PLD1 Expression Research Articles

The molecular mechanism of PLD2-mediated regulation of apoptosis and cell edema in pancreatic cells via the Nrf2/NF-κB pathway

This study aimed to elucidate the molecular mechanisms by which PLD2 controls apoptosis and edema in pancreatic cells via the Nrf2/NF-κB pathway. AR42J rat pancreatic cells were treated with 10 nM mitomycin to create an in vitro pancreatitis model (model group), with a control group receiving phosphate-buffered saline. Cells were transfected with a PLD2 overexpression plasmid using Lipofectamine 3000, forming the PLD2 overexpression group. PLD2 protein expression was assessed by Western blotting, and TNF-α, IL-6, and IL-10 levels were measured by RT-qPCR. Nrf2/NF-κB protein expressions were also analyzed. Apoptosis and necrosis were evaluated using Annexin V-FITC/PI staining and the LDH release test. Cell edema was assessed by cell volume, ion content, and membrane damage. Western blotting was used to analyze pan-apoptosis-related proteins. PLD2 expression was lower in the model group compared to controls (P < 0.05) but higher in the PLD2 overexpression group (P < 0.05). TNF-α, IL-6, and IL-10 levels were elevated in the model group (P < 0.05) and reduced in the PLD2 overexpression group (P < 0.05). Nrf2 expression decreased in the model group but increased with PLD2 overexpression (P < 0.05). NF-κB expression increased in the model group but decreased with PLD2 overexpression (P < 0.05). Apoptosis and necrosis rates were higher in the model group (P < 0.05) but lower in the PLD2 overexpression group (P < 0.05). Cell volume, Na + content, and LDH release increased in the model group (P < 0.05) but decreased with PLD2 overexpression (P < 0.05). RIPK1 expression decreased in the model group (P < 0.05) but increased with PLD2 overexpression (P < 0.05). CASP8, FADD, and ZBP1 levels were higher in the model group (P < 0.05) and reduced with PLD2 overexpression (P < 0.05). PLD2 exerts a protective effect in acute pancreatitis by activating Nrf2 and inhibiting NF-κB, reducing apoptosis, cell swelling, and membrane damage. This highlights potential therapeutic targets for pancreatic inflammation.

Essential role of PLD2 in hypoxia-induced stemness and therapy resistance in ovarian tumors.

Hypoxia in solid tumors is an important source of chemoresistance that can determine poor patient prognosis. Such chemoresistance relies on the presence of cancer stem cells (CSCs), and hypoxia promotes their generation through transcriptional activation by HIF transcription factors. We used ovarian cancer (OC) cell lines, xenograft models, OC patient samples, transcriptional databases, induced pluripotent stem cells (iPSCs) and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). Here, we show that hypoxia induces CSC formation and chemoresistance in ovarian cancer through transcriptional activation of the PLD2 gene. Mechanistically, HIF-1α activates PLD2 transcription through hypoxia response elements, and both hypoxia and PLD2 overexpression lead to increased accessibility around stemness genes, detected by ATAC-seq, at sites bound by AP-1 transcription factors. This in turn provokes a rewiring of stemness genes, including the overexpression of SOX2, SOX9 or NOTCH1. PLD2 overexpression also leads to decreased patient survival, enhanced tumor growth and CSC formation, and increased iPSCs reprograming, confirming its role in dedifferentiation to a stem-like phenotype. Importantly, hypoxia-induced stemness is dependent on PLD2 expression, demonstrating that PLD2 is a major determinant of de-differentiation of ovarian cancer cells to stem-like cells in hypoxic conditions. Finally, we demonstrate that high PLD2 expression increases chemoresistance to cisplatin and carboplatin treatments, both in vitro and in vivo, while its pharmacological inhibition restores sensitivity. Altogether, our work highlights the importance of the HIF-1α-PLD2 axis for CSC generation and chemoresistance in OC and proposes an alternative treatment for patients with high PLD2 expression.

Decreased Expression of PLD2 Promotes EMT in Colorectal Cancer Invasion and Metastasis.

