The proteasome component PSMD14 drives myelomagenesis through a histone deubiquitinase activity.

While 19S proteasome regulatory particle (RP) inhibition is a promising new avenue for treating bortezomib-resistant myeloma, the anti-tumor impact of inhibiting 19S RP component PSMD14 could not be explained by a selective inhibition of proteasomal activity. Here, we report that PSMD14 interacts with NSD2 on chromatin, independent of 19S RP. Functionally, PSMD14 acts as a histone H2AK119 deubiquitinase, facilitating NSD2-directed H3K36 dimethylation. Integrative genomic and epigenomic analyses revealed the functional coordination of PSMD14 and NSD2 in transcriptional activation of target genes (e.g., RELA) linked to myelomagenesis. Reciprocally, RELA transactivates PSMD14, forming a PSMD14/NSD2-RELA positive feedback loop. Remarkably, PSMD14 inhibitors enhance bortezomib sensitivity and fosters anti-myeloma synergy. PSMD14 expression is elevated in myeloma and inversely correlated with overall survival. Our study uncovers an unappreciated function of PSMD14 as an epigenetic regulator and a myeloma driver, supporting the pursuit of PSMD14 as a therapeutic target to overcome the treatment limitation of myeloma.

(p)ppGpp controls stringent factors by exploiting antagonistic allosteric coupling between catalytic domains.

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product-the alarmone nucleotide (p)ppGpp-through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.

RelA-SpoT Homolog toxins pyrophosphorylate the CCA end of tRNA to inhibit protein synthesis.

RelA-SpoT Homolog (RSH) enzymes control bacterial physiology through synthesis and degradation of the nucleotide alarmone (p)ppGpp. We recently discovered multiple families of small alarmone synthetase (SAS) RSH acting as toxins of toxin-antitoxin (TA) modules, with the FaRel subfamily of toxSAS abrogating bacterial growth by producing an analog of (p)ppGpp, (pp)pApp. Here we probe the mechanism of growth arrest used by four experimentally unexplored subfamilies of toxSAS: FaRel2, PhRel, PhRel2, and CapRel. Surprisingly, all these toxins specifically inhibit protein synthesis. To do so, they transfer a pyrophosphate moiety from ATP to the tRNA 3' CCA. The modification inhibits both tRNA aminoacylation and the sensing of cellular amino acid starvation by the ribosome-associated RSH RelA. Conversely, we show that some small alarmone hydrolase (SAH) RSH enzymes can reverse the pyrophosphorylation of tRNA to counter the growth inhibition by toxSAS. Collectively, we establish RSHs as RNA-modifying enzymes.

Activation of the Stringent Response by Loading of RelA-tRNA Complexes at the Ribosomal A-Site.

RelA/SpoT homologs (RSHs) are ubiquitous bacterial enzymes that synthesize and hydrolyze (p)ppGpp in response to environmental challenges. Bacteria cannot survive in hosts and produce infection without activating the (p)ppGpp-mediated stringent response, but it is not yet understood how the enzymatic activities of RSHs are controlled. Using UV crosslinking and deep sequencing, we show that Escherichia coli RelA ((p)ppGpp synthetase I) interacts with uncharged tRNA without being activated. Amino acid starvation leads to loading of cognate tRNA⋅RelA complexes at vacant ribosomal A-sites. In turn, RelA is activated and synthesizes (p)ppGpp. Mutation of a single, conserved residue in RelA simultaneously prevents tRNA binding, ribosome binding, and activation of RelA, showing that all three processes are interdependent. Our results support a model in which (p)ppGpp synthesis occurs by ribosome-bound RelA interacting with the Sarcin-Ricin loop of 23S rRNA.

Global Interactome Mapping Reveals Pro-tumorigenic Interactions of NF-κB in Breast Cancer.

NF-κB pathway is involved in inflammation; however, recent data shows its role also in cancer development and progression, including metastasis. To understand the role of NF-κB interactome dynamics in cancer, we study the complexity of breast cancer interactome in luminal A breast cancer model and its rearrangement associated with NF-κB modulation. Liquid chromatography-mass spectrometry measurement of 160 size-exclusion chromatography fractions identifies 5460 protein groups. Seven thousand five hundred sixty eight interactions among these proteins have been reconstructed by PrInCE algorithm, of which 2564 have been validated in independent datasets. NF-κB modulation leads to rearrangement of protein complexes involved in NF-κB signaling and immune response, cell cycle regulation, and DNA replication. Central NF-κB transcription regulator RELA co-elutes with interactors of NF-κB activator PRMT5, and these complexes are confirmed by AlphaPulldown prediction. A complementary immunoprecipitation experiment recapitulates RELA interactions with other NF-κB factors, associating NF-κB inhibition with lower binding of NF-κB activators to RELA. This study describes a network of pro-tumorigenic protein interactions and their rearrangement upon NF-κB inhibition with potential therapeutic implications in tumors with high NF-κB activity.

