Methyl-CpG binding domain protein 2 (Mbd2) drives breast cancer progression through the modulation of epithelial-to-mesenchymal transition.

Methyl-CpG-binding domain protein 2 (Mbd2), a reader of DNA methylation, has been implicated in different types of malignancies, including breast cancer. However, the exact role of Mbd2 in various stages of breast cancer growth and progression in vivo has not been determined. To test whether Mbd2 plays a causal role in mammary tumor growth and metastasis, we performed genetic knockout (KO) of Mbd2 in MMTV-PyMT transgenic mice and compared mammary tumor progression kinetics between the wild-type (PyMT-Mbd2+/+) and KO (PyMT-Mbd2-/-) groups. Our results demonstrated that deletion of Mbd2 in PyMT mice impedes primary tumor growth and lung metastasis at the experimental endpoint (postnatal week 20). Transcriptomic and proteomic analyses of primary tumors revealed that Mbd2 deletion abrogates the expression of several key determinants involved in epithelial-to-mesenchymal transition, such as neural cadherin (N-cadherin) and osteopontin. Importantly, loss of the Mbd2 gene impairs the activation of the PI3K/AKT pathway, which is required for PyMT-mediated oncogenic transformation, growth, and survival of breast tumor cells. Taken together, the results of this study provide a rationale for further development of epigenetic therapies targeting Mbd2 to inhibit the progression of breast cancer.

Impact of eIF2α phosphorylation on the translational landscape of mouse embryonic stem cells.

The integrated stress response (ISR) is critical for cell survival under stress. In response to diverse environmental cues, eIF2α becomes phosphorylated, engendering a dramatic change in mRNA translation. The activation of ISR plays a pivotal role in the early embryogenesis, but the eIF2-dependent translational landscape in pluripotent embryonic stem cells (ESCs) is largely unexplored. We employ a multi-omics approach consisting of ribosome profiling, proteomics, and metabolomics in wild-type (eIF2α+/+) and phosphorylation-deficient mutant eIF2α (eIF2αA/A) mouse ESCs (mESCs) to investigate phosphorylated (p)-eIF2α-dependent translational control of naive pluripotency. We show a transient increase in p-eIF2α in the naive epiblast layer of E4.5 embryos. Absence of eIF2α phosphorylation engenders an exit from naive pluripotency following 2i (two chemical inhibitors of MEK1/2 and GSK3α/ß) withdrawal. p-eIF2α controls translation of mRNAs encoding proteins that govern pluripotency, chromatin organization, and glutathione synthesis. Thus, p-eIF2α acts as a key regulator of the naive pluripotency gene regulatory network.

mRNA translation in astrocytes controls hippocampal long-term synaptic plasticity and memory.

Activation of neuronal protein synthesis upon learning is critical for the formation of long-term memory. Here, we report that learning in the contextual fear conditioning paradigm engenders a decrease in eIF2α (eukaryotic translation initiation factor 2) phosphorylation in astrocytes in the hippocampal CA1 region, which promotes protein synthesis. Genetic reduction of eIF2α phosphorylation in hippocampal astrocytes enhanced contextual and spatial memory and lowered the threshold for the induction of long-lasting plasticity by modulating synaptic transmission. Thus, learning-induced dephosphorylation of eIF2α in astrocytes bolsters hippocampal synaptic plasticity and consolidation of long-term memories.

Methyl-CpG binding domain 2 (Mbd2) is an epigenetic regulator of autism-risk genes and cognition.

