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.

Expression of the epithelial sodium channel (ENaC) in the endometrium - Implications for fertility in a patient with pseudohypoaldosteronism.

Pseudohypoaldosteronism type 1 (PHA) is a syndrome of unresponsiveness to aldosterone. The severe form of this disease results from mutations in the genes that encode for the epithelial sodium channel subunits, SCNN1A, SCNN1B, and SCNN1G. A PHA patient under our care failed to conceive after many years and IVF trials. Our earlier studies had shown that ENaC is expressed in the female reproductive tract. We hypothesized that a defective ENaC expression may be responsible for the infertility of the patient. To test this hypothesis we examined ENaC expression in endometrial Pipelle biopsy samples from three healthy women and the PHA patient with an Arg508X mutation in the SCNN1A gene. The formalin fixed samples were reacted with anti-ENaCA (alpha subunit) antisera, followed by secondary antibodies to visualize ENaC expression by immunofluorescence. Confocal microscopy imaging of the samples showed strong ENaC immunofluorescence along the luminal border (apical membrane) of the epithelial cells in Pipelle samples from healthy women. In contrast, none of the samples from the PHA patient showed ENaC immunofluorescence. The Arg508X mutation interrupts the transport of ENaC subunits to the cell surface, yet it would not be expected to disrupt ENaC localization in the cytoplasm. In contrast to endometrium where ENaC is localized in the apical membrane of the epithelial cells, in keratinocytes ENaC is expressed in cytoplasmic pools. Therefore, we examined ENaC immunofluorescence in plucked hair follicles. As expected, ENaC immunofluorescence was detected in the cytoplasm of keratinocytes of both normal and PHA samples. Our results support the hypothesis that lack of expression of ENaC on the endometrial surface may be responsible for the infertility of the PHA patient.

SILAC identifies LAD1 as a filamin-binding regulator of actin dynamics in response to EGF and a marker of aggressive breast tumors.

Mutations mimicking growth factor-induced proliferation and motility characterize aggressive subtypes of mammary tumors. To unravel currently unknown players in these processes, we performed phosphoproteomic analysis on untransformed mammary epithelial cells (MCF10A) that were stimulated in culture with epidermal growth factor (EGF). We identified ladinin-1 (LAD1), a largely uncharacterized protein to date, as a phosphorylation-regulated mediator of the EGF-to-ERK pathway. Further experiments revealed that LAD1 mediated the proliferation and migration of mammary cells. LAD1 was transcriptionally induced, phosphorylated, and partly colocalized with actin stress fibers in response to EGF. Yeast two-hybrid, proximity ligation, and coimmunoprecipitation assays revealed that LAD1 bound to actin-cross-linking proteins called filamins. Cosedimentation analyses indicated that LAD1 played a role in actin dynamics, probably in collaboration with the scaffold protein 14-3-3σ (also called SFN). Depletion of LAD1 decreased the expression of transcripts associated with cell survival and inhibited the growth of mammary xenografts in an animal model. Furthermore, LAD1 predicts poor patient prognosis and is highly expressed in aggressive subtypes of breast cancer characterized as integrative clusters 5 and 10, which partly correspond to triple-negative and HER2-positive tumors. Thus, these findings reveal a cytoskeletal component that is critically involved in cell migration and the acquisition of oncogenic attributes in human mammary tumors.

An oligoclonal antibody durably overcomes resistance of lung cancer to third-generation EGFR inhibitors.

Epidermal growth factor receptor (EGFR) mutations identify patients with lung cancer who derive benefit from kinase inhibitors. However, most patients eventually develop resistance, primarily due to the T790M second-site mutation. Irreversible inhibitors (e.g., osimertinib/AZD9291) inhibit T790M-EGFR, but several mechanisms, including a third-site mutation, C797S, confer renewed resistance. We previously reported that a triple mixture of monoclonal antibodies, 3×mAbs, simultaneously targeting EGFR, HER2, and HER3, inhibits T790M-expressing tumors. We now report that 3×mAbs, including a triplet containing cetuximab and trastuzumab, inhibits C797S-expressing tumors. Unlike osimertinib, which induces apoptosis, 3×mAbs promotes degradation of the three receptors and induces cellular senescence. Consistent with distinct mechanisms, treatments combining 3×mAbs plus sub-inhibitory doses of osimertinib synergistically and persistently eliminated tumors. Thus, oligoclonal antibodies, either alone or in combination with kinase inhibitors, might preempt repeated cycles of treatment and rapid emergence of resistance.

Epigenetic mechanisms underlie the crosstalk between growth factors and a steroid hormone.

Crosstalk between growth factors (GFs) and steroid hormones recurs in embryogenesis and is co-opted in pathology, but underlying mechanisms remain elusive. Our data from mammary cells imply that the crosstalk between the epidermal GF and glucocorticoids (GCs) involves transcription factors like p53 and NF-κB, along with reduced pausing and traveling of RNA polymerase II (RNAPII) at both promoters and bodies of GF-inducible genes. Essentially, GCs inhibit positive feedback loops activated by GFs and stimulate the reciprocal inhibitory loops. As expected, no alterations in DNA methylation accompany the transcriptional events instigated by either stimulus, but forced demethylation of regulatory regions broadened the repertoire of GF-inducible genes. We report that enhancers, like some promoters, are poised for activation by GFs and GCs. In addition, within the cooperative interface of the crosstalk, GFs enhance binding of the GC receptor to DNA and, in synergy with GCs, promote productive RNAPII elongation. Reciprocally, within the antagonistic interface GFs hyper-acetylate chromatin at unmethylated promoters and enhancers of genes involved in motility, but GCs hypoacetylate the corresponding regions. In conclusion, unmethylated genomic regions that encode feedback regulatory modules and differentially recruit RNAPII and acetylases/deacetylases underlie the crosstalk between GFs and a steroid hormone.

