LPA suppresses HLA-DR expression in human melanoma cells: a potential immune escape mechanism involving LPAR1 and DR6-mediated release of IL-10.

While immune checkpoint inhibitors (ICIs) are promising in the treatment of metastatic melanoma, about half of patients do not respond well to them. Low levels of human leukocyte antigen-DR (HLA-DR) in tumors have been shown to negatively influence prognosis and response to ICIs. Lysophosphatidic acid (LPA) is produced in large amounts by melanoma and is abundantly present in the tumor microenvironment. LPA induces the release of various cytokines and chemokines from tumor cells, which affect cancer development, metastasis, and tumor immunity. In the present study, we investigated the role of LPA-induced IL-10 release in regulating HLA-DR expression and the underlying mechanisms in human melanoma cells. We showed that LPA (0.001-10 µM) dose-dependently increased DR6 transcript levels through activating LPAR1 in HEK293T cells. Knockdown of NF-κB1 abrogated the LPA-increased DR6 expression without affecting basal DR6 expression in both A2058 and A375 melanoma cell lines. LPA (10 µM) significantly increased IL-10 transcripts in A2058 and A375 melanoma cells, the effect was abolished by pharmacological inhibition of LPAR1 or knockdown of DR6. We found a statistically significant correlation between the expression of LPAR1, DR6 and IL-10 in human melanoma tissue and an association between increased expression of LPAR1 and reduced effectiveness of ICI therapy. We demonstrated that LPA (10 µM) markedly suppressed HLA-DR expression in both A375 and A2058 melanoma cells via activating the LPAR1-DR6-IL-10 pathway. These data suggest that the LPAR1-DR6-IL-10 autocrine loop could constitute a novel mechanism used by tumor cells to evade immunosurveillance by decreasing HLA-DR expression.

Establishing Three-Dimensional Explant Culture of Human Dental Pulp Tissue.

Mesenchymal stem cells in the dental tissue indicate a disposition for differentiation into diverse dental lineages and contain enormous potential as the important means for regenerative medicine in dentistry. Among various dental tissues, the dental pulp contains stem cells, progenitor cells and odontoblasts for maintaining dentin homeostasis. The conventional culture of stem cells holds a limit as the living tissue constitutes the three-dimensional (3D) structure. Recent development in the organoid cultures have successfully recapitulated 3D structure and advanced to the assembling of different types. In the current study, the protocol for 3D explant culture of the human dental pulp tissue has been established by adopting the organoid culture. After isolating dental pulp from human tooth, the intact tissue was placed between two layers for Matrigel with addition of the culture medium. The reticular outgrowth of pre-odontoblast layer continued for a month and the random accumulation of dentin was observed near the end. Electron microscopy showed the cellular organization and in situ development of dentin, and immunohistochemistry exhibited the expression of odontoblast and stem cell markers in the outgrowth area. Three-dimensional explant culture of human dental pulp will provide a novel platform for understanding stem cell biology inside the tooth and developing the regenerative medicine.

LPA-Induced Thromboxane A2-Mediated Vasoconstriction Is Limited to Poly-Unsaturated Molecular Species in Mouse Aortas.

We have previously reported that, in aortic rings, 18:1 lysophosphatidic acid (LPA) can induce both vasodilation and vasoconstriction depending on the integrity of the endothelium. The predominant molecular species generated in blood serum are poly-unsaturated LPA species, yet the vascular effects of these species are largely unexplored. We aimed to compare the vasoactive effects of seven naturally occurring LPA species in order to elucidate their potential pathophysiological role in vasculopathies. Vascular tone was measured using myography, and thromboxane A2 (TXA2) release was detected by ELISA in C57Bl/6 mouse aortas. The Ca2+-responses to LPA-stimulated primary isolated endothelial cells were measured by Fluo-4 AM imaging. Our results indicate that saturated molecular species of LPA elicit no significant effect on the vascular tone of the aorta. In contrast, all 18 unsaturated carbon-containing (C18) LPAs (18:1, 18:2, 18:3) were effective, with 18:1 LPA being the most potent. However, following inhibition of cyclooxygenase (COX), these LPAs induced similar vasorelaxation, primarily indicating that the vasoconstrictor potency differed among these species. Indeed, C18 LPA evoked a similar Ca2+-signal in endothelial cells, whereas in endothelium-denuded aortas, the constrictor activity increased with the level of unsaturation, correlating with TXA2 release in intact aortas. COX inhibition abolished TXA2 release, and the C18 LPA induced vasoconstriction. In conclusion, polyunsaturated LPA have markedly increased TXA2-releasing and vasoconstrictor capacity, implying potential pathophysiological consequences in vasculopathies.

