Securin, Cdc20 and Cdc27 in predicting the outcome of human breast cancer

Deregulated proliferation has been recognized among the most important factors promoting breast cancer development and progression. The aim of the project is to gain understanding of the role of specific cell cycle regulators of metaphase-anaphase transition and evaluate their potential in breast cancer prognostication and treatment decisions. Metaphase-anaphase transition is triggered by activation of anaphase promoting complex (APC) which is activated by a cascade of regulatory proteins, among them securin, Cdc20 and Cdc27. These proteins promote the metaphase–anaphase transition and participate in the timely separation of the chromatids. This study is based on a patient material of approximately 600 breast cancer patients and up to 22 years of follow-up. As the main observation, based on DNA cytometric and immunohistochemical methods, securin, Cdc20 and Cdc27 protein expressions were associated with abnormal DNA content and outcome of breast cancer. In the studied patient material, high securin expression alone and in combination with Cdc20 and Cdc27 predicted up to 9.8-fold odds for aneuploid DNA content in human breast cancer. In Kaplan–Meier analyses, high expression of securin systematically indicated decrease in breast cancer survival as compared to low expression cases. The adverse effect of high securin expression was further strengthened by combining it with Cdc20 or Cdc27 expressions, resulting in up to 6.8-fold risk of breast cancer death. High securin and Cdc20 expression was also associated with triple-negative breast cancer type with high statistical significance. Securin, Cdc20 or Cdc27 have not previously been investigated in a clinically relevant large breast cancer patient material or in association with DNA ploidy. The present findings suggest that the studied proteins may serve as potential biomarkers for identification of aggressive course of disease and unfavourable outcome of human breast cancer, and that they may provide a future research aim for understanding abnormal proliferation in malignant disease.

Choosing the target loci : heat shock factors HSF1 and HSF2 as regulators of cell stress and development

Heat shock factors (HSFs) are an evolutionarily well conserved family of transcription factors that coordinate stress-induced gene expression and direct versatile physiological processes in eukaryote organisms. The essentiality of HSFs for cellular homeostasis has been well demonstrated, mainly through HSF1-induced transcription of heat shock protein (HSP) genes. HSFs are important regulators of many fundamental processes such as gametogenesis, metabolic control and aging, and are involved in pathological conditions including cancer progression and neurodegenerative diseases. In each of the HSF-mediated processes, however, the detailed mechanisms of HSF family members and their complete set of target genes have remained unknown. Recently, rapid advances in chromatin studies have enabled genome-wide characterization of protein binding sites in a high resolution and in an unbiased manner. In this PhD thesis, these novel methods that base on chromatin immunoprecipitation (ChIP) are utilized and the genome-wide target loci for HSF1 and HSF2 are identified in cellular stress responses and in developmental processes. The thesis and its original publications characterize the individual and shared target genes of HSF1 and HSF2, describe HSF1 as a potent transactivator, and discover HSF2 as an epigenetic regulator that coordinates gene expression throughout the cell cycle progression. In male gametogenesis, novel physiological functions for HSF1 and HSF2 are revealed and HSFs are demonstrated to control the expression of X- and Y-chromosomal multicopy genes in a silenced chromatin environment. In stressed human cells, HSF1 and HSF2 are shown to coordinate the expression of a wide variety of genes including genes for chaperone machinery, ubiquitin, regulators of cell cycle progression and signaling. These results highlight the importance of cell type and cell cycle phase in transcriptional responses, reveal the myriad of processes that are adjusted in a stressed cell and describe novel mechanisms that maintain transcriptional memory in mitotic cell division.

