Aspm sustains postnatal cerebellar neurogenesis and medulloblastoma growth in mice.

Alterations in genes that regulate brain size may contribute to both microcephaly and brain tumor formation. Here, we report that Aspm, a gene that is mutated in familial microcephaly, regulates postnatal neurogenesis in the cerebellum and supports the growth of medulloblastoma, the most common malignant pediatric brain tumor. Cerebellar granule neuron progenitors (CGNPs) express Aspm when maintained in a proliferative state by sonic hedgehog (Shh) signaling, and Aspm is expressed in Shh-driven medulloblastoma in mice. Genetic deletion of Aspm reduces cerebellar growth, while paradoxically increasing the mitotic rate of CGNPs. Aspm-deficient CGNPs show impaired mitotic progression, altered patterns of division orientation and differentiation, and increased DNA damage, which causes progenitor attrition through apoptosis. Deletion of Aspm in mice with Smo-induced medulloblastoma reduces tumor growth and increases DNA damage. Co-deletion of Aspm and either of the apoptosis regulators Bax or Trp53 (also known as p53) rescues the survival of neural progenitors and reduces the growth restriction imposed by Aspm deletion. Our data show that Aspm functions to regulate mitosis and to mitigate DNA damage during CGNP cell division, causes microcephaly through progenitor apoptosis when mutated, and sustains tumor growth in medulloblastoma.

Targeting mTOR as a Therapeutic Approach in Medulloblastoma.

Mechanistic target of rapamycin (mTOR) is a master signaling pathway that regulates organismal growth and homeostasis, because of its implication in protein and lipid synthesis, and in the control of the cell cycle and the cellular metabolism. Moreover, it is necessary in cerebellar development and stem cell pluripotency maintenance. Its deregulation has been implicated in the medulloblastoma and in medulloblastoma stem cells (MBSCs). Medulloblastoma is the most common malignant solid tumor in childhood. The current therapies have improved the overall survival but they carry serious side effects, such as permanent neurological sequelae and disability. Recent studies have given rise to a new molecular classification of the subgroups of medulloblastoma, specifying 12 different subtypes containing novel potential therapeutic targets. In this review we propose the targeting of mTOR, in combination with current therapies, as a promising novel therapeutic approach.

Chaperone-Mediated Autophagy Controls Proteomic and Transcriptomic Pathways to Maintain Glioma Stem Cell Activity.

Chaperone-mediated autophagy (CMA) is a homeostatic process essential for the lysosomal degradation of a selected subset of the proteome. CMA activity directly depends on the levels of LAMP2A, a critical receptor for CMA substrate proteins at the lysosomal membrane. In glioblastoma (GBM), the most common and aggressive brain cancer in adulthood, high levels of LAMP2A in the tumor and tumor-associated pericytes have been linked to temozolomide resistance and tumor progression. However, the role of LAMP2A, and hence CMA, in any cancer stem cell type or in glioblastoma stem cells (GSC) remains unknown. In this work, we show that LAMP2A expression is enriched in patient-derived GSCs, and its depletion diminishes GSC-mediated tumorigenic activities. Conversely, overexpression of LAMP2A facilitates the acquisition of GSC properties. Proteomic and transcriptomic analysis of LAMP2A-depleted GSCs revealed reduced extracellular matrix interaction effectors in both analyses. Moreover, pathways related to mitochondrial metabolism and the immune system were differentially deregulated at the proteome level. Furthermore, clinical samples of GBM tissue presented overexpression of LAMP2, which correlated with advanced glioma grade and poor overall survival. In conclusion, we identified a novel role of CMA in directly regulating GSCs activity via multiple pathways at the proteome and transcriptome levels. SIGNIFICANCE: A receptor of chaperone-mediated autophagy regulates glioblastoma stem cells and may serve as a potential biomarker for advanced tumor grade and poor survival in this disease.

Expression of the neuron-specific protein CHD5 is an independent marker of outcome in neuroblastoma.

