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Surface α-enolase promotes extracellular matrix degradation and tumor metastasis and represents a new therapeutic target.

Hsiao KC, Shih NY, Fang HL, Huang TS, Kuo CC, Chu PY, Hung YM, Chou SW, Yang YY, Chang GC, Liu KJ - PLoS ONE (2013)

Bottom Line: Treatment with antibody against α-enolase in vitro suppressed cell-associated plasminogen and matrix metalloproteinase activation, collagen and gelatin degradation, and cell invasion.Examination of the effect of treatment with shRNA plasmids revealed that down regulation of α-enolase decreases extracellular matrix degradation by and the invasion capacity of lung cancer cells.Adoptive transfer of α-enolase-specific antibody to mice resulted in accumulation of antibody in subcutaneous tumor and inhibited the formation of tumor metastasis in lung and bone.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Pharmacy and Pharmaceutical Sciences, National Cheng Kung University, Tainan, Taiwan.

ABSTRACT
In previous research, we found α-enolase to be inversely correlated with progression-free and overall survival in lung cancer patients and detected α-enolase on the surface of lung cancer cells. Based on these findings, we hypothesized that surface α-enolase has a significant role in cancer metastasis and tested this hypothesis in the current study. We found that α-enolase was co-immunoprecipitated with urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen in lung cancer cells and interacted with these proteins in a cell-free dot blotting assay, which can be interrupted by α-enolase-specific antibody. α-Enolase in lung cancer cells co-localized with these proteins and was present at the site of pericellular degradation of extracellular matrix components. Treatment with antibody against α-enolase in vitro suppressed cell-associated plasminogen and matrix metalloproteinase activation, collagen and gelatin degradation, and cell invasion. Examination of the effect of treatment with shRNA plasmids revealed that down regulation of α-enolase decreases extracellular matrix degradation by and the invasion capacity of lung cancer cells. Adoptive transfer of α-enolase-specific antibody to mice resulted in accumulation of antibody in subcutaneous tumor and inhibited the formation of tumor metastasis in lung and bone. This study demonstrated that surface α-enolase promotes extracellular matrix degradation and invasion of cancer cells and that targeting surface α-enolase is a promising approach to suppress tumor metastasis.

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Ab against ENO1 suppressed cell invasion.PE089 (A) or LLC/luc (B) cells were suspended in serum-free medium containing PBS, an ENO1-specific Ab (2 µg/ml), or an isotype-control Ab (2 µg/ml), and then added into transwell chambers consisting of matrigel-coated polycarbonate membranes. The transwell chambers were placed into wells containing medium with 5% FBS as a chemoattractant. After incubation for 24 h, the invading cells on the opposite side of the chambers were determined. (C) The growth of LLC/luc cells in a culture containing PBS, an isotype-control Ab (2 µg/ml), or an Ab against ENO1 (2 µg/ml) was determined by a MTT assay. *p<0.05 and **p<0.01. The error bars were defined as mean±SD.
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pone-0069354-g003: Ab against ENO1 suppressed cell invasion.PE089 (A) or LLC/luc (B) cells were suspended in serum-free medium containing PBS, an ENO1-specific Ab (2 µg/ml), or an isotype-control Ab (2 µg/ml), and then added into transwell chambers consisting of matrigel-coated polycarbonate membranes. The transwell chambers were placed into wells containing medium with 5% FBS as a chemoattractant. After incubation for 24 h, the invading cells on the opposite side of the chambers were determined. (C) The growth of LLC/luc cells in a culture containing PBS, an isotype-control Ab (2 µg/ml), or an Ab against ENO1 (2 µg/ml) was determined by a MTT assay. *p<0.05 and **p<0.01. The error bars were defined as mean±SD.

Mentions: As MMP2 and 9, substrates in the plasmin proteolytic cascade [34], [35], are involved in the degradation of ECM and invasion of tumor cells [36], the effect of blocking ENO1 on the activation and activity of MMP2/9 was next examined. We observed a low MMP2/9 activity in the culture supernatant of these tumor cells with gelatin zymographic gel analysis (data not shown). Using a more sensitive assay, we observed that culturing with an ENO1-specific Ab significantly reduced the degradation of a fluorescence-labeled gelatin by LLC/luc cells (Figure 2E). In vitro transwell assay revealed that addition of ENO-specific Ab leads to a 40% to 50% reduction in the number of PE089 and LLC/luc cells passing through the matrigel-coated membrane (Figures 3A and 3B). These results suggest that surface ENO1 participates in tissue invasion of tumor cells. The in vitro growth curve of the LLC/luc cells cultured with the ENO1-specific Ab was similar to that of cells cultured with the isotype-control Ab within the first 72 h (Figure 3C), suggesting that the results described above cannot be simply attributed to interference of the ENO1-specific Ab on the growth of tumor cells.


