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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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Knockdown or overexpression of SLUG modulates E-cadherin and P-cadherin adhesion molecules.(A) Expression levels of EMT markers in SLUG-depleted cells: 501mel cells were transfected with control siRNA (siCTRL) for 4 days or two SLUG siRNAs (siSLUG #1 and #2) for the indicated times. Expression levels of SLUG, E-cadherin, P-cadherin and Fibronectin were analyzed by immunoblotting. HSP60 was used as loading control. (B) Morphology of SLUG-depleted cells: expression of SLUG (green), E-cadherin (cyan) and actin cytoskeleton (Texas Red-X phalloidin) following siRNA-mediated SLUG depletion in 501mel cells was analyzed by fluorescence staining and confocal microscopy. Bars, 10 µm. (C) Ectopic SLUG expression induces an EMT-like phenotype: control (bl. CTRL) or SLUG-overexpressing (bl. SLUG #1 and #2) 501mel cell populations were analyzed by immunoblotting for expression of SLUG, E-cadherin, P-cadherin, Fibronectin and HSP60 (loading control). (D) Depletion of SLUG increases Ca2+-dependent cell-cell adhesion: adhesion assays were performed as described in the Materials and Methods after treatment of 501mel cells with siCTRL or siSLUG as indicated. The phase-contrast pictures show aggregates formed in presence of 1 mmol/L CaCl2 alone or with 3 mmol/L EDTA. The average of two independent adhesion assays and SD are presented. Columns, average of two independent assays; error bars, SD. *P<0.05 (Student’s test). (E) SLUG regulates E-cadherin mRNA levels: RNAs were prepared from 501mel cells transfected with siCTRL or siSLUG for 4 days, and from bulk selected control or SLUG-overexpressing 501mel cells. The relative mRNA expression levels of SLUG and E-cadherin were measured by SYBR green-based real-time Q-PCR. Columns, mean of three independent amplifications performed in duplicate; error bars, SD. *P<0.05 (Student’s test). (F) E-cadherin promoter activity: 501mel cells were transfected with siCTRL, siSPARC or siSLUG, and 24 hours later with wild-type E-cadherin promoter reporter construct (left). 501mel cells were co-transfected with an empty vector (mock) or vectors expressing SPARC or SLUG and wild-type (−178 wt/luc) or mutant (mE-pal/luc) E-cadherin promoter reporter constructs (right). After 3 days, luciferase activities were measured and normalized to β-galactosidase activities. Columns, mean of triplicates; errors bars, SD.
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pone-0040378-g005: Knockdown or overexpression of SLUG modulates E-cadherin and P-cadherin adhesion molecules.(A) Expression levels of EMT markers in SLUG-depleted cells: 501mel cells were transfected with control siRNA (siCTRL) for 4 days or two SLUG siRNAs (siSLUG #1 and #2) for the indicated times. Expression levels of SLUG, E-cadherin, P-cadherin and Fibronectin were analyzed by immunoblotting. HSP60 was used as loading control. (B) Morphology of SLUG-depleted cells: expression of SLUG (green), E-cadherin (cyan) and actin cytoskeleton (Texas Red-X phalloidin) following siRNA-mediated SLUG depletion in 501mel cells was analyzed by fluorescence staining and confocal microscopy. Bars, 10 µm. (C) Ectopic SLUG expression induces an EMT-like phenotype: control (bl. CTRL) or SLUG-overexpressing (bl. SLUG #1 and #2) 501mel cell populations were analyzed by immunoblotting for expression of SLUG, E-cadherin, P-cadherin, Fibronectin and HSP60 (loading control). (D) Depletion of SLUG increases Ca2+-dependent cell-cell adhesion: adhesion assays were performed as described in the Materials and Methods after treatment of 501mel cells with siCTRL or siSLUG as indicated. The phase-contrast pictures show aggregates formed in presence of 1 mmol/L CaCl2 alone or with 3 mmol/L EDTA. The average of two independent adhesion assays and SD are presented. Columns, average of two independent assays; error bars, SD. *P<0.05 (Student’s test). (E) SLUG regulates E-cadherin mRNA levels: RNAs were prepared from 501mel cells transfected with siCTRL or siSLUG for 4 days, and from bulk selected control or SLUG-overexpressing 501mel cells. The relative mRNA expression levels of SLUG and E-cadherin were measured by SYBR green-based real-time Q-PCR. Columns, mean of three independent amplifications performed in duplicate; error bars, SD. *P<0.05 (Student’s test). (F) E-cadherin promoter activity: 501mel cells were transfected with siCTRL, siSPARC or siSLUG, and 24 hours later with wild-type E-cadherin promoter reporter construct (left). 501mel cells were co-transfected with an empty vector (mock) or vectors expressing SPARC or SLUG and wild-type (−178 wt/luc) or mutant (mE-pal/luc) E-cadherin promoter reporter constructs (right). After 3 days, luciferase activities were measured and normalized to β-galactosidase activities. Columns, mean of triplicates; errors bars, SD.