BACKGROUND: The chromodomain, helicase DNA-binding protein 5 (CHD5) is a potential tumor suppressor gene located on chromosome 1p36, a region recurrently deleted in high risk neuroblastoma (NB). Previous data have shown that CHD5 mRNA is present in normal neural tissues and in low risk NB, nevertheless, the distribution of CHD5 protein has not been explored. The aim of this study was to investigate CHD5 protein expression as an immunohistochemical marker of outcome in NB. With this purpose, CHD5 protein expression was analyzed in normal neural tissues and neuroblastic tumors (NTs). CHD5 gene and protein expression was reexamined after induction chemotherapy in a subset of high risk tumors to identify potential changes reflecting tumor response. RESULTS: We provide evidence that CHD5 is a neuron-specific protein, absent in glial cells, with diverse expression amongst neuron types. Within NTs, CHD5 immunoreactivity was found restricted to differentiating neuroblasts and ganglion-like cells, and absent in undifferentiated neuroblasts and stromal Schwann cells. Correlation between protein and mRNA levels was found, suggesting transcriptional regulation of CHD5. An immunohistochemical analysis of 90 primary NTs highlighted a strong association of CHD5 expression with favorable prognostic variables (age at diagnosis <12 months, low clinical stage, and favorable histology; P < 0.001 for all), overall survival (OS) (P < 0.001) and event-free survival (EFS) (P < 0.001). Multivariate analysis showed that CHD5 prognostic value is independent of other clinical and biologically relevant parameters, and could therefore represent a marker of outcome in NB that can be tested by conventional immunohistochemistry. The prognostic value of CHD5 was confirmed in an independent, blinded set of 32 NB tumors (P < 0.001).Reactivation of CHD5 expression after induction chemotherapy was observed mainly in those high risk tumors with induced tumor cell differentiation features. Remarkably, these NB tumors showed good clinical response and prolonged patient survival. CONCLUSIONS: The neuron-specific protein CHD5 may represent a marker of outcome in NB that can be tested by conventional immunohistochemistry. Re-establishment of CHD5 expression induced by chemotherapy could be a surrogate marker of treatment response.

Erbb4 Is Required for Cerebellar Developmentand Malignant Phenotype of Medulloblastoma.

Medulloblastoma is the most common and malignant pediatric brain tumor in childhood. It originates from dysregulation of cerebellar development, due to an excessive proliferation of cerebellar granule neuron precursor cells (CGNPs). The underlying molecular mechanisms, except for the role of SHH and WNT pathways, remain largely unknown. ERBB4 is a tyrosine kinase receptor whose activity in cancer is tissue dependent. In this study, we characterized the role of ERBB4 during cerebellum development and medulloblastoma progression paying particular interests to its role in CGNPs and medulloblastoma stem cells (MBSCs). Our results show that ERBB4 is expressed in the CGNPs during cerebellum development where it plays a critical role in migration, apoptosis and differentiation. Similarly, it is enriched in the population of MBSCs, where also controls those critical processes, as well as self-renewal and tumor initiation for medulloblastoma progression. These results are translated to clinical samples where high levels of ERBB4 correlate with poor outcome in Group 4 and all medulloblastomas groups. Transcriptomic analysis identified critical processes and pathways altered in cells with knock-down of ERBB4. These results highlight the impact and underlying mechanisms of ERBB4 in critical processes during cerebellum development and medulloblastoma.

Morphine leads to global genome changes in H3K27me3 levels via a Polycomb Repressive Complex 2 (PRC2) self-regulatory mechanism in mESCs.

BACKGROUND: Environmentally induced epigenetic changes can lead to health problems or disease, but the mechanisms involved remain unclear. Morphine can pass through the placental barrier leading to abnormal embryo development. However, the mechanism by which morphine causes these effects and how they sometimes persist into adulthood is not well known. To unravel the morphine-induced chromatin alterations involved in aberrant embryo development, we explored the role of the H3K27me3/PRC2 repressive complex in gene expression and its transmission across cellular generations in response to morphine. RESULTS: Using mouse embryonic stem cells as a model system, we found that chronic morphine treatment induces a global downregulation of the histone modification H3K27me3. Conversely, ChIP-Seq showed a remarkable increase in H3K27me3 levels at specific genomic sites, particularly promoters, disrupting selective target genes related to embryo development, cell cycle and metabolism. Through a self-regulatory mechanism, morphine downregulated the transcription of PRC2 components responsible for H3K27me3 by enriching high H3K27me3 levels at the promoter region. Downregulation of PRC2 components persisted for at least 48 h (4 cell cycles) following morphine removal, though promoter H3K27me3 levels returned to control levels. CONCLUSIONS: Morphine induces targeting of the PRC2 complex to selected promoters, including those of PRC2 components, leading to characteristic changes in gene expression and a global reduction in H3K27me3. Following morphine removal, enhanced promoter H3K27me3 levels revert to normal sooner than global H3K27me3 or PRC2 component transcript levels. We suggest that H3K27me3 is involved in initiating morphine-induced changes in gene expression, but not in their maintenance. Model of Polycomb repressive complex 2 (PRC2) and H3K27me3 alterations induced by chronic morphine exposure. Morphine induces H3K27me3 enrichment at promoters of genes encoding core members of the PRC2 complex and is associated with their transcriptional downregulation.

