Master\u27s Project: A Case Study of the Benet Land Problem in Eastern Uganda

Boundaries are inherently political creations. Boundaries of national parks and other protected conservation areas are one such instance. Social boundary lines are also drawn within communities to determine who is legitimate and who is allowed to access resources as a community member. Boundary lines are also present in the stories people tell about themselves and their environment; the portrayal of their roles as land stewards may leave out certain details. The effects of environmental issues such as deforestation and soil erosion transcend such constructed boundaries. Historically, the Benet, as indigenous peoples of eastern Uganda, had derived their livelihood and cultural identity from land-based activities within the forest of Mount Elgon before being resettled by the Ugandan government in 1983. When Mount Elgon National Park was created in 1993, the government discovered that more land had been distributed than the intended 6,000 hectares. Officially, that surplus land is within the bounds of Mount Elgon National Park, but people continue to reside and make their living there and the High Court of Uganda has put forth a consent judgment that the Benet have a right to this land. Most members of the community currently derive their livelihood from subsistence farming and grazing in this area. Small, fragmented land holdings and population pressures, as well as the movement of others from outside the Benet community into this land area, contribute to members of the community continuing to access resources within the national park boundaries illegally. This illegal access use (notably, firewood gathering and grazing of livestock) creates conflict between the community and the authorities controlling the national park and leads to perceptions by government officials that the Benet community is responsible for environmental degradation. I consider, through the analysis of documents, and of interviews and observations undertaken in May and June of 2014, how the resettlement process (and subsequent lack of resettlement for the Yatui, a sub-group of the Benet) is connected to resource use within the national park. What I deem “the land problem” is the combination of a lack of resettlement (or adequate resettlement) and a lack of access to resources necessary for a subsistence livelihood. Using examples from my interviews and analysis, I identify the connections and relationships that resettlement and resource use have to one another and assess possible responses to the land problem

Stem cell-associated heterogeneity in Glioblastoma results from intrinsic tumor plasticity shaped by the microenvironment

The identity and unique capacity of cancer stem cells (CSC) to drive tumor growth and resistance have been challenged in brain tumors. Here we report that cells expressing CSC-associated cell membrane markers in Glioblastoma (GBM) do not represent a clonal entity defined by distinct functional properties and transcriptomic profiles, but rather a plastic state that most cancer cells can adopt. We show that phenotypic heterogeneity arises from non-hierarchical, reversible state transitions, instructed by the microenvironment and is predictable by mathematical modeling. Although functional stem cell properties were similar in vitro, accelerated reconstitution of heterogeneity provides a growth advantage in vivo, suggesting that tumorigenic potential is linked to intrinsic plasticity rather than CSC multipotency. The capacity of any given cancer cell to reconstitute tumor heterogeneity cautions against therapies targeting CSC-associated membrane epitopes. Instead inherent cancer cell plasticity emerges as a novel relevant target for treatment.publishedVersio

Regulation of hypoxia-induced autophagy in glioblastoma involves ATG9A.

peer reviewedBACKGROUND: Hypoxia is negatively associated with glioblastoma (GBM) patient survival and contributes to tumour resistance. Anti-angiogenic therapy in GBM further increases hypoxia and activates survival pathways. The aim of this study was to determine the role of hypoxia-induced autophagy in GBM. METHODS: Pharmacological inhibition of autophagy was applied in combination with bevacizumab in GBM patient-derived xenografts (PDXs). Sensitivity towards inhibitors was further tested in vitro under normoxia and hypoxia, followed by transcriptomic analysis. Genetic interference was done using ATG9A-depleted cells. RESULTS: We find that GBM cells activate autophagy as a survival mechanism to hypoxia, although basic autophagy appears active under normoxic conditions. Although single agent chloroquine treatment in vivo significantly increased survival of PDXs, the combination with bevacizumab resulted in a synergistic effect at low non-effective chloroquine dose. ATG9A was consistently induced by hypoxia, and silencing of ATG9A led to decreased proliferation in vitro and delayed tumour growth in vivo. Hypoxia-induced activation of autophagy was compromised upon ATG9A depletion. CONCLUSIONS: This work shows that inhibition of autophagy is a promising strategy against GBM and identifies ATG9 as a novel target in hypoxia-induced autophagy. Combination with hypoxia-inducing agents may provide benefit by allowing to decrease the effective dose of autophagy inhibitors