The Methyl-CpG-Binding Domain Protein family has been implicated in neurodevelopmental disorders. The Methyl-CpG-binding domain 2 (Mbd2) binds methylated DNA and was shown to play an important role in cancer and immunity. Some evidence linked this protein to neurodevelopment. However, its exact role in neurodevelopment and brain function is mostly unknown. Here we show that Mbd2-deficiency in mice (Mbd2-/-) results in deficits in cognitive, social and emotional functions. Mbd2 binds regulatory DNA regions of neuronal genes in the hippocampus and loss of Mbd2 alters the expression of hundreds of genes with a robust down-regulation of neuronal gene pathways. Further, a genome-wide DNA methylation analysis found an altered DNA methylation pattern in regulatory DNA regions of neuronal genes in Mbd2-/- mice. Differentially expressed genes significantly overlap with gene-expression changes observed in brains of Autism Spectrum Disorder (ASD) individuals. Notably, downregulated genes are significantly enriched for human ortholog ASD risk genes. Observed hippocampal morphological abnormalities were similar to those found in individuals with ASD and ASD rodent models. Hippocampal Mbd2 knockdown partially recapitulates the behavioral phenotypes observed in Mbd2-/- mice. These findings suggest that Mbd2 is a novel epigenetic regulator of genes that are associated with ASD in humans. Mbd2 loss causes behavioral alterations that resemble those found in ASD individuals.

SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation.

Viruses evade the innate immune response by suppressing the production or activity of cytokines such as type I interferons (IFNs). Here we report the discovery of a mechanism by which the SARS-CoV-2 virus coopts an intrinsic cellular machinery to suppress the production of the key immunostimulatory cytokine IFN-ß. We reveal that the SARS-CoV-2 encoded nonstructural protein 2 (NSP2) directly interacts with the cellular GIGYF2 protein. This interaction enhances the binding of GIGYF2 to the mRNA cap-binding protein 4EHP, thereby repressing the translation of the Ifnb1 mRNA. Depletion of GIGYF2 or 4EHP significantly enhances IFN-ß production, which inhibits SARS-CoV-2 replication. Our findings reveal a target for rescuing the antiviral innate immune response to SARS-CoV-2 and other RNA viruses.

Fibrinolytic System and Cancer: Diagnostic and Therapeutic Applications.

Fibrinolysis is a crucial physiological process that helps to maintain a hemostatic balance by counteracting excessive thrombosis. The components of the fibrinolytic system are well established and are associated with a wide array of physiological and pathophysiological processes. The aberrant expression of several components, especially urokinase-type plasminogen activator (uPA), its cognate receptor uPAR, and plasminogen activator inhibitor-1 (PAI-1), has shown a direct correlation with increased tumor growth, invasiveness, and metastasis. As a result, targeting the fibrinolytic system has been of great interest in the field of cancer biology. Even though there is a plethora of encouraging preclinical evidence on the potential therapeutic benefits of targeting the key oncogenic components of the fibrinolytic system, none of them made it from "bench to bedside" due to a limited number of clinical trials on them. This review summarizes our existing understanding of the various diagnostic and therapeutic strategies targeting the fibrinolytic system during cancer.

Enhanced Anticancer Effect of a Combination of S-adenosylmethionine (SAM) and Immune Checkpoint Inhibitor (ICPi) in a Syngeneic Mouse Model of Advanced Melanoma.

Immune checkpoint inhibitors (ICPi) targeting the PD-1/PD-L1 pathway have shown marked success in patients with advanced melanoma. However, 60-70% of patients fail to respond, warranting a therapeutic intervention that could increase response rates. We and others have shown that S-adenosylmethionine (SAM), a universal methyl donor, has significant anticancer effects in numerous cancers previously; however, its effect on melanoma progression has not been evaluated. Interestingly, SAM was reported to be essential for T cell activation and proliferation and, thus, could potentially cooperate with ICPi and block melanoma progression. In this study, we examined the antitumor effects of SAM and ICPi alone and in combination in a well-established melanoma mouse model wherein syngeneic C57BL/6 mouse were subcutaneously (orthotopic) injected with B16-F1 cells. Treatment of mice with either SAM or anti-PD-1 antibody alone resulted in significant reduction in tumor volumes and weights; effects that were highest in mice treated with a combination of SAM+anti-PD-1. RNA-sequencing analysis of the primary tumors showed numerous differentially expressed genes (DEGs) following treatment with SAM+anti-PD-1, which was shown to downregulate cancer, MAPK, and tyrosine kinase pathways. Indeed, SAM+anti-PD-1 reversed the aberrant expression of some known melanoma genes. Tumor immunophenotyping revealed the SAM+anti-PD-1 combination was significantly more effective than either SAM or anti-PD-1 as the CD8+ T cells had higher activation, proliferation, and cytokine production compared to all other groups. This study shows that the combination of currently approved agents SAM and ICPi can effectively block melanoma via alteration of key genes/pathways implicated in cancer and immune response pathways, providing the rationale for the initiation of clinical trials with SAM and ICPi.