Starvation sensing by mycobacterial RelA/SpoT homologue through constitutive surveillance of translation.

The stringent response, which leads to persistence of nutrient-starved mycobacteria, is induced by activation of the RelA/SpoT homolog (Rsh) upon entry of a deacylated-tRNA in a translating ribosome. However, the mechanism by which Rsh identifies such ribosomes in vivo remains unclear. Here, we show that conditions inducing ribosome hibernation result in loss of intracellular Rsh in a Clp protease-dependent manner. This loss is also observed in nonstarved cells using mutations in Rsh that block its interaction with the ribosome, indicating that Rsh association with the ribosome is important for Rsh stability. The cryo-EM structure of the Rsh-bound 70S ribosome in a translation initiation complex reveals unknown interactions between the ACT domain of Rsh and components of the ribosomal L7/L12 stalk base, suggesting that the aminoacylation status of A-site tRNA is surveilled during the first cycle of elongation. Altogether, we propose a surveillance model of Rsh activation that originates from its constitutive interaction with the ribosomes entering the translation cycle.

C-terminal deletion of RelA protein is suggested as a possible cause of infective endocarditis recurrence with Enterococcus faecium.

Infective endocarditis (IE) caused by Enterococcus spp. represents the third most common cause of IE, with high rates of relapse compared with other bacteria. Interestingly, late relapses (>6 months) have only been described in Enterococcus faecalis, but here we describe the first reported IE relapse with Enterococcus faecium more than a year (17 months) after the initial endocarditis episode. Firstly, by multi locus sequence typing (MLST), we demonstrated that both isolates (EF646 and EF641) belong to the same sequence type (ST117). Considering that EF641 was able to overcome starvation and antibiotic treatment conditions surviving for a long period of time, we performed bioinformatic analysis in identifying potential genes involved in virulence and stringent response. Our results showed a 13-nucleotide duplication (positions 1638-1650) in the gene relA, resulting in a premature stop codon, with a loss of 167 amino acids from the C-terminal domains of the RelA enzyme. RelA mediates the stringent response in bacteria, modulating levels of the alarmone guanosine tetraphosphate (ppGpp). The relA mutant (EF641) was associated with lower growth capacity, the presence of small colony variants, and higher capacity to produce biofilms (compared with the strain EF646), but without differences in antimicrobial susceptibility patterns according to standard procedures during planktonic growth. Instead, EF641 demonstrated tolerance to high doses of teicoplanin when growing in a biofilm. We conclude that all these events would be closely related to the long-term survival of the E. faecium and the late relapse of the IE. These data represent the first clinical evidence of mutations in the stringent response (relA gene) related with E. faecium IE relapse.

Neural-Cell-Intrinsic NF-κB Signaling Enhances Reovirus Virulence.

