PRMT6 methylation of RCC1 regulates mitosis, tumorigenicity, and radiation response of glioblastoma stem cells.

Aberrant cell proliferation is a hallmark of cancer, including glioblastoma (GBM). Here we report that protein arginine methyltransferase (PRMT) 6 activity is required for the proliferation, stem-like properties, and tumorigenicity of glioblastoma stem cells (GSCs), a subpopulation in GBM critical for malignancy. We identified a casein kinase 2 (CK2)-PRMT6-regulator of chromatin condensation 1 (RCC1) signaling axis whose activity is an important contributor to the stem-like properties and tumor biology of GSCs. CK2 phosphorylates and stabilizes PRMT6 through deubiquitylation, which promotes PRMT6 methylation of RCC1, which in turn is required for RCC1 association with chromatin and activation of RAN. Disruption of this pathway results in defects in mitosis. EPZ020411, a specific small-molecule inhibitor for PRMT6, suppresses RCC1 arginine methylation and improves the cytotoxic activity of radiotherapy against GSC brain tumor xenografts. This study identifies a CK2α-PRMT6-RCC1 signaling axis that can be therapeutically targeted in the treatment of GBM.

Stem cell programs in cancer initiation, progression, and therapy resistance.

Over the past few decades, substantial evidence has convincingly revealed the existence of cancer stem cells (CSCs) as a minor subpopulation in cancers, contributing to an aberrantly high degree of cellular heterogeneity within the tumor. CSCs are functionally defined by their abilities of self-renewal and differentiation, often in response to cues from their microenvironment. Biological phenotypes of CSCs are regulated by the integrated transcriptional, post-transcriptional, metabolic, and epigenetic regulatory networks. CSCs contribute to tumor progression, therapeutic resistance, and disease recurrence through their sustained proliferation, invasion into normal tissue, promotion of angiogenesis, evasion of the immune system, and resistance to conventional anticancer therapies. Therefore, elucidation of the molecular mechanisms that drive cancer stem cell maintenance, plasticity, and therapeutic resistance will enhance our ability to improve the effectiveness of targeted therapies for CSCs. In this review, we highlight the key features and mechanisms that regulate CSC function in tumor initiation, progression, and therapy resistance. We discuss factors for CSC therapeutic resistance, such as quiescence, induction of epithelial-to-mesenchymal transition (EMT), and resistance to DNA damage-induced cell death. We evaluate therapeutic approaches for eliminating therapy-resistant CSC subpopulations, including anticancer drugs that target key CSC signaling pathways and cell surface markers, viral therapies, the awakening of quiescent CSCs, and immunotherapy. We also assess the impact of new technologies, such as single-cell sequencing and CRISPR-Cas9 screening, on the investigation of the biological properties of CSCs. Moreover, challenges remain to be addressed in the coming years, including experimental approaches for investigating CSCs and obstacles in therapeutic targeting of CSCs.

LY6K promotes glioblastoma tumorigenicity via CAV-1-mediated ERK1/2 signaling enhancement.

BACKGROUND: Lymphocyte antigen 6 complex, locus K (LY6K) is a putative oncogene in various cancers. Elevated expression of LY6K is correlated with poor patient prognosis in glioblastoma (GBM). The aim of this study is to advance our understanding of the mechanism by which LY6K contributes to GBM tumor biology. METHODS: Bioinformatic data mining was used to investigate LY6K expression in relation to GBM clinical outcome. To understand the role of LY6K in GBM, we utilized patient-derived glioma stemlike cells (GSCs) and U87 cells and employed immunoblotting, immunofluorescent staining, radiation treatment, and orthotopic GBM xenograft models. RESULTS: Our results show that increased expression of LY6K inversely correlates with GBM patient survival. LY6K promotes tumorigenicity in GBM cells both in vitro and in vivo. The mechanism underlying this tumorigenic behavior is enhancement of extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling. Interestingly, we observed that tumor-promoting LY6K-ERK1/2 signaling is mediated by the interaction of LY6K with caveolin-1, rather than through oncogenic receptor tyrosine kinase-mediated signaling. Moreover, association of LY6K with the cell membrane is crucial for its tumorigenic functions. Finally, DNA methylation maintains LY6K silencing, and hypomethylation of the LY6K promoter increases its expression. In GSCs, ionizing radiation leads to demethylation of the LY6K promoter, thereby increasing LY6K expression and GSC resistance to radiation. CONCLUSIONS: Our study highlights the importance of the contribution of LY6K to GBM tumor biology and suggests LY6K as a potential membrane target for treating GBM.

