Delta-like 1 Homologue (DLK1), a transmembrane protein of 383 amino acids, belongs to a family of epidermal growth factor (EGF)-like repeat-containing proteins that include Notch/Delta/Serrate, which are involved in cell fate determination. DLK1 is also known as preadipocyte factor-1, pG2, and FA-1, which are identical or polymorphic products of a single gene. Structural analysis revealed that DLK1 consists of an extracellular domain with six EGF-like repeats, a transmembrane domain, and an intracellular domain. The extracellular EGF-like region of DLK1 (DLK1-EC) can be released to the medium by the action of tumor necrosis factor alpha converting enzyme (TACE). DLK1 participates in various differentiation processes including adipogenesis, hematopoiesis, and adrenal gland differentiation. Besides, DLK1 overexpression was observed in patients with biliary atresia and in glioblastoma. Recently, the extracellular domain of thrombomodulin, which also contains six EGF¡Vlike structures, has been delineated to stimulate angiogenesis in vitro and in vivo. This prompted us to investigate whether DLK1-EC played a role in angiogenesis. To test such hypothesis, recombinant DLK1-EC was expressed and purified in E. coli. Adding DLK1-EC recombinant protein inhibited the adipogenesis of adipocytes-derived stem cells in a dose-dependent manner. Despite marginal effect on matrix-metalloproteinase secretion, exogenous DLK1-EC significantly stimulated the proliferation, motility and tube-forming capability of cultured endothelial cells. Above all, implantation of DLK1-EC-containing hydron pellets induced cornea neovascularization in a dose-dependent manner. Western blot analysis revealed that exogenous DLK1-EC induced angiogenesis through Notch1 activating downstream gene Hes1 and subsequently signaling such as Akt/eNOS, p38 MAPK, and ERK pathway to perform its function. Indeed, blockade of Notch1 signaling using £^-secretase inhibitor leads to decreased angiogenesis and inhibits DLK1 EC-induced endothelial cell tubular formation in vitro and in vivo. These findings indicate that DLK1-EC induced Notch1 activation mediated by £^-secretase and tansactivation Akt/eNOS pathway and that Notch1 is critical for DLK1 EC-induced angiogenesis. These results may bring further insights into the physiological and pathological functions of DLK1

The PI3K-PKB/Akt-PTEN signal transduction pathway orchestrates a variety of fundamental cell processes and its deregulation is implicated in several human diseases, including cancer. While the importance of this pathway to many cellular functions is well established, the mechanisms leading to context-specific physiological outcomes in response to a variety of stimuli remain largely unknown. Spatial restriction of signaling events is one of the means to coordinate specific cellular responses. To investigate the subcellular locus-specific roles of the major PI3K effector PKB/Akt in various cell processes, I have devised a novel experimental system employing cellular compartment-directed PKB/Akt pseudosubstrate inhibitors. The work herein describes the development and characterization of the localized PKB/Akt pseudosubstrate inhibitor system and its application to investigate potential locus-specific functions in established PKB/Akt-regulated cellular processes. Subcellular compartment-restricted PKB/Akt inhibition in the 3T3L1 adipocyte differentiation model revealed that nuclear and plasma membrane, but not cytoplasmic, PKB/Akt activity is required for terminal adipocyte differentiation. Nuclear and plasma membrane pools of PKB/Akt were found to contribute to distinct stages of adipocyte differentiation, revealing that PKB/Akt activity impacts multiple points of this program. The localized PKB/Akt pseudosubstrate inhibitor system was also utilized to investigate the importance of distinct subcellular pools of PKB/Akt in breast epithelial cells. MCF-10A human breast epithelial cells can be grown in three-dimensional culture to form acinar structures that recapitulate in vivo mammary glandular architecture. Expression of the plasma membrane PKB/Akt inhibitor during cell growth in three-dimensional culture severely impaired acinar formation. On the other hand, expression of the nuclear PKB/Akt inhibitor during acinar development resulted in the formation of large, misshapen, multi-acinar structures. Assessment of the migratory capacity of MCF-10A cells upon localized PKB/Akt inhibition revealed that nuclear PKB/Akt inhibition promoted, while plasma membrane PKB/Akt inhibition impaired, MCF-10A cell migration. The development of locus-specific PKB/Akt inhibitors represents the first attempt to prioritize the targets of this kinase based on their subcellular localization. This work and its immediate extensions will further our understanding of the biology of PKB/Akt, a multi-tasking kinase with profound roles in development, cellular and organismal homeostasis and disease.

