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Sphingosine-1-Phosphate Mediates a Reciprocal Signaling Pathway between Stellate Cells and Cancer Cells that Promotes Pancreatic Cancer Growth

Open AccessPublished:August 08, 2014DOI:https://doi.org/10.1016/j.ajpath.2014.06.023
      Sphingosine-1-phosphate (S1P) is produced by sphingosine kinase 1 and is implicated in tumor growth, although the mechanisms remain incompletely understood. Pancreatic stellate cells (PSCs) reside within the tumor microenvironment and may regulate tumor progression. We hypothesized that S1P activates PSCs to release paracrine factors, which, in turn, increase cancer cell invasion and growth. We used a combination of human tissue, in vitro, and in vivo studies to mechanistically evaluate this concept. Sphingosine kinase 1 was overexpressed in human pancreatic tissue, especially within tumor cells. S1P activated PSCs in vitro and conditioned medium from S1P-stimulated PSCs, increased pancreatic cancer cell migration, and invasion, which was dependent on S1P2, ABL1 (alias c-Abl) kinase, and matrix metalloproteinase-9. In vivo studies showed that pancreatic cancer cells co-implanted with S1P2 receptor knockdown PSCs led to less cancer growth and metastasis in s.c. and orthotopic pancreatic cancer models compared with control PSCs. Pancreatic cancer cell–derived S1P activates PSCs to release paracrine factors, including matrix metalloproteinase-9, which reciprocally promotes tumor cell migration and invasion in vitro and cancer growth in vivo.
      Sphingosine-1-phosphate (S1P) is a lipid-signaling molecule that governs growth, survival, and migration of epithelial cells.
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      • Maceyka M.
      • Milstien S.
      • Spiegel S.
      Targeting the sphingosine-1-phosphate axis in cancer, inflammation and beyond.
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      • Pyne S.
      Sphingosine 1-phosphate and cancer.
      S1P is converted from sphingosine through a reaction that is catalyzed by sphingosine kinase (SK), which exists in two isoforms: SK1 and SK2. S1P is produced intracellularly and then exported out, where it binds its receptors S1P1 and S1P2, which couple to distinct G proteins and signaling pathways.
      • Pyne N.J.
      • Pyne S.
      Sphingosine 1-phosphate and cancer.
      Accumulating evidence indicates that excessive S1P signaling correlates with cancer phenotype, including up-regulation of S1P and/or SK1 in several cancer types
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      • Davenne L.
      • Pchejetski D.
      • Saint-Laurent N.
      • Brizuela L.
      • Guilbeau-Frugier C.
      • Delisle M.B.
      • Cuvillier O.
      • Susini C.
      • Bousquet C.
      Targeting the sphingolipid metabolism to defeat pancreatic cancer cell resistance to the chemotherapeutic gemcitabine drug.
      and correlation of the levels of these molecules with patient prognosis. However, the mechanistic relationship of the S1P pathway with cancer growth remains incompletely developed.
      Additional lines of evidence link S1P signaling with fibrosis. Indeed, S1P induces extracellular matrix synthesis and profibrotic marker gene expression. Mice genetically deficient in S1P2 are resistant to liver fibrosis.
      • Ikeda H.
      • Watanabe N.
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      • Fujita R.
      • Omata M.
      • Chun J.
      • Yatomi Y.
      Sphingosine 1-phosphate regulates regeneration and fibrosis after liver injury via sphingosine 1-phosphate receptor 2.
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      • Gatfield J.
      Sphingosine 1-phosphate (S1P) receptor agonists mediate pro-fibrotic responses in normal human lung fibroblasts via S1P2 and S1P3 receptors and Smad-independent signaling.
      These observations are relevant to pancreatic cancer, in which the tumor microenvironment is characterized by dense fibrotic stroma owing to enhanced matrix production by activated pancreatic stellate cells (PSCs).
      • Apte M.V.
      • Wilson J.S.
      • Lugea A.
      • Pandol S.J.
      A starring role for stellate cells in the pancreatic cancer microenvironment.
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      The role of stroma in pancreatic cancer: diagnostic and therapeutic implications.
      • Omary M.B.
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      • Lowe A.W.
      • Pandol S.J.
      The pancreatic stellate cell: a star on the rise in pancreatic diseases.
      Indeed, the activation phenotype of PSCs, characterized by increased proliferation, migration, synthesis of matrix proteins, and secretion of growth factors and cytokines, is thought to occur in response to tumor cell–derived cues and, in turn, contribute to tumor growth through multiple postulated mechanisms.
      • Apte M.V.
      • Wilson J.S.
      • Lugea A.
      • Pandol S.J.
      A starring role for stellate cells in the pancreatic cancer microenvironment.
      • Pandol S.
      • Gukovskaya A.
      • Edderkaoui M.
      • Dawson D.
      • Eibl G.
      • Lugea A.
      Epidemiology, risk factors, and the promotion of pancreatic cancer: role of the stellate cell.
      • Vonlaufen A.
      • Joshi S.
      • Qu C.
      • Phillips P.A.
      • Xu Z.
      • Parker N.R.
      • Toi C.S.
      • Pirola R.C.
      • Wilson J.S.
      • Goldstein D.
      • Apte M.V.
      Pancreatic stellate cells: partners in crime with pancreatic cancer cells.
      This led us to explore whether S1P may mediate reciprocal interactions between tumor cells and PSCs that could sanction tumor growth.
      Herein, we found that SK1 is up-regulated in pancreatic cancer. S1P activates PSCs to produce matrix metalloproteinase-9 (MMP-9) through an S1P2-, c-Abl–, and NF-κB–dependent pathway. In turn, PSC-derived MMP-9 stimulates pancreatic cancer cell migration and invasion. Both s.c. and orthotopic pancreatic cancer models indicate that this molecular pathway regulates tumor growth in vivo. These studies highlight the importance of S1P signaling as a mechanism that links the tumor with its microenvironment to achieve tumor growth in pancreas.

      Materials and Methods

      Cell Culture and Transfection

      Immortalized mouse PSCs,
      • Mathison A.
      • Liebl A.
      • Bharucha J.
      • Mukhopadhyay D.
      • Lomberk G.
      • Shah V.
      • Urrutia R.
      Pancreatic stellate cell models for transcriptional studies of desmoplasia-associated genes.
      immortalized human PSCs,
      • Paulo J.A.
      • Urrutia R.
      • Kadiyala V.
      • Banks P.
      • Conwell D.L.
      • Steen H.
      Cross-species analysis of nicotine-induced proteomic alterations in pancreatic cells.
      human pancreatic cancer cell lines PANC1,
      • Lieber M.
      • Mazzetta J.
      • Nelson-Rees W.
      • Kaplan M.
      • Todaro G.
      Establishment of a continuous tumor-cell line (panc-1) from a human carcinoma of the exocrine pancreas.
      ASPC1,
      • Chen W.H.
      • Horoszewicz J.S.
      • Leong S.S.
      • Shimano T.
      • Penetrante R.
      • Sanders W.H.
      • Berjian R.
      • Douglass H.O.
      • Martin E.W.
      • Chu T.M.
      Human pancreatic adenocarcinoma: in vitro and in vivo morphology of a new tumor line established from ascites.
      and L3.6 cells
      • Bruns C.J.
      • Harbison M.T.
      • Kuniyasu H.
      • Eue I.
      • Fidler I.J.
      In vivo selection and characterization of metastatic variants from human pancreatic adenocarcinoma by using orthotopic implantation in nude mice.
      were obtained from Dr. Debabrata Mukhopadhyay and Dr. Raul A. Urrutia (Mayo Clinic, Rochester, MN). c-Abl−/−/Arg−/− MEF cells were kindly provided by Dr. Edward B. Leof (Mayo Clinic).
      • Daniels C.
      • Wilkes M.
      • Edens M.
      • Kottom T.
      • Murphy S.
      • Limper A.
      • Leof E.
      Imatinib mesylate inhibits the profibrogenic activity of TGF-beta and prevents bleomycin-mediated lung fibrosis.
      Cells were cultured under standard conditions in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics (penicillin and streptomycin) in a 5% CO2-humidified atmosphere. For transfection experiments, cells were seeded onto 6-well plates, and cultured for 24 hours to be 50% to 60% confluent on the day of transfection. siRNA transfection was performed using Oligofectamine (Life Technologies, Grand Island, NY, USA) according to the manufacturer's protocol. Knockdown efficiency was determined by real-time PCR for S1P1, S1P2, and c-Abl mRNA or by Western blot analysis. Sequences of siRNAs (sense) used in this study are as follows: non-target siRNA, 5′-ATGTGCCTATGACTGTTCGTT-3′; S1P1 siRNA, 5′-AAGCAGAAGAGAATATAGTGC-3′; S1P2 siRNA, 5′-CAGGAACACUACAAUUACA-3′; c-Abl siRNA, 5′-GAAGGGAGGGUGUACCAUU-3′; and MMP-9 siRNA, 5′-TGGACGATGCCTGCAACGTG-3′. Lentiviral vectors encoding S1P2 shRNAs were purchased from Sigma-Aldrich (St. Louis, MO) (NM_003870.2-6211s1c1, NM_003870.2-3950s1c1, and NM_003870.2-569s1c1). Lentiviruses were generated by Vira Power Lentiviral Expression Systems (Life Technologies).

