[Show abstract][Hide abstract] ABSTRACT: Neural stem cells (NSCs) can self-renew and give rise to the major cell types of the CNS. Studies of NSC include the investigation of primary, CNS-derived cells as well as animal and human embryonic stem cell (ESC)-derived and induced pluripotent stem cell (iPSC)-derived sources. NSCs provide a means with which to study normal neural development, neurodegeneration and neurological disease and are clinically relevant sources for cellular repair to the damaged and diseased CNS. Proteomics studies of NSCs have the potential to delineate molecules and pathways critical for NSC biology and the means by which NSC can participate in neural repair. In this review, we provide a background to NSC biology, including the means to obtain them and the caveats to these processes. We then focus on advances in the proteomic interrogation of NSCs, from the analysis of posttranslational modifications (PTMs) and crosstalk, of approaches to comparing NSCs, of spatial expression to gain an understanding of, for instance, the secretome, and of temporal expression to elucidate mechanisms of differentiation. We also discuss some of the methods tht will undoubtedly be useful in the investigation of NSC, but which have not yet been applied to the field. While many proteomics studies of NSC have largely catalogued the proteome or post-translational modifications of specific cellular states, without delving into specific functions, some have led to understandings of functional processes or identified markers that could not have been identified via other means. Many challenges remain in the field including the precise identification and standardization of NSCs used for proteomic analyses, as well as how to translate fundamental proteomics studies to functional biology. The next level of investigation will require interdisciplinary approaches, combining the skills of those interested in the biochemistry of proteomics with those interested in modulating NSC function.
[Show abstract][Hide abstract] ABSTRACT: Maternal embryonic leucine zipper kinase (MELK) is a highly conserved serine/threonine kinase initially found to be expressed in a wide range of early embryonic cellular stages, and as a result has been implicated in embryogenesis and cell cycle control. Recent evidence has identified a broader spectrum of tissue expression pattern for this kinase than previously appreciated. MELK is expressed in several human cancers and stem cell populations. Unique spatial and temporal patterns of expression within these tissues suggest that MELK plays a prominent role in cell cycle control, cell proliferation, apoptosis, cell migration, cell renewal, embryogenesis, oncogenesis, and cancer treatment resistance and recurrence. These findings have important implications for our understanding of development, disease, and cancer therapeutics. Furthermore understanding MELK signaling may elucidate an added dimension of stem cell control.
Full-text · Article · Feb 2015 · Clinical and Translational Medicine
[Show abstract][Hide abstract] ABSTRACT: A period of mild brain overgrowth with an unknown etiology has been identified as one of the most common phenotypes in autism. Here, we test the hypothesis that maternal inflammation during critical periods of embryonic development can cause brain overgrowth and autism-associated behaviors as a result of altered neural stem cell function. Pregnant mice treated with low-dose lipopolysaccharide at embryonic day 9 had offspring with brain overgrowth, with a more pronounced effect in PTEN heterozygotes. Exposure to maternal inflammation also enhanced NADPH oxidase (NOX)-PI3K pathway signaling, stimulated the hyperproliferation of neural stem and progenitor cells, increased forebrain microglia, and produced abnormal autism-associated behaviors in affected pups. Our evidence supports the idea that a prenatal neuroinflammatory dysregulation in neural stem cell redox signaling can act in concert with underlying genetic susceptibilities to affect cellular responses to environmentally altered cellular levels of reactive oxygen species.
Full-text · Article · Oct 2014 · Stem Cell Reports
[Show abstract][Hide abstract] ABSTRACT: Neuronal ceroid lipofuscinosis (NCL) diseases consist of a group of genetically inherited neurodegenerative disorders that share common symptoms such as seizures, psychomotor retardation, blindness, and premature death. Although gene defects behind the NCL diseases are well characterized, very little is known how these defects affect normal development of the brain and cause the pathology of the disease. To obtain understanding of the development of the cell types that are mostly affected by defective function of CLN proteins, timing of expression of CLN2, CLN3 and CLN5 genes was investigated in developing mouse brain. The relationship between the expression pattern and the developmental stage of the brain showed that these genes are co-expressed spatially and temporally during brain development. Throughout the development strong expression of the three mRNAs was detected in germinal epithelium and in ventricle regions, hippocampus and cerebellum, all representing regions that are known to be associated with the formation of new neurons. More specifically, RT-PCR studies on developing mouse cortices revealed that the CLN genes were temporally co-expressed in the neural progenitor cells together with known stem cell markers. This suggested that CLN2, CLN3 and CLN5 genes may play an important role in early embryonal neurogenesis.