Pathological effects of apoptosis associated with viral infections of the central nervous system are an important cause of morbidity and mortality. Reovirus is a neurotropic virus that causes apoptosis in neurons, leading to lethal encephalitis in newborn mice. Reovirus-induced encephalitis is diminished in mice with germ line ablation of NF-κB subunit p50. It is not known whether the proapoptotic function of NF-κB is mediated by neural-cell-intrinsic (neural-intrinsic) processes, NF-κB-regulated cytokine production by inflammatory cells, or a combination of both. To determine the contribution of cell type-specific NF-κB signaling in reovirus-induced neuronal injury, we established mice that lack NF-κB p65 expression in neural cells using the Cre/loxP recombination system. Following intracranial inoculation of reovirus, 50% of wild-type (WT) mice succumbed to infection, whereas more than 90% of mice lacking neural cell NF-κB p65 (Nsp65-/-) survived. While viral loads in brains of WT and Nsp65-/- mice were comparable, histological analysis revealed that reovirus antigen-positive areas in the brains of WT mice displayed increased immunoreactivity for cleaved caspase-3, a marker of apoptosis, relative to Nsp65-/- mice. These data suggest that neural-intrinsic NF-κB-dependent factors are essential mediators of reovirus neurovirulence. RNA sequencing analysis of reovirus-infected brain cortices of WT and Nsp65-/- mice suggests that NF-κB activation in neuronal cells upregulates genes involved in innate immunity, inflammation, and cell death following reovirus infection. A better understanding of the contribution of cell type-specific NF-κB-dependent signaling to viral neuropathogenesis could inform development of new therapeutics that target and protect highly vulnerable cell populations. IMPORTANCE Viral encephalitis contributes to illness and death in children and adults worldwide and has limited treatment options. Identifying common host factors upregulated by neurotropic viruses can enhance an understanding of virus-induced neuropathogenesis and aid in development of therapeutics. Although many neurotropic viruses activate NF-κB during infection, mechanisms by which NF-κB regulates viral neuropathogenesis and contributes to viral encephalitis are not well understood. We established mice in which NF-κB expression is ablated in neural tissue to study the function of NF-κB in reovirus neurovirulence and identify genes activated by NF-κB in response to reovirus infection in the central nervous system. Encephalitis following reovirus infection was dampened in mice lacking neural cell NF-κB. Reovirus induced a chemokine profile in the brain that was dependent on NF-κB signaling and was similar to chemokine profiles elicited by other neurotropic viruses. These data suggest common underlying mechanisms of encephalitis caused by neurotropic viruses and potentially shared therapeutic targets.

Insulin-like growth factor 2 mRNA-binding protein 3 enhanced melanoma migration through regulation of AKT1 and RELA expression.

IMP-3 expression is a poor prognostic factor of melanomas and it promotes melanoma cell migration and invasion by a pathway modulating HMGA2 mRNA expression. We tried to identify other putative targets of IMP-3. We identified putative IMP-3-binding RNAs, including AKT1, MAPK3, RB1 and RELA, by RNA immunoprecipitation coupled with next-generation sequencing. IMP-3 overexpression increased AKT and RELA levels in MeWo cells. siRNAs against AKT1 and RELA inhibited MeWo/Full-length IMP-3 cell migration. IMP-3 knockdown of A2058 cells decreased AKT1 and RELA expression and lowered migration ability. Co-transfection of A2058 cells with AKT1- or RELA-expressing plasmids with IMP-3 siRNA restored the inhibitory effects of IMP-3 knockdown on migration. HMGA2 did not influence AKT1 and RELA expression in melanoma cells. Human melanoma samples with high IMP-3 levels also showed high HMGA2, AKT1 and RELA expression. Our results show that IMP-3 enhances melanoma cell migration through the regulation of the AKT1 and RELA axis.

Resistance to African swine fever virus among African domestic pigs appears to be associated with a distinct polymorphic signature in the RelA gene and upregulation of RelA transcription.

African swine fever virus (ASFV) is a highly contagious and fatal hemorrhagic disease of domestic pigs, which poses a major threat to the swine industry worldwide. Studies have shown that indigenous African pigs tolerate ASFV infection better than European pigs. The porcine v-rel avian reticuloendotheliosis viral oncogene homolog A (RelA) encoding a p65 kD protein, a major subunit of the NF-kB transcription factor, plays important roles in controlling both innate and adaptive immunity during infection with ASFV. In the present study, RelA genes from ASFV-surviving and symptomatic pigs were sequenced and found to contain polymorphisms revealing two discrete RelA amino acid sequences. One was found in the surviving pigs, and the other in symptomatic pigs. In total, 16 nonsynonymous SNPs (nsSNPs) resulting in codon changes were identified using bioinformatics software (SIFT and Polyphen v2) and web-based tools (MutPre and PredictSNP). Seven nsSNPs (P374-S, T448-S, P462-R, V464-P, Q478-H, L495-E, and P499-Q) were predicted to alter RelA protein function and stability, while 5 of these (P374-S, T448-S, P462-R, L495-E, and Q499-P) were predicted as disease-related SNPs.Additionally, the inflammatory cytokine levels of IFN-α, IL-10, and TNF-α at both the protein and the mRNA transcript levels were measured using ELISA and Real-Time PCR, respectively. The resulting data was used in correlation analysis to assess the association between cytokine levels and the RelA gene expression. Higher levels of IFN-α and detectable levels of IL-10 protein and RelA mRNA were observed in surviving pigs compared to healthy (non-infected). A positive correlation of IFN-α cytokine levels with RelA mRNA expression was also obtained. In conclusion, 7 polymorphic events in the coding region of the RelA gene may contribute to the tolerance of ASFV in pigs.

