Introduction: Over the past decades, considerable advances in understanding of cell biology at genetic, epigenetic and proteomic levels led to development of new strategies for better outcome of cancer therapy. One of these new strategies is targeting the class I PI3K/Akt/mTOR signaling pathway, in that this pathway plays a key role in regulation of many cellular functions, including proliferation, survival, metabolism, autophagy and motility. Dysregulation of the class I PI3K/Akt/mTOR pathway has been documented in a variety of human tumours and inhibition of this pathway has been observed to hamper tumour proliferation in vitro and prevent tumour progression in vivo and in clinic. More recently, emerging evidence suggests that the class I PI3K/Akt/mTOR pathway is associated with Cancer Stem Cell (CSC) biology, in light of maintenance, viability and conventional therapy resistance of CSCs. The CSC theory conceptualizes that a subset of tumour cells with Stem Cell-like properties, including self-renewal, multipotency, differentiation, and resistance to chemotherapy and radiotherapy, can recapitulate new tumours and resistance to cancer therapy. Materials and Methods: To explore class I PI3K/Akt/mTOR signaling pathway and CSCs as therapeutic targets in canine oncology, in one series of experiments, smallmolecular inhibitors Wortmannin, ZSTK474, KP372-1 and Rapamycin, which selectively target pan-class I PI3K, pan-class I PI3K, Akt and mTOR, respectively, were utilized to treat canine cancer cell lines using inhibitors alone or in combination with conventional therapeutic drugs. The human acute lymphoblastic leukaemia of T-cell origin cell line (Jurkat T cell line) was used as a comparative control. In another, a stem cell culture system was performed to isolate CSCs from canine glioma J3T cell line. Subsequently, microarray analysis of transcriptional expression profiles of J3T spheres (the putative CSCs) versus J3T parental cells was performed. Results: In this study, small molecules ZSTK474 and KP372-1 were found to significantly decrease cell viability at lower micromolar and nanomolar ranges, respectively. Rapamycin decreased cell viability at lower micromolar concentrations. However, the efficacy of Wortmannin varied from one cell line to another. Dissection of the mechanism of these inhibitors using Western Blot analysis and annexin V staining showed that all inhibitors functioned by decreasing phosphorylation of class I PI3K pathway members. Notably, the efficacy of Wortmannin for this pathway inhibition is confined to certain cell lines. In addition, Wortmannin had shorter drug duration than the other three inhibitors. Annexin V staining showed that KP372-1 was a potent inducer of apoptosis, with decreasing potency in hierarchy order, Rapamycin, Wortmannin and ZSTK474. The data obtained from the combination of pan-class I PI3K inhibitor (Wortmannin or ZSTK474) and mTOR inhibitor (Rapamycin) suggested that additive/synergistic effects were, in part, due to inactivation of Akt. The class I PI3K pathway inhibitors enhanced the efficacy of Doxorubicin in SB cells but not in canine REM, 3132 and J3T cells. The CSC colonies of canine glioma J3T cells were successfully isolated and expanded in the neurosphere formation assay. By microarray analysis, several class I PI3K signaling network-associated genes, particularly IGFBP2 (27-fold), FYN (9.3- fold), and DDIT4 (8.5-fold), were found to be highly up-regulated in the J3T CSCs. However, the genes encoding components, such as Akt1 and eIF4E, of class I PI3K/Akt/mTOR axis signaling were either unchanged or down-regulated in the CSCs. The majority of the genes encoding translation initiation factors were also downregulated in the CSCs. Conclusions: This study demonstrates that class I PI3K/Akt/mTOR signaling pathway is critical for proliferation and survival of cell lines derived from human acute lymphoblastic leukemia of T cell origin (Jurkat T cell line) and a variety of canine tumours. However, it appears that this pathway is dispensible for maintainence and viability of the CSCs isolated from canine gloma J3T cell line. This study suggests that the strategy of dual inhibition of class I PI3K and mTOR kinases may have better outcomes than the combination inhibitors of this pathway (such as ZSTK474 and KP372-1) with Doxorubicin in canine oncology.