Comprehensive characterization of neuroblastoma cell line subtypes reveals bilineage potential similar to neural crest stem cells.

BACKGROUND: Neuroblastic tumors (NBT) derive from neural crest stem cells (NCSC). Histologically, NBT are composed by neuroblasts and Schwannian cells. In culture, neuroblastic (N-), substrate-adherent (S-) and intermediate phenotype (I-) cell subtypes arise spontaneously. METHODS: Here, neuroblastoma (NB) cell line subtypes were characterized according to embryonic peripheral nervous system development markers (GAP43, Phox2b, Sox10, c-kit, GD2, NF68, vimentin, S100beta, calcyclin and ABCG2), morphological features, gene expression and differentiation potential. I-type cells were investigated as a bipotential (neuronal and glial) differentiation stage. RESULTS: Positive immunostaining of NCSC (GAP43, c-kit, NF68, vimentin and Phox2b) and undifferentiated cell (ABCG2) markers was observed in all NB subtypes. N- and I-type cells displayed cytoplasmic membrane GD2 staining, while nuclear calcyclin was restricted to S-type. N- and I-type cells showed similar phenotype and immunoreactivity pattern. Differential gene expression was associated with each cell subtype. N- and I-type cells displayed similar differentiation capacity towards neuronal and glial lineage fates. S-type cells, upon induction, did not show a neuronal-like phenotype, despite gene expression changes. CONCLUSION: Results suggest that N- and I-type NB cell subtypes represent an immature bilineage stage, able to progress towards neuronal and glial fates upon induction of differentiation. S-type cells appear irreversibly committed to a glial lineage fate.

Specific gene expression profiles and chromosomal abnormalities are associated with infant disseminated neuroblastoma.

BACKGROUND: Neuroblastoma (NB) tumours have the highest incidence of spontaneous remission, especially among the stage 4s NB subgroup affecting infants. Clinical distinction of stage 4s from lethal stage 4 can be difficult, but critical for therapeutic decisions. The aim of this study was to investigate chromosomal alterations and differential gene expression amongst infant disseminated NB subgroups. METHODS: Thirty-five NB tumours from patients diagnosed at < 18 months (25 stage 4 and 10 stage 4s), were evaluated by allelic and gene expression analyses. RESULTS: All stage 4s patients underwent spontaneous remission, only 48% stage 4 patients survived despite combined modality therapy. Stage 4 tumours were 90% near-diploid/tetraploid, 44% MYCN amplified, 77% had 1p LOH (50% 1p36), 23% 11q and/or 14q LOH (27%) and 47% had 17q gain. Stage 4s were 90% near-triploid, none MYCN amplified and LOH was restricted to 11q. Initial comparison analyses between stage 4s and 4 < 12 months tumours revealed distinct gene expression profiles. A significant portion of genes mapped to chromosome 1 (P < 0.0001), 90% with higher expression in stage 4s, and chromosome 11 (P = 0.0054), 91% with higher expression in stage 4. Less definite expression profiles were observed between stage 4s and 4 < 18m, yet, association with chromosomes 1 (P < 0.0001) and 11 (P = 0.005) was maintained. Distinct gene expression profiles but no significant association with specific chromosomal region localization was observed between stage 4s and stage 4 < 18 months without MYCN amplification. CONCLUSION: Specific chromosomal aberrations are associated with distinct gene expression profiles which characterize spontaneously regressing or aggressive infant NB, providing the biological basis for the distinct clinical behaviour.

SOX2 expression diminishes with ageing in several tissues in mice and humans.