Life without Phd? – Phd Oxygen Sensor Proteins in Hypoxic Carcinoma Cell Survival

The microenvironment within the tumor plays a central role in cellular signaling. Rapidly proliferating cancer cells need building blocks for structures as well as nutrients and oxygen for energy production. In normal tissue, the vasculature effectively transports oxygen, nutrient and waste products, and maintains physiological pH. Within a tumor however, the vasculature is rarely sufficient for the needs of tumor cells. This causes the tumor to suffer from lack of oxygen (hypoxia) and nutrients as well as acidification, as the glycolytic end product lactate is accumulated. Cancer cells harbor mutations enabling survival in the rough microenvironment. One of the best characterized mutations is the inactivation of the von Hippel-Lindau protein (pVHL) in clear cell renal cell carcinoma (ccRCC). Inactivation causes constitutive activation of hypoxia-inducible factor HIF which is an important survival factor regulating glycolysis, neovascularization and apoptosis. HIFs are normally regulated by HIF prolyl hydroxylases (PHDs), which in the presence of oxygen target HIF α-subunit to ubiquitination by pVHL and degradation by proteasomes. In my thesis work, I studied the role of PHDs in the survival of carcinoma cells in hypoxia. My work revealed an essential role of PHD1 and PHD3 in cell cycle regulation through two cyclin-dependent kinase inhibitors (CKIs) p21 and p27. Depletion of PHD1 or PHD3 caused a cell cycle arrest and subjected the carcinoma cells to stress and impaired the survival.

The Integrin Tail: A Tale of Cell Motility and Division

Integrin transmembrane receptor functions are regulated by adaptor molecules binding to their alpha and beta subunit intracellular domains, or tails, thus affecting integrin traffic and adhesion during e.g. cell motility. Interestingly, many cellular proteins function in both cell motility and cell division, thus raising the possibility that integrins might be involved in regulating the cell cycle. A thorough understanding of cell division is essential in cell biology and in human malignancies. It is well established that failures to complete cell cycle can give rise to genetically unstable cells with tumorigenic properties. Transformed cells promote the disruption of intercellular adhesions such as tight junctions, and this correlates with the onset of cell motility, invasion and unfavorable prognosis in cancer. In this study, we analyzed integrin regulation, mediated by adaptor binding to the  subunit tail, during cell motility and cell division. We revealed a novel molecular mechanism by which Rab21, through association with the integrin alpha subunits, drives integrin endosomal traffic during mitotic phases. In addition, we found indications for this finding in vivo, as RAB21 gene deletions were mapped in ovarian and prostate cancer samples. Importantly, the multinucleated phenotype of cultured ovarian cancer cells could be reverted by Rab21 overexpression. In this thesis work, we also show how the tight junction protein ZO-1 unexpectedly interacts with the 5 integrin cytoplasmic domain in the lamellipodia to promote cell motility and at the cleavage furrow to support separation of the daughter cells. The alpha5-ZO-1 complex formation was dependent on PKC which regulates ZO-1 phosphorylation and its subcellular localization. In addition, by an in situ detection method, we showed that a subset of metastatic human lung cancers expressed the alpha5beta-ZO-1 complex. Taken together, we were able to identify new molecular pathways that regulate integrin functions in an alpha tail-mediated fashion. These findings firmly suggest that genetic alterations in integrin traffic may lead to progression of tumorigenesis as a result of failed cell division. Also, the interplay of integrins and ZO-1 in forming spatially regulated adhesive structures broadens our view of crosstalk between pathways and distinct adhesive structures that can be involved in cancer cell biology.

Functional characterization of proteins required for mitotic progression and the spindle assembly checkpoint

