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Dual effects of collagenase-3 on melanoma: metastasis promotion and disruption of vasculogenic mimicry.

Zhao X, Sun B, Li Y, Liu Y, Zhang D, Wang X, Gu Q, Zhao J, Dong X, Liu Z, Che N - Oncotarget (2015)

Bottom Line: These results were confirmed in human and mouse melanoma cell lines.We found that MMP-13 cleaves laminin-5 (Ln-5) into small fragments to accelerate tumor metastasis.In conclusion, MMP-13 has a dual effect in melanoma, as it promotes invasion and metastasis but disrupts VM formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, General Hospital of Tianjin Medical University, Tianjin, China.

ABSTRACT
Vasculogenic mimicry (VM) is a functional microcirculation formed by tumor cells. Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, promote VM formation. Another specific MMP, collagenase-3 (MMP-13), has broad substrate specificity and potentially affects tumor metastasis and invasion. Here we found that MMP-13 was associated with metastasis and poor survival in 79 patients with melanoma. MMP-13 expression was inversely correlated with VM. These results were confirmed in human and mouse melanoma cell lines. We found that MMP-13 cleaves laminin-5 (Ln-5) into small fragments to accelerate tumor metastasis. Degradation of Ln-5 and VE-cadherin by MMP-13 inhibited VM formation. In conclusion, MMP-13 has a dual effect in melanoma, as it promotes invasion and metastasis but disrupts VM formation.

No MeSH data available.


Related in: MedlinePlus

MMP-13 promoted invasion of melanoma cells and cleaved Ln-5 into small molecular weight fragments(A) Transfection efficiency was confirmed by western blotting. (B) Upregulated or knocked-down MMP-13 expression in A375 or B16-F10 melanoma cells promoted or decreased (respectively) invasion of tumor cells through Matrigel-coated transwell membranes. *P < 0.05, compared with the pcDNA3.1 empty vector control; #P < 0.05, compared with pRNAT− control. (C and D) Invasiveness of different ECM substrates cleaved by MMP-13. A375 and B16-F10 melanoma cells were incubated for 24 h with products of collagen I, II, III, IV, aggrecan, gelatin or laminin-5 cleaved by MMP-13, and their invasion abilities through Matrigel were measured. Compared with untreated controls, the MMP-13 cleavage products of collagen II, III, IV, aggrecan or laminin-5 all increased invasiveness of the two melanoma cell lines. However, 3× as many cells incubated with the Ln-5, and 2× as many cells incubated with collagen II, III, IV and aggrecan cleavage products invaded the transwell membrane than untreated cells, which suggests the important role of Ln-5–MMP-13 cleavage fragments in melanoma invasiveness. *P < 0.05 compared with untreated control. (E) Cleavage fragments of human Ln-5γ2 by MMP-2 or MMP-13 at 37°C for 6 h were run on 12% SDS-PAGE gel and detected by western blot using an anti-human Ln-5γ2-chain antibody. The left lane shows intact Ln-5γ2-chain in 140- and 100-kDa forms. MMP-2 generated an 80-kDa Ln-5γ2x fragment and very faint 66-kDa fragment. MMP-13 further generated low molecular weight fragments of ~40 kDa. (F) Adding MMP-13–LN-5 cleavage fragments to the cell culture medium induced melanoma cells to be significantly more invasive than untreated cells and cells treated with MMP-2–Ln-5 fragments. *P < 0.05, compared with untreated controls; **P < 0.05, compared with cells treated with MMP-2–Ln-5 fragments. All of the experiments were repeated three times.
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Figure 2: MMP-13 promoted invasion of melanoma cells and cleaved Ln-5 into small molecular weight fragments(A) Transfection efficiency was confirmed by western blotting. (B) Upregulated or knocked-down MMP-13 expression in A375 or B16-F10 melanoma cells promoted or decreased (respectively) invasion of tumor cells through Matrigel-coated transwell membranes. *P < 0.05, compared with the pcDNA3.1 empty vector control; #P < 0.05, compared with pRNAT− control. (C and D) Invasiveness of different ECM substrates cleaved by MMP-13. A375 and B16-F10 melanoma cells were incubated for 24 h with products of collagen I, II, III, IV, aggrecan, gelatin or laminin-5 cleaved by MMP-13, and their invasion abilities through Matrigel were measured. Compared with untreated controls, the MMP-13 cleavage products of collagen II, III, IV, aggrecan or laminin-5 all increased invasiveness of the two melanoma cell lines. However, 3× as many cells incubated with the Ln-5, and 2× as many cells incubated with collagen II, III, IV and aggrecan cleavage products invaded the transwell membrane than untreated cells, which suggests the important role of Ln-5–MMP-13 cleavage fragments in melanoma invasiveness. *P < 0.05 compared with untreated control. (E) Cleavage fragments of human Ln-5γ2 by MMP-2 or MMP-13 at 37°C for 6 h were run on 12% SDS-PAGE gel and detected by western blot using an anti-human Ln-5γ2-chain antibody. The left lane shows intact Ln-5γ2-chain in 140- and 100-kDa forms. MMP-2 generated an 80-kDa Ln-5γ2x fragment and very faint 66-kDa fragment. MMP-13 further generated low molecular weight fragments of ~40 kDa. (F) Adding MMP-13–LN-5 cleavage fragments to the cell culture medium induced melanoma cells to be significantly more invasive than untreated cells and cells treated with MMP-2–Ln-5 fragments. *P < 0.05, compared with untreated controls; **P < 0.05, compared with cells treated with MMP-2–Ln-5 fragments. All of the experiments were repeated three times.

