Glycolytic Pfkp acts as a Lin41 protein kinase to promote endodermal differentiation of embryonic stem cells.

Unveiling the principles governing embryonic stem cell (ESC) differentiation into specific lineages is critical for understanding embryonic development and for stem cell applications in regenerative medicine. Here, we establish an intersection between LIF-Stat3 signaling that is essential for maintaining murine (m) ESCs pluripotency, and the glycolytic enzyme, the platelet isoform of phosphofructokinase (Pfkp). In the pluripotent state, Stat3 transcriptionally suppresses Pfkp in mESCs while manipulating the cells to lift this repression results in differentiation towards the ectodermal lineage. Pfkp exhibits substrate specificity changes to act as a protein kinase, catalyzing serine phosphorylation of the developmental regulator Lin41. Such phosphorylation stabilizes Lin41 by impeding its autoubiquitination and proteasomal degradation, permitting Lin41-mediated binding and destabilization of mRNAs encoding ectodermal specification markers to favor the expression of endodermal specification genes. This provides new insights into the wiring of pluripotency-differentiation circuitry where Pfkp plays a role in germ layer specification during mESC differentiation.

NIPSNAP1 directs dual mechanisms to restrain senescence in cancer cells.

BACKGROUND: Although the executive pathways of senescence are known, the underlying control mechanisms are diverse and not fully understood, particularly how cancer cells avoid triggering senescence despite experiencing exacerbated stress conditions within the tumor microenvironment. METHODS: Mass spectrometry (MS)-based proteomic screening was used to identify differentially regulated genes in serum-starved hepatocellular carcinoma cells and RNAi employed to determine knockdown phenotypes of prioritized genes. Thereafter, gene function was investigated using cell proliferation assays (colony-formation, CCK-8, Edu incorporation and cell cycle) together with cellular senescence assays (SA-ß-gal, SAHF and SASP). Gene overexpression and knockdown techniques were applied to examine mRNA and protein regulation in combination with luciferase reporter and proteasome degradation assays, respectively. Flow cytometry was applied to detect changes in cellular reactive oxygen species (ROS) and in vivo gene function examined using a xenograft model. RESULTS: Among the genes induced by serum deprivation, NIPSNAP1 was selected for investigation. Subsequent experiments revealed that NIPSNAP1 promotes cancer cell proliferation and inhibits P27-dependent induction of senescence via dual mechanisms. Firstly, NIPSNAP1 maintains the levels of c-Myc by sequestering the E3 ubiquitin ligase FBXL14 to prevent the proteasome-mediated turnover of c-Myc. Intriguingly, NIPSNAP1 levels are restrained by transcriptional repression mediated by c-Myc-Miz1, with repression lifted in response to serum withdrawal, thus identifying feedback regulation between NIPSNAP1 and c-Myc. Secondly, NIPSNAP1 was shown to modulate ROS levels by promoting interactions between the deacetylase SIRT3 and superoxide dismutase 2 (SOD2). Consequent activation of SOD2 serves to maintain cellular ROS levels below the critical levels required to induce cell cycle arrest and senescence. Importantly, the actions of NIPSNAP1 in promoting cancer cell proliferation and preventing senescence were recapitulated in vivo using xenograft models. CONCLUSIONS: Together, these findings reveal NIPSNAP1 as an important mediator of c-Myc function and a negative regulator of cellular senescence. These findings also provide a theoretical basis for cancer therapy where targeting NIPSNAP1 invokes cellular senescence.

P21-Activated Kinase 4 Pak4 Maintains Embryonic Stem Cell Pluripotency via Akt Activation.