Surface α-enolase promotes extracellular matrix degradation and tumor metastasis and represents a new therapeutic target.

Hsiao KC, Shih NY, Fang HL, Huang TS, Kuo CC, Chu PY, Hung YM, Chou SW, Yang YY, Chang GC, Liu KJ - PLoS ONE (2013)

Ab against ENO1 suppressed cell invasion.PE089 (A) or LLC/luc (B) cells were suspended in serum-free medium containing PBS, an ENO1-specific Ab (2 µg/ml), or an isotype-control Ab (2 µg/ml), and then added into transwell chambers consisting of matrigel-coated polycarbonate membranes. The transwell chambers were placed into wells containing medium with 5% FBS as a chemoattractant. After incubation for 24 h, the invading cells on the opposite side of the chambers were determined. (C) The growth of LLC/luc cells in a culture containing PBS, an isotype-control Ab (2 µg/ml), or an Ab against ENO1 (2 µg/ml) was determined by a MTT assay. *p<0.05 and **p<0.01. The error bars were defined as mean±SD.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3716638&req=5

pone-0069354-g003: Ab against ENO1 suppressed cell invasion.PE089 (A) or LLC/luc (B) cells were suspended in serum-free medium containing PBS, an ENO1-specific Ab (2 µg/ml), or an isotype-control Ab (2 µg/ml), and then added into transwell chambers consisting of matrigel-coated polycarbonate membranes. The transwell chambers were placed into wells containing medium with 5% FBS as a chemoattractant. After incubation for 24 h, the invading cells on the opposite side of the chambers were determined. (C) The growth of LLC/luc cells in a culture containing PBS, an isotype-control Ab (2 µg/ml), or an Ab against ENO1 (2 µg/ml) was determined by a MTT assay. *p<0.05 and **p<0.01. The error bars were defined as mean±SD.
Mentions: As MMP2 and 9, substrates in the plasmin proteolytic cascade [34], [35], are involved in the degradation of ECM and invasion of tumor cells [36], the effect of blocking ENO1 on the activation and activity of MMP2/9 was next examined. We observed a low MMP2/9 activity in the culture supernatant of these tumor cells with gelatin zymographic gel analysis (data not shown). Using a more sensitive assay, we observed that culturing with an ENO1-specific Ab significantly reduced the degradation of a fluorescence-labeled gelatin by LLC/luc cells (Figure 2E). In vitro transwell assay revealed that addition of ENO-specific Ab leads to a 40% to 50% reduction in the number of PE089 and LLC/luc cells passing through the matrigel-coated membrane (Figures 3A and 3B). These results suggest that surface ENO1 participates in tissue invasion of tumor cells. The in vitro growth curve of the LLC/luc cells cultured with the ENO1-specific Ab was similar to that of cells cultured with the isotype-control Ab within the first 72 h (Figure 3C), suggesting that the results described above cannot be simply attributed to interference of the ENO1-specific Ab on the growth of tumor cells.

Bottom Line: Treatment with antibody against α-enolase in vitro suppressed cell-associated plasminogen and matrix metalloproteinase activation, collagen and gelatin degradation, and cell invasion.Examination of the effect of treatment with shRNA plasmids revealed that down regulation of α-enolase decreases extracellular matrix degradation by and the invasion capacity of lung cancer cells.Adoptive transfer of α-enolase-specific antibody to mice resulted in accumulation of antibody in subcutaneous tumor and inhibited the formation of tumor metastasis in lung and bone.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Pharmacy and Pharmaceutical Sciences, National Cheng Kung University, Tainan, Taiwan.

ABSTRACT
In previous research, we found α-enolase to be inversely correlated with progression-free and overall survival in lung cancer patients and detected α-enolase on the surface of lung cancer cells. Based on these findings, we hypothesized that surface α-enolase has a significant role in cancer metastasis and tested this hypothesis in the current study. We found that α-enolase was co-immunoprecipitated with urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor, and plasminogen in lung cancer cells and interacted with these proteins in a cell-free dot blotting assay, which can be interrupted by α-enolase-specific antibody. α-Enolase in lung cancer cells co-localized with these proteins and was present at the site of pericellular degradation of extracellular matrix components. Treatment with antibody against α-enolase in vitro suppressed cell-associated plasminogen and matrix metalloproteinase activation, collagen and gelatin degradation, and cell invasion. Examination of the effect of treatment with shRNA plasmids revealed that down regulation of α-enolase decreases extracellular matrix degradation by and the invasion capacity of lung cancer cells. Adoptive transfer of α-enolase-specific antibody to mice resulted in accumulation of antibody in subcutaneous tumor and inhibited the formation of tumor metastasis in lung and bone. This study demonstrated that surface α-enolase promotes extracellular matrix degradation and invasion of cancer cells and that targeting surface α-enolase is a promising approach to suppress tumor metastasis.

Show MeSH
Related in: MedlinePlus