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Down-regulation of CYLD expression by Snail promotes tumor progression in malignant melanoma.

Massoumi R, Kuphal S, Hellerbrand C, Haas B, Wild P, Spruss T, Pfeifer A, Fässler R, Bosserhoff AK - J. Exp. Med. (2009)

Bottom Line: Rescue of CYLD expression in melanoma cells reduced proliferation and invasion in vitro and tumor growth and metastasis in vivo.Importantly, tumor thickness and progression-free and overall survival inversely correlated with CYLD expression.Our data suggest that Snail1-mediated suppression of CYLD plays a key role in melanoma malignancy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

ABSTRACT
High malignancy and early metastasis are hallmarks of melanoma. Here, we report that the transcription factor Snail1 inhibits expression of the tumor suppressor CYLD in melanoma. As a direct consequence of CYLD repression, the protooncogene BCL-3 translocates into the nucleus and activates Cyclin D1 and N-cadherin promoters, resulting in proliferation and invasion of melanoma cells. Rescue of CYLD expression in melanoma cells reduced proliferation and invasion in vitro and tumor growth and metastasis in vivo. Analysis of a tissue microarray with primary melanomas from patients revealed an inverse correlation of Snail1 induction and loss of CYLD expression. Importantly, tumor thickness and progression-free and overall survival inversely correlated with CYLD expression. Our data suggest that Snail1-mediated suppression of CYLD plays a key role in melanoma malignancy.

