- Email: [email protected]
Linking of mPGES-1 and iNOS activates stem-like phenotype in EGFR-driven epithelial tumor cells
Accepted Manuscript Linking of mPGES-1 and iNOS activates stem-like phenotype in EGFR-driven epithelial tumor cells Erika Terzuoli, Federica Finetti, Filomena Costanza, Antonio Giachetti, Marina Ziche, Sandra Donnini PII:
S1089-8603(16)30198-7
DOI:
10.1016/j.niox.2017.02.010
Reference:
YNIOX 1649
To appear in:
Nitric Oxide
Received Date: 12 October 2016 Revised Date:
21 February 2017
Accepted Date: 27 February 2017
Please cite this article as: E. Terzuoli, F. Finetti, F. Costanza, A. Giachetti, M. Ziche, S. Donnini, Linking of mPGES-1 and iNOS activates stem-like phenotype in EGFR-driven epithelial tumor cells, Nitric Oxide (2017), doi: 10.1016/j.niox.2017.02.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT Linking of mPGES-1 and iNOS activates stem-like phenotype in EGFR-driven epithelial tumor cells
Short title: mPGE-1 and iNOS induce tumor stemness
Sandra Donninia,b*
Department of Life Sciences, University of Siena, 53100 Siena, Italy
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Istituto Toscano Tumori (ITT), 50136 Florence, Italy
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Erika Terzuoli,a Federica Finetti,a Filomena Costanza,a Antonio Giachetti,a Marina Ziche,a,b and
[email protected], [email protected], [email protected], [email protected]
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*Corresponding author: Prof. Sandra Donnini,
Department of Life Sciences, University of Siena, via Aldo Moro, 2, 53100, Siena, Italy Phone: +390577235382
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E-mail: [email protected] and [email protected]
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ACCEPTED MANUSCRIPT Abstract Inflammatory prostaglandin E-2 (PGE-2) favors cancer progression in epithelial tumors characterized by persistent oncogene input. However, its effects on tumor cell stemness are poorly understood at molecular level. Here we describe two epithelial tumor cells A431 and A459,
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originating from human lung and skin tumors, in which epithelial growth factor (EGF) induces sequential up-regulation of mPGES-1 and iNOS enzymes, producing an inflammatory intracellular milieu. We demonstrated that concerted action of EGF, mPGES-1 and iNOS causes sharp changes
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in cell phenotype demonstrated by acquisition of stem-cell features and activation of the epithelialmesenchymal transition (EMT). When primed with EGF, epithelial tumor cells transfected with
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mPGES-1 or iNOS to ensure steady enzyme levels display major stem-like and EMT markers, such as reduction in E-cadherin with a concomitant rise in vimentin, ALDH-1, CD133 and ALDH activity. Tumorsphere studies with these cells show increased sphere number and size, enhanced migratory and clonogenic capacity and sharp changes in EMT markers, indicating activation of this
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process. The concerted action of the enzymes forms a well-orchestrated cascade where expression of iNOS depends on overexpression of mPGES-1. Indeed, we show that through its downstream effectors (PGE-2, PKA, PI3K/Akt), mPGES-1 recruits non-canonical transcription factors, thus
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facilitating iNOS production.
In conclusion, we propose that the initial event leading to tumor stem-cell activation may be a
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leveraged intrinsic mechanism in which all players are either inherent constituents (EGF) or highly inducible proteins (mPGES-1, iNOS) of tumor cells. We suggest that incipient tumor aggressiveness may be moderated by reducing pivotal input of mPGES-1.
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ACCEPTED MANUSCRIPT Keywords mPGES-1, iNOS, epithelial tumor cells, stemness, EGF, tumorspheres
Abbreviations: mPGES-1: microsomal prostaglandin E synthase-1; PGE-2: prostaglandin E-2; EGF: epidermal growth factor; EMT: epithelial to mesenchymal transition; ALDH: aldehyde
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dehydrogenase; PKA: protein kinase A; HIF-1α: hypoxia inducible factor-1α; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; AP1: activator protein 1; EP receptors: PGE2 receptors; SNP: sodium nitroprusside; FBS: fetal bovine serum; PI3K: phosphatidylinositol-3-
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kinase; A.D.U. arbitrary densitometric units.
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ACCEPTED MANUSCRIPT 1. Introduction Malignant tumor cell phenotype appears to be driven by a subpopulation of functional heterogeneous stem-like cells having, remarkable self-renewing capacity and the ability to evolve into progenitor cells with pre-metastatic traits by epithelial-mesenchymal transition (EMT) [1]. The
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heterogeneity and plasticity of stem cells in tumors is a result of cell-intrinsic and extrinsic mechanisms [2-4]. Examples of intrinsic factors inducing tumor cell stemness and EMT are cyclooxygenase-2 and microsomal prostaglandin E synthase type 1 (COX-2 and mPGES-1) and in
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certain cases inducible nitric oxide synthase (iNOS), as well as their end products, prostaglandin E2 (PGE-2) and nitric oxide (NO) [5-8]. These enzymes and signals have been shown to strongly
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favor oncogene-driven tumorigenesis (as by epidermal growth factor, EGF), in epithelial tumor cells and tumor xenograft models [8, 9-13]. Here we sought to determine the role and crosstalk of the tumor intrinsic signals PGE-2 and NO, originating from mPGES-1 and iNOS, in inducing cell stemness and EMT in epithelial tumor cells, characterized by constitutive expression of EGF
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receptor (EGFR). The rationale for selecting the above enzymes and signals is based on evidence that expression of these molecules in certain epithelial tumor types gives them a remarkable growth advantage and enhances their aggressive attributes [14-19].
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We postulated that PGE-2 functions as a regulator of tumor cell stemness mediated by activation of EGF/EGF-receptor (EGFR) signaling and works via activation of the iNOS/NO signaling pathway
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activation. We show that A431 and A549 human epithelial cancer cell lines express elevated levels of mPGES-1/PGE-2 in response to stimulation by EGF. The resulting PGE-2 was found to upregulate iNOS expression through activation of AP1 and HIF-1α. The combined action of PGE-2 and NO increases cell clonogenic potential in vitro and induces formation of three-dimensional tumorspheres expressing markers of stem-like and EMT phenotype. Induction of iNOS by PGE-2 is mediated through the EP4 receptor in a PKA and Akt-dependent manner, as suggested by suppression of iNOS expression and tumor cell capacity to form tumorspheres when mPGES-1 or EP4 are inhibited by shRNA or siRNA or by selective pharmacological inhibitors. 4
ACCEPTED MANUSCRIPT Thus our data suggests that EGFR activation followed by mPGES-1-induced activation of the PGE2/EP4 signaling pathway leads to upregulation of iNOS: a cascade that promotes stem-cell and
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EMT phenotype in epithelial cancer cells.
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ACCEPTED MANUSCRIPT Material and methods 2.1. Cell lines A431 squamous carcinoma cells (passages 10-20, ATCC® CRL-1555™) and A549 lung cancer cells (passages 12-20, ATCC® CRL-1555™ and CCL-185™, respectively) were from the
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American Type Culture Collection (ATCC) and certified by STRA and were cultured as recommended. A549 mPGES-1 knockdown (mPGES-1 shRNA, passages 8-20) and non-target shRNA (Scr, passages 8-20) cells were obtained and cultured as described [20]. For details see
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supplementary material (S1 file). Cells were used at passages 8-20 and discarded after 2 months in culture.
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2.2. Reagents
Reagents were as follows: sodium nitroprusside (SNP), crystal violet, PGE-2, H-89, LY294002, anti-β-actin, anti-lamin, anti-tubulin (Sigma Aldrich); anti-mPGES-1, L-NG-nitroarginine methyl ester (L-NAME), 17-phenyl trinor prostaglandin e2 (EP1-EP3 agonist), butaprost (EP2 agonist),
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sulprostone (EP3 agonist), PGE1 alcohol (EP3-EP4 agonist), L-902, 688 (EP4 agonist), L-161, 982 (EP4 antagonist) (Cayman Chemicals); anti-E-cadherin (Dako), EGF (RELIAtech), anti-iNOS, antip65, anti-ALDH1 and anti-CD133 (Santa Cruz); anti- HIF-1α (BD Transduction Laboratories);
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anti-cJUN, anti-KLF4, anti-Sox-2, anti-Oct4, anti-Nanog, anti-c-Myc, anti-SNAIL (Cell Signaling); Lipofectamine 2000 (Life Technologies).
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2.3. Generation of tumor cells overexpressing iNOS and mPGES1 and lentiviral production Lenti vector plasmids for mPGES-1 (p Lenti vector with C-terminal Myc-DDK tag-NM_004878) and iNOS (p Lenti vector with C-terminal Myc-DDK tag-NM_000625) and vehicle (empty vector construct: pLenti-C-Myc-DDK-puro) were from Origene. psPAX2 packaging plasmid (12260) and pMDG.2 envelope plasmid (12259) were from Addgene. All plasmids were sequence verified. To generate cells overexpressing mPGES-1 and iNOS, 1 x 106 HEK293 cells (Life Technologies) were transfected with 2.25 µg PAX2 packaging plasmid, 0.75 µg PMD2G envelope plasmid and 3 µg pLKO.1 hairpin vector using 12 µl Lipofectamine 2000 on 10 cm plates. Polyclonal 6
ACCEPTED MANUSCRIPT populations of transduced cells were generated by infection with 1 MOI (multiplicity of infectious units) of lentiviral particles. Three days after infection, cells were selected with 20 µg/ml neomycin/kanamycin (Sigma Aldrich) for 1 week. 2.4. Invasion assay
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Cell invasion was investigated using a modified Boyden chamber as described previously [21]. Briefly, 1.2x104 cells were stimulated for 40 min with/ without NOS pathway inhibitor (L-NAME, 200 µM) or PKA (H89, 500 nM), PI3K (LY294002, 10 µM), or EP4 inhibitor (1 µM) in 0.1% FBS.
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Cell suspension was added to the upper wells. PGE-2, EGF and EP4 agonist were used as chemoattractive molecules. Invasion was measured by counting the number of cells that moved across the
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filter coated with Matrigel (250 µg/ml) in 16 h. Cells were counted in five random fields/well at a magnification of 20 X. Data (from triplicate experiments) is expressed as total number of cells migrating/well. 2.5. Clonogenic cell survival assay
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Tumor cells grown to confluence were incubated with EGF, PGE-2 or EP4 agonist with/without LNAME, LY294002 or H89, then plated in 60 mm dishes at a density of 500 cells/dish in medium containing 10% FBS as reported [21], and then kept in a humidified incubator at 37° C and 5% CO2
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for 2 weeks. Colonies (>50 cells) were fixed and stained with 1% crystal violet in 10% methanol and counted and photographed [16]. Results are expressed as number of colonies/well.
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2.6. Immunoblot analysis and nuclear/cytoplasm translocation Total protein lysates were obtained as described [22]. Antibodies were: anti-mPGES-1 (1:200, Cayman Chemicals); anti-E-cadherin (1:1000, Dako), anti-iNOS (1:500), anti ALDH1 and antiCD133 (1:1000, Santa Cruz); anti- HIF-1α (1:300, BD Transduction Laboratories). To assess the translocation of AP1 (evaluated as cJUN) and p 65 from cytosol to nucleus, 8x105 cells were plated in 10 cm diameter dishes, maintained in 10% FBS for 18 h, then treated with PGE2 or EP4 agonist for the indicated times, or with PGE-2 with/without EP4 antagonist or PGE-2 or EP4 agonist in presence/absence of H89 or LY294002, then scraped and homogenized on ice in 7
ACCEPTED MANUSCRIPT lysis buffer containing (in mM) 10 HEPES, 1 DTT, 10 KCl, 50 NaF, 0.1 EDTA, 0.1 EGTA, 1 Na3VO4, 0.5 PMSF and 0.1 NP-40 at 4°C, and centrifuged at 1,000 g for 10 min to separate the nuclei. The supernatant was centrifuged (13,200 g, 5 min) to yield the cytosolic fraction. The nuclear fraction was lysed in buffer containing (in mM) 20 HEPES, 1 EDTA, 1 EGTA and 0.5
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PMSF and analyzed for cJUN (Cell Signaling) and, P65 (Santa Cruz). Western blotting was performed as described [22]. Images were digitalized with CHEMI DOC Quantity One program, blots were analyzed in triplicate by densitometry using NIH Image 1.60B5 software, and arbitrary
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densitometric units (ADU) were normalized for β-actin, β-tubulin or lamin (Sigma). 2.7. EIA assay
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PGE-2 was measured with an EIA kit (Prostaglandin E-2 EIA kit-Monoclonal, Cayman Chemical). Cells were exposed to EGF (25 ng/ml) in presence/absence of L-NAME (200 µM) or SNP (10 µM) for 18 h and treated for 30 min with 30 µM arachidonic acid. Cell culture supernatants were assayed directly at a final dilution of 1:50. The least detectable concentration was 1 to 2 pg/ml. PGE-2
2.8. cGMP assay
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concentration was expressed in pg/ml normalized to total protein concentration.
Cyclic GMP (cGMP) levels were measured in cell extracts from confluent A431 and A549 by
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enzyme-immunoassay kit (Cayman Chemical, Italy). Cells were pre-treated with 1 mM 3-isobutyl5-methyl-xanthine (IBMX) for 30 min then stimulated with 25 ng/ml EGF for 18 h. cGMP levels
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were assayed according to the manufacturer's instructions and protein levels were measured by the Bradford procedure.