A dual role of lysophosphatidic acid type 2 receptor (LPAR2) in nonsteroidal anti-inflammatory drug-induced mouse enteropathy.

Lysophosphatidic acid (LPA) is a bioactive phospholipid mediator that has been found to ameliorate nonsteroidal anti-inflammatory drug (NSAID)-induced gastric injury by acting on lysophosphatidic acid type 2 receptor (LPAR2). In this study, we investigated whether LPAR2 signaling was implicated in the development of NSAID-induced small intestinal injury (enteropathy), another major complication of NSAID use. Wild-type (WT) and Lpar2 deficient (Lpar2-/-) mice were treated with a single, large dose (20 or 30 mg/kg, i.g.) of indomethacin (IND). The mice were euthanized at 6 or 24 h after IND treatment. We showed that IND-induced mucosal enteropathy and neutrophil recruitment occurred much earlier (at 6 h after IND treatment) in Lpar2-/- mice compared to WT mice, but the tissue levels of inflammatory mediators (IL-1ß, TNF-α, inducible COX-2, CAMP) remained at much lower levels. Administration of a selective LPAR2 agonist DBIBB (1, 10 mg/kg, i.g., twice at 24 h and 30 min before IND treatment) dose-dependently reduced mucosal injury and neutrophil activation in enteropathy, but it also enhanced IND-induced elevation of several proinflammatory chemokines and cytokines. By assessing caspase-3 activation, we found significantly increased intestinal apoptosis in IND-treated Lpar2-/- mice, but it was attenuated after DBIBB administration, especially in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Finally, we showed that IND treatment reduced the plasma activity and expression of autotaxin (ATX), the main LPA-producing enzyme, and also reduced the intestinal expression of Lpar2 mRNA, which preceded the development of mucosal damage. We conclude that LPAR2 has a dual role in NSAID enteropathy, as it contributes to the maintenance of mucosal integrity after NSAID exposure, but also orchestrates the inflammatory responses associated with ulceration. Our study suggests that IND-induced inhibition of the ATX-LPAR2 axis is an early event in the pathogenesis of enteropathy.

Functional modulation of lysophosphatidic acid type 2 G-protein coupled receptor facilitates alveolar bone formation.

Lipid biosynthesis is recently studied its functions in a range of cellular physiology including differentiation and regeneration. However, it still remains to be elucidated in its precise function. To reveal this, we evaluated the roles of lysophosphatidic acid (LPA) signaling in alveolar bone formation using the LPA type 2 receptor (LPAR2) antagonist AMG-35 (Amgen Compound 35) using tooth loss without periodontal disease model which would be caused by trauma and usually requires a dental implant to restore masticatory function. In this study, in vitro cell culture experiments in osteoblasts and periodontal ligament fibroblasts revealed cell type-specific responses, with AMG-35 modulating osteogenic differentiation in osteoblasts in vitro. To confirm the in vivo results, we employed a mouse model of tooth loss without periodontal disease. Five to 10 days after tooth extraction, AMG-35 facilitated bone formation in the tooth root socket as measured by immunohistochemistry for differentiation markers KI67, Osteocalcin, Periostin, RUNX2, transforming growth factor beta 1 (TGF-ß1) and SMAD2/3. The increased expression and the localization of these proteins suggest that AMG-35 elicits osteoblast differentiation through TGF-ß1 and SMAD2/3 signaling. These results indicate that LPAR2/TGF-ß1/SMAD2/3 represents a new signaling pathway in alveolar bone formation and that local application of AMG-35 in traumatic tooth loss can be used to facilitate bone regeneration and healing for further clinical treatment.

