Figure S2 from The Autotaxin—Lysophosphatidic Acid Axis Promotes Lung Carcinogenesis
Christiana MagkriotiNikos OikonomouEleanna KaffeMarios-Angelos MouratisNikos XylourgidisIliana BarbayianniPetros MegadoukasVaggelis HarokoposChristos ValavanisJerold ChunAlexandra KosmaGeorgios T. StathopoulosEvangelos BourosDemosthenes BourosKonstantinos SyrigosVassilis Aidinis
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<p>Decreased ENPP2 mRNA expression in human lung cancer</p>Keywords:
Autotaxin
Abstract The aberrant processes driving hepatocellular carcinoma (HCC) are not fully understood. Lysophosphatidic acid receptors (LPAR) are commonly overexpressed in HCC, but their contributions to malignant development are not well established. In this report, we show that aberrant expression of LPAR6 sustains tumorigenesis and growth of HCC. Overexpression of LPAR6 in HCC specimens associated with poor survival in a cohort of 128 patients with HCC. We took a genetic approach to elucidate how LPAR6 sustains the HCC tumorigenic process, including through an expression profiling analysis to identify genes under the control of LPAR6. RNAi-mediated attenuation of LPAR6 impaired HCC tumorigenicity in tumor xenograft assays. Expression profiling and mechanistic analyses identified Pim-3 as a pathophysiologically relevant LPAR6 target gene. In nonmalignant cells where LPAR6 overexpression was sufficient to drive malignant character, Pim-3 was upregulated at the level of transcription initiation through a STAT3-dependent mechanism. A further analysis of HCC clinical specimens validated the connection between overexpression of LPAR6 and Pim-3, high proliferation rates, and poorer survival outcomes. Together, our findings establish LPAR6 as an important theranostic target in HCC tumorigenesis. Cancer Res; 75(3); 532–43. ©2014 AACR.
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Autotaxin
Imatinib Mesylate
ABL
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Abstract Glioblastoma multiforme (GBM), a grade IV astrocytoma, is a very aggressive and prevalent primary brain tumor with a median survival rate of 12-15 months. Though the standard treatment relies on combinations of chemotherapy, radiation therapy and surgical resection, GBM remains radio-resistant with a high recurrence rate after resection. Understanding the molecular mechanisms involved in the therapeutic response could lead to the identification of novel targets to improve therapy. Radiation can trigger the activation of cytosolic phospholipase A2 (cPLA2), leading to production of lipid second-messengers such as lysophosphatidylcholine (LPC). Autotaxin (ATX), also known as lysophospholipase D (LysoPLD), is a secreted enzyme that catalyzes the production of lysophosphatidic acid (LPA) in the tumor microenvironment by cleaving the head-group of LPC. Specific G-protein coupled receptors (GPCRs) mediate the autocrine and paracrine effects of LPA such as migration, angiogenesis and proliferation in cancer. ATX is highly expressed in glioblastoma cells and is known to contribute to its invasive properties. We studied ATX as a potential target to enhance radiosensitivity in GBM by targeting ATX with PF-8380, a specific inhibitor developed by Pfizer Inc., and genetically by using shRNA. Pre-treatment with 1μM PF-8380 followed by irradiation with 4Gy resulted in decreased clonogenic survival, decreased migration and decreased invasion in mouse glioblastoma GL-261and human glioblastoma U87-MG cells. We confirmed these results by knocking down ATX with shRNA in GL-261 and U87-MG cells. Inhibition of ATX by PF-8380, or its knockdown by shRNA, lead to attenuation of Akt phosphorylation in GL-261 and U87-MG cells. Inhibition or knockdown of ATX resulted in significantly reduced radiation-induced neovascularization as assayed with GL-261 tumor vascular window model. The effect of ATX inhibition by PF-8380 in-vivo was studied using a heterotopic mouse GL-261 tumor model. Tumor bearing mice were treated with DMSO or 1 mg/kg PF-8380 followed by irradiation with 1.8Gy for 5 consecutive days. Tumor growth delay was analyzed using a non-parametric (Kruskal-Wallis) test to determine the amount of days to reach a tumor volume of 0.7 cm3 and ANOVA to determine the difference between treatments. Tumors treated with DMSO or PF-8380 took 11.2 and 12.2 days respectively to reach a volume of 0.7 cm3. Tumors treated with radiation alone took 23.3 days while the combination of PF-8380 with radiation significantly delayed tumor growth to 32 days. In this study, we found that inhibition of pro-survival cellular signaling pathways, migration, invasion and radiation-induced neovascularization can be achieved by targeting ATX. Thus, ATX could serve as a novel and effective molecular target for the development of radiosensitizers of GBM, and may provide a new approach to treating patients with this lethal form of brain cancer. Citation Format: David Y.A. Dadey, Sandeep R. Bhave, Daniel J. Ferraro, Rowan M. Karvas, Dennis E. Hallahan, Dinesh Thotala. Inhibition of autotaxin enhances radiosensitivity in human and murine glioblastoma cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2136. doi:10.1158/1538-7445.AM2013-2136