SRSF3-Regulated RNA Alternative Splicing Promotes Glioblastoma Tumorigenicity by Affecting Multiple Cellular Processes.

Misregulated alternative RNA splicing (AS) contributes to the tumorigenesis and progression of human cancers, including glioblastoma (GBM). Here, we showed that a major splicing factor, serine and arginine rich splicing factor 3 (SRSF3), was frequently upregulated in clinical glioma specimens and that elevated SRSF3 was associated with tumor progression and a poor prognosis for patients with glioma. In patient-derived glioma stem-like cells (GSC), SRSF3 expression promoted cell proliferation, self-renewal, and tumorigenesis. Transcriptomic profiling identified more than 1,000 SRSF3-affected AS events, with a preference for exon skipping in genes involved with cell mitosis. Motif analysis identified the sequence of CA(G/C/A)CC(C/A) as a potential exonic splicing enhancer for these SRSF3-regulated exons. To evaluate the biological impact of SRSF3-affected AS events, four candidates were selected whose AS correlated with SRSF3 expression in glioma tissues, and their splicing pattern was modified using a CRISPR/Cas9 approach. Two functionally validated AS candidates were further investigated for the mechanisms underlying their isoform-specific functions. Specifically, following knockout of SRSF3, transcription factor ETS variant 1 (ETV1) gene showed exon skipping at exon 7, while nudE neurodevelopment protein 1 (NDE1) gene showed replacement of terminal exon 9 with a mutually exclusive exon 9'. SRSF3-regulated AS of these two genes markedly increased their oncogenic activity in GSCs. Taken together, our data demonstrate that SRSF3 is a key regulator of AS in GBM and that understanding mechanisms of misregulated AS could provide critical insights for developing effective therapeutic strategies against GBMs. SIGNIFICANCE: SRSF3 is a significant regulator of glioma-associated alternative splicing, implicating SRSF3 as an oncogenic factor that contributes to the tumor biology of GBM.

MIR93 (microRNA -93) regulates tumorigenicity and therapy response of glioblastoma by targeting autophagy.

Macroautophagy/autophagy is a natural intracellular process that maintains cellular homeostasis and protects cells from death under stress conditions. Autophagy sustains tumor survival and growth when induced by common cancer treatments, including IR and cytotoxic chemotherapy, thereby contributing to therapeutic resistance of tumors. In this study, we report that the expression of MIR93, noted in two clinically relevant tumor subtypes of GBM, influenced GSC phenotype as well as tumor response to therapy through its effects on autophagy. Our mechanistic studies revealed that MIR93 regulated autophagic activities in GSCs through simultaneous inhibition of multiple autophagy regulators, including BECN1/Beclin 1, ATG5, ATG4B, and SQSTM1/p62. Moreover, two first-line treatments for GBM, IR and temozolomide (TMZ), as well as rapamycin (Rap), the prototypic MTOR inhibitor, decreased MIR93 expression that, in turn, stimulated autophagic processes in GSCs. Inhibition of autophagy by ectopic MIR93 expression, or via autophagy inhibitors NSC (an ATG4B inhibitor) and CQ, enhanced the activity of IR and TMZ against GSCs. Collectively, our findings reveal a key role for MIR93 in the regulation of autophagy and suggest a combination treatment strategy involving the inhibition of autophagy while administering cytotoxic therapy. Abbreviations: ACTB: actin beta; ATG4B: autophagy related 4B cysteine peptidase; ATG5: autophagy related 5; BECN1: beclin 1; CL: classical; CQ: chloroquine diphosphate; CSCs: cancer stem cells; GBM: glioblastoma; GSCs: glioma stem-like cells; HEK: human embryonic kidney; IB: immunoblotting; IF: immunofluorescent staining; IR: irradiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MES: mesenchymal; MIR93: microRNA 93; MIRC: a control miRNA; miRNA/miR: microRNA; MTOR: mechanistic target of rapamycin kinase; NSC: NSC185085; PN: proneural; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; Rap: rapamycin; SQSTM1/p62: sequestosome 1; TCGA: the cancer genome atlas; TMZ: temozolomide; WT: wild type; ZIP93: lentiviral miRZIP targeting MIR93; ZIPC: lentiviral miRZip targeting control miRNA.