2.9. Transient siRNA transfection The day before transfection, cells were trypsinized and 3 x 105 cells were seeded in six-well plates. Transient transfection of siRNA was carried out using Lipofectamin 2000 according to the manufacturer’s instructions. Cells were assayed 48 h after transfection. For siRNA transfection the siRNA sequences targeting: human iNOS (5’-AACCCAGCTGCTGCTCCAAAA-3’), human PKA
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ACCEPTED MANUSCRIPT (5’-AAGCCGGAGAATCTGCT) and human mPGES-1 (5’-CGGGCTAAGAATGCAGACTTT3’) were from Qiagen. Scr siRNA is a random siRNA (Qiagen). 2.10. Tumorsphere formation in vitro This assay tests the ability of single cells to form tumorspheres, the in vitro surrogate of stem-like
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cells [23]. Cells (2 x 105 cells/well in 1.5 ml medium) were distributed in an ultralow attachment six-well plate. All tumorspheres were grown in sphere medium: DME F12 medium (Gibco), supplemented with penicillin/streptomycin, L-glutamine and B27 supplement (1x, Life
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Technologies), bFGF (20 ng/ml, Gibco) and hEGF (20 ng/ml, Gibco), and allowed to grow for 7 to 10 days, or until most spheres reached a diameter of 60 µm. Tumorspheres were counted, harvested,
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and protein extracted [22] or split for second tumorspheres generation [23] and lysed for protein extraction or were reduced to a single cells suspension (with EDTA 10 mM and pipet up and down) for ALDEFLUOR assay.
The tumorsphere formation efficiency (TFE) indicates the percentage of cells within a culture that
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are capable of forming a sphere from a single cell. TFE was determined using the formula: (# of spheres/# of cells plated) x 100. 2.11 ALDEFLUOR assay
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The aldehyde dehydrogenase (ALDH) activity was detected using an ALDEFLUOR assay kit (StemCell Technologies) according to the manufacturer’s protocol. Briefly, the cells were stained
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with bodipy-aminoacetaldehyde (BAAA) and incubated for 45 minutes at 37°C. a specific inhibitor of ALDH, diemethylamino-benzaldehyde (DEAB), was used to control for background fluorescence. The stained cells were analyzed using the Guava easyCyte Single Sample Flow Cytometer (Merk Millipore) and ALDH positive cells (ALDH+) were detected on the green fluorescence channel. The data were analyzed using the InCyte Software program (Millipore). 2.12. Statistical analysis
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ACCEPTED MANUSCRIPT Results are expressed as means ± SD. Statistical analysis was performed using Student's t test, twoway ANOVA, or the Bonferroni post-test for multiple comparisons. P<0.05 was considered
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statistically significant.
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ACCEPTED MANUSCRIPT 2. Results 3.1. PGE-2 and NO induce A431 and A549 tumor cells to express markers of stemness and EMT and to form tumorspheres We examined the ability of mPGES-1 and iNOS expression in A431 and A549 cells to regulate
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stem-cell-like features, by evaluating their capacity to form tumorspheres and express stem-like markers and their proclivity to initiate epithelial to mesenchymal transition (EMT). To do so, we compared A431 and A549 wild type (WT) or transfected with empty vector (vehicle) with cells
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forced to express either mPGES-1 or iNOS by transfection (+mPGES-1 and +iNOS).
Transfection with empty vector did not affect mPGES-1 or iNOS in A431 WT and A549 cells (Fig.
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S1A and B). We found that overexpression of mPGES-1 and iNOS produced a significant increase in expression of stem-like markers such as ALDH1 and CD133 in transfected cells with respect to A431 WT (Fig. 1A and B). Concomitantly, we observed a large increase in vimentin protein expression and decrease in E-cadherin expression, indicating a significant loss of cell-cell adhesion.
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Also in +mPGES-1 A549, we observed a large decrease in E-cadherin and an increase in fibronectin and vimentin, markers of EMT, and an increase in ALDH1 (Fig. S2A). Similar changes in EMT and stem-like markers were reproduced by exposing cells to PGE-2 or
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EGF, an upstream signal to mPGES-1 (Fig. 1C and D). Next, we investigated the ability of tumor cells, forced to express mPGES-1 or iNOS, to form
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tumorspheres under non adherent culture conditions [23]. First, tumorsphere formation efficiency (TFE) for A431 and A549 cancer cell lines was evaluated. The TFE indicates the percentage of cells within a culture that are capable of forming a sphere. A431 and A549 tumor cells were grown in suspension at low density in serum-free sphere medium for 7–10 days. Both cell lines formed tumorspheres (Fig. 2A-C and Fig. S3A-C). A431 cells showed a higher tumorsphere forming efficiency (TFE; means 55% ± SD 2.3 of three triplicate independent experiments) as compared to A549 (TFE; means 13.70 ± SD 5.4 of three duplicate independent experiments) (Fig.2A and Fig.
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ACCEPTED MANUSCRIPT S2A, respectively). A431 WT and vehicle displayed similar TFE (means 55% ± SE 2.3 and 53 % ± SE 1.7 of three triplicate independent experiments) (Fig. S3D). By comparing A431 WT with +mPGES-1 and +iNOS cells, we found that the latter two cell lines displayed a striking increase (~ 3 to 4 fold) in TFE and size of second-generation spheres (means
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182.5.7% ± SE 5.3 and 138 % ± SE 4.7 of three triplicate independent experiments) (Fig 2A, B and C). +mPGES-1 and +iNOS spheres also showed enhanced expression of stem-like markers, such as KLF4, Sox2, Nanog, CD133, c-Myc, Oct 4 and EMT marker SNAIL and ALDH activity, as well as
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lower E-cadherin levels (Fig 2D-F and table S1A and B, for western blot quantification). In the +mPGES-1 and +iNOS cells, the ratio of the ALDH+ cells stained with BAAA was 35.46% and
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39.92% respectively, while that of those stained with BAAA and DEAB as a negative control was 16.1% and 18.6%, respectively (Fig. 2E). In WT cells, the ratio of the ALDH+ cells without DEAB was 14.4% and with DEAB was 13.1% (Fig. 2E). The fold increase of ALDH+ cells versus DEAB was about 2 in +mPGES-1 and +iNOS cells, and 1 in WT cells, indicating a stem-like phenotype in
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+mPGES-1 and +iNOS compared to WT cells.
We also noted a significant increase in cellular invasion and clonogenic potential in +mPGES-1 and +iNOS than in WT cells (Fig. 2G and H). Similar qualitative changes were detected in A431 cells
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exposed to PGE-2, or EGF (Fig. 3A-J and table S2 and table S3 A and B, for western blot quantification, respectively).
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The converse picture was observed in mPGES-1-KD and iNOS-KD cells, as both cell lines displayed a significant decrease in TFE and size of second-generation spheres under basal condition and after treatment with EGF, with respect to WT cells (Fig. 4A-C). In addition, A431 mPGES-1KD and iNOS-KD spheres showed reduced expression of the above stem-like markers and increased E-cadherin expression (Fig 4D and table S4 A and B, for western blot quantification). As well, tumor cell propensity to invade and form colonies under basal condition and in the presence of EGF was greatly reduced in mPGES-1-KD cells (Fig. 4E and F and Fig. S4A and B).
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ACCEPTED MANUSCRIPT 3.2. iNOS/NO signaling controls mPGES-1-induced stemness and EMT We studied whether iNOS silencing influences PGE-2-induced tumor cell stemness and EMT by measuring spheroid formation and stem-like markers in A431 +mPGES-1 cells. In the absence of NO production, these cells displayed a significant decrease in TFE and size of second-generation
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spheres (Fig 5A-C). Moreover, silencing iNOS in +mPGES-1 cells induced reduced expression of canonical stem-like markers and ALDH activity and increased E-cadherin expression compared to +mPGES-1 cells, both in monolayer and three-dimensional (tumorspheres) culture conditions (Fig.
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5D-G and table S5 A and B, for western blot quantification). This indicates that in the absence of iNOS/NO signaling, mPGES-1 is not per se sufficient to sustain the cell phenotype transition.
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Furthermore, cell invasion and clonogenic potential were reduced in cells pretreated with the iNOS inhibitor L-NAME and exposed to PGE-2 or EGF (Fig 5H and J).
These results provide evidence that mPGES-1 and iNOS exert concerted action inducing stemness and EMT in EGF-primed epithelial cancer cells, increasing stem-like and mesenchymal marker
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expression and promoting tumor clonogenic potential and invasiveness. 3.3. Relationship between mPGES-1/PGE-2 and iNOS/NO in epithelial cancer cells In light of the cooperation observed between mPGES-1 and iNOS in promoting tumorspheres
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formation and cell invasion, we investigated the relationship between the two enzymes in epithelial tumor cells exposed to EGF. After exposing A431 cells to EGF (25 ng/ml), we observed a
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significant increase in mPGES-1 levels as early as 4 h, peaking between 8 and 24 h (Fig. 6A), whereas the rise in iNOS was slower (8 h), reaching a maximum at 18 h (Fig. 6A). Blockade of the NO supply either by silencing iNOS or L-NAME application, or conversely increasing NO input by forced overexpression of iNOS (+iNOS), or incubation with the NO donor, sodium nitroprusside (SNP), had no influence on mPGES-1 expression (Fig. 6B-D and F). PGE-2 production was not modified by SNP stimulation, or by L-NAME (Fig. S5A). Similar results were obtained in lung A549 tumor cells (Fig. S5B-D).
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ACCEPTED MANUSCRIPT On the other hand, transient and stable transfection with siRNA or shRNA for mPGES-1, respectively, strongly reduced iNOS expression (Fig. 7A for A431 and Fig.S6A and B for A549 cells), whereas, stable overexpression of mPGES-1 in A431 and A549 cells (+mPGES-1) upregulated iNOS expression (Fig 7B for A431 and Fig.S6C for A549 cells). Moreover, mPGES-1
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knockdown reduced EGF-induced cGMP production in both cell lines (Fig. 7C and Fig. S6D). Collectively, these results indicate that mPGES-1/PGE-2 signaling is an obligate step in promoting EGF-induced iNOS expression.
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Next, we determined which of the multiple receptor subtypes of PGE-2 and its downstream effectors (PKA and PI3K/Akt) induce iNOS expression in A431 cells. As shown in Fig. S7A, the
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EP4 subtype selective agonist was the only one capable of eliciting a large increase in iNOS expression, while selective agonists for another receptors subtype (EP 1-3) were totally ineffective (Fig. 8A). Corroborating evidence for EP4 involvement was obtained in experiments using EP4 antagonist, or inhibitors of its effector signals, i.e. PKA (H89) or PI3K/Akt (LY2942002), or by
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silencing PKA, all of which suppressed agonist-promoted iNOS expression (Fig. S7B-E). The role of the PGE-2/EP4 signaling pathway was also investigated for its contribution to tumor-cell stemness and EMT markers. The EP4 agonist, L-902,688 induced expression of stem-like and EMT
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markers in A431 tumor-cell monolayer, as well as cell invasion and tumor cell colony formation in tumorspheres (Fig. 1 and 3). Inhibition of iNOS reduced EP4 agonist effects (Fig. 5F and G).
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Thus, PGE-2 induces iNOS expression in cancer cells by activating its EP4 receptor subtype and in turn its downstream PKA and Akt signals.
3.4. PGE-2/EP4 upregulates iNOS expression by recruiting transcription factors HIF-1α and AP1 Based on the above results (see Figs 7 and 8) we sought to determine how PGE-2 influences iNOS transcription in cancer cells. Among the wide variety of transcription factors of iNOS, we examined three factors present in the human iNOS promoter [24], i.e. HIF-1α, AP1 and NF-κB. EGF and 14
ACCEPTED MANUSCRIPT PGE-2, as well as EP4 agonist, sharply induced HIF-1α, and promoted AP1 nuclear translocation in both tumor cells (Fig. 9 and 10 A-D and Fig. S8A and B). These effects were abolished by silencing mPGES-1 or by inhibitors of prostanoid receptors and downstream effectors, PKA and Akt, respectively (Fig. 9 and Fig. S7B-E and S8A and B).
was insensitive to silencing mPGES-1 (Fig 9C).
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Although EGF induced a NF-κB component, p65, promoting its nuclear translocation, this effect
Briefly, HIF-1α expression and AP-1 nuclear translocation, appear to be controlled by the PGE-
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2/EP4-Akt and PKA axis, while transcription through NF-κB expression and nuclear translocation are prostanoid- independent. These findings, showing that PGE-2 reveals additional transcription
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factors besides the canonical NF-κB for iNOS, reveal an additional mechanism underlying the
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enhanced iNOS expression observed in epithelial tumor cells (Fig. S9A).
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ACCEPTED MANUSCRIPT 3. Discussion In certain epithelial tumors the strong oncogenic drive exerted by the EGF/EGFR axis promotes upregulation of mPGES-1 and iNOS, two enzymes, which through their end products, PGE-2 and NO, create an inflammatory milieu within tumor cells [13, 25, 26]. This inflammatory condition
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leads to activation of the epithelial-mesenchymal transition (EMT) and to ensuing phenotype changes of cells that acquire distinct stem-like features [27]. In the present study we showed evidence of a well-orchestrated action involving EGF, PGE-2 and NO, fully capable of initiating a
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functional stem-like and mesenchymal transition. An initial event is upregulation of iNOS which appears to depend closely on mPGES-1 and its downstream signals (PGE-2/EP4, PKA, AKT).
