Focal Adhesion Kinase Regulates Syndecan-2–Mediated Tumorigenic Activity of HT1080 Fibrosarcoma Cells

The syndecans are a four-member family of transmembrane cell surface heparan sulfate proteoglycans that function as cell surface receptors in the regulation of adhesion-dependent signal transduction during cell growth, adhesion, migration, and differentiation through binding of the extracellular matrix (ECM) and/or soluble ligands (1–3). Conserved sequences in the transmembrane and cytoplasmic domains are a unifying feature within the family, but the extracellular domain sequences are molecule specific, implying that each syndecan has evolved to carry out similar but nonidentical functions. This functional specificity is particularly obvious in terms of tumorigenesis. Syndecan-1 is down-regulated in several carcinoma cells (4–6), and its loss may be an early genetic event contributing to tumor progression (7), suggesting its role as a tumor suppressor. Syndecan-1 may also function as a promoter of carcinogenesis, as it promotes tumorigenesis of the mouse mammary gland (8) and metastasis of mouse lung squamous carcinoma cells (9). Enhanced syndecan-1 expression has been observed in several other carcinomas, including prostate, lung, and breast cancer (10–12), and this up-regulation has been correlated with increased tumor aggressiveness and poor clinical prognosis (13). This dual role of syndecan-1 in tumorigenesis suggests that there is no simple correlation between syndecan expression and its function in carcinogenesis. Instead, more complex molecular interactions are likely to be involved, perhaps reflecting tissue- and tumor stage–specific functions. Indeed, syndecan-1 expression is characteristic of epithelial and neuronal cells (14) and shows distinct patterns during cell type transitions during development and differentiation (15, 16). In contrast, syndecan-2 is mainly expressed in mesenchymal cells (17), where its function seems to be closely related with cell migration (18, 19). We reported recently that syndecan-2 expression is increased in several colon cancer cell lines and that this up-regulation is crucial for the tumorigenic activity of colon cancer cells (20). Reduced syndecan-2 expression has been correlated with reduced tumorigenic activity in colon carcinoma cells (20, 21), whereas increased syndecan-2 expression is necessary and sufficient to induce tumorigenic activity through the regulation of adhesion and proliferation in these cells (20). Thus, the previous studies indicated that that syndecan-2 acts as a key regulator in the malignant progression of colon carcinoma cells (20, 21). Because syndecan-2 expression is tissue specific, and the distinct functions of the various syndecan molecules may be tissue specific, the function of syndecan-2 in sarcoma cells could differ from that in carcinoma cells. However, although syndecan-2 is highly expressed in mesenchymal cells, the functions of syndecan-2 in mesenchymal-derived sarcoma tumors have not been examined previously.

Focal adhesion kinase (FAK) is an intracellular protein tyrosine kinase that plays a pivotal role in integrin-linked signal transduction, such as that associated with cell migration. High levels of FAK have been found in a variety of carcinomas, including head and neck carcinomas, ovarian carcinomas, thyroid carcinomas, and colon carcinomas (22–26), as well as sarcomas of the muscle and glial cells (24, 27, 28). Therefore, it is likely that FAK plays a critical role in the progression of tumor cells to malignancy and/or the pathogenesis of cancer cells. Interestingly, although FAK signaling has been well studied in cells of mesenchymal origin, such as fibroblasts, little is known regarding FAK signaling in sarcoma cells. Here, we report that syndecan-2 is crucial for the tumorigenic activity of sarcoma cells and that this function relies on FAK-mediated signaling.

Materials and antibodies. Monoclonal antibodies (mAb) to phosphotyrosine (4G10) and p130Cas (8G4-E8) were purchased from UBI (Hauppauge, NY), mAb to T-lymphoma invasion and metastasis gene-1 (Tiam-1; G1604) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA), mAb to integrin β₁ (clone 18) was purchased from BD PharMingen/Transduction Laboratories (San Diego, CA), mAb to Rac1 was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY), and mAb to hemagglutinin (HA; 12CA5) was purchased from Roche Applied Science (Indianapolis, IN). mAb to FAK and polyclonal antibodies to phosphorylation site-specific FAK[PY³⁹⁷] and FAK[PY⁸⁶¹] were purchased from BioSource Quality Controlled Biochemicals, Inc. (Morgan Hill, CA), and Effectene was purchased from Qiagen (Hilden, Germany). FITC-conjugated AffiniPure F(ab′)₂ fragment donkey anti-chicken IgY was purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA).