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Snail1 inhibits transcriptional expression of CYLD in melanocytic cells. (A) Lysates from NHEMs, primary melanoma cells (MM1 and MM2), and melanoma cell lines (Mel Im, Mel Juso) were examined by ChIP assay using an anti-Snail1, anti-Twist, and anti-Snail2–specific antibody, respectively, as well as a PCR primer pair corresponding to the promoter of the CYLD gene. Recruitment to the CYLD promoter was only shown for Snail1. Snail1, Twist, Snail2, immunoprecipitation (IP) using specific antibodies; IgG, IP using negative control rabbit immunoglobulin; Input, 10% of the cell lysate used for the IP is shown. (B) Reporter assays revealing inducible CYLD promoter (−550 to −1 bp) activity in Mel Im cells after transient transfection with asSnail (asSnail), whereas transfection with Snail1 expression plasmid (Snail1) completely abrogated CYLD promoter activity compared with the control (pGL3). Mutation of the consensus Snail1 binding site (CYLD mut-19) led to strong promoter activity that was not affected by transfection with asSnail or Snail1. Data are given as mean ± SEM. *, P < 0.05 compared with CYLD control. (C) Immunoblot analysis showing that CYLD protein is up-regulated in Mel Im cells after stable transfection with asSnail expression plasmids (clone 1 and clone 2; top) and after transient transduction with Snail1 siRNA nucleotides (siRNA Snail1) or scrambled siRNA control (siRNA control; bottom). Nontransduced Mel Im cells were used as control. (D) Quantitative RT-PCR analysis showing increased CYLD mRNA expression in three different melanoma cell lines (Mel Im, Mel Ei, and Mel Juso) and melanoma cells (MM1 and MM2) transiently transfected with asSnail expression plasmids compared with mock-transfected (pcDNA3) cells. Data are given as mean ± SEM. *, P < 0.05. (E) Western blot analysis showing decreased CYLD protein expression in NHEM cells transiently transfected with Snail1 expression plasmids compared with mock-transfected (pcDNA3) or nontransfected (NHEM) cells. (F) ERK inhibition by treatment with MEK inhibitors PD98059 and UO126 significantly affects Snail1 and CYLD mRNA expression in Mel Im cells. JNK inhibition (SP600125) exhibits no effect compared with vehicle (DMSO) control. Data are given as mean ± SEM. *, P < 0.05. ns, not significant, both compared with Control. (G) Quantitative RT-PCR revealing that inhibition of ERK by PD98059 or UO126 further induced CYLD expression in melanocytes. JNK inhibition (SP600125) exhibits no effect compared with vehicle (DMSO) control. Data are given as mean ± SEM. *, P < 0.05. Experiments in A–G were repeated at least three times. (H) Detection of CYLD expression by Western blot analysis. Transfection of NHEMs with mutated BRAF (V600E) induced Snail1 protein expression and, consequently, CYLD protein suppression, whereas no changes were found after transfection of wild-type BRAF (wtBRAF). Experiments were repeated three times with cells from two different donors.
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fig2: Snail1 inhibits transcriptional expression of CYLD in melanocytic cells. (A) Lysates from NHEMs, primary melanoma cells (MM1 and MM2), and melanoma cell lines (Mel Im, Mel Juso) were examined by ChIP assay using an anti-Snail1, anti-Twist, and anti-Snail2–specific antibody, respectively, as well as a PCR primer pair corresponding to the promoter of the CYLD gene. Recruitment to the CYLD promoter was only shown for Snail1. Snail1, Twist, Snail2, immunoprecipitation (IP) using specific antibodies; IgG, IP using negative control rabbit immunoglobulin; Input, 10% of the cell lysate used for the IP is shown. (B) Reporter assays revealing inducible CYLD promoter (−550 to −1 bp) activity in Mel Im cells after transient transfection with asSnail (asSnail), whereas transfection with Snail1 expression plasmid (Snail1) completely abrogated CYLD promoter activity compared with the control (pGL3). Mutation of the consensus Snail1 binding site (CYLD mut-19) led to strong promoter activity that was not affected by transfection with asSnail or Snail1. Data are given as mean ± SEM. *, P < 0.05 compared with CYLD control. (C) Immunoblot analysis showing that CYLD protein is up-regulated in Mel Im cells after stable transfection with asSnail expression plasmids (clone 1 and clone 2; top) and after transient transduction with Snail1 siRNA nucleotides (siRNA Snail1) or scrambled siRNA control (siRNA control; bottom). Nontransduced Mel Im cells were used as control. (D) Quantitative RT-PCR analysis showing increased CYLD mRNA expression in three different melanoma cell lines (Mel Im, Mel Ei, and Mel Juso) and melanoma cells (MM1 and MM2) transiently transfected with asSnail expression plasmids compared with mock-transfected (pcDNA3) cells. Data are given as mean ± SEM. *, P < 0.05. (E) Western blot analysis showing decreased CYLD protein expression in NHEM cells transiently transfected with Snail1 expression plasmids compared with mock-transfected (pcDNA3) or nontransfected (NHEM) cells. (F) ERK inhibition by treatment with MEK inhibitors PD98059 and UO126 significantly affects Snail1 and CYLD mRNA expression in Mel Im cells. JNK inhibition (SP600125) exhibits no effect compared with vehicle (DMSO) control. Data are given as mean ± SEM. *, P < 0.05. ns, not significant, both compared with Control. (G) Quantitative RT-PCR revealing that inhibition of ERK by PD98059 or UO126 further induced CYLD expression in melanocytes. JNK inhibition (SP600125) exhibits no effect compared with vehicle (DMSO) control. Data are given as mean ± SEM. *, P < 0.05. Experiments in A–G were repeated at least three times. (H) Detection of CYLD expression by Western blot analysis. Transfection of NHEMs with mutated BRAF (V600E) induced Snail1 protein expression and, consequently, CYLD protein suppression, whereas no changes were found after transfection of wild-type BRAF (wtBRAF). Experiments were repeated three times with cells from two different donors.

Mentions: Chromatin immunoprecipitation (ChIP) assays demonstrated Snail1 binding to the CYLD promoter in primary melanoma cells and the melanoma cell lines Mel Im and Mel Juso, but not in NHEM (Fig. 2 A). Furthermore, neither Snail2 nor Twist (another E-box binding factor associated with poor prognosis in melanoma [22]) bound to the CYLD promoter in ChIP assays (Fig. 2 A). Snail1 recruitment to the CYLD promoter was observed to the first E-box at position −19 to −14, which resembles the consensus Snail1 binding sequence (CAGGTG; Fig. S1), but not to E-box II (-382 to -377) or III (−429 to −424; unpublished data).