Comprehensive Analysis of Glycolytic Enzymes as Therapeutic Targets in the Treatment of Glioblastoma

<div><p>Major efforts have been put in anti-angiogenic treatment for glioblastoma (GBM), an aggressive and highly vascularized brain tumor with dismal prognosis. However clinical outcome with anti-angiogenic agents has been disappointing and tumors quickly develop escape mechanisms. In preclinical GBM models we have recently shown that bevacizumab, a blocking antibody against vascular endothelial growth factor, induces hypoxia in treated tumors, which is accompanied by increased glycolytic activity and tumor invasiveness. Genome-wide transcriptomic analysis of patient derived GBM cells including stem cell lines revealed a strong up-regulation of glycolysis-related genes in response to severe hypoxia. We therefore investigated the importance of glycolytic enzymes in GBM adaptation and survival under hypoxia, both in vitro and in vivo. We found that shRNA-mediated attenuation of glycolytic enzyme expression interfered with GBM growth under normoxic and hypoxic conditions in all cellular models. Using intracranial GBM xenografts we identified seven glycolytic genes whose knockdown led to a dramatic survival benefit in mice. The most drastic effect was observed for <i>PFKP</i> (PFK1, +21.8%) and <i>PDK1</i> (+20.9%), followed by <i>PGAM1</i> and <i>ENO1</i> (+14.5% each), <i>HK2</i> (+11.8%), <i>ALDOA</i> (+10.9%) and <i>ENO2</i> (+7.2%). The increase in mouse survival after genetic interference was confirmed using chemical inhibition of PFK1 with clotrimazole. We thus provide a comprehensive analysis on the importance of the glycolytic pathway for GBM growth in vivo and propose PFK1 and PDK1 as the most promising therapeutic targets to address the metabolic escape mechanisms of GBM.</p></div

Stem cell-associated heterogeneity in Glioblastoma results from intrinsic tumor plasticity shaped by the microenvironment

The identity and unique capacity of cancer stem cells (CSC) to drive tumor growth and resistance have been challenged in brain tumors. Here we report that cells expressing CSC-associated cell membrane markers in Glioblastoma (GBM) do not represent a clonal entity defined by distinct functional properties and transcriptomic profiles, but rather a plastic state that most cancer cells can adopt. We show that phenotypic heterogeneity arises from non-hierarchical, reversible state transitions, instructed by the microenvironment and is predictable by mathematical modeling. Although functional stem cell properties were similar in vitro, accelerated reconstitution of heterogeneity provides a growth advantage in vivo, suggesting that tumorigenic potential is linked to intrinsic plasticity rather than CSC multipotency. The capacity of any given cancer cell to reconstitute tumor heterogeneity cautions against therapies targeting CSC-associated membrane epitopes. Instead inherent cancer cell plasticity emerges as a novel relevant target for treatment

Glycolysis-related genes are up-regulated in glioblastoma cells under hypoxia.

