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A Transcriptional Circuit Links MAPK activation to EGR1, KLF4, SOX2, and NOTCH. (a) A regression model was fit between significantly enriched transcription factor binding motifs and differentially expressed genes from the 5 GSCs in the differentiation experiment. Dashed lines represent regulatory relationships derived from the regression model. Solid lines represent relationships taken from the literature. The color of each regulator indicates direction of gene expression or protein level change during differentiation (blue for decreased expression and red for increased expression). Downward arrows indicate regulators that have significantly enriched motifs from the set of differentially expressed genes during differentiation. (b) Heatmap of gene expression changes in key regulators.
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Glioblastoma, the most common primary malignant brain tumor, harbors a small population of tumor initiating cells (glioblastoma stem cells) that have many properties similar to neural stem cells. To investigate common regulatory networks in both neural and glioblastoma stem cells, we subjected both cell types to in-vitro differentiation conditions...
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... Core Transcriptional Circuit Links EGR1 to KLF4, NOTCH, and SOX2. Given the importance of the transcription factor KLF4 in NSC biology, we examined the resulting network of TF-Target-Gene interactions for both upstream and downstream regulators of KLF4 (Fig. 4). Upstream transcriptional activators of KLF4 pre- dicted by the model included both STAT3 (a transcription factor previously shown to control KLF4 expression in embryonic stem cells) and EGR1 (a transcription factor and so-called intermediate early gene activated down- stream of RAS/MAPK). The large decrease in EGR1 levels during GSC ...
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... of KLF4 pre- dicted by the model included both STAT3 (a transcription factor previously shown to control KLF4 expression in embryonic stem cells) and EGR1 (a transcription factor and so-called intermediate early gene activated down- stream of RAS/MAPK). The large decrease in EGR1 levels during GSC differentiation shown by both microarray (Fig. 4b) and by western blot (Fig. 2d) and the significant positive correlation between KLF4 and EGR1 gene expression levels in clinical GBM samples led us to examine the relationship between EGR1 and KLF4 protein levels. We constructed GSC827 and GSC923 cell lines stably expressing two different shRNAs directed against EGR1 and demonstrated ...
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... SOX2, DLL1, and NOTCH1 protein levels in-vitro as demonstrated by western blot (Fig. 5d). First, we demonstrated that knockdown of KLF4 by shRNA reduced the phosphorylation of ERK (MAPK) by western blot (Fig. 5c). Since upstream regulators of RAS/MAPK activation including EGFR, PDGFRA, and HRAS were down-regulated following differentiation (Fig. 4b), we explored the regulatory relationship between KLF4 and the expression of these potential target genes. Binding of KLF4 to the upstream promoters (− 1 kb, 0 kb TSS) of EGFR, HRAS, PDGFRA, and MET was confirmed by ChIP-PCR in GSC923 (Fig. 5a). However, overex- pression of KLF4 induced only the upregulation of HRAS by western blot ...
Citations
... To our knowledge, we report the first results of alternating EF stimulation in excess of 5 V/cm. We studied two human GBM cell lines utilized in prior studies: U87 and U118, and two recently validated GBM stem cell lines: GSC827, GSC923 [32]. Primary rat astrocytes were also studied as an example of normal central nervous system cells. ...
... To elucidate the effects of 200 kHz induced EMFs on brain tumor transcriptomic profiles, we performed RNA-Seq on cultures of the GSC827 and GSC923 cell lines, which were originated from the Neuro-Oncology Branch [32], rather than traditional model cell lines; GSC827 was derived from a 60-year-old male GBM patient and GSC923 was from a 56-yearold female GBM patient. These cell lines better represent the heterogeneity that may be found amongst GBM patients. ...
... For primary rat astrocytes, no effect on cell growth was observed. These results complement the U87 and U118 results by providing analogous findings for normal central nervous system cells (astrocytes) and for well-validated stem cell lines which better mimic the behavior of GBM in vivo [32]. ...