Hepatitis C virus (HCV) infects millions of people worldwide and is one of the leading causes of liver damage. Infection with HCV is strongly correlated with an increased risk of steatosis, or fatty liver disease, which is caused by a build-up of fat deposits in hepatocytes. All genotypes of HCV appear to cause some degree of steatosis in approximately 50% of infected individuals, especially in the presence of contributing host factors such as diabetes, obesity and alcoholism. However, approximately 70% of genotype 3a infections exhibit steatosis. Furthermore, successful clearance of the genotype 3a virus results in eradication of the steatosis, suggesting the genotype 3a virus may be able to directly cause steatosis.<p> Research suggests a role for the core protein of HCV, which forms the capsid of the virus, in the alteration of lipid metabolism pathways during infection. As such, I hypothesized that: 1) HCV alters lipid metabolism pathways and causes the build up of lipid in hepatocytes and the development of steatosis; 2) HCV-3a core protein has a differential or increased effect on these pathways in comparison to 1b core protein; and 3) other HCV proteins could also play a role in the altering of lipid metabolism. My research characterized the subcellular localization on lipid droplets of the HCV-3a core protein in comparison to HCV-1b core protein. It was found that HCV-3a core causes increased transcriptional activity from the Fatty Acid Synthase (FAS) promoter, an important enzyme involved in the synthesis of triglycerides in hepatocytes. In addition, one specific amino acid of HCV-3a core was determined to be partially responsible for this effect. Further research determined that the effect of HCV-3a core on FAS was dependent on the transcription factor Sterol Response Element Binding Protein-1 (SREBP-1) and the presence of HCV-3a core increased the processing and activity of SREBP-1. HCV core was also able to increase activity of Akt 1 and Akt2; inhibition of Akt activity resulted in decreased SREBP-1 activity thereby indicating that HCV core partially mediates SREBP-1 via Akt. Further experiments examined the role of another HCV protein, NS2, in these same lipid metabolism pathways. NS2 was also able to increase transcription from the FAS promoter via SREBP-1, suggesting that this HCV protein may also be important in the development of steatosis during HCV infection.<p> The evidence provided in these studies shows a very important role for HCV in altering lipid metabolism during infection that may lead to the development of steatosis. Current research suggests that the SREBP-1 pathway may be critical in the life cycle of the virus and these studies have provided important information on how lipid metabolism pathways are being changed by the virus. Hopefully this work can help identify potential treatment options for HCV that can slow down disease progression by preventing the development of steatosis or by decreasing viral replication.

The PI3K-PKB/Akt-PTEN signal transduction pathway orchestrates a variety of fundamental cell processes and its deregulation is implicated in several human diseases, including cancer. While the importance of this pathway to many cellular functions is well established, the mechanisms leading to context-specific physiological outcomes in response to a variety of stimuli remain largely unknown. Spatial restriction of signaling events is one of the means to coordinate specific cellular responses. To investigate the subcellular locus-specific roles of the major PI3K effector PKB/Akt in various cell processes, I have devised a novel experimental system employing cellular compartment-directed PKB/Akt pseudosubstrate inhibitors. The work herein describes the development and characterization of the localized PKB/Akt pseudosubstrate inhibitor system and its application to investigate potential locus-specific functions in established PKB/Akt-regulated cellular processes. Subcellular compartment-restricted PKB/Akt inhibition in the 3T3L1 adipocyte differentiation model revealed that nuclear and plasma membrane, but not cytoplasmic, PKB/Akt activity is required for terminal adipocyte differentiation. Nuclear and plasma membrane pools of PKB/Akt were found to contribute to distinct stages of adipocyte differentiation, revealing that PKB/Akt activity impacts multiple points of this program. The localized PKB/Akt pseudosubstrate inhibitor system was also utilized to investigate the importance of distinct subcellular pools of PKB/Akt in breast epithelial cells. MCF-10A human breast epithelial cells can be grown in three-dimensional culture to form acinar structures that recapitulate in vivo mammary glandular architecture. Expression of the plasma membrane PKB/Akt inhibitor during cell growth in three-dimensional culture severely impaired acinar formation. On the other hand, expression of the nuclear PKB/Akt inhibitor during acinar development resulted in the formation of large, misshapen, multi-acinar structures. Assessment of the migratory capacity of MCF-10A cells upon localized PKB/Akt inhibition revealed that nuclear PKB/Akt inhibition promoted, while plasma membrane PKB/Akt inhibition impaired, MCF-10A cell migration. The development of locus-specific PKB/Akt inhibitors represents the first attempt to prioritize the targets of this kinase based on their subcellular localization. This work and its immediate extensions will further our understanding of the biology of PKB/Akt, a multi-tasking kinase with profound roles in development, cellular and organismal homeostasis and disease.