The metastatic spread of tumor cells is the major lethal aspect of cancer, and lymphatic metastasis is one of the most important routes. Recent studies indicated that cyclooxygenase-2 (COX-2) expression is frequently associated with lymph node metastasis and over-expression of COX-2 can enhance lymphatic invasion of cancer cells. The interaction of chemokines and their cognate receptors also plays a critical role in cancer metastasis. Previous results of our laboratory demonstrated that CCR7 is a downstream target for COX-2 and COX-2 up-regulated CCR7 expression via the EP2 and EP4 receptor. We also found that protein kinase A (PKA) and AKT kinase are involved in COX-2-induced CCR7. In this study, we provided further evidences that COX-2 directly stimulates CCR7 expression via promoter activation. Promoter deletion and mutation assay indicated that COX-2 stimulated CCR7 promoter via the Sp1 binding site located at the -61/-52 bp region upstream of the transcription start site. Increase of Sp1 binding to CCR7 promoter by COX-2 was confirmed by chromatin immunoprecipitation (ChIP) assay. Furthermore, knockdown of Sp1 expression resulted in inhibition of PGE2-induced CCR7, and over-expression of Sp1 potently up-regulated CCR7 in MCF-7 cells. In vitro kinase assay indicated that AKT could directly phosphorylate Sp1 at S42, T679 and S698 sites. And the phosphorylation of Sp1 by AKT led to enhanced protein stability and DNA binding affinity of Sp1. The results of immunohistochemistry indicated that CCR7 expression was significantly associated with Sp1 and phosphor-AKT. Taken together, COX-2 may act via the EP receptor/PKA/AKT/Sp1 signaling pathway to stimulate CCR7 expression in breast cancer cells to promote lymphatic spread.

Gliomblastoma is a highly malignant tumor of the central nervous system that is resistant to radiation and chemotherapy. Evidences accumulated over recent years have indicated the phosphoinositide 3-kinase/Akt signal transduction pathway as one of the major factors implicated in cancer resistance to conventional therapies. In this study we determined whether inhibition of PI3K/Akt signal pathway through pharmacological and/or genetic manipulation could enhance radiation sensitivity in glioma cells. Our results showed 6 of 12 glioma cell lines with activated Akt mostly due to reciprocal down-regulation of PTEN activity (loss-of-function mutations) but not by PIK3CA gain-of-function mutations. U87 and U373 glioma cell lines with PTEN mutation showing strong Akt Ser473 phopshorylation were treated with PI3K inhibitor LY294002 and irradiated with 0, 2.5, 5 and 7.5 Gy of radiation dosages. The results showed LY294002 inhibited Akt actvation in the glioma cells and decreased clonogenic survival in a radiation dose-dependent manner. Expression of dominant-negative Akt and PTEN through adenovirus mediated gene delivery in U87 and U373 glioma cells sensitized tumor cells to radiation treatment. Furthermore, PDK1 and mTOR inhibitors were also used on radiation sensitivity test. But both inhibitors had no radiosensitization in glioma cells. Glioma invasion was linked to advanced tumor stages. Recently, Type 1 insulin-like growth factor regulates tumor invasion have been showed to be mediated through the PI3K/Akt signaling pathway. In this study, we treated glioma cells with LY294002 to analyze its effects on invasion and migration potentials of the tumor cells. The results showed LY294002 inhibited both abilities in most glioma cell lines in vitro. In addition we used adv-PTEN and adv-dnAkt to confirm these results. Adv-PTEN performed dramatic decrease in glioma cell invasion potentials. Furthermore, we investigated whehter PI3K downstream PDK1, and mTOR involved in tumor cell invasion. We used PDK1 and mTOR inhibitors in glioma and determined their effects on invasion by Boyden chamber assay. Unfortunately, both of inhibitors had only limited inhibition on glioma invasion. Take together, our results indicate the feasibility of using PI3K/Akt inhibiting genetic and pharmacological agents to induce glioma cells to become more sensitive to radiation treatment and reduced invasion potentials. However, glioma radiosensitization and invasion may also be regulated by other signaling pathway.