S-adenosylmethionine in combination with decitabine shows enhanced anti-cancer effects in repressing breast cancer growth and metastasis.

Abnormal DNA methylation orchestrates many of the cancer-related gene expression irregularities such as the inactivation of tumour suppressor genes through hypermethylation as well as activation of prometastatic genes through hypomethylation. The fact that DNA methylation abnormalities can be chemically reversed positions the DNA methylation machinery as an attractive target for anti-cancer drug development. However, although in vitro studies suggested that targeting concordantly hypo- and hypermethylation is of benefit in suppressing both oncogenic and prometastatic functions of breast cancer cells, this has never been tested in a therapeutic setting in vivo. In this context, we investigated the combined therapeutic effects of an approved nutraceutical agent S-adenosylmethionine (SAM) and FDA-approved hypomethylating agent decitabine using the MDA-MB-231 xenograft model of breast cancer and found a pronounced reduction in mammary tumour volume and lung metastasis compared to the animals in the control and monotherapy treatment arms. Immunohistochemical assessment of the primary breast tumours showed a significantly reduced expression of proliferation (Ki-67) and angiogenesis (CD31) markers following combination therapy as compared to the control group. Global transcriptome and methylome analyses have revealed that the combination therapy regulates genes from several key cancer-related pathways that are abnormally expressed in breast tumours. To our knowledge, this is the first preclinical study demonstrating the anti-cancer therapeutic potential of using a combination of methylating (SAM) and demethylating agent (decitabine) in vivo. Results from this study provide a molecularly founded rationale for clinically testing a combination of agents targeting the epigenome to reduce the morbidity and mortality from breast cancer.

An enhanced chemopreventive effect of methyl donor S-adenosylmethionine in combination with 25-hydroxyvitamin D in blocking mammary tumor growth and metastasis.

Therapeutic targeting of metastatic breast cancer still remains a challenge as the tumor cells are highly heterogenous and exploit multiple pathways for their growth and metastatic spread that cannot always be targeted by a single-agent monotherapy regimen. Therefore, a rational approach through simultaneous targeting of several pathways may provide a better anti-cancer therapeutic effect. We tested this hypothesis using a combination of two nutraceutical agents S-adenosylmethionine (SAM) and Vitamin D (Vit. D) prohormone [25-hydroxyvitamin D; '25(OH)D'] that are individually known to exert distinct changes in the expression of genes involved in tumor growth and metastasis. Our results show that both SAM and 25(OH)D monotherapy significantly reduced proliferation and clonogenic survival of a panel of breast cancer cell lines in vitro and inhibited tumor growth, lung metastasis, and breast tumor cell colonization to the skeleton in vivo. However, these effects were significantly more pronounced in the combination setting. RNA-Sequencing revealed that the transcriptomic footprint on key cancer-related signaling pathways is broader in the combination setting than any of the monotherapies. Furthermore, comparison of the differentially expressed genes from our transcriptome analyses with publicly available cancer-related dataset demonstrated that the combination treatment upregulates genes from immune-related pathways that are otherwise downregulated in bone metastasis in vivo. Since SAM and Vit. D are both approved nutraceuticals with known safety profiles, this combination treatment may serve as a novel strategy to reduce breast cancer-associated morbidity and mortality.

uPAR antibody (huATN-658) and Zometa reduce breast cancer growth and skeletal lesions.