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According to the hierarchal order regulating expression of the above enzymes, mPGES-1 is necessary to induce stemness and, iNOS expression seems to complement this effect. This notion is corroborated by observation of incomplete induction of stemness and EMT in experiments involving silencing of iNOS (see Fig 5). Major intracellular changes in stem-like markers were
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detected in tumor cells transfected with mPGES-1 and iNOS. We observed a large reduction in Ecadherin concomitant with a significant rise in ALDH-1 expression and activity and CD133 levels in +mPGES-1 and +iNOS cells. Our studies on tumorsphere formation, an assay of the stemness of
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tumor cells [23], provided compelling evidence that the combined action of mPGES-1 and iNOS elicits stem-like properties and triggers EMT in tumor cells. Indeed, the sharp increase in number
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and size of second-generation spheres, as well as the enhanced invasiveness and colony formation ability reported here, indicate an incipient stem-like state. Moreover, the presence of a mesenchymal cell population, suggested by the pattern of EMT markers, detected in tumorspheres, is indicative of EMT activation. Several studies indicate that in carcinoma cells the EMT can give rise to cells with stem-like properties, suggesting a functional link between the two processes and providing an explanation for how cancer stem cells may arise in tumors and be manipulated therapeutically [28, 29]. At molecular level, mPGES-1 was insensitive to attempts to modify its expression whether by supplying exogenous NO (NO donor) or NO blocking (iNOS silencing, or L16
ACCEPTED MANUSCRIPT NAME), whereas silencing mPGES-1 abolished iNOS expression. Exogenous PGE-2 or its EP4 agonist reproduced all the effects observed in epithelial tumors, causing activation of PKA and PI3K/Akt, while release of endogenous PGE-2 was also detected in tumor cells stimulated by EGF. We observed a fairly fast induction of mPGES-1, followed by a significant rise in cGMP, as well as
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activation of PKA and/or PI3K/Akt, indicative of activation of EP4 [30]. EP4 activity associates with multiple mechanisms involved in cancer progression, including induction of stem-like cell phenotype [30, 31], and in particular, EP4/PI3K/Akt signalling has been reported to promote cancer
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cell stemness in breast cancer [30]. Further, EP4/PI3K/Akt signalling is also involved in maintaining the basal mammary stem cell phenotype, suggesting a key role of this pathway in PGE-
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2-mediated induction of cell stem-like phenotype [32]. Intracellular release of PGE-2 was also involved in inducing nuclear translocation of transcription factors such as HIF-1α and AP1, which were readily blocked by inhibitors of the EP4-Akt and PKA axis. Molecular cross-talk between cell-derived prostaglandins and NO is a well-known phenomenon. Data from the literature
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regarding the effects of PGE-2 on iNOS expression is contradictory. Depending on cell type, PGE-2 has been shown to potentiate or inhibit iNOS expression and activity [33-35]. Like we found here, in the case of PGE-2 stimulation of iNOS expression, the effects are associated with an increase in
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cAMP/PKA signaling [36, 37]. Here we showed that in A431 and A549 cells, Akt activity, upstream of HIF-1α expression and downstream of EP4, is also involved in iNOS expression. HIF-
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1α is involved in maintenance of tumor stemness [38], suggesting that microenvironment-generated -PGE-2 may promotes enhancement of iNOS expression and in turn induction of tumor cell stemness by widening the repertoire of transcription factors available for iNOS expression. The picture emerging from these studies is that a mechanism intrinsic to epithelial tumor cells is fully capable of initiating the cell transition from epithelial to mesenchymal state, and therefore of generating a metastatic cell population endowed with enhanced aggressive/stem-like properties. Although confined to oncogene-driven tumor cells harboring genes inducible by persistent oncogene output (e.g. EGF), this mechanism is distinctly intrinsic since all its players are either 17
ACCEPTED MANUSCRIPT inherent constituents (e.g. EGFR) or highly inducible proteins (mPGES-1, iNOS). The mechanism, a remarkable example of concerted action between its components, is a protein induction cascade characterized by a fast rise in EGF-induced mPGES-1 expression followed by slightly delayed PGE-2/EP4-induced iNOS expression, which appears to be totally dependent on the rise in mPGES-
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1. In this mechanism the prevailing mode of interaction is autocrine, as indicated by the modest levels of PGE-2 released in cells via stimulation of mPGES-1 by EGF. Although, these PGE-2 levels are relatively low, they are nonetheless sufficient to recruit the transcription factors for iNOS,
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which however does not preclude a paracrine-type interaction of gaseous NO with proximal stromal cells [39].
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Nitric oxide and particularly mPGES-1 have been reported to promote stemness and mesenchymal phenotype in a variety of epithelial tumors [5-8]. However, in most of these studies the source of mesenchymal or stemness-inducing mediators was the tumor stroma (a source extrinsic to the tumor), known to produce large quantities of chemokines.
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This study provides evidence that the initial event inducing stemness/EMT may occur within the tumor cell (intrinsic), and may subsequently propagate to the adjoining stroma by a paracrine mechanism to amplify the mesenchymal and/or stemness-inducing response (extrinsic). In
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conclusion, we find that in tumors arising from EGF-oncogene-primed cells, the downstream mPGES-1/PGE-2/EP4 signaling pathway plays a critical role in inducing the iNOS required for
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tumor progression. PGE-2 blockade through mPGES-1 or EP4 inhibitors is a potential therapeutic option for such aggressive cancers.
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ACCEPTED MANUSCRIPT Conflict of interest None Funding: This research was supported by the Associazione Italiana sul Cancro (IG10731, MZ 2010-2013;
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IG15443, MZ, 2015-2017). E.T. was a fellow of the Fondazione Italiana per la Ricerca sul cancro
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(FIRC, 2012-2014).
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ACCEPTED MANUSCRIPT References [1]
G. Driessens, B. Beck, A. Caauwe, BD. Simons, C. Blanpain, Defining the mode of tumour
growth by clonal analysis, Nature. 2012; 488:527-530. [2]
AS. Cleary, TL. Leonard, SA. Gestl, EJ. Gunther, Tumour cell heterogeneity maintained by
P. Greulich, BD. Simons, Dynamic heterogeneity as a strategy of stem cell self-renewal,
Proc Natl Acad Sci U S A. 2016; 113:7509-7514. [4]
K. Eason, A. Sadanandam, Molecular or metabolic reprograming: what triggers tumor
subtypes? Cancer Res. 2016;76:5195-5200.
TH. Chu, HH. Chan, HM. Kuo, LF. Liu, TH. Hu, et al., Celecoxib suppresses hepatoma
M AN U
[5]
SC
[3]
RI PT
cooperating subclones in Wnt-driven mammary cancers, Nature. 2014;508:113-117.
stemness and progression by up-regulating PTEN, Oncotarget. 2014;5:1475-1490. [6]
F. Finetti, E. Terzuoli, A. Giachetti, R. Santi, D. Villari, et al., mPGES-1 in prostate cancer
controls stemness and amplifies epidermal growth factor receptor-driven oncogenicity, Endocr
[7]
TE D
Relat Cancer. 2015;22:665-678.
MA. Puglisi, C. Cenciarelli, V. Tesori, M. Cappellari, M., Martini, et al., High nitric oxide
production, secondary to inducible nitric oxide synthase expression, is essential for regulation of the
[8]
EP
tumour-initiating properties of colon cancer stem cells, J Pathol. 2015;236:479-490. D. Peng, T. Tanikawa, W. Li, L. Zhao, L. Vatan, et al., Myeloid-derived suppressor cells
AC C
endow stem-like qualities to breast cancer cells through IL6/STAT3 and NO/NOTCH cross-talk signaling, Cancer Res. 2016;76:3156-3165. [9]
MD. Castellone, H. Teramoto, BO. Williams, KM. Druey, JS. Gutkind, Prostaglandin E2
promotes colon cancer cell growth through a Gs-axin-beta-catenin signaling axis. Science. 2005;310:1504-1510. [10]
W. Goessling, TE. North, S. Loewer, AM. Lord, S. Lee, et al., Genetic interaction of PGE2
and Wnt signaling regulates developmental specification of stem cells and regeneration, Cell. 2009;136:1136-1147. 20
ACCEPTED MANUSCRIPT [11]
DG. Menter, RL. Schilsky, RN. DuBois, Cyclooxygenase-2 and cancer treatment:
understanding the risk should be worth the reward, Clin Cancer Res. 2010;16:1384-1390. [12]
N. Yongsanguanchai, V. Pongrakhananon, A. Mutirangura, Y. Rojanasakul, P.
Chanvorachote, Nitric oxide induces cancer stem cell-like phenotypes in human lung cancer cells,
[13]
D.
Wang,
RN.
DuBois,
RI PT
Am J Physiol Cell Physiol. 2015;308:C89-100.
The role of prostaglandin
immunosuppression, Trends Mol Med. 2016;22:1-3.
tumor-associated
O. Gallo, E. Masini, L. Morbidelli, A. Franchi, I. Fini-Storchi, et al., Role of nitric oxide in
SC
[14]
e(2) in
angiogenesis and tumor progression in head and neck cancer, J Natl Cancer Inst. 1998;90:587-596. F. Finetti, E. Terzuoli, E. Bocci, I. Coletta, L. Polenzani, et al., Pharmacological inhibition
M AN U
[15]
of microsomal prostaglandin E synthase-1 suppresses epidermal growth factor receptor-mediated tumor growth and angiogenesis, PLoS One. 2012;7:e40576. [16]
S. Donnini, F. Finetti, E. Terzuoli, A. Giachetti, MA. Iñiguez, et al., EGFR signaling
TE D
upregulates expression of microsomal prostaglandin E synthase-1 in cancer cells leading to enhanced tumorigenicity, Oncogene. 2012;31:3457-3466. [17]
D. Wang, RN. DuBois, Urinary PGE-M: a promising cancer biomarker. Cancer Prev Res
[18]
EP
(Phila). 2013;6:507-510.
S. Granados-Principal, Y. Liu, ML. Guevara, E. Blanco, DS. Choi, et al., Inhibition of iNOS
AC C
as a novel effective targeted therapy against triple-negative breast cancer, Breast Cancer Res. 2015;17:25. [19]
E. Della Pietra, F. Simonella, B. Bonavida, LE. Xodo, V. Rapozzi, Repeated sub-optimal
photodynamic treatments with pheophorbide a induce an epithelial mesenchymal transition in prostate cancer cells via nitric oxide, Nitric Oxide. 2015;45:43-53. [20]
H. Hanaka, SC. Pawelzik, JI. Johnsen, M. Rakonjac, K. Terawaki, et al., Microsomal
prostaglandin E synthase 1 determines tumor growth in vivo of prostate and lung cancer cells, Proc Natl Acad Sci U S A. 2009;106:18757-18762. 21
ACCEPTED MANUSCRIPT [21]
S. Donnini, F. Finetti, R. Solito, E. Terzuoli, A. Sacchetti, et al., EP2 prostanoid receptor
promotes squamous cell carcinoma growth through epidermal growth factor receptor transactivation and iNOS and ERK1/2 pathways. FASEB J. 2007;21:2418-2430. [22]
E. Terzuoli, S. Donnini, A. Giachetti, MA. Iñiguez, M. Fresno, et al., Inhibition of hypoxia
RI PT
inducible factor-1alpha by dihydroxyphenylethanol, a product from olive oil, blocks microsomal prostaglandin-E synthase-1/vascular endothelial growth factor expression and reduces tumor angiogenesis, Clin Cancer Res. 2010;16:4207-2016.
E. Pastrana, V. Silva-Vargas, and F. Doetsch. Eyes Wide Open: a critical review of sphere-
SC
[23]
formation as an assay for stem cells, Cell Stem Cell 2011; 486-498.
SC. Chu, J. Marks Konczalik, HP. Wu, TC. Banks, and J. Moss, Analysis of the cytokine-
M AN U
[24]
stimulated human inducible nitric oxide synthase (iNOS) gene: characterization of differences between human and mouse iNOS promoters, Biochem. Biophys. Res. Commun 1998; 248: 871– 878.
674. [26]
D. Hanahan, RA. Weinber, Hallmarks of cancer: the next generation, Cell. 2011;144:646-
TE D
[25]
SS. McAllister, RA. Weinberg, The tumour-induced systemic environment as a critical
[27]
EP
regulator of cancer progression and metastasis, Nat Cell Biol. 2014;16:717-727. DR. Pattabiraman, RA. Weinberg, Tackling the cancer stem cells - what challenges do they
[28]
AC C
pose? Nat Rev Drug Discov. 2014;13:497-512. PB. Gupta, CM. Fillmore, G. Jiang, SD. Shapira, K. Tao, et al., Stochastic state transitions
give rise to phenotypic equilibrium in populations of cancer cells, Cell. 2011; 146:633-644. [29]
SA. Mani, W. Guo, MJ. Liao, EN. Eaton, A. Ayyanan, et al., The epithelial-mesenchymal
transition generates cells with properties of stem cells, Cell. 2008; 133:704-715. [30]
M. Majumder, E. Landman, L. Liu, D. Hess, PK. Lala. COX-2 elevates oncogenic miR-
526b in breast cancer by EP4 activation, Mol Cancer Res 2015;13(6):1022-1033.