Mentions: To assess the role of SLUG in the control of the cell-cell adhesion molecules E- and P-cadherins in melanoma cells, we carried out knockdown experiments using two differents non-overlapping siRNAs specifically targeting SLUG, and gain-of-function experiments where 501mel cells were stably transfected with SLUG. As shown in Figure 5A, knockdown of SLUG led to regained expression of both E- and P-cadherins in a time-dependent manner. A slight decrease in Fibronectin protein expression was also observed in SLUG silenced 501mel cells compared to control siRNA-treated cells. Increased expression of E-cadherin in SLUG knockdown cells was also confirmed by immunofluorescence analysis (Figure 5B). In addition, SLUG-deficient cells adopted a rounded morphology with an altered actin cytoskeleton and a loss of the characteristic fibroblastic-like morphology, as revealed by fluorescent F-actin staining (Figure 5B). In SLUG-overexpressing 501mel cell populations, we found reduced E- and P-cadherins protein levels and increased Fibronectin expression (Figure 5C). We next tested if SLUG depletion would have a functional impact on Ca2+-dependent cell-cell adherence. 501mel cells were transfected with control or SLUG siRNA and an in vitro cell-cell adhesion assay was performed in presence or absence of Ca2+ ions (+ EDTA). The results shown in Figure 5D revealed an increase in cell aggregates from SLUG-depleted cells compared to control cells, and that formation of cell aggregates was prevented upon treatment with the chelating agent EDTA. Thus, knockdown of SLUG increases Ca2+-dependent cell-cell adhesion. In the next series of experiments, we confirmed that SLUG is a transcriptional repressor of E-cadherin in 501mel cells [26], [39]. Analysis of E-cadherin mRNA levels by real-time Q-PCR upon SLUG depletion or ectopic expression showed an increase or decrease of E-cadherin transcript, respectively (Figure 5E). We also examined the effect of SLUG on the activity of the mouse E-cadherin promoter in 501mel cells (Figure 5F). Luciferase reporter assays show that depletion of SPARC or SLUG by siRNA increased the activity of the E-cadherin promoter and conversely, expression of SPARC or SLUG repressed promoter activity. No repression was seen when the E-cadherin promoter is mutated in the E-box elements (mE-pal construct). Similar responses to SLUG knockdown or SLUG ectopic expression were seen in other melanoma cell lines (Figure S2).


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)

Knockdown or overexpression of SLUG modulates E-cadherin and P-cadherin adhesion molecules.(A) Expression levels of EMT markers in SLUG-depleted cells: 501mel cells were transfected with control siRNA (siCTRL) for 4 days or two SLUG siRNAs (siSLUG #1 and #2) for the indicated times. Expression levels of SLUG, E-cadherin, P-cadherin and Fibronectin were analyzed by immunoblotting. HSP60 was used as loading control. (B) Morphology of SLUG-depleted cells: expression of SLUG (green), E-cadherin (cyan) and actin cytoskeleton (Texas Red-X phalloidin) following siRNA-mediated SLUG depletion in 501mel cells was analyzed by fluorescence staining and confocal microscopy. Bars, 10 µm. (C) Ectopic SLUG expression induces an EMT-like phenotype: control (bl. CTRL) or SLUG-overexpressing (bl. SLUG #1 and #2) 501mel cell populations were analyzed by immunoblotting for expression of SLUG, E-cadherin, P-cadherin, Fibronectin and HSP60 (loading control). (D) Depletion of SLUG increases Ca2+-dependent cell-cell adhesion: adhesion assays were performed as described in the Materials and Methods after treatment of 501mel cells with siCTRL or siSLUG as indicated. The phase-contrast pictures show aggregates formed in presence of 1 mmol/L CaCl2 alone or with 3 mmol/L EDTA. The average of two independent adhesion assays and SD are presented. Columns, average of two independent assays; error bars, SD. *P<0.05 (Student’s test). (E) SLUG regulates E-cadherin mRNA levels: RNAs were prepared from 501mel cells transfected with siCTRL or siSLUG for 4 days, and from bulk selected control or SLUG-overexpressing 501mel cells. The relative mRNA expression levels of SLUG and E-cadherin were measured by SYBR green-based real-time Q-PCR. Columns, mean of three independent amplifications performed in duplicate; error bars, SD. *P<0.05 (Student’s test). (F) E-cadherin promoter activity: 501mel cells were transfected with siCTRL, siSPARC or siSLUG, and 24 hours later with wild-type E-cadherin promoter reporter construct (left). 501mel cells were co-transfected with an empty vector (mock) or vectors expressing SPARC or SLUG and wild-type (−178 wt/luc) or mutant (mE-pal/luc) E-cadherin promoter reporter constructs (right). After 3 days, luciferase activities were measured and normalized to β-galactosidase activities. Columns, mean of triplicates; errors bars, SD.
