Cell-lineage controlled epigenetic regulation in glioblastoma stem cells determines functionally distinct subgroups and predicts patient survival.

There is ample support for developmental regulation of glioblastoma stem cells. To examine how cell lineage controls glioblastoma stem cell function, we present a cross-species epigenome analysis of mouse and human glioblastoma stem cells. We analyze and compare the chromatin-accessibility landscape of nine mouse glioblastoma stem cell cultures of three defined origins and 60 patient-derived glioblastoma stem cell cultures by assay for transposase-accessible chromatin using sequencing. This separates the mouse cultures according to cell of origin and identifies three human glioblastoma stem cell clusters that show overlapping characteristics with each of the mouse groups, and a distribution along an axis of proneural to mesenchymal phenotypes. The epigenetic-based human glioblastoma stem cell clusters display distinct functional properties and can separate patient survival. Cross-species analyses reveals conserved epigenetic regulation of mouse and human glioblastoma stem cells. We conclude that epigenetic control of glioblastoma stem cells primarily is dictated by developmental origin which impacts clinically relevant glioblastoma stem cell properties and patient survival.

A molecularly distinct subset of glioblastoma requires serum-containing media to establish sustainable bona fide glioblastoma stem cell cultures.

Glioblastoma (GBM) is the most frequent and deadly primary malignant brain tumor. Hallmarks are extensive intra-tumor and inter-tumor heterogeneity and highly invasive growth, which provide great challenges for treatment. Efficient therapy is lacking and the majority of patients survive less than 1 year from diagnosis. GBM progression and recurrence is caused by treatment-resistant glioblastoma stem cells (GSCs). GSC cultures are considered important models in target identification and drug screening studies. The current state-of-the-art method, to isolate and maintain GSC cultures that faithfully mimic the primary tumor, is to use serum-free (SF) media conditions developed for neural stem cells (NSCs). Here we have investigated the outcome of explanting 218 consecutively collected GBM patient samples under both SF and standard, serum-containing media conditions. The frequency of maintainable SF cultures (SFCs) was most successful, but for a subgroup of GBM specimens, a viable culture could only be established in serum-containing media, called exclusive serum culture (ESC). ESCs expressed nestin and SOX2, and displayed all functional characteristics of a GSC, that is, extended proliferation, sustained self-renewal and orthotopic tumor initiation. Once adapted to the in vitro milieu they were also sustainable in SF media. Molecular analyses showed that ESCs formed a discrete group that was most related to the mesenchymal GBM subtype. This distinct subgroup of GBM that would have evaded modeling in SF conditions only provide unique cell models of GBM inter-tumor heterogeneity.

LGR5 promotes tumorigenicity and invasion of glioblastoma stem-like cells and is a potential therapeutic target for a subset of glioblastoma patients.

Glioblastoma (GBM) is the most common and lethal primary malignant brain tumor which lacks efficient treatment and predictive biomarkers. Expression of the epithelial stem cell marker Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) has been described in GBM, but its functional role has not been conclusively elucidated. Here, we have investigated the role of LGR5 in a large repository of patient-derived GBM stem cell (GSC) cultures. The consequences of LGR5 overexpression or depletion have been analyzed using in vitro and in vivo methods, which showed that, among those with highest LGR5 expression (LGR5high ), there were two phenotypically distinct groups: one that was dependent on LGR5 for its malignant properties and another that was unaffected by changes in LGR5 expression. The LGR5-responding cultures could be identified by their significantly higher self-renewal capacity as measured by extreme limiting dilution assay (ELDA), and these LGR5high -ELDAhigh cultures were also significantly more malignant and invasive compared to the LGR5high -ELDAlow cultures. This showed that LGR5 expression alone would not be a strict marker of LGR5 responsiveness. In a search for additional biomarkers, we identified LPAR4, CCND2, and OLIG2 that were significantly upregulated in LGR5-responsive GSC cultures, and we found that OLIG2 together with LGR5 were predictive of GSC radiation and drug response. Overall, we show that LGR5 regulates the malignant phenotype in a subset of patient-derived GSC cultures, which supports its potential as a predictive GBM biomarker. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Snail regulates BMP and TGFß pathways to control the differentiation status of glioma-initiating cells.

Glioblastoma multiforme is a brain malignancy characterized by high heterogeneity, invasiveness, and resistance to current therapies, attributes related to the occurrence of glioma stem cells (GSCs). Transforming growth factor ß (TGFß) promotes self-renewal and bone morphogenetic protein (BMP) induces differentiation of GSCs. BMP7 induces the transcription factor Snail to promote astrocytic differentiation in GSCs and suppress tumor growth in vivo. We demonstrate that Snail represses stemness in GSCs. Snail interacts with SMAD signaling mediators, generates a positive feedback loop of BMP signaling and transcriptionally represses the TGFB1 gene, decreasing TGFß1 signaling activity. Exogenous TGFß1 counteracts Snail function in vitro, and in vivo promotes proliferation and re-expression of Nestin, confirming the importance of TGFB1 gene repression by Snail. In conclusion, novel insight highlights mechanisms whereby Snail differentially regulates the activity of the opposing BMP and TGFß pathways, thus promoting an astrocytic fate switch and repressing stemness in GSCs.