22
ACCEPTED MANUSCRIPT [31]
M. Majumder, X. Xin, L. Liu, E. Tutunea-Fatan, M. Rodriguez-Torres, et al., COX-2
Induces Breast Cancer Stem Cells via EP4/PI3K/AKT/NOTCH/WNT Axis. Stem Cells 2016;34(9):2290-305. [32]
M-C. Lin, S-Y. Chen, H-M. Tsai, P-L. He, Y-C. Lin et al., PGE-2/EP4 signaling controls
RI PT
the transfer of the mammary stem cell state by lipid rafts in extracellular vesicles, Stem Cells 2016; doi: 10.1002/stem.2476. [33]
M. Di Rosa, A. Ialenti, A. Ianaro, L. Sautebin, Interaction between nitric oxide and
[34]
SC
cyclooxygenase pathways, Prostaglandins Leukot Essent Fatty Acids 1996;54:229–238.
D. Salvemini, Cyclooxygenase: an important transduction system for the multifaceted roles
M AN U
of nitric oxide. In: Rubanyi GM, editor. Patho-physiology and clinical applications of nitric oxide, part A. Amsterdam: Harwood Academic, 1999;155–170. [35]
JB. Weinberg, Nitric oxide synthase 2 and cyclooxygenase 2 interactions in inflammation,
Immunol Res 2000;22:319–341.
E. Galea, DL. Feinstein, Regulation of the expression of the inflammatory nitric oxide
TE D
[36]
synthase (NOS2) by cyclic AMP, FASEB J 1999;13:2125–2137. [37]
AV. Timoshenko, PK. Lala, C. Chakraborty, PGE2-mediated upregulation of iNOS in
[38]
EP
murine breast cancer cells through the activation of EP4 receptors, Int J Cancer. 2004;108:384-389. B. Philip, K. Ito, R. Moreno-Sánchez, SJ. Ralph, HIF expression and the role of hypoxic
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microenvironments within primary tumours as protective sites driving cancer stem cell renewal and metastatic progression, Carcinogenesis. 2013;34:1699-1707. [39]
N. Charles, T. Ozawa, M. Squatrito, AM. Bleau, CW. Brennan, et al., Perivascular nitric
oxide activates notch signaling and promotes stem-like character in PDGF-induced glioma cells, Cell Stem Cell. 2010;6:141-152.
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ACCEPTED MANUSCRIPT Figure legends Fig 1. mPGES-1 affects EMT and stemness marker expression in A431 cells. (A) Western blot and quantification (henceforth analysis in legends of Fig 1-10; arbitrary density unit, A.D.U.) of Ecadherin, Vimentin, CD133 and ALDH1 protein expression in A431 WT cells and +mPGES-1
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cells, or in (B) +iNOS cells, and (C) A431 cells treated with PGE-2 and EP4 agonist (1 µM, 24 h,) or (D) with EGF (25 ng/ml,) N=3. ***P<0.001; **P<0.01; *p<0.05 vs. WT cells or control. Fig 2. mPGES-1 and iNOS affect stemness marker expression, invasiveness and colony
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formation in A431 cells. Comparison of tumorsphere forming efficiency (A) and size (B) of WT, mPGES-1 and iNOS overexpressing cells. The data is the mean of triplicate, independent
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experiments ± SD. *** P<0.001, ** P<0.01 vs. WT cells. (C) Representative images of A431 cells showing morphology of spheroids grown on ultra-low attachment plate. Scale bar, 100 µm. (D) Western blot of E-cadherin, CD133, KLF4, Sox2, Oct4, Nanog, c-Myc and SNAIL protein expression in A431 WT, +mPGES-1 and +iNOS cells maintained in basal condition (10% FBS).
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(E) ALDH+ cells were detected in WT, +mPGES-1 and +iNOS cells treated with DEAB (control: left panel) or without DEAB (right panel) after being stained with BAAA, and then analyzed used Guava easyCyte Single Sample Flow Cytometer with the ALDEFLUOR assay kit. The proportion
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of ALDH+ cells was reported as %. Data represent mean of three independent experiments. (F) Fold increase of ALDH+ cells versus DEAB in WT, +mPGES-1 and +iNOS cells. *** P<0.001 vs.
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WT A431 cells. (G) Effect of mPGES-1 and iNOS overexpression on A431 invasion (H) and colony formation induced by 10% FBS. ***P<0.001 vs. control. Data expressed as total cells/well for invasion and as number of cell colonies (> 50 cells)/well means of three duplicate experiments. Fig 3. EGF, mPGES-1/PGE-2 signaling affect stemness marker expression, invasiveness and colony formation in A431 cells. Comparison of tumorsphere forming efficiency (A) and size (B) of A431 cells treated with PGE-2, EP4 agonist (1 µM) or EGF (25 ng/ml). Results are means of triplicate, independent experiments ± SD. *P<0.05, **P<0.01, *** P<0.001 vs. 1% FBS-treated A431 cells. (C) Representative images of A431 cells showing morphology of spheroids grown on 24
ACCEPTED MANUSCRIPT ultra-low attachment plate. Scale bar, 100 µm. (D) and (E) Western blot of E-cadherin, CD133, KLF4, Sox2, Oct4, Nanog, c-Myc and SNAIL protein expression in A431 cells treated as in (A). βactin was used to normalize loading (Figures 2-8). (F) ALDH+ cells were detected in A431 cells untreated (Ctr) or treated with PGE-2, EP4 agonist (1 µM) or EGF (25 ng/ml) and treated with
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DEAB (control: left panel) or without DEAB (right panel) after being stained with BAAA, and then analyzed used Guava easyCyte Single Sample Flow Cytometer with the ALDEFLUOR assay kit. The proportion of ALDH+ cells was reported as %. Data represent mean of three independent
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experiments. (F) Fold increase of ALDH+ cells versus DEAB in A431 cells Ctr, or treated with PGE-2, EP4 agonist (1 µM) or EGF (25 ng/ml). ** P<0.01, * P<0.05 vs. Ctr cells. (G) Effect of
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compounds as in (A) on A431 invasion (H) and colony formation (J) ***P<0.001; **P<0.01 vs. control; ##P<0.01 vs. EGF-treated cells. Data expressed as total cells/well for invasion and as number of cell colonies (> 50 cells)/well means of three duplicate experiments. Fig 4. mPGES-1 and/or iNOS signaling modulation affects EGF-induced SLC phenotype in
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A431 cells. Comparison of tumorsphere forming efficiency (A) and size (B) of scramble, mPGES-1 and iNOS silenced cells treated or otherwise with EGF. Results are means of triplicate independent experiments ± SD. *** P<0.001, ** P<0.01 vs. basal scramble cells; ### P<0.001 vs. EGF-treated
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scramble cells. (C) Representative images of A431 cells showing morphology of spheroids grown on ultra-low attachment plate. Scale bar, 100 µm. (D) Western blot of E-cadherin, CD133, KLF4,
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Sox2, Oct4, Nanog, c-Myc and SNAIL protein expression in A431 scramble, mPGES-1-KD and iNOS-KD cells with/without EGF (25 ng/ml). (E) Transient transfection with mPGES-1 siRNA or Scramble (Scr) on A431 invasion and (F) colony formation induced by 1% FBS or EGF (25 ng/ml). **P<0.01 vs. control; ##P<0.01 vs. EGF-treated cells. Fig 5. iNOS signaling affects mPGES-1-mediated SLC phenotype in cells. Comparison of tumorsphere forming efficiency (A) and size (B) of +mPGES-1 and +mPGES-1/iNOS-KD cells. Results are means of triplicate, independent experiments ± SD. ***P<0.001; ** P<0.01 vs. +mPGES-1 scramble cells. (C) Representative images of A431 cells showing morphology of 25
ACCEPTED MANUSCRIPT spheroids grown on ultra-low attachment plate. Scale bar, 100 µm. (D) Western blot and quantification (A.D.U.) of E-cadherin, vimentin, CD133 and ALDH1 protein expression in A431 +mPGES-1 cells (cultured in monolayer) transiently silenced for iNOS maintained in basal condition (10% FBS, 48 h). N=3. ***P<0.001 vs. scramble. (E) Western blot of E-cadherin,
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CD133, KLF4, Sox2, Oct4, Nanog, c-Myc and SNAIL protein expression in +mPGES-1 and +mPGES-1/iNOS-KD cells (spheres) maintained in basal condition (10% FBS). (F) ALDH+ cells were detected in A431 +mPGES-1 cells scramble or iNOS KD and treated with DEAB (control: left
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panel) or without DEAB (right panel) after being stained with BAAA, and then analyzed used Guava easyCyte Single Sample Flow Cytometer with the ALDEFLUOR assay kit. The proportion
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of ALDH+ cells was reported as %. Data represent mean of three independent experiments. (F) Fold increase of ALDH+ cells versus DEAB A431 +mPGES-1 cells scramble or iNOS KD. ** P<0.01 vs. scramble cells. (H) L-NAME (200 µM) effect on A431 invasion and (J) colony formation in presence of EGF (25 ng/ml), PGE-2 or EP4 agonist (1 µM). ***P<0.001; **P<0.01 vs
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control; ###P<0.001; ##P<0.01; #P<<0.05 vs. PGE-2, EP4 agonist, or EGF-treated cells. Data expressed as total cells/well for invasion and as number of cell colonies (> 50 cells)/well means of three duplicate experiments.
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Fig 6. iNOS modulation does not affect mPGES-1 expression in A431 cells. (A) Western blot analysis and quantification of iNOS and mPGES-1 expression in cells exposed to EGF (25 ng/ml)
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for the indicated times. (B) mPGES-1 expression in A431 cells exposed to EGF with or without LNAME (200 µM) for 8 h. (C) Analysis of mPGES-1 in A431 cells exposed to SNP (10 µM) for 8 h. (D) A.D.U. of iNOS (WT: 0.13 ± 0.05, iNOS: 0.48 ± 0.06 ***) and mPGES-1 (WT: 0.35 ± 0.07, mPGES-1: 0.36 ± 0.05) expression in A431 cells overexpressing iNOS cultured in 10% FBS for 24 h. (E) mPGES-1 expression in A431 cells transiently transfected with iNOS siRNA and treated with EGF for 8 h. N=3. Numbers are means ± SD. *** P <0.001, ** P < 0.01 vs. untreated cells. Fig 7. mPGES-1 modulation affects iNOS expression in cells. (A) A.D.U. of iNOS (Control: 0.14 ± 0.05, Scr: 0.31 ± 0.06, mPGES-1-KD: 0.02 ± 0.02, EGF: 1.05 ± 0.04 ***, EGF + Scr: 0.92 ± 26
ACCEPTED MANUSCRIPT 0.08 ***, EGF + mPGES-1-KD: 0.06 ± 0.01) expression in A431 cells transiently transfected with mPGES-1 siRNA and exposed to EGF for 18 h. (B) A.D.U. of iNOS (WT: 0.2 ± 0.03, +mPGES-1: 1.28 ± 0.02 ***) and mPGES-1 (WT: 0.04 ± 0.01, +mPGES-1: 1.61 ± 0.09) expression in A431 cells overexpressing mPGES-1 cultured in 10% FBS for 24 h. N=3. Numbers are mean ± SD. *** P
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<0.001 vs. untreated cells. (C) cGMP production in cells transiently transfected with mPGES-1 siRNA and treated with EGF (25 ng/ml). Data expressed in (pg/ml)/mg protein. N=3. *** P <0.001 vs. untreated cells; ### P <0.001 vs. EGF-treated cells.
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Fig 8. EP4 receptor increases iNOS protein expression in A431 cells. (A) Western blot of iNOS protein expression in A431 cells treated for 18 h with EP receptor agonist (1 µM), EP1-EP3: 17-
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phenyl trinor prostaglandin e2, EP2: butaprost, EP3: sulprostone, EP4: L-902,688 and EP3-EP4: PGE1 alcohol. N=3. *** P <0.001 vs. untreated cells.
Fig 9. HIF-1α and AP1 expression in mPGES-1 shRNA cells. (A) Western blot and quantification (A.D.U.) of HIF-1α protein expression in A431 Scr or mPGES-1 shRNA cells treated
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with EGF (25 ng/ml) (shRNA Scr cells: Control: 0.19 ± 0.03, EGF: 0.8 ± 0.07***; shRNA mPGES1 cells: Control: 0.09 ± 0.02, EGF: 0.11 ± 0.04) and (B) in A549 Scr or mPGES-1 shRNA cells treated with EGF (25 ng/ml) (shRNA Scr cells: Control: 0.12 ± 0.02, EGF: 1.8 ± 0.08 ***; shRNA
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mPGES-1 cells: Control: 0.11 ± 0.03, EGF: 0.11 ± 0.03). (C) cJUN and p65 protein expression in nuclear and cytosolic extract expression in A431. (D) cJUN expression in A549 Scr or mPGES-1
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shRNA cells treated with EGF. β-actin, β-tubulin and lamin (for cytosol and nuclear extract, respectively) were used to normalize loading. N=3. Fig 10. HIF-1α and AP1 expression induced by exogenous PGE-2. Western blot analysis and quantification (A.D.U.) of HIF-1α and cJUN expression in A431 cells exposed to PGE-2 (1 µM, A and C, respectively) or EP4 agonist L-902,688 (1 µM, B and D, respectively) for the indicated times. β-actin, β-tubulin and lamin (for total, cytosol and nuclear extract, respectively) were used to normalize loading. N=3. *** P <0.001, ** P <0.01 vs. untreated cells.