During mitotic cell division, the genetic material packed into chromosomes is divided equally between two daughter cells. Before the separation of the two copies of a chromosome (sister chromatids), each chromosome has to be properly connected with microtubules of the mitotic spindle apparatus and aligned to the centre of the cell. The spindle assembly checkpoint (SAC) monitors connections between microtubules and chromosomes as well as tension applied across the centromere. Microtubules connect to a chromosome via kinetochores, which are proteinaceous organelles assembled onto the centromeric region of the sister chromatids. Improper kinetochore-microtubule attachments activate the SAC and block chromosome segregation until errors are corrected and all chromosomes are connected to the mitotic spindle in a bipolar manner. The purpose of this surveillance mechanism is to prevent loss or gain of chromosomes in daughter cells that according to current understanding contributes to cancer formation. Numerous proteins participate in the regulation of mitotic progression. In this thesis, the mitotic tasks of three kinetochore proteins, Shugoshin 1 (Sgo1), INCENP, and p38 MAP kinase (p38 MAPK), were investigated. Sgo1 is a protector of centromeric cohesion. It is also described in the tension-sensing mechanism of the SAC and in the regulation of kinetochore-microtubule connections. Our results revealed a central role for Sgo1 in a novel branch of kinetochore assembly. INCENP constitutes part of the chromosomal passenger complex (CPC). The other members of the core complex are the Aurora B kinase, Survivin and Borealin. CPC is an important regulatory element of cell division having several roles at various stages of mitosis. Our results indicated that INCENP and Aurora B are highly dynamic proteins at the mitotic centromeres and suggested a new role for CPC in regulation of chromosome movements and spindle structure during late mitosis. The p38 MAPK has been implicated in G1 and G2 checkpoints during the cell cycle. However, its role in mitotic progression and control of SAC signaling has been controversial. In this thesis, we discovered a novel function for p38γ MAPK in chromosome orientation and spindle structure as well as in promotion of viability of mitotic cells.

Nuclear Matrix in Apoptotic Cell Death and Cell Proliferation. The role of Nuclear Mitotic Apparatus (NuMA) Protein

The nucleus is a membrane enclosed organelle containing most of the genetic information of the cell in the form of chromatin. The nucleus, which can be divided into many sub-organelles such as the nucleoli, the Cajal bodies and the nuclear lamina, is the site for several essential cellular functions such as the DNA replication and its regulation and most of the RNA synthesis and processing. The nucleus is often affected in disease: the size and the shape of the nucleus, the chromatin distribution and the size of the nucleoli have remained the basis for the grading of several cancers. The maintenance of the vertebrate body shape depends on the skeleton. Similarly, in a smaller context, the shape of the cell and the nucleus are mainly regulated by the cytoskeletal and nucleoskeletal elements. The nuclear matrix, which by definition is a detergent, DNase and salt resistant proteinaceous nuclear structure, has been suggested to form the nucleoskeleton responsible for the nuclear integrity. Nuclear mitotic apparatus protein, NuMA, a component of the nuclear matrix, is better known for its mitotic spindle organizing function. NuMA is one of the nuclear matrix proteins suggested to participate in the maintenance of the nuclear integrity during interphase but its interphase function has not been solved to date. This thesis study concentrated on the role of NuMA and the nuclear matrix as structural and functional components of the interphase nucleus. The first two studies clarified the essential role of caspase-3 in the disintegration of the nuclear structures during apoptosis. The second study also showed NuMA and chromatin to co-elute from cells in significant amounts and the apoptotic cleavage of NuMA was clarified to have an important role in the dissociation of NuMA from the chromatin. The third study concentrated on the interphase function of NuMA showing NuMA depletion to result in cell cycle arrest and the cytoplasmic relocalization of NuMA interaction partner GAS41. We suggest that the relocalization of the transcription factor GAS41 may mediate the cell cycle arrest. Thus, this study has given new aspects in the interactions of NuMA, chromatin and the nuclear matrix.