Urokinase plasminogen activator receptor (uPAR) is implicated in tumor growth and metastasis due to its ability to activate latent growth factors, proteases, and different oncogenic signaling pathways upon binding to different ligands. Elevated uPAR expression is correlated with the increased aggressiveness of cancer cells, which led to its credentialing as an attractive diagnostic and therapeutic target in advanced solid cancer. Here, we examine the antitumor effects of a humanized anti-uPAR antibody (huATN-658) alone and in combination with the approved bisphosphonate Zometa (Zoledronic acid) on skeletal lesion through a series of studies in vitro and in vivo. Treatment with huATN-658 or Zometa alone significantly decreased human MDA-MB-231 cell proliferation and invasion in vitro, effects which were more pronounced when huATN-658 was combined with Zometa. In vivo studies demonstrated that huATN-658 treatment significantly reduced MDA-MB-231 primary tumor growth compared with controls. In a model of breast tumor-induced bone disease, huATN-658 and Zometa were equally effective in reducing skeletal lesions. The skeletal lesions were significantly reduced in animals receiving the combination of huATN-658 + Zometa compared with monotherapy treatment. These effects were due to a significant decrease in osteoclastic activity and tumor cell proliferation in the combination treatment group. Transcriptome analysis revealed that combination treatment significantly changes the expression of genes from signaling pathways implicated in tumor progression and bone remodeling. Results from these studies provide a rationale for the continued development of huATN-658 as a monotherapy and in combination with currently approved agents such as Zometa in patients with metastatic breast cancer.

Targeting DNA Hypomethylation in Malignancy by Epigenetic Therapies.

DNA methylation is a chemically reversible epigenetic modification that regulates the chromatin structure and gene expression, and thereby takes part in various cellular processes like embryogenesis, genomic imprinting, X-chromosome inactivation, and genome stability. Alterations in the normal methylation levels of DNA may contribute to the development of pathological conditions like cancer. Even though both hypo- and hypermethylation-mediated abnormalities are prevalent in the cancer genome, the field of cancer epigenetics has been more focused on targeting hypermethylation. As a result, DNA hypomethylation-mediated abnormalities remained relatively less explored, and currently, there are no approved drugs that can be clinically used to target hypomethylation. Understanding the precise role of DNA hypomethylation is not only crucial from a mechanistic point of view but also for the development of pharmacological agents that can reverse the hypomethylated state of the DNA. This chapter focuses on the causes and impact of DNA hypomethylation in the development of cancer and describes the possible ways to pharmacologically target it, especially by using a naturally occurring physiologic agent S-adenosylmethionine (SAM).

DNA Methylation Readers and Cancer: Mechanistic and Therapeutic Applications.

DNA methylation is a major epigenetic process that regulates chromatin structure which causes transcriptional activation or repression of genes in a context-dependent manner. In general, DNA methylation takes place when methyl groups are added to the appropriate bases on the genome by the action of "writer" molecules known as DNA methyltransferases. How these methylation marks are read and interpreted into different functionalities represents one of the main mechanisms through which the genes are switched "ON" or "OFF" and typically involves different types of "reader" proteins that can recognize and bind to the methylated regions. A tightly balanced regulation exists between the "writers" and "readers" in order to mediate normal cellular functions. However, alterations in normal methylation pattern is a typical hallmark of cancer which alters the way methylation marks are written, read and interpreted in different disease states. This unique characteristic of DNA methylation "readers" has identified them as attractive therapeutic targets. In this review, we describe the current state of knowledge on the different classes of DNA methylation "readers" identified thus far along with their normal biological functions, describe how they are dysregulated in cancer, and discuss the various anti-cancer therapies that are currently being developed and evaluated for targeting these proteins.

Identification of an Epigenetic Signature of Osteoporosis in Blood DNA of Postmenopausal Women.

Osteoporosis is one of the most common age-related progressive bone diseases in elderly people. Approximately one in three women and one in five men are predisposed to developing osteoporosis. In postmenopausal women, a reduction in BMD leads to an increased risk of fractures. In the current study, we delineated the DNA methylation signatures in whole blood samples of postmenopausal osteoporotic women. We obtained whole blood DNA from 22 normal women and 22 postmenopausal osteoporotic women (51 to 89 years old) from the Canadian Multicenter Osteoporosis Study (CaMos) cohort. These DNA samples were subjected to Illumina Infinium human methylation 450 K analysis. Illumina 450K raw data were analyzed by Genome Studio software. Analysis of the female participants with early and advanced osteoporosis resulted in the generation of a list of 1233 differentially methylated CpG sites when compared with age-matched normal women. T test, ANOVA, and post hoc statistical analyses were performed, and 77 significantly differentially methylated CpG sites were identified. From the 13 most significant genes, ZNF267, ABLIM2, RHOJ, CDKL5, and PDCD1 were selected for their potential role in bone biology. A weighted polygenic DNA methylation score of these genes predicted osteoporosis at an early stage with high sensitivity and specificity and correlated with measures of bone density. Pyrosequencing analysis of these genes was performed to validate the results obtained from Illumina 450 K methylation analysis. The current study provides proof of principal for the role of DNA methylation in osteoporosis. Using whole blood DNA methylation analysis, women at risk of developing osteoporosis can be identified before a diagnosis of osteoporosis is made using BMD as a screening method. Early diagnosis will help to select patients who might benefit from early therapeutic intervention. © 2018 American Society for Bone and Mineral Research.