Mentions: MMP-13 expression levels after transfection were confirmed by western blotting (Figure 2A), which showed that MMP-13 expression was efficiently up-regulated by transfection with pcDNA3-MMP-13 or down-regulated after transfection with pRNAT-MMP-13 siRNA. Corresponding active MMP-13 concentrations in culture media are shown in Supplementary Table S2. We found that melanoma cells with up-regulated MMP-13 expression promoted invasion of tumor cells through Matrigel compared with control cells transfected with empty vector. Knocked-down MMP-13 expression also decreased invading cell numbers (Figure 2B).


Dual effects of collagenase-3 on melanoma: metastasis promotion and disruption of vasculogenic mimicry.

Zhao X, Sun B, Li Y, Liu Y, Zhang D, Wang X, Gu Q, Zhao J, Dong X, Liu Z, Che N - Oncotarget (2015)

MMP-13 promoted invasion of melanoma cells and cleaved Ln-5 into small molecular weight fragments(A) Transfection efficiency was confirmed by western blotting. (B) Upregulated or knocked-down MMP-13 expression in A375 or B16-F10 melanoma cells promoted or decreased (respectively) invasion of tumor cells through Matrigel-coated transwell membranes. *P < 0.05, compared with the pcDNA3.1 empty vector control; #P < 0.05, compared with pRNAT− control. (C and D) Invasiveness of different ECM substrates cleaved by MMP-13. A375 and B16-F10 melanoma cells were incubated for 24 h with products of collagen I, II, III, IV, aggrecan, gelatin or laminin-5 cleaved by MMP-13, and their invasion abilities through Matrigel were measured. Compared with untreated controls, the MMP-13 cleavage products of collagen II, III, IV, aggrecan or laminin-5 all increased invasiveness of the two melanoma cell lines. However, 3× as many cells incubated with the Ln-5, and 2× as many cells incubated with collagen II, III, IV and aggrecan cleavage products invaded the transwell membrane than untreated cells, which suggests the important role of Ln-5–MMP-13 cleavage fragments in melanoma invasiveness. *P < 0.05 compared with untreated control. (E) Cleavage fragments of human Ln-5γ2 by MMP-2 or MMP-13 at 37°C for 6 h were run on 12% SDS-PAGE gel and detected by western blot using an anti-human Ln-5γ2-chain antibody. The left lane shows intact Ln-5γ2-chain in 140- and 100-kDa forms. MMP-2 generated an 80-kDa Ln-5γ2x fragment and very faint 66-kDa fragment. MMP-13 further generated low molecular weight fragments of ~40 kDa. (F) Adding MMP-13–LN-5 cleavage fragments to the cell culture medium induced melanoma cells to be significantly more invasive than untreated cells and cells treated with MMP-2–Ln-5 fragments. *P < 0.05, compared with untreated controls; **P < 0.05, compared with cells treated with MMP-2–Ln-5 fragments. All of the experiments were repeated three times.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496190&req=5