The Dual Role of HIF Hydroxylase PHD3 in Cancer Cell Survival

Most advanced tumours face periods of reduced oxygen availability i.e. hypoxia. During these periods tumour cells undergo adaptive changes enabling their survival under adverse conditions. In cancer hypoxia-induced cellular changes cause tumour progression, hinder cancer treatment and are indicative of poor prognosis. Within cells the main regulator of hypoxic responses is the hypoxia-inducible factor (HIF). HIF governs the expression of over a hundred hypoxia-inducible genes that regulate a number of cellular functions such as angiogenesis, glucose metabolism and cell migration. Therefore the activity of HIF must be tightly governed. HIF is regulated by a family of prolyl hydroxylase enzymes, PHDs, which mark HIF for destruction in normoxia. Under hypoxic conditions PHDs lose much of their enzymatic activity as they need molecular oxygen as a cofactor. Out of the three PHDs (PHD1, 2 and 3) PHD2 has been considered to be the main HIF-1 regulator in normoxic conditions. PHD3 on the other hand shows the most robust induction in response to oxygen deprivation and it has been implied as the main HIF-1 regulator under prolonged hypoxia. SQSTM1/p62 (p62) is an adaptor protein that functions through its binding motifs to bring together proteins in order to regulate signal transduction. In non-stressed situations p62 levels are kept low but its expression has been reported to be upregulated in many cancers. It has a definitive role as an autophagy receptor and as such it serves a key function in cancer cell survival decisions. In my thesis work I evaluated the significance of PHD3 in cancer cell and tumour biology. My results revealed that PHD3 has a dual role in cancer cell fate. First, I demonstrated that PHD3 forms subcellular protein aggregates in oxygenated carcinoma cells and that this aggregation promotes apoptosis induction in a subset of cancer cells. In these aggregates an adaptor protein SQSTM1/p62 interacts with PHD3 and in so doing regulates PHD3 expression. SQSTM1/p62 expression is needed to keep PHD3 levels low in normoxic conditions. Its levels rapidly decrease in response to hypoxia allowing PHD3 protein levels to be upregulated and the protein to be diffusely expressed throughout the cell. The interaction between PHD3 and SQSTM1/p62 limits the ability of PHD3 to function on its hydroxylation target protein HIF-1alpha. Second, the results indicate that when PHD3 is upregulated under hypoxia it protects cancer cells by allowing cell cycle to proceed from G1 to S-phase. My data demonstrates that PHD3 may either cause cell death or protect the cells depending on its expression pattern and the oxygen availability of tumours.

Prognostic Factors In Breast Cancer. With Special Reference to Cyclins A, B1, D1 and E, MMP-1 and Decorin

Breast cancer is a highly heterogenous malignancy, which despite of the similar histological type shows different clinical behaviour and response to therapy. Prognostic factors are used to estimate the risk for recurrence and the likelihood of treatment effectiveness. Because breast cancer is one of the most common causes of cancer death in women worldwide, identification of new prognostic markers are needed to develop more specific and targeted therapies. Cancer is caused by uncontrolled cell proliferation. The cell cycle is controlled by specific proteins, which are known as cyclins. They function at important checkpoints by activating cyclin-dependent kinase enzymes. Overexpression of different cyclins has been linked to several cancer types and altered expression of cyclins A, B1, D1 and E has been associated with poor survival. Little is known about the combined expression of cyclins in relation to the tumour grade, breast cancer subtype and other known prognostic factors. In this study cyclins A, B1 and E were shown to correlate with histological grade, Ki-67 and HER2 expression. Overexpression of cyclin D1 correlated with receptor status and non-basal breast cancer suggesting that cyclin D1 might be a marker of good prognosis. Proteolysis in the surrounding tumour stroma is increased during cancer development. Matrix metalloproteinases (MMPs) are proteolytic enzymes that are capable of degrading extracellular matrix proteins. Increased expression and activation of several MMPs have been found in many cancers and MMPs appear to be important regulators of invasion and metastasis. In this study MMP-1 expression was analysed in breast cancer epithelial cells and in cancer associated stromal cells. MMP-1 expression by breast cancer epithelial cells was found to carry an independent prognostic value as did Ki-67 and bcl-2. The results suggest that in addition to stromal cells MMP-1 expression in tumour cells control breast cancer progression. Decorin is a small proteoglycan and an important component of the extracellular matrix. Decorin has been shown to inhibit growth of tumour cells and reduced decorin expression is associated with a poor prognosis in several cancer types. There has been some suspicion wheather different cancer cells express decorin. In this study decorin expression was shown to localize only in the cells of the original stroma, while breast cancer epithelial cells were negative for decorin expression. However, transduction of decorin in decorin-negative human breast cancer cells markedly modulated the growth pattern of these cells. This study provides evidence that targeted decorin transduction to breast cancer cells could be used as a novel adjuvant therapy in breast malignancies.