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ACCEPTED MANUSCRIPT Highlights In epithelial tumor cells iNOS expression is controlled by mPGES-1 overexpression
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EGF/mPGES-1/iNOS signaling is an initial event leading to tumor stem cells activation
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The incipient tumor aggressiveness may be modulated reducing the pivotal PGE-2/NO input
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S1089-8603(16)30198-7
DOI:
10.1016/j.niox.2017.02.010
Reference:
YNIOX 1649
To appear in:
Nitric Oxide
Received Date: 12 October 2016 Revised Date:
21 February 2017
Accepted Date: 27 February 2017
Please cite this article as: E. Terzuoli, F. Finetti, F. Costanza, A. Giachetti, M. Ziche, S. Donnini, Linking of mPGES-1 and iNOS activates stem-like phenotype in EGFR-driven epithelial tumor cells, Nitric Oxide (2017), doi: 10.1016/j.niox.2017.02.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ACCEPTED MANUSCRIPT Linking of mPGES-1 and iNOS activates stem-like phenotype in EGFR-driven epithelial tumor cells
Short title: mPGE-1 and iNOS induce tumor stemness
Sandra Donninia,b*
Department of Life Sciences, University of Siena, 53100 Siena, Italy
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Istituto Toscano Tumori (ITT), 50136 Florence, Italy
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Erika Terzuoli,a Federica Finetti,a Filomena Costanza,a Antonio Giachetti,a Marina Ziche,a,b and
[email protected], [email protected], [email protected], [email protected]
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*Corresponding author: Prof. Sandra Donnini,
Department of Life Sciences, University of Siena, via Aldo Moro, 2, 53100, Siena, Italy Phone: +390577235382
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E-mail: [email protected] and [email protected]
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ACCEPTED MANUSCRIPT Abstract Inflammatory prostaglandin E-2 (PGE-2) favors cancer progression in epithelial tumors characterized by persistent oncogene input. However, its effects on tumor cell stemness are poorly understood at molecular level. Here we describe two epithelial tumor cells A431 and A459,
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originating from human lung and skin tumors, in which epithelial growth factor (EGF) induces sequential up-regulation of mPGES-1 and iNOS enzymes, producing an inflammatory intracellular milieu. We demonstrated that concerted action of EGF, mPGES-1 and iNOS causes sharp changes
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in cell phenotype demonstrated by acquisition of stem-cell features and activation of the epithelialmesenchymal transition (EMT). When primed with EGF, epithelial tumor cells transfected with
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mPGES-1 or iNOS to ensure steady enzyme levels display major stem-like and EMT markers, such as reduction in E-cadherin with a concomitant rise in vimentin, ALDH-1, CD133 and ALDH activity. Tumorsphere studies with these cells show increased sphere number and size, enhanced migratory and clonogenic capacity and sharp changes in EMT markers, indicating activation of this
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process. The concerted action of the enzymes forms a well-orchestrated cascade where expression of iNOS depends on overexpression of mPGES-1. Indeed, we show that through its downstream effectors (PGE-2, PKA, PI3K/Akt), mPGES-1 recruits non-canonical transcription factors, thus
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facilitating iNOS production.
In conclusion, we propose that the initial event leading to tumor stem-cell activation may be a
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leveraged intrinsic mechanism in which all players are either inherent constituents (EGF) or highly inducible proteins (mPGES-1, iNOS) of tumor cells. We suggest that incipient tumor aggressiveness may be moderated by reducing pivotal input of mPGES-1.
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ACCEPTED MANUSCRIPT Keywords mPGES-1, iNOS, epithelial tumor cells, stemness, EGF, tumorspheres
Abbreviations: mPGES-1: microsomal prostaglandin E synthase-1; PGE-2: prostaglandin E-2; EGF: epidermal growth factor; EMT: epithelial to mesenchymal transition; ALDH: aldehyde
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dehydrogenase; PKA: protein kinase A; HIF-1α: hypoxia inducible factor-1α; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; AP1: activator protein 1; EP receptors: PGE2 receptors; SNP: sodium nitroprusside; FBS: fetal bovine serum; PI3K: phosphatidylinositol-3-
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kinase; A.D.U. arbitrary densitometric units.
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ACCEPTED MANUSCRIPT 1. Introduction Malignant tumor cell phenotype appears to be driven by a subpopulation of functional heterogeneous stem-like cells having, remarkable self-renewing capacity and the ability to evolve into progenitor cells with pre-metastatic traits by epithelial-mesenchymal transition (EMT) [1]. The
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heterogeneity and plasticity of stem cells in tumors is a result of cell-intrinsic and extrinsic mechanisms [2-4]. Examples of intrinsic factors inducing tumor cell stemness and EMT are cyclooxygenase-2 and microsomal prostaglandin E synthase type 1 (COX-2 and mPGES-1) and in
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certain cases inducible nitric oxide synthase (iNOS), as well as their end products, prostaglandin E2 (PGE-2) and nitric oxide (NO) [5-8]. These enzymes and signals have been shown to strongly
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favor oncogene-driven tumorigenesis (as by epidermal growth factor, EGF), in epithelial tumor cells and tumor xenograft models [8, 9-13]. Here we sought to determine the role and crosstalk of the tumor intrinsic signals PGE-2 and NO, originating from mPGES-1 and iNOS, in inducing cell stemness and EMT in epithelial tumor cells, characterized by constitutive expression of EGF
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receptor (EGFR). The rationale for selecting the above enzymes and signals is based on evidence that expression of these molecules in certain epithelial tumor types gives them a remarkable growth advantage and enhances their aggressive attributes [14-19].
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We postulated that PGE-2 functions as a regulator of tumor cell stemness mediated by activation of EGF/EGF-receptor (EGFR) signaling and works via activation of the iNOS/NO signaling pathway
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activation. We show that A431 and A549 human epithelial cancer cell lines express elevated levels of mPGES-1/PGE-2 in response to stimulation by EGF. The resulting PGE-2 was found to upregulate iNOS expression through activation of AP1 and HIF-1α. The combined action of PGE-2 and NO increases cell clonogenic potential in vitro and induces formation of three-dimensional tumorspheres expressing markers of stem-like and EMT phenotype. Induction of iNOS by PGE-2 is mediated through the EP4 receptor in a PKA and Akt-dependent manner, as suggested by suppression of iNOS expression and tumor cell capacity to form tumorspheres when mPGES-1 or EP4 are inhibited by shRNA or siRNA or by selective pharmacological inhibitors. 4
ACCEPTED MANUSCRIPT Thus our data suggests that EGFR activation followed by mPGES-1-induced activation of the PGE2/EP4 signaling pathway leads to upregulation of iNOS: a cascade that promotes stem-cell and
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EMT phenotype in epithelial cancer cells.
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ACCEPTED MANUSCRIPT Material and methods 2.1. Cell lines A431 squamous carcinoma cells (passages 10-20, ATCC® CRL-1555™) and A549 lung cancer cells (passages 12-20, ATCC® CRL-1555™ and CCL-185™, respectively) were from the
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American Type Culture Collection (ATCC) and certified by STRA and were cultured as recommended. A549 mPGES-1 knockdown (mPGES-1 shRNA, passages 8-20) and non-target shRNA (Scr, passages 8-20) cells were obtained and cultured as described [20]. For details see
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supplementary material (S1 file). Cells were used at passages 8-20 and discarded after 2 months in culture.
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2.2. Reagents
Reagents were as follows: sodium nitroprusside (SNP), crystal violet, PGE-2, H-89, LY294002, anti-β-actin, anti-lamin, anti-tubulin (Sigma Aldrich); anti-mPGES-1, L-NG-nitroarginine methyl ester (L-NAME), 17-phenyl trinor prostaglandin e2 (EP1-EP3 agonist), butaprost (EP2 agonist),
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sulprostone (EP3 agonist), PGE1 alcohol (EP3-EP4 agonist), L-902, 688 (EP4 agonist), L-161, 982 (EP4 antagonist) (Cayman Chemicals); anti-E-cadherin (Dako), EGF (RELIAtech), anti-iNOS, antip65, anti-ALDH1 and anti-CD133 (Santa Cruz); anti- HIF-1α (BD Transduction Laboratories);
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anti-cJUN, anti-KLF4, anti-Sox-2, anti-Oct4, anti-Nanog, anti-c-Myc, anti-SNAIL (Cell Signaling); Lipofectamine 2000 (Life Technologies).
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2.3. Generation of tumor cells overexpressing iNOS and mPGES1 and lentiviral production Lenti vector plasmids for mPGES-1 (p Lenti vector with C-terminal Myc-DDK tag-NM_004878) and iNOS (p Lenti vector with C-terminal Myc-DDK tag-NM_000625) and vehicle (empty vector construct: pLenti-C-Myc-DDK-puro) were from Origene. psPAX2 packaging plasmid (12260) and pMDG.2 envelope plasmid (12259) were from Addgene. All plasmids were sequence verified. To generate cells overexpressing mPGES-1 and iNOS, 1 x 106 HEK293 cells (Life Technologies) were transfected with 2.25 µg PAX2 packaging plasmid, 0.75 µg PMD2G envelope plasmid and 3 µg pLKO.1 hairpin vector using 12 µl Lipofectamine 2000 on 10 cm plates. Polyclonal 6
ACCEPTED MANUSCRIPT populations of transduced cells were generated by infection with 1 MOI (multiplicity of infectious units) of lentiviral particles. Three days after infection, cells were selected with 20 µg/ml neomycin/kanamycin (Sigma Aldrich) for 1 week. 2.4. Invasion assay
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Cell invasion was investigated using a modified Boyden chamber as described previously [21]. Briefly, 1.2x104 cells were stimulated for 40 min with/ without NOS pathway inhibitor (L-NAME, 200 µM) or PKA (H89, 500 nM), PI3K (LY294002, 10 µM), or EP4 inhibitor (1 µM) in 0.1% FBS.
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Cell suspension was added to the upper wells. PGE-2, EGF and EP4 agonist were used as chemoattractive molecules. Invasion was measured by counting the number of cells that moved across the
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filter coated with Matrigel (250 µg/ml) in 16 h. Cells were counted in five random fields/well at a magnification of 20 X. Data (from triplicate experiments) is expressed as total number of cells migrating/well. 2.5. Clonogenic cell survival assay
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Tumor cells grown to confluence were incubated with EGF, PGE-2 or EP4 agonist with/without LNAME, LY294002 or H89, then plated in 60 mm dishes at a density of 500 cells/dish in medium containing 10% FBS as reported [21], and then kept in a humidified incubator at 37° C and 5% CO2
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for 2 weeks. Colonies (>50 cells) were fixed and stained with 1% crystal violet in 10% methanol and counted and photographed [16]. Results are expressed as number of colonies/well.
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2.6. Immunoblot analysis and nuclear/cytoplasm translocation Total protein lysates were obtained as described [22]. Antibodies were: anti-mPGES-1 (1:200, Cayman Chemicals); anti-E-cadherin (1:1000, Dako), anti-iNOS (1:500), anti ALDH1 and antiCD133 (1:1000, Santa Cruz); anti- HIF-1α (1:300, BD Transduction Laboratories). To assess the translocation of AP1 (evaluated as cJUN) and p 65 from cytosol to nucleus, 8x105 cells were plated in 10 cm diameter dishes, maintained in 10% FBS for 18 h, then treated with PGE2 or EP4 agonist for the indicated times, or with PGE-2 with/without EP4 antagonist or PGE-2 or EP4 agonist in presence/absence of H89 or LY294002, then scraped and homogenized on ice in 7
ACCEPTED MANUSCRIPT lysis buffer containing (in mM) 10 HEPES, 1 DTT, 10 KCl, 50 NaF, 0.1 EDTA, 0.1 EGTA, 1 Na3VO4, 0.5 PMSF and 0.1 NP-40 at 4°C, and centrifuged at 1,000 g for 10 min to separate the nuclei. The supernatant was centrifuged (13,200 g, 5 min) to yield the cytosolic fraction. The nuclear fraction was lysed in buffer containing (in mM) 20 HEPES, 1 EDTA, 1 EGTA and 0.5
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PMSF and analyzed for cJUN (Cell Signaling) and, P65 (Santa Cruz). Western blotting was performed as described [22]. Images were digitalized with CHEMI DOC Quantity One program, blots were analyzed in triplicate by densitometry using NIH Image 1.60B5 software, and arbitrary
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densitometric units (ADU) were normalized for β-actin, β-tubulin or lamin (Sigma). 2.7. EIA assay
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PGE-2 was measured with an EIA kit (Prostaglandin E-2 EIA kit-Monoclonal, Cayman Chemical). Cells were exposed to EGF (25 ng/ml) in presence/absence of L-NAME (200 µM) or SNP (10 µM) for 18 h and treated for 30 min with 30 µM arachidonic acid. Cell culture supernatants were assayed directly at a final dilution of 1:50. The least detectable concentration was 1 to 2 pg/ml. PGE-2
2.8. cGMP assay
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concentration was expressed in pg/ml normalized to total protein concentration.
Cyclic GMP (cGMP) levels were measured in cell extracts from confluent A431 and A549 by
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enzyme-immunoassay kit (Cayman Chemical, Italy). Cells were pre-treated with 1 mM 3-isobutyl5-methyl-xanthine (IBMX) for 30 min then stimulated with 25 ng/ml EGF for 18 h. cGMP levels
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were assayed according to the manufacturer's instructions and protein levels were measured by the Bradford procedure.