Histone variant H2AZ1 drives lung cancer progression through the RELA-HIF1A-EGFR signaling pathway.

BACKGROUND: A growing body of evidence indicates that histone variants play an oncogenic role in cancer progression. However, the role and mechanism of histone variant H2AZ1 in lung cancer remain poorly understood. In this study, we aim to identify novel functions and molecular mechanisms of H2AZ1 in lung cancer. METHODS: We analyzed H2AZ1 expression in lung adenocarcinoma using several RNA-seq and microarray datasets. Immunohistochemistry staining for H2AZ1 was performed on two sets of lung cancer tissue microarrays. To study the function of H2AZ1, we conducted assays for cell proliferation, colony formation, invasion, and migration. We employed CUT&Tag-seq, ATAC-seq, RNA-seq, and Western blotting to explore the regulatory patterns and potential mechanisms of H2AZ1 in lung adenocarcinoma. RESULTS: Our findings reveal that H2AZ1 is highly expressed in lung cancer and high levels of H2AZ1 mRNA are associated with poor patient survival. Silencing H2AZ1 impaired cell proliferation, colony formation, migration, and invasion. Mechanistically, our CUT&Tag-seq, ATAC-seq, and RNA-seq results showed that H2AZ1 is primarily deposited around TSS and affects multiple oncogenic signaling pathways. Importantly, we uncovered that H2AZ1 may drive lung cancer progression through the RELA-HIF1A-EGFR signaling pathway. CONCLUSION: H2AZ1 plays an oncogenic role via several cancer-related pathways, including the RELA-HIF1A-EGFR axis in lung cancer. Intervention targeting H2AZ1 and its related signaling genes may have translational potential for precision therapy.

HYPOXIA induces lncRNA HOTAIR for recruiting RELA in papillary thyroid cancer cells to upregulate miR-181a and promote angiogenesis.

BACKGROUND: Papillary thyroid carcinoma (PTC) is one of the most common subtypes of thyroid carcinoma. Exosomal miR-181a plays an important role in the development of PTC. This study examined the regulatory mechanism of miR-181a under conditions of hypoxia and its impact on angiogenesis. METHODS: A ribonucleoprotein immunoprecipitation (RIP) experiment was conducted to verify the interaction between HOTAIR and RELA. The relationship between RELA and the miR-181a promoter was detected by ChIP-qPCR. Short hairpin (sh) RNA was designed to knock down HOTAIR in TPC cells. The underlying mechanism of miR-181a was verified by use of dual-luciferase assays and rescue experiments. The regulatory effect of GATA6 on angiogenesis was studied using CCK8, EdU, Transwell, and western blot assays. RESULTS: A RIP assay showed that HOTAIR could bind to RELA under hypoxic conditions. ChIP-qPCR and dual luciferase assays showed RELA could interact with the miR181a promoter and upregulate miR-181a. Knockdown of HOTAIR downregulated miR-181a in TPC-1 cells, and the downregulation could be rescued by RELA overexpression. MiR-181a downregulated GATA6 in HUVEC cells. Overexpression of GATA6 inhibited HUVEC proliferation, migration, tube formation, and EGFR expression. Exosomal miR-181a promoted angiogenesis by downregulating GATA6 expression. CONCLUSION: HOTAIR activated RELA to upregulate miR-181a during hypoxia. Exosomal miR-181a promotes tumor angiogenesis by downregulating GATA6.

The Mechanism of Inhibition of Mycobacterial (p)ppGpp Synthetases by a Synthetic Analog of Erogorgiaene.

