lineage of adrenocortical malignant cells and 4 human lineages derived from Ewing sarcomas. We showed that the cytotoxic effect of FGF2 did not involve novel cell cycle regulatory pathways; instead, this cytotoxic effect is a result of sustainedhyper mitogenic stimulation by FGF2. Activation of the KRas/MAPK pathway, the major intracellular mitogenic signaling, at high and sustained levels provokes mitogenic stress, which is propagated to S phase as replicative stress. In this situation, the cell dependence on the ATR protective signaling is enhanced, so that the combination of stimulation with FGF2 and inhibition of ATR was highly lethal for the Ras dependent malignant cells employed in this work. We also analyzed the molecular basis of FGF2 resistance exhibited by Y1 cells previously selected for resistance to FGF2. We found that the selective pressure of FGF2 had no effect on the expression of its receptors but promoted the elimination of one of the two marker chromosomes that carry the K-ras amplified copies, while the second chromosome was maintained because it is the only source of active ribosomal genes; however, its K-ras amplified copies were transcriptionally silenced. In addition, the Y1FRs sublines did not express the main RasGEF, GRP4, found in the parental Y1 cells, which might have played a role in the emergence of the FGF2-resistant phenotype. The resistant Y1FRs sublines showed a large reduction in chromosome numbers and increased frequency of fusions between non-homologous chromosomes in relation to parental cells.

Mutações com ganho de função do proto-oncogene Ras se encontram entre umas das mais freqüentes modificações em cânceres humanos, além disso, tumores com esses caracterísitcas possuem, em geral, mau prognóstico. O objetivo inicial desta tese foi estudar novos mecanismos anti-proliferativos disparados por dois agentesmitogênicos, FGF2 (\"Fibroblast Growth Factor 2\") e PMA (\"Phorbol-12-Myristate-13-Acetate\", (um diéster de forbol), sobre células de camundongos transformadas por Ras e refratárias a apoptose. Para isso utilizamos duas linhagens celulares: uma linhagem naturalmente trtansformada por uma ampliação do gene K-Ras, que é derivada de um tumor de córtex adreno-cortical de camundongo e é denominada Y1, e uma sublinhagem derivada de Balb/c-3T3, transformada em laboratório com o oncogene H-RasV12 humano. A fim de se elucidar o mecanismo de ação de FGF2, foram selecionadas e caracterizadas múltiplas sublinhagens clonais resistentes a FGF2, derivadas das linhagens parentais Y1 e B61. Mostramos assim, que o FGF2 exerce um forte efeito negativo, de forma que os clones resistentes ao mesmo tendem a perder aos altos níveis de expressão da proteína Ras. Mostramos ainda que esses células passam a ser dependentes de FGF2 para crescer em cultura, perdem a capacidade de crescimento em suspensão e são menos tumorigênicas quando comparadas às células parentais. Em uma segunda etapa, caracterizamos o efeito citotóxico de PMA sobre células transformadas por Ras, e vimos que esse efeito é mais acentuado para células transformadas por K-Ras, mas é nulo sobre células imortalizadas não tumorigênicas. Mostramos ainda que esse efeito passa pela ativação da via de PKC. A inibição da proliferação por PMA se deve, ao menos parcialmente, à indução de senescência nessas células. De forma semelhante ao que foi para o estudo com FGF2, foram selecionados clones resistentes a PMA, derivados de Y1. Os clones obtidos se mostraram muito instáveis, pouco resistentes a PMA e dependentes de FGF2 para crescer. Todos os clones testados se mostram tumorigênicos, entretanto, apresentaram maior tempo de latência, estaticamente diferente da célula parental, Y1. Assim, neste trabalho, mostramos que duas substâncias, com caráter mitogênico e potencialmente oncogênico, são capazes de inibir seletivamente a proliferação de células transformadas por Ras, uma vez que elas não têm efeito sobre células não transformadas. Desvendar os mecanismos que causam a citotoxidade dessas substâncias deve trazer informações relevantes com possibilidades de impacto em terapia de tumores dependentes dos oncogenes Ras. / Amplification and gain of function mutations in ras proto-oncogenes are frequent genetic lesions in human cancers of bad prognostic. This thesis aimed to investigate novel anti-proliferative mechanisms induced by two mitogens, FGF2 (\"Fibroblast Growth Factor 2\") and PMA (\"Phorbol-12-Myristate-13-Acetate\", a phorbol diester), in murine cell lines transformed by ras and resistant to apoptosis. To this end, we took two different mouse malignant cell lines: Y1, a cell line derived from an adrenal tumor, naturally transformed by K-ras amplification and another one, 3T3-B61, obtained by transformation of Balb-3T3 fibroblasts with the H-rasV12 oncogene. To elucidate FGF2 mechanisms of action, we selected, isolated and characterized clonal sublines resistant to FGF2 from both Y1 and 3T3-B61 parental lines. FGF2-resistant clones are rare normal-like revertant sublines that no longer display Ras over expression, dependent on FGF2 for growth, do not grow in suspension cultures and exhibit low tumorigenicity in Nude mice. These results show that FGF2 exerts a strong selective pressure against ras-transformed cells, inducing senescence and irreversibly blocking proliferation. Differently from FGF2 , PMA citotoxic effect is completely dependent on PKC activity. In addition, PMA is highly toxic to K-Ras transformed Y1 cells, poorly toxic to H-Ras-transformed 3T3-B61 cells and not toxic to immortalized non tumorigenic cell lines. Attempts to select PMA-resistant cells fropm Y1 parental line have yielded very rare, highly clonal sublines, dependent on FGF2 for proliferation. In conclusion, two mitogens, FGF2 and PMA, can selectively inhibit Ras-driven proliferation, a phenomenon of great interest for biology and therapy of tumors dependent on ras oncogenes.

