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Loss of hepatocyte growth factor activator inhibitor type 1 participates in metastatic spreading of human pancreatic cancer cells in a mouse orthotopic transplantation model.

Ye J, Kawaguchi M, Haruyama Y, Kanemaru A, Fukushima T, Yamamoto K, Lin CY, Kataoka H - Cancer Sci. (2013)

Bottom Line: Previously we reported that S2-CP8 cells, a metastatic variant of the SUIT-2 human pancreatic adenocarcinoma cell line, showed markedly decreased HAI-1 expression.In vitro migration and invasion assays revealed inhibitory effects of HAI-1 on S2-CP8 cell migration and invasion.These data indicate that HAI-1 loss contributes to invasion and dissemination of a highly metastatic subline of SUIT-2, suggesting crucial roles for the balance of pericellular serine proteases/inhibitors in pancreatic cancer progression.

View Article: PubMed Central - PubMed

Affiliation: Section of Oncopathology and Regenerative Biology, Department of Pathology, University of Miyazaki, Miyazaki, Japan; Clinical Research Center, The 2nd Affiliated Hospital School of Medicine, Zhejiang University, Hangzhou, China.

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Establishment of S2-CP8 subclones with Tet-inducible HAI-1 expression. (a) Immunoblot analysis of cellular proteins from two subclones with Tet-inducible HAI-1 expression (S2-CP8_HAI-1tet#1, S2-CP8_HAI-1tet#2) and a mock-transfected subclone (S2-CP8/mock). The same blot was reprobed with an anti-β-actin antibody. (b) RT-PCR for cell surface proteases, HAIs, HGF, c-MET, PAR-2, uPA and GAPDH. (c) Growth curves of S2-CP8_HAI-1tet#1 cells with or without Tet treatment. P = 0.02 (one-way anova). (d) Morphology of cultured S2-CP8_HAI-1tet#1 cells without or with Tet treatment.
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fig01: Establishment of S2-CP8 subclones with Tet-inducible HAI-1 expression. (a) Immunoblot analysis of cellular proteins from two subclones with Tet-inducible HAI-1 expression (S2-CP8_HAI-1tet#1, S2-CP8_HAI-1tet#2) and a mock-transfected subclone (S2-CP8/mock). The same blot was reprobed with an anti-β-actin antibody. (b) RT-PCR for cell surface proteases, HAIs, HGF, c-MET, PAR-2, uPA and GAPDH. (c) Growth curves of S2-CP8_HAI-1tet#1 cells with or without Tet treatment. P = 0.02 (one-way anova). (d) Morphology of cultured S2-CP8_HAI-1tet#1 cells without or with Tet treatment.

Mentions: Previously we reported that S2-CP8 cells, a highly metastatic subline from lungs and selected from SUIT-2, showed markedly decreased HAI-1 expression compared to the parent line. To determine whether the loss of HAI-1 is directly involved in S2-CP8 cell invasion and metastasis or is simply an epiphenomenon that occurred after the selection of the metastatic variant, we generated S2-CP8 cells that expressed HAI-1 under the control of a Tet-inducible promoter using the pLenti6.3 expression vector system (S2-CP8_HAI-1tet). Two sublines (S2-CP8_HAI-1tet#1 and #2) were isolated in which significant HAI-1 protein expression was induced by Tet treatment (Fig.1a). In accordance with the previous report, HAI-1 expression was barely detectable in parent S2-CP8 cells (not shown) and mock-transfected S2-CP8 cells (S2-CP8_mock) (Fig.1a). It should be noted that even in the absence of Tet, very low HAI-1 levels were detectable in S2-CP8_HAI-1tet cells by immunoblotting and RT-PCR (Fig.1a,b). The parent S2-CP8 cells expressed membrane-anchored serine proteases that can be regulated by HAI-1, such as matriptase, TMPRSS13, TMPRSS4 and prostasin. The mRNA levels of these proteases were not altered by the forced overexpression of HAI-1 (Fig.1b). Among presumed matriptase substrates, PAR-2 and uPA were also expressed by S2-CP8 cells. Although HGF, another important matriptase substrate, was not expressed by these cells, its receptor c-MET was expressed (Fig.1b). The Tet-induced overexpression of HAI-1 modestly enhanced the growth rate of S2-CP8 cells (Fig.1c) and resulted in a more epithelial morphology in vitro (Fig.1d).