Exploiting the pluripotent properties of embryonic stem cells (ESCs) holds great promise for regenerative medicine. Nevertheless, directing ESC differentiation into specialized cell lineages requires intricate control governed by both intrinsic and extrinsic factors along with the actions of specific signaling networks. Here, we reveal the involvement of the p21-activated kinase 4 (Pak4), a serine/threonine kinase, in sustaining murine ESC (mESC) pluripotency. Pak4 is highly expressed in R1 ESC cells compared with embryonic fibroblast cells and its expression is progressively decreased during differentiation. Manipulations using knockdown and overexpression demonstrated a positive relationship between Pak4 expression and the clonogenic potential of mESCs. Moreover, ectopic Pak4 expression increases reprogramming efficiency of Oct4-Klf4-Sox2-Myc-induced pluripotent stem cells (iPSCs) whereas Pak4-knockdown iPSCs were largely incapable of generating teratomas containing mesodermal, ectodermal and endodermal tissues, indicative of a failure in differentiation. We further establish that Pak4 expression in mESCs is transcriptionally driven by the core pluripotency factor Nanog which recognizes specific binding motifs in the Pak4 proximal promoter region. In turn, the increased levels of Pak4 in mESCs fundamentally act as an upstream activator of the Akt pathway. Pak4 directly binds to and phosphorylates Akt at Ser473 with the resulting Akt activation shown to attenuate downstream GSK3ß signaling. Thus, our findings indicate that the Nanog-Pak4-Akt signaling axis is essential for maintaining mESC self-renewal potential with further importance shown during somatic cell reprogramming where Pak4 appears indispensable for multi-lineage specification.

LncRNA GUARDIN suppresses cellular senescence through a LRP130-PGC1α-FOXO4-p21-dependent signaling axis.

The long noncoding RNA GUARDIN functions to protect genome stability. Inhibiting GUARDIN expression can alter cell fate decisions toward senescence or apoptosis, but the underlying molecular signals are unknown. Here, we show that GUARDIN is an essential component of a transcriptional repressor complex involving LRP130 and PGC1α. GUARDIN acts as a scaffold to stabilize LRP130/PGC1α heterodimers and their occupancy at the FOXO4 promotor. Destabilizing this complex by silencing of GUARDIN, LRP130, or PGC1α leads to increased expression of FOXO4 and upregulation of its target gene p21, thereby driving cells into senescence. We also found that GUARDIN expression was induced by rapamycin, an agent that suppresses cell senescence. FOS-like antigen 2 (FOSL2) acts as a transcriptional repressor of GUARDIN, and lower FOSL2 levels in response to rapamycin correlate with increased levels of GUARDIN. Together, these results demonstrate that GUARDIN inhibits p21-dependent senescence through a LRP130-PGC1α-FOXO4 signaling axis, and moreover, GUARDIN contributes to the anti-aging activities of rapamycin.

Targeting high circDNA2v levels in colorectal cancer induces cellular senescence and elicits an anti-tumor secretome.

The efficacy of immunotherapy against colorectal cancer (CRC) is impaired by insufficient immune cell recruitment into the tumor microenvironment. Our study shows that targeting circDNA2v, a circular RNA commonly overexpressed in CRC, can be exploited to elicit cytotoxic T cell recruitment. circDNA2v functions through binding to IGF2BP3, preventing its ubiquitination, and prolonging the IGF2BP3 half-life, which in turn sustains mRNA levels of the protooncogene c-Myc. Targeting circDNA2v by gene silencing downregulates c-Myc to concordantly induce tumor cell senescence and the release of proinflammatory mediators. Production of CXCL10 and interleukin-9 by CRC cells is elicited through JAK-STAT1 signaling, in turn promoting the chemotactic and cytolytic activities of CD8+ T cells. Clinical evidence associates increased circDNA2v expression in CRC tissues with reductions in CD8+ T cell infiltration and worse outcomes. The regulatory relationship between circDNA2v, cellular senescence, and tumor-infiltrating lymphocytes thus provides a rational approach for improving immunotherapy in CRC.

Reciprocal regulation of lncRNA MEF and c-Myc drives colorectal cancer tumorigenesis.