Background and Objectives: PLD2 has been identified as playing a critical role in cancer cell motility and migration and other pathophysiological processes. We investigated the expression of PLD2 and its biological functions and clinical implications in human colorectal cancer. Materials and Methods: In this study, the expressions of PLD2 were analyzed in CRC cell lines and CRC samples by RT-PCR, western blot and immunohistochemistry. The PLD enzyme activity was studied using an PLD detection kit. We also performed matrigel invasion assay to evaluate the invasive capabilities in CRC cells. The expressions of EMT-related markers were quantified at mRNA and protein level using RT-PCR and western blot. We performed high-throughput RNA sequencing on PLD2 knockdown and overexpression CRC cell lines to explore the changes in gene expression associated with PLD2. Result: Herein, we showed that PLD2 expression was relatively low in CRC cell lines and CRC samples and PLD2 deficiency was significantly correlated with more advanced clinical phenotype regarding lymphatic and distant metastasis and poor patient survival. We also detected that PLD2 knockdown favored epithelial-mesenchymal transition (EMT) and thus promoted CRC invasion and metastasis. Further exploration uncovered that the expressions of several important genes closely related to metabolic pathways in CRC were noticeably altered due to PLD2 deficiency, including ID1, IFIT4, OASL, IFIT2 and CTAG2. Conclusion: Our results revealed that PLD2 deficiency promotes cell invasion and metastasis in CRC via EMT indicating PLD2 might have an important implication in carcinogenesis and progression and would be a new therapeutic target for cancer treatment.

Inhibition of phospholipase D1 ameliorates hepatocyte steatosis and non-alcoholic fatty liver disease

Background & AimsPhospholipase D1 (PLD1), a phosphatidylcholine-hydrolysing enzyme, is involved in cellular lipid metabolism. However, its involvement in hepatocyte lipid metabolism and consequently non-alcoholic fatty liver disease (NAFLD) has not been explicitly explored. MethodsNAFLD was induced in hepatocyte-specific Pld1 knockout (Pld1(H)-KO) and littermate Pld1flox/flox (Pld1-Flox) control mice feeding a high-fat diet (HFD) for 20 wk. Changes of the lipid composition in the liver were compared. Alpha mouse liver 12 (AML12) cells and mouse primary hepatocytes were incubated with oleic acid or sodium palmitate in vitro to explore the role of PLD1 in the development of hepatic steatosis. Hepatic PLD1 expression was evaluated in liver biopsy samples in patients with NAFLD. ResultsPLD1 expression levels were increased in the hepatocytes of patients with NAFLD and HFD-fed mice. Compared with Pld1-Flox mice, Pld1(H)-KO mice exhibited decreased plasma glucose and lipid levels as well as lipid accumulation in liver tissues after HFD feeding. Transcriptomic analysis showed that hepatocyte-specific deficiency of PLD1 decreased Cd36 expression in steatosis liver tissues, which was confirmed at the protein and gene levels. In vitro, specific inhibition of PLD1 with VU0155069 or VU0359595 decreased CD36 expression and lipid accumulation in oleic acid- or sodium palmitate-treated AML12 cells or primary hepatocytes. Inhibition of hepatocyte PLD1 significantly altered lipid composition, especially phosphatidic acid and lysophosphatidic acid levels in liver tissues with hepatic steatosis. Furthermore, phosphatidic acid, the downstream product of PLD1, increased the expression levels of CD36 in AML12 cells, which was reversed by a PPARγ antagonist. ConclusionsHepatocyte-specific Pld1 deficiency ameliorates lipid accumulation and NAFLD development by inhibiting the PPARγ/CD36 pathway. PLD1 may be a new target for the treatment of NAFLD. Impact and implicationsThe involvement of PLD1 in hepatocyte lipid metabolism and NAFLD has not been explicitly explored. In this study, we found that the inhibition of hepatocyte PLD1 exerted potent protective effects against HFD-induced NAFLD, which were attributable to a reduction in PPARγ/CD36 pathway-mediated lipid accumulation in hepatocytes. Targeting hepatocyte PLD1 may be a new target for the treatment of NAFLD.

