Die EZM bildet ein Netzwerk quervernetzter Proteine, welches alle Zellen im Tumor umgibt. Sie übt direkte Effekte auf die Medikamenteneinbringung und -verteilung aus und somit auch auf die therapeutische Effizienz von Chemotherapeutika. Die LOX(L)-Proteinfamilie katalysiert die oxidative Desaminierung von Lysinresten in Elastin und Kollagenfasern und ermöglicht dadurch eine intra- und intermolekulare Quervernetzung. Diese wird für die Reifung und Stabilisierung der Kollagene in der EZM benötigt. Eine erhöhte LOX(L)-Expression steigert durch eine verstärkte EZM-Quervernetzung die Gewebesteifheit im Tumor und bildet so eine physikalische Diffusionsbarriere. Durch diese Barriere wird die Versorgung mit Sauerstoff und Nährstoffen reduziert. Die resultierende Hypoxie im Tumor kann eine fehlgeleitete Angiogenese triggern und zu einer Aktivierung maligner Signalkaskaden führen. In dieser Arbeit wurden durch eine LOX(L)-Inhibierung mittels βAPN einerseits und eine ektopische LOX-/LOXL2-Überexpression andererseits Auswirkungen solcher Eingriffe auf verschiedene Indikatoren wie Zellproliferation und apoptose, Versorgung mit Sauerstoff und Nährstoffen, Angiogenese, Hypoxie, Makrophageninfiltration und die Expression verschiedener Wachstumsfaktoren analysiert. Die Versuche wurden an fünf verschiedenen Tumoren (4T1-, E0771- und EMT6-Brustkarzinome, LLC-Lungenkarzinome und MT6-Fibrosarkome) durchgeführt. Die Ergebnisse dieser Arbeit demonstrieren eine direkte Verbindung zwischen der EZM und einer Therapieresistenz. Nach βAPN-Behandlung konnte eine verbesserte Versorgung mit Sauerstoff und Nährstoffen beobachtet werden, welches in einer Verringerung maligner Signalkaskaden und folglich auch in einer verbesserten Vaskularisierung resultierte. Als Konsequenz wurde die therapeutische Effizienz von Chemotherapeutika verbessert. Im Gegensatz dazu führte eine LOX-/LOXL2-Überexpression zu einer erhöhten Therapieresistenz. Die vorliegende Studie zeigt, dass die Modifizierung der EZM durch eine Hemmung von LOX(L) das Potenzial birgt, das Ansprechen von Chemotherapeutika in der Behandlung von Krebserkrankungen zu verbessern. / The extracellular matrix (ECM) forms a network of cross-linked proteins, which surrounds all cells in the tumor. The ECM has direct effects on the accumulation and distribution of drugs and therefore also on the therapeutic efficacy of chemotherapeutics. The LOX protein family catalyzes the oxidative deamination of lysin-residues in elastins and collagen fibers and as a consequence allows an intra- and intermolecular crosslinking. This is necessary for the maturation and stabilization of the collagens in the ECM. An elevated LOX(L)-expression increases the stiffness through a higher cross-linked ECM and thus builds a physical diffusion barrier. Because of this barrier the oxygen and nutritional supply is limited. The resulting hypoxia in the tumor could promote errant angiogenesis and lead to an activation of malign signal cascades. In this dissertation, effects on different influences were examined on the one hand through a LOX(L)-inhibition with βAPN and on the other hand through an ectopic LOX /LOXL2-overexpression. These influences were cell proliferation and -apoptosis, supply with oxygen and nutrients, angiogenesis, hypoxia, infiltration of macrophages and the expression of various growth factors. The experiments were performed on five different tumor models (4T1, E0771, EMT6 breast carcinomas, LLC lung carcinomas and MT6 fibrosarcomas). The results of this work demonstrate a direct link between the ECM and a therapeutic resistance of tumors. After βAPN-treatment a better supply with oxygen and nutrients could be determined, which ended up in a reduction of malign signal cascades and thus in a better vascularization. As a consequence the therapeutic efficacy of chemotherapeutics was improved. In contrast, LOX /LOXL2-overexpression lead to a higher therapeutic resistance. This dissertation shows that modification of the ECM through LOX(L)-inhibition has the potential to improve response of chemotherapeutics in the treatment of cancer.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2003. / Includes bibliographical references (leaves 184-233).