<p><b>A</b>. Stem-like (NCH644, NCH421k) and classical adherent (U87, U251) glioma cells were cultured in 0.1% O₂ for short term (12 hours = 12h) and long term (7 days = 7d). Differentially expressed genes (DEGs) were established between hypoxic and normoxic cells (n = 3–6). Venn diagrams (top) represent analysis of DEGs after 12h and 7d respectively (FDR<0.001; any fold change (FC)). Red squares highlight the genes commonly modulated in all four glioma cell lines. 120 genes were commonly deregulated upon 12h and 7d hypoxia (Venn diagram, middle) which were strongly associated with glycolysis (9 genes) and glucose metabolism (11 genes) (Revigo representation of significant GO terms, bottom). <b>B</b>. Schematic representation of the glycolytic pathway and associated enzymes. HK2 = hexokinase 2; PFK1 = phosphofructokinase 1 (encoded by <i>PFKP</i> = <i>Phosphofructokinase</i>, <i>platelet</i>); ALDOA = aldolase A; PGAM1 = phosphoglycerate mutase 1; ENO1 = enolase 1; ENO2 = enolase 2; PDH = pyruvate dehydrogenase; PDK1 = pyruvate dehydrogenase kinase 1. <b>C</b>. Quantitative PCR analysis of glycolytic gene expression in adherent glioma cells (U87 and U251) and glioma stem-like cells (NCH421k, NCH644, NCH660h, NCH465 and NCH601), under normoxia and hypoxia (12h and 7d). Data are presented as mean +/- SEM (n = 3). Data were normalized against <i>EZRIN</i> expression. NCH421k cells were used as an internal calibration (value = ‘1’); * p<0.05; p**<0.01; p***<0.001.</p

In vitro effect of glycolytic gene knockdown in glioblastoma cells.

<p><b>A</b>. Cell viability test of 3D spheres carrying gene knockdowns under long-term (7d) hypoxia. Viable cells = ‘green’, dead cells = ‘red’. Representative images are shown (n = 10). <b>B</b>. Quantification of the percentage of dead cells within 3D spheres in hypoxia (n = 10; mean ± SEM) (* p<0.05; ** p<0.01; *** p<0.001).</p

Mouse survival study revealed key glycolysis-related genes for in vivo tumor growth.

<p><b>A</b>. Targeted in vivo shRNA screen in NCH421k cells. From 11 glycolytic target genes, five shRNA containing clones were depleted after in vivo growth compared to in vitro culture (<i>ALDOA</i>, <i>ENO1</i>, <i>ENO2</i>, <i>HK2</i>, <i>PDK1</i>) (* p<0.05; ** p<0.01; *** p<0.001; n = 3 for in vitro, n = 5 for in vivo). The number of shRNAs in each sample was quantified using NGS and is indicated as percentage of control. As <i>PGAM1</i> and <i>PFKP</i> knockdown clones were strongly depleted both in vivo and in vitro, these results were compared to baseline (original cell pool n = 1, p values not available). <b>B</b>. NCH421k cells with the indicated gene specific shRNAs were implanted intracranially into nude mice (n = 21 for control and n = 6–7 for glycolytic genes). Kaplan-Meier graphs show the effect of glycolytic gene knockdown on mouse survival. C. Table summarizing the effect of glycolytic gene knockdown on mouse survival (* p<0.05; ** p<0.01; *** p<0.001).</p

Glycolysis inhibition with clotrimazole affects glioma cell survival in vitro and delays tumor growth in vivo.

<p><b>A</b>. The IC₅₀ of different glycolysis inhibitors was determined for patient derived GBM cells (P3A) and normal human astrocytes (NHA). N: normoxia, H: hypoxia (0.1% O2). Cells were exposed to indicated compounds for 72h and IC₅₀ was determined with the SRB assay (n = 3). <b>B</b>. The cytotoxic effect of clotrimazole (30μM) was assessed on organotypic spheroids of several patient-derived GBM (P3, P8, T16) and NHA, treated for 72h in normoxia and 0,1% O₂ (n = 5). Representative images showing viable cells in ‘green’, dead cells in ‘red’ fluorescence. <b>C</b>. P3 spheroids were implanted intracranially and clotrimazole (CTZ, 150mg/kg) treatment was started 3 weeks after implantation (n = 7). Kaplan-Meier curve shows significantly improved mouse survival (* p<0.05).</p