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Nestin depletion induces melanoma matrix metalloproteinases and invasion.

Lee CW, Zhan Q, Lezcano C, Frank MH, Huang J, Larson AR, Lin JY, Wan MT, Lin PI, Ma J, Kleffel S, Schatton T, Lian CG, Murphy GF - Lab. Invest. (2014)

Bottom Line: Matrix metalloproteinases (MMPs) are key biological mediators of processes as diverse as wound healing, embryogenesis, and cancer progression.Although MMPs may be induced through multiple signaling pathways, the precise mechanisms for their regulation in cancer are incompletely understood.The results suggest that the heretofore poorly understood intermediate filament, nestin, may serve as a novel mediator of MMPs critical to melanoma virulence.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

ABSTRACT
Matrix metalloproteinases (MMPs) are key biological mediators of processes as diverse as wound healing, embryogenesis, and cancer progression. Although MMPs may be induced through multiple signaling pathways, the precise mechanisms for their regulation in cancer are incompletely understood. Because cytoskeletal changes are known to accompany MMP expression, we sought to examine the potential role of the poorly understood cytoskeletal protein, nestin, in modulating melanoma MMPs. Nestin knockdown (KD) upregulated the expression of specific MMPs and MMP-dependent invasion both through extracellular matrix barriers in vitro and in peritumoral connective tissue of xenografts in vivo. The development of three-dimensional melanospheres that in vitro partially recapitulate noninvasive tumorigenic melanoma growth was inhibited by nestin KD, although ECM invasion by aberrant melanospheres that did form was enhanced. Mechanistically, nestin KD-dependent melanoma invasion was associated with intracellular redistribution of phosphorylated focal adhesion kinase and increased melanoma cell responsiveness to transforming growth factor-beta, both implicated in pathways of melanoma invasion. The results suggest that the heretofore poorly understood intermediate filament, nestin, may serve as a novel mediator of MMPs critical to melanoma virulence.