Functional role of PLD1 signalosome in Tauopathies

AbstractBackgroundPhospholipase D (PLD), a lipolytic enzyme for membrane phospholipids, exists in two isoforms of mammalian phosphatidyl choline (PC) specific PLD, inducible PC‐PLD1 and constitutive PC‐PLD2. Elevated PLD activity is well documented in clinical studies involving Alzheimer’s disease (AD). Our lab was the first to show elevated PLD1 facilitates synaptic dysfunction and underlying memory deficits driven by amyloidogenic insults by Aß/tau. We observed preservation of memory in response to PLD1 specific small molecule inhibitor in transgenic mouse models and documented preservation of dendritic spines as a underlying basis. In this study, we explored a role for PLD1 in Frontotemporal Dementia in an attempt to explore its pathological role in this tauopathy‐driven dementia.MethodWe studied altered expression/activity of key F‐actin signaling elements common to PLD1 signalosome in synaptosomal fractions of human post‐mortem FTD brain regions. Synaptic deficits associated with elevated PLD1 expression in human FTD samples was studied by Fluorescence Analysis of Single Synapse Long Term Potentiation (FASS‐LTP). Using immunofluorescence, we studied the association of PLD1 with tau, presynaptic and post‐synaptic receptors, and neuroinflammation markers.ResultElevated PLD1 expression in synaptosomal fractions of post‐mortem FTD brain tissues was corroborated by synaptic deficits and association of PLD1 with postsynaptic density protein (PSD95). FASS‐LTP assay indicated relatively lower long‐term potentiation in synaptosomal fractions of FTD brains as compared to the control brains implying synaptic deficits in FTD subjects.ConclusionIn the light of these observations, we conclude elevated PLD1 expression is associated with toxicity of tau causing memory deficits in FTD patients. This PLD1‐tau interaction alters PLD signaling partners such as mTOR (mammalian target of rapamycin), cofilin and phosphocofilin. This would implicate the actin‐dependent remodeling of synapse to be a key step in the role of PLD1 signalosome in FTD. Delineating the effects of PLD1 signalosome in tauopathies such as FTD will hugely be beneficial in tau‐targeting therapeutics for a multipronged approach in the treatment of neurodegenerative disorders.

Expression with early postnatal peak and female-dominant sexual dimorphism of phospholipase D (PLD) 2 in submandibular gland ducts in situ of mice.

In the present study, expression and localization of PLD2 were examined in mouse submandibular glands in situ in comparison with those of PLD1 previously reported by the present authors. In immunoblots, PLD2-expression was high at postnatal 0week (P0W) and P2W, and at P4W decreased considerably with the decrease of the male gland more marked, and at P8W it was undetectable in the male gland, but remained faint in the female. In the male gland at P8W after castration at P6W, PLD2-expression reappeared faintly. In the female glands with daily injections of testosterone started at P6W, at P8W the expression was undetectable. In immunohistochemistry, PLD2 was localized throughout immature duct epithelia without distinct regional differences in its intensity at P0W and P2W. PLD2-localization was weak and diffuse in granular convoluted tubules at P4W and negligible there at P8W in the female gland, while it was at negligible levels in the tubules at P4W and at undetectable levels there at P8W in the male gland. The expression changes detected in immunoblots after castration or testosterone injection were duplicated in granular convoluted tubules in immunohistochemistry. The present findings suggest that PLD2 is dominantly involved in proliferation/differentiation/apoptosis of most immature ductal cells at early postnatal and at least perinatal stages, different from PLD1. This also indicates that the female-dominant sexual dimorphism of PLD2-expression occurs in synchrony of differentiation of granular convoluted tubules at puberty and at young adulthood, similar to PLD1 though at much lower levels.

Exosome Secretion and Epithelial-Mesenchymal Transition in Ovarian Cancer Are Regulated by Phospholipase D.

Phospholipase D (PLD) isoenzymes participate in a variety of cellular functions that are mostly attributed to phosphatidic acid (PA) synthesis. Dysregulation of PLD regulates tumor progression and metastasis, yet little is known about the underlying mechanism. We previously reported on the expression and clinical role of the PLD isoenzymes PLD1 and PLD2 in tubo-ovarian high-grade serous carcinoma (HGSC). In the present study, we investigated the biological function of PLD1 and PLD2 using the OVCAR-3 and OVCAR-8 HGSC cell lines. KO cell lines for both PLDs were generated using CRISPR/CAS9 technology and assayed for exosome secretion, spheroid formation, migration, invasion and expression of molecules involved in epithelial-mesenchymal transition (EMT) and intracellular signaling. Significant differences between PLD1 and PLD2 KO cells and controls were observed for all the above parameters, supporting an important role for PLD in regulating migration, invasion, metastasis and EMT.

Targeting Phospholipase D Pharmacologically Prevents Phagocytic Function Loss of Retinal Pigment Epithelium Cells Exposed to High Glucose Levels.

