Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 3 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 99-131).

Protein Kinase C-iota (PKC-é), an atypical protein kinase C isoform manifests its potential as an oncogene by targeting various aspects of cancer cells such as growth, invasion and survival. PKC-é confers resistance to drug-induced apoptosis in cancer cells. The acquisition of drug resistance is a major obstacle to good prognosis in neuroblastoma. The focus of the dissertation was three-fold: First to study the role of PKC-é in the proliferation of neuroblastoma. Secondly, to identify the efficacy of [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate (ICA-1) as a novel PKC-é inhibitor in neuroblastoma cell proliferation and apoptosis. Finally, to analyze whether PKC-é could self-regulate its expression. Cyclin dependent kinase 7 (Cdk7) phosphorylates cyclin dependent kinases (cdks) and promotes cell proliferation. Our data shows that PKC-é is an in-vitro Cdk7 kinase and that neuroblastoma cells proliferate via a PKC-é/Cdk7/cdk2 cell signaling pathway. ICA-1 specifically inhibits the activity of PKC-é but not that of PKC-zeta (PKC-æ), the closely related atypical PKC family member. The IC50 for the kinase activity assay was approximately 0.1µM which is 1000 times less than that of aurothiomalate, a known PKC-é inhibitor. The phosphorylation of Cdk7 by PKC-é was potently inhibited by ICA-1. ICA-1 mediates its antiproliferative effects on neuroblastoma cells by inhibiting the PKC-é/Cdk7/cdk2 signaling pathway. ICA-1 (0.1µM) inhibited the in-vitro proliferation of BE(2)-C neuroblastoma cells by 58% (P=0.01). Additionally, ICA-1 also induced apoptosis in neuroblastoma cells. Interestingly, ICA-1 did not affect the proliferation of normal neuronal cells suggesting its potential as chemotherapeutic with low toxicity. Hence, our results emphasize the potential of ICA-1 as a novel PKC-é inhibitor and chemotherapeutic agent for neuroblastoma. Bcr-Abl has been shown to regulate the activation of the transcription factor ELK-1 which in turn regulates the expression of PKC-é. Alternatively, we hypothesize that PKC-é can self regulate its expression by indirectly regulating the activity of Elk-1 in an ERK1 dependent manner. Our preliminary data shows that there was robust increase in the expression as well as association of PKC-é and Elk-1 in actively proliferating neuroblastoma cells suggesting a potential role of PKC-é in regulating the activity of Elk-1. Analysis of the subcellular fractions also presented a similar increase in the association between PKC-é and Elk-1 in the nuclear fraction of actively proliferating cells as compared to cytoplasm. Interestingly, the nuclear expression of PKC-é was also found to be higher in these cells, suggesting that PKC-é translocated to the nucleus in actively proliferating cells and regulated the transcriptional activity of Elk-1. However, our data from in-vitro kinase activity demonstrated that PKC-é was not an Elk-1 kinase but that it increased the phosphorylation of Elk-1 in the presence of ERK1, an upstream kinase of Elk-1 in the Bcr-Abl mediated regulatory pathway of PKC-é. This suggested that ERK1 was integral to the self-regulatory activity of PKC-é. In conclusion, we hypothesize that the self-regulatory mechanism of PKC-é is initiated by the translocation PKC-é into the nucleus where it activates ERK1. This promotes the activation of its downstream target Elk-1 which subsequently upregulates the expression of PKC-é

Acute oncogenic stress can activate autophagy and facilitate permanent arrest of the cell cycle through a failsafe mechanism known as oncogene-induced senescence (OIS). Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s Sarcoma (KS) and has been reported to encode oncoproteins within its latency program that engage host autophagy and OIS pathways; however, the mechanisms by which KSHV oncoproteins promote KS tumorigenesis remain unclear. Here, I demonstrate that ectopic expression of the latent KSHV protein viral cyclin (v-cyclin) deregulates the cell cycle, induces DNA-damage responses (DDRs) and promotes OIS through an autophagy-dependent mechanism. During latency, v-cyclin is co-expressed from a single transcript with a viral homolog of FLICE-inhibitory protein (v-FLIP) that blocks autophagy by binding and inhibiting Atg3. Co-expression of v-FLIP has no effect on DDRs, but efficiently blocks v-cyclin-induced autophagy and senescence. Remarkably, suppression of v-FLIP function during KSHV latency, through specific inhibitory peptides, rescues host cell autophagy and induces senescence of infected cells. Together, these data reveal a coordinated viral gene-expression program that subverts autophagy, impairs senescence, and facilitates the proliferation of KSHV-infected cells.