Previous studies reported that alternating electric fields (EFs) in the intermediate frequency (100-300 kHz) and low intensity (1-3 V/cm) regime - termed “Tumor Treating Fields” (TTFields) - have a specific, anti-proliferative effect on glioblastoma multiforme (GBM) cells. However, the mechanism(s) of action remain(s) incompletely understood, hindering the clinical adoption of treatments based on TTFields. To advance the study of such treatment in vitro, we developed an inductive device to deliver EFs to cell cultures which improves thermal and osmolar regulation compared to prior devices. Using this inductive device, we applied continuous, 200 kHz electromagnetic fields (EMFs) with a radial EF amplitude profile spanning 0-6.5 V/cm to cultures of primary rat astrocytes and several human GBM cell lines - U87, U118, GSC827, and GSC923 - for a duration of 72 hours. Cell density was assessed via segmented pixel densities from GFP expression (U87, U118) or from staining (astrocytes, GSC827, GSC923). Further RNA-Seq analyses were performed on GSC827 and GSC923 cells. Treated cultures of all cell lines exhibited little to no change in proliferation at lower EF amplitudes (0-3 V/cm). At higher amplitudes (> 4 V/cm), different effects were observed. Apparent cell densities increased (U87), decreased (GSC827, GSC923), or showed little change (U118, astrocytes). RNA-Seq analyses on treated and untreated GSC827 and GSC923 cells revealed differentially expressed gene sets of interest, such as those related to cell cycle control. Up- and down-regulation, however, was not consistent across cell lines nor EF amplitudes. Our results indicate no consistent, anti-proliferative effect of 200 kHz EMFs across GBM cell lines and thus contradict previous in vitro findings. Rather, effects varied across different cell lines and EF amplitude regimes, highlighting the need to assess the effect(s) of TTFields and similar treatments on a per cell line basis.
... As a result, TTFields may, in 34 principle, have direct biophysical effect(s) on the interior of GBM cells. Kirson et 35 al. [20] initially proposed two effects that were deemed specifically anti-mitotic: (1) 36 TTFields may affect the dipole alignment of charged molecules, namely tubulin and 37 septin dimers, and (2) TTFields may result in non-uniform EFs (i.e., EF gradients) 38 in the cleavage furrow of the dividing cell. The first mechanism would disrupt 39 mitotic spindle formation and possibly the localization of the septin complex to 40 the anaphase spindle midline. ...
... To our knowledge, we report 129 the first results of alternating EF stimulation in excess of 5 V/cm. We studied two 130 human GBM cell lines utilized in prior studies: U87 and U118, and two recently 131 validated GBM stem cell lines: GSC827, GSC923 [36]. Primary rat astrocytes were 132 also studied as an example of normal central nervous system cells. ...
... To elucidate the effects of 200 kHz induced EMFs on brain tumor transcriptomic 159 profiles, we performed RNA-Seq on cultures of the GSC827 and GSC923 cell lines, 160 which were originated from the Neuro-Oncology Branch [36], rather than traditional 161 model cell lines; GSC827 was derived from a 60-year-old male GBM patient and 162 GSC923 was from a 56-year-old female GBM patient. These cell lines better represent 163 the heterogeneity that may be found amongst GBM patients. ...
Previous studies reported that alternating electric fields (EFs) in the intermediate frequency (100 - 300 kHz) and low intensity (1-3 V/cm) regime - termed ``Tumor Treating Fields'' (TTFields) - have a specific, anti-proliferative effect on glioblastoma multiforme (GBM) cells. However, the mechanism(s) of action remain(s) incompletely understood, hindering the clinical adoption of treatments based on TTFields. To advance the study of such treatment in vitro, we developed an inductive device to deliver EFs to cell cultures which improves thermal and osmolar regulation compared to prior devices. Using this inductive device, we applied continuous, 200 kHz electromagnetic fields (EMFs) with a radial EF amplitude profile spanning 0 - 6.5 V/cm to cultures of primary rat astrocytes and several human GBM cell lines - U87, U118, GSC827, and GSC923 - for a duration of 72 hours. Cell density was assessed via segmented pixel densities from GFP expression (U87, U118) or from staining (astrocytes, GSC827, GSC923). Further RNA-Seq analyses were performed on GSC827 and GSC923 cells. Treated cultures of all cell lines exhibited little to no change in proliferation at lower EF amplitudes (0 - 3 V/cm). At higher amplitudes (> 4 V/cm), different effects were observed. Apparent cell densities increased (U87), decreased (GSC827, GSC923), or showed little change (U118, astrocytes). RNA-Seq analyses on treated and untreated GSC827 and GSC923 cells revealed differentially expressed gene sets of interest, such as those related to cell cycle control. Up- and down-regulation, however, was not consistent across cell lines nor EF amplitudes. Our results indicate no consistent, anti-proliferative effect of 200 kHz EMFs across GBM cell lines and thus contradict previous in vitro findings. Rather, effects varied across different cell lines and EF amplitude regimes, highlighting the need to assess the effect(s) of TTFields and similar treatments on a per cell line basis.
