George Kannourakis

Neutrophil elastase mutations and risk of leukaemia in severe congenital neutropenia.

Severe congenital neutropenia (SCN) is a heterogeneous bone marrow failure syndrome predisposing to myelodysplastic syndrome and acute myeloid leukaemia (MDS/AML). We studied 82 North American and Australian SCN patients enrolled in the Severe Chronic Neutropenia International Registry who were on long‐term treatment with granulocyte colony‐stimulating factor and for whom the neutrophil elastase (ELA2) gene was sequenced. There was no significant difference in the risk of MDS/AML in patients with mutant versus wild‐type ELA2: the respective cumulative incidences at 15 years were 36% and 25% (P = 0·96). Patients with either mutant or wild‐type ELA2 should be followed closely for leukaemic transformation.

Acute exercise leads to regulation of telomere-associated genes and microRNA expression in immune cells.

Telomeres are specialized nucleoprotein structures that protect chromosomal ends from degradation. These structures progressively shorten during cellular division and can signal replicative senescence below a critical length. Telomere length is predominantly maintained by the enzyme telomerase. Significant decreases in telomere length and telomerase activity are associated with a host of chronic diseases; conversely their maintenance underpins the optimal function of the adaptive immune system. Habitual physical activity is associated with longer leukocyte telomere length; however, the precise mechanisms are unclear. Potential hypotheses include regulation of telomeric gene transcription and/or microRNAs (miRNAs). We investigated the acute exercise-induced response of telomeric genes and miRNAs in twenty-two healthy males (mean age = 24.1±1.55 years). Participants undertook 30 minutes of treadmill running at 80% of peak oxygen uptake. Blood samples were taken before exercise, immediately post-exercise and 60 minutes post-exercise. Total RNA from white blood cells was submitted to miRNA arrays and telomere extension mRNA array. Results were individually validated in white blood cells and sorted T cell lymphocyte subsets using quantitative real-time PCR (qPCR). Telomerase reverse transcriptase (TERT) mRNA (P = 0.001) and sirtuin-6 (SIRT6) (P<0.05) mRNA expression were upregulated in white blood cells after exercise. Fifty-six miRNAs were also differentially regulated post-exercise (FDR <0.05). In silico analysis identified four miRNAs (miR-186, miR-181, miR-15a and miR-96) that potentially targeted telomeric gene mRNA. The four miRNAs exhibited significant upregulation 60 minutes post-exercise (P<0.001). Telomeric repeat binding factor 2, interacting protein (TERF2IP) was identified as a potential binding target for miR-186 and miR-96 and demonstrated concomitant downregulation (P<0.01) at the corresponding time point. Intense cardiorespiratory exercise was sufficient to differentially regulate key telomeric genes and miRNAs in white blood cells. These results may provide a mechanistic insight into telomere homeostasis and improved immune function and physical health.

Unique proteome signature of post-chemotherapy ovarian cancer ascites-derived tumor cells

Eighty % of ovarian cancer patients diagnosed at an advanced-stage have complete remission after initial surgery and chemotherapy. However, most patients die within <5 years due to episodes of recurrences resulting from the growth of residual chemoresistant cells. In an effort to identify mechanisms associated with chemoresistance and recurrence, we compared the expression of proteins in ascites-derived tumor cells isolated from advanced-stage ovarian cancer patients obtained at diagnosis (chemonaive, CN) and after chemotherapy treatments (chemoresistant/at recurrence, CR) by using in-depth, high-resolution label-free quantitative proteomic profiling. A total of 2,999 proteins were identified. Using a stringent selection criterion to define only significantly differentially expressed proteins, we report identification of 353 proteins. There were significant differences in proteins encoding for immune surveillance, DNA repair mechanisms, cytoskeleton rearrangement, cell-cell adhesion, cell cycle pathways, cellular transport, and proteins involved with glycine/proline/arginine synthesis in tumor cells isolated from CR relative to CN patients. Pathway analyses revealed enrichment of metabolic pathways, DNA repair mechanisms and energy metabolism pathways in CR tumor cells. In conclusion, this is the first proteomics study to comprehensively analyze ascites-derived tumor cells from CN and CR ovarian cancer patients.

