Dysregulated COMT Expression in Fragile X Syndrome.

Transcriptional and proteomics analyses in human fragile X syndrome (FXS) neurons identified markedly reduced expression of COMT, a key enzyme involved in the metabolism of catecholamines, including dopamine, epinephrine and norepinephrine. FXS is the most common genetic cause of intellectual disability and autism spectrum disorders. COMT encodes for catechol-o-methyltransferase and its association with neuropsychiatric disorders and cognitive function has been extensively studied. We observed a significantly reduced level of COMT in in FXS human neural progenitors and neurons, as well as hippocampal neurons from Fmr1 null mice. We show that deficits in COMT were associated with an altered response in an assay of dopaminergic activity in Fmr1 null mice. These findings demonstrate that loss of FMRP downregulates COMT expression and affects dopamine signaling in FXS, and supports the notion that targeting catecholamine metabolism may be useful in regulating certain neuropsychiatric aspects of FXS.

Integrative Analysis Identifies Key Molecular Signatures Underlying Neurodevelopmental Deficits in Fragile X Syndrome.

BACKGROUND: Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by epigenetic silencing of FMR1 and loss of FMRP expression. Efforts to understand the molecular underpinnings of the disease have been largely performed in rodent or nonisogenic settings. A detailed examination of the impact of FMRP loss on cellular processes and neuronal properties in the context of isogenic human neurons remains lacking. METHODS: Using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 to introduce indels in exon 3 of FMR1, we generated an isogenic human pluripotent stem cell model of FXS that shows complete loss of FMRP expression. We generated neuronal cultures and performed genome-wide transcriptome and proteome profiling followed by functional validation of key dysregulated processes. We further analyzed neurodevelopmental and neuronal properties, including neurite length and neuronal activity, using multielectrode arrays and patch clamp electrophysiology. RESULTS: We showed that the transcriptome and proteome profiles of isogenic FMRP-deficient neurons demonstrate perturbations in synaptic transmission, neuron differentiation, cell proliferation and ion transmembrane transporter activity pathways, and autism spectrum disorder-associated gene sets. We uncovered key deficits in FMRP-deficient cells demonstrating abnormal neural rosette formation and neural progenitor cell proliferation. We further showed that FMRP-deficient neurons exhibit a number of additional phenotypic abnormalities, including neurite outgrowth and branching deficits and impaired electrophysiological network activity. These FMRP-deficient related impairments have also been validated in additional FXS patient-derived human-induced pluripotent stem cell neural cells. CONCLUSIONS: Using isogenic human pluripotent stem cells as a model to investigate the pathophysiology of FXS in human neurons, we reveal key neural abnormalities arising from the loss of FMRP.

Mitochondrial peptide BRAWNIN is essential for vertebrate respiratory complex III assembly.

The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution. Here, we show that the mitochondrial proteome, particularly the respiratory chain, is enriched for small proteins. Using a prediction and validation pipeline for SEPs, we report the discovery of 16 endogenous nuclear encoded, mitochondrial-localized SEPs (mito-SEPs). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN, a 71 a.a. peptide encoded by C12orf73, is essential for respiratory chain complex III (CIII) assembly. In human cells, BRAWNIN is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In zebrafish, Brawnin deletion causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BRAWNIN is essential for vertebrate oxidative phosphorylation. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism.

MicroRNA-138 is a Prognostic Biomarker for Triple-Negative Breast Cancer and Promotes Tumorigenesis via TUSC2 repression.

