SbMYC2 mediates jasmonic acid signaling to improve drought tolerance via directly activating SbGR1 in sorghum.

KEY MESSAGE: SbMYC2 functions as a key regulator under JA signaling in enhancing drought tolerance of sorghum through direct activating SbGR1. Drought stress is one of the major threats to crop yield. In response to drought stress, functions of basic helix-loop-helix (bHLH) transcription factors (TFs) have been reported in Arabidopsis and rice, but little is known for sorghum. Here, we characterized the function of SbMYC2, a bHLH TF in sorghum, and found that SbMYC2 responded most significantly to PEG-simulated drought stress and JA treatments. Overexpression of SbMYC2 significantly enhanced drought tolerance in Arabidopsis, rice and sorghum. In addition, it reduced reactive oxygen species (ROS) accumulation and increased chlorophyll content in sorghum leaves. While silencing SbMYC2 by virus-induced gene silencing (VIGS) resulted in compromised drought tolerance of sorghum seedlings. Moreover, SbMYC2 can directly activate the expression of GLUTATHIONE-DISULFIDE REDUCTASE gene SbGR1. SbGR1 silencing led to significantly weakened drought tolerance of sorghum, and higher ROS accumulation and lower chlorophyll content in sorghum leaves were detected. In addition, SbMYC2 can interact with SbJAZs, suppressors of JA signaling, and thus can mediate JA signaling to activate SbGR1, thereby regulating sorghum's tolerance to drought stress. Overall, our findings demonstrate that bHLH TF SbMYC2 plays an important role in sorghum's response to drought stress, thus providing one theoretical basis for genetic enhancement of sorghum and even rice.

The inhibition of YTHDF3/m6A/LRP6 reprograms fatty acid metabolism and suppresses lymph node metastasis in cervical cancer.

The lipid synthesis of fatty acid (FA) represents a significant hallmark in the occurrence and progression of malignant tumor, which are associated with lymph node (LN) metastasis. Elucidation of the molecular mechanisms underlying LN metastasis could provide therapeutic strategies for cervical cancer (CCa). N6-Methyladenosine (m6A), the most prevalent and abundant RNA modification, exerts specific regulatory control over a series of oncogene expressions. This study demonstrated a clinical correlation between the upregulation of the m6A reader YTHDF3 and LN metastasis, thereby contributing to poor overall survival probability (OS) among CCa patients. The mechanistic investigation revealed that SREBF1 transcriptionally activated YTHDF3 expression by binding to its promoter. Functional experiments demonstrated that the upregulation of YTHDF3 significantly enhanced the in vitro proliferative, migratory, and invasive capacities of CCa cells, while also promoting lymphangiogenesis and facilitating LN metastasis in vivo. Mechanistically, the upregulation of LRP6 through YTHDF3-mediated m6A modification resulted in increased expression of FASN and ACC1, leading to both lipolysis of lipid droplets and synthesis of free fatty acid. Ultimately, this promoted fatty acid metabolism and enhanced LN metastasis by activating the LRP6-YAP-VEGF-C axis, which could induce lymphangiogenesis in CCa. Our study highlighted that YTHDF3 can serve as a promising therapeutic target and predictive biomarker for CCa patients with LN metastasis.

Programmed Death Ligand-1-Overexpressing Donor Exosomes Mediate Donor-Specific Immunosuppression by Delivering Co-Inhibitory Signals to Donor-Specific T Cells.

Programmed death ligand-1 (PD-L1) and donor antigens are critical for donor immature dendritic cells (DCs) targeting donor-specific T cells to induce transplant tolerance. This study aims to clarify whether DC-derived exosomes (DEX) with donor antigens (H2b) and high levels of PD-L1 expression (DEXPDL1+ ) can help to suppress graft rejection. In this study, it is demonstrated that DEXPDL1+ presents donor antigens, as well as PD-L1 co-inhibitory signals, directly or semi-directly via DCs to H2b-reactive T cells. This dual signal presentation can prolong the survival of heart grafts from B6 (H2b) mice but not from C3H (H2k) mice by inhibiting T cell activation, inducing activated T cell apoptosis, and modulating the balance of T cell differentiation from inflammatory to regulatory. Additionally, even though DEXPDL1+ treatment cannot induce tolerance after short-term treatment, this study provides a new vehicle for presenting co-inhibitory signals to donor-specific T cells. This novel strategy may facilitate the realization of donor-specific tolerance via the further optimization of drug-loading combinations and therapeutic regimens to elevate their killing ability.

FOXD1-dependent RalA-ANXA2-Src complex promotes CTC formation in breast cancer.

