Impaired binding affinity of YTHDC1 with METTL3/METTL14 results in R-loop accumulation in myelodysplastic neoplasms with DDX41 mutation.

DEAD box helicase 41 (DDX41) mutations are the most prevalent predisposition to familial myelodysplastic syndrome (MDS). However, the precise roles of these variants in the pathogenesis of MDS have yet to be elucidated. Here, we discovered a novel mechanism by which DDX41 contributes to R-loop-induced DNA damage responses (DDR) in cooperation with the m6A-METTL complex (MAC) and YTHDC1 using DDX41 knockout (KO) and DDX41 knock-in (KI, R525H, Y259C) cell lines as well as primary samples from MDS patients. Compared to wild type (WT), DDX41 KO and KI led to increased levels of m6A RNA methylated R-loop. Interestingly, we found that DDX41 regulates m6A/R-loop levels by interacting with MAC components. Further, DDX41 promoted the recruitment of YTHDC1 to R-loops by promoting the binding between METTL3 and YTHDC1, which was dysregulated in DDX41-deficient cells, contributing to genomic instability. Collectively, we demonstrated that DDX41 plays a key role in the physiological control of R-loops in cooperation with MAC and YTHDC1. These findings provide novel insights into how defects in DDX41 influence MDS pathogenesis and suggest potential therapeutic targets for the treatment of MDS.

Inhibition of phospholipase D1 induces immunogenic cell death and potentiates cancer immunotherapy in colorectal cancer.

Phospholipase D (PLD) is a potential therapeutic target against cancer. However, the contribution of PLD inhibition to the antitumor response remains unknown. We developed a potent and selective PLD1 inhibitor based on computer-aided drug design. The inhibitor enhanced apoptosis in colorectal cancer (CRC) cells but not in normal colonic cells, and in vitro cardiotoxicity was not observed. The inhibitor downregulated the Wnt/ß-catenin signaling pathway and reduced the migration, invasion, and self-renewal capacity of CRC cells. In cancer, therapeutic engagement of immunogenic cell death (ICD) leads to more effective responses by eliciting the antitumor immunity of T cells. The CRC cells treated with the inhibitor showed hallmarks of ICD, including downregulation of "do not eat-me" signals (CD24, CD47, programmed cell death ligand 1 [PD-L1]), upregulation of "eat-me" signal (calreticulin), release of high-mobility group Box 1, and ATP. PLD1 inhibition subsequently enhanced the phagocytosis of cancer cells by macrophages through the surface expression of costimulatory molecules; as a result, the cancer cells were more susceptible to cytotoxic T-cell-mediated killing. Moreover, PLD1 inhibition attenuated colitis-associated CRC and orthotopically injected tumors, probably by controlling multiple pathways, including Wnt signaling, phagocytosis checkpoints, and immune signaling. Furthermore, combination therapy with a PLD1 inhibitor and an anti-PD-L1 antibody further enhanced tumor regression via immune activation in the tumor environment. Collectively, in this study, PLD1 was identified as a critical regulator of the tumor microenvironment in colorectal cancer, suggesting the potential of PLD1 inhibitors for cancer immunotherapy based on ICD and immune activation. PLD1 inhibitors may act as promising immune modulators in antitumor treatment via ICD.

Cellular and subcellular localization of endogenous phospholipase D6 in seminiferous tubules of mouse testes.

Phospholipase D6 (PLD6) plays pivotal roles in mitochondrial dynamics and spermatogenesis, but the cellular and subcellular localization of endogenous PLD6 in testis germ cells is poorly defined. We examined the distribution and subcellular localization of PLD6 in mouse testes using validated specific anti-PLD6 antibodies. Ectopically expressed PLD6 protein was detected in the mitochondria of PLD6-transfected cells, but endogenous PLD6 expression in mouse testes was localized to the perinuclear region of pachytene spermatocytes, and more prominently, to the round (Golgi and cap phases) and elongating spermatids (acrosomal phase); these results suggest that PLD6 is localized to the Golgi apparatus. The distribution of PLD6 in the round spermatids partially overlapped with that of the cis-Golgi marker GM130, indicating that the PLD6 expression corresponded to the GM130-positive subdomains of the Golgi apparatus. Correlative light and electron microscopy revealed that PLD6 expression in developing spermatids was localized almost exclusively to several flattened cisternae, and these structures might correspond to the medial Golgi subcompartment; neither the trans-Golgi networks nor the developing acrosomal system expressed PLD6. Further, we observed that PLD6 interacted with tesmin, a testis-specific transcript necessary for successful spermatogenesis in mouse testes. To our knowledge, these results provide the first evidence of PLD6 as a Golgi-localized protein of pachytene spermatocytes and developing spermatids and suggest that its subcompartment-specific distribution within the Golgi apparatus may be related to the specific functions of this organelle during spermatogenesis.

