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The epithelial-mesenchymal transition (EMT) regulatory factor SLUG (SNAI2) is a downstream target of SPARC and AKT in promoting melanoma cell invasion.

Fenouille N, Tichet M, Dufies M, Pottier A, Mogha A, Soo JK, Rocchi S, Mallavialle A, Galibert MD, Khammari A, Lacour JP, Ballotti R, Deckert M, Tartare-Deckert S - PLoS ONE (2012)

Bottom Line: Here, we report that SLUG expression and activation is driven by SPARC (also known as osteonectin), a secreted extracellular matrix-associated factor that promotes EMT-like changes.Ectopic expression or knockdown of SPARC resulted in increased or reduced expression of SLUG, respectively.In freshly isolated metastatic melanoma cells, a positive association between SPARC and SLUG mRNA levels was also found.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U1065, Centre Méditerranéen de Médecine Moléculaire (C3M), Biologie et Pathologies des Mélanocytes, Nice, France.

ABSTRACT
During progression of melanoma, malignant melanocytes can be reprogrammed into mesenchymal-like cells through a process similar to epithelial-mesenchymal transition (EMT), which is associated with downregulation of the junctional protein E-cadherin and acquisition of a migratory phenotype. Recent evidence supports a role for SLUG, a transcriptional repressor of E-cadherin, as a melanocyte lineage transcription factor that predisposes to melanoma metastasis. However, the signals responsible for SLUG expression in melanoma are unclear and its role in the invasive phenotype is not fully elucidated. Here, we report that SLUG expression and activation is driven by SPARC (also known as osteonectin), a secreted extracellular matrix-associated factor that promotes EMT-like changes. Ectopic expression or knockdown of SPARC resulted in increased or reduced expression of SLUG, respectively. SLUG increase occurred concomitantly with SPARC-mediated downregulation of E-cadherin and P-cadherin, and induction of mesenchymal traits in human melanocytes and melanoma cells. Pharmacological blockade of PI3 kinase/AKT signaling impeded SPARC-induced SLUG levels and cell migration, whereas adenoviral introduction of constitutively active AKT allowed rescue of SLUG and migratory capabilities of SPARC knockdown cells. We also observed that pharmacological inhibition of oncogenic BRAF(V600E) using PLX4720 did not influence SLUG expression in melanoma cells harboring BRAF(V600E). Furthermore, SLUG is a bona fide transcriptional repressor of E-cadherin as well as a regulator of P-cadherin in melanoma cells and its knockdown attenuated invasive behavior and blocked SPARC-enhanced cell migration. Notably, inhibition of cell migration in SPARC-depleted cells was rescued by expression of a SLUG transgene. In freshly isolated metastatic melanoma cells, a positive association between SPARC and SLUG mRNA levels was also found. These findings reveal that autocrine SPARC maintains heightened SLUG expression in melanoma cells and indicate that SPARC may promote EMT-associated tumor invasion by supporting AKT-dependent upregulation of SLUG.

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siRNA-mediated knockdown of SPARC or SLUG and inhibition of AKT signaling reduce invasion into 3-dimensional collagen matrix.(A) Depletion of SPARC or SLUG decreases tumor spheroid invasion: preformed melanoma spheroids of 501mel, WM9 and WM793 cells transfected with siCTRL, siSPARC or siSLUG as indicated were implanted into a gel of collagen type I. Spheroids were incubated in growth medium for 3 days and tumor cell outgrowth was visualized by phase contrast microscopy. (B) Influence of PI3K/AKT signaling on spheroid invasion: preformed melanoma spheroids of 501mel, WM9 and WM793 were implanted into a gel of collagen type I and incubated in growth medium for 3 days with with DMSO or 10 mmol/L AIIV or LY294002. Tumor cell invasion was visualized as above. Representative example of spheroids from each culture is shown.
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pone-0040378-g006: siRNA-mediated knockdown of SPARC or SLUG and inhibition of AKT signaling reduce invasion into 3-dimensional collagen matrix.(A) Depletion of SPARC or SLUG decreases tumor spheroid invasion: preformed melanoma spheroids of 501mel, WM9 and WM793 cells transfected with siCTRL, siSPARC or siSLUG as indicated were implanted into a gel of collagen type I. Spheroids were incubated in growth medium for 3 days and tumor cell outgrowth was visualized by phase contrast microscopy. (B) Influence of PI3K/AKT signaling on spheroid invasion: preformed melanoma spheroids of 501mel, WM9 and WM793 were implanted into a gel of collagen type I and incubated in growth medium for 3 days with with DMSO or 10 mmol/L AIIV or LY294002. Tumor cell invasion was visualized as above. Representative example of spheroids from each culture is shown.

Mentions: Tumor migration was then analyzed in a more physiological context; 501mel, WM9 or WM793 cells were grown as spheroids embedded in collagen, an assay that mimics 3-dimensional growth and invasion by melanoma cells [40]. Knockdown of SPARC or SLUG had no effect on spheroid growth rate but significantly reduced cell invasion into collagen (Figure 6A). Thus, SPARC and SLUG expression is required for tumor invasion in 3-dimensional cultures. Consistent with the regulation of SLUG by the PI3 kinase/AKT pathway (see Figure 4), pharmacologic inhibition of this signaling pathway by LY294002 or AIIV inhibited the invasive phenotype of melanoma spheroids into collagen (Figure 6B).