Autotaxin-lysophosphatidic acid receptor 5 axis evokes endothelial dysfunction via reactive oxygen species signaling.

Lysophosphatidylcholine (LPC) is a bioactive lipid that has been shown to attenuate endothelium-dependent vasorelaxation contributing to endothelial dysfunction; however, the underlying mechanisms are not well understood. In this study, we investigated the molecular mechanisms involved in the development of LPC-evoked impairment of endothelium-dependent vasorelaxation. In aortic rings isolated from wild-type (WT) mice, a 20-min exposure to LPC significantly reduced the acetylcholine chloride (ACh)-induced vasorelaxation indicating the impairment of normal endothelial function. Interestingly, pharmacological inhibition of autotaxin (ATX) by GLPG1690 partially reversed the endothelial dysfunction, suggesting that lysophosphatidic acid (LPA) derived from LPC may be involved in the effect. Therefore, the effect of LPC was also tested in aortic rings isolated from different LPA receptor knock-out (KO) mice. LPC evoked a marked reduction in ACh-dependent vasorelaxation in Lpar1, Lpar2, and Lpar4 KO, but its effect was significantly attenuated in Lpar5 KO vessels. Furthermore, addition of superoxide dismutase reduced the LPC-induced endothelial dysfunction in WT but not in the Lpar5 KO mice. In addition, LPC increased H2O2 release from WT vessels, which was significantly reduced in Lpar5 KO vessels. Our findings indicate that the ATX-LPA-LPA5 receptor axis is involved in the development of LPC-induced impairment of endothelium-dependent vasorelaxation via LPA5 receptor-mediated reactive oxygen species production. Taken together, in this study, we identified a new pathway contributing to the development of LPC-induced endothelial dysfunction.

Paneth cell dysfunction in radiation injury and radio-mitigation by human α-defensin 5.

Introduction: The mechanism underlying radiation-induced gut microbiota dysbiosis is undefined. This study examined the effect of radiation on the intestinal Paneth cell α-defensin expression and its impact on microbiota composition and mucosal tissue injury and evaluated the radio-mitigative effect of human α-defensin 5 (HD5). Methods: Adult mice were subjected to total body irradiation, and Paneth cell α-defensin expression was evaluated by measuring α-defensin mRNA by RT-PCR and α-defensin peptide levels by mass spectrometry. Vascular-to-luminal flux of FITC-inulin was measured to evaluate intestinal mucosal permeability and endotoxemia by measuring plasma lipopolysaccharide. HD5 was administered in a liquid diet 24 hours before or after irradiation. Gut microbiota was analyzed by 16S rRNA sequencing. Intestinal epithelial junctions were analyzed by immunofluorescence confocal microscopy and mucosal inflammatory response by cytokine expression. Systemic inflammation was evaluated by measuring plasma cytokine levels. Results: Ionizing radiation reduced the Paneth cell α-defensin expression and depleted α-defensin peptides in the intestinal lumen. α-Defensin down-regulation was associated with the time-dependent alteration of gut microbiota composition, increased gut permeability, and endotoxemia. Administration of human α-defensin 5 (HD5) in the diet 24 hours before irradiation (prophylactic) significantly blocked radiation-induced gut microbiota dysbiosis, disruption of intestinal epithelial tight junction and adherens junction, mucosal barrier dysfunction, and mucosal inflammatory response. HD5, administered 24 hours after irradiation (treatment), reversed radiation-induced microbiota dysbiosis, tight junction and adherens junction disruption, and barrier dysfunction. Furthermore, HD5 treatment also prevents and reverses radiation-induced endotoxemia and systemic inflammation. Conclusion: These data demonstrate that radiation induces Paneth cell dysfunction in the intestine, and HD5 feeding prevents and mitigates radiation-induced intestinal mucosal injury, endotoxemia, and systemic inflammation.

Lysophosphatidic acid induces proliferation and osteogenic differentiation of human dental pulp stem cell through lysophosphatidic acid receptor 3/extracellular signal-regulated kinase signaling axis.