The synthesis of (p)ppGpp alarmones plays a vital role in the regulation of metabolism suppression, growth rate control, virulence, bacterial persistence, and biofilm formation. The (p)ppGpp alarmones are synthesized by proteins of the RelA/SpoT homolog (RSH) superfamily, including long bifunctional RSH proteins and small alarmone synthetases. Here, we investigated enzyme kinetics and dose-dependent enzyme inhibition to elucidate the mechanism of 4-(4,7-dimethyl-1,2,3,4-tetrahydronaphthalen-1-yl)pentanoic acid (DMNP) action on the (p)ppGpp synthetases RelMsm and RelZ from Mycolicibacterium smegmatis and RelMtb from Mycobacterium tuberculosis. DMNP was found to inhibit the activity of RelMtb. According to the enzyme kinetics analysis, DMNP acts as a noncompetitive inhibitor of RelMsm and RelZ. Based on the results of molecular docking, the DMNP-binding site is located in the proximity of the synthetase domain active site. This study might help in the development of alarmone synthetase inhibitors, which includes relacin and its derivatives, as well as DMNP - a synthetic analog of the marine coral metabolite erogorgiaene. Unlike conventional antibiotics, alarmone synthetase inhibitors target metabolic pathways linked to the bacterial stringent response. Although these pathways are not essential for bacteria, they regulate the development of adaptation mechanisms. Combining conventional antibiotics that target actively growing cells with compounds that impede bacterial adaptation may address challenges associated with antimicrobial resistance and bacterial persistence.

NF-κB Transcription Factors: Their Distribution, Family Expansion, Structural Conservation, and Evolution in Animals.

The Nuclear Factor Kappa B (NF-κB) transcription factor family consists of five members: RelA (p65), RelB, c-Rel, p50 (p105/NF-κB1), and p52 (p100/NF-κB2). This family is considered a master regulator of classical biochemical pathways such as inflammation, immunity, cell proliferation, and cell death. The proteins in this family have a conserved Rel homology domain (RHD) with the following subdomains: DNA binding domain (RHD-DBD) and dimerization domain (RHD-DD). Despite the importance of the NF-κB family in biology, there is a lack of information with respect to their distribution patterns, evolution, and structural conservation concerning domains and subdomains in animals. This study aims to address this critical gap regarding NF-κB proteins. A comprehensive analysis of NF-κB family proteins revealed their distinct distribution in animals, with differences in protein sizes, conserved domains, and subdomains (RHD-DBD and RHD-DD). For the first time, NF-κB proteins with multiple RHD-DBDs and RHD-DDs have been identified, and in some cases, this is due to subdomain duplication. The presence of RelA/p65 exclusively in vertebrates shows that innate immunity originated in fishes, followed by amphibians, reptiles, aves, and mammals. Phylogenetic analysis showed that NF-κB family proteins grouped according to animal groups, signifying structural conservation after speciation. The evolutionary analysis of RHDs suggests that NF-κB family members p50/p105 and c-Rel may have been the first to emerge in arthropod ancestors, followed by RelB, RelA, and p52/p100.

Gold Nanoparticles Downregulate IL-6 Expression/Production by Upregulating microRNA-26a-5p and Deactivating the RelA and NF-κBp50 Transcription Pathways in Activated Breast Cancer Cells.

MicroRNAs (miRNAs) are involved in the modulation of pathogenic genes by binding to their mRNA sequences' 3' untranslated regions (3'UTR). Interleukin-6 (IL-6) is known to promote cancer progression and treatment resistance. In this study, we aimed to explore the therapeutic effects of gold nanoparticles (GNP) against IL-6 overexpression and the modulation of miRNA-26a-5p in breast cancer (BC) cells. GNP were synthesized using the trisodium citrate method and characterized through UV-Vis spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). To predict the binding of miR-26a-5p in the IL-6 mRNA's 3'UTR, we utilized bioinformatics algorithms. Luciferase reporter clone assays and anti-miRNA-26a-5p transfection were employed to validate the binding of miR26a-5p in the IL-6 mRNA's 3'UTR. The activity of RelA and NF-κBp50 was assessed and confirmed using Bay 11-7082. The synthesized GNP were spherical with a mean size of 28.3 nm, exhibiting high stability, and were suitable for BC cell treatment. We found that miR-26a-5p directly regulated IL-6 overexpression in MCF-7 cells activated with PMA. Treatment of MCF-7 cells with GNP resulted in the inhibition of IL-6 overexpression and secretion through the increase of miR26a-5p. Furthermore, GNP deactivated NF-κBp65/NF-κBp50 transcription activity. The newly engineered GNP demonstrated safety and showed promise as a therapeutic approach for reducing IL-6 overexpression. The GNP suppressed IL-6 overexpression and secretion by deactivating NF-κBp65/NF-κBp50 transcription activity and upregulating miR-26a-5p expression in activated BC cells. These findings suggest that GNP have potential as a therapeutic intervention for BC by targeting IL-6 expression and associated pathways.

NF-κB in the Radiation Response of A549 Non-Small Cell Lung Cancer Cells to X-rays and Carbon Ions under Hypoxia.