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Nestin KD enhances melanoma invasion in vitro and decreases sphere formation(a) Note significant diminished cell migration of nestin KD cells (NesKD, solid lines) as compared to the vector control cells (Vec, dash lines) of A2058 (left; p<0.00001 at 6 and 8 hours, respectively) and A375 (right; p<0.00001 at 6 and 8 hours, respectively). (b) Representative fields of migrating A2058 (left) and A375 (right) cells in Matrigel inserts (top panel) and control inserts (bottom panel). Scale bars: 50 µm. (c) Nestin KD cells (black bars) had significantly higher Matrigel invasion than vector control cells (white bars) of A2058 (left, 2.54-fold, p<0.001) and A375 (right, 4.02-fold, p<0.001). (d) Matrigel invasion of A2058 and A375 nestin KD cells was characterized in the presence of MMP3-specific inhibitor UK356618 or the broad spectrum MMP inhibitor Marimastat. Compared to untreated A2058 nestin KD cells (black bar), UK356618 (20 nM, 33% decrease, p<0.05) and Marimastat (20 nM, 43% decrease, p<0.01) significantly suppressed Matrigel invasion of A2058 nestin KD cells. Combinatorial treatment of UK356618 (20 nM) and Marimastat (20 nM) additively suppressed Matrigel invasion of A2058 Nestin KD cells (56% decrease, p<0.001). Additionally, Matrigel invasion of A2058 nestin KD cells with combinatorial treatment of UK356618 and Marimastat was lower than monotherapy with UK356618 (65%, p=0.097). Likewise, in A375 nestin KD cells, Matrigel invasion of A375 nestin KD cells was not altered by UK356618 (20 nM), but was significantly suppressed by Marimastat (20 nM) (45% decrease, p<0.01) or by combinatorial treatment of Marimastat (20 nM) and UK356618 (20 nM) (55% decrease, p<0.001), as compared to untreated cells (black bar). Additionally, Marimastat monotherapy and combinatorial treatment of Marimastat and UK356618 had comparable suppressive effects on A375 nestin KD Matrigel invasion. Results were compared by one-way ANOVA test with Tukey’s multiple comparison test. Representative cell migration and invasion results are shown from at least three independent assays. (e) Representative images of A2058 and A375 melanospheres in spherogenic suspension culture in DMEM supplemented with 10% FBS and 0.5% methyl cellulose for 21 days. Melanospheres were visualized with p-iodonitrotetrazolium violet staining. Note marked decreased of sphere formation in both A2058 (top) and A375 (bottom) nestin KD cells as compared to vector control control cells (both p<0.01, respectively). Representative results are shown from two independent experiments. (f) H&E and IHC images of spheres growing over Matrigel. A2058 vector control spheres grew mainly on the surface of Matrigel (top), while nestin KD spheres invaded into the lattice of Matrigel (bottom). Scale bars: 50 µm Location of spheres on Matrigel. Majority (83.3%) of A2058 vector control spheres grew on the surface of Matrigel (white bar), while 16.7% partially invaded into Matrigel (gray bar). In contrast, 28.3% nestin KD spheres grew on the surface of Matrigel (white bar), 25% partially invaded into Matrigel (gray bar), and 46.7% completely invaded and embedded into Matrigel (black bar). Difference in Matrigel invasion between melanospheres derived from A2058 vector control (N=60) and Nestin KD (N=60) cells was significant (p<0.0001, Chi-square test). Data is represented from two independent Matrigel sphere experiments. Nestin was abundantly expressed in the cytoplasm of vector control A2058-derived melanospheres grown in association with Matrigel (top), whereas decreased nestin protein levels in nestin KD A2058-derived spheres correlated with a localized submembranous pattern (bottom, arrows). Left panels: low magnification; right panels: high magnification. Scale bars: 10 µm. Note: * p<0.05, ** p<0.01, *** p<0.001.
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Figure 2: Nestin KD enhances melanoma invasion in vitro and decreases sphere formation(a) Note significant diminished cell migration of nestin KD cells (NesKD, solid lines) as compared to the vector control cells (Vec, dash lines) of A2058 (left; p<0.00001 at 6 and 8 hours, respectively) and A375 (right; p<0.00001 at 6 and 8 hours, respectively). (b) Representative fields of migrating A2058 (left) and A375 (right) cells in Matrigel inserts (top panel) and control inserts (bottom panel). Scale bars: 50 µm. (c) Nestin KD cells (black bars) had significantly higher Matrigel invasion than vector control cells (white bars) of A2058 (left, 2.54-fold, p<0.001) and A375 (right, 4.02-fold, p<0.001). (d) Matrigel invasion of A2058 and A375 nestin KD cells was characterized in the presence of MMP3-specific inhibitor UK356618 or the broad spectrum MMP inhibitor Marimastat. Compared to untreated A2058 nestin KD cells (black bar), UK356618 (20 nM, 33% decrease, p<0.05) and Marimastat (20 nM, 43% decrease, p<0.01) significantly suppressed Matrigel invasion of A2058 nestin KD cells. Combinatorial treatment of UK356618 (20 nM) and Marimastat (20 nM) additively suppressed Matrigel invasion of A2058 Nestin KD cells (56% decrease, p<0.001). Additionally, Matrigel invasion of A2058 nestin KD cells with combinatorial treatment of UK356618 and Marimastat was lower than monotherapy with UK356618 (65%, p=0.097). Likewise, in A375 nestin KD cells, Matrigel invasion of A375 nestin KD cells was not altered by UK356618 (20 nM), but was significantly suppressed by Marimastat (20 nM) (45% decrease, p<0.01) or by combinatorial treatment of Marimastat (20 nM) and UK356618 (20 nM) (55% decrease, p<0.001), as compared to untreated cells (black bar). Additionally, Marimastat monotherapy and combinatorial treatment of Marimastat and UK356618 had comparable suppressive effects on A375 nestin KD Matrigel invasion. Results were compared by one-way ANOVA test with Tukey’s multiple comparison test. Representative cell migration and invasion results are shown from at least three independent assays. (e) Representative images of A2058 and A375 melanospheres in spherogenic suspension culture in DMEM supplemented with 10% FBS and 0.5% methyl cellulose for 21 days. Melanospheres were visualized with p-iodonitrotetrazolium violet staining. Note marked decreased of sphere formation in both A2058 (top) and A375 (bottom) nestin KD cells as compared to vector control control cells (both p<0.01, respectively). Representative results are shown from two independent experiments. (f) H&E and IHC images of spheres growing over Matrigel. A2058 vector control spheres grew mainly on the surface of Matrigel (top), while nestin KD spheres invaded into the lattice of Matrigel (bottom). Scale bars: 50 µm Location of spheres on Matrigel. Majority (83.3%) of A2058 vector control spheres grew on the surface of Matrigel (white bar), while 16.7% partially invaded into Matrigel (gray bar). In contrast, 28.3% nestin KD spheres grew on the surface of Matrigel (white bar), 25% partially invaded into Matrigel (gray bar), and 46.7% completely invaded and embedded into Matrigel (black bar). Difference in Matrigel invasion between melanospheres derived from A2058 vector control (N=60) and Nestin KD (N=60) cells was significant (p<0.0001, Chi-square test). Data is represented from two independent Matrigel sphere experiments. Nestin was abundantly expressed in the cytoplasm of vector control A2058-derived melanospheres grown in association with Matrigel (top), whereas decreased nestin protein levels in nestin KD A2058-derived spheres correlated with a localized submembranous pattern (bottom, arrows). Left panels: low magnification; right panels: high magnification. Scale bars: 10 µm. Note: * p<0.05, ** p<0.01, *** p<0.001.