Background/purpose: Human dental pulp stem cells (hDPSCs) possess excellent proliferative and osteogenic differentiation potentials. This study aimed to elucidate the role of lysophosphatidic acid (LPA) signaling in the proliferation and osteogenic differentiation of hDPSCs. Materials and methods: hDPSCs were treated with LPA and proliferation was measured using the cell counting kit-8 assay. Following the osteogenic differentiation of hDPSCs using osteogenic medium in the presence or absence of LPA, alkaline phosphatase (ALP) staining, ALP activity measurements, and RT-qPCR were performed to analyze the osteoblast differentiation. Small interfering RNA (siRNA)-mediated LPAR3 silencing and extracellular signal-regulated (ERK)/mitogen-activated protein (MAP) kinase inhibitors were used to elucidate the molecular mechanisms underlying LPA-induced proliferation and differentiation of hDPSCs. Results: LPA treatment significantly induced proliferation and osteogenic differentiation of hDPSCs. The depletion of LPAR3 expression by LPAR3-speicifc siRNA in hDPSCs diminished LPA-induced proliferation and osteogenic differentiation. The LPAR3-mediated proliferation and osteogenic differentiation of hDPSCs in response to LPA were significantly suppressed by U0126, a selective inhibitor of ERK. Conclusion: These findings suggest that LPA induces the proliferation and osteogenic differentiation of hDPSCs via LPAR3-ERK-dependent pathways.

E2F7 drives autotaxin/Enpp2 transcription via chromosome looping: Repression by p53 in murine but not in human carcinomas.

Dysregulation of the autotaxin (ATX, Enpp2)-lysophosphatidic acid (LPA) signaling in cancerous cells contributes to tumorigenesis and therapy resistance. We previously found that ATX activity was elevated in p53-KO mice compared to wild-type (WT) mice. Here, we report that ATX expression was upregulated in mouse embryonic fibroblasts from p53-KO and p53R172H mutant mice. ATX promoter analysis combined with yeast one-hybrid testing revealed that WT p53 directly inhibits ATX expression via E2F7. Knockdown of E2F7 reduced ATX expression and chromosome immunoprecipitation showed that E2F7 promotes Enpp2 transcription through cooperative binding to two E2F7 sites (promoter region -1393 bp and second intron 996 bp). Using chromosome conformation capture, we found that chromosome looping brings together the two E2F7 binding sites. We discovered a p53 binding site in the first intron of murine Enpp2, but not in human ENPP2. Binding of p53 disrupted the E2F7-mediated chromosomal looping and repressed Enpp2 transcription in murine cells. In contrast, we found no disruption of E2F7-mediated ENPP2 transcription via direct p53 binding in human carcinoma cells. In summary, E2F7 is a common transcription factor that upregulates ATX in human and mouse cells but is subject to steric interference by direct intronic p53 binding only in mice.

Enhancement of Sphingomyelinase-Induced Endothelial Nitric Oxide Synthase-Mediated Vasorelaxation in a Murine Model of Type 2 Diabetes.

Sphingolipids are important biological mediators both in health and disease. We investigated the vascular effects of enhanced sphingomyelinase (SMase) activity in a mouse model of type 2 diabetes mellitus (T2DM) to gain an understanding of the signaling pathways involved. Myography was used to measure changes in the tone of the thoracic aorta after administration of 0.2 U/mL neutral SMase in the presence or absence of the thromboxane prostanoid (TP) receptor antagonist SQ 29,548 and the nitric oxide synthase (NOS) inhibitor L-NAME. In precontracted aortic segments of non-diabetic mice, SMase induced transient contraction and subsequent weak relaxation, whereas vessels of diabetic (Leprdb/Leprdb, referred to as db/db) mice showed marked relaxation. In the presence of the TP receptor antagonist, SMase induced enhanced relaxation in both groups, which was 3-fold stronger in the vessels of db/db mice as compared to controls and could not be abolished by ceramidase or sphingosine-kinase inhibitors. Co-administration of the NOS inhibitor L-NAME abolished vasorelaxation in both groups. Our results indicate dual vasoactive effects of SMase: TP-mediated vasoconstriction and NO-mediated vasorelaxation. Surprisingly, in spite of the general endothelial dysfunction in T2DM, the endothelial NOS-mediated vasorelaxant effect of SMase was markedly enhanced.