Osteosarcoma is the most common bone malignancy in children and adolescents with poorly understood aetiology. Recently, disease susceptibility and aetiology in several cancers have been associated with genomic copy-number (CN) change. We therefore studied the contribution of CN change in osteosarcoma. We report that individuals with osteosarcoma have increased germline structural variation compared to controls. These CN variants (CNVs) preferentially localize to genes implicated in control of osteoblast differentiation, bone mineralization and ossification. We propose that germline CNVs contribute to osteosarcoma susceptibility through deregulation of developmental processes controlled by genes contained within CNVs. Further supporting the notion that germline CNVs in individuals with osteosarcoma are pathogenic, we demonstrate that CNVs are associated with poor patient survival. Finally, we characterize two germline CNVs, at chromosome 1q43 and 2p11.2, which are overrepresented in osteosarcoma patients and propose that they contribute to osteosarcoma susceptibility through effect on neighbouring genes, which could be involved in control of microtubule dynamics and tumour suppression. We further characterize two regions in the tumour genome of osteosarcoma patients that harbour recurrent CN alterations (CNAs). These include deletions at chromosome 3q13.31 and vi ii amplifications at chromosome 7p14.1, which are the most altered regions in osteosarcoma and contest the view that CNAs in osteosarcoma are non-recurrent. Both chromosome 3q13.31 and 7p14.1 CNAs involve genes implicated in carcinogenesis, including LASMP at 3q13.31 and TARP at 7p14.1, while 3q13.31 CNAs also involve two non-coding RNAs. We further show that expression of 3q13.31 genes correlates with the presence of 3q13.31 CNAs. We report that chromosome 3q13.31 and 7p14.1 CNAs are also common in other cancers, identifying these loci as candidates with a global role in carcinogenesis. Supporting the notion that 3q13.31 deletions play a role in osteosarcomagenesis, we find that depletion of 3q13.31 genes promotes proliferation of osteoblasts by regulation of apoptotic and cell-cycle transcripts and also VEGF receptor 1 and that genetic deletions of 3q13.31 are associated with poor survival of osteosarcoma patients. In summary, our study implicates germline and somatic CN changes in osteosarcoma and represents a model approach for elucidation of elements contributing to disease susceptibility and aetiology in human cancer.

Osteosarcoma is the most common bone malignancy in children and adolescents with poorly understood aetiology. Recently, disease susceptibility and aetiology in several cancers have been associated with genomic copy-number (CN) change. We therefore studied the contribution of CN change in osteosarcoma. We report that individuals with osteosarcoma have increased germline structural variation compared to controls. These CN variants (CNVs) preferentially localize to genes implicated in control of osteoblast differentiation, bone mineralization and ossification. We propose that germline CNVs contribute to osteosarcoma susceptibility through deregulation of developmental processes controlled by genes contained within CNVs. Further supporting the notion that germline CNVs in individuals with osteosarcoma are pathogenic, we demonstrate that CNVs are associated with poor patient survival. Finally, we characterize two germline CNVs, at chromosome 1q43 and 2p11.2, which are overrepresented in osteosarcoma patients and propose that they contribute to osteosarcoma susceptibility through effect on neighbouring genes, which could be involved in control of microtubule dynamics and tumour suppression. We further characterize two regions in the tumour genome of osteosarcoma patients that harbour recurrent CN alterations (CNAs). These include deletions at chromosome 3q13.31 and vi ii amplifications at chromosome 7p14.1, which are the most altered regions in osteosarcoma and contest the view that CNAs in osteosarcoma are non-recurrent. Both chromosome 3q13.31 and 7p14.1 CNAs involve genes implicated in carcinogenesis, including LASMP at 3q13.31 and TARP at 7p14.1, while 3q13.31 CNAs also involve two non-coding RNAs. We further show that expression of 3q13.31 genes correlates with the presence of 3q13.31 CNAs. We report that chromosome 3q13.31 and 7p14.1 CNAs are also common in other cancers, identifying these loci as candidates with a global role in carcinogenesis. Supporting the notion that 3q13.31 deletions play a role in osteosarcomagenesis, we find that depletion of 3q13.31 genes promotes proliferation of osteoblasts by regulation of apoptotic and cell-cycle transcripts and also VEGF receptor 1 and that genetic deletions of 3q13.31 are associated with poor survival of osteosarcoma patients. In summary, our study implicates germline and somatic CN changes in osteosarcoma and represents a model approach for elucidation of elements contributing to disease susceptibility and aetiology in human cancer.