We previously described the participation of canonical phospholipase D isoforms (PLD1 and PLD2) in the inflammatory response of retinal pigment epithelium (RPE) cells exposed to high glucose concentrations (HG). Here, we studied the role of the PLD pathway in RPE phagocytic function. For this purpose, ARPE-19 cells were exposed to HG (33 mM) or to normal glucose concentration (NG, 5.5 mM) and phagocytosis was measured using pHrodo™ green bioparticles® or photoreceptor outer segments (POS). HG exposure for 48 and 72 h reduced phagocytic function of ARPE-19 cells, and this loss of function was prevented when cells were treated with 5 μM of PLD1 (VU0359595 or PLD1i) or PLD2 (VU0285655-1 or PLD2i) selective inhibitors. Furthermore, PLD1i and PLD2i did not affect RPE phagocytosis under physiological conditions and prevented oxidative stress induced by HG. In addition, we demonstrated PLD1 and PLD2 expression in ABC cells, a novel human RPE cell line. Under physiological conditions, PLD1i and PLD2i did not affect ABC cell viability, and partial silencing of both PLDs did not affect ABC cell POS phagocytosis. In conclusion, PLD1i and PLD2i prevent the loss of phagocytic function of RPE cells exposed to HG without affecting RPE function or viability under non-inflammatory conditions.

Phospholipase D1 promotes cervical cancer progression by activating the RAS pathway.

This study aimed to further investigate the effect of PLD1 on the biological characteristics of human cervical cancer (CC) cell line, CASKI and the potential related molecular mechanism. CRISPR/Cas9 genome editing technology was used to knock out the PLD1 gene in CASKI cells. Cell function assays were performed to evaluate the effect of PLD1 on the biological function of CASKI cells in vivo and in vitro. A PLD1‐overexpression rescue experiment in these knockout cells was performed to further confirm its function. Two PLD1‐knockout CASKI cell lines (named PC‐11 and PC‐40, which carried the ins1/del4 mutation and del1/del2/ins1 mutation, respectively), were constructed by CRISPR/Cas9. PLD1 was overexpressed in these knockout cells (named PC11‐PLD1 and PC40‐PLD1 cells), which rescued the expression of PLD1 by approximately 71.33% and 74.54%, respectively. In vivo, the cell function assay results revealed that compared with wild‐type (WT)‐CASKI cells, the ability of PC‐11 and PC‐40 cells to proliferate, invade and migrate was significantly inhibited. The expression of H‐Ras and phosphorylation of Erk1/2 (p‐Erk1/2) was decreased in PC‐11 and PC‐40 cells compared with WT‐CASKI cells. PC‐11 and PC‐40 cells could sensitize CASKI cells to cisplatin. More importantly, the proliferation, migration and invasion of PC11‐PLD1 and PC40‐PLD1 cells with PLD1 overexpression were significantly improved compared with those of the two types of PLD1 knockout cells. The sensitivity to cisplatin was decreased in PC11‐PLD1 and PC40‐PLD1 cells compared with PC‐11 and PC‐40 cells. In vivo, in the PC‐11 and PC‐40 tumour groups, tumour growth was significantly inhibited and tumour weight (0.95 ± 0.27 g and 0.66 ± 0.43 g vs. 1.59 ± 0.67 g, p = 0.0313 and 0.0108) and volume (1069.41 ± 393.84 and 1077.72 mm3 ± 815.07 vs. 2142.94 ± 577.37 mm3, p = 0.0153 and 0.0128) were significantly reduced compared to those in the WT‐CASKI group. Tumour differentiation of the PC‐11 and PC40 cells was significantly better than that of the WT‐CASKI cells. The immunohistochemistry results confirmed that the expression of H‐Ras and p‐Erk1/2 was decreased in PC‐11 and PC‐40 tumour tissues compared with WT‐CASKI tumour tissues. PLD1 promotes CC progression by activating the RAS pathway. Inhibition of PLD1 may serve as an attractive therapeutic modality for CC.

Differential expression patterns of phospholipase D isoforms 1 and 2 in the mammalian brain and retina

Phosphatidic acid is a key signaling molecule heavily implicated in exocytosis due to its protein-binding partners and propensity to induce negative membrane curvature. One phosphatidic acid-producing enzyme, phospholipase D (PLD), has also been implicated in neurotransmission. Unfortunately, due to the unreliability of reagents, there has been confusion in the literature regarding the expression of PLD isoforms in the mammalian brain which has hampered our understanding of their functional roles in neurons. To address this, we generated epitope-tagged PLD1 and PLD2 knockin mice using CRISPR/Cas9. Using these mice, we show that PLD1 and PLD2 are both localized at synapses by adulthood, with PLD2 expression being considerably higher in glial cells and PLD1 expression predominating in neurons. Interestingly, we observed that only PLD1 is expressed in the mouse retina, where it is found in the synaptic plexiform layers. These data provide critical information regarding the localization and potential role of PLDs in the central nervous system.