Um passo limitante no desenvolvimento de fármacos para terapias do câncer está na descoberta de vulnerabilidades específicas de células tumorais que sirvam à identificação de alvos moleculares apropriados à intervenção farmacológica. Esta é a motivação central desta tese, cuja abordagem experimental focaliza a ação oncogênica das proteínas Ras. Amplificação ou mutação ativadora nos proto-oncogenes ras estão entre as alterações genéticas mais frequentes em cânceres. Essas lesões genéticas aparecem na origem etiológica de múltiplas formas de fenótipos malignos. Mas, essas lesões oncogênicas também conferem susceptibilidades letais às células malignamente transformadas frente a determinados agentes que não interferem significativamente nas funções vitais de células normais. Nos últimos anos nosso laboratório vem estudando os mecanismos moleculares da ação antiproliferativa do fator de crescimento FGF2 (Fibroblast Growth Factor2) e do éster de forbol PMA (Phorbol-12-Myristate-13-Acetate) em linhagens de células murinas malignas dependentes de ras oncogênico. Nesta tese investigamos quanto de nossas observações anteriores com células murinas são aplicáveis a células humanas. Nesse sentido focalizamos a linhagem HaCaT de queratinócitos humanos imortalizados e seus subclones malignizados por expressão ectópica de H-RasV12; além disso, numa triagem inicial também examinamos treze linhagens celulares humanas derivadas de tumores naturais portadores de mutação ativadora em H-Ras, N-Ras ou K-Ras. Nossos resultados mostram que os queratinócitos da linhagem parental HaCaT expressam receptores de FGFs e respondem mitogenicamente tanto a FGF2 como a PMA; portanto, ambos FGF2 e PMA são benéficos aos queratinócitos HaCaT. Por outro lado, o FGF2 mostrou-se citotóxico para subclones HaCaT que expressam H-RasV12 induzível, mas sublinhagens HaCaT com expressão constitutiva de H-RasV12 mostraram-se resistentes à ação citotóxica de FGF2. Diferentemente de FGF2, PMA bloqueou a proliferação de sublinhagens clonais HaCaT-H-RasV12 em ambos substrato sólido e suspensão de agarose e, também, reduziu a estratificação dos queratinócitos HaCaT-H-RasV12 em culturas organotípicas. PMA foi citotóxico e não citostático, pois induziu morte apoptótica sem causar arresto em nenhuma fase específica do ciclo celular. Em HaCaT parental, PMA induziu aumento transitório dos níveis intracelulares de espécies reativas de oxigênio (ROS), mas nos queratinócitos HaCaT-H-RasV12, PMA causou aumentos mais altos e persistentes de ROS, o que promove forte estresse oxidativo, provavelmente responsável pela toxidez deste ester de forbol. Entre as treze linhagens celulares humanas malignas com H-Ras, N-Ras ou K-Ras mutados, onze foram vulneráveis à ação citotóxica de PMA; mas apenas uma delas, a linhagem de tumor urotelial UM-UC-3, foi sensível ao efeito anti-proliferativo de FGF2. Em conclusão, células malignas humanas com Ras mutado parecem superar rapidamente uma possível toxidez de FGF2, mas não ultrapassam a toxidez causada por PMA. / A challenge in drug development for cancer therapy is the discovering of molecular targets suitable for pharmacological interference. This challenge was the main motivation of the present thesis. Amplification or activating mutation in ras proto-oncogenes are among the most frequent genetic lesions in human cancer. Actually, mutated Ras onco-proteins are in the etiological roots of multiple malignant phenotypes; however these onco-proteins also cause specific lethal vulnerabilities even in robust malignant cells. Recently, our laboratory reported that malignant murine cell lines dependent on oncogenic Ras are prone to toxicity initiated by FGF2 (Fibroblast Growth Factor 2) and