      Boyden Chamber Assay and Transwell Assay

      Migration assays were conducted using a modified Boyden chamber assay with 8-μm porosity polyvinylpyrrolidone-free polycarbonate filters precoated with 10 μg/mL type I collagen. Briefly, 1 × 104 PSCs or PANC1 cells were seeded in 50-μL culture medium in the upper chambers with 50-μL serum-free Dulbecco's modified Eagle's medium containing vehicle or S1P, or conditioned medium, in the lower chambers. After 5 hours of incubation, cells on the upper surface of the inserts were removed using a cotton swab, and those that had migrated through the filter were fixed, permeabilized, and stained with DAPI. Transwell assays were done in 12-well plates with Transwell inserts (Corning, Corning, NY) equipped with 5-μm pores (Corning) coated with 20 μg/mL Matrigel (BD Biosciences, San Jose, CA) at 37°C overnight. Cells (1 to 2 × 105) were seeded in the upper well and serum-free medium with vehicle or S1P or conditioned medium, as indicated, in the lower well. After completion, membranes were removed, wiped on the side facing the upper well, and stained with crystal violet. At least six representative images of each well were taken, and cell numbers were counted using ImageJ software version 1.47 (NIH, Bethesda, MD). The experiments were performed in triplicate.

      RNA Preparation, PCR Array, and qPCR

      Total RNA was extracted using the TRIzol method (Life Technologies) and cleaned with the RNeasy Mini kit (Qiagen, Crawley, UK), according to the manufacturer's instructions. RNA was reverse transcribed with SuperScript II Reverse Transcriptase (Life Technologies) using OligoDT primers. cDNA was quantified using a spectrophotometer (Beckman Coulter, Brea, CA). RT2 Profiler PCR Array (Qiagen) was performed per manufacturer’s instruction. Reverse transcription–quantitative real-time PCR (RT-qPCR) was performed (model 7500 Realtime PCR System; Applied Biosystems, Foster City, CA) to quantify the steady-state concentration of RNA using a Bio-Rad iQ SYBR Green Supermix (Bio-Rad, Hercules, CA). Primers used for this study are as given below. Human primers were as follows: MMP-9, 5′-CGACGTCTTCCAGTACCGA-3′ (forward) and 5′-CTCAGGGCACTGCAGGAT-3′ (reverse); SK1, 5′-AATTTCAAATATTGAACAGCTCGGAA-3′ (forward) and 5′-TTTATAATGTTTGACATGGTCTCCTTT-3′ (reverse); smooth muscle actin (SMA), 5′-GGAGATCACGGCCCTAGCAC-3′ (forward) and 5′-AGGCCCGGCTTCATCGTAT-3′ (reverse); colony-stimulating factor (CSF)1, 5′-GCTGTTGTTGGTCTGTCTC-3′ (forward) and 5′-CATGCTCTTCATAATCCTTG-3′ (reverse); and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-CCAGGGCTGCTTTTAACTCT-3′ (forward) and 5′-GGACTCCACGACGTACTCA-3′ (reverse). Mouse primers were as follows: CSF1r, 5′-CCACCATCCACTTGTATGTCAAAGAT-3′ (forward) and 5′-CTCAACCACTGTCACCTCCTGT-3′ (reverse); and GAPDH, 5′-ACCACAGTCCATGCCATCAC-3′ (forward) and 5′-CACCACCCTGTTGCTGTAGCC-3′ (reverse). The reaction contained 10 ng cDNA and 0.5 μm primers. The relative expression (fold of GAPDH) of each gene was calculated using the ΔCT method.

      Construction of Luciferase Reporter Plasmid and Luciferase Activity Assay

      Wild-type MMP9 promoter luciferase reporter, and MMP9 promoter luciferase reporters with a point mutation in an NF-κB binding site were generous gifts from Dr. Hiroshi Sato (Kanazawa University, Kanazawa, Japan).
      • Sato H.
      • Seiki M.
      Regulatory mechanism of 92 kDa type IV collagenase gene expression which is associated with invasiveness of tumor cells.
      The constructs were cloned into a pWPXLd vector and then transfected 293T cells to generate lentivirus. PSCs were transformed with the lentivirus expressing wild-type MMP9 promoter luciferase reporter or reporters harboring an NF-κB binding site mutation for 72 hours and then split into 96-well plates before the cells were starved and then stimulated with agonists for 6 hours, as indicated. Luciferase activities were measured using a Luciferase Reporter Assay System (Promega, Madison, WI) with a TD-20/20 luminometer (Tuner Designs, Sunnyvale, CA), according to the manufacturer's instructions. Adenovirus expressing NF-κB activity luciferase reporter was described previously.
      • Gaiser S.
      • Daniluk J.
      • Liu Y.
      • Tsou L.
      • Chu J.
      • Lee W.
      • Longnecker D.S.
      • Logsdon C.D.
      • Ji B.
      Intracellular activation of trypsinogen in transgenic mice induces acute but not chronic pancreatitis.

      Western Blot Analysis and Zymography

      Cells were washed with ice-cold phosphate-buffered saline (PBS) and then lysed in radioimmunoprecipitation assay buffer: 10 mmol/L Tris-Cl (pH 8.0), 1 mmol/L EDTA, 0.5 mmol/L EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, and 140 mmol/L NaCl. Protein concentration in lysates was used to normalize loading of gels. Cell extracts and conditioned medium were diluted fourfold in lysis buffer, reduced with 5% β-mercaptoethanol, and then fractionated by SDS-PAGE and analyzed by Western blot analysis. Detection was performed using enhanced chemiluminescence. MMP-9 metalloproteinase activity in conditioned medium was evaluated by zymography, as described.
      • Das A.
      • Yaqoob U.
      • Mehta D.
      • Shah V.H.
      FXR promotes endothelial cell motility through coordinated regulation of FAK and MMP-9.
      Briefly, medium was collected and centrifuged at 270 × g for 3 minutes to remove cellular debris. Polyacrylamide gels (7.5%) containing 2 mg/mL gelatin were subjected to electrophoresis under nonreducing conditions. After electrophoresis, SDS was removed by washing in 2.5% Triton X-100, and gels were incubated at 37°C for 18 hours in 50 mmol/L Tris-HCl, pH 8.0, 50 mmol/L NaCl, 10 mmol/L Ca2Cl, and 0.05% Triton X-100. Gels were then stained in 0.2% Coomassie Brilliant Blue. Gelatinase activity was detected as clear bands on a dark background. Densitometric analysis of bands was performed using ImageJ software.