Cellular hypoxia, detectable in up to 80% of non-small cell lung carcinoma (NSCLC) tumors, is a known cause of radioresistance. High linear energy transfer (LET) particle radiation might be effective in the treatment of hypoxic solid tumors, including NSCLC. Cellular hypoxia can activate nuclear factor κB (NF-κB), which can modulate radioresistance by influencing cancer cell survival. The effect of high-LET radiation on NF-κB activation in hypoxic NSCLC cells is unclear. Therefore, we compared the effect of low (X-rays)- and high (12C)-LET radiation on NF-κB responsive genes' upregulation, as well as its target cytokines' synthesis in normoxic and hypoxic A549 NSCLC cells. The cells were incubated under normoxia (20% O2) or hypoxia (1% O2) for 48 h, followed by irradiation with 8 Gy X-rays or 12C ions, maintaining the oxygen conditions until fixation or lysis. Regulation of NF-κB responsive genes was evaluated by mRNA sequencing. Secretion of NF-κB target cytokines, IL-6 and IL-8, was quantified by ELISA. A greater fold change increase in expression of NF-κB target genes in A549 cells following exposure to 12C ions compared to X-rays was observed, regardless of oxygenation status. These genes regulate cell migration, cell cycle, and cell survival. A greater number of NF-κB target genes was activated under hypoxia, regardless of irradiation status. These genes regulate cell migration, survival, proliferation, and inflammation. X-ray exposure under hypoxia additionally upregulated NF-κB target genes modulating immunosurveillance and epithelial-mesenchymal transition (EMT). Increased IL-6 and IL-8 secretion under hypoxia confirmed NF-κB-mediated expression of pro-inflammatory genes. Therefore, radiotherapy, particularly with X-rays, may increase tumor invasiveness in surviving hypoxic A549 cells.

ZFTA::RELA fusion in a distinct liposarcoma morphologically overlapping with chondroid lipoma.

Chondroid lipoma is a rare benign adipose tumor characterized by a recurrent ZFTA::MRTFB fusion. Herein, we report an unusual liposarcoma that partly exhibited overlapping features with those of chondroid lipoma and harbored a ZFTA::RELA fusion. A 59-year-old man presented with a shoulder mass that had existed for approximately 8 years and with increasing pain due to a pelvic mass. The 5.8-cm resected shoulder tumor partly consisted of nests and strands of variably lipogenic epithelioid cells within a hyalinized or focally chondromyxoid stroma, indistinguishable from chondroid lipoma. The histological pattern gradually transitioned to highly cellular, stroma-poor, diffuse sheets of cells with greater nuclear atypia and mitotic activity. Vascular invasion and necrosis were present. The metastatic pelvic tumor revealed a similar histology. Despite multimodal treatment, the patient developed multiple bone metastases and succumbed to the disease 14 months after presentation. Targeted RNA sequencing identified an in-frame ZFTA (exon 3)::RELA (exon 2) fusion, which was confirmed by reverse transcription-polymerase chain reaction, Sanger sequencing, and break-apart fluorescent in situ hybridization assays. The tumor showed a different histology from that of ependymoma, no brain involvement, and no match with any sarcoma types or ZFTA::RELA-positive ependymomas according to DNA methylation analysis. p65 and L1CAM were diffusely expressed, and a CDKN2A/B deletion was present. This is the first report of an extra-central nervous system tumor with a ZFTA::RELA fusion. The tumor partly displayed an overlapping histology with that of chondroid lipoma, suggesting that it may represent a hitherto undescribed malignant chondroid lipoma with an alternative ZFTA fusion.

RelA binding of mRNAs modulates translation or sRNA-mRNA basepairing depending on the position of the GGAG site.

