Bisphosphonate Inhibition of Prostate Cancer Cell Invasion, Migration and Cytoskeletal Organization

Metastatic bone lesions are commonly associated with prostate cancer affecting approximately 60-80% of the patients. The progression of prostate cancer into an advanced stage is a complex process and its molecular mechanisms are poorly understood. So far, no curative treatment is available for advanced stages of prostate cancer. Bisphosphonates (BPs) are synthetic pyrophosphate analogues, which are used as therapeutics for various metabolic bone diseases because of their ability to inhibit osteoclastic bone resorption. Nitrogen-containing bisphosphonates block the function of osteoclasts by disturbing the vesicular traffic and the mevalonate pathway -related enzymes, for example farnesyl diphosphate synthase, which is involved in post-translational isoprenylation of small GTPases. In addition, the anti-proliferative, anti-invasive and pro-apoptotic effects of nitrogen-containing bisphosphonates on various cancer cell lines have been reported. The aim of this thesis work was to clarify the effects of bisphosphonates on prostate cancer cells, focusing on the mechanisms of adhesion, invasion and migration. Furthermore, the role of the mevalonate pathway and prenylation reactions in invasion and regulation of the cytoskeleton of prostate cancer cells were examined. Finally, the effects of alendronate on cytoskeleton- and actin-related proteins in prostate cancer cells were studied in vitro and in vivo. The results showed that the nitrogen-containing bisphosphonate alendronate inhibited the adhesion of prostate cancer cells to various extracellular matrix proteins and migration and invasion in vitro. Inhibition of invasion and migration was reversed by mevalonate pathway intermediates. The blockage of the prenylation transferases GGTase I and FTase inhibited the invasion, migration and actin organization of prostate cancer cells. The marked decrease of cofilin was observed by the prenylation inhibitors used. Inhibition of GGTase I also disrupted the regulation of focal adhesion kinase and paxillin. In addition, alendronate disrupted the cytoskeletal organization and decreased the level of cofilin in vitro and in vivo. The decrease of the cofilin level by alendronate could be one of the key mechanisms behind the observed inhibition of migration and invasion. Based on the effects of nitrogen-containing bisphosphonates on tumor cell invasion and cytoskeletal organization, they can be suggested to be developed as therapeutics for inhibiting prostate cancer metastasis.

Integrins in Tumorigenesis and Cancer Cell Invasion

The integrin family of transmembrane receptors are important for cell-matrix adhesion and signal transmission to the interior of the cell. Integrins are essential for many physiological processes and defective integrin function can consequently result in a multitude of diseases, including cancer. Integrin traffic is needed for completion of cytokinesis and cell division failure has been proposed to be an early event in the formation of chromosomally aberrant and transformed cells. Impaired integrin traffic and changes in integrin expression are known to promote invasion of malignant cells. However, the direct roles of impaired integrin traffic in tumorigenesis and increased integrin expression in oncogene driven invasion have not been examined. In this study we have investigated both of these aspects. We found that cells with reduced integrin endocytosis become binucleate and subsequently aneuploid. These aneuploid cells display characteristics of transformed cells; they are anchorage-independent, resistant to apoptosis and invasive in vitro. Importantly, subcutaneous injection of the aneuploid cells into athymic nude mice produced highly malignant tumors. Through gene expression profiling and analysis of integrin-triggered signaling pathways we have identified several molecules involved in the malignancy of these cells, including Src kinase and the transcription factor Twist2. Thus, even though chromosomal aberrations are associated with reduced cell fitness, we show that aneuploidy can facilitate tumor evolution and selection of transformed cells. Invasion and metastasis are the primary reason for deaths caused by cancer and the molecular pathways responsible for invasion are therefore attractive targets in cancer therapy. In addition to integrins, another major family of adhesion receptors are the proteoglycans syndecans. Integrins and syndecans are known to signal in a synergistic manner in controlling cell adhesion on 2D matrixes. Here we explored the role of syndecans as α2β1 integrin co-receptors in 3D collagen. We show that in breast cancer cells harbouring mutant K-Ras, increased levels of integrins, their co-receptors syndecans and matrix cleaving proteases are necessary for the invasive phenotype of these cells. Together, these findings increase our knowledge of the complicated changes that occur during tumorigenesis and the pathways that control the ability of cancer cells to invade and metastasize.