Mentions: The effects of nestin expression on cell migration and the ability of melanoma cells to traverse an ECM barrier facilitated by serum-mediated chemotaxis were next assessed. To control loading error, cells migrated through microporous transwell or Matrigel were normalized to total loaded cells or to non-Matrigel control insert, respectively. The migratory kinetics of both control melanoma cells and nestin KD melanoma cells through microporous transwell assays were similar, with increments for the first 6 hours, followed by gradual decrements thereafter, although a significantly lower percentage of nestin KD cells migrated by the 6 and 8 hour timepoints.(Figure 2a). However, nestin KD cells exhibited significantly greater ability to invade through ECM barriers containing collagen and basement membrane proteins (Matrigel-coated transwell membranes) when compared to vector controls after 18 hours (Figure 2b and c). Additional nestin shRNA construct transfected into A2058 cells further validated significantly enhanced Matrigel invasion in nestin KD cells and further ruled out the possibility of off-target effects of nestin shRNA on invasion (supplemental Figure 1d). The potential contribution of MMPs to nestin KD-enhanced invasion next was addressed using the MMP3-specific inhibitor UK356618 31, which suppressed invasion of A2058 nestin KD cells by 33%. The broad-spectrum MMP inhibitor, Marimastat 32, had a slightly greater effect (43% inhibition), and combination of UK356618 and Marimastat resulted in further enhancement of inhibition (56%) (Figure 2d). In A375 nestin KD cells where MMP3 protein was not upregulated in comparison to A2058 (Figure 1F), Matrigel invasion was suppressed by Marimastat by 45% but not by the MMP3 inhibitor, UK356618 (Figure 2d). These data collectively support a role for MMP induction in nestin KD cells in facilitating in vitro ECM invasion.


Nestin depletion induces melanoma matrix metalloproteinases and invasion.

Lee CW, Zhan Q, Lezcano C, Frank MH, Huang J, Larson AR, Lin JY, Wan MT, Lin PI, Ma J, Kleffel S, Schatton T, Lian CG, Murphy GF - Lab. Invest. (2014)