PMA (Phorbol-12-Myristate-13-Acetate), which are not harmful to normal cells. This cytotoxicity of FGF2 and PMA very likely follows different molecular mechanisms, which, however, are not yet completely understood. The aim of this thesis was to investigate whether these vulnerabilities found in murine malignant cells were also valid for human malignant cell lines dependent on oncogenic Ras. To this end the experimental approach was focused on the HaCaT cell line of immortalized human keratinocytes and its sublines transformed by H-RasV12 ectopic expression. In addition thirteen human cell lines derived from natural tumor carrying mutated H-Ras, N-Ras or K-Ras oncogenes were also screened. The results showed that HaCaT keratinocytes express FGF receptors and respond mitogenically to both FGF2 and PMA. On the other hand, FGF2 was cytotoxic to HaCaT subclones expressing inducible H-RasV12. But, HaCaT sublines constitutively expressing H-RasV12 were resistant to FGF2 toxicity. However, PMA was toxic to all HaCaT-H-RasV12 sublines, inhibiting proliferation in both solid substrate and agarose suspension cultures and, also reducing stratification in organotypic cultures. Furthermore, in HaCaT-H-RasV12 sublines, but not in the parental HaCaT line, PMA caused a persistently high increase in intracellular levels of reactive oxygen species (ROS) and concomitantly induced apoptosis. Moreover, eleven of the thirteen human tumor cell lines with mutated H-Ras, N-Ras or K-Ras, were growth inhibited by PMA, whereas only one of them was inhibited by FGF2, the urothelial tumor cell line UM-UC-3. In conclusion, human malignant cells driven by Ras oncogenes very likely rapidly overcome FGF2 toxicity, whereas they remain stably vulnerable to PMA cytotoxicity.

O FGF2 (Fibroblast Growth Factor 2) é um clássico fator peptídico de crescimento que ativa vias intracelulares de sinalização molecular promovendo a transição G0 &#8594; G1 e o comprometimento com o ciclo celular. Não surpreendentemente, seus papéis pró-tumoral e angiogênico estão bem caracterizados e estabelecidos na literatura. No entanto, um crescente corpo de evidências tem indicado que o FGF2 também pode exercer efeitos anti-tumorais in vitro e in vivo, em modelos murinos e também humanos. Neste contexto, nosso grupo publicou em 2008 que o FGF2 exerce um efeito antiproliferativo seletivo em células murinas malignas dependentes de alta atividade de K-Ras e H-Ras. Os genes ras compõem a família de oncogenes mais frequentemente mutada em tumores malignos humanos, alcançando aproximadamente 30% de todos os casos. O desenvolvimento de terapias contra tumores dependentes de Ras fracassou, apesar dos intensos esforços e investimentos desde a descoberta em 1982 de suas mutações ativadoras em múltiplos cânceres. O objetivo deste trabalho foi desvendar os mecanismos moleculares pelo quais o FGF2 inibe irreversivelmente a proliferação de células malignas dependentes da atividade de Ras, empregando como modelos experimentais a linhagem murina Y1 de células adrenocorticais, e 4 linhagens humanas derivadas de sarcomas de Ewing. Identificamos que o efeito citotóxico do FGF2 não se processa por um mecanismo novo e independente das viasproliferativas classicamente ativadas por fatores peptídicos de crescimento. Ao contrário, seu efeito tóxico é resultado de sinalização mitogênica exagerada decorrente de estimulação sustentada por FGF2. A ativação da via de MAPK, principal sinalização mitogênica intracelular, a níveis elevados e sustentados provoca estresse mitogênico, que se propaga para a fase S na forma de estresse replicativo. Nesta situação, a célula passa a depender exageradamente da sinalização protetora de ATR, de modo que a