(理学療法基礎系32)

Hepatitis C virus (HCV) infects millions of people worldwide and is one of the leading causes of liver damage. Infection with HCV is strongly correlated with an increased risk of steatosis, or fatty liver disease, which is caused by a build-up of fat deposits in hepatocytes. All genotypes of HCV appear to cause some degree of steatosis in approximately 50% of infected individuals, especially in the presence of contributing host factors such as diabetes, obesity and alcoholism. However, approximately 70% of genotype 3a infections exhibit steatosis. Furthermore, successful clearance of the genotype 3a virus results in eradication of the steatosis, suggesting the genotype 3a virus may be able to directly cause steatosis.<p> Research suggests a role for the core protein of HCV, which forms the capsid of the virus, in the alteration of lipid metabolism pathways during infection. As such, I hypothesized that: 1) HCV alters lipid metabolism pathways and causes the build up of lipid in hepatocytes and the development of steatosis; 2) HCV-3a core protein has a differential or increased effect on these pathways in comparison to 1b core protein; and 3) other HCV proteins could also play a role in the altering of lipid metabolism. My research characterized the subcellular localization on lipid droplets of the HCV-3a core protein in comparison to HCV-1b core protein. It was found that HCV-3a core causes increased transcriptional activity from the Fatty Acid Synthase (FAS) promoter, an important enzyme involved in the synthesis of triglycerides in hepatocytes. In addition, one specific amino acid of HCV-3a core was determined to be partially responsible for this effect. Further research determined that the effect of HCV-3a core on FAS was dependent on the transcription factor Sterol Response Element Binding Protein-1 (SREBP-1) and the presence of HCV-3a core increased the processing and activity of SREBP-1. HCV core was also able to increase activity of Akt 1 and Akt2; inhibition of Akt activity resulted in decreased SREBP-1 activity thereby indicating that HCV core partially mediates SREBP-1 via Akt. Further experiments examined the role of another HCV protein, NS2, in these same lipid metabolism pathways. NS2 was also able to increase transcription from the FAS promoter via SREBP-1, suggesting that this HCV protein may also be important in the development of steatosis during HCV infection.<p> The evidence provided in these studies shows a very important role for HCV in altering lipid metabolism during infection that may lead to the development of steatosis. Current research suggests that the SREBP-1 pathway may be critical in the life cycle of the virus and these studies have provided important information on how lipid metabolism pathways are being changed by the virus. Hopefully this work can help identify potential treatment options for HCV that can slow down disease progression by preventing the development of steatosis or by decreasing viral replication.

<p>Human cancer is a leading cause of morbidity and mortality in the developed world. Contrary to the classical model in which tumors are homogeneously composed of malignant cells, accumulating evidence suggests that subpopulations of highly malignant cells play a dominant role in tumor initiation and growth. These cells have the capacity for prolonged self-renewal and they efficiently generate tumors that phenotypically resemble the parental tumor in transplantation assays. Such characteristics are reminiscent of normal stem cells, and these potently tumorigenic cells have therefore been called cancer stem cells (CSCs). Importantly, studies have shown that CSCs are central mediators of therapeutic resistance, tumor angiogenesis, and metastatic or invasive potential. In the case of malignant glioma, poor patient survival and the paucity of effective therapeutic advances have been attributed to inherent CSC growth potential and treatment resistance, respectively. For this reason, there is great interest in elucidating the molecular features of CSCs, with the ultimate hope of developing CSC-directed therapies.</p><p>Given the overlap between the highly malignant characteristics exhibited by CSCs and those promoted by the PI3K/AKT pathway, we hypothesized that AKT activity within CSCs could represent a reasonable therapeutic target for CSC-directed therapies. Indeed, a pharmacological inhibitor of AKT preferentially targeted glioma CSCs versus non-CSCs and was associated with increased apoptosis and impaired tumorigenesis. These data suggest that interventions targeting AKT could effectively target glioma CSCs. </p><p>Quite distinct from the PI3K/AKT pathway, we hypothesized that the pro-survival and pro-growth features of nitric oxide (NO) might also operate in glioma CSCs. Our experiments found that glioma CSCs produced more NO than non-CSCs, which is attributed to inducible nitric oxide synthase (iNOS) expression and activity within the CSCs. Interference with iNOS activity or expression, as well as selective NO consumption, attenuated CSC growth and tumorigenicity. The mechanism behind iNOS-mediated survival appears to involve, at least in part, suppression of the cell cycle inhibitor CDA1. iNOS inhibition decreased glioma growth in murine xenografts and human expression studies demonstrate an inverse correlation between iNOS expression and patient survival.</p><p>To more fully evaluate the biological effects of NO in CSCs, we designed a novel strategy to consume NO within mammalian cells through heterologous expression of E. coli flavohemoglobin (FlavoHb). This enzyme is a highly specific NO dioxygenase which converts NO to inert nitrate several orders of magnitude faster than iNOS synthesizes NO. Expression of FlavoHb in mammalian cells is therefore a novel and functional tool to interrogate the role of NO in cellular stress and signaling. </p><p>In summary, this doctoral thesis focuses on several molecular characteristics that define malignant CSCs and describes a novel strategy for studying NO, which is one of the CSC-specific molecular effectors.</p> / Dissertation