Loss of hepatocyte growth factor activator inhibitor type 1 participates in metastatic spreading of human pancreatic cancer cells in a mouse orthotopic transplantation model.

Ye J, Kawaguchi M, Haruyama Y, Kanemaru A, Fukushima T, Yamamoto K, Lin CY, Kataoka H - Cancer Sci. (2013)

Establishment of S2-CP8 subclones with Tet-inducible HAI-1 expression. (a) Immunoblot analysis of cellular proteins from two subclones with Tet-inducible HAI-1 expression (S2-CP8_HAI-1tet#1, S2-CP8_HAI-1tet#2) and a mock-transfected subclone (S2-CP8/mock). The same blot was reprobed with an anti-β-actin antibody. (b) RT-PCR for cell surface proteases, HAIs, HGF, c-MET, PAR-2, uPA and GAPDH. (c) Growth curves of S2-CP8_HAI-1tet#1 cells with or without Tet treatment. P = 0.02 (one-way anova). (d) Morphology of cultured S2-CP8_HAI-1tet#1 cells without or with Tet treatment.
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Related In: Results  -  Collection

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fig01: Establishment of S2-CP8 subclones with Tet-inducible HAI-1 expression. (a) Immunoblot analysis of cellular proteins from two subclones with Tet-inducible HAI-1 expression (S2-CP8_HAI-1tet#1, S2-CP8_HAI-1tet#2) and a mock-transfected subclone (S2-CP8/mock). The same blot was reprobed with an anti-β-actin antibody. (b) RT-PCR for cell surface proteases, HAIs, HGF, c-MET, PAR-2, uPA and GAPDH. (c) Growth curves of S2-CP8_HAI-1tet#1 cells with or without Tet treatment. P = 0.02 (one-way anova). (d) Morphology of cultured S2-CP8_HAI-1tet#1 cells without or with Tet treatment.
Mentions: Previously we reported that S2-CP8 cells, a highly metastatic subline from lungs and selected from SUIT-2, showed markedly decreased HAI-1 expression compared to the parent line. To determine whether the loss of HAI-1 is directly involved in S2-CP8 cell invasion and metastasis or is simply an epiphenomenon that occurred after the selection of the metastatic variant, we generated S2-CP8 cells that expressed HAI-1 under the control of a Tet-inducible promoter using the pLenti6.3 expression vector system (S2-CP8_HAI-1tet). Two sublines (S2-CP8_HAI-1tet#1 and #2) were isolated in which significant HAI-1 protein expression was induced by Tet treatment (Fig.1a). In accordance with the previous report, HAI-1 expression was barely detectable in parent S2-CP8 cells (not shown) and mock-transfected S2-CP8 cells (S2-CP8_mock) (Fig.1a). It should be noted that even in the absence of Tet, very low HAI-1 levels were detectable in S2-CP8_HAI-1tet cells by immunoblotting and RT-PCR (Fig.1a,b). The parent S2-CP8 cells expressed membrane-anchored serine proteases that can be regulated by HAI-1, such as matriptase, TMPRSS13, TMPRSS4 and prostasin. The mRNA levels of these proteases were not altered by the forced overexpression of HAI-1 (Fig.1b). Among presumed matriptase substrates, PAR-2 and uPA were also expressed by S2-CP8 cells. Although HGF, another important matriptase substrate, was not expressed by these cells, its receptor c-MET was expressed (Fig.1b). The Tet-induced overexpression of HAI-1 modestly enhanced the growth rate of S2-CP8 cells (Fig.1c) and resulted in a more epithelial morphology in vitro (Fig.1d).

Bottom Line: Previously we reported that S2-CP8 cells, a metastatic variant of the SUIT-2 human pancreatic adenocarcinoma cell line, showed markedly decreased HAI-1 expression.In vitro migration and invasion assays revealed inhibitory effects of HAI-1 on S2-CP8 cell migration and invasion.These data indicate that HAI-1 loss contributes to invasion and dissemination of a highly metastatic subline of SUIT-2, suggesting crucial roles for the balance of pericellular serine proteases/inhibitors in pancreatic cancer progression.

View Article: PubMed Central - PubMed

Affiliation: Section of Oncopathology and Regenerative Biology, Department of Pathology, University of Miyazaki, Miyazaki, Japan; Clinical Research Center, The 2nd Affiliated Hospital School of Medicine, Zhejiang University, Hangzhou, China.

Show MeSH
Related in: MedlinePlus