To divide or differentiate? : nestin-mediated regulation of Cdk5 in cell fate decisions

The molecular functions of the non-cell cycle-related Cyclin-dependent kinase 5 (Cdk5) have been of primary interest within the neuroscience field, but novel undertakings are constantly emerging for the kinase in tissue homeostasis, as well as in diseases such as diabetes and cancer. Although Cdk5 activation is predominantly regulated by specific non-cyclin activator protein binding, additional mechanisms have proved to orchestrate Cdk5 signaling in cells. For example, the interaction between the intermediate filament protein nestin and Cdk5 has been proposed to determine cellular fate during neuronal apoptosis through nestin-dependent adjustment of the sensitive balance and turnover of Cdk5 activators. While nestin constitutes a crucial regulatory scaffold for appropriate Cdk5 activation in apoptosis, Cdk5 itself phosphorylates nestin with the consequence of filament reorganization in both neuronal progenitors and differentiating muscle cells. Interestingly, the two proteins are often found coexpressed in various tissues and cell types, proposing that nestin-mediated scaffolding of Cdk5 and its activators may be applicable to other tissue systems as well. In the literature, the molecular functions of nestin have remained in the shade, as it is mostly exploited as a marker protein for progenitor cells. In light of these studies, the aim of this thesis was to assess the importance of the nestin scaffold in regulation of Cdk5 actions in cell fate decisions. This thesis can be subdivided into two major projects: one that studied the nature of the Cdk5-nestin interplay in muscle, and one that assessed their role in prostate cancer. During differentiation of a myoblast cell line, the filament formation properties of nestin was found to be crucial in directing Cdk5 activity, with direct consequences on the process of differentiation. Also the genetic knockout of nestin was found to influence Cdk5 activity, although differentiation per se was not affected. Instead, the genetic ablation of nestin had broad consequences on muscle homeostasis and regeneration. While the nestin-mediated regulation of Cdk5 in muscle was found to act in multiple ways, the connection remained more elusive in cancer models. Cdk5 was, however, established as a significant determinant of prostate cancer proliferation; a behavior uncharacteristic for this differentiation-associated kinase. Through complex and simultaneous regulation of two major prostate cancer pathways, Cdk5 was placed upstream of both Akt kinase and the androgen receptor. Its action on proliferation was nonetheless mainly exerted through the Akt signaling pathway in various cancer models. In summary, this thesis contributed to the knowledge of Cdk5 regulation and functions in two atypical settings; proliferation (in a cancer framework) and muscle differentiation, which is a poorly understood model system in the Cdk5 field. This balance between proliferation and differentiation implemented by Cdk5 is ultimately regulated (where present) by the dynamics of the cytoskeletal nestin scaffold.

Cellular responses to stress and kinase signaling activation : apoptosis and differentiation

Stressignaler avkänns många gånger av membranbundna proteiner som översätter signalerna till kemisk modifiering av molekyler, ofta proteinkinaser Dessa kinaser överför de avkodade budskapen till specifika transkriptionsfaktorer genom en kaskad av sekventiella fosforyleringshändelser, transkriptionsfaktorerna aktiverar i sin tur de gener som behövs för att reagera på stressen. En av de mest kända måltavlorna för stressignaler är transkriptionsfaktor AP-1 familjemedlemen c-Jun. I denna studie har jag identifierat den nukleolära proteinet AATF som en ny regulator av c-Jun-medierad transkriptionsaktivitet. Jag visar att stresstimuli inducerar omlokalisering av AATF vilket i sin tur leder till aktivering av c-Jun. Den AATF-medierad ökningen av c-Jun-aktiviteten leder till en betydande ökning av programmerad celldöd. Parallellt har jag vidarekarakteriserat Cdk5/p35 signaleringskomplexet som tidigare har identifierats i vårt laboratorium som en viktig faktor för myoblastdifferentiering. Jag identifierade den atypiska PKCξ som en uppströms regulator av Cdk5/p35-komplexet och visar att klyvning och aktivering av Cdk5 regulatorn p35 är av fysiologisk betydelse för differentieringsprocessen och beroende av PKCξ aktivitet. Jag visar att vid induktion av differentiering fosforylerar PKCξ p35 vilket leder till calpain-medierad klyvning av p35 och därmed ökning av Cdk5-aktiviteten. Denna avhandling ökar förståelsen för de regulatoriska mekanismer som styr c-Jun-transkriptionsaktiviteten och c-Jun beroende apoptos genom att identifiera AATF som en viktig faktor. Dessutom ger detta arbete nya insikter om funktionen av Cdk5/p35-komplexet under myoblastdifferentiering och identifierar PKCξ som en uppströms regulator av Cdk5 aktivitet och myoblast differentiering.