2.9. Transient siRNA transfection The day before transfection, cells were trypsinized and 3 x 105 cells were seeded in six-well plates. Transient transfection of siRNA was carried out using Lipofectamin 2000 according to the manufacturer’s instructions. Cells were assayed 48 h after transfection. For siRNA transfection the siRNA sequences targeting: human iNOS (5’-AACCCAGCTGCTGCTCCAAAA-3’), human PKA
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ACCEPTED MANUSCRIPT (5’-AAGCCGGAGAATCTGCT) and human mPGES-1 (5’-CGGGCTAAGAATGCAGACTTT3’) were from Qiagen. Scr siRNA is a random siRNA (Qiagen). 2.10. Tumorsphere formation in vitro This assay tests the ability of single cells to form tumorspheres, the in vitro surrogate of stem-like
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cells [23]. Cells (2 x 105 cells/well in 1.5 ml medium) were distributed in an ultralow attachment six-well plate. All tumorspheres were grown in sphere medium: DME F12 medium (Gibco), supplemented with penicillin/streptomycin, L-glutamine and B27 supplement (1x, Life
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Technologies), bFGF (20 ng/ml, Gibco) and hEGF (20 ng/ml, Gibco), and allowed to grow for 7 to 10 days, or until most spheres reached a diameter of 60 µm. Tumorspheres were counted, harvested,
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and protein extracted [22] or split for second tumorspheres generation [23] and lysed for protein extraction or were reduced to a single cells suspension (with EDTA 10 mM and pipet up and down) for ALDEFLUOR assay.
The tumorsphere formation efficiency (TFE) indicates the percentage of cells within a culture that
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are capable of forming a sphere from a single cell. TFE was determined using the formula: (# of spheres/# of cells plated) x 100. 2.11 ALDEFLUOR assay
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The aldehyde dehydrogenase (ALDH) activity was detected using an ALDEFLUOR assay kit (StemCell Technologies) according to the manufacturer’s protocol. Briefly, the cells were stained
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with bodipy-aminoacetaldehyde (BAAA) and incubated for 45 minutes at 37°C. a specific inhibitor of ALDH, diemethylamino-benzaldehyde (DEAB), was used to control for background fluorescence. The stained cells were analyzed using the Guava easyCyte Single Sample Flow Cytometer (Merk Millipore) and ALDH positive cells (ALDH+) were detected on the green fluorescence channel. The data were analyzed using the InCyte Software program (Millipore). 2.12. Statistical analysis
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ACCEPTED MANUSCRIPT Results are expressed as means ± SD. Statistical analysis was performed using Student's t test, twoway ANOVA, or the Bonferroni post-test for multiple comparisons. P<0.05 was considered
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statistically significant.
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ACCEPTED MANUSCRIPT 2. Results 3.1. PGE-2 and NO induce A431 and A549 tumor cells to express markers of stemness and EMT and to form tumorspheres We examined the ability of mPGES-1 and iNOS expression in A431 and A549 cells to regulate
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stem-cell-like features, by evaluating their capacity to form tumorspheres and express stem-like markers and their proclivity to initiate epithelial to mesenchymal transition (EMT). To do so, we compared A431 and A549 wild type (WT) or transfected with empty vector (vehicle) with cells
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forced to express either mPGES-1 or iNOS by transfection (+mPGES-1 and +iNOS).
Transfection with empty vector did not affect mPGES-1 or iNOS in A431 WT and A549 cells (Fig.
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S1A and B). We found that overexpression of mPGES-1 and iNOS produced a significant increase in expression of stem-like markers such as ALDH1 and CD133 in transfected cells with respect to A431 WT (Fig. 1A and B). Concomitantly, we observed a large increase in vimentin protein expression and decrease in E-cadherin expression, indicating a significant loss of cell-cell adhesion.
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Also in +mPGES-1 A549, we observed a large decrease in E-cadherin and an increase in fibronectin and vimentin, markers of EMT, and an increase in ALDH1 (Fig. S2A). Similar changes in EMT and stem-like markers were reproduced by exposing cells to PGE-2 or
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EGF, an upstream signal to mPGES-1 (Fig. 1C and D). Next, we investigated the ability of tumor cells, forced to express mPGES-1 or iNOS, to form
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tumorspheres under non adherent culture conditions [23]. First, tumorsphere formation efficiency (TFE) for A431 and A549 cancer cell lines was evaluated. The TFE indicates the percentage of cells within a culture that are capable of forming a sphere. A431 and A549 tumor cells were grown in suspension at low density in serum-free sphere medium for 7–10 days. Both cell lines formed tumorspheres (Fig. 2A-C and Fig. S3A-C). A431 cells showed a higher tumorsphere forming efficiency (TFE; means 55% ± SD 2.3 of three triplicate independent experiments) as compared to A549 (TFE; means 13.70 ± SD 5.4 of three duplicate independent experiments) (Fig.2A and Fig.
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ACCEPTED MANUSCRIPT S2A, respectively). A431 WT and vehicle displayed similar TFE (means 55% ± SE 2.3 and 53 % ± SE 1.7 of three triplicate independent experiments) (Fig. S3D). By comparing A431 WT with +mPGES-1 and +iNOS cells, we found that the latter two cell lines displayed a striking increase (~ 3 to 4 fold) in TFE and size of second-generation spheres (means
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182.5.7% ± SE 5.3 and 138 % ± SE 4.7 of three triplicate independent experiments) (Fig 2A, B and C). +mPGES-1 and +iNOS spheres also showed enhanced expression of stem-like markers, such as KLF4, Sox2, Nanog, CD133, c-Myc, Oct 4 and EMT marker SNAIL and ALDH activity, as well as
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lower E-cadherin levels (Fig 2D-F and table S1A and B, for western blot quantification). In the +mPGES-1 and +iNOS cells, the ratio of the ALDH+ cells stained with BAAA was 35.46% and
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39.92% respectively, while that of those stained with BAAA and DEAB as a negative control was 16.1% and 18.6%, respectively (Fig. 2E). In WT cells, the ratio of the ALDH+ cells without DEAB was 14.4% and with DEAB was 13.1% (Fig. 2E). The fold increase of ALDH+ cells versus DEAB was about 2 in +mPGES-1 and +iNOS cells, and 1 in WT cells, indicating a stem-like phenotype in
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+mPGES-1 and +iNOS compared to WT cells.
We also noted a significant increase in cellular invasion and clonogenic potential in +mPGES-1 and +iNOS than in WT cells (Fig. 2G and H). Similar qualitative changes were detected in A431 cells
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exposed to PGE-2, or EGF (Fig. 3A-J and table S2 and table S3 A and B, for western blot quantification, respectively).
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The converse picture was observed in mPGES-1-KD and iNOS-KD cells, as both cell lines displayed a significant decrease in TFE and size of second-generation spheres under basal condition and after treatment with EGF, with respect to WT cells (Fig. 4A-C). In addition, A431 mPGES-1KD and iNOS-KD spheres showed reduced expression of the above stem-like markers and increased E-cadherin expression (Fig 4D and table S4 A and B, for western blot quantification). As well, tumor cell propensity to invade and form colonies under basal condition and in the presence of EGF was greatly reduced in mPGES-1-KD cells (Fig. 4E and F and Fig. S4A and B).
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ACCEPTED MANUSCRIPT 3.2. iNOS/NO signaling controls mPGES-1-induced stemness and EMT We studied whether iNOS silencing influences PGE-2-induced tumor cell stemness and EMT by measuring spheroid formation and stem-like markers in A431 +mPGES-1 cells. In the absence of NO production, these cells displayed a significant decrease in TFE and size of second-generation
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spheres (Fig 5A-C). Moreover, silencing iNOS in +mPGES-1 cells induced reduced expression of canonical stem-like markers and ALDH activity and increased E-cadherin expression compared to +mPGES-1 cells, both in monolayer and three-dimensional (tumorspheres) culture conditions (Fig.
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5D-G and table S5 A and B, for western blot quantification). This indicates that in the absence of iNOS/NO signaling, mPGES-1 is not per se sufficient to sustain the cell phenotype transition.
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Furthermore, cell invasion and clonogenic potential were reduced in cells pretreated with the iNOS inhibitor L-NAME and exposed to PGE-2 or EGF (Fig 5H and J).
These results provide evidence that mPGES-1 and iNOS exert concerted action inducing stemness and EMT in EGF-primed epithelial cancer cells, increasing stem-like and mesenchymal marker
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expression and promoting tumor clonogenic potential and invasiveness. 3.3. Relationship between mPGES-1/PGE-2 and iNOS/NO in epithelial cancer cells In light of the cooperation observed between mPGES-1 and iNOS in promoting tumorspheres
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formation and cell invasion, we investigated the relationship between the two enzymes in epithelial tumor cells exposed to EGF. After exposing A431 cells to EGF (25 ng/ml), we observed a
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significant increase in mPGES-1 levels as early as 4 h, peaking between 8 and 24 h (Fig. 6A), whereas the rise in iNOS was slower (8 h), reaching a maximum at 18 h (Fig. 6A). Blockade of the NO supply either by silencing iNOS or L-NAME application, or conversely increasing NO input by forced overexpression of iNOS (+iNOS), or incubation with the NO donor, sodium nitroprusside (SNP), had no influence on mPGES-1 expression (Fig. 6B-D and F). PGE-2 production was not modified by SNP stimulation, or by L-NAME (Fig. S5A). Similar results were obtained in lung A549 tumor cells (Fig. S5B-D).
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ACCEPTED MANUSCRIPT On the other hand, transient and stable transfection with siRNA or shRNA for mPGES-1, respectively, strongly reduced iNOS expression (Fig. 7A for A431 and Fig.S6A and B for A549 cells), whereas, stable overexpression of mPGES-1 in A431 and A549 cells (+mPGES-1) upregulated iNOS expression (Fig 7B for A431 and Fig.S6C for A549 cells). Moreover, mPGES-1
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knockdown reduced EGF-induced cGMP production in both cell lines (Fig. 7C and Fig. S6D). Collectively, these results indicate that mPGES-1/PGE-2 signaling is an obligate step in promoting EGF-induced iNOS expression.
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Next, we determined which of the multiple receptor subtypes of PGE-2 and its downstream effectors (PKA and PI3K/Akt) induce iNOS expression in A431 cells. As shown in Fig. S7A, the
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EP4 subtype selective agonist was the only one capable of eliciting a large increase in iNOS expression, while selective agonists for another receptors subtype (EP 1-3) were totally ineffective (Fig. 8A). Corroborating evidence for EP4 involvement was obtained in experiments using EP4 antagonist, or inhibitors of its effector signals, i.e. PKA (H89) or PI3K/Akt (LY2942002), or by
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silencing PKA, all of which suppressed agonist-promoted iNOS expression (Fig. S7B-E). The role of the PGE-2/EP4 signaling pathway was also investigated for its contribution to tumor-cell stemness and EMT markers. The EP4 agonist, L-902,688 induced expression of stem-like and EMT
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markers in A431 tumor-cell monolayer, as well as cell invasion and tumor cell colony formation in tumorspheres (Fig. 1 and 3). Inhibition of iNOS reduced EP4 agonist effects (Fig. 5F and G).
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Thus, PGE-2 induces iNOS expression in cancer cells by activating its EP4 receptor subtype and in turn its downstream PKA and Akt signals.
3.4. PGE-2/EP4 upregulates iNOS expression by recruiting transcription factors HIF-1α and AP1 Based on the above results (see Figs 7 and 8) we sought to determine how PGE-2 influences iNOS transcription in cancer cells. Among the wide variety of transcription factors of iNOS, we examined three factors present in the human iNOS promoter [24], i.e. HIF-1α, AP1 and NF-κB. EGF and 14
ACCEPTED MANUSCRIPT PGE-2, as well as EP4 agonist, sharply induced HIF-1α, and promoted AP1 nuclear translocation in both tumor cells (Fig. 9 and 10 A-D and Fig. S8A and B). These effects were abolished by silencing mPGES-1 or by inhibitors of prostanoid receptors and downstream effectors, PKA and Akt, respectively (Fig. 9 and Fig. S7B-E and S8A and B).
was insensitive to silencing mPGES-1 (Fig 9C).
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Although EGF induced a NF-κB component, p65, promoting its nuclear translocation, this effect
Briefly, HIF-1α expression and AP-1 nuclear translocation, appear to be controlled by the PGE-
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2/EP4-Akt and PKA axis, while transcription through NF-κB expression and nuclear translocation are prostanoid- independent. These findings, showing that PGE-2 reveals additional transcription
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factors besides the canonical NF-κB for iNOS, reveal an additional mechanism underlying the
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enhanced iNOS expression observed in epithelial tumor cells (Fig. S9A).
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ACCEPTED MANUSCRIPT 3. Discussion In certain epithelial tumors the strong oncogenic drive exerted by the EGF/EGFR axis promotes upregulation of mPGES-1 and iNOS, two enzymes, which through their end products, PGE-2 and NO, create an inflammatory milieu within tumor cells [13, 25, 26]. This inflammatory condition
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leads to activation of the epithelial-mesenchymal transition (EMT) and to ensuing phenotype changes of cells that acquire distinct stem-like features [27]. In the present study we showed evidence of a well-orchestrated action involving EGF, PGE-2 and NO, fully capable of initiating a
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functional stem-like and mesenchymal transition. An initial event is upregulation of iNOS which appears to depend closely on mPGES-1 and its downstream signals (PGE-2/EP4, PKA, AKT).