Down-regulation of CYLD expression by Snail promotes tumor progression in malignant melanoma.

Massoumi R, Kuphal S, Hellerbrand C, Haas B, Wild P, Spruss T, Pfeifer A, Fässler R, Bosserhoff AK - J. Exp. Med. (2009)

Snail1 inhibits transcriptional expression of CYLD in melanocytic cells. (A) Lysates from NHEMs, primary melanoma cells (MM1 and MM2), and melanoma cell lines (Mel Im, Mel Juso) were examined by ChIP assay using an anti-Snail1, anti-Twist, and anti-Snail2–specific antibody, respectively, as well as a PCR primer pair corresponding to the promoter of the CYLD gene. Recruitment to the CYLD promoter was only shown for Snail1. Snail1, Twist, Snail2, immunoprecipitation (IP) using specific antibodies; IgG, IP using negative control rabbit immunoglobulin; Input, 10% of the cell lysate used for the IP is shown. (B) Reporter assays revealing inducible CYLD promoter (−550 to −1 bp) activity in Mel Im cells after transient transfection with asSnail (asSnail), whereas transfection with Snail1 expression plasmid (Snail1) completely abrogated CYLD promoter activity compared with the control (pGL3). Mutation of the consensus Snail1 binding site (CYLD mut-19) led to strong promoter activity that was not affected by transfection with asSnail or Snail1. Data are given as mean ± SEM. *, P < 0.05 compared with CYLD control. (C) Immunoblot analysis showing that CYLD protein is up-regulated in Mel Im cells after stable transfection with asSnail expression plasmids (clone 1 and clone 2; top) and after transient transduction with Snail1 siRNA nucleotides (siRNA Snail1) or scrambled siRNA control (siRNA control; bottom). Nontransduced Mel Im cells were used as control. (D) Quantitative RT-PCR analysis showing increased CYLD mRNA expression in three different melanoma cell lines (Mel Im, Mel Ei, and Mel Juso) and melanoma cells (MM1 and MM2) transiently transfected with asSnail expression plasmids compared with mock-transfected (pcDNA3) cells. Data are given as mean ± SEM. *, P < 0.05. (E) Western blot analysis showing decreased CYLD protein expression in NHEM cells transiently transfected with Snail1 expression plasmids compared with mock-transfected (pcDNA3) or nontransfected (NHEM) cells. (F) ERK inhibition by treatment with MEK inhibitors PD98059 and UO126 significantly affects Snail1 and CYLD mRNA expression in Mel Im cells. JNK inhibition (SP600125) exhibits no effect compared with vehicle (DMSO) control. Data are given as mean ± SEM. *, P < 0.05. ns, not significant, both compared with Control. (G) Quantitative RT-PCR revealing that inhibition of ERK by PD98059 or UO126 further induced CYLD expression in melanocytes. JNK inhibition (SP600125) exhibits no effect compared with vehicle (DMSO) control. Data are given as mean ± SEM. *, P < 0.05. Experiments in A–G were repeated at least three times. (H) Detection of CYLD expression by Western blot analysis. Transfection of NHEMs with mutated BRAF (V600E) induced Snail1 protein expression and, consequently, CYLD protein suppression, whereas no changes were found after transfection of wild-type BRAF (wtBRAF). Experiments were repeated three times with cells from two different donors.
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Related In: Results  -  Collection