The Ser/Thr kinase Akt plays an important role in many of insulin's actions including GLUT4 translocation to the plasma membrane (PM). However, there are several features of Akt's regulation of GLUT4 translocation that remain unclear. The goal of my thesis was to resolve some of the following questions: Is activation of Akt sufficient to stimulate GLUT4 translocation? What is the quantitative relationship in signal transmission between individual components within the Akt cascade? What is the role of Akt in insulin resistance? To determine if activation of Akt is sufficient to mediate GLUT4 translocation, I developed a drug-inducible heterodimerisation strategy to activate Akt2 independently of other potential insulin signalling pathways. These studies revealed that activation of Akt2 resulted in rapid stimulation of GLUT4 translocation to a similar extent with maximum insulin, indicating that Akt2 is sufficient for this event. It was previously observed that maximum effect of insulin on GLUT4 translocation was obtained with minimum activation of Akt. To resolve this discrepancy, the relationship between Akt signalling components was examined using a quantitative kinetic and dose response approach combined with hierarchical cluster analysis. Most notably I observed a strong relationship between Akt at the PM, but not Akt in the whole cell lysate, with its substrate phosphorylation. Active pools of phospho-Akt and -AS160, a major substrate involved in GLUT4 translocation, were found in the lipid raft, highlighting the importance of subcellular partitioning of key signalling components for achieving biological specificity. The involvement of Akt in insulin resistance was investigated using the heterodimerisation strategy. These studies revealed that insulin itself initiates a pathway that causes insulin resistance by converging on target(s) downstream of Akt. This inhibitory pathway emanates from PI3-kinase and is likely induced by a range of insults including chronic insulin and dexamethasone. In conclusion, Akt is a crucial element in the insulin action pathway that exhibits precise spatial regulation. While the role of this nanoregulation of Akt in disease remains to be evaluated, my studies suggest that the major defect contributing to insulin resistance occurs downstream of Akt. The elucidation of this target will have major implications for metabolic diseases.

As resveratrol derivatives, resveratrol aliphatic acids were synthesized in our laboratory. Previously, we reported the improved pharmaceutical properties of the compounds compared to resveratrol, including better solubility in water and much tighter binding with human serum albumin. Here, we investigate the role of resveratrol aliphatic acids in Toll-like receptor 2 (TLR2)-mediated apoptosis. We showed that resveratrol aliphatic acid (R6A) significantly inhibits the expression of TLR2. In addition, overexpression of TLR2 in HEK293 cells caused a significant decrease in apoptosis after R6A treatment. Moreover, inhibition of TLR2 by R6A decreases serum deprivation-reduced the levels of phosphorylated Akt and phosphorylated glycogen synthase kinase 3β (GSK3β). Our study thus demonstrates that the resveratrol aliphatic acid inhibits cell apoptosis through TLR2 by the involvement of Akt/GSK3β pathway.

In order to support rampant cell growth, tumor cells must reprogram metabolism to simultaneously drive macromolecular biosynthesis and energy production. Mitochondrial uncoupling proteins (UCPs) oppose this phenotype by inducing futile mitochondrial respiration that is disengaged from ATP synthesis. We found that uncoupling protein 3 (UCP3) was normally expressed in follicular and epidermal keratinocytes and that its levels were augmented by calcium-induced differentiation in vitro. Over-expression of a UCP3 transgene targeted to the basal epidermis by the keratin-5 promoter (K5-UCP3) led to increased differentiation of both epidermal and bulge stem cells, the progenitors of most squamous carcinomas. Consistent with this phenotype, K5-UCP3 mice were completely protected from chemically induced skin carcinogenesis. To define the mechanisms by which UCP3 conferred such strong tumor resistance, we interbred K5-UCP3 mice with a “pre-initiated” mouse model, and found that UCP3 over-expression blocked tumor promotion. Uncoupled epidermis displayed reduced proliferation after treatment with tumor promoter, along with diminished activation of Akt signaling. This effect corresponded to decreased Akt activation by epidermal growth factor (EGF) in K5-UCP3 cells, along with UCP3 overexpressing primary human keratinocytes. Mechanistic studies revealed that uncoupling drove global lipid catabolism, along with impaired recruitment of Akt to the plasma membrane. Over-expression of wild type Akt rescued tumor promoter-induced proliferation and two-stage chemical carcinogenesis in bi-transgenic mice. Collectively, these findings demonstrate that mitochondrial uncoupling is an effective strategy to limit cell proliferation and tumorigenesis through inhibition of Akt, and suggest a novel mechanism of crosstalk between mitochondrial metabolism and growth signaling. / text