Breast cancer manifests as a spectrum of subtypes with distinct molecular signatures, and different responses to treatment. Of these subtypes, triple-negative breast cancer (TNBC) has the worst prognoses and limited therapeutic options. Here we report aberrant expression of microRNA-138 (miR-138) in TNBC. Increased miR-138 expression is highly specific to this subtype, correlates with poor prognosis in patients, and is functionally relevant to cancer progression. Our findings establish miR-138 as a specific diagnostic and prognostic biomarker for TNBC. OncomiR-138 is pro-survival; sequence-specific miR-138 inhibition blocks proliferation, promotes apoptosis and inhibits tumour growth in-vivo. miR-138 directly targets a suite of pro-apoptotic and tumour suppressive genes, including tumour suppressor candidate 2 (TUSC2). miR-138 silences TUSC2 by binding to a unique 5'-UTR target-site, which overlaps with the translation start-site of the transcript. Over-expression of TUSC2 mimics the phenotype of miR-138 knockdown and functional rescue experiments confirm that TUSC2 is a direct downstream target of miR-138. Our report of miR-138 as an oncogenic driver in TNBC, positions it as a viable target for oligonucleotide therapeutics and we envision the potential value of using antimiR-138 as an adjuvant therapy to alleviate this therapeutically intractable cancer.

C/EBPß mediates RNA polymerase III-driven transcription of oncomiR-138 in malignant gliomas.

MicroRNA-138 (miR-138) is a pro-survival oncomiR for glioma stem cells. In malignant gliomas, dysregulated expression of microRNAs, such as miR-138, promotes Tumour initiation and progression. Here, we identify the ancillary role of the CCAAT/enhancer binding protein ß (C/EBPß) as a transcriptional activator of miR-138. We demonstrate that a short 158 bp DNA sequence encoding the precursor of miR-138-2 is essential and sufficient for transcription of miR-138. This short sequence includes the A-box and B-box elements characteristic of RNA Polymerase III (Pol III) promoters, and is also directly bound by C/EBPß via an embedded 'C/EBPß responsive element' (CRE). CRE and the Pol III B-box element overlap, suggesting that C/EBPß and transcription factor 3C (TFIIIC) interact at the miR-138-2 locus. We propose that this interaction is essential for the recruitment of the RNA Pol III initiation complex and associated transcription of the oncomiR, miR-138 in malignant gliomas.

Cytoplasmic polyadenylation-mediated translational control of maternal mRNAs directs maternal-to-zygotic transition.

In the earliest stages of animal development following fertilization, maternally deposited mRNAs direct biological processes to the point of zygotic genome activation (ZGA). These maternal mRNAs undergo cytoplasmic polyadenylation (CPA), suggesting translational control of their activation. To elucidate the biological role of CPA during embryogenesis, we performed genome-wide polysome profiling at several stages of zebrafish development. Our analysis revealed a correlation between CPA and polysome-association dynamics, demonstrating a coupling of translation to the CPA of maternal mRNAs. Pan-embryonic CPA inhibition disrupted the maternal-to-zygotic transition (MZT), causing a failure of developmental progression beyond the mid-blastula transition and changes in global gene expression that indicated a failure of ZGA and maternal mRNA clearance. Among the genes that were differentially expressed were those encoding chromatin modifiers and key transcription factors involved in ZGA, including nanog, pou5f3 and sox19b, which have distinct CPA dynamics. Our results establish the necessity of CPA for ensuring progression of the MZT. The RNA-seq data generated in this study represent a valuable zebrafish resource for the discovery of novel elements of the early embryonic transcriptome.

EGF hijacks miR-198/FSTL1 wound-healing switch and steers a two-pronged pathway toward metastasis.

Epithelial carcinomas are well known to activate a prolonged wound-healing program that promotes malignant transformation. Wound closure requires the activation of keratinocyte migration via a dual-state molecular switch. This switch involves production of either the anti-migratory microRNA miR-198 or the pro-migratory follistatin-like 1 (FSTL1) protein from a single transcript; miR-198 expression in healthy skin is down-regulated in favor of FSTL1 upon wounding, which enhances keratinocyte migration and promotes re-epithelialization. Here, we reveal a defective molecular switch in head and neck squamous cell carcinoma (HNSCC). This defect shuts off miR-198 expression in favor of sustained FSTL1 translation, driving metastasis through dual parallel pathways involving DIAPH1 and FSTL1. DIAPH1, a miR-198 target, enhances directional migration through sequestration of Arpin, a competitive inhibitor of Arp2/3 complex. FSTL1 blocks Wnt7a-mediated repression of extracellular signal-regulated kinase phosphorylation, enabling production of MMP9, which degrades the extracellular matrix and facilitates metastasis. The prognostic significance of the FSTL1-DIAPH1 gene pair makes it an attractive target for therapeutic intervention.