Open Environmental Information Upon Disclosure Request in China: The Paradox of Legal Mobilization

Taking a realist view that law is one form of politics, this dissertation studies the roles of citizens and organizations in mobilizing the law to request government agencies to disclose environmental information in China, and during this process, how the socio-legal field interacts with the political-legal sphere, and what changes have been brought about during their interactions. This work takes a socio-legal approach and applies methodologies of social science and legal analysis. It aims to understand the paradox of why and how citizens and entities have been invoking the law to access environmental information despite the fact that various obstacles exist and the effectiveness of the new mechanism of environmental information disclosure still remains low. The study is largely based on the 28 cases and eight surveys of environmental information disclosure requests collected by the author. The cases and surveys analysed in this dissertation all occurred between May 2008, when the OGI Regulations and the OEI Measures came into effect, and August 2012 when the case collection was completed. The findings of this study have shown that by invoking the rules of law made by the authorities to demand government agencies disclosing environmental information, the public, including citizens, organizations, law firms, and the media, have strategically created a repercussive pressure upon the authorities to act according to the law. While it is a top-down process that has established the mechanism of open government information in China, it is indeed the bottom-up activism of the public that makes it work. Citizens and organizations’ use of legal tactics to push government agencies to disclose environmental information have formed not only an end of accessing the information but more a means of making government agencies accountable to their legal obligations. Law has thus played a pivotal role in enabling citizen participation in the political process. Against the current situation in China that political campaigns, or politicization, from general election to collective actions, especially contentious actions, are still restrained or even repressed by the government, legal mobilization, or judicialization, that citizens and organizations use legal tactics to demand their rights and push government agencies to enforce the law, become de facto an alternative of political participation. During this process, legal actions have helped to strengthen the civil society, make government agencies act according to law, push back the political boundaries, and induce changes in the relationship between the state and the public. In the field of environmental information disclosure, citizens and organizations have formed a bottom-up social activism, though limited in scope, using the language of law, creating progressive social, legal and political changes. This study emphasizes that it is partial and incomplete to understand China’s transition only from the top-down policy-making and government administration; it is also important to observe it from the bottom-up perspective that in a realistic view law can be part of politics and legal mobilization, even when utterly apolitical, can help to achieve political aims as well. This study of legal mobilization in the field of environmental information disclosure also helps us to better understand the function of law: law is not only a tool for the authorities to regulate and control, but inevitably also a weapon for the public to demand government agencies to work towards their obligations stipulated by the laws issued by themselves.

Tuning cell motility : roles of nestin and vimentin in cancer cell invasion and motility

The cytoskeleton is a key feature of both prokaryotic and eukaryotic cells. Itis comprised of three protein families, one of which is the intermediate filaments (IFs). Of these, the IFs are the largest and most diverse. The IFs are expressed throughout life, and are involved in the regulation of cell differentiation, homeostasis, ageing and pathogenesis. The IFs not only provide structural integrity to the cell, they are also involved in a range of cellular functions from organelle trafficking and cell migration to signalling transduction. The IFs are highly dynamic proteins, able to respond and adapt their network rapidly in response to intra- and extra- cellular cues. Consequently they interact with a whole host of cellular signalling proteins, regulating function, and activity, and cellular localisation. While the function of some of the better-known IFs such as the keratins is well studied, the understanding of the function of two IFs, nestin and vimentin, is poor. Nestin is well known as a marker of differentiation and is expressed in some cancers. In cancer, nestin is primarily described as is a promoter of cell motility, however, how it fulfils this role remains undefined. Vimentin too is expressed in cancer, and is known to promote cell motility and is used as a marker for epithelial to mesenchymal transition (EMT). It is only in the last decade that studies have addressed the role that vimentin plays in cell motility and EMT. This work provides novel insight into how the IFs, nestin and vimentin regulate cell motility and invasion. In particular we show that nestin regulates the cellular localisation and organisation of two key facilitators of cell migration, focal adhesion kinase and integrins. We identify nestin as a regulator of extracellular matrix degradation and integrin-mediated cell invasion. Two further studies address the specific regulation of vimentin by phosphorylation. A detailed characterisation study identified key phosphorylation sites on vimentin, which are critical for proper organisation of the vimentin network. Furthermore, we show that the bioactive sphingolipids are vimentin network regulators. Specifically, the sphingolipids induced RhoA kinasedependent (ROCK) phosphorylation at vimentin S71, which lead to filament reorganisation and inhibition of cell migration. Together these studies shed new light into the regulation of nestin and vimentin during cell motility.

Service paradox in a product oriented company: A service business development case

The master´s thesis had three aims; to develop a service portfolio, to support the management of services through the developed portfolio, and evaluate effects of service differentiation strategy on the future selection of services. The product oriented case company in service paradox is Hilti (Suomi) Oy, which is entering systematic service management era, supported by the late strategic change. Low return on service business investments is referred as service paradox. The project was carried out as a case study, where the primary information source was twenty-one conducted interviews. The theory part focuses on marketing logics, service strategies, and categorization of services. The empirical part contributes in solving the aim related research questions. As a result of the case study a service portfolio was created, next further steps in service management were suggested, and the effect on selection of services by service differentiation strategy was evaluated. The main goal of creating service portfolio contributes to systematic management of services, which required revising at the case company.