... Paediatric cancers exhibit few mutations genome-wide, but typically harbour sentinel mutations that alter TF proteins [2][3][4][5] . Mutant TFs can hijack wild-type lineage-specific TFs into self-reinforcing, feed-forward core regulatory circuits (CRCs) [6][7][8][9][10][11][12][13][14] . For example, MYCN in MYCN-amplified neuroblastoma and the PAX3-FOXO1 and PAX7-FOXO1 fusion proteins in alveolar rhabdomyosarcoma promote tumour growth by hijacking tumour-type-specific CRC TFs [15][16][17][18][19] . ...
Transcription factors (TFs) are frequently mutated in cancer. Paediatric cancers exhibit few mutations genome-wide but frequently harbour sentinel mutations that affect TFs, which provides a context to precisely study the transcriptional circuits that support mutant TF-driven oncogenesis. A broadly relevant mechanism that has garnered intense focus involves the ability of mutant TFs to hijack wild-type lineage-specific TFs in self-reinforcing transcriptional circuits. However, it is not known whether this specific type of circuitry is equally crucial in all mutant TF-driven cancers. Here we describe an alternative yet central transcriptional mechanism that promotes Ewing sarcoma, wherein constraint, rather than reinforcement, of the activity of the fusion TF EWS–FLI supports cancer growth. We discover that ETV6 is a crucial TF dependency that is specific to this disease because it, counter-intuitively, represses the transcriptional output of EWS–FLI. This work discovers a previously undescribed transcriptional mechanism that promotes cancer.
... Similarly, knockdown experiments in mice uncovered Egr3 as an inhibitor of HSC proliferation . Besides, EGR1 was associated with proliferation and stem cell identity of glioblastoma stem cells (Riddick et al., 2017;Sakakini et al., 2016). In T-cells, Egr2 and Egr3 act as repressors of T-cell activation through upregulation of Fas ligand Ashwell, 1999, 1998) and the E3 ubiquitin ligase Cbl-b which subsequently ubiquitinates factors involved in T-cell receptor signaling including PI3KR1 (Safford et al., 2005). ...
KMT2A-rearrangements are causative for 70-80% all infant acute lymphoblastic leukemias (Pieters et al., 2019, 2007). Among these, the translocation t(4;11)(q21;23) generating the oncogenic fusion genes KMT2A::AFF1 and AFF1::KMT2A is the most frequent one, accounting for almost every second case of KMT2A-r infant ALL (Meyer et al., 2018). Despite passing a multimodal chemotherapy, 64% of patients achieve an event including relapse or death within four years from diagnosis, and overall survival three years from relapse remains poor with only 17% (Driessen et al., 2016; Pieters et al., 2019, 2007). Various studies have shown that relapse and therapy resistance were not mediated by chemotherapy-induced mutagenesis as there was no accumulation of secondary mutations in the dominant leukemic clone between diagnosis and relapse (Agraz-Doblas et al., 2019; Andersson et al., 2015; Bardini et al., 2011; Dobbins et al., 2013; Driessen et al., 2013; Mullighan et al., 2007).
Intriguingly, exclusively infant t(4;11) ALL patients were reported to subdivide in two groups depending on the level of HOXA gene cluster expression (Trentin et al., 2009). The HOXA-lo group displayed a high expression of IRX1 and the HOXA-hi group a low expression of IRX1 (Symeonidou and Ottersbach, 2021; Trentin et al., 2009). Importantly, the HOXA-lo/IRX1-hi group was characterized to possess a strongly elevated relapse incidence compared to the HOXA-hi/IRX1-lo group (Kang et al., 2012; Stam et al., 2010). IRX1 was identified to upregulate the Early growth response genes EGR1, EGR2 and EGR3 (Kühn et al., 2016).
The doctoral project “EGR-mediated relapse mechanisms in infant t(4;11) acute lymphoblastic leukemia” aimed to investigate a potential correlation between the HOXA-lo-IRX1-EGR axis and relapse development in infant t(4;11) ALL. The primary objective was to clarify through which molecular mechanism(s) relapse development despite continuous chemotherapy could be achieved. In this context, the role of the EGR genes has been investigated. In addition, this project aimed to disclose molecular targets which could offer novel therapeutic interventions to interfere with therapy resistance and relapse formation.
... For example, KLF5 has been proven to be collaborated with ELF3, GATA6 and EHF in EAC (46), while cooperated with TP63 and SOX2 in ESCC to form CRC (12), respectively ( Figure 2C). And SOX2 has also been identified co-regulated with KLF4, EGR1 and NOTCH1 in Glioblastoma (48). These cancer-type and -subtype specific CRCs driven by SEs orchestrate the oncogenic transcriptional addiction, while offers therapeutic vulnerabilities due to perturbation sensitivity of their own SEs. ...