Cell culture and transfection. HT1080 fibrosarcoma cell line was maintained in MEM (Life Technologies, Rockville, MD) supplemented with 10% (v/v) fetal bovine serum (FBS) together with sodium pyruvate (1 mmol/L), penicillin (100 units/mL), and streptomycin (10 μg/mL; Life Technologies) at 37°C in 5% CO₂ in a humidified atmosphere. Transient transfections were carried out using Effectene reagent according to the provided protocol.

Molecular constructs. Wild-type syndecan-2 (S2W) was subcloned into a pcDNA3 vector. Antisense syndecan-2 (S2as) cDNA was constructed as described previously (20). Site-directed mutagenesis of full-length cDNA encoding FAK in the pRC/cytomegalovirus (CMV) vector was done using the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). FAK mutants D395A, D396A, Y397F, and Y861F were inserted into pRC/CMV vector at the NotI/XbaI cloning sites, which generated in-frame fusions of a sequence encoding three HA epitopes (YPYDVPDYA) at the 3′ end of the FAK coding sequences. All mutations were confirmed by sequencing the constructs.

Synthesis of small interfering RNA constructs. Oligonucleotides were designed targeting the human Tiam-1 RNA, containing a 9-bp hairpin loop. Oligonucleotides were annealed and cloned into the BglII/EcoRI sites of pSUPER vector. Sequences of the primers are as follows: siTiam1-1 sense primer, 5′-GATCCCCAGACGGCGAGCTTTAAGAATTCAAGAGATTCTTAAAGCTCGCCGTCTTTTTTGGAAA-3′; siTiam1-1 antisense primer, 5′-AGCTTTTCCAAAAAAGACGGCGCGCTTTAAGAATCTCTTGAATTCTTAAAGCTCGCCGTCTGGG-3′; siTiam1-2 sense primer, 5′-GATCCCCGAACCGAAGCTGTAAAGAATTCAAGAGATTCTTTACAGCTTCGGTTCTTTTTGGAAA-3′; and siTiam1-2 antisense primer, 5′-AGCTTTTCCAAAAAGAACCGAAGCTGTAAAGAATCTCTTGAATTCTTTACAGCTTCGGTTCGGG-3′. Bold characters indicate Tiam-1 mRNA targeting sequences; italics indicate the hairpin loop. HT1080 cells were transfected with each siTiam-1.

RNA extraction and reverse transcription-PCR. Total RNA extracted from cultured cells was used as template for reverse transcriptase reaction. Aliquots of cDNA were amplified using the following primers: human syndecan-2, forward 5′-ACATCTCCCCTTTGCTAACGGC-3′ and backward 5′-TAACTCCATCTCCTTCCCCAGG-3′ and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward 5′-CCACCCATGGCAAATTCCATGGCA-3′ and backward 5′-TCTAGACGGCAGGTCAGGTCCACC-3′. After an initial denaturation at 94°C for 5 minutes, 30 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds (except syndecan-2 at 55°C), and extension at 72°C for 60 seconds were carried out. The reaction products were analyzed in 1.5% agarose gels.

Immunoprecipitation and immunoblotting. The cultures were washed twice with PBS and the cells were lysed in radioimmunoprecipitation assay buffer [RIPA; 50 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, 1% NP40, 10 mmol/L NaF, 2 mmol/L Na₃VO₄] containing a protease inhibitor cocktail (1 μg/mL aprotinin, 1 μg/mL antipain, 5 μg/mL leupeptin, 1 μg/mL pepstatin A, 20 μg/mL phenylmethylsulfonyl fluoride). The lysates were clarified by centrifugation at 13,000 × g for 15 minutes at 4°C, denatured with SDS sample buffer, boiled, and analyzed by SDS-PAGE. For immunoprecipitations, each sample containing 200 to 1,000 μg total protein was incubated with the relevant antibody for 2 hours at 4°C followed by incubation with protein G-Sepharose beads for 1 hour. Immune complexes were collected by centrifugation. The proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Amersham Pharmacia Biotech Inc., Piscataway, NJ) and probed with appropriate antibodies followed by species-specific horseradish peroxidase–conjugated secondary antibodies (Amersham Life Science, Little Chalfont, United Kingdom). The signals were detected by enhanced chemiluminescence (Amersham Life Science).