      Tissue Immunofluorescence and Immunohistochemistry

      Specimens containing pancreatic cancer or normal pancreatic tissue samples were obtained from Mayo Clinic Gastroenterology tissue bank and Mayo Clinic Pancreas Spore. The protocol was approved by the Mayo Clinic Institutional Review Board. Tissue frozen sections were subjected to double immunofluorescence for cytokeratin 19 and SK1. Images were acquired by an LSM 5 Pascal Laser Scanning Microscope (Zeiss, Thornwood, NY). Animal tissues from orthotopic and s.c. pancreatic cancer models were embedded in optimum cutting temperature, and tissue sections were fixed with 4% paraformaldehyde and blocked with 5% bovine serum albumin, incubated with indicated primary antibodies at 4°C overnight and with fluoro-conjugated secondary antibody for 1 hour. TOTO3 was incubated with cells for 30 minutes before the slides were washed and mounted onto a glass slide. Images were acquired and processed using Zeiss LSM image programs. Ten random paraffin sections of liver were immunostained for STEM21 (StemCells, Inc., Newark, CA) to identify tumor micrometastasis. The number of animals with micrometastasis detected by microscopy under a high-power field was recorded for each group, and the percentage of positive animals was reported.

      Subcutaneous and Orthotopic Pancreatic Cancer Models

      Eight-week-old male nude mice were used to generate s.c. and orthotopic pancreatic cancer models, as described earlier.
      • Vonlaufen A.
      • Joshi S.
      • Qu C.
      • Phillips P.A.
      • Xu Z.
      • Parker N.R.
      • Toi C.S.
      • Pirola R.C.
      • Wilson J.S.
      • Goldstein D.
      • Apte M.V.
      Pancreatic stellate cells: partners in crime with pancreatic cancer cells.
      • Liu C.
      • Billadeau D.D.
      • Abdelhakim H.
      • Leof E.
      • Kaibuchi K.
      • Bernabeu C.
      • Bloom G.S.
      • Yang L.
      • Boardman L.
      • Shah V.H.
      • Kang N.
      IQGAP1 suppresses TbetaRII-mediated myofibroblastic activation and metastatic growth in liver.
      Briefly, human pancreatic cancer cells (0.5 × 106 L3.6 or 0.25 × 106 ASPC1) (50 μL in PBS) were mixed with human PSCs (1:1), transformed with non-target control siRNA (NTsh) or specific shRNA targeting S1P2 (S1P2sh) for 72 hours (50 μL in PBS), and were co-injected into the lower flank of nude mice for the s.c. model or pancreas tail for the orthotopic model using a 0.5-mL syringe and a 27-gauge needle (12 mice were in each group). To facilitate the observation of tumor growth in vivo, ASPC1 and L3.6 cells were labeled with luciferase, and PSCs were stably transfected with green fluorescent protein. For the s.c. pancreatic cancer model, tumor diameters were measured by a caliper every 3 days after implantation and tumor volume was calculated by the following formula: tumorvolume=width2×length/2.
      For the orthotopic model, tumor size was monitored in vivo after administration of 150 μL of 15 mg/mL d-luciferin to the mice at days 6 and 12 after tumor implantation and in vivo xenogen imaging was performed using a Xenogen IVIS 200 machine (Caliper Life Sciences, Waltham, MA). Mice were sacrificed 14 or 28 days after co-implantation. Tumors were isolated and weighted. Tissue sections were subjected to RT-qPCR, immunofluorescence (IF), immunohistochemistry, and in situ zymography. Each group consisted of 5 to 10 mice.

      In Situ Zymography

      In situ zymography was performed, as previously described,
      • Galis Z.S.
      • Sukhova G.K.
      • Libby P.
      Microscopic localization of active proteases by in situ zymography: detection of matrix metalloproteinase activity in vascular tissue.
      with modification. Frozen section tissue slides were overlaid with 1 mg/mL DQ collagen (DQ Collagen, type I; Bovine Skin; Life Technologies) in 1% low melting agarose. Slides were kept in 4°C for 30 minutes to allow agarose to solidify and then incubated at 37°C for 1 hour. Under confocal fluorescent examination, collagenolytic/gelatinolytic activity was detected as bright green–appearing zones.

      Statistical Analysis

      Results are expressed as means ± SE. Significance was established using the Student's t-test and analysis of variance, when appropriate. P < 0.05 was considered significant.

      Results

      Human Pancreatic Cancer Cells Overexpress SK1 to Generate S1P, Which, in Turn, Activates PSCs