Full-text · Article · Oct 2014 · Experimental and Molecular Pathology
[Show abstract][Hide abstract] ABSTRACT: The acute response of the rodent subventricular zone (SVZ) to traumatic brain injury (TBI) involves a physical expansion through increased cell proliferation. However, the cellular underpinnings of these changes are not well understood. Our analyses have revealed that there are two distinct transit-amplifying cell populations that respond in opposite ways to injury. Mash1+ transit-amplifying cells are the primary SVZ cell type that is stimulated to divide following TBI. In contrast, the EGFR+ population, which has been considered to be a functionally equivalent progenitor population to Mash1+ cells in the uninjured brain, becomes significantly less proliferative after injury. Although normally quiescent GFAP+ stem cells are stimulated to divide in SVZ ablation models, we found that the GFAP+ stem cells do not divide more after TBI. We found, instead, that TBI results in increased numbers of GFAP+/EGFR+ stem cells via non-proliferative means-potentially through the dedifferentiation of progenitor cells. EGFR+ progenitors from injured brains only were competent to revert to a stem cell state following brief exposure to growth factors. Thus, our results demonstrate previously unknown changes in lineage relationships that differ from conventional models and likely reflect an adaptive response of the SVZ to maintain endogenous brain repair after TBI.
[Show abstract][Hide abstract] ABSTRACT: Glioblastoma multiforme (GBM) is a highly lethal brain tumor. Due to resistance to current therapies, patient prognosis remains poor and development of novel and effective GBM therapy is crucial. Glioma stem cells (GSCs) have gained attention as a therapeutic target in GBM due to their relative resistance to current therapies and potent tumor-initiating ability. Previously, we identified that the mitotic kinase maternal embryonic leucine-zipper kinase (MELK) is highly expressed in GBM tissues, specifically in GSCs, and its expression is inversely correlated with the post-surgical survival period of GBM patients. In addition, patient-derived GSCs depend on MELK for their survival and growth both in vitro and in vivo. Here, we demonstrate evidence that the role of MELK in the GSC survival is specifically dependent on its kinase activity. With in silico structure-based analysis for protein-compound interaction, we identified the small molecule Compound 1 (C1) is predicted to bind to the kinase-active site of MELK protein. Elimination of MELK kinase activity was confirmed by in vitro kinase assay in nano-molar concentrations. When patient-derived GSCs were treated with C1, they underwent mitotic arrest and subsequent cellular apoptosis in vitro, a phenotype identical to that observed with shRNA-mediated MELK knockdown. In addition, C1 treatment strongly induced tumor cell apoptosis in slice cultures of GBM surgical specimens and attenuated growth of mouse intracranial tumors derived from GSCs in a dose-dependent manner. Lastly, C1 treatment sensitizes GSCs to radiation treatment. Collectively, these data indicate that targeting MELK kinase activity is a promising approach to attenuate GBM growth by eliminating GSCs in tumors.
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Targeting amino acid metabolism has therapeutic implications for aggressive brain tumors. Asparagine is an amino acid that is synthesized by normal cells. However, some cancer cells lack asparagine synthetase (ASNS), the key enzyme for asparagine synthesis. Asparaginase (ASNase) contributes to eradication of acute leukemia by decreasing asparagine levels in serum and cerebrospinal fluid. However, leukemic cells may become ASNase-resistant by upregulating ASNS. High expression of ASNS has also been associated with biologic aggressiveness of other cancers, including gliomas. Here, the impact of enzymatic depletion of asparagine on proliferation of brain tumor cells was determined. ASNase was used as monotherapy or in combination with conventional chemotherapeutic agents. Viability assays for ASNase-treated cells demonstrated significant growth reduction in multiple cell lines. This effect was reversed by glutamine in a dose-dependent manner--as expected, because glutamine is the main amino group donor for asparagine synthesis. ASNase treatment also reduced sphere formation by medulloblastoma and primary glioblastoma cells. ASNase-resistant glioblastoma cells exhibited elevated levels of ASNS mRNA. ASNase cotreatment significantly enhanced gemcitabine or etoposide cytotoxicity against glioblastoma cells. Xenograft tumors in vivo showed no significant response to ASNase monotherapy and little response to temozolomide alone. However, combinatorial therapy with ASNase and temozolomide resulted in significant growth suppression for an extended duration of time. Taken together, these findings indicate that amino acid depletion warrants further investigation as adjunctive therapy for brain tumors.
Findings have potential impact for providing adjuvant means to enhance brain tumor chemotherapy.