Sexual dimorphism in gut microbiota dictates therapeutic efficacy of intravenous immunoglobulin on radiotherapy complications

IntroductionWith the mounting number of cancer survivors, the complications following cancer treatment become novel conundrums and starve for countermeasures. Intravenous immunoglobulin (IVIg) is a purified preparation for immune-deficient and autoimmune conditions. ObjectivesHere, we investigated whether IVIg could be employed to fight against radiation injuries and explored the underlying mechanism. MethodsHematopoietic or gastrointestinal (GI) tract toxicity was induced by total body or abdominal local irradiation. High-throughput sequencing was performed to analyze the gut microbiota configurations and gene expression profile of small intestine. The untargeted metabolomics of gut microbiome was assessed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analyses. Hydrodynamic-based gene delivery was used to knockdown the target genes in vivo. ResultsIntravenous injection of IVIg protected against radiation-induced hematopoietic and GI tract toxicity in female mice but not in males. IVIg structured sex-characteristic gut microbiota configurations in abdominal irradiated mice. The irradiation enriched gut Lachnospiraceae in female mice but reduced those in males. IVIg injection combined with oral gavage of Lachnospiraceae or its metabolite hypoxanthine, alleviated radiation toxicity in male mice however, Lachnospiraceae or hypoxanthine alone failed to ameliorate the injuries. Abdominal local irradiation drove sex-distinct gene expression signatures in small intestine. Mechanistic investigation showed that replenishment of Lachnospiraceae or hypoxanthine offset abdominal radiation-reduced PLD1 expression in male mice. In females, irradiation elevated PLD1 expression. Deletion of PLD1 in GI tract of female mice erased the radioprotective effects of IVIg. ConclusionIVIg battles against radiation injuries in a sex-specific, gut microbiome-dependent way through Lachnospiraceae/hypoxanthine/PLD1 axis. Our findings provide a sex-precise therapeutic avenue to improve the prognosis of cancer patients with radiotherapy in pre-clinical settings.

Specific small-molecule inhibition of PLD1 prevents ADRD-like memory deficits by preserving dendritic spine integrity driven by amyloidogenic proteins.

Phosphatidyl choline phospholipase D (PC-PLD), a lipolytic enzyme that breaks down membrane phospholipids via two isoforms - a constitutively expressed PLD2 and an inducible PLD1 isoform - are also involved in developmentally important signaling mechanisms that regulate synaptic function. We were the first to propose and present a systematic study that established PLD1 as the aberrantly elevated isoform in AD and related dementia using human clinical samples and provided functional proof using mouse models to demonstrate the underlying synaptic dysfunction and memory deficits. Synaptosomal Western blot analysis on 12 month 3xTg-AD mice hippocampi were used to investigate neuronal PLD1 expression and function. Long term potentiation of PLD1 dependent changes using pharmacological approaches in ex vivo slice preparations from wildtype and transgenic mouse models were used to assess synaptic perturbations that were first studied using the novel object recognition memory (NOR) and fear conditioning (FC) paradigms. Chronic PLD1 small molecule inhibitor treatment was assessed to ascertain the efficacy against Aβ and tau-driven AD-like memory deficit progression. Lastly, brain tissues from these animals were subjected to Western Blot analyses, Golgi analysis, immunohistochemical analysis to ascertain the potential signaling mechanism(s) that is/are perturbed/altered by overexpression of PLD1 in such diseased states. Chronic PLD1 inhibition ameliorates the synaptic dysfunction and underlying memory deficits in the 3xTg-AD mouse model by specific action on preserving mushroom spine dendritic spine integrity. Further analysis using Western Blots revealed an underlying mechanism. Using chronic administration of a well-tolerated halopemide derivative of the specific PLD1 isoform inhibitor in the preclinical mouse models of synaptic dysfunction and memory deficits associated with amyloidogenic effects of Aβ and tau, we demonstrated neuroprotective aspects involving changes in the dendritic spine integrity that contributes to the preservation of memory.

2-Deoxy-D-glucose impedes T cell-induced apoptosis of keratinocytes in oral lichen planus.

