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.

Role and regulatory mechanisms of heat shock factor 2

Protein homeostasis is essential for cells to prosper and survive. Various forms of stress, such as elevated temperatures, oxidative stress, heavy metals or bacterial infections cause protein damage, which might lead to improper folding and formation of toxic protein aggregates. Protein aggregation is associated with serious pathological conditions such as Alzheimer’s and Huntington’s disease. The heat shock response is a defense mechanism that protects the cell against protein-damaging stress. Its ancient origin and high conservation among eukaryotes suggest that the response is crucial for survival. The main regulator of the heat shock response is the transcription factor heat shock factor 1 (HSF1), which induces transcription of genes encoding protective molecular chaperones. In vertebrates, a family of four HSFs exists (HSF1-4), with versatile functions not only in coping with acute stress, but also in development, longevity and cancer. Thus, knowledge of the HSFs will aid in our understanding on how cells survive suboptimal circumstances, but will also provide insights into normal physiological processes as well as diseaseassociated conditions. In this study, the function and regulation of HSF2 have been investigated. Earlier gene inactivation experiments in mice have revealed roles for HSF2 in development, particularly in corticogenesis and spermatogenesis. Here, we demonstrate that HSF2 holds a role also in the heat shock response and influences stress-induced expression of heat shock proteins. Intriguingly, DNA-binding activity of HSF2 upon stress was dependent on the presence of intact HSF1, suggesting functional interplay between HSF1 and HSF2. The underlying mechanism for this phenomenon could be configuration of heterotrimers between the two factors, a possibility that was experimentally verified. By changing the levels of HSF2, the expression of HSF1-HSF2 heterotrimer target genes was altered, implementing HSF2 as a modulator of HSF-mediated transcription. The results further indicate that HSF2 activity is dependent on its concentration, which led us to ask the question of how accurate HSF2 levels are achieved. Using mouse spermatogenesis as a model system, HSF2 was found to be under direct control of miR-18, a miRNA belonging to the miR-17~92 cluster/Oncomir-1 and whose physiological function had remained unclear. Investigations on spermatogenesis are severely hampered by the lack of cell systems that would mimic the complex differentiation processes that constitute male germ cell development. Therefore, to verify that HSF2 is regulated by miR-18 in spermatogenesis, a novel method named T-GIST (Transfection of Germ cells in Intact Seminiferous Tubules) was developed. Employing this method, the functional consequences of miR-18-mediated regulation in vivo were demonstrated; inhibition of miR- 18 led to increased expression of HSF2 and altered the expression of HSF2 target genes Ssty2 and Speer4a. Consequently, the results link miR-18 to HSF2-mediated processes such as germ cell maturation and quality control and provide miR-18 with a physiological role in gene expression during spermatogenesis.Taken together, this study presents compelling evidence that HSF2 is a transcriptional regulator in the heat shock response and establishes the concept of physical interplay between HSF2 and HSF1 and functional consequences thereof. This is also the first study describing miRNA-mediated regulation of an HSF.

The interplay of JNK and SCG10 during cortical development and in excitotoxic responses in brain

Initially identified as stress activated protein kinases (SAPKs), the c-Jun Nterminal kinases (JNKs) are currently accepted as potent regulators of various physiologically important cellular events. Named after their competence to phosphorylate transcription factor c-Jun in response to UVtreatment, JNKs play a key role in cell proliferation, cell death or cell migration. Interestingly, these functions are crucial for proper brain formation. The family consists of three JNK isoforms, JNK1, JNK2 and JNK3. Unlike brain specific JNK3 isoform, JNK1 and JNK2 are ubiquitously expressed. It is estimated that ten splice variants exist. However, the detailed cellular functions of these remain undetermined. In addition, physiological conditions keep the activities of JNK2 and JNK3 low in comparison with JNK1, whereas cellular stress raises the activity of these isoforms dramatically. Importantly, JNK1 activity is constitutively high in neurons, yet it does not stimulate cell death. This suggests a valuable role for JNK1 in brain development, but also as an important mediator of cell wellbeing. The aim of this thesis was to characterize the functional relationship between JNK1 and SCG10. We found that SCG10 is a bona fide target for JNK. By employing differential centrifugation we showed that SCG10 co-localized with active JNK, MKK7 and JIP1 in a fraction containing endosomes and Golgi vesicles. Investigation of JNK knockout tissues using phosphospecific antibodies recognizing JNK-specific phosphorylation sites on SCG10 (Ser 62/Ser 73) showed that phosphorylation of endogenous SCG10 was dramatically decreased in Jnk1-/- brains. Moreover, we found that JNK and SCG10 co-express during early embryonic days in brain regions that undergo extensive neuronal migration. Our study revealed that selective inhibition of JNK in the cytoplasm significantly increased both the frequency of exit from the multipolar stage and radial migration rate. However, as a consequence, it led to ill-defined cellular organization. Furthermore, we found that multipolar exit and radial migration in Jnk1 deficient mice can be connected to changes in phosphorylation state of SCG10. Also, the expression of a pseudo-phosphorylated mutant form of SCG10, mimicking the JNK1- phopshorylated form, brings migration rate back to normal in Jnk1 knockout mouse embryos. Furthermore, we investigated the role of SCG10 and JNK in regulation of Golgi apparatus (GA) biogenesis and whether pathological JNK action could be discernible by its deregulation. We found that SCG10 maintains GA integrity as with the absence of SCG10 neurons present more compact fragmented GA structure, as shown by the knockdown approach. Interestingly, neurons isolated from Jnk1-/- mice show similar characteristics. Block of ER to GA is believed to be involved in development of Parkinson's disease. Hence, by using a pharmacological approach (Brefeldin A treatment), we showed that GA recovery is delayed upon removal of the drug in Jnk1-/- neurons to an extent similar to the shRNA SCG10-treated cells. Finally, we investigated the role of the JNK1-SCG10 duo in the maintenance of GA biogenesis following excitotoxic insult. Although the GA underwent fragmentation in response to NMDA treatment, we observed a substantial delay in GA disintegration in neurons lacking either JNK1 or SCG10.