Figure 2: MMP-13 promoted invasion of melanoma cells and cleaved Ln-5 into small molecular weight fragments(A) Transfection efficiency was confirmed by western blotting. (B) Upregulated or knocked-down MMP-13 expression in A375 or B16-F10 melanoma cells promoted or decreased (respectively) invasion of tumor cells through Matrigel-coated transwell membranes. *P < 0.05, compared with the pcDNA3.1 empty vector control; #P < 0.05, compared with pRNAT− control. (C and D) Invasiveness of different ECM substrates cleaved by MMP-13. A375 and B16-F10 melanoma cells were incubated for 24 h with products of collagen I, II, III, IV, aggrecan, gelatin or laminin-5 cleaved by MMP-13, and their invasion abilities through Matrigel were measured. Compared with untreated controls, the MMP-13 cleavage products of collagen II, III, IV, aggrecan or laminin-5 all increased invasiveness of the two melanoma cell lines. However, 3× as many cells incubated with the Ln-5, and 2× as many cells incubated with collagen II, III, IV and aggrecan cleavage products invaded the transwell membrane than untreated cells, which suggests the important role of Ln-5–MMP-13 cleavage fragments in melanoma invasiveness. *P < 0.05 compared with untreated control. (E) Cleavage fragments of human Ln-5γ2 by MMP-2 or MMP-13 at 37°C for 6 h were run on 12% SDS-PAGE gel and detected by western blot using an anti-human Ln-5γ2-chain antibody. The left lane shows intact Ln-5γ2-chain in 140- and 100-kDa forms. MMP-2 generated an 80-kDa Ln-5γ2x fragment and very faint 66-kDa fragment. MMP-13 further generated low molecular weight fragments of ~40 kDa. (F) Adding MMP-13–LN-5 cleavage fragments to the cell culture medium induced melanoma cells to be significantly more invasive than untreated cells and cells treated with MMP-2–Ln-5 fragments. *P < 0.05, compared with untreated controls; **P < 0.05, compared with cells treated with MMP-2–Ln-5 fragments. All of the experiments were repeated three times.
Mentions: MMP-13 expression levels after transfection were confirmed by western blotting (Figure 2A), which showed that MMP-13 expression was efficiently up-regulated by transfection with pcDNA3-MMP-13 or down-regulated after transfection with pRNAT-MMP-13 siRNA. Corresponding active MMP-13 concentrations in culture media are shown in Supplementary Table S2. We found that melanoma cells with up-regulated MMP-13 expression promoted invasion of tumor cells through Matrigel compared with control cells transfected with empty vector. Knocked-down MMP-13 expression also decreased invading cell numbers (Figure 2B).

Bottom Line: These results were confirmed in human and mouse melanoma cell lines.We found that MMP-13 cleaves laminin-5 (Ln-5) into small fragments to accelerate tumor metastasis.In conclusion, MMP-13 has a dual effect in melanoma, as it promotes invasion and metastasis but disrupts VM formation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, General Hospital of Tianjin Medical University, Tianjin, China.

ABSTRACT
Vasculogenic mimicry (VM) is a functional microcirculation formed by tumor cells. Matrix metalloproteinases (MMPs), especially MMP-2 and MMP-9, promote VM formation. Another specific MMP, collagenase-3 (MMP-13), has broad substrate specificity and potentially affects tumor metastasis and invasion. Here we found that MMP-13 was associated with metastasis and poor survival in 79 patients with melanoma. MMP-13 expression was inversely correlated with VM. These results were confirmed in human and mouse melanoma cell lines. We found that MMP-13 cleaves laminin-5 (Ln-5) into small fragments to accelerate tumor metastasis. Degradation of Ln-5 and VE-cadherin by MMP-13 inhibited VM formation. In conclusion, MMP-13 has a dual effect in melanoma, as it promotes invasion and metastasis but disrupts VM formation.

No MeSH data available.


Related in: MedlinePlus