Coalition of Oct4A and β1 integrins in facilitating metastasis in ovarian cancer

BackgroundOvarian cancer is a metastatic disease and one of the leading causes of gynaecology malignancy-related deaths in women. Cancer stem cells (CSCs) are key contributors of cancer metastasis and relapse. Integrins are a family of cell surface receptors which allow interactions between cells and their surrounding microenvironment and play a fundamental role in promoting metastasis. This study investigates the molecular mechanism which associates CSCs and integrins in ovarian cancer metastasis.MethodsThe expression of Oct4A in high-grade serous ovarian tumors and normal ovaries was determined by immunofluorescence analysis. The functional role of Oct4A was evaluated by generating stable knockdown (KD) of Oct4A clones in an established ovarian cancer cell line HEY using shRNA-mediated silencing. The expression of integrins in cell lines was evaluated by flow cytometry. Spheroid forming ability, adhesion and the activities of matrix metalloproteinases 9/2 (MMP-9/2) was measured by in vitro functional assays and gelatin zymography. These observations were further validated in in vivo mouse models using Balb/c nu/nu mice.ResultsWe report significantly elevated expression of Oct4A in high-grade serous ovarian tumors compared to normal ovarian tissues. The expression of Oct4A in ovarian cancer cell lines correlated with their CSC-related sphere forming abilities. The suppression of Oct4A in HEY cells resulted in a significant diminution of integrin β1 expression and associated α5 and α2 subunits compared to vector control cells. This was associated with a reduced adhesive ability on collagen and fibronectin and decreased secretion of pro-MMP2 in Oct4A KD cells compared to vector control cells. In vivo, Oct4A knock down (KD) cells produced tumors which were significantly smaller in size and weight compared to tumors derived from vector control cells. Immunohistochemical analyses of Oct4A KD tumor xenografts demonstrated a significant loss of cytokeratin 7 (CK7), Glut-1 as well as CD34 and CD31 compared to vector control cell-derived xenografts.ConclusionThe expression of Oct4A may be crucial to promote and sustain integrin-mediated extracellular matrix (ECM) remodeling requisite for tumor metastasis in ovarian cancer patients.

Knockdown of stem cell regulator Oct4A in ovarian cancer reveals cellular reprogramming associated with key regulators of cytoskeleton-extracellular matrix remodelling

Oct4A is a master regulator of self-renewal and pluripotency in embryonic stem cells. It is a well-established marker for cancer stem cell (CSC) in malignancies. Recently, using a loss of function studies, we have demonstrated key roles for Oct4A in tumor cell survival, metastasis and chemoresistance in in vitro and in vivo models of ovarian cancer. In an effort to understand the regulatory role of Oct4A in tumor biology, we employed the use of an ovarian cancer shRNA Oct4A knockdown cell line (HEY Oct4A KD) and a global mass spectrometry (MS)-based proteomic analysis to investigate novel biological targets of Oct4A in HEY samples (cell lysates, secretomes and mouse tumor xenografts). Based on significant differential expression, pathway and protein network analyses, and comprehensive literature search we identified key proteins involved with biologically relevant functions of Oct4A in tumor biology. Across all preparations of HEY Oct4A KD samples significant alterations in protein networks associated with cytoskeleton, extracellular matrix (ECM), proliferation, adhesion, metabolism, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and drug resistance was observed. This comprehensive proteomics study for the first time presents the Oct4A associated proteome and expands our understanding on the biological role of this stem cell regulator in carcinomas.

Tumour Microenvironment and Metabolic Plasticity in Cancer and Cancer Stem Cells: Perspectives on Metabolic and Immune Regulatory Signatures in Chemoresistant Ovarian Cancer Stem Cells

Cancer stem cells (CSCs) are a sub-population of tumour cells, which are responsible to drive tumour growth, metastasis and therapy resistance. It has recently been proposed that enhanced glucose metabolism and immune evasion by tumour cells are linked, and are modulated by the changing tumour microenvironment (TME) that creates a competition for nutrient consumption between tumour and different sub-types of cells attracted to the TME. To facilitate efficient nutrient distribution, oncogene-induced inflammatory milieu in the tumours facilitate adaptive metabolic changes in the surrounding non-malignant cells to secrete metabolites that are used as alternative nutrient sources by the tumours to sustain its increasing energy needs for growth and anabolic functions. This scenario also affects CSCs residing at the primary or metastatic niches. This review summarises recent advances in our understanding of the metabolic phenotypes of cancer cells and CSCs and how these processes are affected by the TME. We also discuss how the evolving TME modulates tumour cells and CSCs in cancer progression. Using previously described proteomic and genomic platforms, ovarian cancer cell lines and a mouse xenograft model we highlight the existence of metabolic and immune regulatory signatures in chemoresistant ovarian CSCs, and discuss how these processes may affect recurrence in ovarian tumours. We propose that progress in cancer control and eradication may depend not only on the elimination of highly chemoresistant CSCs, but also in designing novel strategies which would intervene with the tumour-promoting TME factors.

The Many Facets of Metzincins and Their Endogenous Inhibitors: Perspectives on Ovarian Cancer Progression

Approximately sixty per cent of ovarian cancer patients die within the first five years of diagnosis due to recurrence associated with chemoresistance. The metzincin family of metalloproteinases is enzymes involved in matrix remodeling in response to normal physiological changes and diseased states. Recently, there has been a mounting awareness of these proteinases and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), as superb modulators of cellular communication and signaling regulating key biological processes in cancer progression. This review investigates the role of metzincins and their inhibitors in ovarian cancer. We propose that understanding the metzincins and TIMP biology in ovarian cancer may provide valuable insights in combating ovarian cancer progression and chemoresistance-mediated recurrence in patients.

A potentially important role for T cells and regulatory T cells in Langerhans cell histiocytosis

Langerhans cell histiocytosis is characterized by lesions containing inflammatory immune cells, including myeloid cells and T cells. Patient mortality remains unacceptably high and new treatment options are required. Several LCH studies have identified aberrant frequencies of T cell subsets with potential immune regulatory properties. High numbers of Foxp3+ regulatory T cells and gamma-delta T cells have been reported in patients with LCH, although, the cause of their presence or their significance is not yet clear. This review describes the current understanding of how LCH develops and progresses, focusing on the growing evidence that regulatory T cell subsets may be important and discussing the exciting potential for harnessing these cells to treat LCH using immune based therapies.