BACKGROUND: Early metastasis is a key factor contributing to poor breast cancer (BC) prognosis. Circulating tumor cells (CTCs) are regarded as the precursor cells of metastasis, which are ultimately responsible for the main cause of death in BC. However, to date molecular mechanisms underlying CTC formation in BC have been insufficiently defined. METHODS: RNA-seq was carried out in primary tissues from early-stage BC patients (with CTCs≥5 and CTCs = 0, respectively) and the validation study was conducted in untreated 80 BC patients. Multiple in vitro and in vivo models were used in functional studies. Luciferase reporter, ChIP-seq, CUT&Tag-seq, and GST-pulldown, etc. were utilized in mechanistic studies. CTCs were counted by the CanPatrol™ CTC classification system or LiquidBiospy™ microfluidic chips. ERK1/2 inhibitor SCH772984 was applied to in vivo treatment. RESULTS: Highly expressed FOXD1 of primary BC tissues was observed to be significantly associated with increased CTCs in BC patients, particularly in early BC patients. Overexpressing FOXD1 enhanced the migration capability of BC cells, CTC formation and BC metastasis, via facilitating epithelial-mesenchymal transition of tumor cells. Mechanistically, FOXD1 was discovered to induce RalA expression by directly bound to RalA promotor. Then, RalA formed a complex with ANXA2 and Src, promoting the interaction between ANXA2 and Src, thus increasing the phosphorylation (Tyr23) of ANXA2. Inhibiting RalA-GTP form attenuated the interaction between ANXA2 and Src. This cascade culminated in the activation of ERK1/2 signal that enhanced metastatic ability of BC cells. In addition, in vivo treatment with SCH772984, a specific inhibitor of ERK1/2, was used to dramatically inhibit the CTC formation and BC metastasis. CONCLUSION: Here, we report a FOXD1-dependent RalA-ANXA2-Src complex that promotes CTC formation via activating ERK1/2 signal in BC. FOXD1 may serve as a prognostic factor in evaluation of BC metastasis risks. This signaling cascade is druggable and effective for overcoming CTC formation from the early stages of BC.

Cytoplasm or Supernatant: Where Is the Treasury of the Bioactive Antiaging Factor from Mesenchymal Stem Cells?

Cell-free compounds of mesenchymal stem cells (MSCs) could be a safer and cheaper substitution for MSC transplantation and have gained substantial research interest for antiaging skin treatments. However, whether those bioactive components should be obtained from the cytoplasm or supernatant is yet to be determined. In this study, we examined the ingredients of the MSC cytoplasm extract (MSC-ex) and MSC supernatant (MSC-s) and evaluated their effect in a photoaging model. Although MSC-ex has a richer protein composition than MSC-s, the latter has a proteome associated with wound healing and blood vessel development. Over 85% of the proteins in MSC-s were also found in MSC-ex, including extracellular matrix protein and various growth factors. The results of real-time PCR and western blot also demonstrate that both MSC-s and MSC-ex can upregulate collagen, transforming growth factor ß (TGF-ß), and vascular endothelial growth factor (VEGF) and downregulate IL-1ß and matrix metalloproteinase-1 (MMP-1), which were considered critical for antiphotoaging. This supports our observations in the Hematoxylin and Eosin (HE) and Masson staining assay that they have a comparable effect as MSCs in terms of enhancing dermal thickness, and stimulating collagen regeneration. Although MSC-s and MSC-ex showed a weaker immunosuppression effect than MSCs, moisture measurement showed that they repair damage more rapidly than MSCs. Furthermore, the histological results showed that MSC-s maintains a super effect on immunosuppression, epidermal repair, and angiogenesis. That may be associated with the higher content of laminin, TGF-ß, and VEGF in MSC-s, as well as its super cytokine transcriptional regulation ability. Thus, both MSC-s and MSC-ex can safely and effectively promote the repair of skin light injury, similar to MSCs. Our findings can broaden the range of active factors available in cell-free treatment, determine the difference between MSC-s and MSC-ex, and provide a reference for the development of similar products in regenerative medicine.

Exosomal Delivery of AntagomiRs Targeting Viral and Cellular MicroRNAs Synergistically Inhibits Cancer Angiogenesis.

Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated cancer characterized by a high degree of recurrence, angiogenesis, and metastasis. The importance of alternative pro-angiogenesis pathways including viral factors has emerged after decades of directly targeting various signaling components. Using NPC as a model, we identified an essential oncogenic pathway underlying angiogenesis regulation that involves the inhibition of a tumor suppressor, Spry3, and its downstream targets by EBV-miR-BART10-5p (BART10-5p) and hsa-miR-18a (miR-18a). Overexpression of EBV-miR-BART10-5p and hsa-miR-18a strongly promotes angiogenesis in vitro and in vivo by regulating the expression of VEGF and HIF1-α in a Spry3-dependent manner. In vitro or in vivo treatment with iRGD-tagged exosomes containing antagomiR-BART10-5p and antagomiR-18a preferentially suppressed the angiogenesis and growth of NPC. Our findings first highlight the role of EBV-miR-BART10-5p and oncogenic hsa-miR-18a in NPC angiogenesis and also shed new insights into the clinical intervention and therapeutic strategies for nasopharyngeal carcinoma and other virus-associated tumors.

Lnc-M2 controls M2 macrophage differentiation via the PKA/CREB pathway.

Long noncoding RNAs (lncRNAs) play an indispensable role in the process of M1 macrophage via regulating the development of macrophages and their responses to bacterial pathogens and viral infections. However, there are few studies on the lncRNA-mediated functions and regulatory mechanisms of M2 macrophage polarization. In this study, we found a number of differentially expressed lncRNAs between human monocyte derived M0 and M2 macrophages according to array analysis and quantitative polymerase chain reaction (qPCR) validation. The lncRNA RP11-389C8.2 (we named lnc-M2 in this study) was observed to be highly expressed in M2 macrophages. In Situ Localization and Quantification Analysis showed that lnc-M2 was expressed in the nucleus and cytosolic compartments of M2 macrophages. Notably, lnc-M2 knockdown enhanced the phagocytic ability of M2 macrophages. Ulteriorly, the results of RNA-Protein interaction experiments indicated that protein kinase A (PKA) was a lnc-M2 associated RNA-binding protein (RBP). Western blot showed that phosphorylated cAMP response element binding protein (p-CREB), a well-known key downstream transcription factor of PKA, was lowly phosphorylated in lnc-M2-silencing M2 macrophages. Furthermore, we found that transcriptional factor Signal Transducer And Activator Of Transcription 3 (STAT3) promoted lnc-M2 transcription along with the up-regulation of epigenetic histone modification markers at the lnc-M2 promoter locus, indicating that STAT3 activated lnc-M2 and eventually facilitated the process of M2 macrophage differentiation via the PKA/CREB pathway. Collectively, our date provide evidence that the transcription factor STAT3 can promote the transcription of lnc-M2 and facilitated the process of M2 macrophage differentiation via the PKA/CREB pathway. This study highlights a novel mechanism underlying the M2 macrophage differentiation.

Correction: EBV-miR-BART1-5P activates AMPK/mTOR/HIF1 pathway via a PTEN independent manner to promote glycolysis and angiogenesis in nasopharyngeal carcinoma.

[This corrects the article DOI: 10.1371/journal.ppat.1007484.].

EBV-miR-BART7-3p Imposes Stemness in Nasopharyngeal Carcinoma Cells by Suppressing SMAD7.

Cancer stem-like cells, possessing "stemness" properties, play crucial roles in progression, metastasis, and drug resistance in various cancers. Viral microRNAs (such as EBV-miR-BART7-3p), as exogenous regulators, have been discovered to regulate malignant progression of nasopharyngeal carcinoma (NPC), suggesting a possible role of viral microRNAs in imposing stemness. In this study, we found that EBV-miR-BART7-3p induce stemness of NPC cells. We firstly reported that EBV-miR-BART7-3p increased the percentage of side population cells, the development of tumor spheres, and the expression level of stemness markers in vitro. This viral microRNA also enhanced stem-like or cancer-initiating properties of NPC cells in vivo. Besides, we identified SMAD7 as a novel target gene of EBV-miR-BART7-3p in addition to PTEN gene we previously reported; this viral microRNA suppressed SMAD7, led to activation of TGF-ß signaling, and eventually enhanced the stemness of NPC cells. Silencing of SMAD7 resembled the effects generated by EBV-miR-BART7-3p in NPC cells. After reconstitution of SMAD7, EBV-miR-BART7-3p-expressing cells underwent a phenotypic reversion. EBV-positive NPC cells were used to enable experimental validation. Finally, we further discovered that EBV-miR-BART7-3p increased chemo-resistance of NPC in vitro and in vivo, supporting that EBV-miR-BART7-3 resulted in increased stemness of NPC cells and lead to drug resistance and cancer recurrence. Overall, this study uncovered a novel mechanism underlying viral microRNA-associated stemness of NPC cells. This viral microRNA and its associated cellular genes may be potential therapeutic targets for restraining chemo-resistance and recurrence of NPC.