Human Cytomegalovirus (HCMV), the prototypic β-herpesvirus, is the most common cause of congenital infections as well as morbidity and mortality in immunocompromised patients. The anti-HCMV drugs currently available have a number of drawbacks (i.e. detrimental side-effects and/or the appearance of drug resistant strains), which limit their clinical usefulness. Therefore, a better understanding of host-virus interactions is important to develop new, safe and effective ways to treat HCMV. HCMV has evolved various strategies to make the host cell more conducive for the replication process, many of these involve modulation of host signalling pathways through proteins or non-coding RNAs. The focus of this thesis is on the regulation of one class of non-coding RNA, microRNAs (miRNA) by HCMV as well as murine CMV (MCMV). miRNAs are short ~22 nucleotide RNA sequences, which negatively regulate the stability and translational efficiency of specific target messenger RNAs (mRNAs). It has been previously shown that three host-encoded miRNAs, miR-199-3p, miR-199-5p and miR-214, are down-regulated in both MCMV and HCMV infected cells. Despite the biological and genomic differences between the two viruses, this down-regulation occurs in both infections, suggesting a possible conserved antiviral role of the miRNAs in mouse and human cells. Consistent with this, miR-199-3p and miR-214 manifest antiviral properties against MCMV and HCMV when over-expressed in vitro. This thesis investigates two hypotheses: 1) CMV down-regulates the expression of these host miRNAs through a mechanism involving viral factors, 2) The down-regulation of miR-199-3p leads to the up-regulation of its targets and this influences the cell in a way that favours some aspect of the viral life cycle. The first part of this project examined the regulation of miR-199-3p, miR-199-5p, and miR-214, which derive from a single primary transcript (pri-miRNA). The down-regulation of all three miRNAs was found to occur at the transcriptional level by 4 hours post infection. The promoter of the miR-199a/214 cluster was therefore cloned into a reporter vector in order to interrogate the factors regulating transcription of pri-miRNA in infection; this was carried out in the murine model based on availability of reagents. The reduction in the pri-miRNA was found to correlate with a decrease in the transcriptional activity of miR-199a/214 promoter in infected cells. Further analysis revealed the presence of a sequence between -421 to -273 relative to the transcription start site (TSS) that was critical for promoter activity. This sequence contains a putative serum response element (SRE), which includes two binding sites for the SRF dimer (serum response factor) and a binding site for a molecule of TCF (ternary complex factor), ELK-1. Initial knock-down studies suggest that these transcription factors are required for basal activity but it remains unknown whether they are involved in the differential expression of miR-199a/214 observed during infection. Another binding site for the transcription factor TWIST-1 was found outside this region, which is known to regulate the miR-199a/214 cluster in other cell types. Western blot analysis showed reduced expression of TWIST-1 in cells infected with HCMV and MCMV infections, by 24 and 48 hours, respectively, suggesting a role of TWIST-1 in regulating miR-199a/214 cluster during these infections. This regulation seems to be dependent on viral gene expression, as a replication deficient viral mutant fails to repress the promoter function and subsequent pri-miRNA production. Taken together, these results suggest an active viral mechanism for transcriptional repression of the miR-199a/214 promoter. To understand the antiviral function of miR-199-3p, the second part of this thesis examined whether miR-199-3p regulates host signalling pathways important for CMV replication and/or the life cycle. A microarray analysis was carried out with samples from cells transfected with miR- 199-3p mimic versus inhibitor. This revealed 198 genes significantly down-regulated by the miRNA. From the 198 genes, Ingenuity pathway analysis (IPA) software identified several host pathways with a potential role in HCMV infection including: PI3K/AKT signalling, the ERK-MAPK cascade, and prostaglandin production. This thesis examined the role of miR-199-3p in regulating the PI3K/AKT pathway in HCMV infection. It was found that miR-199-3p modulates the phosphorylation of the central regulator of PI3K/AKT signalling, AKT. Transfection of miR-199-3p before the infection impedes the complete phosphorylation of AKT, which is known to be required for the immediate early viral gene expression and replication. This provides an explanation for the antiviral function of miR-199-3p, through its ability to modulate AKT phosphorylation. An open question, however, is how the natural down-regulation of miR-199-3p from 24 to 72 hours post infection naturally affects AKT phosphorylation. Several predicted targets of miR-199-3p, such as PIK3CB, ITGA3, and ITGA6 were shown to be up-regulated at these late time points, correlating with the miR-199-3p down-regulation. The interaction of miR-199-3p with target sites in the 3′UTRs of PIK3CB and ITGA3 was validated by luciferase reporter assays and western blotting and qRT-PCR results indicated that protein and mRNA levels of ITGA6 were regulated by miR-199-3p mimic transfection. However, the knock-down of these three targets did not result in a significant decrease of the viral growth, and thus cannot alone explain the antiviral function of miR-199-3p. Overall, this study suggests that the transcriptional repression of miR- 199a/214 is likely a strategy employed by CMV to support its own growth through attenuating the biological effect of miR-199-3p within the host cell.