Sphingosine-1-phosphate-evoked invasion of follicular thyroid cancer cells : evidence for the involvement of HIF-Iα, MMP2 and MMP9

Sphingolipids are widely expressed molecules, which traditionally were considered to have majorly structural properties. Nowadays, however, they are implicated in a wide range of different biological processes. The bioactive lipid sphingosine 1-phosphate (S1P) has emerged during the past decade as one of the most studied molecules due to its proliferative and pro-migratory abilities both during normal physiology and in the pathology of a subset of different diseases. Migration and invasion of cancer cells require changes in cell behavior and modulation of the tissue microenvironment. Tumor aggressiveness is markedly enhanced by hypoxia, in which hypoxia inducible transcription factors 1-2α (HIF-1-2α) are activated to promote metabolism, proliferation and migration. Invasion requires degradation of the extracellular matrix (ECM) achieved by several degrading and remodeling enzymes. Matrix metalloproteinases (MMPs) are broadly expressed and well accepted as proteolytic enzymes with essential roles both in normal physiology and in pathology. Previously, S1P was shown to strongly evoke migration of follicular ML-1 thyroid cancer cells. The objective of this study was to further investigate and understand the mechanisms behind this regulation. In the first project it was demonstrated that S1P enhances the expression and activity of HIF-1α. S1P enhanced the expression of HIF-1α by increasing its synthesis and stability. The S1P-increased HIF-1α was mediated via S1P3, Gi/0, PI3K, PKCβI, ERK1/2, mTOR and translation factors p70S6K and eIF4E. Finally, it was shown that HIF-1α mediated S1P-induced migration. The ECM is constituted of a complex and coordinated assembly of many types of proteins. In order to be able to invade, cells need to break down the ECM, therefore several key players in this event were investigated in the second project. S1P increased the secretion and activity of MMP2 and MMP9 via S1P-receptor 1 and 3 and that these MMPs participated in the S1P-facilitated invasion of ML-1 cells. In this interplay, calpains and Rac1 were involved, both of which are crucial players in migration and invasion. The prognosis for some types of thyroid cancer is relatively good. However, there are forms of thyroid cancers, for which there are no treatments or the current available treatments are inefficient. Thus, new medical interventions are urgently needed. In the third project the significance of the S1P-receptor modulating drug FTY720, which is currently used for the treatment of multiple sclerosis (MS), was studied. The effect of FTY720 was tested on several thyroid cancer cell lines, and it inhibited the proliferation and invasion of all cancer cell lines tested. In ML-1 cells, FTY720 attenuated invasion by blocking signaling intermediates important for migration and invasion of the cells. Moreover, FTY720 inhibited the proliferation of ML-1 cells by increasing the expression of p21 and p27, hence, inducing cell arrest in G1 phase of the cell cycle. Thus, it can be suggested that FTY720 could be used in the treatment of thyroid cancer.

Standing before a sentence : Moore’s paradox and a perspective from within language