Cell fractionation experiment. After washing twice with PBS (500 μL/10 cm diameter plate), hypo-osmotic solution [150 μL; 20 mmol/L Tris-HCl (pH 7.5), 2 mmol/L 2-mercaptoethanol, 5 mmol/L EGTA, 2 mmol/L EDTA] containing a protease inhibitor cocktail (1 μg/mL aprotinin, 1 μg/mL antipain, 5 μg/mL leupeptin, 1 μg/mL pepstatin A, 20 μg/mL phenylmethylsulfonyl fluoride) was added to the culture plates. Cells were subsequently scraped off the plates and homogenized on ice. The homogenate was centrifuged at 13,000 × g for 15 minutes at 4°C to prepare the cytosolic fraction. The membrane fraction was collected by solubilizing the remaining pellet in RIPA containing a protease inhibitor cocktail, and RIPA lysates were centrifuged at 15,000 × g for 15 minutes at 4°C. Equal amounts of the cytosol or the membrane fractions were resolved by 6% SDS-PAGE, transferred onto PVDF membranes, and probed with an anti-Tiam-1 antibody.

Invasion and migration assay. Gelatin, fibronectin, or collagen I (10 μg/mL) was added to each well of a Transwell plate (Costar, Corning, NY; 8-μm pore size), and the membranes were allowed to dry at 25°C for 1 hour. The Transwell plates were assembled in a 24-well plate, and the lower chambers were filled with the culture medium containing 0.1% bovine serum albumin and basic fibroblast growth factor (bFGF; 10 μg/mL). Cells (5 × 10⁴) were added to each upper chamber, and the plates were incubated at 37°C in 5% CO₂ for 4 hours. The cells that had migrated to the lower surface of the filters were stained with 0.6% hematoxylin and 0.5% eosin and were counted. For in vitro invasion assays, 24-well Transwell plates (8-μm pore size) were coated with each substrate (10 μg/mL) on the lower side of the membrane and with Matrigel (30 μg/μL) on the upper side.

Glutathione S-transferase-PAK-PBD binding assay. Glutathione S-transferase (GST)-PBD binding assay was done essentially as described previously (29). Briefly, the p21-binding domain of PAK1 (PBD) was expressed in Escherichia coli as a GST-PAK-PBD fusion protein, purified using glutathione-Sepharose beads, and added to cell lysates. Bounding proteins were collected by centrifugation and suspended in SDS sample buffer. Proteins were fractionated by SDS-PAGE and transferred onto PVDF membranes, and the amount of precipitated Rac1 was estimated by Western blotting with an anti-Rac1 antibody.

Flow cytometry. HT1080 cells transfected with 4 μg vector, S2W, or S2as cDNA. Cells were washed with PBS and released trypsin (w/v)/1 mmol/L EDTA followed by the addition of PBS. After pelleting, cells were resuspended in PBS and counted. Cells (1 × 10⁵/mL) were incubated anti-syndecan-2 in 10% FBS in PBS for 1 hour at 4°C followed by PBS containing 0.05% Tween 20, washed thrice, and incubated with FITC-conjugated anti-chicken in 10% FBS in PBS for 30 minutes. Syndecan-2 expressions were analyzed by flow cytometry.

Anchorage-independent growth in soft agarose. Each well of a six-well culture plate was coated with 3 mL bottom agar mixture (MEM/10% FBS/0.5% agar). After the bottom layer had solidified, top agar mixture (2 mL; MEM/10% FBS/0.3% agar) containing HT1080 transfectants (1 × 10⁵ cells) was added to each well, and the cultures were incubated at 37°C in 5% CO₂. Every 5 days, normal growth medium was gently layered over the cultures. Colony formation was monitored daily with a light microscope, and the formation of colonies was scored after 14 days.