More than half of all cancers demonstrate aberrant c-Myc expression, making this arguably the most important human oncogene. Deregulated long non-coding RNAs (lncRNAs) are also commonly implicated in tumorigenesis, and some limited examples have been established where lncRNAs act as biological tuners of c-Myc expression and activity. Here, we demonstrate that the lncRNA denoted c-Myc Enhancing Factor (MEF) enjoys a cooperative relationship with c-Myc, both as a transcriptional target and driver of c-Myc expression. Mechanistically, MEF functions by binding to and stabilizing the expression of hnRNPK in colorectal cancer cells. The MEF-hnRNPK interaction serves to disrupt binding between hnRNPK and the E3 ubiquitin ligase TRIM25, which attenuates TRIM25-dependent hnRNPK ubiquitination and proteasomal destruction. In turn, the stabilization of hnRNPK through MEF enhances c-Myc expression by augmenting the translation c-Myc. Moreover, modulating the expression of MEF in shRNA-mediated knockdown and overexpression studies revealed that MEF expression is essential for colorectal cancer cell proliferation and survival, both in vitro and in vivo. From the clinical perspective, we show that MEF expression is differentially increased in colorectal cancer tissues compared to normal adjacent tissues. Further, correlations exist between MEF, c-Myc, and hnRNPK suggesting the MEF-c-Myc positive feedback loop is active in patients. Together these data demonstrate that MEF is a pivotal partner of the c-Myc network and propose MEF as a valuable therapeutic target for colorectal cancer.

IFRD1 promotes tumor cells "low-cost" survival under glutamine starvation via inhibiting histone H1.0 nucleophagy.

Glutamine addiction represents a metabolic vulnerability of cancer cells; however, effective therapeutic targeting of the pathways involved remains to be realized. Here, we disclose the critical role of interferon-related developmental regulator 1 (IFRD1) in the adaptive survival of hepatocellular carcinoma (HCC) cells during glutamine starvation. IFRD1 is induced under glutamine starvation to inhibit autophagy by promoting the proteasomal degradation of the key autophagy regulator ATG14 in a TRIM21-dependent manner. Conversely, targeting IFRD1 in the glutamine-deprived state increases autophagy flux, triggering cancer cell exhaustive death. This effect largely results from the nucleophilic degradation of histone H1.0 and the ensuing unchecked increases in ribosome and protein biosynthesis associated with globally enhanced chromatin accessibility. Intriguingly, IFRD1 depletion in preclinical HCC models synergizes with the treatment of the glutaminase-1 selective inhibitor CB-839 to potentiate the effect of limiting glutamine. Together, our findings reveal how IFRD1 supports the adaptive survival of cancer cells under glutamine starvation, further highlighting the potential of IFRD1 as a therapeutic target in anti-cancer applications.

Clinical implications of lncRNA LINC-PINT in cancer.

Long noncoding RNAs (lncRNAs) possess the potential for therapeutic targeting to treat many disorders, including cancers. Several RNA-based therapeutics (ASOs and small interfering RNAs) have gained FDA approval over the past decade. And with their potent effects, lncRNA-based therapeutics are of emerging significance. One important lncRNA target is LINC-PINT, with its universalized functions and relationship with the famous tumor suppressor gene TP53. Establishing clinical relevance, much like p53, the tumor suppressor activity of LINC-PINT is implicated in cancer progression. Moreover, several molecular targets of LINC-PINT are directly or indirectly used in routine clinical practice. We further associate LINC-PINT with immune responses in colon adenocarcinoma, proposing the potential utility of LINC-PINT as a novel biomarker of immune checkpoint inhibitors. Collectively, current evidence suggests LINC-PINT can be considered for use as a diagnostic/prognostic marker for cancer and several other diseases.

Sestrin2-mediated disassembly of stress granules dampens aerobic glycolysis to overcome glucose starvation.