Autophagy and Hallmarks of Cancer.

Autophagy is a catabolic program that is responsible for the degradation of dysfunctional or unnecessary proteins and organelles to maintain cellular homeostasis. Mechanistically, it involves the formation of double-membrane autophagosomes that sequester cytoplasmic material and deliver it to lysosomes for degradation. Eventually, the material is recycled back to the cytoplasm. Abnormalities of autophagy often lead to human diseases, such as neurodegeneration and cancer. In the case of cancer, increasing evidence has revealed the paradoxical roles of autophagy in both tumor inhibition and tumor promotion. Here, we summarize the context-dependent role of autophagy and its complicated molecular mechanisms in the hallmarks of cancer. Moreover, we discuss how therapeutics targeting autophagy can counter malignant transformation and tumor progression. Overall, the findings of studies discussed here shed new light on exploiting the complicated mechanisms of the autophagic machinery and relevant small-molecule modulators as potential antitumor agents to improve therapeutic outcomes.

Genome-wide methylomic and transcriptomic analyses identify subtype-specific epigenetic signatures commonly dysregulated in glioma stem cells and glioblastoma.

Glioma stem cells (GSCs), a subpopulation of tumor cells, contribute to tumor heterogeneity and therapy resistance. Gene expression profiling classified glioblastoma (GBM) and GSCs into four transcriptomically-defined subtypes. Here, we determined the DNA methylation signatures in transcriptomically pre-classified GSC and GBM bulk tumors subtypes. We hypothesized that these DNA methylation signatures correlate with gene expression and are uniquely associated either with only GSCs or only GBM bulk tumors. Additional methylation signatures may be commonly associated with both GSCs and GBM bulk tumors, i.e., common to non-stem-like and stem-like tumor cell populations and correlating with the clinical prognosis of glioma patients. We analyzed Illumina 450K methylation array and expression data from a panel of 23 patient-derived GSCs. We referenced these results with The Cancer Genome Atlas (TCGA) GBM datasets to generate methylomic and transcriptomic signatures for GSCs and GBM bulk tumors of each transcriptomically pre-defined tumor subtype. Survival analyses were carried out for these signature genes using publicly available datasets, including from TCGA. We report that DNA methylation signatures in proneural and mesenchymal tumor subtypes are either unique to GSCs, unique to GBM bulk tumors, or common to both. Further, dysregulated DNA methylation correlates with gene expression and clinical prognoses. Additionally, many previously identified transcriptionally-regulated markers are also dysregulated due to DNA methylation. The subtype-specific DNA methylation signatures described in this study could be useful for refining GBM sub-classification, improving prognostic accuracy, and making therapeutic decisions.

MST4 Phosphorylation of ATG4B Regulates Autophagic Activity, Tumorigenicity, and Radioresistance in Glioblastoma.

ATG4B stimulates autophagy by promoting autophagosome formation through reversible modification of ATG8. We identify ATG4B as a substrate of mammalian sterile20-like kinase (STK) 26/MST4. MST4 phosphorylates ATG4B at serine residue 383, which stimulates ATG4B activity and increases autophagic flux. Inhibition of MST4 or ATG4B activities using genetic approaches or an inhibitor of ATG4B suppresses autophagy and the tumorigenicity of glioblastoma (GBM) cells. Furthermore, radiation induces MST4 expression, ATG4B phosphorylation, and autophagy. Inhibiting ATG4B in combination with radiotherapy in treating mice with intracranial GBM xenograft markedly slows tumor growth and provides a significant survival benefit. Our work describes an MST4-ATG4B signaling axis that influences GBM autophagy and malignancy, and whose therapeutic targeting enhances the anti-tumor effects of radiotherapy.