Deletion of phospholipase D1 decreases bone mass and increases fat mass via modulation of Runx2, ß-catenin-osteoprotegerin, PPAR-γ and C/EBPα signaling axis.

In osteoporosis, mesenchymal stem cells (MSCs) prefer to differentiate into adipocytes at the expense of osteoblasts. Although the balance between adipogenesis and osteogenesis has been closely examined, the mechanism of commitment determination switch is unknown. Here we demonstrate that phospholipase D1 (PLD1) plays a key switch in determining the balance between bone and fat mass. Ablation of Pld1 reduced bone mass but increased fat in mice. Mechanistically, Pld1/- MSCs inhibited osteoblast differentiaion with diminished Runx2 expression, while osteoclast differentiation was accelerated in Pld1-/- bone marrow-derived macrophages. Pld1-/- osteoblasts showed decreased expression of osteogenic makers. Increased number and resorption activity of osteoclasts in Pld1-/- mice were corroborated with upregulation of osteoclastogenic markers. Moreover, Pld1-/- osteoblasts reduced ß-catenin mediated-osteoprotegerin (OPG) with increased RANKL/OPG ratio which resulted in accelerated osteoclast differentiation. Thus, low bone mass with upregulated osteoclasts could be due to the contribution of both osteoblasts and osteoclasts during bone remodeling. Moreover, ablation of Pld1 further increased bone loss in ovariectomized mice, suggesting that PLD1 is a negative regulator of osteoclastogenesis. Furthermore, loss of PLD1 increased adipogenesis, body fat mass, and hepatic steatosis along with upregulation of PPAR-γ and C/EBPα. Interestingly, adipocyte-specific Pld1 transgenic mice rescued the compromised phenotypes of fat mass and adipogenesis in Pld1 knockout mice. Collectively, PLD1 regulated the bifurcating pathways of mesenchymal cell lineage into increased osteogenesis and decreased adipogenesis, which uncovered a previously unrecognized role of PLD1 in homeostasis between bone and fat mass.

PLD1 and PLD2 differentially regulate the balance of macrophage polarization in inflammation and tissue injury.

Phospholipase D (PLD) isoforms PLD1 and PLD2 serve as the primary nodes where diverse signaling pathways converge. However, their isoform-specific functions remain unclear. We showed that PLD1 and PLD2 selectively couple to toll-like receptor 4 (TLR4) and interleukin 4 receptor (IL-4R) and differentially regulate macrophage polarization of M1 and M2 via the LPS-MyD88 axis and the IL-4-JAK3 signaling, respectively. Lipopolysaccharide (LPS) enhanced TLR4 or MyD88 interaction with PLD1; IL-4 induced IL-4R or JAK3 association with PLD2, indicating isozyme-specific signaling events. PLD1 and PLD2 are indispensable for M1 polarization and M2 polarization, respectively. Genetic and pharmacological targeting of PLD1 conferred protection against LPS-induced sepsis, cardiotoxin-induced muscle injury, and skin injury by promoting the shift toward M2; PLD2 ablation intensified disease severity by promoting the shift toward M1. Enhanced Foxp3+ regulatory T cell recruitment also influenced the anti-inflammatory phenotype of Pld1LyzCre macrophages. We reveal a previously uncharacterized role of PLD isoforms in macrophage polarization, signifying potential pharmacological interventions for macrophage modulation.

Phospholipase D1 is upregulated by vorinostat and confers resistance to vorinostat in glioblastoma.

Glioblastoma (GBM) is an aggressive brain tumor and drug resistance remains a major barrier for therapeutics. Epigenetic alterations are implicated in GBM pathogenesis, and epigenetic modulators including histone deacetylase (HDAC) inhibitors are exploited as promising anticancer therapies. Here, we demonstrate that phospholipase D1 (PLD1) is a transcriptional target of HDAC inhibitors and confers resistance to HDAC inhibitor in GBM. Treatment of vorinostat upregulates PLD1 through PKCζ-Sp1 axis. Vorinostat induces dynamic changes in the chromatin structure and transcriptional machinery associated with PLD1 promoter region. Cotreatment of vorinostat with PLD1 inhibitor further attenuates invasion, angiogenesis, colony-forming capacity, and self-renewal capacity, compared with those of either treatment. PLD1 inhibitor overcomes resistance to vorinostat in GBM cells intracranial GBM tumors. Our finding provides new insight into the role of PLD1 as a target of resistance to vorinostat, and PLD1 inhibitor might provide the basis for therapeutic combinations with improved efficacy of HDAC inhibitor.