Thesis (Ph. D.)--University of Hawaii at Manoa, 2005. / Includes bibliographical references (leaves 162-188). / Also available by subscription via World Wide Web / xvi, 188 leaves, bound ill. (some col.) 29 cm

Thesis (Ph. D.)--University of Hawaii at Manoa, 2005. / Includes bibliographical references (leaves 162-188).

INTRODUCTION: The lysyl oxidase family consists of 5 members and oxidizes specific lysine residues in biosynthetic collagen and elastin maturation. Lysyl oxidase like-2 (LOXL2) is elevated in oral cancer and promotes metastasis and correlates with poor prognosis. The objective of this study was to determine the mechanism by which LOXL2 promotes the progression and invasiveness of oral squamous cell carcinoma. METHODS: In vitro: The effects of LOXL2 inhibitor (PXS-S1C) on human gingival fibroblasts treated with tumor cell conditioned medium (CM) were investigated. Cell proliferation assays, signaling arrays, gene knock down and western blots were used to evaluate the effect of PXS-S1C on CM-treated fibroblasts. The effects of PXS-S1C on cancer cell expression of LOXL2 and proliferation were determined. To find potential LOXL2 substrates, carbonyl-containing proteins of gingival fibroblasts treated with CM +/- PXS-S1C were affinity-labeled and then purified by affinity chromatography and identified by western blot. In vivo: The effects of PXS-S1C on cancer cell growth and metastasis in vivo were investigated using orthotopic oral tongue cancer mouse models in both immunodeficient and immunocompetent mice. PXS-S1C at 10 mg/kg and 30 mg/kg was injected immediately following tumor cell injections. Tumor growth was monitored by both caliper measurement and in vivo imaging (IVIS). The mice were sacrificed and their organs were subjected to immunohistochemical staining with proliferation markers. RESULTS: PXS-S1C significantly inhibited gingival fibroblast proliferation stimulated by tumor cell CM and attenuated phosphorylation of PDGFRβ at the Y771 and Y857, but not Y751 residues in response to CM treatment. PXS-S1C inhibited ERK1/2-signaling in fibroblasts but not AKT pathway in response to CM treatment. PDGFR activation by oral tumor cells was mimicked by PDGF-AB but not PDGF-BB. PXS-S1C decreased the expression of LOXL2 in HSC3 oral cancer cells in vitro, suggesting the existence of a positive autoregulatory loop. Assessing for direct LOXL2 substrates in fibroblasts with functional consequences identified PDGFR. In vivo studies: Caliper measurements, IVIS, and immunohistochemistry demonstrated that inhibition of LOXL2 by injections of PXS-S1C significantly decreased both progression and metastasis of oral cancers in vivo in both mouse models. Mice without PXS-S1C treatment developed larger tongue volumes (p<0.05), and in immunocompetent mice larger lymph nodes (9 out 12) were observed compared to the PXS-S1C-treated mice (4 out of 12). IVIS imaging of immunodeficient mice revealed inhibition of metastasis by PXS-S1C treatment. The expression of proliferation marker (Ki-67 or PCNA) and LOXL2 was lower in tongue tumors treated with PXS-S1C in both in vivo models (p<0.05). CONCLUSIONS: LOXL2 secreted by cancer cells stimulates fibroblast proliferation by oxidizing PDGFR and thereby enhancing PDGF-mediated signaling. Inhibition of LOXL2 can be used as a therapeutic strategy to suppress the growth and metastasis of oral cancers by modulating tumor microenvironment. / 2019-10-24T00:00:00Z

O microambiente tumoral é composto por células, como fibroblastos, células do sistema imune, células endoteliais e pericitos, envoltas por uma matriz extracelular, além de possuir fatores solúveis que participam da comunicação celular. Nas últimas décadas, têm-se entendido cada vez melhor seu papel na iniciação e progressão dos tumores. É de fundamental importância, portanto, entender a biologia dos seus componentes e como podem agir em favor do desenvolvimento tumoral. Diversos trabalhos demonstram que há uma associação entre a presença dos pericitos nos vasos tumorais com a agressividade e prognóstico de alguns tipos de câncer. Uma vez ativadas, além do papel estrutural, essas células modulam as atividades das células endoteliais durante a formação de novos vasos, além de adquirirem propriedades como proliferação e migração. Neste