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Syndecan-1 enhances proliferation, migration and metastasis of HT-1080 cells in cooperation with syndecan-2.

Péterfia B, Füle T, Baghy K, Szabadkai K, Fullár A, Dobos K, Zong F, Dobra K, Hollósi P, Jeney A, Paku S, Kovalszky I - PLoS ONE (2012)

Bottom Line: These effects were accompanied by a marked increase in syndecan-2 protein expression.The pro-migratory and pro-proliferative effects of truncated syndecan-1 were not observable when syndecan-2 was silenced.Based on our in vitro results, we conclude that the tumour promoter role of syndecan-1 observed in HT-1080 cells is independent of its ectodomain; however, in vivo the presence of the ectodomain further increases tumour proliferation.

View Article: PubMed Central - PubMed

Affiliation: 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary.

ABSTRACT
Syndecans are transmembrane heparan sulphate proteoglycans. Their role in the development of the malignant phenotype is ambiguous and depends upon the particular type of cancer. Nevertheless, syndecans are promising targets in cancer therapy, and it is important to elucidate the mechanisms controlling their various cellular effects. According to earlier studies, both syndecan-1 and syndecan-2 promote malignancy of HT-1080 human fibrosarcoma cells, by increasing the proliferation rate and the metastatic potential and migratory ability, respectively. To better understand their tumour promoter role in this cell line, syndecan expression levels were modulated in HT-1080 cells and the growth rate, chemotaxis and invasion capacity were studied. For in vivo testing, syndecan-1 overexpressing cells were also inoculated into mice. Overexpression of full length or truncated syndecan-1 lacking the entire ectodomain but containing the four juxtamembrane amino acids promoted proliferation and chemotaxis. These effects were accompanied by a marked increase in syndecan-2 protein expression. The pro-migratory and pro-proliferative effects of truncated syndecan-1 were not observable when syndecan-2 was silenced. Antisense silencing of syndecan-2, but not that of syndecan-1, inhibited cell migration. In vivo, both full length and truncated syndecan-1 increased tumour growth and metastatic rate. Based on our in vitro results, we conclude that the tumour promoter role of syndecan-1 observed in HT-1080 cells is independent of its ectodomain; however, in vivo the presence of the ectodomain further increases tumour proliferation. The enhanced migratory ability induced by syndecan-1 overexpression is mediated by syndecan-2. Overexpression of syndecan-1 also leads to activation of IGF1R and increased expression of Ets-1. These changes were not evident when syndecan-2 was overexpressed. These findings suggest the involvement of IGF1R and Ets-1 in the induction of syndecan-2 synthesis and stimulation of proliferation by syndecan-1. This is the first report demonstrating that syndecan-1 enhances malignancy of a mesenchymal tumour cell line, via induction of syndecan-2 expression.

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Effects of FullEGFP and 78Sig on the malignancy of HT-1080 cells in vivo.(A) Size of primary tumours in the footpads of SCID mice on the 24th day after injection of HT-1080 cells expressing EGFP, FullEGFP or 78Sig. Photographs and results of morphometric analysis are shown. *p<0.05 (t-test), #p<0.05 (χ2-test)(n = 5). (B) Histological appearance of lung metastases (arrows) of HT-1080 transfectants. Images do not represent the area percentage of metastases; rather, the only one small EGFP tumour found is shown. HE-stained sections are shown, scale bar: 200 µm. (C) CDK2, phospho-retinoblastoma (at T373 position) and GAPDH immunoblots from HT-1080 primary tumours stably expressing EGFP, FullEGFP or 78Sig. (D) Relative syndecan-2 mRNA levels of the transfectants. Values are expressed as mean±s.d calculated by relative quantification of three independent qRT-PCR results using GAPDH as reference gene and EGFP transfected control as calibrator. *p<0.05 (non-parametric Mann-Whitney test) compared to EGFP control cells. (E) Immunofluorescent staining of methanol-fixed frozen sections by the syndecan-2 specific antibody L-18. Identical exposure times and background corrections were applied Scale bar: 50 µm.
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pone-0039474-g005: Effects of FullEGFP and 78Sig on the malignancy of HT-1080 cells in vivo.(A) Size of primary tumours in the footpads of SCID mice on the 24th day after injection of HT-1080 cells expressing EGFP, FullEGFP or 78Sig. Photographs and results of morphometric analysis are shown. *p<0.05 (t-test), #p<0.05 (χ2-test)(n = 5). (B) Histological appearance of lung metastases (arrows) of HT-1080 transfectants. Images do not represent the area percentage of metastases; rather, the only one small EGFP tumour found is shown. HE-stained sections are shown, scale bar: 200 µm. (C) CDK2, phospho-retinoblastoma (at T373 position) and GAPDH immunoblots from HT-1080 primary tumours stably expressing EGFP, FullEGFP or 78Sig. (D) Relative syndecan-2 mRNA levels of the transfectants. Values are expressed as mean±s.d calculated by relative quantification of three independent qRT-PCR results using GAPDH as reference gene and EGFP transfected control as calibrator. *p<0.05 (non-parametric Mann-Whitney test) compared to EGFP control cells. (E) Immunofluorescent staining of methanol-fixed frozen sections by the syndecan-2 specific antibody L-18. Identical exposure times and background corrections were applied Scale bar: 50 µm.

