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Non-Hodgkin’s lymphoma is the fifth most common cancer in the UK, accounting for 4% of all new cases. The control of lymphomagenesis still remains a challenge. Early growth response gene (Egr) 2 and 3 are zinc finger transcription factors. Egr2 plays an important role in the development of both central nervous system and lymphocytes. However the mechanism of action in lymphocytes is still unknown. In order to fully understand the function of Egr2, in lymphocytes, we developed Egr2 and 3 double knockout mice (Egr2-/-Egr3-/-) by crossbreeding lymphocyte specific Egr2 knockout mice (CD2-Egr2-/-) with Egr3 knockout mice (Egr3-/-), as previous reports suggested that Egr3 compensates for the role of Egr2. In the absence of Egr2 and 3, the homeostasis of T cells is dysregulated with hyper-homeostatic proliferation of effector like phenotype cells. More importantly the development of spontaneous B and T cell lymphoma was found in more than 70% of Egr2-/-Egr3-/- mice. The lymphoma cells from Egr2-/-Egr3-/- mice were highly proliferative and metastatically spread into other non-lymphoid organs, such as lung, liver and kidney. In additional to this lymphoma development the Egr2-/-Egr3-/- mice showed signs of chronic inflammatory disorder. This inflammatory disorder was characterised by glomerulonephritis and an increase in serum cytokines, which may provide the microenvironment for the lymphoma development. To explore the molecular mechanism of tumour development in Egr2-/-Egr3-/- mice, the transcriptional profile of Egr2 was studied by microarray and ChIP-on-chip. We found firstly that Egr2 directly binds to the promoter regions of Ikaros and FOXO3. The deletion of Egr2 and 3 in lymphocytes led to the downregulation of Ikaros, Aiolos and FOXO3 expression. The impaired expression was found to be associated with proliferative disorder and the development of T and B cell lymphoma. Secondly Egr2 strongly inhibits STAT3 transcriptional activity by regulating SOCS3, which is a known inhibitor of STAT3. The breakdown of this regulation could be an important mechanism in lymphomagenesis. A model is proposed which defines Egr2 and Egr3 as the backbone of important tumour suppressor genes that control cell fate decision and regulates homeostasis in the lymphoid system. Thus, our results suggest that Egr2 and 3 are important regulators of lymphocyte function by their involvement in multiple cell signalling pathways, which could potentially be key genes for future cancer therapy.

Cells adapt their metabolism to satisfy changing bioenergetic and biosynthetic needs. Investigation of metabolic reprogramming in cancer has provided insight into the metabolic control of proliferation and survival. While the predominant focus of this field has been aerobic glycolysis (the Warburg effect), increasing evidence points to a complex landscape of tumor metabolic circuitries beyond aerobic glycolysis, including varied degrees of mitochondrial contribution to tumor metabolism. To investigate alternative metabolic programs compatible with tumor growth, we turned to Diffuse Large B-cell Lymphoma (DLBCL), a highly heterogeneous disease encompassing discrete clusters or subtypes defined by tumor-intrinsic genetic distinctions. In one classification scheme, a B-cell receptor (BCR)/proliferation cluster identified BCR-dependent DLBCLs with elevated expression of BCR signaling components. A second subset, OxPhos-DLBCL, displayed increased expression of mitochondrial oxidative phosphorylation genes, and was insensitive to BCR inhibition. However, the functional attributes of OxPhos-DLBCLs and the nature of their BCR-independent survival were unknown. Upon integrative analyses of DLBCL subtypes, we uncovered quantitative proteome- and metabolome-level signatures associated with differences in nutrient and energy metabolism. Specifically, BCR-DLBCLs have greater glycolytic flux typical of the Warburg phenotype. Unlike BCR-DLBCLs, OxPhos-DLBCLs channel the majority of glucose-derived pyruvate into mitochondria, display elevated mitochondrial electron transport chain (ETC) activity, ATP production, and fatty acid oxidation (FAO). Importantly, these metabolic distinctions are associated with subtype-selective survival mechanisms. Moreover, acute inhibition of BCR signaling in BCR-DLBCLs increased their FAO capacity, thus revealing a reciprocal relationship between BCR and FAO. Further dissection of mitochondrial function in OxPhos-DLBCLs indicates that increased mitochondrial metabolism is integrated with at least two homeostatic mechanisms that help maintain ETC activity and FAO capacity. In particular, OxPhos-DLBCLs harbor robust protein-level enrichment of mitochondrial translation factors required for the synthesis of mitochondrial-DNA-encoded ETC subunits. Inhibition of the mitochondrial translation pathway is selectively toxic to OxPhos-DLBCLs. A second mitochondrial homeostatic pathway, mitochondrial network dynamics, also proved relevant to OxPhos-DLBCLs. Compared to BCR-DLBCLs, OxPhos-DLBCLs display a fragmented mitochondrial network that supports their FAO capacity. Overall, these findings demonstrate previously unappreciated metabolic heterogeneity in molecular subsets of DLBCL and uncover BCR-independent survival mechanisms linked to mitochondrial FAO, protein translation, and network architecture.