Preview · Article · Feb 2014 · Molecular Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Numerous studies and case reports show co-morbidity of autism and epilepsy, suggesting some common molecular underpinnings of the two phenotypes. However, the relationship between the two on the molecular level remains unclear. Here, whole exome sequencing was performed on a family with identical twins affected with autism and severe, intractable seizures. A de novo variant was identified in the KCND2 gene, which encodes the Kv4.2 potassium channel. Kv4.2 is a major pore-forming subunit in somatodendritic subthreshold A-type potassium current (ISA) channels. The de novo mutation p.Val404Met is novel and occurs at a highly conserved residue within the C-terminal end of the transmembrane helix S6 region of the ion permeation pathway. Functional analysis revealed the likely pathogenicity of the variant in that the p.Val404Met mutant construct showed significantly slowed inactivation, either by itself or after equimolar co-expression with the wild-type Kv4.2 channel construct consistent with a dominant effect. Further, the effect of the mutation on closed-state inactivation was evident in the presence of auxiliary subunits that associate with Kv4 subunits to form ISAchannels in vivo. Discovery of a functionally relevant novel de novo variant, coupled with physiological evidence that the mutant protein disrupts potassium current inactivation, strongly supports KCND2 as the causal gene for epilepsy in this family. Interaction of KCND2 with other genes implicated in autism, and the role of KCND2 in synaptic plasticity provide suggestive evidence of an etiological role in autism.
No preview · Article · Feb 2014 · Human Molecular Genetics
[Show abstract][Hide abstract] ABSTRACT: Intratumoral heterogeneity contributes to cancer drug resistance, but the underlying mechanisms are not understood. Single-cell
analyses of patient-derived models and clinical samples from glioblastoma patients treated with epidermal growth factor receptor
(EGFR) tyrosine kinase inhibitors (TKIs) demonstrate that tumor cells reversibly up-regulate or suppress mutant EGFR expression,
conferring distinct cellular phenotypes to reach an optimal equilibrium for growth. Resistance to EGFR TKIs is shown to occur
by elimination of mutant EGFR from extrachromosomal DNA. After drug withdrawal, reemergence of clonal EGFR mutations on extrachromosomal DNA follows. These results indicate a highly specific, dynamic, and adaptive route by which
cancers can evade therapies that target oncogenes maintained on extrachromosomal DNA.
[Show abstract][Hide abstract] ABSTRACT: Glioblastoma multiforme (GBM) is a life-threatening brain tumor. Accumulating evidence suggests that eradication of glioma stem-like cells (GSCs) in GBM is essential to achieve cure. The transcription factor FOXM1 has recently gained attention as a master regulator of mitotic progression of cancer cells in various organs. Here, we demonstrate that FOXM1 forms a protein complex with the mitotic kinase MELK in GSCs, leading to phosphorylation and activation of FOXM1 in a MELK kinase-dependent manner. This MELK-dependent activation of FOXM1 results in a subsequent increase in mitotic regulatory genes in GSCs. MELK-driven FOXM1 activation is regulated by the binding and subsequent trans-phosphorylation of FOXM1 by another kinase PLK1. Using mouse neural progenitor cells (NPCs), we found that transgenic expression of FOXM1 enhances, while siRNA-mediated gene silencing diminishes neurosphere formation, suggesting that FOXM1 is required for NPC growth. During tumorigenesis, FOXM1 expression sequentially increases as cells progress from NPCs, to pretumorigenic progenitors and GSCs. The antibiotic Siomycin A disrupts MELK-mediated FOXM1 signaling with a greater sensitivity in GSC compared to neural stem cell. Treatment with the first-line chemotherapy agent for GBM, Temozolomide, paradoxically enriches for both FOXM1 (+) and MELK (+) cells in GBM cells, and addition of Siomycin A to Temozolomide treatment in mice harboring GSC-derived intracranial tumors enhances the effects of the latter. Collectively, our data indicate that FOXM1 signaling through its direct interaction with MELK regulates key mitotic genes in GSCs in a PLK1-dependent manner and thus, this protein complex is a potential therapeutic target for GBM. Stem Cells
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Acquired resistance to tyrosine kinase inhibitors (TKI) represents a major challenge for personalized cancer therapy. Multiple genetic mechanisms of acquired TKI resistance have been identified in several types of human cancer. However, the possibility that cancer cells may also evade treatment by co-opting physiologically regulated receptors has not been addressed. Here, we show the first example of this alternate mechanism in brain tumors by showing that EGF receptor (EGFR)-mutant glioblastomas (GBMs) evade EGFR TKIs by transcriptionally de-repressing platelet-derived growth factor receptor β (PDGFRβ). Mechanistic studies show that EGFRvIII signaling actively suppresses PDGFRβ transcription in an mTORC1- and extracellular signal-regulated kinase-dependent manner. Genetic or pharmacologic inhibition of oncogenic EGFR renders GBMs dependent on the consequently de-repressed PDGFRβ signaling for growth and survival. Importantly, combined inhibition of EGFR and PDGFRβ signaling potently suppresses tumor growth in vivo. These data identify a novel, nongenetic TKI resistance mechanism in brain tumors and provide compelling rationale for combination therapy.