The tumorigenesis of esophageal carcinoma arises from transcriptional dysregulation would become exceptionally dependent on specific regulators of gene expression, which could be preferentially attributed to the larger non-coding cis-regulatory elements, i.e. super-enhancers (SEs). SEs, large genomic regulatory entity in close genomic proximity, are underpinned by control cancer cell identity. As a consequence, the transcriptional addictions driven by SEs could offer an Achilles’ heel for molecular treatments on patients of esophageal carcinoma and other types of cancer as well. In this review, we summarize the recent findings about the oncogenic SEs upon which esophageal cancer cells depend, and discuss why SEs could be seen as the hallmark of cancer, how transcriptional dependencies driven by SEs, and what opportunities could be supplied based on this cancer-specific SEs.
... For example, e2f1 expression induction might be responsible for the increased expression of klf4 and egr1 (Riverso et al., 2017;Zhang et al., 2014;Zheng et al., 2009), but not foxj1 in the pituitary of the vgll3*EE genotype. Moreover, it has been shown that egr1 can act directly upstream of klf4 and induces its expression (Lai et al., 2012;Riddick et al., 2017). On the other hand, the similar expression patterns of the four identified TFs here could also indicate cooperative interactions among them in transcriptional regulation of jun and the other downstream coexpressed genes. ...
Age at maturity is a key life history trait and a significant contributor to life history strategy variation. The maturation process is influenced by genetic and environmental factors, but specific causes of variation in maturation timing remain elusive. In many species, the increase in the regulatory gonadotropin-releasing hormone 1 (GnRH1) marks the onset of puberty. Atlantic salmon, however, lacks gnrh1 gene, suggesting gnrh3 and/or other regulatory factors are involved in the maturation process. Earlier research in Atlantic salmon has found a strong association between alternative alleles of vgll3 and maturation timing. Recently we reported strong induction of gonadotropin genes (fshb and lhb) in the pituitary of Atlantic salmon homozygous for the early maturation allele (E) of vgll3. The induction of gonadotropins was accompanied by increased expression of their direct upstream regulators, c-jun and sf1 (nr5a1b) but the regulatory connection between vgll3 and these regulators has never been investigated in any organism. In this study, we investigated the potential regulatory connection between vgll3 genotypes and these regulators through a stepwise approach of identifying a gene regulatory network (GRN) containing c-jun and sf1, and transcription factor motif enrichment analysis. We found a GRN containing c-jun with predicted upstream regulators, e2f1, egr1, foxj1 and klf4, to be differentially expressed in the pituitary. Finally, we suggest a vgll3 and Hippo pathway -dependent model for transcriptional regulation of c-jun and sf1 in the pituitary, which may have broader implications across vertebrates.
... Based on the molecular signatures observed in our study, this link could be relevant for EGR1 in melanoma, being further induced by 3D culturing in 0.6% (w/v) alginate and therefore enhancing the number of quiescent, stem cell-like cells. In glioma, EGR1 has been linked to the self-renewal of brain tumor-initiating cells [89][90][91], indicating an implication in the maintenance of niche populations. Due to the same embryonic origin of the neural crest, this role could be also assumed for melanoma supported by the data of this study. ...
Alginate hydrogels have been used as a biomaterial for 3D culturing for several years. Here, gene expression patterns in melanoma cells cultivated in 3D alginate are compared to 2D cultures. It is well-known that 2D cell culture is not resembling the complex in vivo situation well. However, the use of very intricate 3D models does not allow performing high-throughput screening and analysis is highly complex. 3D cell culture strategies in hydrogels will better mimic the in vivo situation while they maintain feasibility for large-scale analysis. As alginate is an easy-to-use material and due to its favorable properties, it is commonly applied as a bioink component in the growing field of cell encapsulation and biofabrication. Yet, only a little information about the transcriptome in 3D cultures in hydrogels like alginate is available. In this study, changes in the transcriptome based on RNA-Seq data by cultivating melanoma cells in 3D alginate are analyzed and reveal marked changes compared to cells cultured on usual 2D tissue culture plastic. Deregulated genes represent valuable cues to signaling pathways and molecules affected by the culture method. Using this as a model system for tumor cell plasticity and heterogeneity, EGR1 is determined to play an important role in melanoma progression.