Nestin KD enhances melanoma invasion in vitro and decreases sphere formation(a) Note significant diminished cell migration of nestin KD cells (NesKD, solid lines) as compared to the vector control cells (Vec, dash lines) of A2058 (left; p<0.00001 at 6 and 8 hours, respectively) and A375 (right; p<0.00001 at 6 and 8 hours, respectively). (b) Representative fields of migrating A2058 (left) and A375 (right) cells in Matrigel inserts (top panel) and control inserts (bottom panel). Scale bars: 50 µm. (c) Nestin KD cells (black bars) had significantly higher Matrigel invasion than vector control cells (white bars) of A2058 (left, 2.54-fold, p<0.001) and A375 (right, 4.02-fold, p<0.001). (d) Matrigel invasion of A2058 and A375 nestin KD cells was characterized in the presence of MMP3-specific inhibitor UK356618 or the broad spectrum MMP inhibitor Marimastat. Compared to untreated A2058 nestin KD cells (black bar), UK356618 (20 nM, 33% decrease, p<0.05) and Marimastat (20 nM, 43% decrease, p<0.01) significantly suppressed Matrigel invasion of A2058 nestin KD cells. Combinatorial treatment of UK356618 (20 nM) and Marimastat (20 nM) additively suppressed Matrigel invasion of A2058 Nestin KD cells (56% decrease, p<0.001). Additionally, Matrigel invasion of A2058 nestin KD cells with combinatorial treatment of UK356618 and Marimastat was lower than monotherapy with UK356618 (65%, p=0.097). Likewise, in A375 nestin KD cells, Matrigel invasion of A375 nestin KD cells was not altered by UK356618 (20 nM), but was significantly suppressed by Marimastat (20 nM) (45% decrease, p<0.01) or by combinatorial treatment of Marimastat (20 nM) and UK356618 (20 nM) (55% decrease, p<0.001), as compared to untreated cells (black bar). Additionally, Marimastat monotherapy and combinatorial treatment of Marimastat and UK356618 had comparable suppressive effects on A375 nestin KD Matrigel invasion. Results were compared by one-way ANOVA test with Tukey’s multiple comparison test. Representative cell migration and invasion results are shown from at least three independent assays. (e) Representative images of A2058 and A375 melanospheres in spherogenic suspension culture in DMEM supplemented with 10% FBS and 0.5% methyl cellulose for 21 days. Melanospheres were visualized with p-iodonitrotetrazolium violet staining. Note marked decreased of sphere formation in both A2058 (top) and A375 (bottom) nestin KD cells as compared to vector control control cells (both p<0.01, respectively). Representative results are shown from two independent experiments. (f) H&E and IHC images of spheres growing over Matrigel. A2058 vector control spheres grew mainly on the surface of Matrigel (top), while nestin KD spheres invaded into the lattice of Matrigel (bottom). Scale bars: 50 µm Location of spheres on Matrigel. Majority (83.3%) of A2058 vector control spheres grew on the surface of Matrigel (white bar), while 16.7% partially invaded into Matrigel (gray bar). In contrast, 28.3% nestin KD spheres grew on the surface of Matrigel (white bar), 25% partially invaded into Matrigel (gray bar), and 46.7% completely invaded and embedded into Matrigel (black bar). Difference in Matrigel invasion between melanospheres derived from A2058 vector control (N=60) and Nestin KD (N=60) cells was significant (p<0.0001, Chi-square test). Data is represented from two independent Matrigel sphere experiments. Nestin was abundantly expressed in the cytoplasm of vector control A2058-derived melanospheres grown in association with Matrigel (top), whereas decreased nestin protein levels in nestin KD A2058-derived spheres correlated with a localized submembranous pattern (bottom, arrows). Left panels: low magnification; right panels: high magnification. Scale bars: 10 µm. Note: * p<0.05, ** p<0.01, *** p<0.001.
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Figure 2: Nestin KD enhances melanoma invasion in vitro and decreases sphere formation(a) Note significant diminished cell migration of nestin KD cells (NesKD, solid lines) as compared to the vector control cells (Vec, dash lines) of A2058 (left; p<0.00001 at 6 and 8 hours, respectively) and A375 (right; p<0.00001 at 6 and 8 hours, respectively). (b) Representative fields of migrating A2058 (left) and A375 (right) cells in Matrigel inserts (top panel) and control inserts (bottom panel). Scale bars: 50 µm. (c) Nestin KD cells (black bars) had significantly higher Matrigel invasion than vector control cells (white bars) of A2058 (left, 2.54-fold, p<0.001) and A375 (right, 4.02-fold, p<0.001). (d) Matrigel invasion of A2058 and A375 nestin KD cells was characterized in the presence of MMP3-specific inhibitor UK356618 or the broad spectrum MMP inhibitor Marimastat. Compared to untreated A2058 nestin KD cells (black bar), UK356618 (20 nM, 33% decrease, p<0.05) and Marimastat (20 nM, 43% decrease, p<0.01) significantly suppressed Matrigel invasion of A2058 nestin KD cells. Combinatorial treatment of UK356618 (20 nM) and Marimastat (20 nM) additively suppressed Matrigel invasion of A2058 Nestin KD cells (56% decrease, p<0.001). Additionally, Matrigel invasion of A2058 nestin KD cells with combinatorial treatment of UK356618 and Marimastat was lower than monotherapy with UK356618 (65%, p=0.097). Likewise, in A375 nestin KD cells, Matrigel invasion of A375 nestin KD cells was not altered by UK356618 (20 nM), but was significantly suppressed by Marimastat (20 nM) (45% decrease, p<0.01) or by combinatorial treatment of Marimastat (20 nM) and UK356618 (20 nM) (55% decrease, p<0.001), as compared to untreated cells (black bar). Additionally, Marimastat monotherapy and combinatorial treatment of Marimastat and UK356618 had comparable suppressive effects on A375 nestin KD Matrigel invasion. Results were compared by one-way ANOVA test with Tukey’s multiple comparison test. Representative cell migration and invasion results are shown from at least three independent assays. (e) Representative images of A2058 and A375 melanospheres in spherogenic suspension culture in DMEM supplemented with 10% FBS and 0.5% methyl cellulose for 21 days. Melanospheres were visualized with p-iodonitrotetrazolium violet staining. Note marked decreased of sphere formation in both A2058 (top) and A375 (bottom) nestin KD cells as compared to vector control control cells (both p<0.01, respectively). Representative results are shown from two independent experiments. (f) H&E and IHC images of spheres growing over Matrigel. A2058 vector control spheres grew mainly on the surface of Matrigel (top), while nestin KD spheres invaded into the lattice of Matrigel (bottom). Scale bars: 50 µm Location of spheres on Matrigel. Majority (83.3%) of A2058 vector control spheres grew on the surface of Matrigel (white bar), while 16.7% partially invaded into Matrigel (gray bar). In contrast, 28.3% nestin KD spheres grew on the surface of Matrigel (white bar), 25% partially invaded into Matrigel (gray bar), and 46.7% completely invaded and embedded into Matrigel (black bar). Difference in Matrigel invasion between melanospheres derived from A2058 vector control (N=60) and Nestin KD (N=60) cells was significant (p<0.0001, Chi-square test). Data is represented from two independent Matrigel sphere experiments. Nestin was abundantly expressed in the cytoplasm of vector control A2058-derived melanospheres grown in association with Matrigel (top), whereas decreased nestin protein levels in nestin KD A2058-derived spheres correlated with a localized submembranous pattern (bottom, arrows). Left panels: low magnification; right panels: high magnification. Scale bars: 10 µm. Note: * p<0.05, ** p<0.01, *** p<0.001.
Mentions: The effects of nestin expression on cell migration and the ability of melanoma cells to traverse an ECM barrier facilitated by serum-mediated chemotaxis were next assessed. To control loading error, cells migrated through microporous transwell or Matrigel were normalized to total loaded cells or to non-Matrigel control insert, respectively. The migratory kinetics of both control melanoma cells and nestin KD melanoma cells through microporous transwell assays were similar, with increments for the first 6 hours, followed by gradual decrements thereafter, although a significantly lower percentage of nestin KD cells migrated by the 6 and 8 hour timepoints.(Figure 2a). However, nestin KD cells exhibited significantly greater ability to invade through ECM barriers containing collagen and basement membrane proteins (Matrigel-coated transwell membranes) when compared to vector controls after 18 hours (Figure 2b and c). Additional nestin shRNA construct transfected into A2058 cells further validated significantly enhanced Matrigel invasion in nestin KD cells and further ruled out the possibility of off-target effects of nestin shRNA on invasion (supplemental Figure 1d). The potential contribution of MMPs to nestin KD-enhanced invasion next was addressed using the MMP3-specific inhibitor UK356618 31, which suppressed invasion of A2058 nestin KD cells by 33%. The broad-spectrum MMP inhibitor, Marimastat 32, had a slightly greater effect (43% inhibition), and combination of UK356618 and Marimastat resulted in further enhancement of inhibition (56%) (Figure 2d). In A375 nestin KD cells where MMP3 protein was not upregulated in comparison to A2058 (Figure 1F), Matrigel invasion was suppressed by Marimastat by 45% but not by the MMP3 inhibitor, UK356618 (Figure 2d). These data collectively support a role for MMP induction in nestin KD cells in facilitating in vitro ECM invasion.