SOX2 (Sex-determining region Y box 2) is a transcription factor expressed in several foetal and adult tissues and its deregulated activity has been linked to chronic diseases associated with ageing. Nevertheless, the level of SOX2 expression in aged individuals at the tissue level has not previously been examined. In this work, we show that SOX2 expression decreases significantly in the brain with ageing, in both humans and rodents. The administration of resveratrol for 6 months in mice partly attenuated this reduction. We also identified an age-related decline in SOX2 mRNA and protein expression in several other organs, namely, the lung, heart, kidney, spleen and liver. Moreover, peripheral blood mononuclear cells (PBMCs) from elderly expressed lower levels of SOX2 than those from young individuals. Mechanistically, SOX2 expression inversely correlates with p16Ink4a levels. Together, these data show a widespread decrease in SOX2 with age, suggesting that the decline in SOX2 expression might be used as a biomarker of ageing.

SOX3 can promote the malignant behavior of glioblastoma cells.

PURPOSE: Glioblastoma is the most common and lethal adult brain tumor. Despite current therapeutic strategies, including surgery, radiation and chemotherapy, the median survival of glioblastoma patients is 15 months. The development of this tumor depends on a sub-population of glioblastoma stem cells governing tumor propagation and therapy resistance. SOX3 plays a role in both normal neural development and carcinogenesis. However, little is known about its role in glioblastoma. Thus, the aim of this work was to elucidate the role of SOX3 in glioblastoma. METHODS: SOX3 expression was assessed using real-time quantitative PCR (RT-qPCR), Western blotting and immunohistochemistry. MTT, immunocytochemistry and Transwell assays were used to evaluate the effects of exogenous SOX3 overexpression on the viability, proliferation, migration and invasion of glioblastoma cells, respectively. The expression of Hedgehog signaling pathway components and autophagy markers was assessed using RT-qPCR and Western blot analyses, respectively. RESULTS: Higher levels of SOX3 expression were detected in a subset of primary glioblastoma samples compared to those in non-tumoral brain tissues. Exogenous overexpression of this gene was found to increase the proliferation, viability, migration and invasion of glioblastoma cells. We also found that SOX3 up-regulation was accompanied by an enhanced activity of the Hedgehog signaling pathway and by suppression of autophagy in glioblastoma cells. Additionally, we found that SOX3 expression was elevated in patient-derived glioblastoma stem cells, as well as in oncospheres derived from glioblastoma cell lines, compared to their differentiated counterparts, implying that SOX3 expression is associated with the undifferentiated state of glioblastoma cells. CONCLUSION: From our data we conclude that SOX3 can promote the malignant behavior of glioblastoma cells.

PR-LncRNA signature regulates glioma cell activity through expression of SOX factors.

Long non-coding RNAs (LncRNAs) have emerged as a relevant class of genome regulators involved in a broad range of biological processes and with important roles in tumor initiation and malignant progression. We have previously identified a p53-regulated tumor suppressor signature of LncRNAs (PR-LncRNAs) in colorectal cancer. Our aim was to identify the expression and function of this signature in gliomas. We found that the expression of the four PR-LncRNAs tested was high in human low-grade glioma samples and diminished with increasing grade of disease, being the lowest in glioblastoma samples. Functional assays demonstrated that PR-LncRNA silencing increased glioma cell proliferation and oncosphere formation. Mechanistically, we found an inverse correlation between PR-LncRNA expression and SOX1, SOX2 and SOX9 stem cell factors in human glioma biopsies and in glioma cells in vitro. Moreover, knock-down of SOX activity abolished the effect of PR-LncRNA silencing in glioma cell activity. In conclusion, our results demonstrate that the expression and function of PR-LncRNAs are significantly altered in gliomagenesis and that their activity is mediated by SOX factors. These results may provide important insights into the mechanisms responsible for glioblastoma pathogenesis.