Syndecan-2 regulates migration and invasion of HT1080 cells. To examine the tumorigenic activity of syndecan-2 in sarcomas, we investigated the functions of syndecan-2 in HT1080 fibrosarcoma cells. We transfected either S2W or S2as cDNA into HT1080 cells (Fig. 1A) and found that expression of S2W or S2as did not affect adhesion of HT1080 cells to substrates, including poly-l-lysine, fibronectin, laminin, and collagen I (data not shown). Proliferation of HT1080 cells was not affected by expression of S2W or S2as either (data not shown), suggesting that syndecan-2 plays different functional roles in fibrosarcoma and colon carcinoma cells.

We then investigated whether syndecan-2 regulates HT1080 cell migration/invasion. Compared with vector-transfected cells, both cell migration and invasion on various substrate were markedly increased in HT1080 cells transfected with S2W and decreased in S2as-transfected cells (Fig. 1B). Furthermore, addition of recombinant polypeptide corresponding to the extracellular domain of syndecan-2 (1 μg/mL), which has been shown to significantly inhibit syndecan-2 functions (20) but not that of syndecan-4 (1 μg/mL), inhibited the migration and invasion of HT1080 fibrosarcoma cells (Fig. 1C). These results suggest that syndecan-2 likely regulates cancer cell migration and invasion in sarcoma cells.

Syndecan-2 regulates migration/invasion of HT1080 cells in a focal adhesion kinase/phosphatidylinositol 3-kinase–dependent manner. Tyrosine phosphorylation of FAK is one of the key signaling events during cell migration. Consistent with our observation of increased migration activity, HT1080 cells overexpressing S2W showed increased tyrosine phosphorylation of FAK, whereas those expressing S2as showed decreased FAK phosphorylation (Fig. 2A). Expression of S2W resulted in increased autophosphorylation at Tyr³⁹⁷ (Y397; Fig. 2A,, top) and enhanced interaction with phosphatidylinositol 3-kinase (PI3K), another key regulator of cell migration (Fig. 2B). In addition, both phosphorylation of FAK at Tyr⁸⁶¹ (Y861), which regulates cell transformation via an interaction with p130Cas (Fig. 2A,, top), and interaction of FAK with p130Cas were increased in cells transfected with S2W (Fig. 2C). These data indicate that syndecan-2–induced migration activity is intimately associated with activation of FAK-mediated signaling.

Because the ability of FAK to integrate adhesion-mediated signals requires the integrity of Tyr³⁹⁷, a major autophosphorylation site, we replaced Tyr³⁹⁷ with a nonphosphorylatable phenylalanine residue (Y397F) and investigated the effect of this construct cotransfected with S2W (Fig. 3A) on cell migration. As shown in Fig. 3B, cotransfection of S2W and FAK Y397F abolished syndecan-2–mediated migration and invasion of HT1080 cells, implying the importance of FAK phosphorylation at Tyr³⁹⁷. As the phosphorylation of FAK Tyr³⁹⁷ creates a high-affinity binding site for cytoskeletal and/or scaffolding proteins, such as PI3K and Src family kinases (30, 31), we next sought to identify the downstream effector molecules through the use of additional FAK mutants. We substituted Asp³⁹⁵ with Ala (D395A), abolishing FAK binding to PI3K but retaining its interaction with Src, and also substituted Asp³⁹⁶ to Ala (D396A), which abolished FAK binding to both Src and PI3K (32). Both FAK D395A and FAK D396A mutant cDNAs were cotransfected into HT1080 cells along with S2W, and syndecan-2–mediated cell migration was investigated. Both mutants inhibited syndecan-2–mediated migration/invasion to the same degree as seen with mutant Y397F (Fig. 3B), implying that PI3K is involved in syndecan-2-mediated regulation of HT1080 cell migration/invasion. Consistent with this, syndecan-2–induced enhancement of cell migration was dose-dependently inhibited by the PI3K inhibitor LY294002 (Fig. 3C). In contrast, FAK Y861F did not affect syndecan-2–mediated migration activity (Fig. 3B), suggesting that p130Cas might not contribute to syndecan-2–mediated HT1080 cell migration. Taken together, these data show that syndecan-2–mediated cell migration/invasion is regulated in a FAK-PI3K–dependent manner.

