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.

A cellular defense mechanism counteracts the deleterious effects of misfolded protein accumulation by eliciting a stress response. The cytoplasmic deacetylase HDAC6 (histone deacetylase 6) was previously shown to be a key element in this response by coordinating the clearance of protein aggregates through aggresome formation and their autophagic degradation. Here, for the first time, we demonstrate that HDAC6 is involved in another crucial cell response to the accumulation of ubiquitinated protein aggregates, and unravel its molecular basis. Indeed, our data show that HDAC6 senses ubiquitinated cellular aggregates and consequently induces the expression of major cellular chaperones by triggering the dissociation of a repressive HDAC6/HSF1 (heat-shock factor 1)/HSP90 (heat-shock protein 90) complex and a subsequent HSF1 activation. HDAC6 therefore appears as a master regulator of the cell protective response to cytotoxic protein aggregate formation.[Keywords: HSP25/27; HSP70; acetylation; microtubules; heat shock; p97/VCP] Supplemental material is available at http://www.genesdev.org.

Heterozygous mutations in NADP‐dependent isocitrate dehydrogenases (IDH) define the large majority of diffuse gliomas and are associated with hypermethylation of DNA and chromatin. The metabolic dysregulations imposed by these mutations, whether dependent or not on the oncometabolite D‐2‐hydroxyglutarate (D2HG), are less well understood. Here, we applied mass spectrometry imaging on intracranial patient‐derived xenografts of IDH‐mutant versus IDH wild‐type glioma to profile the distribution of metabolites at high anatomical resolution in situ. This approach was complemented by in vivo tracing of labeled nutrients followed by liquid chromatography–mass spectrometry (LC‐MS) analysis. Selected metabolites were verified on clinical specimen. Our data identify remarkable differences in the phospholipid composition of gliomas harboring the IDH1 mutation. Moreover, we show that these tumors are characterized by reduced glucose turnover and a lower energy potential, correlating with their reduced aggressivity. Despite these differences, our data also show that D2HG overproduction does not result in a global aberration of the central carbon metabolism, indicating strong adaptive mechanisms at hand. Intriguingly, D2HG shows no quantitatively important glucose‐derived label in IDH‐mutant tumors, which suggests that the synthesis of this oncometabolite may rely on alternative carbon sources. Despite a reduction in NADPH, glutathione levels are maintained. We found that genes coding for key enzymes in de novo glutathione synthesis are highly expressed in IDH‐mutant gliomas and the expression of cystathionine‐β‐synthase (CBS) correlates with patient survival in the oligodendroglial subtype. This study provides a detailed and clinically relevant insight into the in vivo metabolism of IDH1‐mutant gliomas and points to novel metabolic vulnerabilities in these tumors.

The histopathological and molecular heterogeneity of glioblastomas represents a major obstacle for effective therapies. Glioblastomas do not develop autonomously, but evolve in a unique environment that adapts to the growing tumour mass and contributes to the malignancy of these neoplasms. Here, we show that patient-derived glioblastoma xenografts generated in the mouse brain from organotypic spheroids reproducibly give rise to three different histological phenotypes: (i) a highly invasive phenotype with an apparent normal brain vasculature, (ii) a highly angiogenic phenotype displaying microvascular proliferation and necrosis and (iii) an intermediate phenotype combining features of invasion and vessel abnormalities. These phenotypic differences were visible during early phases of tumour development suggesting an early instructive role of tumour cells on the brain parenchyma. Conversely, we found that tumour-instructed stromal cells differentially influenced tumour cell proliferation and migration in vitro, indicating a reciprocal crosstalk between neoplastic and non-neoplastic cells. We did not detect any transdifferentiation of tumour cells into endothelial cells. Cell type-specific transcriptomic analysis of tumour and endothelial cells revealed a strong phenotype-specific molecular conversion between the two cell types, suggesting co-evolution of tumour and endothelial cells. Integrative bioinformatic analysis confirmed the reciprocal crosstalk between tumour and microenvironment and suggested a key role for TGFβ1 and extracellular matrix proteins as major interaction modules that shape glioblastoma progression. These data provide novel insight into tumour-host interactions and identify novel stroma-specific targets that may play a role in combinatorial treatment strategies against glioblastoma.

Long non-coding RNAs play critical roles in tumour progression. Through analysis of publicly available genomic datasets, we found that MIR22HG, the host gene of microRNAs miR-22-3p and miR-22-5p, is ranked among the most dysregulated long non-coding RNAs in glioblastoma. The main purpose of this work was to determine the impact of MIR22HG on glioblastoma growth and invasion and to elucidate its mechanistic function. The MIR22HG/miR-22 axis was highly expressed in glioblastoma as well as in glioma stem-like cells compared to normal neural stem cells. In glioblastoma, increased expression of MIR22HG is associated with poor prognosis. Through a number of functional studies, we show that MIR22HG silencing inhibits the Wnt/β-catenin signalling pathway through loss of miR-22-3p and -5p. This leads to attenuated cell proliferation, invasion and in vivo tumour growth. We further show that two genes, SFRP2 and PCDH15, are direct targets of miR-22-3p and -5p and inhibit Wnt signalling in glioblastoma. Finally, based on the 3D structure of the pre-miR-22, we identified a specific small-molecule inhibitor, AC1L6JTK, that inhibits the enzyme Dicer to block processing of pre-miR-22 into mature miR-22. AC1L6JTK treatment caused an inhibition of tumour growth in vivo. Our findings show that MIR22HG is a critical inducer of the Wnt/β-catenin signalling pathway, and that its targeting may represent a novel therapeutic strategy in glioblastoma patients.