combinação de estimulação com FGF2 e inibição de ATR foi altamente letal para as células malignas dependentes de Ras empregadas neste trabalho. Também analisamos as bases moleculares de resistência a FGF2 exibida por células Y1 anteriormente selecionadas para resistir ao efeito tóxico do FGF2 (Y1FRs). Descobrimos que a pressão seletiva do FGF2 não teve efeito na expressão de seus receptores, mas provocou a eliminação de um dos dois cromossomos que portam a amplificação gênica de ras nesta linhagem, enquanto o segundo cromossomo foi mantido por ser a única fonte de genes ribossomais ativos. Suas cópias de ras, no entanto, mostraram-se transcricionalmente silenciadas. Além disso, as sublinhagens Y1FRs não expressam o principal RasGEF, GRP4, encontrado nas células parentais Y1, o que pode ter influenciado o surgimento do fenótipo resistente ao FGF2. As linhagens resistentes mostraram grande redução no número de cromossomos e aumento da frequência de fusões entre cromossomos não homólogos em relação às células parentais. / FGF2 (Fibroblast Growth Factor 2) is a classic peptide growth factor that activates intracellular molecular signaling pathways promoting the G0 &#8594; G1 transition and cell cycle commitment. Not surprisingly, its pro-tumor and angiogenic roles are well characterized and established in the literature. However, a growing body of evidence has indicated that FGF2 may also exert anti-tumor effects in vitro and in vivo in murine and human models. In this context, our group reported in 2008 that FGF2 exerts a selective antiproliferative effect in murine cells dependent on high activity of K-Ras and H-Ras. Ras genes make up the most frequently mutated oncogene family in human malignant tumors, reaching approximately 30% of all cases. The development of therapies against Ras-dependent tumors has failed despite intense efforts and investments since the discovery in 1982 of its activating mutations in multiple cancers. The objective of this work was to uncover the molecular mechanisms by which FGF2 irreversibly inhibits the proliferation of malignant cells dependent on Ras activity, using as experimental models the Y1 murine lineage of adrenocortical malignant cells and 4 human lineages derived from Ewing sarcomas. We showed that the cytotoxic effect of FGF2 did not involve novel cell cycle regulatory pathways; instead, this cytotoxic effect is a result of sustainedhyper mitogenic stimulation by FGF2. Activation of the KRas/MAPK pathway, the major intracellular mitogenic signaling, at high and sustained levels provokes mitogenic stress, which is propagated to S phase as replicative stress. In this situation, the cell dependence on the ATR protective signaling is enhanced, so that the combination of stimulation with FGF2 and inhibition of ATR was highly lethal for the Ras dependent malignant cells employed in this work. We also analyzed the molecular basis of FGF2 resistance exhibited by Y1 cells previously selected for resistance to FGF2. We found that the selective pressure of FGF2 had no effect on the expression of its receptors but promoted the elimination of one of the two marker chromosomes that carry the K-ras amplified copies, while the second chromosome was maintained because it is the only source of active ribosomal genes; however, its K-ras amplified copies were transcriptionally silenced. In addition, the Y1FRs sublines did not express the main RasGEF, GRP4, found in the parental Y1 cells, which might have played a role in the emergence of the FGF2-resistant phenotype. The resistant Y1FRs sublines showed a large reduction in chromosome numbers and increased frequency of fusions between non-homologous chromosomes in relation to parental cells.