Vesicular Trafficking in Osteoclasts

Osteoclasts are multinucleated bone-degrading cells that undergo large changes in their polarisation and vesicular trafficking during the bone resorption cycle. Rab proteins are small GTPases that offer both temporal and spatial regulation to the transport between membranous organelles. Previously the presence and function of only few of the currently known 60 Rab proteins in osteoclasts have been reported. In this study, the expression of 26 Rab genes in bone-resorbing osteoclasts was demonstrated with gene-specific primer pairs. The further analysis of three Rab genes during human osteoclast differentiation revealed that Rab13 gene is highly induced during osteoclastogenesis. The presence of Rab13 protein in the secretory vesicles directed towards the ruffled border and in the endocytotic or transcytotic pathways in resorbing osteoclasts was excluded. The localisation of Rab13 suggests that that it is associated with a previously unknown vesicle population travelling between the trans-Golgi network and the basolateral membrane in bone resorbing osteoclasts. Rab proteins convey their functions by binding to specific effector proteins. We found a novel Rab13 interaction with endospanins-1 and -2 that are yet poorly characterised small transmembrane proteins. The Rab13 subfamily member Rab8 also bound to endospanins, while Rab10 and unrelated Rabs did not. Rab13 and endospanin-2 co-localised in perinuclear vesicles in transfected cells, demonstrating the interaction also in vivo. The inhibition of Rab13 did not interfere with the localisation of endospanin-2 nor did it affect the cell surface expression of growth hormone receptor, as has been previously described for endospanins. The physiological role of this novel protein-protein interaction thus remains to be clarified. The analysis of the transcytotic route in bone resorbing osteoclasts revealed that multiple vesicle populations arise from the ruffled border and transport the bone degradation products for exocytosis. These vesicles are directed to the functional secretory domain that is encircled by an actin-based molecular barrier. Furthermore, the transcytotic vesicles contain abundant Helix pomatia lectin binding sites and represent lipid raft concentrates. Finally, autophagosomal compartments may also be involved in the transcytosis.