The epithelial-mesenchymal transition (EMT) regulatory factor SLUG (SNAI2) is a downstream target of SPARC and AKT in promoting melanoma cell invasion.

Fenouille N, Tichet M, Dufies M, Pottier A, Mogha A, Soo JK, Rocchi S, Mallavialle A, Galibert MD, Khammari A, Lacour JP, Ballotti R, Deckert M, Tartare-Deckert S - PLoS ONE (2012)

siRNA-mediated knockdown of SPARC or SLUG and inhibition of AKT signaling reduce invasion into 3-dimensional collagen matrix.(A) Depletion of SPARC or SLUG decreases tumor spheroid invasion: preformed melanoma spheroids of 501mel, WM9 and WM793 cells transfected with siCTRL, siSPARC or siSLUG as indicated were implanted into a gel of collagen type I. Spheroids were incubated in growth medium for 3 days and tumor cell outgrowth was visualized by phase contrast microscopy. (B) Influence of PI3K/AKT signaling on spheroid invasion: preformed melanoma spheroids of 501mel, WM9 and WM793 were implanted into a gel of collagen type I and incubated in growth medium for 3 days with with DMSO or 10 mmol/L AIIV or LY294002. Tumor cell invasion was visualized as above. Representative example of spheroids from each culture is shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3401237&req=5

pone-0040378-g006: siRNA-mediated knockdown of SPARC or SLUG and inhibition of AKT signaling reduce invasion into 3-dimensional collagen matrix.(A) Depletion of SPARC or SLUG decreases tumor spheroid invasion: preformed melanoma spheroids of 501mel, WM9 and WM793 cells transfected with siCTRL, siSPARC or siSLUG as indicated were implanted into a gel of collagen type I. Spheroids were incubated in growth medium for 3 days and tumor cell outgrowth was visualized by phase contrast microscopy. (B) Influence of PI3K/AKT signaling on spheroid invasion: preformed melanoma spheroids of 501mel, WM9 and WM793 were implanted into a gel of collagen type I and incubated in growth medium for 3 days with with DMSO or 10 mmol/L AIIV or LY294002. Tumor cell invasion was visualized as above. Representative example of spheroids from each culture is shown.
Mentions: Tumor migration was then analyzed in a more physiological context; 501mel, WM9 or WM793 cells were grown as spheroids embedded in collagen, an assay that mimics 3-dimensional growth and invasion by melanoma cells [40]. Knockdown of SPARC or SLUG had no effect on spheroid growth rate but significantly reduced cell invasion into collagen (Figure 6A). Thus, SPARC and SLUG expression is required for tumor invasion in 3-dimensional cultures. Consistent with the regulation of SLUG by the PI3 kinase/AKT pathway (see Figure 4), pharmacologic inhibition of this signaling pathway by LY294002 or AIIV inhibited the invasive phenotype of melanoma spheroids into collagen (Figure 6B).

Bottom Line: Here, we report that SLUG expression and activation is driven by SPARC (also known as osteonectin), a secreted extracellular matrix-associated factor that promotes EMT-like changes.Ectopic expression or knockdown of SPARC resulted in increased or reduced expression of SLUG, respectively.In freshly isolated metastatic melanoma cells, a positive association between SPARC and SLUG mRNA levels was also found.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U1065, Centre Méditerranéen de Médecine Moléculaire (C3M), Biologie et Pathologies des Mélanocytes, Nice, France.

ABSTRACT
During progression of melanoma, malignant melanocytes can be reprogrammed into mesenchymal-like cells through a process similar to epithelial-mesenchymal transition (EMT), which is associated with downregulation of the junctional protein E-cadherin and acquisition of a migratory phenotype. Recent evidence supports a role for SLUG, a transcriptional repressor of E-cadherin, as a melanocyte lineage transcription factor that predisposes to melanoma metastasis. However, the signals responsible for SLUG expression in melanoma are unclear and its role in the invasive phenotype is not fully elucidated. Here, we report that SLUG expression and activation is driven by SPARC (also known as osteonectin), a secreted extracellular matrix-associated factor that promotes EMT-like changes. Ectopic expression or knockdown of SPARC resulted in increased or reduced expression of SLUG, respectively. SLUG increase occurred concomitantly with SPARC-mediated downregulation of E-cadherin and P-cadherin, and induction of mesenchymal traits in human melanocytes and melanoma cells. Pharmacological blockade of PI3 kinase/AKT signaling impeded SPARC-induced SLUG levels and cell migration, whereas adenoviral introduction of constitutively active AKT allowed rescue of SLUG and migratory capabilities of SPARC knockdown cells. We also observed that pharmacological inhibition of oncogenic BRAF(V600E) using PLX4720 did not influence SLUG expression in melanoma cells harboring BRAF(V600E). Furthermore, SLUG is a bona fide transcriptional repressor of E-cadherin as well as a regulator of P-cadherin in melanoma cells and its knockdown attenuated invasive behavior and blocked SPARC-enhanced cell migration. Notably, inhibition of cell migration in SPARC-depleted cells was rescued by expression of a SLUG transgene. In freshly isolated metastatic melanoma cells, a positive association between SPARC and SLUG mRNA levels was also found. These findings reveal that autocrine SPARC maintains heightened SLUG expression in melanoma cells and indicate that SPARC may promote EMT-associated tumor invasion by supporting AKT-dependent upregulation of SLUG.

Show MeSH
Related in: MedlinePlus