Increased expression of syndecan-2 lead to activation of Rac via T-lymphoma invasion and metastasis gene-1. Diverse signaling cascades associated with migration/invasion are dependent on small GTPases (such as Rac), which are essential for regulating the actin assembly/disassembly required for cell movement (33). Activation of Rac in HT1080 cells was determined using a Rac pull-down assay, which uses the PBD of PAK to precipitate GTP-bound Rac from cell lysates. As expected, the activity of Rac was higher in HT1080 cells transfected with S2W (Fig. 4, left) and lower in S2as-transfected cells (Fig. 4 right,) versus cells transfected with empty vector.

Because Rac is activated by the exchange of GDP for GTP by guanine nucleotide exchange factors (GEF) and Tiam-1 is a Rac-specific GEF (34), we investigated whether Tiam-1 mediates the activation of Rac in syndecan-2–transfected cells. Although total levels of Tiam-1 remained unchanged (data not shown), our results revealed that overexpression of S2W enhanced the membrane localization of Tiam-1 (Fig. 5A). This increased membrane localization of Tiam-1 was inhibited when cells were cotransfected with S2W and with the FAK D395A, D396A, or Y397F mutants but not with FAK Y861F (Fig. 5A). Furthermore, addition of LY294002 inhibited the syndecan-2–induced membrane localization of Tiam-1 (Fig. 5B). These data indicate that syndecan-2–stimulated membrane localization of Tiam-1 requires PI3K activity mediated by FAK phosphorylation at Tyr³⁹⁷.

We next investigated whether syndecan-2 requires Tiam-1–induced Rac activation to regulate HT1080 cell migration. Two unique 21-bp small interfering RNA (siRNA) sequences targeted against human Tiam-1 mRNA were cloned and used to knockdown Tiam-1 expression (Fig. 5C). HT1080 cells transfected with Tiam-1 siRNA showed decreased expression of Tiam-1 protein (Fig. 5C,, top) as well as decreased cell migration and invasion activity (Fig. 5C , bottom), showing that Tiam-1 regulates syndecan-2–induced cell migration/invasion in HT1080 cells.

Focal adhesion kinase/phosphatidylinositol 3-kinase–mediated signaling regulates syndecan-2–mediated tumorigenic activity of HT1080 fibrosarcoma cells. Finally, we investigated whether FAK-PI3K–mediated signaling is involved in the syndecan-2–mediated tumorigenic activity of HT1080 cells. Overexpression of S2W, but not S2as, enhanced colony growth on soft agar, whereas the colony-forming ability of cells was dramatically reduced by cotransfection of S2W with the FAK D395A, D396A, or Y397F mutants (Fig. 6). These results imply that syndecan-2 is crucial for the tumorigenic activity of HT1080 cells and that this function is mediated by activation of FAK-PI3K signaling.

We reported previously that elevated expression of syndecan-2 is crucial for the tumorigenic activity in colon carcinoma cells (20). Here, we provide the first evidence for the function of syndecan-2 in fibrosarcoma cells. Consistent with its effects in colon carcinoma cells, overexpression of syndecan-2 increased the tumorigenic activity of HT1080 fibrosarcoma cells based on increased HT1080 cell migration/invasion (Fig. 1) and increased anchorage-independent growth (Fig. 6), whereas expression of a S2as construct decreased these processes. Our results indicate that syndecan-2 plays a critical role as an adhesion receptor during tumorigenesis. However, we observed clear differences in the roles of syndecan-2 in colon carcinoma versus sarcoma cells in terms of regulating cell adhesion and proliferation, which required syndecan-2 expression in colon carcinoma cells (20) but not in HT1080 cells (data not shown). It is not yet known how syndecan-2 regulates tumorigenic activity in sarcoma cells and why this functionality differs, but it seems that, whereas syndecan-2 acts as a general protumorigenic adhesion receptor, more complex molecular regulatory mechanisms may reflect tissue-specific functions.