EBV-miR-BART1-5P activates AMPK/mTOR/HIF1 pathway via a PTEN independent manner to promote glycolysis and angiogenesis in nasopharyngeal carcinoma.

Abnormal metabolism and uncontrolled angiogenesis are two important characteristics of malignant tumors. The occurrence of both events involves many key molecular changes including miRNA. However, EBV encoded miRNAs are rarely mentioned as capable of regulating tumor metabolism and tumor angiogenesis. Here, we reported that one of the key miRNAs encoded by EBV, EBV-miR-Bart1-5P, can significantly promote nasopharyngeal carcinoma (NPC) cell glycolysis and induces angiogenesis in vitro and in vivo. Mechanistically, EBV-miR-Bart1-5P directly targets the α1 catalytic subunit of AMP-activated protein kinase (AMPKα1) and consequently regulates the AMPK/mTOR/HIF1 pathway which impelled NPC cell anomalous aerobic glycolysis and angiogenesis, ultimately leads to uncontrolled growth of NPC. Our findings provide new insights into metabolism and angiogenesis of NPC and new opportunities for the development of targeted NPC therapy in the future.

Nuclear expression of CDK4 correlates with disease progression and poor prognosis in human nasopharyngeal carcinoma.

AIMS: The purpose of this study was to examine the correlation between nuclear expression of cyclin-dependent kinase 4 (CDK4) and clinicopathological data in nasopharyngeal carcinoma (NPC), including patient survival. METHODS AND RESULTS: Using real-time PCR and immunohistochemistry, the expression of CDK4 was examined in NPC and nasopharyngeal (NP) tissues. We observed that mRNA expression of CDK4 was elevated significantly in NPC tissues compared to NP tissues. Further, we found that CDK4 protein was expressed in both the nucleus and cytoplasm. Nuclear expression of CDK4 was correlated positively with clinical stage (P = 0.048), but not associated with other clinical features. Patients with tumours showing nuclear expression of CDK4 had poorer overall survival rates than those without nuclear tumour expression of CDK4. Nuclear expression of CDK4 was associated inversely with survival time for NPC patients in stages T1-2, stages N2-3 and clinical stages III-IV, and after treatment with radiotherapy or chemotherapy. Nuclear expression of CDK4 was an independent and unfavourable prognostic factor for patients with NPC. CONCLUSIONS: Our findings suggest that nuclear expression of CDK4 is a potential marker for the progression and poor prognosis of NPC.

Nuclear p27 expression confers a favorable outcome for nasopharyngeal carcinoma patients.

OBJECTIVE: The purpose of the present study is to explore the correlation between nuclear expression of cyclin-dependent kinase inhibitor 1B (p27) and clinicopathologic features in nasopharyngeal carcinoma (NPC), including patient survival. METHODS: Immunohistochemistry was used to examine the expression of p27 in 130 primary NPC tissues. The relationship between the levels of p27 expression and clinicopathologic characteristics was analyzed. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. The significance of various survival variables was analyzed using multivariate Cox proportional hazards model. RESULTS: p27 was expressed in both nuclear and cytoplasmic compartments. Nuclear expression of p27 was inversely correlated with T classification and clinical stage. Patients with nuclear p27 expression had better overall survival rates than those without nuclear expression of p27. Further, we observed that nuclear expression of p27 was positively associated with survival time of NPC patients not only in N0-1 and M0 classifications but also in radiotherapy and chemotherapy treatment groups. Finally, we found that nuclear expression of p27 was not an independent prognostic factor for patients with NPC. CONCLUSIONS: Our findings hint that nuclear expression of p27 is a potentially favorable factor in the progression and prognosis of NPC.

Conserved arginine residue in the membrane-spanning domain of HIV-1 gp41 is required for efficient membrane fusion.

Despite the high mutation rate of HIV-1, the amino acid sequences of the membrane-spanning domain (MSD) of HIV-1 gp41 are well conserved. Arginine residues are rarely found in single membrane-spanning domains, yet an arginine residue, R(696) (the numbering is based on that of HXB2), is highly conserved in HIV-1 gp41. To examine the role of R(696), it was mutated to K, A, I, L, D, E, N, and Q. Most of these substitutions did not affect the expression, processing or surface distribution of the envelope protein (Env). However, a syncytia formation assay showed that the substitution of R(696) with amino acid residues other than K, a naturally observed mutation in the gp41 MSD, decreased fusion activity. Substitution with hydrophobic amino acid residues (A, I, and L) resulted in a modest decrease, while substitution with D or E, potentially negatively-charged residues, almost abolished the syncytia formation. All the fusion-defective mutants showed slower kinetics with the cell-based dual split protein (DSP) assay that scores the degree of membrane fusion based on pore formation between fusing cells. Interestingly, the D and E substitutions did show some fusion activity in the DSP assays, suggesting that proteins containing D or E substitutions retained some fusion pore-forming capability. However, nascent pores failed to develop, due probably to impaired activity in the pore enlargement process. Our data show the importance of this conserved arginine residue for efficient membrane fusion.