Regulation of HSF2 and its function in mitosis

The cell is continuously subjected to various forms of external and intrinsic proteindamaging stresses, including hyperthermia, pathophysiological states, as well as cell differentiation and proliferation. Proteindamaging stresses result in denaturation and improper folding of proteins, leading to the formation of toxic aggregates that are detrimental for various pathological conditions, including Alzheimer’s and Huntington’s diseases. In order to maintain protein homeostasis, cells have developed different cytoprotective mechanisms, one of which is the evolutionary well-conserved heat shock response. The heat shock response results in the expression of heat shock proteins (Hsps), which act as molecular chaperones that bind to misfolded proteins, facilitate their refolding and prevent the formation of protein aggregates. Stress-induced expression of Hsps is mediated by a family of transcription factors, the heat shock factors, HSFs. Of the four HSFs found in vertebrates, HSF1-4, HSF1 is the major stress-responsive factor that is required for the induction of the heat shock response. HSF2 cannot alone induce Hsps, but modulates the heat shock response by forming heterotrimers with HSF1. HSFs are not only involved in the heat shock response, but they have also been found to have a function in development, neurodegenerative disorders, cancer, and longevity. Therefore, insight into how HSFs are regulated is important for the understanding of both normal physiological and disease processes. The activity of HSF1 is mainly regulated by intricate post-translational modifications, whereas the activity of HSF2 is concentrationdependent. However, there is only limited understanding of how the abundance of HSF2 is regulated. This study describes two different means of how HSF2 levels are regulated. In the first study it was shown that microRNA miR-18, a member of the miR-17~92 cluster, directly regulates Hsf2 mRNA stability and thus protein levels. HSF2 has earlier been shown to play a profound role in the regulation of male germ cell maturation during the spermatogenesis. The effect on miR-18 on HSF2 was examined in vivo by transfecting intact seminiferous tubules, and it was found that inhibition of miR-18 resulted in increased HSF2 levels and modified expression of the HSF2 targets Ssty2 and Speer4a. HSF2 has earlier been reported to modulate the heat shock response by forming heterotrimers with HSF1. In the second study, it was shown that HSF2 is cleared off the Hsp70 promoter and degraded by the ubiquitinproteasome pathway upon acute stress. By silencing components of the anaphase promoting complex/cyclosome (APC/C), including the co-activators Cdc20 and Cdh1, it was shown that APC/C mediates the heatinduced ubiquitylation of HSF2. Furthermore, down-regulation of Cdc20 was shown to alter the expression of heat shock-responsive genes. Next, we studied if APC/C-Cdc20, which controls cell cycle progression, also regulates HSF2 during the cell cycle. We found that both HSF2 mRNA and protein levels decreased during mitosis in several but not all human cell lines, indicating that HSF2 has a function in mitotic cells. Interestingly, although transcription is globally repressed during mitosis, mainly due to the displacement of RNA polymerase II and transcription factors, including HSF1, from the mitotic chromatin, HSF2 is capable of binding DNA during mitosis. Thus, during mitosis the heat shock response is impaired, leaving mitotic cells vulnerable to proteotoxic stress. However, in HSF2-deficient mitotic cells the Hsp70 promoter is accessible to both HSF1 and RNA polymerase II, allowing for stress-inducible Hsp expression to occur. As a consequence HSF2-deficient mitotic cells have a survival advantage upon acute heat stress. The results, presented in this thesis contribute to the understanding of the regulatory mechanisms of HSF2 and its function in the heat shock response in both interphase and mitotic cells.

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.

Family rivalry in the testis: Retinoblastoma protein and E2F transcription factors in testicular development and adult spermatogenesis

Disorders of male reproductive health are becoming increasingly prevalent globally. These defects, ranging from decreasing sperm counts to an increasing rate of infertility and testicular cancer, have a common origin in the early phases of testicular development, but the exact mechanisms that cause them remain unknown. Testicular development and adult spermatogenesis are complex processes in which different cell types undergo mitosis, meiosis, differentiation and apoptosis. The retinoblastoma protein family and its associated E2F transcription factors are key regulators of these cellular events. In the present study, the functions of these factors in postnatal testicular development and adult spermatogenesis were explored using different animal models. In addition, a new application of flow cytometry to study testicular cell dynamics was developed. An ablation of retinoblastoma protein in mouse Sertoli cells resulted in their cell cycle re-entry in adult testes, dedifferentiation and a severe spermatogenic defect. We showed that deregulated E2F3 contributed to these changes. Our results indicated that the E2F1 transcription factor is critical for the control of apoptosis in the developing postnatal testis. In the adult testis, E2F1 controls the maintenance of the spermatogonial stem cell pool, in addition to inhibiting apoptosis of spermatocytes. In summary, this study elucidated the complex interdependencies of the RB and E2F transcription factor families in the control of postnatal testicular development and adult spermatogenesis. Furthermore, this study provided a new methodology for the analysis of testicular cells.