contexto, os pericitos passam a secretar fatores importantes na comunicação célula-a-célula e liberam enzimas moduladoras na matriz extracelular. A lisil oxidase (LOX) é uma das principais enzimas que atuam sobre a matriz extracelular. Já está bem descrito que, quando superexpressa em células tumorais, a LOX pode alterar a migração e invasão dessas células, promovendo a geração de metástases. Entretanto, pouco se sabe a respeito da atuação dessa enzima sobre os demais componentes celulares do estroma tumoral, como os pericitos. Sendo assim, o presente trabalho teve como objetivo principal verificar se enzima LOX é relevante para a ativação de propriedades dos pericitos que possam contribuir para suas funções pró-tumorigênicas, como migração, proliferação e formação de vasos. Os resultados foram gerados avaliando essas atividades dos pericitos após pré-tratamento de 24 horas com &beta;-aminopropionitrile (&beta;APN), um inibidor irreversível da LOX. Foram utilizadas duas linhagens de pericitos derivados de tecido normal (adiposo e muscular) e duas linhagens de pericitos provenientes de tecido tumores do sistema nervoso central (neuroblastoma e ependimoma). Este composto foi capaz de diminuir a capacidade de migração das células de todas as linhagens testadas e, de maneira geral, tornou o processo de formação de estruturas tubulares in vitro menos eficiente. Entretanto, não foram observadas alterações na proliferação celular. Os dados indicam, portanto, que a enzima LOX pode ser importante para a ativação dos pericitos e, possivelmente, influenciem no seu comportamento no microambiente tumoral / The tumor microenvironment is composed of non-cancer cells, such as fibroblasts, immune cells, endothelial cells and pericytes, surrounded by an extracellular matrix, in addition to soluble factors involved in cellular crosstalk. In the last decades, it has been better understood its role in the initiation and progression of tumors. It is critical, therefore, to understand the biology of its components and how they can act in favor of tumor development. Several studies show an association between the presence of pericytes in tumor vessels with aggressiveness and prognosis of some cancers. Once activated, these cells modulate the activities of endothelial cells during the new vessels formation, and acquire properties as proliferation and migration. In this context, pericytes triggers the secretion of important factors in cell-to-cell communication and release modulating enzymes of extracellular matrix. The lysyl oxidase (LOX) is one of the main enzymes that act on the extracelular matrix. It is well described that when overexpressed in tumor cells, LOX can alter the migration and invasion of these cells, promoting the generation of metastases. However, little is known about the role of this enzyme over other cellular components of the tumor stroma, such as pericytes. Therefore, the aim of this study was to verify whether LOX enzyme is relevant to the activation of properties of the pericytes that could contribute to its pro-tumorigenic functions such as migration, proliferation and vessel formation. All the results were generated by evaluation of the activities of these pericytes after 24 hours pretreatment with &beta;-aminopropionitrile (&beta;APN), an irreversible inhibitor of LOX. This study used two cell lines of pericytes derived from normal tissue (fat and muscle) and two isolated from tissue of the central nervous system. The &beta;APN was able to reduce the migration of cells of all tested cell lines and, in general, alter the tubular formation in vitro. However, changes in cell proliferation weren&prime;t observed. The data showed, that the LOX family may be important for the activation of pericytes and possibly influence on their behavior in the tumor microenvironment