Retinoblastoma development is a multistep process, and inactivation of the RB1 gene is not sufficient for tumorigenesis. Previous studies suggest that the p53-tumor suppressor is inactivated due to overexpression of p53-antagonists MDM4 and MDM2. This thesis evaluates the importance of ARF, a p53-activator that inhibits MDM2. In retinoblastomas, ARF protein is nearly undetectable despite robust mRNA expression. Chemical inhibition of the proteasome, which regulates ARF protein-turnover, did not result in ARF accumulation in retinoblastoma cells, indicating that ARF protein was not aberrantly degraded by the proteasome. During mouse retinoblastoma development, Arf protein was expressed at low level, and p53-target genes involved in cell cycle arrest and autoregulation were not activated. Overexpression of ARF in retinoblastoma cells led to growth inhibition, accompanied by increased expression of p53 and p53-transcriptional targets. Taken together, our data suggests that low ARF protein is an important factor in silencing of the p53-pathway during retinoblastoma development.

Retinoblastoma development is a multistep process, and inactivation of the RB1 gene is not sufficient for tumorigenesis. Previous studies suggest that the p53-tumor suppressor is inactivated due to overexpression of p53-antagonists MDM4 and MDM2. This thesis evaluates the importance of ARF, a p53-activator that inhibits MDM2. In retinoblastomas, ARF protein is nearly undetectable despite robust mRNA expression. Chemical inhibition of the proteasome, which regulates ARF protein-turnover, did not result in ARF accumulation in retinoblastoma cells, indicating that ARF protein was not aberrantly degraded by the proteasome. During mouse retinoblastoma development, Arf protein was expressed at low level, and p53-target genes involved in cell cycle arrest and autoregulation were not activated. Overexpression of ARF in retinoblastoma cells led to growth inhibition, accompanied by increased expression of p53 and p53-transcriptional targets. Taken together, our data suggests that low ARF protein is an important factor in silencing of the p53-pathway during retinoblastoma development.

Self-renewal is central to the expansion of normal and cancerous stem cells. Its understanding is therefore critical for future advances in transplantation-based therapies and cancer treatment. Although the molecular machinery controlling stem cell self-renewal remains poorly defined, a number of genes important to this process have recently been identified. Two prominent genes in this group are Hoxb4 and Bmi1. Members of our group led the way to demonstrate important regulatory functions of these genes in hematopoietic stem cell (HSC) self-renewal and expansion. / The major goal of my thesis project is to dissect mechanisms that regulate self-renewal of HSCs. Our starting hypothesis was that HSC activity is regulated by complementary and independent self-renewal mechanisms: self-renewal of expansion and self-renewal of maintenance (Chapters 1-2). In order to further verify this theory, we have analyzed the genetic interaction between Hoxb4 and Bmi1. While Hoxb4 overexpression triggers HSC expansion, Bmi1 proper expression is essential to sustain long-term stem cell activity. We have also demonstrated that Hoxb4 and Bmi1 regulate distinct gene targets, likely suggesting a complementary and independent function for these two regulators in HSC activity (Chapter 3). / The second part of this thesis highlights efforts that were made in order to get a better understanding of self-renewal mechanisms. We have identified potential new regulators of stem cell activity by characterizing a stem cell leukemia population (Chapter 4) and by assessing the expression of asymmetrical distributed factors (Chapter 5) and selected nuclear factors of the Hematopoietic Stem Cell Nuclear Factor Database (Chapter 6) in stem cell-enriched sub-fractions. / This project will lead to a better understanding of the cellular basis regulating self-renewal of both normal and cancer stem cells and potentially to the future identification of new self-renewal determinants.