Pleckstrin homology domain of phospholipase D2 is a negative regulator of focal adhesion kinase.

Phospholipase D2 (PLD2) has been implicated in the tyrosine kinase-mediated signaling pathways, but the regulation events are yet to be identified. Herein, we demonstrate that pleckstrin homology (PH) domain of PLD2 (PLD2-PH) exerts an antitumorigenic effect via the suppression of PLD2 and focal adhesion kinase (FAK). The kinase domain of FAK interacts with PLD2-PH and induces tyrosine phosphorylation and activation of PLD2. Furthermore, PLD2 increased tyrosine phosphorylation of FAK. However, ectopic expression of the PLD2-PH competes for binding to FAK and reduces the interaction between PLD2 and FAK, thereby suppressing FAK-induced PLD activation and tyrosine phosphorylation of FAK. The PLD2-PH suppressed the migration and invasion of glioblastoma cells, as well as tumor formation in a xenograft mouse model. This study uncovers a novel role of PLD2-PH as a negative regulator of PLD2 and FAK. [BMB Reports 2021; 54(2): 112-117].

Inhibition of phospholipase D2 augments histone deacetylase inhibitor-induced cell death in breast cancer cells.

BACKGROUND: Histone deacetylase (HDAC) inhibitors are promising anticancer drugs but their effect on tumor treatment has been disappointing mainly due to the acquisition of HDAC inhibitor resistance. However, the mechanisms underlying such resistance remain unclear. METHODS: In this study, we performed Western blot, q-PCR, and promoter assay to examine the expression of HDAC inhibitor-induced phospholipase D2 (PLD2) in MDA-MB231and MDA-MB435 breast cancer cells. Apoptosis and proliferation were analyzed by flow cytometry. In addition to invasion and migration assay, angiogenesis was further measured using in vitro tube formation and chick embryo chorioallantoic membrane model. RESULTS: HDAC inhibitors including suberoylanilide hydroxamic acid (SAHA), trichostatin, and apicidin, induce expression of PLD2 in a transcriptional level. SAHA upregulates expression of PLD2 via protein kinase C-ζ in breast cancer cells and increases the enzymatic activity of PLD. The combination treatment of SAHA with PLD2 inhibitor significantly enhances cell death in breast cancer cells. Phosphatidic acid, a product of PLD activity, prevented apoptosis promoted by cotreatment with SAHA and PLD2 inhibitor, suggesting that SAHA-induced PLD2 expression and subsequent activation of PLD2 might confers resistance of breast cancer cells to HDAC inhibitor. The combinational treatment of the drugs significantly suppressed invasion, migration, and angiogenesis, compared with that of either treatment. CONCLUSION: These findings provide further insight into elucidating the advantages of combination therapy with HDAC and PLD2 inhibitors over single-agent strategies for the treatment of cancer.

Phospholipase D1 inhibition sensitizes glioblastoma to temozolomide and suppresses its tumorigenicity.

Resistance of glioblastoma to the chemotherapeutic compound temozolomide is associated with the presence of glioblastoma stem cells in glioblastoma and is a key obstacle for the poor prognosis of glioblastoma. Here, we show that phospholipase D1 is elevated in CD44High glioblastoma stem cells and in glioblastoma, especially recurring glioblastoma. Phospholipase D1 elevation positively correlated with the level of CD44 and poor prognosis in glioblastoma patients. Temozolomide significantly upregulated the expression of phospholipase D1 in the low and moderate CD44 populations of glioblastoma stem cells, but not in the CD44High population in which phospholipase D1 is highly expressed. Phospholipase D1 conferred resistance to temozolomide in CD44High glioblastoma stem cells and increased their self-renewal capacity and maintenance. Phospholipase D1 expression significantly correlated with levels of temozolomide resistance factors, which were suppressed by microRNA-320a and -4496 induced by phospholipase D1 inhibition. Genetic and pharmacological targeting of phospholipase D1 attenuated glioblastoma stem cell-derived intracranial tumors of glioblastoma using the microRNAs, and improved survival. Treatment solely with temozolomide produced no benefits on the glioblastoma, whereas in combination, phospholipase D1 inhibition sensitized glioblastoma stem cells to temozolomide and reduced glioblastoma tumorigenesis. Together, these findings indicate that phospholipase D1 inhibition might overcome resistance to temozolomide and represents a potential treatment strategy for glioblastoma. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Targeting of Phospholipase D1 Ameliorates Collagen-Induced Arthritis via Modulation of Treg and Th17 Cell Imbalance and Suppression of Osteoclastogenesis.