Synaptic vesicle glycoprotein 2C (SV2C) modulates dopamine release and is disrupted in Parkinson disease.

Members of the synaptic vesicle glycoprotein 2 (SV2) family of proteins are involved in synaptic function throughout the brain. The ubiquitously expressed SV2A has been widely implicated in epilepsy, although SV2C with its restricted basal ganglia distribution is poorly characterized. SV2C is emerging as a potentially relevant protein in Parkinson disease (PD), because it is a genetic modifier of sensitivity to l-DOPA and of nicotine neuroprotection in PD. Here we identify SV2C as a mediator of dopamine homeostasis and report that disrupted expression of SV2C within the basal ganglia is a pathological feature of PD. Genetic deletion of SV2C leads to reduced dopamine release in the dorsal striatum as measured by fast-scan cyclic voltammetry, reduced striatal dopamine content, disrupted α-synuclein expression, deficits in motor function, and alterations in neurochemical effects of nicotine. Furthermore, SV2C expression is dramatically altered in postmortem brain tissue from PD cases but not in Alzheimer disease, progressive supranuclear palsy, or multiple system atrophy. This disruption was paralleled in mice overexpressing mutated α-synuclein. These data establish SV2C as a mediator of dopamine neuron function and suggest that SV2C disruption is a unique feature of PD that likely contributes to dopaminergic dysfunction.

NEDD4-mediated HSF1 degradation underlies α-synucleinopathy.

Cellular protein homeostasis is achieved by a delicate network of molecular chaperones and various proteolytic processes such as ubiquitin-proteasome system (UPS) to avoid a build-up of misfolded protein aggregates. The latter is a common denominator of neurodegeneration. Neurons are found to be particularly vulnerable to toxic stress from aggregation-prone proteins such as α-synuclein. Induction of heat-shock proteins (HSPs), such as through activated heat shock transcription factor 1 (HSF1) via Hsp90 inhibition, is being investigated as a therapeutic option for proteinopathic diseases. HSF1 is a master stress-protective transcription factor which activates genes encoding protein chaperones (e.g. iHsp70) and anti-apoptotic proteins. However, whether and how HSF1 is dysregulated during neurodegeneration has not been studied. Here, we discover aberrant HSF1 degradation by aggregated α-synuclein (or α-synuclein-induced proteotoxic stress) in transfected neuroblastoma cells. HSF1 dysregulation via α-synuclein was confirmed by in vivo assessment of mouse and in situ studies of human specimens with α-synucleinopathy. We demonstrate that elevated NEDD4 is implicated as the responsible ubiquitin E3 ligase for HSF1 degradation through UPS. Furthermore, pharmacologically induced SIRT1-mediated deacetylation can attenuate aberrant NEDD4-mediated HSF1 degradation. Indeed, we define the acetylation status of the Lys 80 residue located in the DNA-binding domain of HSF1 as a critical factor in modulating HSF1 protein stability in addition to its previously identified role in the transcriptional activity. Together with the finding that preserving HSF1 can alleviate α-synuclein toxicity, this study strongly suggests that aberrant HSF1 degradation is a key neurodegenerative mechanism underlying α-synucleinopathy.

No apparent transmission of transgenic α-synuclein into nigrostriatal dopaminergic neurons in multiple mouse models.

BACKGROUND: α-synuclein (α-syn) is the main component of intracytoplasmic inclusions deposited in the brains of patients with Parkinson's disease (PD) and certain other neurodegenerative disorders. Recent studies have explored the ability of α-syn to propagate between or across neighboring neurons and supposedly "infect" them with a prion-like mechanism. However, much of this research has used stereotaxic injections of heterologous α-syn fibrils to induce the spreading of inclusions in the rodent brains. Whether α-syn is able to transmit from the host cells to their neighboring cells in vivo is unclear. METHODS: Using immunestaining, we examined the potential propagation of α-syn into nigrostriatal dopaminergic (DA) neurons in three lines of transgenic mice that overexpress human wild-type α-syn (hα-syn) in different neuron populations. RESULTS: After testing for three different routes by which hα-syn propagation might occur, we were unable to find any evidence that hα-syn behaved like a prion and could be transmitted overtime into the DA neurons initially lack of hα-syn expression. CONCLUSIONS: In transgenic mice hα-syn does not have the ability to propagate at pathologically significant levels between or across neurons. It must be noted that these observations do not disprove the studies that show its prion-like qualities, but rather that propagation is not detectable in transgenic models that do not use any injections of heterologous proteins or viral vectors to induce a spreading state.