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According to the hierarchal order regulating expression of the above enzymes, mPGES-1 is necessary to induce stemness and, iNOS expression seems to complement this effect. This notion is corroborated by observation of incomplete induction of stemness and EMT in experiments involving silencing of iNOS (see Fig 5). Major intracellular changes in stem-like markers were
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detected in tumor cells transfected with mPGES-1 and iNOS. We observed a large reduction in Ecadherin concomitant with a significant rise in ALDH-1 expression and activity and CD133 levels in +mPGES-1 and +iNOS cells. Our studies on tumorsphere formation, an assay of the stemness of
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tumor cells [23], provided compelling evidence that the combined action of mPGES-1 and iNOS elicits stem-like properties and triggers EMT in tumor cells. Indeed, the sharp increase in number
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and size of second-generation spheres, as well as the enhanced invasiveness and colony formation ability reported here, indicate an incipient stem-like state. Moreover, the presence of a mesenchymal cell population, suggested by the pattern of EMT markers, detected in tumorspheres, is indicative of EMT activation. Several studies indicate that in carcinoma cells the EMT can give rise to cells with stem-like properties, suggesting a functional link between the two processes and providing an explanation for how cancer stem cells may arise in tumors and be manipulated therapeutically [28, 29]. At molecular level, mPGES-1 was insensitive to attempts to modify its expression whether by supplying exogenous NO (NO donor) or NO blocking (iNOS silencing, or L16
ACCEPTED MANUSCRIPT NAME), whereas silencing mPGES-1 abolished iNOS expression. Exogenous PGE-2 or its EP4 agonist reproduced all the effects observed in epithelial tumors, causing activation of PKA and PI3K/Akt, while release of endogenous PGE-2 was also detected in tumor cells stimulated by EGF. We observed a fairly fast induction of mPGES-1, followed by a significant rise in cGMP, as well as
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activation of PKA and/or PI3K/Akt, indicative of activation of EP4 [30]. EP4 activity associates with multiple mechanisms involved in cancer progression, including induction of stem-like cell phenotype [30, 31], and in particular, EP4/PI3K/Akt signalling has been reported to promote cancer
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cell stemness in breast cancer [30]. Further, EP4/PI3K/Akt signalling is also involved in maintaining the basal mammary stem cell phenotype, suggesting a key role of this pathway in PGE-
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2-mediated induction of cell stem-like phenotype [32]. Intracellular release of PGE-2 was also involved in inducing nuclear translocation of transcription factors such as HIF-1α and AP1, which were readily blocked by inhibitors of the EP4-Akt and PKA axis. Molecular cross-talk between cell-derived prostaglandins and NO is a well-known phenomenon. Data from the literature
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regarding the effects of PGE-2 on iNOS expression is contradictory. Depending on cell type, PGE-2 has been shown to potentiate or inhibit iNOS expression and activity [33-35]. Like we found here, in the case of PGE-2 stimulation of iNOS expression, the effects are associated with an increase in
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cAMP/PKA signaling [36, 37]. Here we showed that in A431 and A549 cells, Akt activity, upstream of HIF-1α expression and downstream of EP4, is also involved in iNOS expression. HIF-
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1α is involved in maintenance of tumor stemness [38], suggesting that microenvironment-generated -PGE-2 may promotes enhancement of iNOS expression and in turn induction of tumor cell stemness by widening the repertoire of transcription factors available for iNOS expression. The picture emerging from these studies is that a mechanism intrinsic to epithelial tumor cells is fully capable of initiating the cell transition from epithelial to mesenchymal state, and therefore of generating a metastatic cell population endowed with enhanced aggressive/stem-like properties. Although confined to oncogene-driven tumor cells harboring genes inducible by persistent oncogene output (e.g. EGF), this mechanism is distinctly intrinsic since all its players are either 17
ACCEPTED MANUSCRIPT inherent constituents (e.g. EGFR) or highly inducible proteins (mPGES-1, iNOS). The mechanism, a remarkable example of concerted action between its components, is a protein induction cascade characterized by a fast rise in EGF-induced mPGES-1 expression followed by slightly delayed PGE-2/EP4-induced iNOS expression, which appears to be totally dependent on the rise in mPGES-
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1. In this mechanism the prevailing mode of interaction is autocrine, as indicated by the modest levels of PGE-2 released in cells via stimulation of mPGES-1 by EGF. Although, these PGE-2 levels are relatively low, they are nonetheless sufficient to recruit the transcription factors for iNOS,
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which however does not preclude a paracrine-type interaction of gaseous NO with proximal stromal cells [39].
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Nitric oxide and particularly mPGES-1 have been reported to promote stemness and mesenchymal phenotype in a variety of epithelial tumors [5-8]. However, in most of these studies the source of mesenchymal or stemness-inducing mediators was the tumor stroma (a source extrinsic to the tumor), known to produce large quantities of chemokines.
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This study provides evidence that the initial event inducing stemness/EMT may occur within the tumor cell (intrinsic), and may subsequently propagate to the adjoining stroma by a paracrine mechanism to amplify the mesenchymal and/or stemness-inducing response (extrinsic). In
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conclusion, we find that in tumors arising from EGF-oncogene-primed cells, the downstream mPGES-1/PGE-2/EP4 signaling pathway plays a critical role in inducing the iNOS required for
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tumor progression. PGE-2 blockade through mPGES-1 or EP4 inhibitors is a potential therapeutic option for such aggressive cancers.
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ACCEPTED MANUSCRIPT Conflict of interest None Funding: This research was supported by the Associazione Italiana sul Cancro (IG10731, MZ 2010-2013;
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IG15443, MZ, 2015-2017). E.T. was a fellow of the Fondazione Italiana per la Ricerca sul cancro
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(FIRC, 2012-2014).
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ACCEPTED MANUSCRIPT References [1]
G. Driessens, B. Beck, A. Caauwe, BD. Simons, C. Blanpain, Defining the mode of tumour
growth by clonal analysis, Nature. 2012; 488:527-530. [2]
AS. Cleary, TL. Leonard, SA. Gestl, EJ. Gunther, Tumour cell heterogeneity maintained by
P. Greulich, BD. Simons, Dynamic heterogeneity as a strategy of stem cell self-renewal,
Proc Natl Acad Sci U S A. 2016; 113:7509-7514. [4]
K. Eason, A. Sadanandam, Molecular or metabolic reprograming: what triggers tumor
subtypes? Cancer Res. 2016;76:5195-5200.
TH. Chu, HH. Chan, HM. Kuo, LF. Liu, TH. Hu, et al., Celecoxib suppresses hepatoma
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[5]
SC
[3]
RI PT
cooperating subclones in Wnt-driven mammary cancers, Nature. 2014;508:113-117.
stemness and progression by up-regulating PTEN, Oncotarget. 2014;5:1475-1490. [6]
F. Finetti, E. Terzuoli, A. Giachetti, R. Santi, D. Villari, et al., mPGES-1 in prostate cancer
controls stemness and amplifies epidermal growth factor receptor-driven oncogenicity, Endocr
[7]
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Relat Cancer. 2015;22:665-678.
MA. Puglisi, C. Cenciarelli, V. Tesori, M. Cappellari, M., Martini, et al., High nitric oxide
production, secondary to inducible nitric oxide synthase expression, is essential for regulation of the
[8]
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tumour-initiating properties of colon cancer stem cells, J Pathol. 2015;236:479-490. D. Peng, T. Tanikawa, W. Li, L. Zhao, L. Vatan, et al., Myeloid-derived suppressor cells
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endow stem-like qualities to breast cancer cells through IL6/STAT3 and NO/NOTCH cross-talk signaling, Cancer Res. 2016;76:3156-3165. [9]
MD. Castellone, H. Teramoto, BO. Williams, KM. Druey, JS. Gutkind, Prostaglandin E2
promotes colon cancer cell growth through a Gs-axin-beta-catenin signaling axis. Science. 2005;310:1504-1510. [10]
W. Goessling, TE. North, S. Loewer, AM. Lord, S. Lee, et al., Genetic interaction of PGE2
and Wnt signaling regulates developmental specification of stem cells and regeneration, Cell. 2009;136:1136-1147. 20
ACCEPTED MANUSCRIPT [11]
DG. Menter, RL. Schilsky, RN. DuBois, Cyclooxygenase-2 and cancer treatment:
understanding the risk should be worth the reward, Clin Cancer Res. 2010;16:1384-1390. [12]
N. Yongsanguanchai, V. Pongrakhananon, A. Mutirangura, Y. Rojanasakul, P.
Chanvorachote, Nitric oxide induces cancer stem cell-like phenotypes in human lung cancer cells,
[13]
D.
Wang,
RN.
DuBois,
RI PT
Am J Physiol Cell Physiol. 2015;308:C89-100.
The role of prostaglandin
immunosuppression, Trends Mol Med. 2016;22:1-3.
tumor-associated
O. Gallo, E. Masini, L. Morbidelli, A. Franchi, I. Fini-Storchi, et al., Role of nitric oxide in
SC
[14]
e(2) in
angiogenesis and tumor progression in head and neck cancer, J Natl Cancer Inst. 1998;90:587-596. F. Finetti, E. Terzuoli, E. Bocci, I. Coletta, L. Polenzani, et al., Pharmacological inhibition
M AN U
[15]
of microsomal prostaglandin E synthase-1 suppresses epidermal growth factor receptor-mediated tumor growth and angiogenesis, PLoS One. 2012;7:e40576. [16]
S. Donnini, F. Finetti, E. Terzuoli, A. Giachetti, MA. Iñiguez, et al., EGFR signaling
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upregulates expression of microsomal prostaglandin E synthase-1 in cancer cells leading to enhanced tumorigenicity, Oncogene. 2012;31:3457-3466. [17]
D. Wang, RN. DuBois, Urinary PGE-M: a promising cancer biomarker. Cancer Prev Res
[18]
EP
(Phila). 2013;6:507-510.
S. Granados-Principal, Y. Liu, ML. Guevara, E. Blanco, DS. Choi, et al., Inhibition of iNOS
AC C
as a novel effective targeted therapy against triple-negative breast cancer, Breast Cancer Res. 2015;17:25. [19]
E. Della Pietra, F. Simonella, B. Bonavida, LE. Xodo, V. Rapozzi, Repeated sub-optimal
photodynamic treatments with pheophorbide a induce an epithelial mesenchymal transition in prostate cancer cells via nitric oxide, Nitric Oxide. 2015;45:43-53. [20]
H. Hanaka, SC. Pawelzik, JI. Johnsen, M. Rakonjac, K. Terawaki, et al., Microsomal
prostaglandin E synthase 1 determines tumor growth in vivo of prostate and lung cancer cells, Proc Natl Acad Sci U S A. 2009;106:18757-18762. 21
ACCEPTED MANUSCRIPT [21]
S. Donnini, F. Finetti, R. Solito, E. Terzuoli, A. Sacchetti, et al., EP2 prostanoid receptor
promotes squamous cell carcinoma growth through epidermal growth factor receptor transactivation and iNOS and ERK1/2 pathways. FASEB J. 2007;21:2418-2430. [22]
E. Terzuoli, S. Donnini, A. Giachetti, MA. Iñiguez, M. Fresno, et al., Inhibition of hypoxia
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inducible factor-1alpha by dihydroxyphenylethanol, a product from olive oil, blocks microsomal prostaglandin-E synthase-1/vascular endothelial growth factor expression and reduces tumor angiogenesis, Clin Cancer Res. 2010;16:4207-2016.
E. Pastrana, V. Silva-Vargas, and F. Doetsch. Eyes Wide Open: a critical review of sphere-
SC
[23]
formation as an assay for stem cells, Cell Stem Cell 2011; 486-498.
SC. Chu, J. Marks Konczalik, HP. Wu, TC. Banks, and J. Moss, Analysis of the cytokine-
M AN U
[24]
stimulated human inducible nitric oxide synthase (iNOS) gene: characterization of differences between human and mouse iNOS promoters, Biochem. Biophys. Res. Commun 1998; 248: 871– 878.
674. [26]
D. Hanahan, RA. Weinber, Hallmarks of cancer: the next generation, Cell. 2011;144:646-
TE D
[25]
SS. McAllister, RA. Weinberg, The tumour-induced systemic environment as a critical
[27]
EP
regulator of cancer progression and metastasis, Nat Cell Biol. 2014;16:717-727. DR. Pattabiraman, RA. Weinberg, Tackling the cancer stem cells - what challenges do they
[28]
AC C
pose? Nat Rev Drug Discov. 2014;13:497-512. PB. Gupta, CM. Fillmore, G. Jiang, SD. Shapira, K. Tao, et al., Stochastic state transitions
give rise to phenotypic equilibrium in populations of cancer cells, Cell. 2011; 146:633-644. [29]
SA. Mani, W. Guo, MJ. Liao, EN. Eaton, A. Ayyanan, et al., The epithelial-mesenchymal
transition generates cells with properties of stem cells, Cell. 2008; 133:704-715. [30]
M. Majumder, E. Landman, L. Liu, D. Hess, PK. Lala. COX-2 elevates oncogenic miR-
526b in breast cancer by EP4 activation, Mol Cancer Res 2015;13(6):1022-1033.