Kindlin-3 interacts with the ribosome and regulates c-Myc expression required for proliferation of chronic myeloid leukemia cells.

Kindlins are FERM-containing cytoplasmic proteins that regulate integrin-mediated cell-cell and cell-extracellular matrix (ECM) attachments. Kindlin-3 is expressed in hematopoietic cells, platelets, and endothelial cells. Studies have shown that kindlin-3 stabilizes cell adhesion mediated by ß1, ß2, and ß3 integrins. Apart from integrin cytoplasmic tails, kindlins are known to interact with other cytoplasmic proteins. Here we demonstrate that kindlin-3 can associate with ribosome via the receptor for activated-C kinase 1 (RACK1) scaffold protein based on immunoprecipitation, ribosome binding, and proximity ligation assays. We show that kindlin-3 regulates c-Myc protein expression in the human chronic myeloid leukemia cell line K562. Cell proliferation was reduced following siRNA reduction of kindlin-3 expression and a significant reduction in tumor mass was observed in xenograft experiments. Mechanistically, kindlin-3 is involved in integrin α5ß1-Akt-mTOR-p70S6K signaling; however, its regulation of c-Myc protein expression could be independent of this signaling axis.

ELABELA Is an Endogenous Growth Factor that Sustains hESC Self-Renewal via the PI3K/AKT Pathway.

ELABELA (ELA) is a peptide hormone required for heart development that signals via the Apelin Receptor (APLNR, APJ). ELA is also abundantly secreted by human embryonic stem cells (hESCs), which do not express APLNR. Here we show that ELA signals in a paracrine fashion in hESCs to maintain self-renewal. ELA inhibition by CRISPR/Cas9-mediated deletion, shRNA, or neutralizing antibodies causes reduced hESC growth, cell death, and loss of pluripotency. Global phosphoproteomic and transcriptomic analyses of ELA-pulsed hESCs show that it activates PI3K/AKT/mTORC1 signaling required for cell survival. ELA promotes hESC cell-cycle progression and protein translation and blocks stress-induced apoptosis. INSULIN and ELA have partially overlapping functions in hESC medium, but only ELA can potentiate the TGFß pathway to prime hESCs toward the endoderm lineage. We propose that ELA, acting through an alternate cell-surface receptor, is an endogenous secreted growth factor in human embryos and hESCs that promotes growth and pluripotency.

Contrasting expression patterns of coding and noncoding parts of the human genome upon oxidative stress.

Oxidative stress (OS) is caused by an imbalance between pro- and anti-oxidant reactions leading to accumulation of reactive oxygen species within cells. We here investigate the effect of OS on the transcriptome of human fibroblasts. OS causes a rapid and transient global induction of transcription characterized by pausing of RNA polymerase II (PolII) in both directions, at specific promoters, within 30 minutes of the OS response. In contrast to protein-coding genes, which are commonly down-regulated, this novel divergent, PolII pausing-phenomenon leads to the generation of thousands of long noncoding RNAs (lncRNAs) with promoter-associated antisense lncRNAs transcripts (si-paancRNAs) representing the major group of stress-induced transcripts. OS causes transient dynamics of si-lncRNAs in nucleus and cytosol, leading to their accumulation at polysomes, in contrast to mRNAs, which get depleted from polysomes. We propose that si-lncRNAs represent a novel component of the transcriptional stress that is known to determine the outcome of immediate-early and later cellular stress responses and we provide insights on the fate of those novel mature lncRNA transcripts by showing that their association with polysomal complexes is significantly increased in OS.