      Accumulating evidence supports a role for the S1P pathway in cancer; levels of SK1 mRNA and/or protein are increased in tumors of the stomach,
      • Li W.
      • Yu C.P.
      • Xia J.T.
      • Zhang L.
      • Weng G.X.
      • Zheng H.Q.
      • Kong Q.L.
      • Hu L.J.
      • Zeng M.S.
      • Zeng Y.X.
      • Li M.
      • Li J.
      • Song L.B.
      Sphingosine kinase 1 is associated with gastric cancer progression and poor survival of patients.
      lung,
      • Johnson K.R.
      • Johnson K.Y.
      • Crellin H.G.
      • Ogretmen B.
      • Boylan A.M.
      • Harley R.A.
      • Obeid L.M.
      Immunohistochemical distribution of sphingosine kinase 1 in normal and tumor lung tissue.
      brain astrocytoma,
      • Li J.
      • Guan H.Y.
      • Gong L.Y.
      • Song L.B.
      • Zhang N.
      • Wu J.
      • Yuan J.
      • Zheng Y.J.
      • Huang Z.S.
      • Li M.
      Clinical significance of sphingosine kinase-1 expression in human astrocytomas progression and overall patient survival.
      colon,
      • Kawamori T.
      • Kaneshiro T.
      • Okumura M.
      • Maalouf S.
      • Uflacker A.
      • Bielawski J.
      • Hannun Y.A.
      • Obeid L.M.
      Role for sphingosine kinase 1 in colon carcinogenesis.
      non-Hodgkin lymphoma,
      • Bayerl M.G.
      • Bruggeman R.D.
      • Conroy E.J.
      • Hengst J.A.
      • King T.S.
      • Jimenez M.
      • Claxton D.F.
      • Yun J.K.
      Sphingosine kinase 1 protein and mRNA are overexpressed in non-Hodgkin lymphomas and are attractive targets for novel pharmacological interventions.
      and breast.
      • Ruckhaberle E.
      • Rody A.
      • Engels K.
      • Gaetje R.
      • von Minckwitz G.
      • Schiffmann S.
      • Grosch S.
      • Geisslinger G.
      • Holtrich U.
      • Karn T.
      • Kaufmann M.
      Microarray analysis of altered sphingolipid metabolism reveals prognostic significance of sphingosine kinase 1 in breast cancer.
      Furthermore, increased SK1 expression correlated with increased tumor grade and reduced patient survival.
      • Huang W.C.
      • Nagahashi M.
      • Terracina K.P.
      • Takabe K.
      Emerging role of sphingosine-1-phosphate in inflammation, cancer, and lymphangiogenesis.
      However, the links between S1P signaling and pancreatic cancer remain poorly understood.
      • Guillermet-Guibert J.
      • Davenne L.
      • Pchejetski D.
      • Saint-Laurent N.
      • Brizuela L.
      • Guilbeau-Frugier C.
      • Delisle M.B.
      • Cuvillier O.
      • Susini C.
      • Bousquet C.
      Targeting the sphingolipid metabolism to defeat pancreatic cancer cell resistance to the chemotherapeutic gemcitabine drug.
      To identify the cellular source of SK1 from pancreatic cancer, first, we used IF microscopy to show that, although SK1 expression in normal human pancreatic ducts was minimal (Figure 1A), there was high expression of SK1 in cytokeratin 19–positive pancreatic cancer cells (Figure 1A). On the basis of this observation, we examined SK1 mRNA levels in pancreatic cancer tissues. RT-qPCR showed increased SK1 mRNA levels in pancreatic cancer tissue compared with control pancreas (Figure 1B).
      Figure thumbnail gr1
      Figure 1Sphingosine kinase 1 (SK1) is overexpressed in human pancreatic cancer cells. A: Immunofluorescence of cytokeratin 19 (red) and SK1 (green) on normal human pancreas and pancreatic cancer tissue frozen sections. Tumor cells are positive for both cytokeratin and SK1. mRNA was extracted from normal human pancreatic tissue and human pancreatic cancer tissues and then subjected to real-time PCR for SK1. Relative mRNA expression level of human pancreatic cancer tissue was compared with normal pancreas. B: Data shown represent human pancreatic cancer SK1 mRNA fold changes compared with normal pancreas tissue (n = 5). C: Sphingosine 1-phosphate (S1P) induces human PSC Erk/AKT activation. Overnight starved pancreatic stellate cells (PSCs) were treated with vehicle or 0.5 μm S1P for the indicated time before proteins were subjected to Western blot analysis. D: S1P increases actin stress fiber and focal adhesion formation. PSCs were plated on poly-d-lysine–coated coverslips and starved overnight before 0.5 μm S1P treatment for 15 minutes. Actin stress fibers were stained with rhodamine phalloidin (red), and focal adhesions were stained with vinculin conjugated with secondary Alex 488 Fluro antibody (green; Life Technologies). E: S1P increases PSC proliferation. PSCs were plated in 96-well plates and starved overnight before being treated with vehicle or S1P at the indicated concentration. An MTS proliferation assay was performed 48 hours later (n = 4). F: S1P stimulates fibronectin (FN) and collagen 1α mRNA production. Serum-starved PSCs were subjected to vehicle or S1P treatment, and mRNA was extracted for RT-qPCR. Data shown were fold changes compared with vehicle treatment (n = 3). P < 0.05. Ctrl, control.
      Reciprocal interactions between stellate cells and cancer cells are critical for pancreatic cancer progression and metastasis. To examine if pancreatic cancer cell–derived S1P can activate PSCs, we examined effects of S1P on PSCs. Indeed, S1P stimulation increased extracellular signal-regulated kinase and AKT phosphorylation in both human and mouse immortalized PSCs, which peaked at 5 to 10 minutes and persisted for at least 60 minutes (Figure 1C and data not shown). Furthermore, S1P mobilized actin re-organization, stimulated cell proliferation, and promoted profibrotic gene expression, including fibronectin and collagen 1α (Figure 1, D–F). Thus, these data indicate that S1P can activate PSCs.

      S1P-Activated PSCs Stimulate PANC1 Cell Migration and Invasion

      After we showed that pancreatic cancer cells can activate PSCs through effects of S1P, we next explored if S1P-activated PSCs could reciprocally regulate tumor cell behavior and growth using conditioned media experiments. We applied conditioned media collected from S1P-treated PSCs onto the human pancreatic cancer cell line, PANC1, and assessed cancer cell migration and invasion. Conditioned media from S1P-stimulated PSCs significantly increased PANC1 cell migration and invasion (Figure 2A) by 6.7- and 5.8-fold compared with vehicle-treated PSC-conditioned media using Boyden and Transwell assays, respectively. Similarly, conditioned medium from S1P-treated PSCs also stimulated cell migration in another human pancreatic cancer cell line L3.6 by 3.8-fold compared with vehicle-treated conditioned medium (Figure 2B).
      Figure thumbnail gr2
      Figure 2Sphingosine 1-phosphate (S1P)-activated pancreatic stellate cells (PSCs) stimulate PANC1 cell migration and invasion. PSCs were treated with vehicle (bovine serum albumin) or 0.5 μm S1P for 4 hours before being washed, and fresh serum-free medium was added. Conditioned medium was collected 48 hours later. A: Representative micrographs of PANC1 cell migration after 3-hour treatment with vehicle or S1P-treated PSC-conditioned medium using Boyden chamber assay; representative micrographs of PANC1 cell invasion 24 hours after conditioned medium was collected from vehicle-treated PSCs or S1P-treated PSCs using Transwell assay. Quantitative data of Boyden chamber and Transwell assay. B: Representative micrographs of L3.6 cell migration after 3-hour treatment with vehicle or S1P-treated PSC-conditioned medium using Boyden chamber assay. Quantitative data of Boyden chamber. C and D: S1P up-regulates MMP-9 mRNA. mRNA was extracted from human (C) or mouse (D) PSCs, stimulated with vehicle or 0.5 μm S1P for 2 hours and subjected to RT-qPCR for MMP-9. E: S1P up-regulates MMP-9 zymographic activity. PSCs were treated with vehicle or the indicated concentration of S1P for 2 hours. Medium was collected 24 hours later for gelatin zymography. MMP-9 standards were used to identify the correct MMP-9 band. E: Representative image and quantitation of zymography. F: S1P up-regulates MMP9 promoter activity. PSCs were infected with lentivirus expressing wild-type MMP-9 receptor luciferase reporter and then subjected to vehicle or S1P treatment for 6 hours. F: Luciferase activity fold increase. G: S1P does not up-regulate MMP-9 mRNA levels in human pancreatic cancer cell lines PANC1 and ASPC1. PANC1 and ASPC1 were stimulated with vehicle or 0.5 μm S1P for 2 hours, and mRNA was extracted and subjected to RT-qPCR for MMP-9 (n = 3). n = 3 (CE); n = 4 (B and F). P < 0.05, ∗∗P < 0.01. BSA, bovine serum albumin; HPF, high-power field.

      S1P Transcriptionally Activates MMP9 in PSCs

      We next investigated the molecular mechanisms of how S1P-stimulated PSCs affect cancer cells. As an initial step to identify factors secreted from PSCs that may activate the tumor microenvironment and tumor growth, we treated PSCs with S1P and performed a screening with an angiogenesis pathway–specific PCR array. Among 84 factors that were included in the PCR array, 46 were up-regulated by more than twofold (Supplemental Table S1). We further focused on MMP-9, which was increased by 4.2-fold, on the basis of the knowledge that it is critical for cancer cell invasion and metastasis.
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      • Itohara S.
      • Werb Z.
      • Hanahan D.
      Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis.
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      • Hanahan D.
      • Werb Z.
      MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis.
      The array result for MMP-9 was confirmed by RT-qPCR showing S1P up-regulation of MMP-9 mRNA levels by fourfold in human PSCs (Figure 2C) and 3.2-fold in mouse PSCs (Figure 2D). MMP-9 zymographic activity also increased in a dose-dependent manner that peaked at 2.6-fold with 1 μm of S1P (Figure 2E). The lack of further increase of MMP-9 by high S1P concentrations beyond 5 μm is likely due to off-target effects on other processes, such as cell apoptosis and proliferation.
      • Sultan A.
      • Ling B.
      • Zhang H.
      • Ma B.
      • Michel D.
      • Alcorn J.
      • Yang J.
      Synergistic effect between sphingosine-1-phosphate and chemotherapy drugs against human brain-metastasized breast cancer MDA-MB-361 cells.
      In PSCs transduced with a lentivirus construct expressing a luciferase reporter driven by a wild-type MMP9 promoter, S1P increased MMP9 promoter activity by 2.5-fold (Figure 2F). These data indicate that S1P stimulates MMP9 gene expression and active MMP-9 protein production in PSCs. In contrast, S1P failed to induce MMP-9 expression in pancreatic cancer cells, PANC1, and ASPC1 (Figure 2G).