These results provide the fi rst clinical and biologic evidence for receptor tyrosinekinase (RTK) "switching" as a mechanism of resistance to EGFR inhibitors in GBM and provide a molecular explanation of how tumors can become "addicted" to a non amplified, nonmutated, physiologically regulated RTK to evade targeted treatment.
[Show abstract][Hide abstract] ABSTRACT: Levels of reactive oxygen species (ROS) in neural stem cells (NSCs) can regulate cell proliferation, self-renewal and neurogenesis. The levels of ROS in NSCs are dependent on the enzyme nicotinamide adenine dinucleotide phosphate-oxidase (NOX). Previous studies have indicated the existence of cancer stem cells that initiate tumors and share many similarities with normal stem cells. Although it has been observed that most cancer cells show an increased level of NOX-generated ROS, the functional role that ROS plays in these cancer cells has not been determined. Therefore, we are investigating the role that NOX-generated ROS plays in neural cancer stem cell function. Two main questions are how the inhibition of the NOX enzyme affects the cancer cells’ proliferation, and how the cells respond after being released from this inhibition. We are currently examining the effects of different ROS levels on various neural cancer cell lines that originated from glial brain tumors obtained from patients after surgical resection. Cell culture assays of self-renewal and western blots will be performed to observe the signaling pathways that are activated in the cells due to changes in cellular ROS, such as phospho-AKT and phospho-S6, which play key roles in cell proliferation. By studying the activation of these pathways, we will be able to determine the differences caused by ROS in cancer stem cells compared to normal NSCs, which will contribute to developing new targets for glioblastoma cancer treatment.
[Show abstract][Hide abstract] ABSTRACT: Background Mutations in isocitrate dehydrogenase 1 (IDH1) and associated CpG island hypermethylation represent early events in the development of low-grade gliomas and secondary glioblastomas. To identify candidate tumor suppressor genes whose promoter methylation may contribute to gliomagenesis, we compared methylation profiles of IDH1 mutant (MUT) and IDH1 wild-type (WT) tumors using massively parallel reduced representation bisulfite sequencing.Methods
Reduced representation bisulfite sequencing was performed on ten pathologically matched WT and MUT glioma samples and compared with data from a methylation-sensitive restriction enzyme technique and data from The Cancer Genome Atlas (TCGA). Methylation in the gene retinol-binding protein 1 (RBP1) was identified in IDH1 mutant tumors and further analyzed with primer-based bisulfite sequencing. Correlation between IDH1/IDH2 mutation status and RBP1 methylation was evaluated with Spearman correlation. Survival data were collected retrospectively and analyzed with Kaplan-Meier and Cox proportional hazards analysis. All statistical tests were two-sided.ResultsMethylome analysis identified coordinated CpG island hypermethylation in IDH1 MUT gliomas, consistent with previous reports. RBP1, important in retinoic acid metabolism, was found to be hypermethylated in 76 of 79 IDH1 MUT, 3 of 3 IDH2 MUT, and 0 of 116 IDH1/IDH2 WT tumors. IDH1/IDH2 mutation was highly correlated with RBP1 hypermethylation (n 198; Spearman R 0.94, 95% confidence interval 0.92 to 0.95, P <. 001). The Cancer Genome Atlas showed IDH1 MUT tumors (n 23) to be RBP1-hypermethylated with decreased RBP1 expression compared with WT tumors (n 124). Among patients with primary glioblastoma, patients with RBP1-unmethylated tumors (n 102) had decreased median overall survival compared with patients with RBP1-methylated tumors (n 22) (20.3 months vs 36.8 months, respectively; hazard ratio of death 2.48, 95% confidence interval 1.30 to 4.75, P . 006).ConclusionRBP1 promoter hypermethylation is found in nearly all IDH1 and IDH2 mutant gliomas and is associated with improved patient survival. Because RBP1 is involved in retinoic acid synthesis, our results suggest that dysregulation of retinoic acid metabolism may contribute to glioma formation along the IDH1/IDH2-mutant pathway.
Full-text · Article · Sep 2012 · Journal of the National Cancer Institute