Sestrins are a small gene family of pleiotropic factors whose actions promote cell adaptation to a range of stress conditions. In this report we disclose the selective role of Sestrin2 (SESN2) in dampening aerobic glycolysis to adapt to limiting glucose conditions. Removal of glucose from hepatocellular carcinoma (HCC) cells inhibits glycolysis associated with the downregulation of the rate-limiting glycolytic enzyme hexokinase 2 (HK2). Moreover, the accompanying upregulation of SESN2 through an NRF2/ATF4-dependent mechanism plays a direct role in HK2 regulation by destabilizing HK2 mRNA. We show SESN2 competes with insulin like growth factor 2 mRNA binding protein 3 (IGF2BP3) for binding with the 3'-UTR region of HK2 mRNA. Interactions between IGF2BP3 and HK2 mRNA result in their coalescence into stress granules via liquid-liquid phase separation (LLPS), a process which serves to stabilize HK2 mRNA. Conversely, the enhanced expression and cytoplasmic localization of SESN2 under glucose deprivation conditions favors the downregulation of HK2 levels via decreases in the half-life of HK2 mRNA. The resulting dampening of glucose uptake and glycolytic flux inhibits cell proliferation and protect cells from glucose starvation-induced apoptotic cell death. Collectively, our findings reveal an intrinsic survival mechanism allowing cancer cells to overcome chronic glucose shortages, also providing new mechanistic insights into SESN2 as an RNA-binding protein with a role in reprogramming of cancer cell metabolism.

The diapause-like colorectal cancer cells induced by SMC4 attenuation are characterized by low proliferation and chemotherapy insensitivity.

In response to adverse environmental conditions, embryonic development may reversibly cease, a process termed diapause. Recent reports connect this phenomenon with the non-genetic responses of tumors to chemotherapy, but the mechanisms involved are poorly understood. Here, we establish a multifarious role for SMC4 in the switching of colorectal cancer cells to a diapause-like state. SMC4 attenuation promotes the expression of three investment phase glycolysis enzymes increasing lactate production while also suppressing PGAM1. Resultant high lactate levels increase ABC transporter expression via histone lactylation, rendering tumor cells insensitive to chemotherapy. SMC4 acts as co-activator of PGAM1 transcription, and the coordinate loss of SMC4 and PGAM1 affects F-actin assembly, inducing cytokinesis failure and polyploidy, thereby inhibiting cell proliferation. These insights into the mechanisms underlying non-genetic chemotherapy resistance may have significant implications for the field, advancing our understanding of aerobic glycolysis functions in tumor and potentially informing future therapeutic strategies.

Vanguard is a Glucose Deprivation-Responsive Long Non-Coding RNA Essential for Chromatin Remodeling-Reliant DNA Repair.

Glucose metabolism contributes to DNA damage response pathways by regulating chromatin remodeling, double-strand break (DSB) repair, and redox homeostasis, although the underlying mechanisms are not fully established. Here, a previously uncharacterized long non-coding RNA is revealed that is call Vanguard which acts to promote HMGB1-dependent DNA repair in association with changes in global chromatin accessibility. Vanguard expression is maintained in cancer cells by SP1-dependent transcription according to glucose availability and cellular adenosine triphosphate (ATP) levels. Vanguard promotes complex formation between HMGB1 and HDAC1, with the resulting deacetylation of HMGB1 serving to maintain its nuclear localization and DSB repair function. However, Vanguard downregulation under glucose limiting conditions promotes HMGB1 translocation from the nucleus, increasing DNA damage, and compromising cancer cell growth and viability. Moreover, Vanguard silencing increases the effectiveness of poly (ADP-ribose) polymerase inhibitors against breast cancer cells with wild-type breast cancer gene-1 status, suggesting Vanguard as a potential therapeutic target.

PINTology: A short history of the lncRNA LINC-PINT in different diseases.