Oral lichen planus (OLP) is a T cell–mediated immunoinflammatory disease. Glycolysis plays an essential role in T‐cell immune responses. Blocking glycolytic pathway in activated T cells represents a therapeutic strategy for restraint of immunologic process in autoimmune disorders. 2‐Deoxy‐D‐glucose (2‐DG) has been widely used to probe into glycolysis in immune cells. This study was aimed to explore the role of glycolysis inhibition by 2‐DG on regulating immune responses of OLP‐derived T cells. We observed that lactic dehydrogenase A (LDHA) expression was elevated in OLP lesions and local T cells. 2‐DG inhibited the expression of LDHA, p‐mTOR, Hif1α and PLD2 in T cells; meanwhile, it decreased proliferation and increased apoptosis of T cells. T cells treated by 2‐DG showed lower LDHA expression and elevated apoptosis, resulting in a reduced apoptotic population of keratinocytes that were co‐cultured with them, which was related to the decreased levels of IFN‐γ in co‐culture system. Rapamycin enhanced the effects of 2‐DG on immune responses between T cells and keratinocytes. Thus, these findings indicated that OLP‐derived T cells might be highly dependent upon high glycolysis for proliferation, and 2‐DG treatment combined with rapamycin might be an option to alleviate T‐cell responses, contributing to reducing apoptosis of keratinocytes.

The phospholipase D inhibitor FIPI potently blocks EGF-induced calcium signaling in human breast cancer cells

BackgroundPhosphotyrosine kinase (PTK)-mediated phospholipase C-γ1 (PLC-γ1) signaling plays a crucial role in the release of the universal second messenger calcium from intracellular stores, which is mandatory for several cellular processes, including cell migration. However, PLC-γ1 could also be activated in a PTK-independent manner by phospholipase D (PLD)-derived phosphatidic acid (PA). Because both higher PLD expression levels and PLD activity have also been associated with breast cancer cell invasion and migration, we wondered whether there might be a link between PLD and PLC-γ1, which was investigated in this study.MaterialsMDA-MB-468-NEO (EGFR positive) and MDA-MB-468-HER2 (EGFR and HER2 positive) human breast cancer cells were used in this study. The migratory behavior of the cells in the presence of epidermal growth factor (EGF) and the PLD inhibitor 5-fluoro-2-indolyl-des-chlorohalopemide (FIPI) was analyzed using the 3D collagen matrix migration assay. Changes in cytosolic calcium levels in the presence of EGF, FIPI and Sig-1R agonists and antagonists as well as in PLD1 siRNA knockdown cells were determined by flow cytometry. Western blot analyses were performed to determine the basal expression levels and phosphorylation patterns of EGFR, HER2, AKT, MAPKp42/44, PLC-γ1 and Sig-1R.ResultsThe EGF-induced migration of MDA-MB-468-NEO and MDA-MB-468-HER2 cells was significantly impaired by FIPI. Likewise, FIPI also significantly abolished EGF-induced calcium release in both cell lines. However, neither the expression levels nor the phosphorylation patterns of EGFR, HER2, AKT, MAPKp42/44 and PLC-γ1 were markedly changed by FIPI. Knockdown of PLD1 expression by siRNA also significantly impaired EGF-induced calcium release in both cell lines. Targeting Sig-1R, which interacts with IP3R, with the antagonist BD1047 also abrogated EGF-induced calcium release. However, EGF-induced calcium release was also impaired if cells were treated with the Sig-1R agonists PRE084 and PPBP maleate.ConclusionIn summary, blocking PLD activity with the specific inhibitor FIPI or knocking down PDL1 expression by siRNA significantly impaired EGF-induced calcium release in MDA-MB-468-NEO and MDA-MB-468-HER2 cells, likely indicating a connection between PLD activity and PLC-γ1-mediated calcium signaling. However, how PLD activity interferes with the release of calcium from intracellular stores remains unclear.5hw_Xw1J4QoujjE82ULAZvVideo

Inhibition of chaperone‑mediated autophagy reduces tumor growth and metastasis and promotes drug sensitivity in colorectal cancer.

Chaperone-mediated autophagy (CMA) is a selective type of autophagy whereby a specific subset of intracellular proteins is targeted to the lysosome for degradation. The present study investigated the mechanisms underlying the response and resistance to 5-fluorouracil (5-FU) in colorectal cancer (CRC) cell lines. In engineered 5-FU-resistant CRC cell lines, a significant elevation of lysosome-associated membrane protein 2A (LAMP2A), which is the key molecule in the CMA pathway, was identified. High expression of LAMP2A was found to be responsible for 5-FU resistance and to enhance PLD2 expression through the activation of NF-κB pathway. Accordingly, loss or gain of function of LAMP2A in 5-FU-resistant CRC cells rendered them sensitive or resistant to 5-FU, respectively. Taken together, the results of the present study suggested that chemoresistance in patients with CRC may be mediated by enhancing CMA. Thus, CMA is a promising predictor of chemosensitivity to 5-FU treatment and anti-CMA therapy may be a novel therapeutic option for patients with CRC.