Emerging roles of lysophosphatidic acid receptor subtype 5 (LPAR5) in inflammatory diseases and cancer.

Lysophosphatidic acid (LPA) is a bioactive lipid mediator that regulates a variety of cellular functions such as cell proliferation, migration, survival, calcium mobilization, cytoskeletal rearrangements, and neurite retraction. The biological actions of LPA are mediated by at least six G protein-coupled receptors known as LPAR1-6. Given that LPAR1-3 were among the first LPARs identified, the majority of research efforts have focused on understanding their biology. This review provides an in-depth discussion of LPAR5, which has recently emerged as a key player in regulating normal intestinal homeostasis and modulating pathological conditions such as pain, itch, inflammatory diseases, and cancer. We also present a chronological overview of the efforts made to develop compounds that target LPAR5 for use as tool compounds to probe or validate LPAR5 biology and therapeutic agents for the treatment of inflammatory diseases and cancer.

Cryptotanshinone inhibits ovarian tumor growth and metastasis by degrading c-Myc and attenuating the FAK signaling pathway.

Cryptotanshinone (CT), a natural compound derived from Salvia miltiorrhiza Bunge that is also known as the traditional Chinese medicine Danshen, exhibits antitumor activity in various cancers. However, it remains unclear whether CT has a potential therapeutic benefit against ovarian cancers. The aim of this study was to test the efficacy of CT in ovarian cancer cells in vitro and using a xenograft model in NSG mice orthotopically implanted with HEY A8 human ovarian cancer cells and to explore the molecular mechanism(s) underlying CT's antitumor effects. We found that CT inhibited the proliferation, migration, and invasion of OVCAR3 and HEY A8 cells, while sensitizing the cell responses to the chemotherapy drugs paclitaxel and cisplatin. CT also suppressed ovarian tumor growth and metastasis in immunocompromised mice orthotopically inoculated with HEY A8 cells. Mechanistically, CT degraded the protein encoded by the oncogene c-Myc by promoting its ubiquitination and disrupting the interaction with its partner protein Max. CT also attenuated signaling via the nuclear focal adhesion kinase (FAK) pathway and degraded FAK protein in both cell lines. Knockdown of c-Myc using lentiviral CRISPR/Cas9 nickase resulted in reduction of FAK expression, which phenocopies the effects of CT and the c-Myc/Max inhibitor 10058-F4. Taken together, our studies demonstrate that CT inhibits primary ovarian tumor growth and metastasis by degrading c-Myc and FAK and attenuating the FAK signaling pathway.

Designing Dual Inhibitors of Autotaxin-LPAR GPCR Axis.

The ATX-LPA-LPAR1 signaling pathway plays a universal role in stimulating diverse cellular responses, including cell proliferation, migration, survival, and invasion in almost every cell type. The ATX-LPAR1 axis is linked to several metabolic and inflammatory diseases including cancer, fibrosis, and rheumatoid arthritis. Numerous selective ATX or LPAR1 inhibitors have been developed and so far, their clinical efficacy has only been evaluated in idiopathic pulmonary fibrosis. None of the ATX and LPAR1 inhibitors have advanced to clinical trials for cancer and rheumatoid arthritis. Nonetheless, several research groups, including ours, have shown considerable benefit of simultaneous ATX and LPAR1 inhibition through combination therapy. Recent research suggests that dual-targeting therapies are superior to combination therapies that use two selective inhibitors. However, limited reports are available on ATX-LPAR1 dual inhibitors, potentially due to co-expression of multiple different LPARs with close structural similarities at the same target. In this review, we discuss rational design and future directions of dual ATX-LPAR1 inhibitors.

Prometastatic Effect of ATX Derived from Alveolar Type II Pneumocytes and B16-F10 Melanoma Cells.

