“Left in the Forest and Forgotten” Land Policy and the Plight of the Benet

In carrying out my independent study project on the issue of the Benet being displaced by the creation of Mount Elgon National Park, I had three main objectives in my mind. My first objective was to investigate and gain a balanced perspective of the effects of the Benet’s most recent eviction from Mount Elgon National Park. Secondly, I wished to analyze the government’s role as a duty bearer in compensating and resettling the Benet for their land. Lastly, I wished to explore the Benet Community’s options for redress within the legal system. The methods I utilized included a literature review, informal conversations, semi-structured interviews and observation. I undertook the literature review mainly at the Uganda Land Alliance and the Foundation for Human Rights Initiative, in addition to reading news articles and reports. In living with a Benet family in Kapchorwa for two weeks, I was able to directly observe the living conditions of those with uncertain land tenure and physically see the different boundaries during a transect walk. I was also able to utilize the knowledge of community members in drawing a map of the disputed areas. I conducted semi-structured interviews and also engaged in informal conversations with both Benet and non-Benet community members, including an e-mail correspondence with the Uganda Wildlife Authority. My findings included a better understanding of the reasons for evictions and the problems the Benet have faced historically as a marginalized group. I discovered that although legally the Benet are entitled to the land as indigenous inhabitants, what is put down on paper and what is actually being upheld often differs. My findings also included the government’s plans for permanent resettlement and the Benet community’s own desires for permanent resettlement. There is an overarching need for the disputed land area to be officially degazetted in order for social development such as roads and schools to occur and for the Benet to have land tenure security

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

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

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