22
ACCEPTED MANUSCRIPT [31]
M. Majumder, X. Xin, L. Liu, E. Tutunea-Fatan, M. Rodriguez-Torres, et al., COX-2
Induces Breast Cancer Stem Cells via EP4/PI3K/AKT/NOTCH/WNT Axis. Stem Cells 2016;34(9):2290-305. [32]
M-C. Lin, S-Y. Chen, H-M. Tsai, P-L. He, Y-C. Lin et al., PGE-2/EP4 signaling controls
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the transfer of the mammary stem cell state by lipid rafts in extracellular vesicles, Stem Cells 2016; doi: 10.1002/stem.2476. [33]
M. Di Rosa, A. Ialenti, A. Ianaro, L. Sautebin, Interaction between nitric oxide and
[34]
SC
cyclooxygenase pathways, Prostaglandins Leukot Essent Fatty Acids 1996;54:229–238.
D. Salvemini, Cyclooxygenase: an important transduction system for the multifaceted roles
M AN U
of nitric oxide. In: Rubanyi GM, editor. Patho-physiology and clinical applications of nitric oxide, part A. Amsterdam: Harwood Academic, 1999;155–170. [35]
JB. Weinberg, Nitric oxide synthase 2 and cyclooxygenase 2 interactions in inflammation,
Immunol Res 2000;22:319–341.
E. Galea, DL. Feinstein, Regulation of the expression of the inflammatory nitric oxide
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[36]
synthase (NOS2) by cyclic AMP, FASEB J 1999;13:2125–2137. [37]
AV. Timoshenko, PK. Lala, C. Chakraborty, PGE2-mediated upregulation of iNOS in
[38]
EP
murine breast cancer cells through the activation of EP4 receptors, Int J Cancer. 2004;108:384-389. B. Philip, K. Ito, R. Moreno-Sánchez, SJ. Ralph, HIF expression and the role of hypoxic
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microenvironments within primary tumours as protective sites driving cancer stem cell renewal and metastatic progression, Carcinogenesis. 2013;34:1699-1707. [39]
N. Charles, T. Ozawa, M. Squatrito, AM. Bleau, CW. Brennan, et al., Perivascular nitric
oxide activates notch signaling and promotes stem-like character in PDGF-induced glioma cells, Cell Stem Cell. 2010;6:141-152.
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ACCEPTED MANUSCRIPT Figure legends Fig 1. mPGES-1 affects EMT and stemness marker expression in A431 cells. (A) Western blot and quantification (henceforth analysis in legends of Fig 1-10; arbitrary density unit, A.D.U.) of Ecadherin, Vimentin, CD133 and ALDH1 protein expression in A431 WT cells and +mPGES-1
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cells, or in (B) +iNOS cells, and (C) A431 cells treated with PGE-2 and EP4 agonist (1 µM, 24 h,) or (D) with EGF (25 ng/ml,) N=3. ***P<0.001; **P<0.01; *p<0.05 vs. WT cells or control. Fig 2. mPGES-1 and iNOS affect stemness marker expression, invasiveness and colony
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formation in A431 cells. Comparison of tumorsphere forming efficiency (A) and size (B) of WT, mPGES-1 and iNOS overexpressing cells. The data is the mean of triplicate, independent
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experiments ± SD. *** P<0.001, ** P<0.01 vs. WT cells. (C) Representative images of A431 cells showing morphology of spheroids grown on ultra-low attachment plate. Scale bar, 100 µm. (D) Western blot of E-cadherin, CD133, KLF4, Sox2, Oct4, Nanog, c-Myc and SNAIL protein expression in A431 WT, +mPGES-1 and +iNOS cells maintained in basal condition (10% FBS).
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(E) ALDH+ cells were detected in WT, +mPGES-1 and +iNOS cells treated with DEAB (control: left panel) or without DEAB (right panel) after being stained with BAAA, and then analyzed used Guava easyCyte Single Sample Flow Cytometer with the ALDEFLUOR assay kit. The proportion
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of ALDH+ cells was reported as %. Data represent mean of three independent experiments. (F) Fold increase of ALDH+ cells versus DEAB in WT, +mPGES-1 and +iNOS cells. *** P<0.001 vs.
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WT A431 cells. (G) Effect of mPGES-1 and iNOS overexpression on A431 invasion (H) and colony formation induced by 10% FBS. ***P<0.001 vs. control. Data expressed as total cells/well for invasion and as number of cell colonies (> 50 cells)/well means of three duplicate experiments. Fig 3. EGF, mPGES-1/PGE-2 signaling affect stemness marker expression, invasiveness and colony formation in A431 cells. Comparison of tumorsphere forming efficiency (A) and size (B) of A431 cells treated with PGE-2, EP4 agonist (1 µM) or EGF (25 ng/ml). Results are means of triplicate, independent experiments ± SD. *P<0.05, **P<0.01, *** P<0.001 vs. 1% FBS-treated A431 cells. (C) Representative images of A431 cells showing morphology of spheroids grown on 24
ACCEPTED MANUSCRIPT ultra-low attachment plate. Scale bar, 100 µm. (D) and (E) Western blot of E-cadherin, CD133, KLF4, Sox2, Oct4, Nanog, c-Myc and SNAIL protein expression in A431 cells treated as in (A). βactin was used to normalize loading (Figures 2-8). (F) ALDH+ cells were detected in A431 cells untreated (Ctr) or treated with PGE-2, EP4 agonist (1 µM) or EGF (25 ng/ml) and treated with
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DEAB (control: left panel) or without DEAB (right panel) after being stained with BAAA, and then analyzed used Guava easyCyte Single Sample Flow Cytometer with the ALDEFLUOR assay kit. The proportion of ALDH+ cells was reported as %. Data represent mean of three independent
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experiments. (F) Fold increase of ALDH+ cells versus DEAB in A431 cells Ctr, or treated with PGE-2, EP4 agonist (1 µM) or EGF (25 ng/ml). ** P<0.01, * P<0.05 vs. Ctr cells. (G) Effect of
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compounds as in (A) on A431 invasion (H) and colony formation (J) ***P<0.001; **P<0.01 vs. control; ##P<0.01 vs. EGF-treated cells. Data expressed as total cells/well for invasion and as number of cell colonies (> 50 cells)/well means of three duplicate experiments. Fig 4. mPGES-1 and/or iNOS signaling modulation affects EGF-induced SLC phenotype in
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A431 cells. Comparison of tumorsphere forming efficiency (A) and size (B) of scramble, mPGES-1 and iNOS silenced cells treated or otherwise with EGF. Results are means of triplicate independent experiments ± SD. *** P<0.001, ** P<0.01 vs. basal scramble cells; ### P<0.001 vs. EGF-treated
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scramble cells. (C) Representative images of A431 cells showing morphology of spheroids grown on ultra-low attachment plate. Scale bar, 100 µm. (D) Western blot of E-cadherin, CD133, KLF4,
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Sox2, Oct4, Nanog, c-Myc and SNAIL protein expression in A431 scramble, mPGES-1-KD and iNOS-KD cells with/without EGF (25 ng/ml). (E) Transient transfection with mPGES-1 siRNA or Scramble (Scr) on A431 invasion and (F) colony formation induced by 1% FBS or EGF (25 ng/ml). **P<0.01 vs. control; ##P<0.01 vs. EGF-treated cells. Fig 5. iNOS signaling affects mPGES-1-mediated SLC phenotype in cells. Comparison of tumorsphere forming efficiency (A) and size (B) of +mPGES-1 and +mPGES-1/iNOS-KD cells. Results are means of triplicate, independent experiments ± SD. ***P<0.001; ** P<0.01 vs. +mPGES-1 scramble cells. (C) Representative images of A431 cells showing morphology of 25
ACCEPTED MANUSCRIPT spheroids grown on ultra-low attachment plate. Scale bar, 100 µm. (D) Western blot and quantification (A.D.U.) of E-cadherin, vimentin, CD133 and ALDH1 protein expression in A431 +mPGES-1 cells (cultured in monolayer) transiently silenced for iNOS maintained in basal condition (10% FBS, 48 h). N=3. ***P<0.001 vs. scramble. (E) Western blot of E-cadherin,
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CD133, KLF4, Sox2, Oct4, Nanog, c-Myc and SNAIL protein expression in +mPGES-1 and +mPGES-1/iNOS-KD cells (spheres) maintained in basal condition (10% FBS). (F) ALDH+ cells were detected in A431 +mPGES-1 cells scramble or iNOS KD and treated with DEAB (control: left
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panel) or without DEAB (right panel) after being stained with BAAA, and then analyzed used Guava easyCyte Single Sample Flow Cytometer with the ALDEFLUOR assay kit. The proportion
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of ALDH+ cells was reported as %. Data represent mean of three independent experiments. (F) Fold increase of ALDH+ cells versus DEAB A431 +mPGES-1 cells scramble or iNOS KD. ** P<0.01 vs. scramble cells. (H) L-NAME (200 µM) effect on A431 invasion and (J) colony formation in presence of EGF (25 ng/ml), PGE-2 or EP4 agonist (1 µM). ***P<0.001; **P<0.01 vs
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control; ###P<0.001; ##P<0.01; #P<<0.05 vs. PGE-2, EP4 agonist, or EGF-treated cells. Data expressed as total cells/well for invasion and as number of cell colonies (> 50 cells)/well means of three duplicate experiments.
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Fig 6. iNOS modulation does not affect mPGES-1 expression in A431 cells. (A) Western blot analysis and quantification of iNOS and mPGES-1 expression in cells exposed to EGF (25 ng/ml)
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for the indicated times. (B) mPGES-1 expression in A431 cells exposed to EGF with or without LNAME (200 µM) for 8 h. (C) Analysis of mPGES-1 in A431 cells exposed to SNP (10 µM) for 8 h. (D) A.D.U. of iNOS (WT: 0.13 ± 0.05, iNOS: 0.48 ± 0.06 ***) and mPGES-1 (WT: 0.35 ± 0.07, mPGES-1: 0.36 ± 0.05) expression in A431 cells overexpressing iNOS cultured in 10% FBS for 24 h. (E) mPGES-1 expression in A431 cells transiently transfected with iNOS siRNA and treated with EGF for 8 h. N=3. Numbers are means ± SD. *** P <0.001, ** P < 0.01 vs. untreated cells. Fig 7. mPGES-1 modulation affects iNOS expression in cells. (A) A.D.U. of iNOS (Control: 0.14 ± 0.05, Scr: 0.31 ± 0.06, mPGES-1-KD: 0.02 ± 0.02, EGF: 1.05 ± 0.04 ***, EGF + Scr: 0.92 ± 26
ACCEPTED MANUSCRIPT 0.08 ***, EGF + mPGES-1-KD: 0.06 ± 0.01) expression in A431 cells transiently transfected with mPGES-1 siRNA and exposed to EGF for 18 h. (B) A.D.U. of iNOS (WT: 0.2 ± 0.03, +mPGES-1: 1.28 ± 0.02 ***) and mPGES-1 (WT: 0.04 ± 0.01, +mPGES-1: 1.61 ± 0.09) expression in A431 cells overexpressing mPGES-1 cultured in 10% FBS for 24 h. N=3. Numbers are mean ± SD. *** P
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<0.001 vs. untreated cells. (C) cGMP production in cells transiently transfected with mPGES-1 siRNA and treated with EGF (25 ng/ml). Data expressed in (pg/ml)/mg protein. N=3. *** P <0.001 vs. untreated cells; ### P <0.001 vs. EGF-treated cells.
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Fig 8. EP4 receptor increases iNOS protein expression in A431 cells. (A) Western blot of iNOS protein expression in A431 cells treated for 18 h with EP receptor agonist (1 µM), EP1-EP3: 17-
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phenyl trinor prostaglandin e2, EP2: butaprost, EP3: sulprostone, EP4: L-902,688 and EP3-EP4: PGE1 alcohol. N=3. *** P <0.001 vs. untreated cells.
Fig 9. HIF-1α and AP1 expression in mPGES-1 shRNA cells. (A) Western blot and quantification (A.D.U.) of HIF-1α protein expression in A431 Scr or mPGES-1 shRNA cells treated
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with EGF (25 ng/ml) (shRNA Scr cells: Control: 0.19 ± 0.03, EGF: 0.8 ± 0.07***; shRNA mPGES1 cells: Control: 0.09 ± 0.02, EGF: 0.11 ± 0.04) and (B) in A549 Scr or mPGES-1 shRNA cells treated with EGF (25 ng/ml) (shRNA Scr cells: Control: 0.12 ± 0.02, EGF: 1.8 ± 0.08 ***; shRNA
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mPGES-1 cells: Control: 0.11 ± 0.03, EGF: 0.11 ± 0.03). (C) cJUN and p65 protein expression in nuclear and cytosolic extract expression in A431. (D) cJUN expression in A549 Scr or mPGES-1
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shRNA cells treated with EGF. β-actin, β-tubulin and lamin (for cytosol and nuclear extract, respectively) were used to normalize loading. N=3. Fig 10. HIF-1α and AP1 expression induced by exogenous PGE-2. Western blot analysis and quantification (A.D.U.) of HIF-1α and cJUN expression in A431 cells exposed to PGE-2 (1 µM, A and C, respectively) or EP4 agonist L-902,688 (1 µM, B and D, respectively) for the indicated times. β-actin, β-tubulin and lamin (for total, cytosol and nuclear extract, respectively) were used to normalize loading. N=3. *** P <0.001, ** P <0.01 vs. untreated cells.
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ACCEPTED MANUSCRIPT Highlights In epithelial tumor cells iNOS expression is controlled by mPGES-1 overexpression
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EGF/mPGES-1/iNOS signaling is an initial event leading to tumor stem cells activation
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The incipient tumor aggressiveness may be modulated reducing the pivotal PGE-2/NO input
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