Mentions: To estimate in vivo malignancy of the stable transfectants, they were injected into foot pads of SCID mice (5 per group). Twenty-four days after injection, the average volume of primary tumours increased as follows: EGFP (35.0±11.7 mm3), FullEGFP (156.3±86.0 mm3), and 78Sig (92.8±44.2 mm3) (Figure 5A). After 1 day, significant differences between the EGFP control and the syndecan-1 transfectant groups were found in the size of primary tumours by t-test (*p<0.05). Moreover, in the FullEGFP group, a significantly higher number of primary tumours reached the size of 150 mm3 than those in the 78Sig group, according to the χ2-test (#p<0.05) indicating that the FullEGFP construct promoted proliferation more effectively (Table 3). While the EGFP transfected group required 34 days to reach 150 mm3 average tumour volume, for 78Sig and FullEGFP it was only 29 and 24 days, respectively. Fifty days after injection, mice were sacrificed and the area fraction of lung metastases (Table 4) was calculated by morphometric analysis of 5 different, random planes of HE-stained sections (Figure 5B). In the EGFP group only one animal developed metastasis in its lung with negligible size. This number was 4 and 3 for FullEGFP and 78Sig groups, respectively (Table 4). The area of lung metastases in animals having tumours in the lung was 19.92±16.60% and 28.76±22.97% for FullEGFP and 78Sig, respectively, significantly more than the 0.02±0.15% of the control EGFP (p<0.05).


Syndecan-1 enhances proliferation, migration and metastasis of HT-1080 cells in cooperation with syndecan-2.

Péterfia B, Füle T, Baghy K, Szabadkai K, Fullár A, Dobos K, Zong F, Dobra K, Hollósi P, Jeney A, Paku S, Kovalszky I - PLoS ONE (2012)

Effects of FullEGFP and 78Sig on the malignancy of HT-1080 cells in vivo.(A) Size of primary tumours in the footpads of SCID mice on the 24th day after injection of HT-1080 cells expressing EGFP, FullEGFP or 78Sig. Photographs and results of morphometric analysis are shown. *p<0.05 (t-test), #p<0.05 (χ2-test)(n = 5). (B) Histological appearance of lung metastases (arrows) of HT-1080 transfectants. Images do not represent the area percentage of metastases; rather, the only one small EGFP tumour found is shown. HE-stained sections are shown, scale bar: 200 µm. (C) CDK2, phospho-retinoblastoma (at T373 position) and GAPDH immunoblots from HT-1080 primary tumours stably expressing EGFP, FullEGFP or 78Sig. (D) Relative syndecan-2 mRNA levels of the transfectants. Values are expressed as mean±s.d calculated by relative quantification of three independent qRT-PCR results using GAPDH as reference gene and EGFP transfected control as calibrator. *p<0.05 (non-parametric Mann-Whitney test) compared to EGFP control cells. (E) Immunofluorescent staining of methanol-fixed frozen sections by the syndecan-2 specific antibody L-18. Identical exposure times and background corrections were applied Scale bar: 50 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3383727&req=5

