Arbuscular mycorrhizal fungi form symbiotic associations with most land plants and deliver mineral nutrients, in particular phosphate, to the host. Therefore, understanding the mechanisms of ...phosphate acquisition and delivery in the fungi is critical for full appreciation of the mutualism in this association. Here, we provide updates on physical, chemical, and biological strategies of the fungi for phosphate acquisition, including interactions with phosphate-solubilizing bacteria, and those on the regulatory mechanisms of phosphate homeostasis based on resurveys of published genome sequences and a transcriptome with reference to the latest findings in a model fungus. For the mechanisms underlying phosphate translocation and export to the host, which are major research frontiers in this field, not only recent advances but also testable hypotheses are proposed. Lastly, we briefly discuss applicability of the latest tools to gene silencing in the fungi, which will be breakthrough techniques for comprehensive understanding of the molecular basis of fungal phosphate metabolism.
Arbuscular mycorrhizal fungi provide plants with soil mineral nutrients, particularly phosphorus. In this symbiotic association, the arbuscular interface is the main site for nutrient exchange. To ...understand phosphorus transfer at the interface, we analyzed the subcellular localization of polyphosphate (polyP) in mature arbuscules of
Rhizophagus irregularis
colonizing roots of
Lotus japonicus
wild-type (WT) and H
+
-ATPase
ha1-1
mutant, which is defective in phosphorus acquisition through the mycorrhizal pathway. In both, the WT and the
ha1-1
mutant, polyP accumulated in the cell walls of trunk hyphae and inside fine branch modules close to the trunk hyphae. However, many fine branches lacked polyP. In the mutant, most fine branch modules showed polyP signals compared to the WT. Notably, polyP was also observed in the cell walls of some fine branches formed in the
ha1-1
mutant, indicating phosphorus release from fungal cells to the apoplastic regions. Intense acid phosphatase (ACP) activity was detected in the periarbuscular spaces around the fine branches. Furthermore, double staining of ACP activity and polyP revealed that these had contrasting distribution patterns in arbuscules. These observations suggest that polyP in fungal cell walls and apoplastic phosphatases may play an important role in phosphorus transfer at the symbiotic interface in arbuscules.
Background
Although immunotherapy with immune checkpoint inhibitors (ICIs) has become a standard therapeutic strategy in colorectal cancer (CRC) exhibiting microsatellite instability-high, limited ...patients benefit from this new approach. To increase the efficacy of ICIs in CRC patients, it is crucial to control the function of immunosuppressive cells in the tumor microenvironment. M2-tumor-associated macrophages (TAMs) are key immunosuppressive cells and promote tumor growth, angiogenesis, and epithelial-mesenchymal transition. In the present study, we focused on the VEGF signaling pathway in M2-TAMs to control their inhibitory function.
Methods
We evaluated the population of M2-TAMs, the VEGF receptor 2 (VEGFR2) expression on M2-TAMs, and the correlation between HIF-1α-positive cells and VEGFR2 expression levels on M2-TAMs in CRC using the analysis of The Cancer Genome Atlas colorectal adenocarcinoma dataset (
n
= 592), the flow cytometry of freshly resected surgical specimens of CRC (
n
= 20), and the immunofluorescence staining of formalin-fixed paraffin-embedded whole tissue samples of CRC (
n
= 20). Furthermore, we performed a functional assay of M2 macrophages through the VEGF/VEGFR2 signaling pathway in vitro.
Results
The population of M2-TAMs and their VEGFR2 expression significantly increased in the tumor compared to the normal mucosa in the CRC patients. HIF1-α-positive cells significantly correlated with the VEGFR2 expression level of M2-TAMs. M2 macrophages induced by cytokines in vitro produced TGF-β1 through the VEGF/VEGFR2 signaling pathway.
Conclusions
Our results suggest that anti-VEGFR2 therapy may have therapeutic potential to control the immune inhibitory functions of M2-TAMs in CRC, resulting in enhanced efficacy of immunotherapy with ICIs.
Background
AT-rich interactive domain 1A (
ARID1A
) is a tumor suppressor gene that is frequently mutated in gastric cancer (GC). Although
ARID1A
mutations are not a druggable target for conventional ...treatments, novel therapeutic strategies based on a synthetic lethal approach are effective for ARID1A-deficient cancers. The histone methyltransferase EZH2 acts in a synthetic lethal manner in
ARID1A
-mutated ovarian cancer, although its role in GC remains unknown.
Methods
The selective sensitivity of the EZH2 inhibitors for ARID1A-deficient GC cells was evaluated using cell viability and colony formation assays. The expression of PI3K/AKT signaling genes were investigated using TCGA’s cBioPortal database to determine whether the homeostasis between ARID1A and EZH2 is related to cell proliferation and survival via the PI3K/AKT signaling pathway. We also evaluated the phosphorylation of PI3K/AKT signaling proteins in
ARID1A
knock downed
ARID1A
-WT GC cells.