Recent studies support the association between metabolic syndrome (MetS), a cluster of cardiovascular risk factors, and diastolic dysfunction. Disproportionate collagen accumulation, particularly cross-linking of collagen, plays a key role in translating interstitial fibrosis into mechanical chamber stiffness and diastolic dysfunction. Characteristic changes in the expression and activity of myocardial lysyl oxidase (LOX), a matrix modifying enzyme that catalyzes cross-linked collagen, are unclear in MetS. We established a diet-induced MetS model to study diastolic dysfunction by treating male C57BL/6 mice a high-fat high-simple carbohydrate (HFHSC) diet for 6 months. Despite blunted gene expression of LOX isoforms, MetS mice demonstrated significant increase in the ratio of protein expression of mature to proenzyme LOX, enhanced LOX activity, and increased cardiac cross-linked collagen compared with controls. This fibrotic response coincided with marked increase in left ventricular end-diastolic pressure and stiffness and impaired diastolic filling pattern. Our data demonstrate that diet-induced MetS alters the remodeling enzyme LOX, thereby increasing the amount of crosslinking and inducing diastolic dysfunction.Furthermore we examined the role of T-lymphocytes in myocardial LOX regulation in diet-induced fibrotic hearts. Female SCID mice which are devoid of functional T-lymphocytes and C57BL/6 mice were treated with HFHSC diet for 12 months. Similar to male C67BL/6, female HFHSC-fed C57BL/6 mice demonstrated significant increase in maturation and catalytic activity of myocardial LOX, cross-linking, ventricular stiffness and diastolic dysfunction. Whereas induction of LOX protein was minimal in SCID mice compared with wild-type counterparts. Correspondingly fibrillar cross-linked collagen formation and diastolic dysfunction were less prominent in SCID mice. Our results suggest a potential role of T-lymphocytes in induction of myocardial stiffness and diastolic dysfunction through modulation of LOX-dependent collagen maturation.Moreover we studied the role of leptin, an adipokine over-produced in MetS with fibrotic effects in non-cardiac tissues, as a key mediator of profibrogenic responses in the heart by administrating leptin to C57BL/6 and leptin-deficient ob/ob mice. With exogenous leptin administration ob/ob mice displayed passive diastolic filling dysfunction that coincided with increase in myocardial collagen compared with ob/ob controls. Our findings suggest profibrotic effects of leptin in the heart, primarily through predominance of collagen synthesis over degradation.

Incretins are gastric hormones released by intestinal K-cells in response to food consumption and stimulate insulin secretion from pancreatic beta cells. One of these hormones, glucose-dependent insulinotropic peptide (GIP) is also anabolic in bone. Individuals with diabetes experience diminished bone quality caused by a low bone formation osteopenia. The present study seeks to identify a mechanism for diabetic osteopenia in which diabetes interferes with GIP-stimulated increases in the collagen cross-linking enzyme lysyl oxidase (LOX) in osteoblasts, leading to decreased collagen integrity and the trabecular abnormalities seen in diabetic bone. Micro-CT analysis and picrosirius red histology of long bones from LOX +/- and wild type mice made diabetic by low dose streptozotocin induction revealed a profound exacerbation of the decreased bone volume, impaired trabecular structure, and disorganized collagen matrix seen in diabetic mice when the mice were also haploinsufficient for LOX. Furthermore, qPCR of RNA isolated from diabetic long bones revealed a more than 20 fold decrease in LOX expression in diabetic bone from wild type mice. Treatment of wild type osteoblasts in culture with GIP results in a significant increase in LOX transcript and protein levels. Interestingly in our diabetic mice there is a decrease in osteoblast derived LOX and an abnormal increase in serum levels of the anti-incretin gut-derived dopamine, which is known to inhibit the effects of GIP in the pancreas. Therefore the ability of dopamine to inhibit GIP-stimulated signaling in osteoblasts was examined. Data indicate a strong dose-dependent inhibition of GIP-stimulated LOX expression when primary osteoblast cultures are pretreated with dopamine. Finally, pretreatment of primary osteoblasts with the dopamine receptor inhibitor amisulpride restored the impaired GIP stimulated increases in LOX expression in osteoblasts isolated from diabetic mice. This study defines a potential mechanism for diabetic bone disease and suggests that interference with dopamine signaling would likely restore bone health in diabetes.