<p>Normal cells require input from extrinsic growth factors to control proliferation and survival. Recent studies have demonstrated that these same extrinsic signals also regulate cellular metabolism to ensure that metabolism adequately supports the demands of cell function, proliferation, and cell survival. The PI3K/Akt pathway is downstream of many growth factors and can promote both glucose metabolism and cell survival. Aberrant activation of the PI3K/Akt pathway is common in cancer, and its activation can contribute to the growth factor independence that is a hallmark of neoplastic cells. Metabolic demand is high in stimulated and leukemic T cells, and activation of Akt can increase glucose metabolism to meet these requirements. There is great interest in targeting the unique metabolism of cancer cells for cancer therapy, thus making an understanding of the interaction of metabolism and cell death essential. </p><p>Akt is also anti-apoptotic and can inhibit cell death by regulating members of the Bcl-2 family. Interestingly, the ability of Akt to prevent cell death is inextricably linked to its metabolic function. Several recent studies have demonstrated that glucose metabolism can affect Bcl-2 to family members to promote cell survival, but the role of Akt-dependent glucose metabolism in the regulation of Bcl-2 family members is not understood. Using a model of growth factor withdrawal-induced apoptosis, we show that Akt prevents cell death by maintaining glucose metabolism to regulate the Bcl-2 family members Puma and Mcl-1, and demonstrate the importance of this pathway in the survival of stimulated T lymphocytes and leukemia.</p><p>After growth factor withdrawal, active Akt suppressed Puma induction in abundant glucose, but Puma was rapidly upregulated in glucose-deficient conditions and was necessary and sufficient to promote efficient cell death. Importantly, glucose was not uniquely required, as provision of alternative mitochondrial fuels allowed Akt to suppress Puma and maintain survival. This metabolic regulation of Puma was mediated through partially p53-dependent transcriptional induction as well as control of Puma protein stability. </p><p>In addition to inhibiting Puma expression, active Akt prevented the loss of Mcl-1 after growth factor withdrawal by sustaining Mcl-1 protein synthesis in a glucose-dependent manner. Mcl-1 was essential for preventing Bim-induced apoptosis, as Akt could not inhibit Bim induction after growth factor deprivation. Slowing of Mcl-1 synthesis by inhibiting glucose metabolism reversed Mcl-1-mediated resistance of leukemic cells to the Bcl-2 inhibitor ABT-737. Importantly, Akt and glucose-reliant Mcl-1 expression required mTOR-dependent phosphorylation of 4EBP, and treatment with mTOR inhibitors also reversed ABT-737 resistance. </p><p>Together, this study demonstrates that Akt promotes cell survival by preventing metabolic checkpoints that stimulate Puma expression and stability and inhibit Mcl-1 synthesis, advancing our understanding of the links between metabolism and cell death. These studies highlight the importance of cellular metabolism--including a potential role for the alternative sugar fructose--in cancer cell survival that may provide a mechanistic understanding to drive development of metabolism-targeted cancer therapies.</p> / Dissertation