Results
EZH2 inhibitors decreased the viability of ARID1A-deficient cells in a dose-dependent manner and demonstrated the selective sensitivity to ARID1A-deficient cells in vitro experiment system. Bioinformatics approach revealed that the PI3K/AKT signaling was tended to be activated in ARID1A-deficient GC enhancing cell viability and, furthermore, down-regulation of EZH2 in ARID1A-deficient GC was related to normalization of PI3K/AKT signaling pathway. The cell experiment revealed that phosphorylated AKT was upregulated in ARID1A-deficent GC cells.
Conclusions
The present findings provide a rationale for the selective sensitivity of EZH2 inhibitors against ARID1A-deficient GC and suggest the potential efficacy of targeted therapy using EZH2 inhibitors in this patient population.
Epstein-Barr virus-positive gastric cancer EBV (+) GC is a distinct GC subtype with unique genetic and epigenetic aberrations. Here, we examined resected GC samples and publicly available microarray ...data and The Cancer Genome Atlas (TCGA) database to identify the mechanism underlying overexpression of PD-L1 in EBV (+) GC. We found that high levels of PD-L1 overexpression in EBV (+) GC were caused by focal amplification of CD274. By contrast, relatively high expression of PD-L1 in tumor tissue and infiltrating immune cells correlated with CD8 lymphocyte infiltration and IFN-γ expression via IRF3 activation. Since we reported previously that PD-L1 expression is associated both with the presence of CD8 T cells in the tumor microenvironment and with IFN-γ expression in GC, we examined a database to see whether IFN-γ-associated overexpression of PD-L1 plays a significant role in EBV (+) GC. Immunohistochemical staining showed that expression of the IRF3 signature in clinical GC samples was higher in EBV (+) than in EBV (-) cases. The data presented herein reveal a unique dual mechanism underlying PD-L1 overexpression in EBV (+) GC: high focal amplification of CD274 or IFN-γ-mediated signaling via activation of IRF3.
The development of arbuscular mycorrhiza (AM) is strongly suppressed under high-phosphate (Pi) conditions. To investigate AM fungal responses during the suppression of AM by high Pi, we performed an ...RNA-seq analysis of
Rhizophagus irregularis
colonizing
Lotus japonicus
roots at different levels of Pi (20, 100, 300, and 500 μM). AM fungal colonization decreased markedly under high-Pi conditions. In total, 163 fungal genes were differentially expressed among the four Pi treatments. Among these genes, a cell cycle-regulatory gene, cyclin-dependent kinase
CDK1
, and several DNA replication- and mitosis-related genes were repressed under high-Pi conditions. More than 20 genes encoding secreted proteins were also downregulated by high-Pi conditions, including the strigolactone-induced putative secreted protein 1 gene that enhances AM fungal colonization. In contrast, the expression of genes related to aerobic respiration and transport in
R. irregularis
were largely unaffected. Our data suggest that high Pi suppresses the expression of genes associated with fungal cell cycle progression or that encode secreted proteins that may be required for intercellular hyphal growth and arbuscule formation. However, high Pi has little effect on the transcriptional regulation of the primary metabolism or transport in preformed fungal structures.
Arbuscular mycorrhizal fungi translocate polyphosphate through hyphae over a long distance to deliver to the host. More than three decades ago, suppression of host transpiration was found to ...decelerate phosphate delivery of the fungal symbiont, leading us to hypothesize that transpiration provides a primary driving force for polyphosphate translocation, probably via creating hyphal water flow in which fungal aquaporin(s) may be involved.
The impact of transpiration suppression on polyphosphate translocation through hyphae of Rhizophagus clarus was evaluated. An aquaporin gene expressed in intraradical mycelia was characterized and knocked down by virus-induced gene silencing to investigate the involvement of the gene in polyphosphate translocation.
Rhizophagus clarus aquaporin 3 (RcAQP3) that was most highly expressed in intraradical mycelia encodes an aquaglyceroporin responsible for water transport across the plasma membrane. Knockdown of RcAQP3 as well as the suppression of host transpiration decelerated polyphosphate translocation in proportion to the levels of knockdown and suppression, respectively.
These results provide the first insight into the mechanism underlying long-distance polyphosphate translocation in mycorrhizal associations at the molecular level, in which host transpiration and the fungal aquaporin play key roles. A hypothetical model of the translocation is proposed for further elucidation of the mechanism.