published_or_final_version / Medicine / Master / Master of Philosophy

Non-Hodgkin lymphoma is commonly associated with chronic infection and inflammation. Such conditions are characterized by chronic oxidative stress. Because apoptosis signaling is often mediated by reactive oxygen species, lymphoma arising in the context of oxidative stress may become resistant to these apoptosis signals. Resistance to oxidative stress could contribute to tumorigenesis and limit response to chemotherapy, as apoptosis induced by many drugs involves reactive oxygen species. We used a cell culture model to understand how changes in the ability to handle oxidative stress contribute to apoptosis resistance. WEHI7.2 murine thymic lymphoma cells transfected with catalase or selected for resistance to hydrogen peroxide acquire a concomitant resistance to apoptosis induced by glucocorticoids. Cytochrome c release is delayed in these variants, demonstrating that apoptosis resistance lies upstream, in the signaling phase, or in the mitochondria themselves. By comparing the apoptosis-sensitive WEHI7.2 parental cells with the oxidative stress- and apoptosis-resistant variant cells, we investigated the contribution of cytosolic and mitochondrial changes to glucocorticoid-induced apoptosis. We showed that neither JNK kinase signaling, nor GSTπ, a redox sensor protein which regulates JNK, is activated during glucocorticoid-induced apoptosis. Our work using isolated mitochondria and recombinant tBid protein in cell-free apoptosis assays showed that the apoptosisresistant variants are intrinsically resistant to the release of cytochrome c and other intermembrane space proteins. The resistance was mediated upstream and within the mitochondria, and occurred at both steps controlling cytochrome c release. Given that the resistant variants demonstrated alterations in mitochondrial apoptotic function, we investigated mitochondrial protein changes that could explain these differences. An increased expression of cytochrome c was observed in the resistant variants, but selective reduction of cytochrome c expression showed that this change alone was not sufficient to affect sensitivity. The balance of pro- and anti-apoptotic Bcl-2 family members in untreated cells also did not explain intrinsic resistance. Alterations in Bcl-2 protein levels following treatment could contribute to glucocorticoid resistance, but additional work to test Bcl-2 family interactions will be required. We have identified points of resistance that are important in glucocorticoid-induced apoptosis and may also contribute to resistance to novel mitochondrial-targeting drugs.

No description available.

The oncogene Ahi-1 was recently identified through provirus insertional mutagenesis in murine leukemias and lymphomas. Its involvement in human leukemogenesis is demonstrated by gross perturbations in its expression in several leukemic cells lines, particularly in cutaneous T-cell lymphoma (CTCL) cell lines (Hut 78 and Hut 102). Hut 78 is derived from a patient with Sezary syndrome, a common leukemic variant of the human CTCL mycosis fungoides. Aberrant expression of AHI-1 mRNA and protein has been found in CD4⁺CD7⁻ leukemic Sezary cells from patients with Sezary syndrome. Moreover, stable suppression of AHI-1 using retroviral-mediated RNA interference in Hut 78 cells inhibits their transforming activity in vitro and in vivo. In an effort to identify genes involved in AHI-1-mediated leukemic transformation in CTCL, microarray analysis was performed to compare six RNA samples from AHI-1 suppressed Hut 78/sh4 cells to five samples from Hut 78 control cells. Limma and dChip analyses identified 218 and 95 differentially expressed genes, respectively, using a fold change criteria of > or < 2 and a p-value threshold of ≤ 0.01. After evaluation of both analyses, 21 genes were selected based upon interesting structural and functional information, specificity to hematopoietic cells or T-cells, and previous connections to cancer. Expression patterns of these 21 genes were validated by qRT-PCR with p-values < 0.05 ranging from 1.97 x 10⁻¹⁰ to 6.55 x 10⁻³, with the exception of BRDG1 at 5.88 x 10⁻². The observed up-regulation of both BIN1 and HCK in AHI-1 suppressed Hut 78/sh4 cells as compared to control cells further confirmed at the protein level. The tumor suppressor BIN1 is known to physically interact with c-MYC, which also exhibits differential protein expression in these cells. Characterization of BIN1 identified 4 isoforms all of which contain exon 10 and demonstrate alternative splicing of exons 12A and 13. Additionally, qRT-PCR results from primary Sezary samples indicate there is clinical significance in the expression changes detected for BIN1, HCK, REPS2, BRDG1, NKG7 and SPIB. These findings identify several new differentially expressed genes that may play critical roles in AHI-1-mediated leukemic transformation of human CTCL cells.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年3月25日 水野紘樹氏の博士論文として提出された

Follicular lymphoma (FL) is an attractive model for discovering biomarkers and elucidating mechanisms of tumour progression. We hypothesized that alterations in the expression of proteins with known roles in cancer biology and hematological cells might correlate with clinical outcome and thereby shed light on biological mechanisms. Sections from a tissue microarray (TMA) containing FL samples from 67 patients were immunostained for candidate biomarkers, including p53, p16INK4a, Bcl-2, Bcl-6, MUM1, PML, phospho-ERK, and p27Kip1. The Kaplan-Meier method and log-rank test were used to identify markers that correlate significantly (p<0.05) with overall survival (OS). The chi-squared or Fisher exact test were used to examine associations between histological markers and baseline clinical features, including the Follicular Lymphoma International Prognostic Index (FLIPI) score. Expression of p16INK4a or p53, or absent CD10 expression correlated with poor survival. Patients with p16INK4a-negative tumours had a median OS of 13.4 years compared to 8.3 years for those with p16INK4a-positive tumours (p=0.006). Expression of p16INK4a was significantly associated with low hemoglobin, elevated serum lactate dehydrogenase (LDH), high histological grade, high cell proliferation index, presence of associated diffuse large B-cell lymphoma (DLBCL) and high-risk FLIPI classification. Our observation of a positive association between p16INK4a expression and indicators of tumour aggressiveness is novel and perhaps surprising since loss of the INK4a tumour suppressor gene is one of the most frequently observed lesions in human cancers, including lymphoma. Expression of p16INK4a may be part of a cellular response to unidentified pro-mitotic mutations, such as deleterious mutations of the RB tumour suppressor gene, associated with more aggressive instances of FL. Immunostaining FL diagnostic biopsies for expression of p16INK4a may serve as an informative prognostic biomarker to aid clinicians managing FL patients. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2008-07-04 15:55:16.121

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