The high mobility group A2 protein epigenetically silences the Cdh1 gene during epithelial-to-mesenchymal transition.

The loss of the tumour suppressor E-cadherin (Cdh1) is a key event during tumourigenesis and epithelial-mesenchymal transition (EMT). Transforming growth factor-ß (TGFß) triggers EMT by inducing the expression of non-histone chromatin protein High Mobility Group A2 (HMGA2). We have previously shown that HMGA2, together with Smads, regulate a network of EMT-transcription factors (EMT-TFs) like Snail1, Snail2, ZEB1, ZEB2 and Twist1, most of which are well-known repressors of the Cdh1 gene. In this study, we show that the Cdh1 promoter is hypermethylated and epigenetically silenced in our constitutive EMT cell model, whereby HMGA2 is ectopically expressed in mammary epithelial NMuMG cells and these cells are highly motile and invasive. Furthermore, HMGA2 remodels the chromatin to favour binding of de novo DNA methyltransferase 3A (DNMT3A) to the Cdh1 promoter. E-cadherin expression could be restored after treatment with the DNA de-methylating agent 5-aza-2'-deoxycytidine. Here, we describe a new epigenetic role for HMGA2, which follows the actions that HMGA2 initiates via the EMT-TFs, thus achieving sustained silencing of E-cadherin expression and promoting tumour cell invasion.

Reprogramming during epithelial to mesenchymal transition under the control of TGFß.

Epithelial-mesenchymal transition (EMT) refers to plastic changes in epithelial tissue architecture. Breast cancer stromal cells provide secreted molecules, such as transforming growth factor ß (TGFß), that promote EMT on tumor cells to facilitate breast cancer cell invasion, stemness and metastasis. TGFß signaling is considered to be abnormal in the context of cancer development; however, TGFß acting on breast cancer EMT resembles physiological signaling during embryonic development, when EMT generates or patterns new tissues. Interestingly, while EMT promotes metastatic fate, successful metastatic colonization seems to require the inverse process of mesenchymal-epithelial transition (MET). EMT and MET are interconnected in a time-dependent and tissue context-dependent manner and are coordinated by TGFß, other extracellular proteins, intracellular signaling cascades, non-coding RNAs and chromatin-based molecular alterations. Research on breast cancer EMT/MET aims at delivering biomolecules that can be used diagnostically in cancer pathology and possibly provide ideas for how to improve breast cancer therapy.

p53 regulates epithelial-mesenchymal transition induced by transforming growth factor ß.

Epithelial plasticity characterizes embryonic development and diseases such as cancer. Epithelial-mesenchymal transition (EMT) is a reversible and guided process of plasticity whereby embryonic or adult epithelia acquire mesenchymal properties. Multiple signaling pathways control EMT, and the transforming growth factor ß (TGFß) pathway plays a central role as its inducer. Here, we analyzed the role of the tumor suppressor protein p53 in TGFß-induced EMT in a well-established mammary epithelial cell model. We found that diploid NMuMG mammary cells bi-allelically express a wild type and a missense mutant (R277C) form of p53. Global reduction of both forms of p53 led to an enhanced EMT response to TGFß. Conversely, stabilization of wild type p53 using the compound nutlin had a negative impact on EMT. After silencing both p53 forms, rescue experiments using either wild type or R277C mutant p53 revealed that wild type p53 inhibited, whereas the R277C mutant did not significantly affect, the TGFß-driven EMT response. Under serum-free culture conditions, silencing of total p53 levels led to higher numbers of mammospheres characterized by larger size. Rescue of the silenced endogenous p53 with R277C mutant p53, in contrast, suppressed both size and numbers of the mammospheres. This work proposes that wild type p53 controls the efficiency by which mammary epithelial cells undergo EMT in response to TGFß.

Regulation of transcription factor Twist expression by the DNA architectural protein high mobility group A2 during epithelial-to-mesenchymal transition.

Deciphering molecular mechanisms that control epithelial-to-mesenchymal transition (EMT) contributes to our understanding of how tumor cells become invasive and competent for intravasation. We have established that transforming growth factor ß activates Smad proteins, which induce expression of the embryonic factor high mobility group A2 (HMGA2), which causes mesenchymal transition. HMGA2 associates with Smad complexes and induces expression of an established regulator of EMT, the zinc finger transcription factor Snail. We now show that HMGA2 can also induce expression of a second regulator of EMT, the basic helix-loop-helix transcription factor Twist. Silencing of endogenous Twist demonstrated that this protein acts in a partially redundant manner together with Snail. Double silencing of Snail and Twist reverts mesenchymal HMGA2-expressing cells to a more epithelial phenotype when compared with single silencing of Snail or Twist. Furthermore, HMGA2 can directly associate with A:T-rich sequences and promote transcription from the Twist promoter. The new evidence proposes a model whereby HMGA2 directly induces multiple transcriptional regulators of the EMT program and, thus, is a potential biomarker for carcinomas displaying EMT during progression to more advanced stages of malignancy.