Targeting glioma stem cells by functional inhibition of a prosurvival oncomiR-138 in malignant gliomas.

Malignant gliomas are the most aggressive forms of brain tumors, associated with high rates of morbidity and mortality. Recurrence and tumorigenesis are attributed to a subpopulation of tumor-initiating glioma stem cells (GSCs) that are intrinsically resistant to therapy. Initiation and progression of gliomas have been linked to alterations in microRNA expression. Here, we report the identification of microRNA-138 (miR-138) as a molecular signature of GSCs and demonstrate a vital role for miR-138 in promoting growth and survival of bona fide tumor-initiating cells with self-renewal potential. Sequence-specific functional inhibition of miR-138 prevents tumorsphere formation in vitro and impedes tumorigenesis in vivo. We delineate the components of the miR-138 regulatory network by loss-of-function analysis to identify specific regulators of apoptosis. Finally, the higher expression of miR-138 in GSCs compared to non-neoplastic tissue and association with tumor recurrence and survival highlights the clinical significance of miR-138 as a prognostic biomarker and a therapeutic target for treatment of malignant gliomas.

HDAC inhibitor valproic acid enhances tumor cell kill in adenovirus-HSVtk mediated suicide gene therapy in HNSCC xenograft mouse model.

Safety, efficacy and enhanced transgene expression are the primary concerns while using any vector for gene therapy. One of the widely used vectors in clinical trials is adenovirus which provides a safe way to deliver the therapeutic gene. However, adenovirus has poor transduction efficiency in vivo since most tumor cells express low coxsackie and adenovirus receptors. Similarly transgene expression remains low, possibly because of the chromatization of adenoviral genome upon infection in eukaryotic cells, an effect mediated by histone deacetylases (HDACs). Using a recombinant adenovirus (Ad-HSVtk) carrying the herpes simplex thymidine kinase (HSVtk) and GFP genes we demonstrate that HDAC inhibitor valproic acid can bring about an increase in CAR expression on host cells and thereby enhanced Ad-HSVtk infectivity. It also resulted in an increase in transgene (HSVtk and GFP) expression. This, in turn, resulted in increased cell kill of HNSCC cells, following ganciclovir treatment in vitro as well as in vivo in a xenograft nude mouse model.

p73 beta-expressing recombinant adenovirus: a potential anticancer agent.

Tumor suppressor p53-based gene therapy strategy is ineffective in certain conditions. p73, a p53 homologue, could be a potential alternative gene therapy agent as it has been found to be an important determinant of chemosensitivity in cancer cells. Previously, we have reported the generation of a replication-deficient adenovirus expressing p73 beta (Ad-p73). In this study, we evaluated the therapeutic potential of Ad-p73 against a panel of cancer cells (n=12) of different tissue origin. Ad-p73 infected all the cell lines tested very efficiently resulting in several-fold increase in p73 beta levels, which is also functional as it activated the known target gene p21(WAF1/CIP1). Infection with Ad-p73 resulted in potent cytotoxicity in all the cell lines tested. The mechanism of p73-induced cytotoxicity in these cell lines is found to be due to a combination of cell cycle arrest and induction of apoptosis. In addition, exogenous overexpression of p73 by Ad-p73 infection increased the chemosensitivity of cancer cells by many fold to commonly used drug adriamycin. Moreover, Ad-p73 is more efficient than Ad-p53 in enhancing the chemosensitivity of mutant p53 harboring cells. Furthermore, Ad-p73 infection did not induce apoptosis in human normal lung fibroblasts (HEL 299) and human immortalized keratinocytes (HaCaT). These results suggest that Ad-p73 is a potent cytotoxic agent specifically against cancer cells and could be developed as a cancer gene therapy agent either alone or in combination with chemotherapeutic agents.