D-series Resolvins activate Phospholipase D in phagocytes during inflammation and resolution.

A successful acute inflammatory response results in the elimination of infectious agents by neutrophils and monocytes, followed by resolution and repair through tissue-resident and recruited macrophages. Resolvins (D-series and E-series) are pro-resolving lipid mediators involved in resolution and tissue repair, whose intracellular signaling remains of interest. Here, we report that D-series resolvins (RvD1- RvD5) activate phospholipase D (PLD), a ubiquitously expressed membrane lipase enzyme activity in modulating phagocyte functions. The mechanism for PLD-mediated actions of Resolvin-D5 (RvD5) in polarizing macrophages (M1-like toward M2-like) was found to be two-pronged: (a) RvD5 inhibits post-transcriptional modifications, by miRs and 3'exonucleases that process PLD2 mRNA, thus increasing PLD2 expression and activity; and (b) RvD5 enhances PLD2-S6Kinase signaling required for membrane expansion and efferocytosis. In an in vivo model of second organ reflow injury, we found that RvD5 did not reduce lung neutrophil myeloperoxidase levels in PLD2-/- mice compared to WT and PLD1-/- mice, confirming a novel role of PLD2 as the isoform in RvD5-mediated resolution processes. These results demonstrate that RvD5-PLD2 are attractive targets for therapeutic interventions in vascular inflammation such as ischemia-reperfusion injury and cardiovascular diseases.

Increased phospholipase D activity contributes to tumorigenesis in prostate cancer cell models.

Prostate cancer (PCa) is the most frequent cancer among men and the first cause of death over 65. Approximately 90% of patients with advanced disease will develop bone metastasis, which dramatically reduces long-term survival. Therefore, effective therapies need to be developed, especially when disease is still well-localized. Phospholipase D (PLD), an enzyme that hydrolyzes phosphatidylcholine to yield phosphatidic acid, regulates several cellular functions as proliferation, survival, migration or vesicular trafficking. PLD is implicated in numerous diseases such as neurodegenerative, cardiovascular, autoimmune disorders or cancer. Indeed, PLD controls different aspects of oncogenesis including tumor progression and resistance to targeted therapies such as radiotherapy. PLD1 and PLD2 are the only isoforms with catalytic activity involved in cancer. Surprisingly, studies deciphering the role of PLD in the pathophysiology of PCa are scarce. Here we describe the correlation between PLD activity and PLD1 and PLD2 expression in PCa bone metastasis-derived cell lines C4-2B and PC-3. Next, by using PLD pharmacological inhibitors and RNA interference strategy, we validate the implication of PLD1 and PLD2 in cell viability, clonogenicity and proliferation of C4-2B and PC-3 cells and in migration capacity of PC-3 cells. Last, we show an increase in PLD activity as well as PLD2 protein expression during controlled starvation of PC-3 cells, concomitant with an augmentation of its migration capacity. Specifically, upregulation of PLD activity appears to be PKC-independent. Taken together, our results indicate that PLD, and in particular PLD2, could be considered as a potential therapeutic target for the treatment of PCa-derived bone metastasis.

Abstract A10: Fusobacteria upregulates PD-L1 in head and neck cancer cell lines: Potential mechanism for immune evasion

