Microarray analysis reveals ONC201 mediated differential mechanisms of CHOP gene regulation in metastatic and nonmetastatic colorectal cancer cells

The imipramine ONC201 has antiproliferative effects in several cancer cell types and activates integrated stress response pathway associated with the induction of Damage Inducible Transcript 3 (DDIT3, also known as C/EBP homologous protein or CHOP). We investigated the signaling pathways through which ONC201/CHOP crosstalk is regulated in ONC201-treated nonmetastatic and metastatic cancer cell lines (Dukes' type B colorectal adenocarcinoma nonmetastatic SW480 and metastatic LS-174T cells, respectively). Cell proliferation and apoptosis were evaluated by MTT assays and flow cytometry, gene expression was assessed by Affymetrix microarray, signaling pathway perturbations were assessed in silico, and key regulatory proteins were validated by Western blotting. Unlike LS-174T cells, SW480 cells were resistant to ONC201 treatment; Gene Ontology analysis of differentially expressed genes showed that cellular responsiveness to ONC201 treatment also differed substantially. In both ONC201-treated cell lines, CHOP expression was upregulated; however, its upstream regulatory mechanisms were perturbed. Although, PERK, ATF6 and IRE1 ER-stress pathways upregulated CHOP in both cell types, the Bak/Bax pathway regulated CHOP only LS-174T cells. Additionally, CHOP RNA splicing profiles varied between cell lines; these were further modified by ONC201 treatment. In conclusion, we delineated the signaling mechanisms by which CHOP expression is regulated in ONC201-treated non-metastatic and metastatic colorectal cell lines. The observed differences could be related to cellular plasticity and metabolic reprogramming, nevertheless, detailed mechanistic studies are required for further validations.

Vaccine responses in ageing and chronic viral infection

Lay Summary Improved life expectancy in recent years has led to a growing population of adults over the age of 60. Age is commonly associated with increased inflammatory conditions and infections. Similar immunological changes have been observed during chronic infections, in particular HIV, where this is compounded by the success of antiretroviral therapy that has increased the number of people living with HIV into their sixties and beyond. The increased susceptibility of these groups to infection makes vaccination all the more important. However, the alterations to their immune systems call into question how effective those vaccinations may be. Here we discuss vaccine efficacy within elderly and chronically infected populations and investigate the immunological changes that may impact vaccine responsiveness. Over the last few decades, changing population demographics have shown that there is a growing number of individuals living past the age of 60. With this expanding older population comes an increase in individuals that are more susceptible to chronic illness and disease. An important part of maintaining health in this population is through prophylactic vaccination, however, there is growing evidence that vaccines may be less effective in the elderly. Furthermore, with the success of anti-viral therapies, chronic infections such as HIV are becoming increasingly prevalent in older populations and present a relatively unstudied population with respect to the efficacy of vaccination. Here we will examine the evidence for age-associated reduction in antibody and cellular responsiveness to a variety of common vaccines, and investigate the underlying causes attributed to this phenomenon, such as inflammation and senescence. We will also discuss the impact of chronic viral infections on immune responses in both young and elderly patients, particularly those living with HIV, and how this affects vaccinations in these populations.

MNK1 and MNK2 enforce expression of E2F1, FOXM1, and WEE1 to drive soft tissue sarcoma

Soft tissue sarcoma (STS) is a heterogeneous disease that arises from connective tissues. Clinical outcome of patients with advanced tumors especially de-differentiated liposarcoma and uterine leiomyosarcoma remains unsatisfactory, despite intensive treatment regimens including maximal surgical resection, radiation, and chemotherapy. MAP kinase-interacting serine/threonine-protein kinase 1 and 2 (MNK1/2) have been shown to contribute to oncogenic translation via phosphorylation of eukaryotic translation initiation factor 4E (eIF4E). However, little is known about the role of MNK1/2 and their downstream targets in STS. In this study, we show that depletion of either MNK1 or MNK2 suppresses cell viability, anchorage-independent growth, and tumorigenicity of STS cells. We also identify a compelling antiproliferative efficacy of a novel, selective MNK inhibitor ETC-168. Cellular responsiveness of STS cells to ETC-168 correlates positively with that of phosphorylated ribosomal protein S6 (RPS6). Mirroring MNK1/2 silencing, ETC-168 treatment strongly blocks eIF4E phosphorylation and represses expression of sarcoma-driving onco-proteins including E2F1, FOXM1, and WEE1. Moreover, combination of ETC-168 and MCL1 inhibitor S63845 exerts a synergistic antiproliferative activity against STS cells. In summary, our study reveals crucial roles of MNK1/2 and their downstream targets in STS tumorigenesis. Our data encourage further clinical translation of MNK inhibitors for STS treatment.

Endosomal cAMP production broadly impacts the cellular phosphoproteome

Endosomal signaling from G protein-coupled receptors (GPCRs) has emerged as a novel paradigm with important pharmacological and physiological implications. Yet, our knowledge of the functional consequences of activating intracellular GPCRs is incomplete. To address this gap, we combined an optogenetic approach for site-specific generation of the prototypical second messenger cyclic AMP (cAMP) with unbiased mass spectrometry-based analysis of phosphoproteomic effects. We identified 218 unique, high-confidence sites whose phosphorylation is either increased or decreased in response to cAMP production. We next determined that cAMP produced from endosomes led to more robust changes in phosphorylation than cAMP produced from the plasma membrane. Remarkably, this was true for the entire repertoire of identified targets, and irrespective of their annotated sub-cellular localization. Furthermore, we identified a particularly strong endosome bias for a subset of proteins that are dephosphorylated in response to cAMP. Through bioinformatics analysis, we established these targets as putative substrates for protein phosphatase 2A (PP2A), and we propose compartmentalized activation of PP2A-B56δ as the likely underlying mechanism. Altogether, our study extends the concept that endosomal signaling is a significant functional contributor to cellular responsiveness by establishing a unique role for localized cAMP production in defining categorically distinct phosphoresponses.