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pone-0040378-g005: Knockdown or overexpression of SLUG modulates E-cadherin and P-cadherin adhesion molecules.(A) Expression levels of EMT markers in SLUG-depleted cells: 501mel cells were transfected with control siRNA (siCTRL) for 4 days or two SLUG siRNAs (siSLUG #1 and #2) for the indicated times. Expression levels of SLUG, E-cadherin, P-cadherin and Fibronectin were analyzed by immunoblotting. HSP60 was used as loading control. (B) Morphology of SLUG-depleted cells: expression of SLUG (green), E-cadherin (cyan) and actin cytoskeleton (Texas Red-X phalloidin) following siRNA-mediated SLUG depletion in 501mel cells was analyzed by fluorescence staining and confocal microscopy. Bars, 10 µm. (C) Ectopic SLUG expression induces an EMT-like phenotype: control (bl. CTRL) or SLUG-overexpressing (bl. SLUG #1 and #2) 501mel cell populations were analyzed by immunoblotting for expression of SLUG, E-cadherin, P-cadherin, Fibronectin and HSP60 (loading control). (D) Depletion of SLUG increases Ca2+-dependent cell-cell adhesion: adhesion assays were performed as described in the Materials and Methods after treatment of 501mel cells with siCTRL or siSLUG as indicated. The phase-contrast pictures show aggregates formed in presence of 1 mmol/L CaCl2 alone or with 3 mmol/L EDTA. The average of two independent adhesion assays and SD are presented. Columns, average of two independent assays; error bars, SD. *P<0.05 (Student’s test). (E) SLUG regulates E-cadherin mRNA levels: RNAs were prepared from 501mel cells transfected with siCTRL or siSLUG for 4 days, and from bulk selected control or SLUG-overexpressing 501mel cells. The relative mRNA expression levels of SLUG and E-cadherin were measured by SYBR green-based real-time Q-PCR. Columns, mean of three independent amplifications performed in duplicate; error bars, SD. *P<0.05 (Student’s test). (F) E-cadherin promoter activity: 501mel cells were transfected with siCTRL, siSPARC or siSLUG, and 24 hours later with wild-type E-cadherin promoter reporter construct (left). 501mel cells were co-transfected with an empty vector (mock) or vectors expressing SPARC or SLUG and wild-type (−178 wt/luc) or mutant (mE-pal/luc) E-cadherin promoter reporter constructs (right). After 3 days, luciferase activities were measured and normalized to β-galactosidase activities. Columns, mean of triplicates; errors bars, SD.
Mentions: To assess the role of SLUG in the control of the cell-cell adhesion molecules E- and P-cadherins in melanoma cells, we carried out knockdown experiments using two differents non-overlapping siRNAs specifically targeting SLUG, and gain-of-function experiments where 501mel cells were stably transfected with SLUG. As shown in Figure 5A, knockdown of SLUG led to regained expression of both E- and P-cadherins in a time-dependent manner. A slight decrease in Fibronectin protein expression was also observed in SLUG silenced 501mel cells compared to control siRNA-treated cells. Increased expression of E-cadherin in SLUG knockdown cells was also confirmed by immunofluorescence analysis (Figure 5B). In addition, SLUG-deficient cells adopted a rounded morphology with an altered actin cytoskeleton and a loss of the characteristic fibroblastic-like morphology, as revealed by fluorescent F-actin staining (Figure 5B). In SLUG-overexpressing 501mel cell populations, we found reduced E- and P-cadherins protein levels and increased Fibronectin expression (Figure 5C). We next tested if SLUG depletion would have a functional impact on Ca2+-dependent cell-cell adherence. 501mel cells were transfected with control or SLUG siRNA and an in vitro cell-cell adhesion assay was performed in presence or absence of Ca2+ ions (+ EDTA). The results shown in Figure 5D revealed an increase in cell aggregates from SLUG-depleted cells compared to control cells, and that formation of cell aggregates was prevented upon treatment with the chelating agent EDTA. Thus, knockdown of SLUG increases Ca2+-dependent cell-cell adhesion. In the next series of experiments, we confirmed that SLUG is a transcriptional repressor of E-cadherin in 501mel cells [26], [39]. Analysis of E-cadherin mRNA levels by real-time Q-PCR upon SLUG depletion or ectopic expression showed an increase or decrease of E-cadherin transcript, respectively (Figure 5E). We also examined the effect of SLUG on the activity of the mouse E-cadherin promoter in 501mel cells (Figure 5F). Luciferase reporter assays show that depletion of SPARC or SLUG by siRNA increased the activity of the E-cadherin promoter and conversely, expression of SPARC or SLUG repressed promoter activity. No repression was seen when the E-cadherin promoter is mutated in the E-box elements (mE-pal construct). Similar responses to SLUG knockdown or SLUG ectopic expression were seen in other melanoma cell lines (Figure S2).

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