Neuronal p38α mediates synaptic and cognitive dysfunction in an Alzheimer's mouse model by controlling ß-amyloid production.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a severe and progressive neuronal loss leading to cognitive dysfunctions. Previous reports, based on the use of chemical inhibitors, have connected the stress kinase p38α to neuroinflammation, neuronal death and synaptic dysfunction. To explore the specific role of neuronal p38α signalling in the appearance of pathological symptoms, we have generated mice that combine expression of the 5XFAD transgenes to induce AD symptoms with the downregulation of p38α only in neurons (5XFAD/p38α∆-N). We found that the neuronal-specific deletion of p38α improves the memory loss and long-term potentiation impairment induced by 5XFAD transgenes. Furthermore, 5XFAD/p38α∆-N mice display reduced amyloid-ß accumulation, improved neurogenesis, and important changes in brain cytokine expression compared with 5XFAD mice. Our results implicate neuronal p38α signalling in the synaptic plasticity dysfunction and memory impairment observed in 5XFAD mice, by regulating both amyloid-ß deposition in the brain and the relay of this accumulation to mount an inflammatory response, which leads to the cognitive deficits.

Oncogenic activity of SOX1 in glioblastoma.

Glioblastoma remains the most common and deadliest type of brain tumor and contains a population of self-renewing, highly tumorigenic glioma stem cells (GSCs), which contributes to tumor initiation and treatment resistance. Developmental programs participating in tissue development and homeostasis re-emerge in GSCs, supporting the development and progression of glioblastoma. SOX1 plays an important role in neural development and neural progenitor pool maintenance. Its impact on glioblastoma remains largely unknown. In this study, we have found that high levels of SOX1 observed in a subset of patients correlate with lower overall survival. At the cellular level, SOX1 expression is elevated in patient-derived GSCs and it is also higher in oncosphere culture compared to differentiation conditions in conventional glioblastoma cell lines. Moreover, genetic inhibition of SOX1 in patient-derived GSCs and conventional cell lines decreases self-renewal and proliferative capacity in vitro and tumor initiation and growth in vivo. Contrarily, SOX1 over-expression moderately promotes self-renewal and proliferation in GSCs. These functions seem to be independent of its activity as Wnt/ß-catenin signaling regulator. In summary, these results identify a functional role for SOX1 in regulating glioma cell heterogeneity and plasticity, and suggest SOX1 as a potential target in the GSC population in glioblastoma.

High expression of MKP1/DUSP1 counteracts glioma stem cell activity and mediates HDAC inhibitor response.

The elucidation of mechanisms involved in resistance to therapies is essential to improve the survival of patients with malignant gliomas. A major feature possessed by glioma cells that may aid their ability to survive therapy and reconstitute tumors is the capacity for self-renewal. We show here that glioma stem cells (GSCs) express low levels of MKP1, a dual-specificity phosphatase, which acts as a negative inhibitor of JNK, ERK1/2, and p38 MAPK, while induction of high levels of MKP1 expression are associated with differentiation of GSC. Notably, we find that high levels of MKP1 correlate with a subset of glioblastoma patients with better prognosis and overall increased survival. Gain of expression studies demonstrated that elevated MKP1 impairs self-renewal and induces differentiation of GSCs while reducing tumorigenesis in vivo. Moreover, we identified that MKP1 is epigenetically regulated and that it mediates the anti-tumor activity of histone deacetylase inhibitors (HDACIs) alone or in combination with temozolomide. In summary, this study identifies MKP1 as a key modulator of the interplay between GSC self-renewal and differentiation and provides evidence that the activation of MKP1, through epigenetic regulation, might be a novel therapeutic strategy to overcome therapy resistance in glioblastoma.

SOX9-regulated cell plasticity in colorectal metastasis is attenuated by rapamycin.

The cancer stem cell (CSC) hypothesis proposes a hierarchical organization of tumors, in which stem-like cells sustain tumors and drive metastasis. The molecular mechanisms underlying the acquisition of CSCs and metastatic traits are not well understood. SOX9 is a transcription factor linked to stem cell maintenance and commonly overexpressed in solid cancers including colorectal cancer. In this study, we show that SOX9 levels are higher in metastatic (SW620) than in primary colorectal cancer cells (SW480) derived from the same patient. This elevated expression correlated with enhanced self-renewal activity. By gain and loss-of-function studies in SW480 and SW620 cells respectively, we reveal that SOX9 levels modulate tumorsphere formation and self-renewal ability in vitro and tumor initiation in vivo. Moreover, SOX9 regulates migration and invasion and triggers the transition between epithelial and mesenchymal states. These activities are partially dependent on SOX9 post-transcriptional modifications. Importantly, treatment with rapamycin inhibits self-renewal and tumor growth in a SOX9-dependent manner. These results identify a functional role for SOX9 in regulating colorectal cancer cell plasticity and metastasis, and provide a strong rationale for a rapamycin-based therapeutic strategy.