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fig2: Snail1 inhibits transcriptional expression of CYLD in melanocytic cells. (A) Lysates from NHEMs, primary melanoma cells (MM1 and MM2), and melanoma cell lines (Mel Im, Mel Juso) were examined by ChIP assay using an anti-Snail1, anti-Twist, and anti-Snail2–specific antibody, respectively, as well as a PCR primer pair corresponding to the promoter of the CYLD gene. Recruitment to the CYLD promoter was only shown for Snail1. Snail1, Twist, Snail2, immunoprecipitation (IP) using specific antibodies; IgG, IP using negative control rabbit immunoglobulin; Input, 10% of the cell lysate used for the IP is shown. (B) Reporter assays revealing inducible CYLD promoter (−550 to −1 bp) activity in Mel Im cells after transient transfection with asSnail (asSnail), whereas transfection with Snail1 expression plasmid (Snail1) completely abrogated CYLD promoter activity compared with the control (pGL3). Mutation of the consensus Snail1 binding site (CYLD mut-19) led to strong promoter activity that was not affected by transfection with asSnail or Snail1. Data are given as mean ± SEM. *, P < 0.05 compared with CYLD control. (C) Immunoblot analysis showing that CYLD protein is up-regulated in Mel Im cells after stable transfection with asSnail expression plasmids (clone 1 and clone 2; top) and after transient transduction with Snail1 siRNA nucleotides (siRNA Snail1) or scrambled siRNA control (siRNA control; bottom). Nontransduced Mel Im cells were used as control. (D) Quantitative RT-PCR analysis showing increased CYLD mRNA expression in three different melanoma cell lines (Mel Im, Mel Ei, and Mel Juso) and melanoma cells (MM1 and MM2) transiently transfected with asSnail expression plasmids compared with mock-transfected (pcDNA3) cells. Data are given as mean ± SEM. *, P < 0.05. (E) Western blot analysis showing decreased CYLD protein expression in NHEM cells transiently transfected with Snail1 expression plasmids compared with mock-transfected (pcDNA3) or nontransfected (NHEM) cells. (F) ERK inhibition by treatment with MEK inhibitors PD98059 and UO126 significantly affects Snail1 and CYLD mRNA expression in Mel Im cells. JNK inhibition (SP600125) exhibits no effect compared with vehicle (DMSO) control. Data are given as mean ± SEM. *, P < 0.05. ns, not significant, both compared with Control. (G) Quantitative RT-PCR revealing that inhibition of ERK by PD98059 or UO126 further induced CYLD expression in melanocytes. JNK inhibition (SP600125) exhibits no effect compared with vehicle (DMSO) control. Data are given as mean ± SEM. *, P < 0.05. Experiments in A–G were repeated at least three times. (H) Detection of CYLD expression by Western blot analysis. Transfection of NHEMs with mutated BRAF (V600E) induced Snail1 protein expression and, consequently, CYLD protein suppression, whereas no changes were found after transfection of wild-type BRAF (wtBRAF). Experiments were repeated three times with cells from two different donors.
Mentions: Chromatin immunoprecipitation (ChIP) assays demonstrated Snail1 binding to the CYLD promoter in primary melanoma cells and the melanoma cell lines Mel Im and Mel Juso, but not in NHEM (Fig. 2 A). Furthermore, neither Snail2 nor Twist (another E-box binding factor associated with poor prognosis in melanoma [22]) bound to the CYLD promoter in ChIP assays (Fig. 2 A). Snail1 recruitment to the CYLD promoter was observed to the first E-box at position −19 to −14, which resembles the consensus Snail1 binding sequence (CAGGTG; Fig. S1), but not to E-box II (-382 to -377) or III (−429 to −424; unpublished data).

Bottom Line: Rescue of CYLD expression in melanoma cells reduced proliferation and invasion in vitro and tumor growth and metastasis in vivo.Importantly, tumor thickness and progression-free and overall survival inversely correlated with CYLD expression.Our data suggest that Snail1-mediated suppression of CYLD plays a key role in melanoma malignancy.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

ABSTRACT
High malignancy and early metastasis are hallmarks of melanoma. Here, we report that the transcription factor Snail1 inhibits expression of the tumor suppressor CYLD in melanoma. As a direct consequence of CYLD repression, the protooncogene BCL-3 translocates into the nucleus and activates Cyclin D1 and N-cadherin promoters, resulting in proliferation and invasion of melanoma cells. Rescue of CYLD expression in melanoma cells reduced proliferation and invasion in vitro and tumor growth and metastasis in vivo. Analysis of a tissue microarray with primary melanomas from patients revealed an inverse correlation of Snail1 induction and loss of CYLD expression. Importantly, tumor thickness and progression-free and overall survival inversely correlated with CYLD expression. Our data suggest that Snail1-mediated suppression of CYLD plays a key role in melanoma malignancy.

Show MeSH
Related in: MedlinePlus