LIMT is a novel metastasis inhibiting lncRNA suppressed by EGF and downregulated in aggressive breast cancer.

Long noncoding RNAs (lncRNAs) are emerging as regulators of gene expression in pathogenesis, including cancer. Recently, lncRNAs have been implicated in progression of specific subtypes of breast cancer. One aggressive, basal-like subtype associates with increased EGFR signaling, while another, the HER2-enriched subtype, engages a kin of EGFR Based on the premise that EGFR-regulated lncRNAs might control the aggressiveness of basal-like tumors, we identified multiple EGFR-inducible lncRNAs in basal-like normal cells and overlaid them with the transcriptomes of over 3,000 breast cancer patients. This led to the identification of 11 prognostic lncRNAs. Functional analyses of this group uncovered LINC01089 (here renamed LncRNA Inhibiting Metastasis; LIMT), a highly conserved lncRNA, which is depleted in basal-like and in HER2-positive tumors, and the low expression of which predicts poor patient prognosis. Interestingly, EGF rapidly downregulates LIMT expression by enhancing histone deacetylation at the respective promoter. We also find that LIMT inhibits extracellular matrix invasion of mammary cells in vitro and tumor metastasis in vivo In conclusion, lncRNAs dynamically regulated by growth factors might act as novel drivers of cancer progression and serve as prognostic biomarkers.

Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor.

Circular RNAs (circRNAs) are widespread circles of non-coding RNAs with largely unknown function. Because stimulation of mammary cells with the epidermal growth factor (EGF) leads to dynamic changes in the abundance of coding and non-coding RNA molecules, and culminates in the acquisition of a robust migratory phenotype, this cellular model might disclose functions of circRNAs. Here we show that circRNAs of EGF-stimulated mammary cells are stably expressed, while mRNAs and microRNAs change within minutes. In general, the circRNAs we detected are relatively long-lived and weakly expressed. Interestingly, they are almost ubiquitously co-expressed with the corresponding linear transcripts, and the respective, shared promoter regions are more active compared to genes producing linear isoforms with no detectable circRNAs. These findings imply that altered abundance of circRNAs, unlike changes in the levels of other RNAs, might not play critical roles in signaling cascades and downstream transcriptional networks that rapidly commit cells to specific outcomes.

Diurnal suppression of EGFR signalling by glucocorticoids and implications for tumour progression and treatment.

Signal transduction by receptor tyrosine kinases (RTKs) and nuclear receptors for steroid hormones is essential for body homeostasis, but the cross-talk between these receptor families is poorly understood. We observed that glucocorticoids inhibit signalling downstream of EGFR, an RTK. The underlying mechanism entails suppression of EGFR's positive feedback loops and simultaneous triggering of negative feedback loops that normally restrain EGFR. Our studies in mice reveal that the regulation of EGFR's feedback loops by glucocorticoids translates to circadian control of EGFR signalling: EGFR signals are suppressed by high glucocorticoids during the active phase (night-time in rodents), while EGFR signals are enhanced during the resting phase. Consistent with this pattern, treatment of animals bearing EGFR-driven tumours with a specific kinase inhibitor was more effective if administered during the resting phase of the day, when glucocorticoids are low. These findings support a circadian clock-based paradigm in cancer therapy.

Epithelial sodium channels (ENaC) are uniformly distributed on motile cilia in the oviduct and the respiratory airways.

Epithelial sodium channels (ENaCs) are located on the apical surface of cells and funnel Na(+) ions from the lumen into the cell. ENaC function also regulates extracellular fluid volume as water flows across membranes accompanying Na(+) ions to maintain osmolarity. To examine the sites of expression and intracellular localization of ENaC, we generated polyclonal antibodies against the extracellular domain of human α-ENaC subunit that we expressed in E. coli. Three-dimensional (3D) confocal microscopy of immunofluorescence using these antibodies for the first time revealed that ENaCs are uniformly distributed on the ciliary surface in all epithelial cells with motile cilia lining the bronchus in human lung and female reproductive tract, all along the fimbrial end of the fallopian tube, the ampulla and rare cells in the uterine glands. Quantitative analysis indicated that cilia increase cell surface area >70-fold and the amount of ENaC on cilia is >1,000-fold higher than on non-ciliated cell surface. These findings indicate that ENaC functions as a regulator of the osmolarity of the periciliary fluid bathing the cilia. In contrast to ENaC, cystic fibrosis transmembrane conductance regulator (CFTR) that channels chloride ions from the cytoplasm to the lumen is located mainly on the apical side, but not on cilia. The cilial localization of ENaC requires reevaluation of the mechanisms of action of CFTR and other modulators of ENaC function. ENaC on motile cilia should be essential for diverse functions of motile cilia, such as germ cell transport, fertilization, implantation, clearance of respiratory airways and cell migration.