Although metastases are the principal cause of cancer-related deaths, the molecular aspects of the role of stromal cells in the establishment of the metastatic niche remain poorly understood. One of the most prevalent sites for cancer metastasis is the lungs. According to recent research, lung stromal cells such as bronchial epithelial cells and resident macrophages secrete autotaxin (ATX), an enzyme with lysophospholipase D activity that promotes cancer progression. In fact, several studies have shown that many cell types in the lung stroma could provide a rich source of ATX in diseases. In the present study, we sought to determine whether ATX derived from alveolar type II epithelial (ATII) pneumocytes could modulate the progression of lung metastasis, which has not been evaluated previously. To accomplish this, we used the B16-F10 syngeneic melanoma model, which readily metastasizes to the lungs when injected intravenously. Because B16-F10 cells express high levels of ATX, we used the CRISPR-Cas9 technology to knock out the ATX gene in B16-F10 cells, eliminating the contribution of tumor-derived ATX in lung metastasis. Next, we used the inducible Cre/loxP system (Sftpc-CreERT2/Enpp2fl/fl) to generate conditional knockout (KO) mice in which ATX is specifically deleted in ATII cells (i.e., Sftpc-KO). Injection of ATX-KO B16-F10 cells into Sftpc-KO or Sftpc-WT control littermates allowed us to investigate the specific contribution of ATII-derived ATX in lung metastasis. We found that targeted KO of ATX in ATII cells significantly reduced the metastatic burden of ATX-KO B16-F10 cells by 30% (unpaired t-test, p = 0.028) compared to Sftpc-WT control mice, suggesting that ATX derived from ATII cells could affect the metastatic progression. We detected upregulated levels of cytokines such as IFNγ (unpaired t-test, p < 0.0001) and TNFα (unpaired t-test, p = 0.0003), which could favor the increase in infiltrating CD8+ T cells observed in the tumor regions of Sftpc-KO mice. Taken together, our results highlight the contribution of host ATII cells as a stromal source of ATX in the progression of melanoma lung metastasis.

A Luminacin D Analog HL142 Inhibits Ovarian Tumor Growth and Metastasis by Reversing EMT and Attenuating the TGFß and FAK Pathways.

Epithelial to mesenchymal transition (EMT) is known to contribute to tumor metastasis and chemoresistance. Reversing EMT using small molecule inhibitors to target EMT associated gene expression represents an effective strategy for cancer treatment. The purpose of this study is to test whether a new luminacin D analog HL142 reverses EMT in ovarian cancer (OC) and has the therapeutic potential for OC. We chemically synthesized HL142 and tested its functions in OC cells in vitro and its efficacy in inhibiting ovarian tumor growth and metastasis in vivo using orthotopic OC mouse models. We first demonstrate that ASAP1 is co-amplified and interacts with the focal adhesion kinase (FAK) protein in serous ovarian carcinoma. HL142 inhibits ASAP1 and its interaction protein FAK in highly invasive OVCAR8 and moderately invasive OVCAR3 cells. HL142 inhibits EMT phenotypic switch, accompanied by upregulating epithelial marker E-cadherin and cytokeratin-7 and downregulating mesenchymal markers vimentin, ß-catenin, and snail2 in both cell lines. Functionally, HL142 inhibits proliferation, colony formation, migration, and invasion. HL142 also sensitizes cell responses to chemotherapy drug paclitaxel treatment and inhibits ovarian tumor growth and metastasis in orthotopic OC mouse models. We further show that HL142 attenuates the TGFß and FAK pathways in vitro using OC cells and in vivo using orthotopic mouse models.

Revisiting the role of lysophosphatidic acid in stem cell biology.

Stem cells possess unique biological characteristics such as the ability to self-renew and to undergo multilineage differentiation into specialized cells. Whereas embryonic stem cells (ESC) can differentiate into all cell types of the body, somatic stem cells (SSC) are a population of stem cells located in distinct niches throughout the body that differentiate into the specific cell types of the tissue in which they reside in. SSC function mainly to restore cells as part of normal tissue homeostasis or to replenish cells that are damaged due to injury. Cancer stem-like cells (CSC) are said to be analogous to SSC in this manner where tumor growth and progression as well as metastasis are fueled by a small population of CSC that reside within the corresponding tumor. Moreover, emerging evidence indicates that CSC are inherently resistant to chemo- and radiotherapy that are often the cause of cancer relapse. Hence, major research efforts have been directed at identifying CSC populations in different cancer types and understanding their biology. Many factors are thought to regulate and maintain cell stemness, including bioactive lysophospholipids such as lysophosphatidic acid (LPA). In this review, we discuss some of the newly discovered functions of LPA not only in the regulation of CSC but also normal SSC, the similarities in these regulatory functions, and how these discoveries can pave way to the development of novel therapies in cancer and regenerative medicine.