      PSC-Derived MMP-9 Promotes Tumor Cell Migration and Invasion

      Up-regulation of MMP-9 is associated with increased cell migration, invasion, and cancer metastasis. We next examined whether MMP-9 contributes to the enhanced migratory and invasive properties of the PANC1 cells, conferred by S1P-stimulated PSC-conditioned media. Therefore, we pretreated PANC1 cells with an MMP-9 inhibitor before exposing the cells to the conditioned media collected from S1P-treated PSCs. PANC1 cells pretreated with MMP-9 inhibitor, compared with vehicle pretreated PANC1 cells, showed less migration and invasion on stimulation with conditioned media from S1P-treated PSCs (Figure 3, A–C). In addition, conditioned medium from S1P-treated PSCs with MMP-9 knockdown with MMP-9 siRNA also demonstrated decreased PANC1 cell invasion compared with control (Figure 3D). The data suggested that MMP-9 released from PSCs on S1P treatment mediated the stimulatory effect of PSCs on pancreatic cancer cell migration and invasion.
      Figure thumbnail gr3
      Figure 3Matrix metalloproteinase-9 (MMP-9) inhibition blocks PANC1 cell migration and invasion induced by sphingosine 1-phosphate (S1P)–stimulated pancreatic stellate cells (PSCs). A: PANC1 cells were pretreated with MMP-9 inhibitor or vehicle for 30 minutes before conditioned medium from S1P-treated PSCs was applied for Boyden chamber assay or Transwell assay. Quantitative data of Boyden chamber (B) and Transwell (C) assays. PSCs were transfected with NTsi or MMP-9si and then treated with S1P. D: Conditioned medium from S1P-treated PSCs was applied to PANC1 cells for Transwell assay; quantification of Transwell assay. n = 4 (B); n = 3 (C and D). P < 0.05, ∗∗P < 0.01. HPF, high-power field.

      S1P2 Receptor Is Required for S1P-Induced MMP-9 Production in PSCs

      After we confirmed that MMP-9 was up-regulated by S1P and was required for PSC-conditioned media stimulation of tumor cell migration, we next investigated the mechanism of MMP-9 transcriptional regulation by S1P at different levels in PSCs from receptor to nucleus. We first determined which S1P receptor was responsible for MMP-9 production on S1P treatment. We used two distinct siRNAs and/or shRNAs specifically targeting S1P1 or S1P2 gene transcripts as well as pharmacological inhibitors selectively targeting S1P1 or S1P2, respectively. siRNA transfection effectively decreased levels of S1P1 and S1P2 mRNA by 60% to 80%, as assessed by RT-qPCR (Figure 4A). S1P-induced up-regulation of MMP-9 luciferase reporter was markedly inhibited by siRNA targeting S1P2, but not S1P1 (Figure 4A). In addition, S1P-induced MMP-9 luciferase activity was blocked by pretreatment of PSCs with JTE-013, an antagonist of S1P2, but not by W146, an antagonist of S1P1 (Figure 4B). These data indicate that S1P2 receptor mediates S1P-induced MMP-9 up-regulation. To examine if S1P2 on PSCs is responsible for the stimulatory effect of S1P-conditioned media on PANC1 cell migration and invasion, we incubated conditioned media from S1P2-knockdown PSCs (S1P2sh PSCs) and control PSCs (NTsh PSCs) with tumor cells. RT-qPCR showed that S1P2sh decreased the S1P2 level by 60% compared with NTsh (Figure 4C). Conditioned media from S1P2sh PSCs stimulated less tumor cell migration and invasion compared with control PSCs (Figure 4, D–F). These data indicate that S1P2 on PSC mediates the stimulating effect of S1P-treated PSC-conditioned media on tumor cell migration and invasion.
      Figure thumbnail gr4
      Figure 4Sphingosine 1-phosphate (S1P)2 receptor mediates S1P-induced matrix metalloproteinase-9 (MMP-9) production in PSC cells. A: S1P2 knockdown decreases MMP-9 luciferase reporter activity. PSCs expressing wild-type MMP-9 luciferase reporter were transfected with S1P1si, S1P2si, or NTsi for 72 hours, and luciferase assay was performed 6 hours after S1P treatment. RT-qPCR shows that S1P1 and S1P2 are effectively knocked down by respective siRNA. Knockdown of S1P2, but not S1P1, decreases MMP-9 luciferase reporter activity. B: Inhibition of S1P2 decreases MMP-9 luciferase reporter activity. PSCs stably expressing wild-type MMP-9 luciferase reporter activity were pretreated with W146 or JTE for 30 minutes before S1P stimulation for 6 hours. CF: S1P2 receptor is involved in PANC1 cell migration induced by the conditioned media from S1P-stimulated PSCs. PSCs were transfected with lentivirus expressing NTshRNA or S1P2shRNA for 24 hours. Cells were starved overnight and treated with 0.5 μm S1P for 4 hours before being washed, and fresh serum-free medium was added. Conditioned medium was collected 48 hours later and applied on PANC1 cells for Boyden chamber assay and Transwell assay. C: RT-qPCR shows S1P2 is effectively knocked down by S1P2shRNA. D: Representative micrographs of PANC1 cell migration after conditioned medium was collected from S1P-treated NTsh PSCs or S1P-treated S1P2sh PSCs for 3 hours using Boyden chamber assay. Representative micrographs of PANC1 cell invasion in response to conditioned medium collected from S1P-treated NTshPSCs or S1P-treated S1P2sh PSCs using Transwell assay. Quantification data of Boyden chamber (E) and Transwell (F) assays. n = 4 (A and B); n = 3 (C, E, and F). P < 0.05, ∗∗P < 0.01. BSA, bovine serum albumin; HPF, high-power field.