In this study, we investigated the mechanisms of HDGF on cell migration in non-transformed NIH/3T3 cells. HDGF promoted the migration and the formation of dorsal ruffles and podosome rosettes. Besides, HDGF supply increased the PI3K expression, Akt phosphorylation and PTEN phosphorylation as well as stimulated the RhoA, Rac1, and Cdc42 activities. Furthermore, Adenoviral gene transfer of PTEN attenuated migration and PI3K/Akt/Rho GTPases signaling in HDGF-overexpressing transfectants. Pharmaceutical intervention using the PI3K inhibitor, LY294002, potently reversed HDGF-stimulated cell migration, dorsal ruffles formation and podosome formation as well as the RhoA, Rac1, and Cdc42 activities. Thus, HDGF elicits the activation of PI3K/Akt /Rho GTPases signaling cascade and promotes cytoskeleton remodeling to stimulate cellular migration. Moreover, we investigate the expression profile of HDGF during breast carcinogenesis. Immunohistochemical studies revealed elevated HDGF expression in human breast cancer. Nuclear HDGF labelling index was positively correlated with tumour grade, stage and proliferation index, but negatively correlated with survival rate in breast cancer patients. Our data also showed that HDGF over-expression was associated with lymph node metastasis and represented an independent prognostic factor for tumor recurrence. Furthermore, Immunoblot study revealed that elevated HDGF expression significantly higher in breast cancer cells (MDA-MB-231 cells) than that in non-transformed breast cells (MCF-7 cells). Consistently, higher invasive potency and colony formation also observed in MDA-MB-231 cells than in MCF-7 cells. Adenovirus-mediated HDGF over-expression and exogenous HDGF treatment stimulated the invasiveness and colony formation as well as E-cadherin down-regulation and Vimentin up-regulation. Conversely, either HDGF knockdown by RNA interference, HDGF antibody neutralization or BITC-induced EMT suppression in MDA-MB-231 cells attenuated the malignant behavior and elicited EMT reversal by enhancing E-cadherin expression while depleting Vimentin expression. In summary, HDGF elicits the activation of PI3K/Akt/Rho GTPases signaling cascade, thereby promoting cytoskeleton remodeling to stimulate cellular migration. Moreover, the formation of podosome rosettes is correlated with cell invasion, the podosome-stimulating capability of HDGF is consistent with HDGF regulates the metastasis of breast cancer through modulating of epithelial-mesenchymal transition. Therefore, our results provide not only novel insights into the role of the HDGF in cell migration and tumor metastasis, but also validate a novel prognostic indicator for breast cancer. Key words: Hepatoma-derived growth factor, PI3K, Akt, epithelal-mesenchemal transition, E-cadherin, Vimentin

The maintenance of normal cell function and tissue homeostasis is dependent on the precise regulation of multiple signaling pathways that control cellular decisions to either proliferate, differentiate, arrest cell growth, or initiate programmed cell death (apoptosis). Cancer arises when clones of mutated cells escape this balance and proliferate inappropriately without compensatory apoptosis. Deregulated cell growth occurs as a result of perturbed signal transduction that modulates or alters cellular behavior or function to keep the critical balance between the rate of cell-cycle progression (cell division) and cell growth (cell mass) on one hand, and programmed cell death (apoptosis, autophagy) on the other. AGC kinases are activated downstream of a wide range of extracellular stimuli by distinct mechanisms. AGC kinase members such as Aurora-A and Akt regulate fundamental cellular functions including cell cycle, cell growth and survival. Inappropriate activation of those kinases has been associated with the development of diseases such as diabetes, autoimmunity, and cancer. The molecular mechanism of AGC kinases including Aurora-A and Akt involved in human cancers indicates that Aurora-A and Akt are important targets for cancer therapeutic strategies. We demonstrate, for the first time, that Aurora-A interacts with AR and phosphorylates AR at Thr282 and Ser293 in vitro and in vivo. Aurora-A induces AR transactivation activity in a phosphorylation-dependent manner. Ectopic expression Aurora-A in LNCaP cells induces the PSA expression and cell survival whereas knockdown of Aurora-A sensitizes LNCaP-RF cells to apoptosis and cell growth arrest. These data indicate that AR is a substrate of Aurora-A and that elevated Aurora-A could contribute to androgen-independent cell growth by phosphorylation and activation of AR. The NACHT leucine-rich repeat protein 1 (NALP1) is a member of the Ced-4 family and locates at chromosome 17p13.2 near TP53 locus. Here we demonstrated frequent somatic mutations and epigenetic silence of the NALP1 in human non-small cell lung, breast, ovarian and colon cancer. Restoration of NALP1 resulted in the inhibition of tumorigenic activity of the cell lines with NALP1 alterations. In addition to apoptosis, the cells expressing NALP1 largely undergo autophagy. Expression of NALP1 induces PI3KC3 kinase activity through directly interacts with Beclin 1, a protein required for activation of PI3KC3. Moreover, Akt phosphorylates NALP1 and disrupts the interaction between NALP1 and Beclin 1, leading to abrogation of NALP1-induced PI3KC3 activation and autophagy. Taken collectively, these data indicate that the NALP1 is a novel tumor suppressor gene on chromosome 17p13 and plays an important role in tumorigenesis by regulation of Beclin 1/PI3KC3 autophagic pathway and that Akt inhibits autophagy through regulation of NALP1/Beclin/ PI3KC3 cascade.