LINC-PINT is a p53-induced long intergenic noncoding transcript that plays a crucial role in many diseases, especially cancer. This long noncoding RNA (lncRNA) gene produces in total 102 (LNCipedia) alternatively spliced variants (LINC-PINT:1 to LINC-PINT:102). The functions of known variants include RNA transcripts, host transcripts for circular RNA (circRNA) generation and as sources for the translation of short peptides. In most human tumors, LINC-PINT is down-regulated where it serves as a tumor suppressor. However, the diversity of its functions in other maladies signifies its general clinical importance. Current LINC-PINT molecular functions include RNA-protein interactions, miRNA sponging and epigenetic modulation with these mechanisms operating in different cellular contexts to exert effects on biological processes ranging from DNA damage responses, cell cycle and growth arrest, senescence, cell migration and invasion, and apoptosis. Genetic polymorphisms in LINC-PINT have also been functionally associated with cancer and other pathologies including the autoimmune diseases pemphigus foliaceus and arthritis. Hence, LINC-PINT shows great potential as a clinical biomarker, especially for the diagnosis and prognosis of cancer. In this review, we explore the current knowledge highlighting the distinctive molecular functions of LINC-PINT in specific cancers and other disease states. This article is categorized under: RNA in Disease and Development > RNA in Disease.

LncRNA GIRGL drives CAPRIN1-mediated phase separation to suppress glutaminase-1 translation under glutamine deprivation.

Glutamine constitutes an essential source of both carbon and nitrogen for numerous biosynthetic processes. The first and rate-limiting step of glutaminolysis involves the generation of glutamate from glutamine, catalyzed by glutaminase-1 (GLS1). Shortages of glutamine result in reductions in GLS1, but the underlying mechanisms are not fully known. Here, we characterize a long noncoding RNA, GIRGL (glutamine insufficiency regulator of glutaminase lncRNA), that is induced upon glutamine starvation. Manipulating GIRGL revealed a relationship between its expression and the translational suppression of GLS1. Cellular GIRGL levels are balanced by a combination of transactivation by c-JUN together with negative stability regulation via HuR/Ago2. Increased levels of GIRGL in the absence of glutamine drive formation of a complex between dimers of CAPRIN1 and GLS1 mRNA, serving to promote liquid-liquid phase separation of CAPRIN1 and inducing stress granule formation. Suppressing GLS1 mRNA translation enables cancer cells to survive under prolonged glutamine deprivation stress.

lncRNA TRMP-S directs dual mechanisms to regulate p27-mediated cellular senescence.

Long noncoding RNAs (lncRNAs) undergo extensive alternative splicing, but little is known about isoform functions. A prior investigation of lncRNA RP11-369C8.1 reported that its splice variant TRMP suppressed p27 translation through PTBP1. Here we characterize a second major splice variant, TRMP-S (short variant), whose enforced loss promotes cancer cell-cycle arrest and p27-dependent entry into cellular senescence. Remarkably, despite sharing a single common exon with TRMP, TRMP-S restrains p27 expression through distinct mechanisms. First, TRMP-S stabilizes UHRF1 protein levels, an epigenetic inhibitor of p27, by promoting interactions between UHRF1 and its deubiquitinating enzyme USP7. Alternatively, binding interactions between TRMP-S and FUBP3 prevent p53 mRNA interactions with RPL26 ribosomal protein, the latter essential for promoting p53 translation with ensuing suppression of p53 translation limiting p27 expression. Significantly, as TRMP-S is itself transactivated by p53, this identifies negative feedback regulation between p53 and TRMP-S. Different splicing variants of the RP11-369C8.1 gene thereby exert distinct roles that converge on the homeostatic control of p27 expression, providing an important precedent for understanding the actions of alternatively spliced lncRNAs.

DDIT3 Directs a Dual Mechanism to Balance Glycolysis and Oxidative Phosphorylation during Glutamine Deprivation.