      c-Abl Is Phosphorylated by S1P and Is Critical for MMP-9 Production

      In light of the in vitro and in vivo data showing that S1P2-mediated MMP-9 from PSCs is critical for pancreatic cancer migration, invasion, and growth, we next investigated the mechanisms by which S1P induces MMP-9 production in PSCs. c-Abl is a nonreceptor tyrosine kinase that engages in a variety of intracellular processes, including MMP activities.
      • Greuber E.K.
      • Smith-Pearson P.
      • Wang J.
      • Pendergast A.M.
      Role of ABL family kinases in cancer: from leukaemia to solid tumours.
      We, therefore, hypothesized that c-Abl may be a candidate protein that could mediate S1P stimulation of MMP-9 production in PSCs. We first tested if S1P phosphorylates c-Abl at specific tyrosine residues that regulate its activity.
      • Greuber E.K.
      • Smith-Pearson P.
      • Wang J.
      • Pendergast A.M.
      Role of ABL family kinases in cancer: from leukaemia to solid tumours.
      • Tanis K.Q.
      • Veach D.
      • Duewel H.S.
      • Bornmann W.G.
      • Koleske A.J.
      Two distinct phosphorylation pathways have additive effects on Abl family kinase activation.
      • Colicelli J.
      ABL tyrosine kinases: evolution of function, regulation, and specificity.
      Y245 of c-Abl showed minimal basal phosphorylation, which increased 5 minutes after S1P stimulation (Figure 5, A and B). In addition, S1P increased phosphorylation of CrkII, a c-Abl substrate, at tyrosine 221 (Figure 5, A and C). Phosphorylation at Y89 and Y412 was also increased 5 minutes after S1P stimulation (Figure 5D).
      Figure thumbnail gr5
      Figure 5c-Abl phosphorylation is required for sphingosine 1-phosphate (S1P) activation of matrix metalloproteinase-9 (MMP-9) production. A: S1P stimulates c-Abl phosphorylation at Y245. Human PSCs were treated with vehicle or 0.5 μm S1P or 10 pmol/L platelet-derived growth factor (PDGF) for the indicated time and immunoblotted with Y245 phosphorylation-specific c-Abl, total c-Abl, phospho-Crk, and total Crk. Quantification of the immunoblots of phospho-specific c-Abl (B) and phospho-specific Crk (C). D: S1P stimulates c-Abl phosphorylation at Y87 and Y412. Human PSCs were treated with vehicle or 0.5 μm S1P for 5 minutes and immunoblotted with Y412 or Y89 phosphorylation c-Abl and total c-Abl. E: Zymography of MEF or MEF c-Abl−/−/Arg−/− cells on treatment with S1P for 24 hours. F: Knockdown c-Abl with two different siRNAs blocks MMP-9 luciferase reporter activity induced by S1P. Human PSCs stably expressing MMP-9 luciferase reporter were transfected with NTsi or c-Ablsi for 72 hours and then subjected to S1P or vehicle treatment for 6 hours before luciferase assay was performed. G: NF-κB activation is required for S1P-induced MMP9 promoter activation. MEF or MEF c-Abl−/−/Arg−/− cells were infected with an NF-κB luciferase reporter adenovirus for 16 hours and then subjected to vehicle or S1P treatment for 6 hours before luciferase assay. n = 3 (B and C); n = 4 (F and G). P < 0.05 versus BSA group (B and C) or versus NTsi (F) or versus BSA (G). BSA, bovine serum albumin.
      We next investigated a causative role of c-Abl in S1P-induced MMP-9 production in PSCs. We used a c-Abl−/−/Arg−/− MEF cell line derived from c-Abl and Arg double-knockout mice (due to the functional redundancy between c-Abl and Arg). In contrast to wild-type MEF, the c-Abl−/−/Arg−/− MEF cells failed to show increased MMP-9 activity after S1P stimulation (Figure 5E). In addition, knockdown of c-Abl with two distinct siRNAs diminished S1P-induced MMP9 promoter luciferase reporter activity (Figure 5F). Thus, the data indicate that c-Abl is phosphorylated by S1P and is required for S1P-induced MMP-9 production in PSCs.

      NF-κB Activation Is Required for c-Abl–Mediated S1P-Induced MMP9 Promoter Activation

      To further delineate the mechanism of how c-Abl promotes MMP-9 production, we determined if NF-κB was required for the transcriptional activation of the MMP9 promoter induced by S1P, because prior studies by us and others have shown that functional NF-κB binding sites reside on the MMP9 promoter.
      • Sato H.
      • Kita M.
      • Seiki M.
      v-Src activates the expression of 92-kDa type IV collagenase gene through the AP-1 site and the GT box homologous to retinoblastoma control elements: a mechanism regulating gene expression independent of that by inflammatory cytokines.
      • Das A.
      • Fernandez Zapico M.
      • Cao S.
      • Yao J.
      • Fiorucci S.
      • Hebbel R.
      • Urrutia R.
      • Shah V.
      Disruption of an SP2/KLF6 repression complex by SHP is required for farnesoid X receptor-induced endothelial cell migration.
      To this end, we used two strategies: an adenovirus expressing an NF-κB activity luciferase reporter driven by five copies of an NF-κB response element; and a lentivirus expressing a human MMP-9 reporter luciferase construct with a point mutation in the NF-κB binding site. S1P significantly increased NF-κB luciferase activity by 1.9-fold in human PSCs but failed to increase MMP9 promoter activity in the NF-κB mutant MMP-9 luciferase reporter (data not shown). We further investigated if c-Abl could act as the intermediary to activate NF-κB using the NF-κB activity luciferase reporter. In wild-type MEF cells, 0.1 μm S1P significantly stimulated NF-κB activity by 2.8-fold and 0.5 μm S1P by 4.8-fold (Figure 5G). In contrast to wild-type MEF cells, S1P failed to increase NF-κB activity in the c-Abl−/−/Arg−/− MEF cells (Figure 5G). These data indicate that S1P stimulation of c-Abl can activate NF-κB and thereby lead to MMP-9 production, thus providing mechanisms for the previously mentioned functional and in vivo studies.

      S1P2 Knockdown in PSCs Reduces Pancreatic Tumor Growth in Vivo in s.c. and Orthotopic Pancreatic Cancer Models