DNA methylation signatures of breast cancer in peripheral T-cells.

BACKGROUND: Immune surveillance acts as a defense mechanism in cancer, and its disruption is involved in cancer progression. DNA methylation reflects the phenotypic identity of cells and recent data suggested that DNA methylation profiles of T cells and peripheral blood mononuclear cells (PBMC) are altered in cancer progression. METHODS: We enrolled 19 females with stage 1 and 2, nine with stage 3 and 4 and 9 age matched healthy women. T cells were isolated from peripheral blood and extracted DNA was subjected to Illumina 450 K DNA methylation array analysis. Raw data was analyzed by BMIQ, ChAMP and ComBat followed by validation of identified genes by pyrosequencing. RESULTS: Analysis of data revealed ~ 10,000 sites that correlated with breast cancer progression and established a list of 89 CG sites that were highly correlated (p < 0.01, r > 0.7, r < - 0.7) with breast cancer progression. The vast majority of these sites were hypomethylated and enriched in genes with functions in the immune system. CONCLUSIONS: The study points to the possibility of using DNA methylation signatures as a noninvasive method for early detection of breast cancer and its progression which need to be tested in clinical studies.

Multifaceted Role of the Urokinase-Type Plasminogen Activator (uPA) and Its Receptor (uPAR): Diagnostic, Prognostic, and Therapeutic Applications.

The plasminogen activator (PA) system is an extracellular proteolytic enzyme system associated with various physiological and pathophysiological processes. A large body of evidence support that among the various components of the PA system, urokinase-type plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitor-1 and -2 (PAI-1 and PAI-2) play a major role in tumor progression and metastasis. The binding of uPA with uPAR is instrumental for the activation of plasminogen to plasmin, which in turn initiates a series of proteolytic cascade to degrade the components of the extracellular matrix, and thereby, cause tumor cell migration from the primary site of origin to a distant secondary organ. The components of the PA system show altered expression patterns in several common malignancies, which have identified them as ideal diagnostic, prognostic, and therapeutic targets to reduce cancer-associated morbidity and mortality. This review summarizes the various components of the PA system and focuses on the role of uPA-uPAR in different biological processes especially in the context of malignancy. We also discuss the current state of knowledge of uPA-uPAR-targeted diagnostic and therapeutic strategies for various malignancies.

Methyl donor S-adenosylmethionine (SAM) supplementation attenuates breast cancer growth, invasion, and metastasis in vivo; therapeutic and chemopreventive applications.

DNA hypomethylation coordinately targets various signaling pathways involved in tumor growth and metastasis. At present, there are no approved therapeutic modalities that target hypomethylation. In this regard, we examined the therapeutic plausibility of using universal methyl group donor S-adenosylmethionine (SAM) to block breast cancer development, growth, and metastasis through a series of studies in vitro using two different human breast cancer cell lines (MDA-MB-231 and Hs578T) and in vivo using an MDA-MB-231 xenograft model of breast cancer. We found that SAM treatment caused a significant dose-dependent decrease in cell proliferation, invasion, migration, anchorage-independent growth and increased apoptosis in vitro. These results were recapitulated in vivo where oral administration of SAM reduced tumor volume and metastasis in green fluorescent protein (GFP)-tagged MDA-MB-231 xenograft model. Gene expression analyses validated the ability of SAM to decrease the expression of several key genes implicated in cancer progression and metastasis in both cell lines and breast tumor xenografts. SAM was found to be bioavailable in the serum of experimental animals as determined by enzyme-linked immunosorbent assay and no notable adverse side effects were seen including any change in animal behavior. The results of this study provide compelling evidence to evaluate the therapeutic potential of methylating agents like SAM in patients with breast cancer to reduce cancer-associated morbidity and mortality.