Previously, we reported that RelA protein facilitates Hfq-mediated mRNA-sRNA regulation by binding sRNAs carrying a Shine-Dalgarno-like GGAG sequence. In turn, sRNA-Hfq monomers are stabilized, enabling the attachment of more Hfq subunits to form a functional hexamer. Here, using CLIP-seq, we present a global analysis of RelA-bound RNAs showing that RelA interacts with sRNAs as well as with mRNAs carrying a GGAG motif. RelA binding of mRNAs carrying GGAG at position -7 relative to the initiation codon (AUG) inhibits translation by interfering with the binding of 30S ribosomes. The extent of inhibition depends on the distance of GGAG relative to the AUG, as shortening the spacing between GGAG and AUG abrogates RelA-mediated inhibition. Interestingly, RelA binding of target mRNAs carrying GGAG in the coding sequence or close to AUG facilitates target gene regulation by sRNA partners that lack GGAG. However, translation inhibition caused by RelA binding of mRNAs carrying GGAG at position -7 relative to the AUG renders sRNA-mRNA basepairing regulation ineffective. Our study indicates that by binding RNAs carrying GGAG the ribosome-associated RelA protein inhibits translation of specific newly synthesized incoming mRNAs or enables basepairing regulation by their respective sRNA partners, thereby introducing a new regulatory concept for the bacterial response.

HOXA1 participates in VSMC-to-macrophage-like cell transformation via regulation of NF-κB p65 and KLF4: a potential mechanism of atherosclerosis pathogenesis.

BACKGROUND: Macrophage-like transformation of vascular smooth muscle cells (VSMCs) is a risk factor of atherosclerosis (AS) progression. Transcription factor homeobox A1 (HOXA1) plays functional roles in differentiation and development. This study aims to explore the role of HOXA1 in VSMC transformation, thereby providing evidence for the potential mechanism of AS pathogenesis. METHODS: High fat diet (HFD)-fed apolipoprotein E knockout (ApoE-/-) mice were applied as an in vivo model to imitate AS, while 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POV-PC)-treated VSMCs were applied as an in vitro model. Recombinant adeno-associated-virus-1 (AAV-1) vectors that express short-hairpin RNAs targeting HOXA1, herein referred as AAV1-shHOXA1, were generated for the loss-of-function experiments throughout the study. RESULTS: In the aortic root of AS mice, lipid deposition was severer and HOXA1 expression was higher than the wide-type mice fed with normal diet or HFD. Silencing of HOXA1 inhibited the AS-induced weight gain, inflammatory response, serum and liver lipid metabolism disorder and atherosclerotic plaque formation. Besides, lesions from AS mice with HOXA1 knockdown showed less trans-differentiation of VSMCs to macrophage-like cells, along with a suppression of krüppel-like factor 4 (KLF4) and nuclear factor (NF)-κB RelA (p65) expression. In vitro experiments consistently confirmed that HOXA1 knockdown suppressed lipid accumulation, VSMC-to-macrophage phenotypic switch and inflammation in POV-PC-treated VSMCs. Mechanism investigations further illustrated that HOXA1 transcriptionally activated RelA and KLF4 to participate in the pathological manifestations of VSMCs. CONCLUSIONS: HOXA1 participates in AS progression by regulating VSMCs plasticity via regulation of NF-κB p65 and KLF4. HOXA1 has the potential to be a biomarker or therapeutic target for AS.

Nuclear localization of p65 reverses therapy-induced senescence.

Senescence is the arrest of cell proliferation and is a tumor suppressor phenomenon. In a previous study, we have shown that therapy-induced senescence of glioblastoma multiforme (GBM) cells can prevent relapse of GBM tumors. Here, we demonstrate that ciprofloxacin-induced senescence in glioma-derived cell lines and primary glioma cultures is defined by SA-ß-gal positivity, a senescence-associated secretory phenotype (SASP), a giant cell (GC) phenotype, increased levels of reactive oxygen species (ROS), γ-H2AX and a senescence-associated gene expression signature, and has three stages of senescence -initiation, pseudo-senescence and permanent senescence. Ciprofloxacin withdrawal during initiation and pseudo-senescence reinitiated proliferation in vitro and tumor formation in vivo Importantly, prolonged treatment with ciprofloxacin induced permanent senescence that failed to reverse following ciprofloxacin withdrawal. RNA-seq revealed downregulation of the p65 (RELA) transcription network, as well as incremental expression of SMAD pathway genes from initiation to permanent senescence. Ciprofloxacin withdrawal during initiation and pseudo-senescence, but not permanent senescence, increased the nuclear localization of p65 and escape from ciprofloxacin-induced senescence. By contrast, permanently senescent cells showed loss of nuclear p65 and increased apoptosis. Pharmacological inhibition or genetic knockdown of p65 upheld senescence in vitro and inhibited tumor formation in vivo Our study demonstrates that levels of nuclear p65 define the window of reversibility of therapy-induced senescence and that permanent senescence can be induced in GBM cells when the use of senotherapeutics is coupled with p65 inhibitors.