      On the basis of these in vitro data, we hypothesized that knockdown of S1P2 in PSCs might modulate tumor cell growth in vivo. Although PANC1 cells were used for in vitro migration studies, these cells are not as aggressive as L3.6 cells for in vivo tumor studies on the basis of data in our model and corroborative reports from the literature.
      • Schmidt R.L.
      • Park C.H.
      • Ahmed A.U.
      • Gundelach J.H.
      • Reed N.R.
      • Cheng S.
      • Knudsen B.E.
      • Tang A.H.
      Inhibition of RAS-mediated transformation and tumorigenesis by targeting the downstream E3 ubiquitin ligase seven in absentia homologue.
      Therefore, L3.6 cells were used for in vivo orthotopic and s.c. pancreatic cancer models. We generated an s.c. pancreatic cancer model by co-implanting 0.5 × 106 L3.6 human pancreatic cancer cells and control NTsh PSCs or S1P2sh PSCs, respectively, in a 1:1 ratio into 8-week-old nude mice. It was previously shown that PSC co-implantation augments tumor cell growth in this model.
      • Xu Z.
      • Vonlaufen A.
      • Phillips P.A.
      • Fiala-Beer E.
      • Zhang X.
      • Yang L.
      • Biankin A.V.
      • Goldstein D.
      • Pirola R.C.
      • Wilson J.S.
      • Apte M.V.
      Role of pancreatic stellate cells in pancreatic cancer metastasis.
      • Schneiderhan W.
      • Diaz F.
      • Fundel M.
      • Zhou S.
      • Siech M.
      • Hasel C.
      • Moller P.
      • Gschwend J.E.
      • Seufferlein T.
      • Gress T.
      • Adler G.
      • Bachem M.G.
      Pancreatic stellate cells are an important source of MMP-2 in human pancreatic cancer and accelerate tumor progression in a murine xenograft model and CAM assay.
      • Hwang R.F.
      • Moore T.
      • Arumugam T.
      • Ramachandran V.
      • Amos K.D.
      • Rivera A.
      • Ji B.
      • Evans D.B.
      • Logsdon C.D.
      Cancer-associated stromal fibroblasts promote pancreatic tumor progression.
      Tumor size was measured by a caliper every 3 days after implantation, and tumor weight was measured at day 14. L3.6 cells alone showed the slowest growth curve and the lowest tumor weight (Figure 6, A and B). Co-implantation of PSCs with L3.6 cells significantly promoted the tumor growth curve and tumor weight (Figure 6, A and B). However, tumor growth kinetics (Figure 6A) from S1P2sh PSCs and L3.6 co-implantation were significantly slower compared with NTsh PSCs and L3.6 co-implantation (Figure 6, A and B), and the final tumor weight was reduced significantly from 145 to 64 mg (Figure 6B). Staining for blood vessel endothelial cells with von Willebrand factor (Figure 6C) or aquaporin (data not shown) showed decreased angiogenesis in the tumor from S1P2sh PSCs and L3.6 co-implantation compared with that from NTsh PSCs and L3.6 co-implantation. Quantification of angiogenesis suggested >65% reduction in the tumor from S1P2sh PSCs and L3.6 co-implantation compared with control (Figure 7C). In addition, knockdown of S1P2 in PSCs was associated with decreased PSC CSF1 mRNA levels (Figure 6D), and the tumors from S1P2sh PSCs and L3.6 co-implantation showed decreased CSF1r mRNA levels (Figure 6D). Moreover, IF with F4/80 for macrophages showed less macrophage infiltration in the tumor from S1P2sh PSCs and L3.6 co-implantation compared with NTsh PSCs and L3.6 co-implantation (Figure 6E). These data indicated potentially important effects of PSCs on other tumor microenvironment cells, including endothelial cells and macrophages. In addition, tumors from L3.6 cells alone had significantly lower MMP-9 mRNA levels compared with those of NTsh PSCs and L3.6 co-implantation. In situ zymography suggested that protease activity was mainly located in the stroma region with colocalization of SMA-positive cells (Figure 6F). These data suggested that stroma cells, rather than the tumor cells, are the main source of MMP activity in tumors in vivo. Tumors from S1P2sh PSCs and L3.6 co-implantation showed decreased MMP-9 mRNA compared with NTsh (Figure 6G), suggesting that S1P2 knockdown in PSCs modulates MMP-9 production in the tumor microenvironment, which may, in turn, regulate cancer growth in vivo.
      Figure thumbnail gr6
      Figure 6Knockdown of sphingosine 1-phosphate (S1P) 2 in pancreatic stellate cells (PSCs) reduces pancreatic tumor growth in the PSC pancreatic cancer s.c. co-implantation model. L3.6 cells (0.5 × 106) were mixed 1:1 with NTsh PSCs or S1P2sh PSCs, respectively, and co-implanted into nude mice via s.c. injection. Tumor nodules were measured by a caliper, and tumor volume was calculated. Tumor weight was measured after sacrifice. A: Tumor growth curves, generated by monitoring mice carefully for 14 days (D), reveal that S1P2sh PSCs and L3.6 co-implantation have slower tumor growth compared with NTsh PSCs and L3.6. B: Representative images of tumors, and average tumor weight. C: Less tumor angiogenesis is observed in tumor from S1P2sh PSC and L3.6 co-implantation compared with control. Left panel: Tumor fresh-frozen sections were subjected to immunofluorescence for von Willebrand factor (VWF; green), SMA (red), or TOTO3 (blue). Right panel: Quantification of tumor angiogenesis. D: RT-qPCR shows decreased CSF1 in S1P2sh PSCs versus NTsh PSCs. RT-qPCR shows that tumors from S1P2sh PSC and L3.6 co-implantation have lower CSF1r compared with NTsh PSC and L3.6. E: Representative micrograph of IF of F4/80 macrophage in tumor derived from co-implantation with NTsh PSCs or S1P2sh PSCs. F: In situ gelatinolytic activity colocalizes with SMA-positive cells. Frozen tissue slides were subjected to in situ zymography with DQ-collagen (green). Tissue sections were subjected to IF for SMA (red) for colocalization analysis. Representative micrograph is shown. G: MMP-9 mRNA is lower in tumors from S1P2sh PSC and L3.6 co-implantation compared with NTsh PSC and L3.6 co-implantation. Ratios of mRNA level to GAPDH are shown. P < 0.05 versus S1P2sh and L3.6 (n = 10; A); P < 0.05 versus NTsh PSC and L3.6 (n = 10; B); P < 0.05 versus NTsh group (n = 10; C); P < 0.05 (n = 6; D); P < 0.05 (n = 10; G).
      Figure thumbnail gr7
      Figure 7S1P2 knockdown in PSCs reduces pancreatic tumor growth and metastasis in an orthotopic pancreatic cancer model. Orthotopic pancreatic cancer model was generated by injecting NTsh or S1P2sh PSCs with luciferase-labeled L3.6 (A and B) or ASPC1 (C and D) cells into the tail of pancreas of nude mice. A and C: Bioluminescence of the cancer cells was quantitated by in vivo xenogene imaging at day 7 after implantation. B and D: Representative photographs of pancreatic cancer from the orthotopic model. Average tumor weight from tumors co-implanted with S1P2sh PSCs is significantly lower compared with that from control PSCs. P < 0.05, versus NTsh PSC + L3.6 (n = 6; B) or versus NTsh PSC + ASPC (n = 6; D).
      Pancreatic cancer is highly metastatic and, therefore, we also sought to determine whether knockdown of S1P2 in PSCs might decrease tumor metastasis in vivo. To this end, we used an orthotopic pancreatic cancer model in which PSCs and cancer cells are directly injected into the tail of the pancreas of nude mice, with subsequent metastasis to the liver. This model also has the added advantage of better recapitulating the pancreatic tumor microenvironment compared with the s.c. model. Given the heterogeneity of pancreatic cancer, we used two different pancreatic cancer cell lines, AsPC1 and L3.6, in the orthotopic model. To facilitate the observation of tumor growth in vivo, ASPC1 and L3.6 cells were labeled with luciferase, and PSCs were stably transfected with green fluorescent protein. In both ASPC1 and L3.6 models, cancer cells co-implanted with S1P2sh PSCs showed slower tumor growth and smaller tumor weight (Figure 7, A–D). One animal in the orthotopic ASPC1 and NTsh PSC co-implantation group developed obstructive jaundice and visible nodules throughout the omentum. Another two animals from the same group had visible nodules on the surface of the liver. None of the animals from the S1P2sh PSC and ASPC1 co-implantation group had jaundice or visible nodules. Furthermore, by using a STEM21 antibody that specifically recognized human cell but not murine cell intracellular protein, we detected higher rates of microscopic metastasis to the liver in the orthotopic model with co-implantation of cancer cells and NTsh PSCs [5 (83.3%) of 6] compared with S1P2sh PSCs [1 (16.7%) of 6].

