ABSTRACT
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.
Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Brain Neoplasms/genetics , Cell Plasticity/drug effects , Glioblastoma/genetics , Tumor Microenvironment/drug effects , Animals , Antineoplastic Agents, Alkylating/therapeutic use , Biopsy , Brain Neoplasms/drug therapy , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Plasticity/genetics , Cohort Studies , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Gene Expression Profiling , Glioblastoma/drug therapy , Glioblastoma/pathology , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Temozolomide/pharmacology , Temozolomide/therapeutic use , Treatment Outcome , Tumor Microenvironment/genetics , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: 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.
Subject(s)
Autophagy-Related Proteins/physiology , Autophagy/drug effects , Bevacizumab/pharmacology , Brain Neoplasms/drug therapy , Chloroquine/pharmacology , Glioblastoma/drug therapy , Membrane Proteins/physiology , Neoplasm Proteins/physiology , Tumor Hypoxia/physiology , Vesicular Transport Proteins/physiology , Angiogenesis Inhibitors/pharmacology , Animals , Autophagy/physiology , Autophagy-Related Proteins/metabolism , Brain Neoplasms/blood supply , Brain Neoplasms/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/physiology , Drug Synergism , Gene Expression Profiling , Gene Knockdown Techniques , Gene Silencing , Glioblastoma/blood supply , Glioblastoma/metabolism , Heterografts , Humans , Membrane Proteins/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Molecular Targeted Therapy/methods , Neoplasm Proteins/metabolism , Neoplasm Transplantation , Random Allocation , Spheroids, Cellular/pathology , Vesicular Transport Proteins/metabolismABSTRACT
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 PFKP (PFK1, +21.8%) and PDK1 (+20.9%), followed by PGAM1 and ENO1 (+14.5% each), HK2 (+11.8%), ALDOA (+10.9%) and ENO2 (+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.