FGF2 (Fibroblast Growth Factor 2) é o membro fundador de uma grande família de fatores de crescimento protéicos. Sua atividade se dá através da ligação e ativação de receptores específicos de membrana (FGFRs) com atividade de tirosina quinase. No organismo adulto, a sinalização de FGF2 está envolvida na indução de processos de sobrevivência, proliferação e diferenciação celular; além de cicatrização e angiogênese. Por atuar como um clássico fator de crescimento, a atividade de FGF2 está freqüentemente implicada em mecanismos pró-tumorais. Entretanto, alguns grupos, incluindo o nosso, têm reportado que FGF2 também pode apresentar efeitos antiproliferativos a até citotóxicos seletivamente em células malignas. Em 2008, publicamos um compreensivo relato mostrando que FGF2 bloqueia irreversivelmente a proliferação de linhagens murinas malignas dependentes de Ras. Alterações que levem a atividade aumentada de proteínas Ras estão presentes em diversos cânceres humanos e, freqüentemente, resultando em problemas no tratamento e prognóstico ruim. No presente trabalho, utilizamos principalmente a linhagem murina maligna dependente de Ras Y1 D1G, que apresenta um controle estrito de quiescência/proliferação em função da presença de soro; e é por isso mesmo um bom modelo para a análise dos efeitos de FGF2 sobre o ciclo celular. Análises por citometria de fluxo mostraram que, nessas células, apesar de disparar a transição G0&#8594;G1&#8594;S, FGF2 provoca um atraso na fase S seguido de um bloqueio do ciclo em G2. Embora bloqueie a progressão no ciclo (proliferação), FGF2 induz em Y1 D1G o crescimento celular em termos de massa e volume. Assim, nessas células FGF2 \"desconecta\" crescimento celular de proliferação. Esse desarranjo do ciclo celular provocado por FGF2 nas células Y1 D1G tem como resultado a instabilidade genotípica e morte celular; evidenciada pela perda da integridade de membrana plasmática e altas taxas de fragmentação de DNA observadas após o estímulo por esse fator. Esse efeito tóxico de FGF2 depende da atividade da proteína Src; porque a inibição química dessa proteína apresentou proteção total frente aos efeitos tóxicos de FGF2. Análises por espectrometria de massas mostraram que FGF2 induz aumento dos níveis de proteínas relacionadas à síntese protéica, e também de proteínas relacionadas ao estresse proteotóxico. Sabe-se que células malignas lidam com níveis basais altos de diferentes tipos de estresse; incluindo o estresse proteotóxico. Esse quadro mostra que o efeito tóxico disparado por FGF2 em Y1 D1G está relacionado a um acumulo de proteínas/célula, perda da homeostase de proteínas e estresse proteotóxico. Corrobora essas proposições o fato de que a inibição química de Src, que protege totalmente as células do efeito tóxico de FGF2, impede completamente o acúmulo de proteínas/célula. Além disso, em células Y1 D1G resistentes ao efeito tóxico de FGF2, e que inclusive dependem deste para proliferar em cultura, a atividade de FGF2 tem efeito oposto; ou seja, provoca diminuição dos níveis estacionários de proteínas/célula. Juntos, esses resultados demonstram que FGF2 é capaz de atacar uma vulnerabilidade de células malignas dependentes de Ras; e no caso estudado, essa vulnerabilidade decorre do desequilíbrio na homeostase de proteínas. / FGF2 is the first member of a large family of peptide growth factors. It binds and activates specific membrane receptors (FGFRs) belonging to a family of tyrosine kinase receptors (RTK). In adult organisms, FGF2 signaling is involved in the induction of cell surveillance, proliferation and differentiation; and also wound healing and angiogenesis. FGF2 is a bona fide growth factor and, as such, it is often implicated in pro-tumor mechanisms. However, several groups, including ours, have reported that FGF2 can also display antiproliferative and even cytotoxic effects selectively in malignant cells. In 2008, we fully reported that FGF2 irreversibly blocks the proliferation of Ras-driven mouse malignant lineages. Alterations leading to Ras proteins overactivity are present in many human cancers frequently with bad prognosis. In the present work, we used mainly the Ras-driven mouse malignant lineage Y1 D1G that shows a strict control of quiescence/proliferation by serum factors, making it a great model to analyze the FGF2 effects upon cell cycle control. Flow cytometry analyses showed that in these cells, in spite of triggering G0&#8594;G1&#8594;S transition, FGF2 causes a delay on S phase followed by cell cycle arrest in G2. Despite blocking cell division, FGF2 induces cell growth in terms of mass and volume. Therefore, in these cells FGF2 \"disconnects\" cell growth from proliferation. This malfunction of cell cycle control caused by FGF2 on Y1 D1G cells leads to genotypic instability and cell death, highlighted by loss of plasma membrane integrity and high rates of DNA fragmentation. This FGF2 toxic effect depends on the activity of Src protein, because Src chemical inhibition completely protects cells from the FGF2 toxic effects. Mass spec analyses showed that FGF2 increases the levels of proteins involved in the protein synthesis machinery, and also of proteins active in proteostasis, indicating proteotoxic stress. It is known that malignant cells deal with high basal levels of different stresses, including the proteotoxic stress. This picture shows that the toxic effects triggered by FGF2 in Y1 D1G involve accumulation of proteins/cell, loss of protein homeostasis and proteotoxic stress. Corroborating these propositions, chemical inhibition of Src, which completely protects the cells from FGF2 toxic effects, totally abrogates the accumulation of proteins/cell. Moreover, in FGF2-resistant Y1 D1G cells, which depend on this factor for proliferation, FGF2 shows the opposite effect, causing decrease in steady state levels of protein/cell. Altogether, these results show that FGF2 causes a severe proteostasis imbalance in these Ras-driven mouse malignant cells.