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U87
Radiosensitivity
Clonogenic assay
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We determined that signal transducer and activator of transcription 3 (Stat3) is tyrosine phosphorylated in 37% of primary breast tumors and 63% of paired metastatic axillary lymph nodes. Examination of the distribution of tyrosine phosphorylated (pStat3) in primary tumors revealed heterogenous expression within the tumor with the highest levels found in cells on the edge of tumors with relatively lower levels in the central portion of tumors. In order to determine Stat3 target genes that may be involved in migration and metastasis, we identified those genes that were differentially expressed in primary breast cancer samples as a function of pStat3 levels. In addition to known Stat3 transcriptional targets (Twist, Snail, Tenascin-C and IL-8), we identified ENPP2 as a novel Stat3 regulated gene, which encodes autotaxin (ATX), a secreted lysophospholipase which mediates mammary tumorigenesis and cancer cell migration. A positive correlation between nuclear pStat3 and ATX was determined by immunohistochemical analysis of primary breast cancer samples and matched axillary lymph nodes and in several breast cancer derived cell lines. Inhibition of pStat3 or reducing Stat3 expression led to a decrease in ATX levels and cell migration. An association between Stat3 and the ATX promoter, which contains a number of putative Stat3 binding sites, was determined by chromatin immunoprecipitation. These observations suggest that activated Stat3 may regulate the migration of breast cancer cells through the regulation of ATX.
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Lysophosphatidic acid (LPA) and its G-protein-coupled receptors (Lpar1-Lpar6) mediate a plethora of activities associated with cancer growth and progression. However, there is no systematic study about whether and how LPA promotes esophageal squamous cell carcinoma (ESCC). Here, we show that autotaxin (ATX), a primary LPA-producing enzyme, is highly expressed in ESCC, and overexpressed ATX is associated with the poor outcome of ESCC patients. Meanwhile, the expression of Lpar1 was much higher in ESCC cells compared with Het-1a (human esophagus normal epithelial cells). Functional experiments showed that LPA remarkably increased the proliferation and migration of ESCC cells. Furthermore, Lpar1 knockdown abolished the effect of LPA on ESCC cell proliferation and migration. Mechanistic studies revealed that LPA promoted ESCC cell lines proliferation and migration through PI3K/Akt pathway. Treatment of KYSE30 cell xenografts with Lpar1 inhibitor BMS-986020 significantly repressed tumor growth. Our results shed light on the important role of LPA in ESCC, and Lpar1 might be a potential treatment target for ESCC.
Squamous cell cancer
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Aggressive Schwann cell‐derived sarcomas known as malignant peripheral nerve sheath tumors (MPNSTs) are the leading cause of death in patients with neurofibromatosis type 1 (NF1). It has been proposed that growth factors such as lysophosphatidic acid (LPA) interact with NF1 loss to drive MPNST pathogenesis. LPA promotes Schwann cell proliferation, migration and survival; LPA induced proliferation and survival is further enhanced in Nf1 −/− Schwann cells. As autotaxin (ATX)/ENPP2, the enzyme that generates LPA via conversion of lysophosphatidylcholine, is overexpressed in several other tumor types, we hypothesized that ATX overexpression promotes the pathogenesis of MPNSTs and the benign neurofibromas from which they are derived. To test this hypothesis, we examined ATX expression in human neurofibromas, MPNSTs and MPNST cell lines and MPNSTs arising in a novel mouse model (P 0 ‐GGFβ3 mice). Immunohistochemical studies showed that ATX was overexpressed in human and murine tumors compared to normal nerve. Real time PCR and immunoblots further demonstrated increased ATX expression in a panel of MPNST cell lines; the ATX transcripts present in these cells encoded several functionally distinct splice variants of ATX including α, β and γ isoforms. We are currently knocking down ATX expression and generating ATX overexpressing transgenic mice to validate ATX as a therapeutic target in MPNSTs. Funded by NIH and DOD.