Comparative genomics of two jute species and insight into fibre biogenesis.

Jute (Corchorus sp.) is one of the most important sources of natural fibre, covering ∼80% of global bast fibre production1. Only Corchorus olitorius and Corchorus capsularis are commercially cultivated, though there are more than 100 Corchorus species2 in the Malvaceae family. Here we describe high-quality draft genomes of these two species and their comparisons at the functional genomics level to support tailor-designed breeding. The assemblies cover 91.6% and 82.2% of the estimated genome sizes for C. olitorius and C. capsularis, respectively. In total, 37,031 C. olitorius and 30,096 C. capsularis genes are identified, and most of the genes are validated by cDNA and RNA-seq data. Analyses of clustered gene families and gene collinearity show that jute underwent shared whole-genome duplication ∼18.66 million years (Myr) ago prior to speciation. RNA expression analysis from isolated fibre cells reveals the key regulatory and structural genes involved in fibre formation. This work expands our understanding of the molecular basis of fibre formation laying the foundation for the genetic improvement of jute.

Analyses of Physcomitrella patens Ankyrin Repeat Proteins by Computational Approach.

Ankyrin (ANK) repeat containing proteins are evolutionary conserved and have functions in crucial cellular processes like cell cycle regulation and signal transduction. In this study, through an entirely in silico approach using the first release of the moss genome annotation, we found that at least 54 ANK proteins are present in P. patens. Based on their differential domain composition, the identified ANK proteins were classified into nine subfamilies. Comparative analysis of the different subfamilies of ANK proteins revealed that P. patens contains almost all the known subgroups of ANK proteins found in the other angiosperm species except for the ones having the TPR domain. Phylogenetic analysis using full length protein sequences supported the subfamily classification where the members of the same subfamily almost always clustered together. Synonymous divergence (dS) and nonsynonymous divergence (dN) ratios showed positive selection for the ANK genes of P. patens which probably helped them to attain significant functional diversity during the course of evolution. Taken together, the data provided here can provide useful insights for future functional studies of the proteins from this superfamily as well as comparative studies of ANK proteins.

An endogenous 'non-specific' protein detected by a His-tag antibody is human transcription regulator YY1.

Histidine-tags have been used for a wide variety of experiments including protein purification, Western blots, immunoprecipitation and immunohistochemistry. In our previous studies, we have repeatedly detected a 'non-specific' endogenous protein of about 60 kD in Western blots of protein lysates from HEK293T or HeLa cells using the anti-His-tag antibody (His-probe (H3), catalogue #, SC-8036, Santa Cruz Biotech. Co.) (Yu et al., J. Biol. Chem. 284 (2009) 1505-1513). Here we have immunoprecipitated the protein from HeLa nuclear extracts using the anti-His-tag antibody, excised the 60 kD band and subjected it to LC-MS/MS (Fig. 1). The deduced sequences of two peptides of the protein match the human transcriptional regulator YY1 (Yin and Yang 1, UniProt ID, P25490, Fig. 2), which contains 11 histidine residues in a stretch (from amino acid 70 to 80) at its NH2-terminal region without known functions (Lee et al., Nucleic Acids Res. 23 (1995) 925-931; Bushmeyer et al., J. Biol. Chem. 270 (1995) 30213-30220). Since genes encoding other Histidine-repeat proteins also exist in the genome (Salichs et al., PLoS Genet. 5 (2009) e1000397), it is possible that YY1 might not be the only endogenous protein that could be expressed and recognized by the antibody in different sources of samples in future experiments. The presence of various endogenous histidine-repeat proteins suggests that data from experiments particularly immunostaining using His-tag antibodies need to be interpreted with caution. This might also be useful to the broader scientific community by providing an example for the interpretation of 'non-specific' bands in Western blots.