Bottom Line: Matrix metalloproteinases (MMPs) are key biological mediators of processes as diverse as wound healing, embryogenesis, and cancer progression.Although MMPs may be induced through multiple signaling pathways, the precise mechanisms for their regulation in cancer are incompletely understood.The results suggest that the heretofore poorly understood intermediate filament, nestin, may serve as a novel mediator of MMPs critical to melanoma virulence.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

ABSTRACT
Matrix metalloproteinases (MMPs) are key biological mediators of processes as diverse as wound healing, embryogenesis, and cancer progression. Although MMPs may be induced through multiple signaling pathways, the precise mechanisms for their regulation in cancer are incompletely understood. Because cytoskeletal changes are known to accompany MMP expression, we sought to examine the potential role of the poorly understood cytoskeletal protein, nestin, in modulating melanoma MMPs. Nestin knockdown (KD) upregulated the expression of specific MMPs and MMP-dependent invasion both through extracellular matrix barriers in vitro and in peritumoral connective tissue of xenografts in vivo. The development of three-dimensional melanospheres that in vitro partially recapitulate noninvasive tumorigenic melanoma growth was inhibited by nestin KD, although ECM invasion by aberrant melanospheres that did form was enhanced. Mechanistically, nestin KD-dependent melanoma invasion was associated with intracellular redistribution of phosphorylated focal adhesion kinase and increased melanoma cell responsiveness to transforming growth factor-beta, both implicated in pathways of melanoma invasion. The results suggest that the heretofore poorly understood intermediate filament, nestin, may serve as a novel mediator of MMPs critical to melanoma virulence.

Show MeSH
Related in: MedlinePlus