Selective expression of Parkinson's disease-related Leucine-rich repeat kinase 2 G2019S missense mutation in midbrain dopaminergic neurons impairs dopamine release and dopaminergic gene expression.

Preferential dysfunction/degeneration of midbrain substantia nigra pars compacta (SNpc) dopaminergic (DA) neurons contributes to the main movement symptoms manifested in Parkinson's disease (PD). Although the Leucine-rich repeat kinase 2 (LRRK2) G2019S missense mutation (LRRK2 G2019S) is the most common causative genetic factor linked to PD, the effects of LRRK2 G2019S on the function and survival of SNpc DA neurons are poorly understood. Using a binary gene expression system, we generated transgenic mice expressing either wild-type human LRRK2 (WT mice) or the LRRK2 G2019S mutation (G2019S mice) selectively in the midbrain DA neurons. Here we show that overexpression of LRRK2 G2019S did not induce overt motor abnormalities or substantial SNpc DA neuron loss. However, the LRRK2 G2019S mutation impaired dopamine homeostasis and release in aged mice. This reduction in dopamine content/release coincided with the degeneration of DA axon terminals and decreased expression of DA neuron-enriched genes tyrosine hydroxylase (TH), vesicular monoamine transporter 2, dopamine transporter and aldehyde dehydrogenase 1. These factors are responsible for dopamine synthesis, transport and degradation, and their expression is regulated by transcription factor paired-like homeodomain 3 (PITX3). Levels of Pitx3 mRNA and protein were similarly decreased in the SNpc DA neurons of aged G2019S mice. Together, these findings suggest that PITX3-dependent transcription regulation could be one of the many potential mechanisms by which LRRK2 G2019S acts in SNpc DA neurons, resulting in downregulation of its downstream target genes critical for dopamine homeostasis and release.

Aldehyde dehydrogenase 1 defines and protects a nigrostriatal dopaminergic neuron subpopulation.

Subpopulations of dopaminergic (DA) neurons within the substantia nigra pars compacta (SNpc) display a differential vulnerability to loss in Parkinson's disease (PD); however, it is not clear why these subsets are preferentially selected in PD-associated neurodegeneration. In rodent SNpc, DA neurons can be divided into two subpopulations based on the expression of aldehyde dehydrogenase 1 (ALDH1A1). Here, we have shown that, in α-synuclein transgenic mice, a murine model of PD-related disease, DA neurodegeneration occurs mainly in a dorsomedial ALDH1A1-negative subpopulation that is also prone to cytotoxic aggregation of α-synuclein. Notably, the topographic ALDH1A1 pattern observed in α-synuclein transgenic mice was conserved in human SNpc. Postmortem evaluation of brains of patients with PD revealed a severe reduction of ALDH1A1 expression and neurodegeneration in the ventral ALDH1A1-positive DA subpopulations. ALDH1A1 expression was also suppressed in α-synuclein transgenic mice. Deletion of Aldh1a1 exacerbated α-synuclein-mediated DA neurodegeneration and α-synuclein aggregation, whereas Aldh1a1-null and control DA neurons were comparably susceptible to 1-methyl-4-phenylpyridinium-, glutamate-, or camptothecin-induced cell death. ALDH1A1 overexpression appeared to preferentially protect against α-synuclein-mediated DA neurodegeneration but did not rescue α-synuclein-induced loss of cortical neurons. Together, our findings suggest that ALDH1A1 protects subpopulations of SNpc DA neurons by preventing the accumulation of dopamine aldehyde intermediates and formation of cytotoxic α-synuclein oligomers.