Interstitial cystitis is a chronic debilitating disease that causes pain and increased frequency of micturition, amongst other symptoms, without any identifiable cause. This disease affects a large number of the population, yet the etiology is still unknown. The present study aimed to characterize BDNF and CGRP—two neuropeptides that have both been proven to play an important role in the transmission of pain as well as in hypersensitivity. The signaling pathways regulating the expression of the two neuropeptides were also examined. Results revealed that BDNF protein expression levels increased in both L1 and L6 DRG following 48 hours post CYP-induced cystitis. CGRP protein expression levels decreased in L1 DRG, but increased in L6 DRG following 48 hours post CYP-induced cystitis. Examination of mRNA levels revealed an increase in the mRNA levels of both BDNF and CGRP in L6 DRG. NGF, a member of the neurotrophin family, mRNA levels also increased following 48 hour CYP-induced cystitis in the urinary bladder. Retrograde analysis revealed NGF possibly retrograde signaled to the DRG to increase BDNF and CGRP expression. Co-localization immunohistochemistry results revealed phospho-Akt co-localized with BDNF, but not with CGRP. Thus NGF retrograde signaling may activate the PI3-K/Akt cascade which may be involved in BDNF expression. CGRP expression may be via another signaling cascade.

No description available.

This thesis defines a novel role for the Herpes Simplex Virus (HSV) tegument protein, virion protein (VP) 11/12 as a modulator of host cell signalling. Studies aimed at examining infection induced lymphocyte inactivation, revealed that VP11/12 is tyrosine phosphorylated in three lymphocyte lineages (T cell, B cell and NK cell) following exposure to HSV-1 or HSV-2 infected fibroblasts. Tyrosine phosphorylation of VP11/12 was greater in lymphocytes compared to fibroblasts or epithelial cells and phosphorylation was enhanced by the lymphocyte specific Src family kinase (SFK) Lck during transfection- or infection-based assays. This suggested that VP11/12 is a substrate of Lck or a kinase activated by Lck. Lck is best known for initiating intracellular signalling downstream of the T cell receptor (TCR) and NK cell receptors. However, VP11/12 null HSV mutants retained the ability to block TCR signalling and NK cell cytotoxicity. Phosphorylation of VP11/12 occurred in the absence of any known Lck stimulus, like TCR ligation. Infection alone may activate Lck since Lck in infected Jurkat cells displayed features characteristic of activation: a reduced electrophoretic mobility in sodium dodecyl sulphate polyacrylamide gel and a marked increase in phosphorylation at the activation loop tyrosine (Y394). SFK substrates sometimes activate their cognate kinase through high affinity binding of the SFK Src homology (SH) 2 or SH3 domains. VP11/12 may serve this dual function since it interacts with Lck or Lck signalling complexes and is strictly required for Lck activation during infection. SFKs including Lck lie upstream of the canonical phosphoinositide 3-kinase (PI3K)-Akt pathway in signalling emanating from immune receptors, growth factor receptors and polyoma middle T antigen (MTAg). In HSV infection of Jurkat T cells and human embryonic lung fibroblasts, we find that VP11/12 interacts with PI3K either directly or indirectly and is required for infection induced activation of the PI3K-Akt signalling pathway. SFK activity is required for tyrosine phosphorylation of VP11/12, VP11/12-PI3K interactions, and Akt activation in infected fibroblasts. This data suggests that VP11/12 orchestrates signalling analogous to that of MTAg. In this model, VP11/12 activates SFKs to induce its own phosphorylation, subsequently allowing for interactions with PI3K and activation of Akt. / Virology