Autotaxin
Pathogenesis
Schwann cell
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Abstract Autotaxin (ATX), a lysophospholipase D that generates the growth factor-like mediator lysophosphatidic acid LPA, is up-regulated in metastatic and chemotherapy-resistant carcinomas. Many cancer cells secrete ATX, and it contributes to their invasive properties. The biological functions of ATX are mediated by LPA acting on G protein-coupled receptors (LPAR1-8). Many cancers overexpress multiple subtypes of LPAR and the overexpression of LPAR leads to malignant transformation, metastasis, and resistance to therapy. We have previously shown that knockdown of ATX expression in B16F10 melanoma cells decreased tumor invasion in vitro and lung metastasis in vivo. In our present study, we sought to understand the roles of LPAR both in the tumor cells and stromal cells of the tumor microenvironment. B16F10 melanoma cells predominantly express the LPA5 receptor. We found that these cells do not invade across a matrigel layer in response to LPA. shRNA knockdown of LPA5 receptors in B16F10 attenuates this inhibition, suggesting that the LPA5 receptor acts as an anti-invasive receptor in these tumor cells. Interestingly, we identified a different role for host LPA5 receptor. We found that the incidence of B16F10 lung metastasis was significantly reduced in the LPA5 knockout mice compared to wild type mice. In addition, LPA1 knockout mice also showed diminished B16F10 lung metastasis suggesting that host LPA1 and LPA5 receptors play critical roles in the seeding of metastasis. The decrease in tumor cell residence in the lungs of these knockout animals was apparent at 24 hours after injection. However, deletion of LPA1 or LPA5 did not affect the subcutaneous growth of B16F10 tumors. In summary, our results suggest that LPAR, in particular LPA5 and LPA1 receptors in tumor and stromal cells may play different roles in invasion and the seeding of metastasis. Citation Format: Sue-Chin Lee, Yuko Fujiwara, Jianxiong Liu, Junming Yue, Ryoko Tsukahara, Erzsebet Szabo, Renukadevi Patil, Duane D. Miller, Louisa Balazs, Tamas Oravecz, Gabor J. Tigyi. Autotaxin, LPA1 and LPA5 receptors in the tumor microenvironment determine melanoma invasion and metastasis. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B28. doi:10.1158/1538-7445.CHTME14-B28
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Knockout mouse
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The lipid mediator lysophosphatidic acid (LPA) signals via six distinct G protein-coupled receptors to mediate both unique and overlapping biological effects, including cell migration, proliferation and survival. LPA is produced extracellularly by autotaxin (ATX), a secreted lysophospholipase D, from lysophosphatidylcholine. ATX-LPA receptor signaling is essential for normal development and implicated in various (patho)physiological processes, but underlying mechanisms remain incompletely understood. Through gene targeting approaches in zebrafish and mice, we show here that loss of ATX-LPA1 signaling leads to disorganization of chondrocytes, causing severe defects in cartilage formation. Mechanistically, ATX-LPA1 signaling acts by promoting S-phase entry and cell proliferation of chondrocytes both in vitro and in vivo, at least in part through β1-integrin translocation leading to fibronectin assembly and further extracellular matrix deposition; this in turn promotes chondrocyte-matrix adhesion and cell proliferation. Thus, the ATX-LPA1 axis is a key regulator of cartilage formation.
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Lymph nodes (LNs) are highly confined environments with a cell-dense three-dimensional meshwork, in which lymphocyte migration is regulated by intracellular contractile proteins. However, the molecular cues directing intranodal cell migration remain poorly characterized. Here we demonstrate that lysophosphatidic acid (LPA) produced by LN fibroblastic reticular cells (FRCs) acts locally to LPA2 to induce T-cell motility. In vivo, either specific ablation of LPA-producing ectoenzyme autotaxin in FRCs or LPA2 deficiency in T cells markedly decreased intranodal T cell motility, and FRC-derived LPA critically affected the LPA2-dependent T-cell motility. In vitro, LPA activated the small GTPase RhoA in T cells and limited T-cell adhesion to the underlying substrate via LPA2. The LPA-LPA2 axis also enhanced T-cell migration through narrow pores in a three-dimensional environment, in a ROCK-myosin II-dependent manner. These results strongly suggest that FRC-derived LPA serves as a cell-extrinsic factor that optimizes T-cell movement through the densely packed LN reticular network.
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Melanoma is the deadliest form of skin cancer in which patients with metastatic disease have a 5-year survival rate of less than 10%. Recently, the overexpression of a β-galactoside binding protein, galectin-3 (LGALS3), has been correlated with metastatic melanoma in patients. We have previously shown that silencing galectin-3 in metastatic melanoma cells reduces tumor growth and metastasis. Gene expression profiling identified the protumorigenic gene autotaxin (ENPP2) to be downregulated after silencing galectin-3. Here we report that galectin-3 regulates autotaxin expression at the transcriptional level by modulating the expression of the transcription factor NFAT1 (NFATC2). Silencing galectin-3 reduced NFAT1 protein expression, which resulted in decreased autotaxin expression and activity. Reexpression of autotaxin in galectin-3 silenced melanoma cells rescues angiogenesis, tumor growth, and metastasis in vivo. Silencing NFAT1 expression in metastatic melanoma cells inhibited tumor growth and metastatic capabilities in vivo. Our data elucidate a previously unidentified mechanism by which galectin-3 regulates autotaxin and assign a novel role for NFAT1 during melanoma progression.
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Galectin-3
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