Phospholipase D1 (PLD1) plays a crucial role in various inflammatory and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease. However, the role of PLD1 in the pathogenesis of RA remains unknown. Here, we first investigated the role and effects of PLD1 in collagen-induced arthritis (CIA) and found that genetic and pharmacological inhibition of PLD1 in DBA1/J mice with CIA reduced the incidence of CIA, decreased the clinical score, and abrogated disease symptoms including infiltration of leukocytes, synovial inflammation, bone erosion, and cartilage destruction. Moreover, ablation and inhibition of PLD1 suppressed the production of type II collagen-specific IgG2a autoantibody and proinflammatory cytokines, accompanied by an increase in the regulatory T (Treg) cell population and a decrease in the Th17 cell population in CIA mice. The PLD1 inhibitor also promoted differentiation of Treg cells and suppressed differentiation of Th17 cells in vitro. Furthermore, the PLD1 inhibitor attenuated pathologic bone destruction in CIA mice by suppressing osteoclastogenesis and bone resorption. Thus, our findings indicate that the targeting of PLD1 can ameliorate CIA by modulating the imbalance of Treg and Th17 cells and suppressing osteoclastogenesis, which might be a novel strategy to treat autoimmune diseases, such as RA.

MicroRNA-320a and microRNA-4496 attenuate Helicobacter pylori cytotoxin-associated gene A (CagA)-induced cancer-initiating potential and chemoresistance by targeting ß-catenin and ATP-binding cassette, subfamily G, member 2.

Infection with Helicobacter pylori is closely linked to an increased risk of gastric cancer. Although cytotoxin-associated gene A (CagA), a major virulence factor of H. pylori, is known to be a causal factor for gastric carcinogenesis, the molecular link between CagA and gastric cancer-initiating cell (CIC)-like properties remains elusive. Here, we demonstrate that CagA is required for increased expression of ß-catenin and its target CIC markers via downregulation of microRNA (miR)-320a and miR-4496. CagA promoted gastric CIC properties and was responsible for chemoresistance. miR-320a and miR-4496 attenuated the in vitro self-renewal and tumour-initiating capacity of CagA-expressing CICs by targeting ß-catenin. Moreover, miR-320a and miR-4496 decreased CagA-induced chemoresistance by targeting ATP-binding cassette, subfamily G, member 2 (ABCG2) at the transcriptional and post-transcriptional levels, respectively. Combination therapy with 5-fluorouracil and miR-320a/miR-4496 suppressed gastric tumourigenesis and metastatic potential in an orthotopic mouse model, probably via suppression of CagA-induced CIC properties and chemoresistance. Our results provide novel evidence that CIC properties, chemoresistance and tumourigenesis associated with H. pylori are linked to CagA-induced upregulation of ß-catenin and ABCG2. These data provide novel insights into the molecular mechanisms of CagA-induced carcinogenisis and the therapeutic potential of of miR-320a and miR-4496. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Phospholipase D1 Acts through Akt/TopBP1 and RB1 to Regulate the E2F1-Dependent Apoptotic Program in Cancer Cells.

The RB1/E2F1 signaling pathway is frequently deregulated in colorectal cancer and has been suggested to intersect with Wnt/ß-catenin and PI3K/Akt pathways, but molecular evidence for this link is lacking. In this study, we demonstrate that phospholipase D1 (PLD1), a transcriptional target of ß-catenin/TCF4, orchestrates functional interactions between these pathways during intestinal tumor development. Overexpression of PLD1 in intestinal epithelial cells protected cells from apoptosis induced by PLD1 ablation in the Apcmin/+ mouse model of intestinal tumorigenesis. Mechanistic investigations revealed that genetic and pharmacologic targeting of PLD1 promote the E2F1-dependent apoptotic program via both miR-192/4465-mediated downregulation of RB1 and inhibition of Akt-TopBP1 pathways. Moreover, the miRNA-RB1 axis and Akt pathway also contributed to the PLD1-mediated self-renewal capacity of colon cancer-initiating cells. Finally, PLD1-driven E2F1 target gene expression positively correlated with tumor stage in patients with colorectal cancer. Overall, our findings suggest that PLD1 mediates cross-talk between multiple major signaling pathways to promote the survival and malignancy of colon cancer cells and may therefore represent an ideal signaling node for therapeutic targeting. Cancer Res; 77(1); 142-52. ©2016 AACR.