Arbuscular mycorrhizal (AM) fungi form symbiotic associations with diverse plant species. The AM fungi enhance mineral uptake from the soil, which benefits the growth of the host plants. Previous ...microarray and RNA-seq analyses have identified a large number of AM-responsive plant genes. However, little is known whether the gene expression profile of mycorrhiza is different among genetically distant plant species. The aim of this study was to assess the conservation and divergence of AM-responsive genes between two different hosts, Solanum lycopersicum L. and Lotus japonicus L., during AM development using RNA-seq data. In each host plant, gene expression was compared between AM roots and non-mycorrhizal (NM) roots. Potential orthologs of AM-responsible genes between S. lycopersicum and L. japonicus were identified with reciprocal BLAST searches. Only one quarter to one third of the AM-inducible genes in each plant species were co-upregulated in both species. The co-upregulated genes included those known to be essential for AM development and function. The co-upregulated genes exhibited a wide range of fold changes in the AM symbiosis, and the fold change value for individual co-upregulated genes was positively correlated between the two hosts. Most of the species-dependent upregulated genes exhibited low levels of induction. We also analyzed gene expression in AM fungi colonizing roots of S. lycopersicum and L. japonicus. Overall, the gene expression profiles of Rhizophagus irregularis were similar among the roots of the two different hosts, although hundreds of fungal genes were differentially expressed between the two hosts. In particular, genes related to the mitochondrial electron transport chain were highly expressed in AM fungi colonizing L. japonicus roots, indicating that adenosine triphosphate (ATP) production was enhanced in the L. japonicus-R. irregularis symbiosis. Overall, these results show that a certain proportion of AM-responsive genes is conserved across plant species. The species-dependent AM-responsive genes may be related to the physiological differences between AM and NM roots in each plant species.
AT-rich interactive domain 1A (ARID1A) functions as a tumor suppressor and several therapeutic targets in ARID1A-mutated cancers are under development. Here, we investigated the prognostic value of ...ARID1A for gastric cancer and its association with expression of PD-L1 and p53. ARID1A expression was examined by immunohistochemistry and negative expression of ARID1A was detected in 39 (19.5%) of 200 cases in a test cohort and in 40 (18.2%) of 220 cases in a validation cohort. Negative expression of ARID1A was associated with worse overall survival in undifferentiated cases, particularly early-stage cases. Negative expression of ARID1A was detected in 11 (50%) of 22 PD-L1-positive cases and in 68 (17.1%) of 398 PD-L1-negative cases in a combined cohort. Negative expression of ARID1A was detected in 45 (22%) of 205 p53-positive cases and in 34 (15.8%) of 215 p53-negative cases in a combined cohort. In addition, expression of EZH2, a potential synthetic lethal target in ARID1A-mutated tumors, was detected in 79 ARID1A-negative cases. An ARID1A-knockdown gastric cancer cell line was subjected to microarray analysis, but no actionable targets or pathways were identified. The present results indicate that ARID1A may serve as an early-stage prognostic biomarker for undifferentiated gastric cancer.
Immunotherapy against the interaction between programmed cell death 1/programmed cell death ligand 1 (PD-L1) has emerged as a promising strategy for colorectal cancer with mismatch repair deficiency ...(dMMR) or microsatellite instability-high (MSI-H). The study aimed to identify miRNAs that posttranscriptionally control PD-L1 expression on tumor cells and also regulate immune evasion. A comprehensive miRNA screening using The Cancer Genome Atlas (TCGA) dataset (
= 260) combined with eight different miRNA target prediction programs resulted in the identification of a tumor suppressive miRNA, miR-148a-3p, as a potential negative regulator of PD-L1 expression, particularly in dMMR/MSI-H colorectal cancer. Using multiple cohorts of colorectal cancer, including TCGA data, a microarray dataset (
= 148), and formalin-fixed, paraffin-embedded samples (
= 395), we found that the expression of miR-148a-3p was decreased in dMMR/MSI-H tumors, correlating inversely with PD-L1 levels. We demonstrate that miR-148a-3p directly binds to the 3'-untranslated region of PD-L1, thereby reducing whole-cell and cell surface PD-L1 levels in HCT116 and SW837 cell lines. Overexpression of miR-148a-3p repressed IFNγ-induced PD-L1 expression on tumor cells and consequently diminished T-cell apoptosis in a coculture model of IL2-activated T cells and IFNγ-treated tumor cells. In conclusion, our data support a regulatory mechanism of PD-L1 expression on tumor cells and immune suppression via miR-148a-3p downregulation in colorectal cancer. IMPLICATIONS: This study provides novel evidence that miR-148a-3p negatively regulates tumor cell PD-L1 expression and decreased levels of miR-148a-3p contributes to the immunosuppressive tumor microenvironment.
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