Abstract DPOC-001: CANCER STEM CELLS: THE SEEDS FOR RECURRENT OVARIAN CANCER

PURPOSE: The treatment of ovarian cancer (OC) with chemotherapy leaves resistant cancer cells which in a short time re–grow as recurrent cancer. A diverse array of resistance mechanisms for chemotherapy in tumor cells has been described but none has proven to be a viable target in a clinical setting. Cancer stem cells (CSCs) are increasingly accepted as the putative mediators of chemoresistance and relapse of cancer. This study aimed to understand the molecular mechanisms involved with chemoresistance and recurrence by investigating the roles of CSCs and their associated pathways in OC cell lines and tumor cells isolated from the ascites of OC patients. METHODS: Tumor cells were collected from chemonaive (CN) and recurrent (CR) OC patients diagnosed with advanced–stage serous OC using a novel in vitro method to obtain a distinct population of epithelial tumor cells. Flow cytometry and immunofluorescence were used to characterize the tumor population. High–resolution label–free quantitative proteomic profiling was used to define significantly differentially expressed proteins between CN and CR tumor cells. KEGG and DAVID software9s were used to determine pathways associated with CR cells. The mechanisms of survival of in vitro cisplatin or paclitaxel treated ascites–derived tumor cells as well as cultured OC cell lines were determined by in vitro assays and in mouse xenografts. In another approach, the expression of embryonic stem cell factor Oct4A was determined in primary ovarian tumors and its functional role was investigated using in vitro assays and in vivo mouse models with stable knockdown (shRNA) of Oct4A in OC cell lines. RESULTS: Proteomic profiling of CN and CR tumor cells showed significant differences in proteins encoding for immune surveillance, DNA repair mechanisms, cytoskeleton rearrangement, cell–cell adhesion, cell cycle pathways, cellular transport, and proteins involved with glycine/proline/arginine synthesis in tumor cells isolated from CR relative to CN patients. Pathway analyses revealed enrichment of metabolic pathways, DNA repair mechanisms and energy metabolism in CR tumor cells. The treatment of ascites–derived OC cells with chemotherapy in vitro resulted in a CSC–like residual population with increased activation of JAK2/STAT3 pathway. Both JAK2/STAT3 activation and CSC–like characteristics were suppressed by a low dose JAK2 specific inhibitor, Momelotinib, in vitro and in vivo. This also resulted in a significantly reduced tumor burden and increased disease–free survival periods in mice in vivo. In another approach, stable knockdown of Oct4A resulted in the decreased expression of CSCs in OC cells and was consistent with decreased cell proliferation, migration and chemoresistance in vitro. In vivo Oct4A knockdown cells produced a significantly reduced tumor burden in mice resulting in a significantly increased disease–free survival periods compared to vector control cells. CONCLUSION: The above studies suggest that targeting the CSCs may prove a therapeutic option for advanced–stage OC patients. Citation Format: Nuzhat Ahmed, Emily Chan, Chantel Samardzija, Khalid Abubaker, Ardian Latifi, George Kannourakis & Jock Findlay. CANCER STEM CELLS: THE SEEDS FOR RECURRENT OVARIAN CANCER [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr DPOC-001.

Report on Patients with Shwachman-Diamond Syndrome with Severe Chronic Neutropenia |[diams]| 751

Patients with Shwachman-Diamond syndrome (SDS) may exhibit a variety of hematologic manifestations including varying degrees of neutropenia. Although these patients have a known predisposition to leukemic transformation, the relationship of the predisposition to the severity of the bone marrow dysfunction or treatment is unclear. The Severe Chronic Neutropenia International Registry (SCNIR) has data from 13 patients with SDS associated with severe chronic neutropenia (SCN) which have been submitted to the SCNIR since the establishment of the registry in 1994. The age range was 10 months to 20 years at the time of registration. Eight patients received G-CSF therapy ranging from 0.4 mcg/kg/day to 10 mcg/kg/day. The duration of therapy ranged from 1 to 72 months. The patients responded to G-CSF therapy with an increase in the absolute neutrophil count (ANC before therapy, mean ± S.D., 0.43± 0.20 to 3.25 ± 2.50 109/L after therapy). Splenomegaly was reported in 3 of the 13 patients, in 1 case the finding was noted after initiation of G-CSF. Of the 13 SDS patients with data in the Registry to date, 3 patients (23%) have developed deletion of chromosome 7, one with transformation to AML. Two of these patients subsequently died and the third has survived 3 years following matched unrelated donor bone marrow transplant. No clonal chromosome abnormalities have been reported in the remaining 5 other patients with SDS who have been treated with G-CSF for varying lengths of time. Prospective studies need to be developed to better document the risk of leukemia and myelodysplastic transformation in SDS irrespective of whether the patients are receiving G-CSF.