O microambiente tumoral é composto por células, como fibroblastos, células do sistema imune, células endoteliais e pericitos, envoltas por uma matriz extracelular, além de possuir fatores solúveis que participam da comunicação celular. Nas últimas décadas, têm-se entendido cada vez melhor seu papel na iniciação e progressão dos tumores. É de fundamental importância, portanto, entender a biologia dos seus componentes e como podem agir em favor do desenvolvimento tumoral. Diversos trabalhos demonstram que há uma associação entre a presença dos pericitos nos vasos tumorais com a agressividade e prognóstico de alguns tipos de câncer. Uma vez ativadas, além do papel estrutural, essas células modulam as atividades das células endoteliais durante a formação de novos vasos, além de adquirirem propriedades como proliferação e migração. Neste contexto, os pericitos passam a secretar fatores importantes na comunicação célula-a-célula e liberam enzimas moduladoras na matriz extracelular. A lisil oxidase (LOX) é uma das principais enzimas que atuam sobre a matriz extracelular. Já está bem descrito que, quando superexpressa em células tumorais, a LOX pode alterar a migração e invasão dessas células, promovendo a geração de metástases. Entretanto, pouco se sabe a respeito da atuação dessa enzima sobre os demais componentes celulares do estroma tumoral, como os pericitos. Sendo assim, o presente trabalho teve como objetivo principal verificar se enzima LOX é relevante para a ativação de propriedades dos pericitos que possam contribuir para suas funções pró-tumorigênicas, como migração, proliferação e formação de vasos. Os resultados foram gerados avaliando essas atividades dos pericitos após pré-tratamento de 24 horas com &beta;-aminopropionitrile (&beta;APN), um inibidor irreversível da LOX. Foram utilizadas duas linhagens de pericitos derivados de tecido normal (adiposo e muscular) e duas linhagens de pericitos provenientes de tecido tumores do sistema nervoso central (neuroblastoma e ependimoma). Este composto foi capaz de diminuir a capacidade de migração das células de todas as linhagens testadas e, de maneira geral, tornou o processo de formação de estruturas tubulares in vitro menos eficiente. Entretanto, não foram observadas alterações na proliferação celular. Os dados indicam, portanto, que a enzima LOX pode ser importante para a ativação dos pericitos e, possivelmente, influenciem no seu comportamento no microambiente tumoral / The tumor microenvironment is composed of non-cancer cells, such as fibroblasts, immune cells, endothelial cells and pericytes, surrounded by an extracellular matrix, in addition to soluble factors involved in cellular crosstalk. In the last decades, it has been better understood its role in the initiation and progression of tumors. It is critical, therefore, to understand the biology of its components and how they can act in favor of tumor development. Several studies show an association between the presence of pericytes in tumor vessels with aggressiveness and prognosis of some cancers. Once activated, these cells modulate the activities of endothelial cells during the new vessels formation, and acquire properties as proliferation and migration. In this context, pericytes triggers the secretion of important factors in cell-to-cell communication and release modulating enzymes of extracellular matrix. The lysyl oxidase (LOX) is one of the main enzymes that act on the extracelular matrix. It is well described that when overexpressed in tumor cells, LOX can alter the migration and invasion of these cells, promoting the generation of metastases. However, little is known about the role of this enzyme over other cellular components of the tumor stroma, such as pericytes. Therefore, the aim of this study was to verify whether LOX enzyme is relevant to the activation of properties of the pericytes that could contribute to its pro-tumorigenic functions such as migration, proliferation and vessel formation. All the results were generated by evaluation of the activities of these pericytes after 24 hours pretreatment with &beta;-aminopropionitrile (&beta;APN), an irreversible inhibitor of LOX. This study used two cell lines of pericytes derived from normal tissue (fat and muscle) and two isolated from tissue of the central nervous system. The &beta;APN was able to reduce the migration of cells of all tested cell lines and, in general, alter the tubular formation in vitro. However, changes in cell proliferation weren&prime;t observed. The data showed, that the LOX family may be important for the activation of pericytes and possibly influence on their behavior in the tumor microenvironment

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