      Discussion

      Pancreatic cancer remains one of the most devastating solid tumors, despite intense research in this area. Strategies to target the S1P pathway are emerging as a promising approach for cancer therapy for multiple solid tumors. In the current study, we make several observations that expand our understanding of the S1P signaling pathway, with associated therapeutic implications in the context of pancreatic cancer and tumor microenvironment. We identify important paracrine interactions between tumor cells and PSCs, whereby S1P activates PSCs to generate MMP-9, which, in turn, stimulates pancreatic tumor cell migration and invasion. Mechanistically, S1P stimulation of MMP-9 production in PSCs occurs through a pathway dependent on the nonreceptor tyrosine kinase c-Abl. Additional in vivo and human studies provide evidence that the proposed pathway is relevant in an integrative biological context. In total, the data indicate that S1P promotes pancreatic cancer growth through modifying tumor microenvironment, specifically through the interactions of PSCs with cancer cells, and supports the concept that targeting the PSC S1P axis may provide therapeutic benefit for pancreatic cancer.
      Excessive activity of the S1P pathway has been detected in various cancer types, including colon, breast, and lymphoma, and, in turn, efforts to inhibit this pathway have been associated with cancer regression.
      • Kunkel G.T.
      • Maceyka M.
      • Milstien S.
      • Spiegel S.
      Targeting the sphingosine-1-phosphate axis in cancer, inflammation and beyond.
      • Pyne N.J.
      • Pyne S.
      Sphingosine 1-phosphate and cancer.
      For example, the specific monoclonal antibody sphingomab suppresses lung metastasis, and sonepcizumab (LPath, Inc., San Diego, CA), a humanized version of sphingomab, has advanced to phase 2 clinical trials.
      • Ponnusamy S.
      • Selvam S.P.
      • Mehrotra S.
      • Kawamori T.
      • Snider A.J.
      • Obeid L.M.
      • Shao Y.
      • Sabbadini R.
      • Ogretmen B.
      Communication between host organism and cancer cells is transduced by systemic sphingosine kinase 1/sphingosine 1-phosphate signalling to regulate tumour metastasis.
      However, the underlying mechanisms of how S1P promotes tumor growth remain incompletely understood. Although previous studies have shown salutary effects of S1P on cancer cells themselves,
      • Liang J.
      • Nagahashi M.
      • Kim E.Y.
      • Harikumar K.B.
      • Yamada A.
      • Huang W.C.
      • Hait N.C.
      • Allegood J.C.
      • Price M.M.
      • Avni D.
      • Takabe K.
      • Kordula T.
      • Milstien S.
      • Spiegel S.
      Sphingosine-1-phosphate links persistent STAT3 activation, chronic intestinal inflammation, and development of colitis-associated cancer.
      • Thamilselvan V.
      • Li W.
      • Sumpio B.E.
      • Basson M.D.
      Sphingosine-1-phosphate stimulates human Caco-2 intestinal epithelial proliferation via p38 activation and activates ERK by an independent mechanism.
      herein we focused on the role of S1P as a relay switch that links cancer cells with their microenvironment, especially PSCs. These cells transform into tumor-associated myofibroblasts during neoplastic growth, and increasing data indicate that they contribute to tumor growth in important ways.
      • Apte M.V.
      • Wilson J.S.
      • Lugea A.
      • Pandol S.J.
      A starring role for stellate cells in the pancreatic cancer microenvironment.
      • Pandol S.
      • Gukovskaya A.
      • Edderkaoui M.
      • Dawson D.
      • Eibl G.
      • Lugea A.
      Epidemiology, risk factors, and the promotion of pancreatic cancer: role of the stellate cell.
      • Vonlaufen A.
      • Joshi S.
      • Qu C.
      • Phillips P.A.
      • Xu Z.
      • Parker N.R.
      • Toi C.S.
      • Pirola R.C.
      • Wilson J.S.
      • Goldstein D.
      • Apte M.V.
      Pancreatic stellate cells: partners in crime with pancreatic cancer cells.
      Indeed, we found that knockdown of S1P2 from PSCs co-implanted with tumor cells diminishes tumor growth not only through direct effects on tumor cells but also through promoting dormancy of the tumor microenvironment, as evidenced by diminished angiogenesis and macrophage infiltration.
      Pancreatic cancers undergo dynamic remodeling, in part through the MMP family of proteases. MMPs, including MMP-9, promote tumor progression through matrix degradation, stimulation of angiogenesis, and ligation-mediated activation of cancer cell signaling pathways.
      • Bergers G.
      • Brekken R.
      • McMahon G.
      • Vu T.H.
      • Itoh T.
      • Tamaki K.
      • Tanzawa K.
      • Thorpe P.
      • Itohara S.
      • Werb Z.
      • Hanahan D.
      Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis.
      • Coussens L.M.
      • Fingleton B.
      • Matrisian L.M.
      Matrix metalloproteinase inhibitors and cancer: trials and tribulations.
      Indeed, increased expression of MMP-9 has been linked to increased cancer invasiveness in humans and in experimental models.
      • Deryugina E.I.
      • Quigley J.P.
      Matrix metalloproteinases and tumor metastasis.
      • Stetler-Stevenson W.G.
      Dynamics of matrix turnover during pathologic remodeling of the extracellular matrix.
      However, MMP inhibition in human trials has been disappointing.
      • Coussens L.M.
      • Fingleton B.
      • Matrisian L.M.
      Matrix metalloproteinase inhibitors and cancer: trials and tribulations.
      • Overall C.M.
      • Kleifeld O.
      Tumour microenvironment - opinion: validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy.
      • Zucker S.
      • Cao J.
      • Chen W.T.
      Critical appraisal of the use of matrix metalloproteinase inhibitors in cancer treatment.
      Our study suggests that stromal cells are a major source of MMP activity in pancreatic cancer and influence tumor cell biological characteristics. Thus, future clinical trials of specific MMP-9 inhibitors could be modified to focus on stromal cell MMPs and their paracrine effects on tumor cells.
      c-Abl kinase is activated in several tumor types and contributes to tumor cell motility, invasion, and metastasis
      • Sirvent A.
      • Benistant C.
      • Roche S.
      Cytoplasmic signalling by the c-Abl tyrosine kinase in normal and cancer cells.
      ; it has represented a successful therapeutic target for some cancers.
      • Hantschel O.
      • Grebien F.
      • Superti-Furga G.
      The growing arsenal of ATP-competitive and allosteric inhibitors of BCR-ABL.
      • Lo Y.H.
      • Ho P.C.
      • Zhao H.
      • Wang S.C.
      Inhibition of c-ABL sensitizes breast cancer cells to the dual ErbB receptor tyrosine kinase inhibitor lapatinib (GW572016).
      The present study focuses on an alternative and indirect mechanism by which c-Abl in PSC promotes tumor invasion and metastasis by stimulating MMP-9 production that affects tumor cell aggressiveness. Our studies indicate that the mechanism by which c-Abl mediates MMP-9 production is mediated, at least in part, through activation of c-Abl with phosphorylation of c-Abl at Y412, Y245, and Y89 in PSCs. In turn, c-Abl may act through NF-κB to mediate MMP-9 production. Indeed, NF-κB binding sites have been identified on MMP9 promoter and evaluated by us and others in prior studies.
      • Das A.
      • Yaqoob U.
      • Mehta D.
      • Shah V.H.
      FXR promotes endothelial cell motility through coordinated regulation of FAK and MMP-9.
      • Sato H.
      • Kita M.
      • Seiki M.
      v-Src activates the expression of 92-kDa type IV collagenase gene through the AP-1 site and the GT box homologous to retinoblastoma control elements: a mechanism regulating gene expression independent of that by inflammatory cytokines.
      • Van den Steen P.E.
      • Dubois B.
      • Nelissen I.
      • Rudd P.M.
      • Dwek R.A.
      • Opdenakker G.
      Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9).
      Recent studies have also linked c-Abl with NF-κB through a noncanonical mechanism.
      • Kawai H.
      • Nie L.
      • Yuan Z.M.
      Inactivation of NF-kappaB-dependent cell survival, a novel mechanism for the proapoptotic function of c-Abl.
      Although a clinical trial of the c-Abl inhibitor, imatinib, failed to improve pancreatic cancer outcomes,
      • Moss R.A.
      • Moore D.
      • Mulcahy M.F.
      • Nahum K.
      • Saraiya B.
      • Eddy S.
      • Kleber M.
      • Poplin E.A.
      A multi-institutional phase 2 study of imatinib mesylate and gemcitabine for first-line treatment of advanced pancreatic cancer.
      the present studies highlight several potential molecular targets situated upstream and downstream from c-Abl, which may warrant further attention.
      Overall, our study shows that pancreatic cancers overexpress SK1 to produce S1P, which, in turn, acts on PSCs to secrete multiple molecules, including MMP-9, to further promote pancreatic cancer growth as a feed-forward loop. Our data support the concept that targeting the S1P axis may provide therapeutic benefit for pancreatic cancer.

      Acknowledgments

      We thank Drs. Raul Urrutia and Debabrata Mukhopadhyay (Mayo Clinic, Rochester, MN) for PSC lines.
      Y.B. and V.S. developed the concept, designed experiments, and wrote the manuscript; Y.B., J.L., N.K., and B.J. performed animal work; M.K. performed immunofluorescence experiments; L.Y. and D.A.S. helped with data analysis; and S.C. and J.H.K. helped with manuscript writing.

      Supplemental Data

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