An optimized toolbox for the optogenetic control of intracellular transport

Cellular functioning relies on active transport of organelles by molecular motors. To explore how intracellular organelle distributions affect cellular functions, several optogenetic approaches enable organelle repositioning through light-inducible recruitment of motors to specific organelles. Nonetheless, robust application of these methods in cellular populations without side effects has remained challenging. Here, we introduce an improved toolbox for optogenetic control of intracellular transport that optimizes cellular responsiveness and limits adverse effects. To improve dynamic range, we employed improved optogenetic heterodimerization modules and engineered a photosensitive kinesin-3, which is activated upon blue light–sensitive homodimerization. This opto-kinesin prevented motor activation before experimental onset, limited dark-state activation, and improved responsiveness. In addition, we adopted moss kinesin-14 for efficient retrograde transport with minimal adverse effects on endogenous transport. Using this optimized toolbox, we demonstrate robust reversible repositioning of (endogenously tagged) organelles within cellular populations. More robust control over organelle motility will aid in dissecting spatial cell biology and transport-related diseases.

Feedback regulation of BMP signaling by C. elegans cuticle collagens

Cellular responsiveness to environmental cues, including changes in extracellular matrix (ECM), is critical for normal processes such as development and wound healing, but can go awry, as in oncogenesis and fibrosis. One type of molecular pathway allowing this responsiveness is the bone morphogenetic protein (BMP) signaling pathway. Due to their broad and potent functions, BMPs and their signaling pathways are highly regulated at multiple levels. In Caenorhabditis elegans, the BMP ligand DBL-1 is a major regulator of body size. We have previously shown that DBL-1/BMP signaling determines body size through transcriptional regulation of cuticle collagen genes. We have now obtained evidence of feedback regulation of DBL-1/BMP by collagen genes. We analyzed four DBL-1-regulated collagen genes that affect body size. Here we show that inactivation of any one of these cuticle collagen genes reduces DBL-1/BMP signaling, as measured by a Smad activity reporter. Furthermore, we find that depletion of these collagens reduces GFP::DBL-1 fluorescence, and acts unexpectedly at the level of dbl-1 transcription. We therefore conclude that cuticle, a type of ECM, impinges on DBL-1/BMP expression and signaling. In contrast to other characterized examples, however, the feedback regulation of DBL-1/BMP signaling by collagens is likely to be contact-independent, due to the physical separation of the cuticle from DBL-1-expressing cells in the ventral nerve cord. Our results provide an entry point into a novel mechanism of regulation of BMP signaling, with broader implications for mechanical regulation of gene expression in general.

Casein Kinase 1α Mediates the Degradation of Receptors for Type I and Type II Interferons Caused by Hemagglutinin of Influenza A Virus

ABSTRACTAlthough influenza A virus (IAV) evades cellular defense systems to effectively propagate in the host, the viral immune-evasive mechanisms are incompletely understood. Our recent data showed that hemagglutinin (HA) of IAV induces degradation of type I IFN receptor 1 (IFNAR1). Here, we demonstrate that IAV HA induces degradation of type II IFN (IFN-γ) receptor 1 (IFNGR1), as well as IFNAR1, via casein kinase 1α (CK1α), resulting in the impairment of cellular responsiveness to both type I and II IFNs. IAV infection or transient HA expression induced degradation of both IFNGR1 and IFNAR1, whereas HA gene-deficient IAV failed to downregulate the receptors. IAV HA caused the phosphorylation and ubiquitination of IFNGR1, leading to the lysosome-dependent degradation of IFNGR1. Influenza viral HA strongly decreased cellular sensitivity to type II IFNs, as it suppressed the activation of STAT1 and the induction of IFN-γ-stimulated genes in response to exogenously supplied recombinant IFN-γ. Importantly, CK1α, but not p38 MAP kinase or protein kinase D2, was proven to be critical for HA-induced degradation of both IFNGR1 and IFNAR1. Pharmacologic inhibition of CK1α or small interfering RNA (siRNA)-based knockdown of CK1α repressed the degradation processes of both IFNGR1 and IFNAR1 triggered by IAV infection. Further, CK1α was shown to be pivotal for proficient replication of IAV. Collectively, the results suggest that IAV HA induces degradation of IFN receptors via CK1α, creating conditions favorable for viral propagation. Therefore, the study uncovers a new immune-evasive pathway of influenza virus.IMPORTANCEInfluenza A virus (IAV) remains a grave threat to humans, causing seasonal and pandemic influenza. Upon infection, innate and adaptive immunity, such as the interferon (IFN) response, is induced to protect hosts against IAV infection. However, IAV seems to be equipped with tactics to evade the IFN-mediated antiviral responses, although the detailed mechanisms need to be elucidated. In the present study, we show that IAV HA induces the degradation of the type II IFN receptor IFNGR1 and thereby substantially attenuates cellular responses to IFN-γ. Of note, a cellular kinase, casein kinase 1α (CK1α), is crucial for IAV HA-induced degradation of both IFNGR1 and IFNAR1. Accordingly, CK1α is proven to positively regulate IAV propagation. Thus, this study unveils a novel strategy employed by IAV to evade IFN-mediated antiviral activities. These findings may provide new insights into the interplay between IAV and host immunity to impact influenza virus pathogenicity.