... Specifically, KLF5 cooperates with ELF3, GATA6 and EHF in the EAC subtype CRC program [7], while in ESCC, KLF5 forms a CRC with TP63 and SOX2 [11]. Similarly, SOX2-containing CRCs have been wellcharacterized in ESCs [5,6] (together with NANOG and OCT4), ESCC [11] (with TP63 and KLF5) and Glioblastoma [65] (with KLF4, EGR1 and NOTCH1). These observations suggest that the same TFs may be involved in different CRC complex by collaboration and cooperation with different master TFs to regulate cell-type-specific transcriptional programs. ...
Super-enhancers (SEs) are congregated enhancer clusters with high level of loading of transcription factors (TFs), cofactors and epigenetic modifications. Through direct co-occupancy at their own SEs as well as each other’s, a small set of so called “master” TFs form interconnected core regulatory circuitry (CRCs) to orchestrate transcriptional programs in both normal and malignant cells. These master TFs can be predicted mathematically using epigenomic methods. In this Review, we summarize the identification of SEs and CRCs in cancer cells, the mechanisms by which master TFs and SEs cooperatively regulate cancer-type-specific expression programs, and the cancer-type- and subtype-specificity of CRC and the significance in cancer biology.
... 56,57 Likewise, genomic alterations inducing constitutive activation of MAPK have been linked to master regulators of stemness in glioblastoma. 58 Interestingly, also FAK inhibition has been established in vitro as a strategy to approach glioblastoma stem-like cells. 59 Harnessing the potential of miRNAs, such as miR-216, could have a broader impact than conventional small molecule inhibitors. ...
Glioblastoma is the most prevalent and aggressive brain cancer. With a median overall survival of ~15–20 months under standard therapy, novel treatment approaches are desperately needed. A recent phase II clinical trial with a personalized immunotherapy based on tumor lysate-charged dendritic cell (DC) vaccination, however, failed to prolong survival. Here, we investigated tumor tissue from trial patients to explore glioblastoma survival-related factors. We followed an innovative approach of combining mass spectrometry-based quantitative proteomics (n = 36) with microRNA sequencing plus RT-qPCR (n = 38). Protein quantification identified, e.g., huntingtin interacting protein 1 (HIP1), retinol-binding protein 1 (RBP1), ferritin heavy chain (FTH1) and focal adhesion kinase 2 (FAK2) as factor candidates correlated with a dismal prognosis. MicroRNA analysis identified miR-216b, miR-216a, miR-708 and let-7i as molecules potentially associated with favorable tissue characteristics as they were enriched in patients with a comparably longer survival. To illustrate the utility of integrated miRNomics and proteomics findings, focal adhesion was studied further as one example for a pathway of potential general interest.
Taken together, we here mapped possible drivers of glioblastoma outcome under immunotherapy in one of the largest DC vaccination tissue analysis cohorts so far—demonstrating usefulness and feasibility of combined proteomics/miRNomics approaches. Future research should investigate agents that sensitize glioblastoma to (immuno)therapy—potentially building on insights generated here.
... Based on findings from biologically verified CRCs, core TFs are vital for cell identity and transcriptional homeostasis under both physiological and pathological conditions. Many other studies have also implemented CRC analysis in uncovering critical transcriptional programs in various cancer types (Table 1) [27,30,[79][80][81][82][83][84]. For instance, by analyzing publically available ChIP-seq data from the ENCODE project [85], Fournier et al. modeled core TF connectivity including ESR1, FOXA1, FOSL2, and JUND in MCF7 breast cancer cells; HNF4A, FOXA1, FOXA2, and CEBPB in HEPG2 liver cancer cells; as well as FOXA1, FOXA2, FOSL2, JUND, and ATF3 in A549 lung carcinoma cells [83]. ...
Transcription factors (TFs) coordinate the on-and-off states of gene expression typically in a combinatorial fashion. Studies from embryonic stem cells and other cell types have revealed that a clique of self-regulated core TFs control cell identity and cell state. These core TFs form interconnected feed-forward transcriptional loops to establish and reinforce the cell-type-specific gene-expression program; the ensemble of core TFs and their regulatory loops constitutes core transcriptional regulatory circuitry (CRC). Here, we summarize recent progress in computational reconstitution and biologic exploration of CRCs across various human malignancies, and consolidate the strategy and methodology for CRC discovery. We also discuss the genetic basis and therapeutic vulnerability of CRC, and highlight new frontiers and future efforts for the study of CRC in cancer. Knowledge of CRC in cancer is fundamental to understanding cancer-specific transcriptional addiction, and should provide important insight to both pathobiology and therapeutics.