Extracellular glutamine represents an important energy source for many cancer cells and its metabolism is intimately involved in maintaining redox homeostasis. The heightened metabolic activity within tumor tissues can result in glutamine deficiency, necessitating metabolic reprogramming responses. Here, dual mechanisms involving the stress-responsive transcription factor DDIT3 (DNA damage induced transcript 3) that establishes an interrelationship between glycolysis and mitochondrial respiration are revealed. DDIT3 is induced during glutamine deprivation to promote glycolysis and adenosine triphosphate production via suppression of the negative glycolytic regulator TIGAR. In concert, a proportion of the DDIT3 pool translocates to the mitochondria and suppresses oxidative phosphorylation through LONP1-mediated down-regulation of COQ9 and COX4. This in turn dampens the sustained levels of reactive oxygen species that follow glutamine withdrawal. Together these mechanisms constitute an adaptive survival mechanism permitting tumor cells to survive metabolic stress induced by glutamine starvation.

ASIC1 and ASIC3 mediate cellular senescence of human nucleus pulposus mesenchymal stem cells during intervertebral disc degeneration.

Stem cell approaches have become an attractive therapeutic option for intervertebral disc degeneration (IVDD). Nucleus pulposus mesenchymal stem cells (NP-MSCs) participate in the regeneration and homeostasis of the intervertebral disc (IVD), but the molecular mechanisms governing these processes remain to be elucidated. Acid-sensing ion channels (ASICs) which act as key receptors for extracellular protons in central and peripheral neurons, have been implicated in IVDD where degeneration is associated with reduced microenvironmental pH. Here we show that ASIC1 and ASIC3, but not ASIC2 and ASIC4 are upregulated in human IVDs according to the degree of clinical degeneration. Subjecting IVD-derived NP-MSCs to pH 6.6 culture conditions to mimic pathological IVD changes resulted in decreased cell proliferation that was associated with cell cycle arrest and induction of senescence. Key molecular changes observed were increased expression of p53, p21, p27, p16 and Rb1. Instructively, premature senescence in NP-MSCs could be largely alleviated using ASIC inhibitors, suggesting both ASIC1 and ASIC3 act decisively upstream to activate senescence programming pathways including p53-p21/p27 and p16-Rb1 signaling. These results highlight the potential of ASIC inhibitors as a therapeutic approach for IVDD and broadly define an in vitro system that can be used to evaluate other IVDD therapies.

Antimicrobial Activity of Lemongrass Essential Oil (Cymbopogon flexuosus) and Its Active Component Citral Against Dual-Species Biofilms of Staphylococcus aureus and Candida Species.

Compared to mono-species biofilm, biofilms formed by cross-kingdom pathogens are more refractory to conventional antibiotics, thus complicating clinical treatment and causing significant morbidity. Lemongrass essential oil and its bioactive component citral were previously demonstrated to possess strong antimicrobial efficacy against pathogenic bacteria and fungi. However, their effects on polymicrobial biofilms remain to be determined. In this study, the efficacy of lemongrass (Cymbopogon flexuosus) essential oil and its bioactive part citral against dual-species biofilms formed by Staphylococcus aureus and Candida species was evaluated in vitro. Biofilm staining and viability test showed both lemongrass essential oil and citral were able to reduce biofilm biomass and cell viability of each species in the biofilm. Microscopic examinations showed these agents interfered with adhesive characteristics of each species and disrupted biofilm matrix through counteracting nucleic acids, proteins and carbohydrates in the biofilm. Moreover, transcriptional analyses indicated citral downregulated hyphal adhesins and virulent factors of Candida albicans, while also reducing expression of genes involved in quorum sensing, peptidoglycan and fatty acids biosynthesis of S. aureus. Taken together, our results demonstrate the potential of lemongrass essential oil and citral as promising agents against polymicrobial biofilms as well as the underlying mechanisms of their activity in this setting.

TP53LNC-DB, the database of lncRNAs in the p53 signalling network.

The TP53 gene product, p53, is a pleiotropic transcription factor induced by stress, which functions to promote cell cycle arrest, apoptosis and senescence. Genome-wide profiling has revealed an extensive system of long noncoding RNAs (lncRNAs) that is integral to the p53 signalling network. As a research tool, we implemented a public access database called TP53LNC-DB that annotates currently available information relating lncRNAs to p53 signalling in humans.