PIG3: a novel link between oxidative stress and DNA damage response in cancer.

Reactive oxygen species (ROS), the most prominent free radicals produced in cells, can have both beneficial and detrimental effects on them. Many genes are known to be involved in ROS regulation. P53 inducible gene 3 (PIG3 or TP53I3) was identified in an analysis of genes induced by p53 before the onset of apoptosis. It is a widely conserved gene between many species. Until now it has been shown to exert two disparate cellular roles. The first is that of ROS producer linked to p53 induced apoptosis. In this context, it exhibits a NADPH dependent reductase activity with orthoquinones. The second is that of a component of the DNA damage response pathway. While it is considered as a p53 dependent pro-apoptotic gene, it is rarely affected in cancer. This data does not support an anti-tumor activity. In the present review we present and discuss aspects on the regulation and function of this factor and how it is implicated in cancer. We conclude by proposing that PIG3 may possibly have a role in cancer cell survival.

HMGA2 and Smads co-regulate SNAIL1 expression during induction of epithelial-to-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is important during embryonic cell layer movement and tumor cell invasiveness. EMT converts adherent epithelial cells to motile mesenchymal cells, favoring metastasis in the context of cancer progression. Transforming growth factor-beta (TGF-beta) triggers EMT via intracellular Smad transducers and other signaling proteins. We previously reported that the high mobility group A2 (HMGA2) gene is required for TGF-beta to elicit EMT in mammary epithelial cells. In the present study we investigated the molecular mechanisms by which HMGA2 induces EMT. We found that HMGA2 regulates expression of many important repressors of E-cadherin. Among these, we analyzed in detail the zinc-finger transcription factor SNAIL1, which plays key roles in tumor progression and EMT. We demonstrate that HMGA2 directly binds to the SNAIL1 promoter and acts as a transcriptional regulator of SNAIL1 expression. Furthermore, we observed that HMGA2 cooperates with the TGF-beta/Smad pathway in regulating SNAIL1 gene expression. The mechanism behind this cooperation involves physical interaction between these factors, leading to an increased binding of Smads to the SNAIL1 promoter. SNAIL1 seems to play the role of a master effector downstream of HMGA2 for induction of EMT, as SNAIL1 knock-down partially reverts HMGA2-induced loss of epithelial differentiation. The data propose that HMGA2 acts in a gene-specific manner to orchestrate the transcriptional network necessary for the EMT program.

Functional interactions between phosphatase POPX2 and mDia modulate RhoA pathways.

Rho GTPases and their downstream effectors regulate changes in the actin cytoskeleton that underlie cell motility and adhesion. They also participate, with RhoA, in the regulation of gene transcription by activating serum response factor (SRF)-mediated transcription from the serum response element (SRE). SRF-mediated transcription is also promoted by several proteins that regulate the polymerization or stability of actin. We have previously identified a family of PP2C phosphatases, POPXs, which can dephosphorylate the CDC42/RAC-activated kinase PAK and downregulate its enzymatic and actin cytoskeletal activity. We now report that POPX2 interacts with the formin protein mDia1 (DIAPH1). This interaction is enhanced when mDia1 is activated by RhoA. The binding of POPX2 to mDia1 or to an mDia-containing complex greatly decreases the ability of mDia1 to activate transcription from the SRE. We propose that the interaction between mDia1 and POPX2 (PPM1F) serves to regulate both the actin cytoskeleton and SRF-mediated transcription, and to link the CDC42/RAC1 pathways with those of RhoA.

The p21-activated kinase PAK is negatively regulated by POPX1 and POPX2, a pair of serine/threonine phosphatases of the PP2C family.

The Rho GTPases are involved in many signaling pathways and cellular functions, including the organization of the actin cytoskeleton, regulation of transcription, cell motility, and cell division. The p21 (Cdc42/Rac)-activated kinase PAK mediates a number of biological effects downstream of these Rho GTPases (reviewed by [1]). The phosphorylation state of mammalian PAK is highly regulated: upon binding of GTPases, PAK is potently activated by autophosphorylation at multiple sites, although the mechanisms of PAK downregulation are not known. We now report two PP2C-like serine/threonine phosphatases (POPX1 and POPX2) that efficiently inactivate PAK. POPX1 was isolated as a binding partner for the PAK interacting guanine nucleotide exchange factor PIX. The dephosphorylating activity of POPX correlates with an ability to block the in vivo effects of active PAK. Consonant with these effects on PAK, POPX can also inhibit actin stress fiber breakdown and morphological changes driven by active Cdc42(V12). The association of the POPX phosphatases with PAK complexes may allow PAK to cycle rapidly between active and inactive states; it represents a unique regulatory component of the signaling pathways of the PAK kinase family.