EIF5A2 controls ovarian tumor growth and metastasis by promoting epithelial to mesenchymal transition via the TGFß pathway.

BACKGROUND: Epithelial to mesenchymal transition (EMT) contributes to tumor metastasis and chemoresistance. Eukaryotic initiation factor 5A2 (EIF5A2) is highly expressed in a variety of human cancers but rarely expressed in normal tissues. While EIF5A2 has oncogenic activity in several cancers and contributes to tumor metastasis, its role in ovarian cancer is unknown. In this study, we investigate whether EIF5A2 contributes to ovarian tumor metastasis by promoting EMT. METHODS: To investigate the role of EIF5A2, we knocked out (KO) EIF5A2 using lentiviral CRISPR/Cas9 nickase in high invasive SKOV3 and OVCAR8 cells and overexpressed EIF5A2 in low invasive OVCAR3 cells using lentiviral vector. Cell proliferation, migration and invasion was examined in vitro ovarian cancer cells and tumor metastasis was evaluated in vivo using orthotopic ovarian cancer mouse models. RESULTS: Here we report that EIF5A2 is highly expressed in ovarian cancers and associated with patient poor survival. Lentiviral CRISPR/Cas9 nickase vector mediated knockout (KO) of EIF5A2 inhibits epithelial to mesenchymal transition (EMT) in SKOV3 and OVCAR8 ovarian cancer cells that express high levels of EIF5A2. In contrast, overexpression of EIF5A2 promotes EMT in OVCAR3 epithelial adenocarcinoma cells that express relatively low EIF5A2 levels. KO of EIF5A2 in SKOV3 and OVCAR8 cells inhibits ovarian cancer cell migration and invasion, while its overexpression promotes cell migration and invasion in OVCAR3 adenocarcinoma cells. We further demonstrate that EIF5A2 promotes EMT by activating the TGFß pathway and KO of EIF5A2 inhibits ovarian tumor growth and metastasis in orthotopic ovarian cancer mouse models. CONCLUSION: Our results indicate that EIF5A2 is an important controller of ovarian tumor growth and metastasis by promoting EMT and activating the TGFß pathway.

Evaluation of In-cage Filter Paper as a Replacement for Sentinel Mice in the Detection of Murine Pathogens.

Recent studies have evaluated alternatives to the use of live animals in colony health monitoring. Currently, an alternative method that is suitable for all rack types and that has been verified to detect the infectious agents most commonly excluded from mouse colonies is unavailable. We compared the use of filter paper placed on the inside floor of mouse cages to the traditional use of sentinel mice in the detection of several prevalent murine pathogens including mouse hepatitis virus (MHV), murine norovirus (MNV), minute virus of mice (MVM), mouse parvovirus (MPV), Theiler murine encephalomyelitis virus (TMEV), Helicobacter spp., Syphacia obvelata, and Aspiculuris tetraptera. Experimental groups comprised 7 cages containing either 2 pieces of filter paper on the cage floor or 2 ICR sentinel mice. Soiled bedding from pet-store mice was transferred to the experimental cages weekly for 8 wk. At 1 and 2 mo after bedding transfer, the filter papers were evaluated by PCR and sentinel mice were tested by serology and fecal PCR. Filter papers detected all pathogens as effectively (MHV, MNV, MPV, MVM, TMEV S. obvelata, and A. tetraptera) or more effectively (Helicobacter spp.) than sentinel mice at both time points. Filter papers more readily detected pathogens with a high copy number per RT-PCR analysis than a low copy number. Helicobacter spp. were not detected by sentinel mice at either time point. These results indicate that the use of filter paper placed on the interior floor of empty mouse cages and exposed to soiled bedding is efficient in detecting bacteria, endoparasites, and most of the common mouse viruses included in an animal health monitoring program.