pone-0039474-g005: Effects of FullEGFP and 78Sig on the malignancy of HT-1080 cells in vivo.(A) Size of primary tumours in the footpads of SCID mice on the 24th day after injection of HT-1080 cells expressing EGFP, FullEGFP or 78Sig. Photographs and results of morphometric analysis are shown. *p<0.05 (t-test), #p<0.05 (χ2-test)(n = 5). (B) Histological appearance of lung metastases (arrows) of HT-1080 transfectants. Images do not represent the area percentage of metastases; rather, the only one small EGFP tumour found is shown. HE-stained sections are shown, scale bar: 200 µm. (C) CDK2, phospho-retinoblastoma (at T373 position) and GAPDH immunoblots from HT-1080 primary tumours stably expressing EGFP, FullEGFP or 78Sig. (D) Relative syndecan-2 mRNA levels of the transfectants. Values are expressed as mean±s.d calculated by relative quantification of three independent qRT-PCR results using GAPDH as reference gene and EGFP transfected control as calibrator. *p<0.05 (non-parametric Mann-Whitney test) compared to EGFP control cells. (E) Immunofluorescent staining of methanol-fixed frozen sections by the syndecan-2 specific antibody L-18. Identical exposure times and background corrections were applied Scale bar: 50 µm.
Mentions: To estimate in vivo malignancy of the stable transfectants, they were injected into foot pads of SCID mice (5 per group). Twenty-four days after injection, the average volume of primary tumours increased as follows: EGFP (35.0±11.7 mm3), FullEGFP (156.3±86.0 mm3), and 78Sig (92.8±44.2 mm3) (Figure 5A). After 1 day, significant differences between the EGFP control and the syndecan-1 transfectant groups were found in the size of primary tumours by t-test (*p<0.05). Moreover, in the FullEGFP group, a significantly higher number of primary tumours reached the size of 150 mm3 than those in the 78Sig group, according to the χ2-test (#p<0.05) indicating that the FullEGFP construct promoted proliferation more effectively (Table 3). While the EGFP transfected group required 34 days to reach 150 mm3 average tumour volume, for 78Sig and FullEGFP it was only 29 and 24 days, respectively. Fifty days after injection, mice were sacrificed and the area fraction of lung metastases (Table 4) was calculated by morphometric analysis of 5 different, random planes of HE-stained sections (Figure 5B). In the EGFP group only one animal developed metastasis in its lung with negligible size. This number was 4 and 3 for FullEGFP and 78Sig groups, respectively (Table 4). The area of lung metastases in animals having tumours in the lung was 19.92±16.60% and 28.76±22.97% for FullEGFP and 78Sig, respectively, significantly more than the 0.02±0.15% of the control EGFP (p<0.05).

Bottom Line: These effects were accompanied by a marked increase in syndecan-2 protein expression.The pro-migratory and pro-proliferative effects of truncated syndecan-1 were not observable when syndecan-2 was silenced.Based on our in vitro results, we conclude that the tumour promoter role of syndecan-1 observed in HT-1080 cells is independent of its ectodomain; however, in vivo the presence of the ectodomain further increases tumour proliferation.

View Article: PubMed Central - PubMed

Affiliation: 1st Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary.

ABSTRACT
Syndecans are transmembrane heparan sulphate proteoglycans. Their role in the development of the malignant phenotype is ambiguous and depends upon the particular type of cancer. Nevertheless, syndecans are promising targets in cancer therapy, and it is important to elucidate the mechanisms controlling their various cellular effects. According to earlier studies, both syndecan-1 and syndecan-2 promote malignancy of HT-1080 human fibrosarcoma cells, by increasing the proliferation rate and the metastatic potential and migratory ability, respectively. To better understand their tumour promoter role in this cell line, syndecan expression levels were modulated in HT-1080 cells and the growth rate, chemotaxis and invasion capacity were studied. For in vivo testing, syndecan-1 overexpressing cells were also inoculated into mice. Overexpression of full length or truncated syndecan-1 lacking the entire ectodomain but containing the four juxtamembrane amino acids promoted proliferation and chemotaxis. These effects were accompanied by a marked increase in syndecan-2 protein expression. The pro-migratory and pro-proliferative effects of truncated syndecan-1 were not observable when syndecan-2 was silenced. Antisense silencing of syndecan-2, but not that of syndecan-1, inhibited cell migration. In vivo, both full length and truncated syndecan-1 increased tumour growth and metastatic rate. Based on our in vitro results, we conclude that the tumour promoter role of syndecan-1 observed in HT-1080 cells is independent of its ectodomain; however, in vivo the presence of the ectodomain further increases tumour proliferation. The enhanced migratory ability induced by syndecan-1 overexpression is mediated by syndecan-2. Overexpression of syndecan-1 also leads to activation of IGF1R and increased expression of Ets-1. These changes were not evident when syndecan-2 was overexpressed. These findings suggest the involvement of IGF1R and Ets-1 in the induction of syndecan-2 synthesis and stimulation of proliferation by syndecan-1. This is the first report demonstrating that syndecan-1 enhances malignancy of a mesenchymal tumour cell line, via induction of syndecan-2 expression.

Show MeSH
Related in: MedlinePlus