Rebamipide attenuates Helicobacter pylori CagA-induced self-renewal capacity via modulation of ß-catenin signaling axis in gastric cancer-initiating cells.

Rebamipide, a mucosal-protective agent, is used clinically for treatment of gastritis and peptic ulcers induced by Helicobacter pylori (H. pylori) which is associated with increased risk of gastric cancer. Although rebamipide is known to inhibit the growth of gastric cancer cells, the action mechanisms of rebamipide in gastric carcinogenesis remains elusive. Here, we show that rebamipide suppresses H. pylori CagA-induced ß-catenin and its target cancer-initiating cells (C-IC) marker gene expression via upregulation of miRNA-320a and -4496. Rebamipide attenuated in vitro self-renewal capacity of H. pylori CagA-infected gastric C-IC via modulation of miRNA-320a/-4496-ß-catenin signaling axis. Moreover, rebamipide enhanced sensitivity to chemotherapeutic drugs in CagA-expressed gastric C-IC. Furthermore, rebamipide suppressed tumor-initiating capacity of gastric C-IC, probably via suppression of CagA-induced C-IC properties. These data provide novel insights for the efficacy of rebamipide as a chemoprotective drug against H. pylori CagA-induced carcinogenic potential.

A translocator protein 18 kDa ligand, Ro5-4864, inhibits ATP-induced NLRP3 inflammasome activation.

Ro5-4864 and PK11195, prototypical synthetic ligands of translocator protein 18 kDa (TSPO), have shown anti-inflammatory effects in several models of inflammatory diseases; however, their biochemical mechanisms remain poorly understood. Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation as a part of the innate immune system, has been implicated in a variety of inflammatory diseases. Here, we demonstrate for the first time that TSPO ligands, especially Ro5-4864, potently suppressed ATP-induced NLRP3 inflammasome activation in THP-1 and BMDM cells. Detailed action mechanism was further investigated in THP-1 cells. Ro5-4864 efficiently attenuated NLRP3 translocation to mitochondria, inflammasome assembly/oligomerization, activation of caspase-1, and subsequent secretion of the mature forms of interleukin-1ß and -18. Ro5-4864 also reduced the production of mitochondrial superoxide and preserved the mitochondrial membrane potential in ATP-treated cells, suggesting that Ro5-4864 may act on mitochondria or more upstream targets in NLRP3 inflammasome signaling. We also observed the distinct effects of the TSPO ligands between THP-1 monocytes and macrophages, which suggested different NLRP3 inflammasome signaling depending on cell type. Collectively, our novel findings demonstrate that Ro5-4864 effectively inhibited ATP-induced NLRP3 inflammasome activation through the prevention of mitochondrial perturbation. Our results indicate Ro5-4864 as a promising candidate for the treatment of NLRP3 inflammasome-related diseases.

Inhibition of phospholipase D2 induces autophagy in colorectal cancer cells.

Autophagy is a conserved lysosomal self-digestion process used for the breakdown of long-lived proteins and damaged organelles, and it is associated with a number of pathological processes, including cancer. Phospholipase D (PLD) isozymes are dysregulated in various cancers. Recently, we reported that PLD1 is a new regulator of autophagy and is a potential target for cancer therapy. Here, we investigated whether PLD2 is involved in the regulation of autophagy. A PLD2-specific inhibitor and siRNA directed against PLD2 were used to treat HT29 and HCT116 colorectal cancer cells, and both inhibition and genetic knockdown of PLD2 in these cells significantly induced autophagy, as demonstrated by the visualization of light chain 3 (LC3) puncta and autophagic vacuoles as well as by determining the LC3-II protein level. Furthermore, PLD2 inhibition promoted autophagic flux via the canonical Atg5-, Atg7- and AMPK-Ulk1-mediated pathways. Taken together, these results suggest that PLD2 might have a role in autophagy and that its inhibition might provide a new therapeutic basis for targeting autophagy.