Paradoxical role of SOX2 in gastric cancer.

Sox2 is a critical regulator of embryogenesis and necessary for cellular reprogramming. It also plays an important role in tissue homeostasis and regeneration, maintaining the population of undifferentiated adult stem cells. Like various developmental and stem cell genes, SOX2 is aberrantly expressed and amplified in several human cancers. Moreover, functional studies have shown that it regulates many biological processes including cell proliferation, apoptosis, self-renewal and invasion. While it is oncogenic in most cancers, SOX2 activity is controversial in gastric cancer, where it might behave as a tumor suppressor in some situations. In this review, we discuss its role in cancer biology, with particular attention to what is known about the involvement of SOX2 in gastric cancer biology.

Essential Function of Dicer in Resolving DNA Damage in the Rapidly Dividing Cells of the Developing and Malignant Cerebellum.

Maintenance of genomic integrity is critical during neurodevelopment, particularly in rapidly dividing cerebellar granule neuronal precursors that experience constitutive replication-associated DNA damage. As Dicer was recently recognized to have an unexpected function in the DNA damage response, we examined whether Dicer was important for preserving genomic integrity in the developing brain. We report that deletion of Dicer in the developing mouse cerebellum resulted in the accumulation of DNA damage leading to cerebellar progenitor degeneration, which was rescued with p53 deficiency; deletion of DGCR8 also resulted in similar DNA damage and cerebellar degeneration. Dicer deficiency also resulted in DNA damage and death in other rapidly dividing cells including embryonic stem cells and the malignant cerebellar progenitors in a mouse model of medulloblastoma. Together, these results identify an essential function of Dicer in resolving the spontaneous DNA damage that occurs during the rapid proliferation of developmental progenitors and malignant cells.

Targeting SOX2 as a Therapeutic Strategy in Glioblastoma.

Glioblastoma is the most common and malignant brain cancer in adults. Current therapy consisting of surgery followed by radiation and temozolomide has a moderate success rate and the tumor reappears. Among the features that a cancer cell must have to survive the therapeutic treatment and reconstitute the tumor is the ability of self-renewal. Therefore, it is vital to identify the molecular mechanisms that regulate this activity. Sex-determining region Y (SRY)-box 2 (SOX2) is a transcription factor whose activity has been associated with the maintenance of the undifferentiated state of cancer stem cells in several tissues, including the brain. Several groups have detected increased SOX2 levels in biopsies of glioblastoma patients, with the highest levels associated with poor outcome. Therefore, SOX2 silencing might be a novel therapeutic approach to combat cancer and particularly brain tumors. In this review, we will summarize the current knowledge about SOX2 in glioblastoma and recapitulate several strategies that have recently been described targeting SOX2 in this malignancy.

mTOR inhibition decreases SOX2-SOX9 mediated glioma stem cell activity and temozolomide resistance.

BACKGROUND: SOX2 and SOX9 are commonly overexpressed in glioblastoma, and regulate the activity of glioma stem cells (GSCs). Their specific and overlapping roles in GSCs and glioma treatment remain unclear. METHODS: SOX2 and SOX9 levels were examined in human biopsies. Gain and loss of function determined the impact of altering SOX2 and SOX9 on cell proliferation, senescence, stem cell activity, tumorigenesis and chemoresistance. RESULTS: SOX2 and SOX9 expression correlates positively in glioma cells and glioblastoma biopsies. High levels of SOX2 bypass cellular senescence and promote resistance to temozolomide. Mechanistic investigations revealed that SOX2 acts upstream of SOX9. mTOR genetic and pharmacologic (rapamycin) inhibition decreased SOX2 and SOX9 expression, and reversed chemoresistance. CONCLUSIONS: Our findings reveal SOX2-SOX9 as an oncogenic axis that regulates stem cell properties and chemoresistance. We identify that rapamycin abrogate SOX protein expression and provide evidence that a combination of rapamycin and temozolomide inhibits tumor growth in cells with high SOX2/SOX9.