Med avstamp i ett satsbegrepp som inspirerats av Ludwig Wittgenstein, där sats och kontext betraktas som internt förbundna och där den verkliga användningen av en sats är central för dess mening, visar avhandlingen hur filosofers olika uppfattning om den filosofiska terminologins roll och möjligheter har följder för deras sätt att uppfatta och behandla filosofiska frågeställningar. Moores paradox fungerar som testfall. Denna kända filosofiska frågeställning har diskuterats sedan 1940-talet och kretsar kring hävdandet av satsen ”Jag tror att det regnar och det regnar inte”. Problemet är att det vore märkligt för en talare att hävda satsen om sig själv: det verkar finnas ett logiskt hinder för att hävda en sats även om den är välformad, kunde vara sann och inte på ett entydigt sätt innehåller en kontradiktion. Moores paradox behandlas genom att granska några lösningsförslag (framförda av bl.a. G.E. Moore, J. L. Austin, J. Searle) och de uppfattningar om språket som de förutsätter. Genom att kontrastera dessa med ett användarperspektiv där språkets användning i meningsfulla sammanhang förväntas spela en central roll i behandlingen av paradoxen visar författaren hur också den intellektuella kontext inom vilken en filosof betraktar paradoxen spelar en avgörande roll för vilka mått som krävs för att upplösa den. Istället för att föreslå en egen lösning presenteras en behandling av paradoxen genom en diskussion och utredning av de grundvillkor som leder till att den uppstår. Här intar satsbegreppet en central plats. Avhandlingen är författad inom traditionen efter den senare Wittgenstein (närmare bestämt den gren som ansluter sig till en s.k. terapeutisk filosofisyn) men går i dialog med filosofiskt arbete som ligger utanför traditionen. Författaren strävar både till att medla mellan, sammanföra och särskilja olika temata som behandlats på ett sätt inom traditionen och på andra sätt utanför den och använder sig här av metaforen att filosofera inifrån i kontrast till utanför språket. Avhandlingens huvudsakliga bidrag är till metafilosofin genom dess fokus på filosofins metod. ------------------------------------------------------------ Mooren paradoksi on tunnettu filosofinen ongelma, josta on keskusteltu 1940-luvulta alkaen. Paradoksin keskeisenä osana esiintyy väitelause ”Uskon, että ulkona sataa, mutta ulkona ei sada”. Ongelman lähtökohta on, että olisi merkillistä jos puhuja esittäisi lauseen itseään koskevana väitteenä: väite näyttää olevan loogisesti mahdoton esittää, vaikka se on hyvinmuodostettu, mahdollisesti tosi, eikä yksiselitteisellä tavalla sisällä kontradiktiota. Tässä väitöskirjassa Mooren paradoksi esiintyy esimerkkinä filosofisesta ongelmasta, jossa mm. lausekäsitteellä on keskeinen rooli ja jonka avulla voidaan muodostella ns. kielensisäinen filosofinen näkökulma. Mooren paradoksia käsitellään tarkastelemalla muutamia ratkaisuehdotuksia (mm. G.E. Mooren, J.L. Austinin, J. Searlen esittämät) ja niitä kielikäsityksiä joita nämä ratkaisuehdotukset edellyttävät. Asettamalla vastakkain ratkaisuehdotukset ja käyttäjänäkymä, jossa kielenkäyttö mielekkäissä konteksteissa asetetaan keskeiseen asemaan paradoksin käsittelyssä, kirjoittaja osoittaa miten myös paradoksin filosofisen tarkastelun intellektuaalinen konteksti on hyväksyttävien toimenpiteiden löytämisen kannalta ratkaisevaa. Uuden ratkaisun sijaan kirjoittaja ehdottaa paradoksin terapeuttista käsittelyä: keskustelua ja selontekoa niistä perusehdoista, jotka johtavat sen syntyyn. Tässä käsittelyssä lausekäsite on keskipisteenä – erityisesti Ludwig Wittgensteinin inspiroima lausekäsitys, jossa lauseen mielekkyyden kannalta sen todellinen käyttö mielekkäässä kontekstissa on ratkaiseva tekijä. Väitöskirja liittyy myöhäisiwittgensteinilaiseen perinteeseen, johon kuuluu ns. terapeuttinen filosofiankäsitys, mutta teos keskustelee myös tradition ulkopuolisten ajattelijoiden kanssa yrittäen yhdistää, sovitella ja myös eritellä teemoja, joita käsitellään yhdellä tavalla tradition sisällä ja toisella tavalla sen ulkopuolella. Väitöskirjan pääasiallinen panos sijoittuu metafilosofiaan, koska sen mielenkiinnon kohteena ovat erityisesti filosofian menetelmät.

Filopodia and stromal extracellular matrix as regulators of cancer cell invasion and growth

Bidirectional exchange of information between the cancer cells and their environment is essential for cancer to evolve. Cancer cells lose the ability to regulate their growth, gain the ability to detach from neighboring cells and finally some of the cells disseminate from the primary tumor and invade to the adjacent tissue. During cancer progression, cells acquire features that promote cancer motility and proliferation one of them being increased filopodia number. Filopodia are dynamic actin-rich structures extending from the leading edge of migrating cells and the main function of these structures is to serve as environmental sensors. It is nowadays widely appreciated, that not only the cancer cells, but also the surrounding of the tumor – the tumor microenvironment- contribute to cancer cell dissemination and tumor growth. Activated stromal fibroblasts, also known as cancer-associated fibroblasts (CAFs) actively participate on tumor progression. CAFs are the most abundant cell type surrounding the cancer cells and they are the main cell type producing the extracellular matrix (ECM) within tumor stroma. CAFs secrete growth factors to promote tumor growth, direct cancer cell invasion as well as modify the stromal ECM architecture. The aim of this thesis was to investigate the function of filopodia, particularly the role of filopodia-inducing protein Myosin-X (Myo10), in breast cancer cell invasion and metastasis. We found that Myo10 is an important regulator of basal type breast cancer spreading downstream of mutant p53. In addition, I investigated the role of CAFs and their secreted matrix on tumor growth. According to the results, CAF-derived matrix has altered organization and stiffness which induces the carcinoma cell proliferation via epigenetic mechanisms. I identified histone demethylase enzyme JMJD1a to be regulated by the stiffness and to participate in stiffness induced growth control.