Membrane topology analysis of HIV-1 envelope glycoprotein gp41.

BACKGROUND: The gp41 subunit of the HIV-1 envelope glycoprotein (Env) has been widely regarded as a type I transmembrane protein with a single membrane-spanning domain (MSD). An alternative topology model suggested multiple MSDs. The major discrepancy between the two models is that the cytoplasmic Kennedy sequence in the single MSD model is assigned as the extracellular loop accessible to neutralizing antibodies in the other model. We examined the membrane topology of the gp41 subunit in both prokaryotic and mammalian systems. We attached topological markers to the C-termini of serially truncated gp41. In the prokaryotic system, we utilized a green fluorescent protein (GFP) that is only active in the cytoplasm. The tag protein (HaloTag) and a membrane-impermeable ligand specific to HaloTag was used in the mammalian system. RESULTS: In the absence of membrane fusion, both the prokaryotic and mammalian systems (293FT cells) supported the single MSD model. In the presence of membrane fusion in mammalian cells (293CD4 cells), the data obtained seem to support the multiple MSD model. However, the region predicted to be a potential MSD is the highly hydrophilic Kennedy sequence and is least likely to become a MSD based on several algorithms. Further analysis revealed the induction of membrane permeability during membrane fusion, allowing the membrane-impermeable ligand and antibodies to cross the membrane. Therefore, we cannot completely rule out the possible artifacts. Addition of membrane fusion inhibitors or alterations of the MSD sequence decreased the induction of membrane permeability. CONCLUSIONS: It is likely that a single MSD model for HIV-1 gp41 holds true even in the presence of membrane fusion. The degree of the augmentation of membrane permeability we observed was dependent on the membrane fusion and sequence of the MSD.

The membrane-spanning domain of gp41 plays a critical role in intracellular trafficking of the HIV envelope protein.

BACKGROUND: The sequences of membrane-spanning domains (MSDs) on the gp41 subunit are highly conserved among many isolates of HIV-1. The GXXXG motif, a potential helix-helix interaction motif, and an arginine residue (rare in hydrophobic MSDs) are especially well conserved. These two conserved elements are expected to locate on the opposite sides of the MSD, if the MSD takes a α-helical secondary structure. A scanning alanine-insertion mutagenesis was performed to elucidate the structure-function relationship of gp41 MSD. RESULTS: A circular dichroism analysis of a synthetic gp41 MSD peptide determined that the secondary structure of the gp41 MSD was α-helical. We then performed a scanning alanine-insertion mutagenesis of the entire gp41 MSD, progressively shifting the relative positions of MSD segments around the helix axis. Altering the position of Gly694, the last residue of the GXXXG motif, relative to Arg696 (the number indicates the position of the amino acid residues in HXB2 Env) around the axis resulted in defective fusion. These mutants showed impaired processing of the gp160 precursor into gp120 and gp41. Furthermore, these Env mutants manifested inefficient intracellular transport in the endoplasmic reticulum and Golgi regions. Indeed, a transplantation of the gp41 MSD portion into the transmembrane domain of another membrane protein, Tac, altered its intracellular distribution. Our data suggest that the intact MSD α-helix is critical in the intracellular trafficking of HIV-1 Env. CONCLUSIONS: The relative position between the highly conserved GXXXG motif and an arginine residue around the gp41 MSD α-helix is critical for intracellular trafficking of HIV-1 Env. The gp41 MSD region not only modulates membrane fusion but also controls biosynthesis of HIV-1 Env.

Monitoring of HIV-1 envelope-mediated membrane fusion using modified split green fluorescent proteins.

A simple, cell-based, membrane fusion assay system that uses split green fluorescent proteins (spGFPs) as an indicator was developed. The attachment of the pleckstrin homology (PH) domain to the N-termini of each spGFP not only localized the reporter signal to the plasma membrane but also helped the stable expression of the smaller spGFP of seventeen amino acid residues. It was shown that this system allowed real-time monitoring of membrane fusion by HIV-1 envelope protein (Env) without the addition of external substrates. This method can be adapted to the analyses of other viral membrane fusion.