Cancer cells can acquire motile and invasive phenotypes through alterations in the expression and/or activity of ECM proteins and cell surface receptors. For example, the surfaces of cancer cells often show decreased levels of heparan sulfate proteoglycans associated with increased expression of heparanase (35, 36). Secreted heparanase can cleave heparan sulfate chains, thereby modifying the heparan sulfate proteoglycans and activating heparan-binding molecules, such as fibroblast growth factors (1, 37). Syndecan-2 is expressed in normal mesenchymal cells but not in normal epithelial cells. Thus, one possible explanation for our cancer type–specific finding is that, although HT1080 sarcoma cells already express relatively high amounts of syndecan-2, colon carcinoma cells must acquire syndecan-2 expression during carcinogenesis. As these expressions are due to different regulatory mechanisms, it is possible that the resulting cell surface syndecan-2 may be differently modified in each cancer type. A second possibility is that syndecan-2 is expressed at different levels over different time courses in colon carcinoma versus sarcoma cells. Altered syndecan-2 expression did not affect cell adhesion in fibrosarcoma cells, which had initially high levels of syndecan-2, whereas these changes were more significant in colon carcinoma cells, which start with lower levels of syndecan-2. The third possibility is that the cells may use different molecular machineries for controlling adhesion, motility, or other tumorigenic activities. We herein showed that without affecting expression of integrin α₂, α₅, and β₁ (data not shown), overexpression of syndecan-2 resulted in increased phosphorylation of FAK and PI3K (Fig. 2A and B), which led to further activation of Rac through Tiam-1 activation (Figs. 4 and 5). Thus, syndecan-2 regulates cell migration/invasion of HT1080 fibrosarcoma cells through FAK-PI3K-Rac signaling. However, it remains unknown whether syndecan-2 regulates migration and invasion of colon carcinoma cells via the same pathway. Rac is known to regulate cell migration in fibroblasts but tends to increase adhesion in epithelial cells (38–41). In addition, Tiam-1 increases cellular migration in fibroblasts but increases cellular adhesion in some epithelial cells (39, 40, 42). Thus, our results collectively indicate that syndecan-2 plays multiple roles in regulating cellular functions likely dependent on the cell and cancer types.

In addition to dramatic cytoskeletal changes, tumor cells must regulate changes in cell-ECM adhesion, because cancer cells are less adhesive to the ECM. This decreased adherence is usually associated with decreased deposition of ECM molecules and/or increased secretion of matrix-degrading enzymes. Matrix metalloproteinases (MMP) are a family of proteases that participate in degradation of ECM molecules (43); the expression and activation of these molecules is carefully regulated to prevent uncontrolled destruction of body tissues (43, 44). We reported recently that phosphorylation of FAK Tyr⁸⁶¹ is crucial for H-Ras–induced transformation through changes in the association of FAK with p130Cas, another essential molecule in the regulation of fibroblast migration/invasion (45). This interaction activates Rac and MMP-9 expression, leading to ECM modulation. Similarly, FAK expression has been associated with increased phosphorylation of p130Cas and increased migration in human malignant astrocytic tumor cells (46). Several studies have shown that FAK signaling mediates MMP-9 secretion in carcinoma cells (47–49), but our data revealed that expression of FAK Y861F had no effect on syndecan-2–induced cell migration and invasion of HT1080 cells. In addition, expressions of MMP-2/MMP-9 were not much altered in syndecan-2–transfected HT1080 cells (data not shown). This seems to indicate that syndecan-2–mediated signaling in HT1080 fibrosarcoma cells regulates tumorigenic activities primarily through cytoskeletal reorganization, not MMP expression. Further work will be required to determine whether syndecan-2 regulates MMP expression in carcinoma cells.

In summary, the cell surface heparan sulfate proteoglycan adhesion receptor, syndecan-2, plays a critical role in regulating the tumorigenic activity of HT1080 fibrosarcoma cells via FAK-PI3K. However, the effect of syndecan-2 in HT1080 fibrosarcoma cells differs from that colon carcinoma cells in terms of cell-ECM adhesion and proliferation perhaps due to differing tissue-specific regulatory mechanisms. Further studies will be required to clarify the precise regulatory mechanisms at work in the two cancer cell types.

Grant support: National R&D Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea grant 0420070-1 and Molecular and Cellular BioDiscovery Research Program grant CBM-01B-2-1 (E-S. Oh) and Brain Korea 21 Project fellowship (H. Park).

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