Heparanase stimulation of protease expression implicates it as a master regulator of the aggressive tumor phenotype in myeloma - PubMed (original) (raw)

Heparanase stimulation of protease expression implicates it as a master regulator of the aggressive tumor phenotype in myeloma

Anurag Purushothaman et al. J Biol Chem. 2008.

Abstract

High levels of heparanase are an indicator of poor prognosis in myeloma patients, and up-regulation of the enzyme enhances tumor growth, angiogenesis, and metastasis in animal models. At least part of the impact of heparanase in driving the aggressive tumor phenotype is due to its effect on increasing the expression and shedding of the heparan sulfate proteoglycan syndecan-1, a molecule known to promote myeloma progression. The present work demonstrated that elevation in heparanase expression in myeloma cells stimulates sustained ERK phosphorylation that in turn drives MMP-9 expression. In addition, urokinase-type plasminogen activator (uPA) and uPA receptor expression levels increased, and blocking the proteolytic activation of either MMP-9 or uPA inhibited the heparanase-induced increase in syndecan-1 shedding. Together these data provide a mechanism for heparanase-induced syndecan-1 shedding and, more importantly, demonstrate that heparanase activity in myeloma cells can lead to increased levels of proteases that are known to play important roles in the aggressive behavior of myeloma tumors. This in addition to its other known biological roles, indicates that heparanase acts as a master regulator of the aggressive tumor phenotype by up-regulating protease expression and activity within the tumor microenvironment.

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Figures

FIGURE 1.

Heparanase enhances MMP-9 expression and activity in myeloma cells. A, Western blot of MMP-9 in serum-free conditioned media from CAG cells demonstrates that pro-MMP-9 protein levels are up-regulated in CAG cells expressing high levels of heparanase as compared with cells expressing low levels of heparanase. (Heparanase high cells were transfected with vector containing the cDNA for human heparanase; heparanase low cells were transfected with empty vector (21).) Gelatin zymography of conditioned medium from either CAG or ARH-77 cells confirms Western blotting data by showing an elevation in levels of MMP-9 activity in cells expressing high levels of heparanase. (Note that the MMP-9 seen in both the Western blot and zymogram is the 92-kDa inactive proform of the enzyme rather than the cleaved 82-kDa active form. The gelatinolytic activity of MMP-9 routinely seen in zymograms is thought to be due to activation of the uncleaved enzyme resulting from the gel electrophoresis procedure.) B, CAG cells expressing heparanase that is mutated at amino acids 225 or 343 (M225 or M343) and thus lacking heparan sulfate-degrading activity do not show enhanced MMP-9 levels in zymograms as compared with cells expressing enzymatically active heparanase. C, addition of recombinant heparanase to CAG cells enhances levels of MMP-9. Recombinant heparanase (rHPSE) was added to wild-type CAG cells at the indicated concentrations, and conditioned medium was collected after 24 h and subjected to gelatin zymography. Quantification of MMP-9 gelatinolytic activity in these zymograms revealed an almost 3-fold higher level in cells treated with 10 ng/ml recombinant heparanase_versus_ untreated cells. Shown are results of densitometric analysis of a single, representative gel.

FIGURE 2.

Down-regulation of heparanase reduces MMP-9 expression levels. A, wild-type CAG myeloma cells were infected with lentiviral vectors coding for control or heparanase knockdown shRNAs. Analysis by both reverse transcription (RT)-PCR and Western blotting from extracts of stably infected cells demonstrates effective knockdown of heparanase (HPSE) expression. B, densitometric quantification of Western blots and gelatin zymograms of MMP-9 levels in conditioned medium of control or heparanase knockdown cells indicates that MMP-9 protein and activity levels are reduced when heparanase is knocked down as compared with controls. Shown are results from single, representative gels. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

FIGURE 3.

Expression of enzymatically active heparanase enhances ERK phosphorylation and up-regulation of MMP-9. A, cell lysates from CAG cells expressing low or high levels of heparanase were subjected to immunoblotting with antibodies against the phosphorylated forms of Src, p38, or ERK. Cell lysates from cells expressing the mutated, enzymatically inactive form of heparanase (M343) were also probed for ERK. Blots were subsequently stripped and probed for total Src, p38, or ERK. B, CAG cells expressing high levels of heparanase were treated with MAPK/ERK pathway inhibitor PD98059 (50 μ

), and MMP-9 activity in the serum-free conditioned medium was assessed by gelatin zymography.

FIGURE 4.

Heparanase induces expression of MMP-9 that becomes activated in vivo. A, CAG myeloma cells expressing low or high levels of heparanase were injected subcutaneously into severe combined immunodeficient mice as described previously (19). After tumors formed, they were removed and immunostained for MMP-9 (original magnification, ×200). B, Western blot of an extract from a tumor formed by heparanase high cells demonstrates that MMP-9 is present predominantly in its enzymatically active (82-kDa) form.

FIGURE 5.

MMP-9 mediates enhanced syndecan-1 shedding by heparanase high cells. A, CAG cells expressing low or high levels of heparanase were plated at equal density, and 0.5 μg/ml MMP-9 function blocking antibody 6-6B was added. After 48 h, conditioned media were harvested, and the level of shed syndecan-1 was determined by enzyme-linked immunoadsorbent assay (values represents means of triplicate determination ±S.D.).*, p < 0.001 versus heparanase (HPSE) high cells. B, cells expressing high levels of heparanase were grown in the presence of varying levels of MMP-9 inhibitor 1, and syndecan-1 shedding was measured by enzyme-linked immunoadsorbent assay. Values represent means ± S.D. of triplicate determinations. *, p < 0.05 versus 0 n

; **, p < 0.01_versus_ 0 n

FIGURE 6.

Enhanced expression of heparanase up-regulates uPA and uPAR. A, immunoblotting of serum-free conditioned media with an antibody to uPA or cell lysates with an antibody to uPAR or actin reveals up-regulation of uPA/uPAR by cells expressing high levels of heparanase. B, heparanase-mediated up-regulation of syndecan-1 shedding is inhibited by an antibody that blocks activation of uPA. Cells were plated at equal density, and 5 μg/ml antibody was added to cells expressing high levels of heparanase. After 48 h conditioned media was harvested, and the level of syndecan-1 was determined by enzyme-linked immunoadsorbent assay (values represents means of triplicate determination ±S.D.). *,p < 0.001 versus heparanase (HPSE) high cells.C, myeloma invasion is enhanced by expression of heparanase and is blocked by the antibody that inhibits uPA activation. An equal number of CAG cells were seeded on invasion chambers coated with matrigel, and cells were allowed to migrate in the presence or absence of antibody. Cell invasion data represent the mean ± S.D. of three independent experiments.*, p < 0.01 versus heparanase high cells.

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Heparanase stimulation of protease expression implicates it as a master regulator of the aggressive tumor phenotype in myeloma - PubMed (original) (raw)