Abstract Objectives: Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer worldwide with a poor prognosis of ~40% at 5 years for HPV-negative disease. Recently, immune checkpoint inhibitors (specifically, anti-PD-1 checkpoint inhibitors that block the PD-L1/PD-1 immunosuppressive axis) have been approved as a treatment for patients with recurrent/metastatic HNSCC. However, most patients (&amp;gt;80%) fail to respond to checkpoint inhibitors. PD-L1 expression on tumors plays an important role in immune evasion and can also influence response to checkpoint inhibitors. Tumor PD-L1 expression has been shown to correlate with checkpoint response. Factors that might affect PDL1 expression in HNSCC remain unclear. In this study, we explored the effect of periodontal bacteria, which has been associated with oral cancer, on PD-L1 expression in oral cancer cells. Materials and Methods: 303 HNSCC were compared to 37 adjacent control tissues in the TCGA mRNA expression database (RNASeq V2); the analysis was done based on tumor types. RT-PCR and flow cytometry were used to measure the base line expression of PD-L1 in seven head and neck cancer cell lines and human oral tissues. OQ01, an oral cancer cell line, was infected with four periodontal bacteria (Tanneralla forsynthia, Treponema denticola, Porphyromonas gingivalis, and Fusobacterium nucleatum). OQ01 and RPMI2650 cells were infected with two different strains of Fusobacteria (F. periodonticum, F. vincentii). The relative changes in expression of PLD1 was measured using RT-PCR in cell lysate after RNA isolation. PD-L1 protein level on the cell surface was measured using flow cytometry. Results: TCGA data analysis revealed that PDL1 was significantly (P&amp;lt; 0.0005) elevated in total cancer tissues compared to total controls. In the same data analysis, PDL1 expression was significantly (P&amp;lt; 0.05) elevated in oral cavity tumor compared to normal control. Expression of PD-L1 was variable among head and neck cancer patients and cell lines. Both F. nucleatum and P. gingivalis were able to enhance PD-L1 expression in OQ01 cells (10 and 15 fold, respectively). Two strains of Fusobacteria tested (F. periodonticum, F. vincentii) were also able to induce PD-L1 both at the mRNA (P&amp;lt; 0.005 and P&amp;lt; 0.05 respectively) and protein level in OQ01 and RPMI 2650 cells. Interestingly, the same strains of Fusobacteria enhanced MYC expression in OQ01 and RPMI 2650 cells. As a transcription factor MYC has been reported to regulate PDL1 transcription. These data along with our results suggest that Fusobacterial upregulation of PD-L1 expression might be through MYC enhanced expression. Conclusions: This study revealed that Fusobacteria enhance PD-L1 expression on HNSCC cancer cell lines, possibly through upregulation of MYC. This may have implications on cancer cell immune evasion and checkpoint inhibitor treatment response. Note: This abstract was not presented at the conference. Citation Format: Amani Harrandah, Rekha Rani Grag, Yuk Pheel Park, Sasanka S. Chukkapalli, Nikhita Yadlapalli, Edward K. L. Chan, Natalie Silver. Fusobacteria upregulates PD-L1 in head and neck cancer cell lines: Potential mechanism for immune evasion [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr A10.

The in vitro effects of phospholipase D1-mTOR axis in liver fibrogenesis

AimsThe activation of hepatic stellate cells (HSCs) plays a central role in liver fibrosis progression. Phospholipase D (PLD) enzymes participate in multiple cellular activities. However, whether and how PLD regulates HSCs activation remain elusive. Main methodsThe expression of intrahepatic PLD1 and PLD2 was determined in CCl4-induced mouse liver fibrosis models by western blot and immunohistochemistry. Cell model of liver fibrogenesis was constructed using rat HSCs line (HSC-T6) treated with recombinant transforming growth factor β1 (TGFβ1). Fibrogenesis was evaluated on the aspects of proliferation, expression of pro-fibrogenic markers and migration. The effects mediated by PLD1-mTOR axis on TGFβ1-induced fibrogenesis were evaluated using HSC-T6 treated with small-molecular PLD1 inhibitors, PLD1-SiRNA, rapamycin (mTOR inhibitor) and MHY1485 (mTOR activator). Key findingsSignificant increase of PLD1, not PLD2 was documented in CCl4-induced cirrhotic compared to normal liver tissues. Suppression of PLD1 activities by PLD inhibitors or down-regulation of PLD1 expression in HSC-T6 could significantly restrain TGFβ1-induced fibrogenesis, as reflected by decreased cell proliferation and reduced expression of pro-fibrogenic markers. Besides, either PLD1 inhibitor or PLD1-SiRNA significantly inhibited mTOR activity of HSC-T6. Moreover, PLD1 inhibitors not only exhibited similar effects with rapamycin in TGFβ1-induced fibrogenesis, but also blunted MHY1485 enhanced cell proliferation of HSC-T6. SignificanceThe PLD1-mTOR axis of HSCs could be therapeutically targeted in advanced liver fibrosis.

Phospholipase D as a key modulator of cancer progression.

The phospholipase D (PLD) family has a ubiquitous expression in cells. PLD isoforms (PLDs) and their hydrolysate phosphatidic acid (PA) have been demonstrated to engage in multiple stages of cancer progression. Aberrant expression of PLDs, especially PLD1 and PLD2, has been detected in various cancers. Inhibition or elimination of PLDs activity has been shown to reduce tumour growth and metastasis. PLDs and PA also serve as downstream effectors of various cell-surface receptors, to trigger and regulate propagation of intracellular signals in the process of tumourigenesis and metastasis. Here, we discuss recent advances in understanding the functions of PLDs and PA in discrete stages of cancer progression, including cancer cell growth, invasion and migration, and angiogenesis, with special emphasis on the tumour-associated signalling pathways mediated by PLDs and PA and the functional importance of PLDs and PA in cancer therapy.