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Methylation-dependent SOX9 expression mediates invasion in human melanoma cells and is a negative prognostic factor in advanced melanoma.

Cheng PF, Shakhova O, Widmer DS, Eichhoff OM, Zingg D, Frommel SC, Belloni B, Raaijmakers MI, Goldinger SM, Santoro R, Hemmi S, Sommer L, Dummer R, Levesque MP - Genome Biol. (2015)

Bottom Line: SOX9 overexpression results in decreased proliferation but increased invasion in vitro.Additional survival analysis on the targets of SOX9 reveals that most SOX9 downregulated genes have survival benefit for metastatic patients.A number of genes downregulated by SOX9 have a negative impact on patient survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Faculty of Medicine, University Hospital Zürich, and University of Zürich, Wagistrasse 14, CH-8952, Zürich, Switzerland. phil.cheng@usz.ch.

ABSTRACT

Background: Melanoma is the most fatal skin cancer displaying a high degree of molecular heterogeneity. Phenotype switching is a mechanism that contributes to melanoma heterogeneity by altering transcription profiles for the transition between states of proliferation/differentiation and invasion/stemness. As phenotype switching is reversible, epigenetic mechanisms, like DNA methylation, could contribute to the changes in gene expression.

Results: Integrative analysis of methylation and gene expression datasets of five proliferative and five invasion melanoma cell cultures reveal two distinct clusters. SOX9 is methylated and lowly expressed in the highly proliferative group. SOX9 overexpression results in decreased proliferation but increased invasion in vitro. In a B16 mouse model, sox9 overexpression increases the number of lung metastases. Transcriptional analysis of SOX9-overexpressing melanoma cells reveals enrichment in epithelial to mesenchymal transition (EMT) pathways. Survival analysis of The Cancer Genome Atlas melanoma dataset shows that metastatic patients with high expression levels of SOX9 have significantly worse survival rates. Additional survival analysis on the targets of SOX9 reveals that most SOX9 downregulated genes have survival benefit for metastatic patients.

Conclusions: Our genome-wide DNA methylation and gene expression study of 10 early passage melanoma cell cultures reveals two phenotypically distinct groups. One of the genes regulated by DNA methylation between the two groups is SOX9. SOX9 induces melanoma cell invasion and metastasis and decreases patient survival. A number of genes downregulated by SOX9 have a negative impact on patient survival. In conclusion, SOX9 is an important gene involved in melanoma invasion and negatively impacts melanoma patient survival.

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Validation of SOX9 methylation. (A) Lollipop diagrams of bisulphite sequencing of SOX9 promoter. Black lollipops are methylated CpGs; white lollipops are unmethylated CpGs. A minimum of five clones were sequenced for each cell culture. (B) mRNA expression of SOX9 normalized to the housekeeping gene RPL28 across 10 melanoma cell cultures. Results are presented as mean +/− s.d., n = 3. Statistical significance of differential expression between the proliferative and invasive phenotype cell cultures was determined by Student’s t-test. (C) Western blot for SOX9 in 10 melanoma cell cultures. GAPDH served as loading control. (D) Western blot for SOX9 of five proliferative melanoma cell cultures treated with 5-aza-2-deoxycytidine (Aza).
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Fig2: Validation of SOX9 methylation. (A) Lollipop diagrams of bisulphite sequencing of SOX9 promoter. Black lollipops are methylated CpGs; white lollipops are unmethylated CpGs. A minimum of five clones were sequenced for each cell culture. (B) mRNA expression of SOX9 normalized to the housekeeping gene RPL28 across 10 melanoma cell cultures. Results are presented as mean +/− s.d., n = 3. Statistical significance of differential expression between the proliferative and invasive phenotype cell cultures was determined by Student’s t-test. (C) Western blot for SOX9 in 10 melanoma cell cultures. GAPDH served as loading control. (D) Western blot for SOX9 of five proliferative melanoma cell cultures treated with 5-aza-2-deoxycytidine (Aza).

Mentions: From the methylation array, the area that had the most enrichment for methylation was about 2 kb upstream of the SOX9 transcriptional start site thus we validated the CpG island located there via sequencing of bisulfite-treated genomic DNA in the 10 melanoma cell cultures (Figure 2A). There are three predicted transcription factor binding sites in that upstream promoter region of SOX9 for MEF2, E2F, and HNF3B. We analyzed the DNA methylation status of a cluster of 17 CpGs across a 283-bp region and 15 CpGs across a 256-bp region of a CpG island, located approximately −2,500 bp to −2,000 bp upstream of the SOX9 transcriptional start site. The majority of CpGs in both regions of the SOX9 promoter were consistently methylated in the proliferative phenotype melanoma cell cultures and consistently unmethylated in the invasive phenotype melanoma cell cultures.Figure 2


Methylation-dependent SOX9 expression mediates invasion in human melanoma cells and is a negative prognostic factor in advanced melanoma.

Cheng PF, Shakhova O, Widmer DS, Eichhoff OM, Zingg D, Frommel SC, Belloni B, Raaijmakers MI, Goldinger SM, Santoro R, Hemmi S, Sommer L, Dummer R, Levesque MP - Genome Biol. (2015)

Validation of SOX9 methylation. (A) Lollipop diagrams of bisulphite sequencing of SOX9 promoter. Black lollipops are methylated CpGs; white lollipops are unmethylated CpGs. A minimum of five clones were sequenced for each cell culture. (B) mRNA expression of SOX9 normalized to the housekeeping gene RPL28 across 10 melanoma cell cultures. Results are presented as mean +/− s.d., n = 3. Statistical significance of differential expression between the proliferative and invasive phenotype cell cultures was determined by Student’s t-test. (C) Western blot for SOX9 in 10 melanoma cell cultures. GAPDH served as loading control. (D) Western blot for SOX9 of five proliferative melanoma cell cultures treated with 5-aza-2-deoxycytidine (Aza).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4378455&req=5

Fig2: Validation of SOX9 methylation. (A) Lollipop diagrams of bisulphite sequencing of SOX9 promoter. Black lollipops are methylated CpGs; white lollipops are unmethylated CpGs. A minimum of five clones were sequenced for each cell culture. (B) mRNA expression of SOX9 normalized to the housekeeping gene RPL28 across 10 melanoma cell cultures. Results are presented as mean +/− s.d., n = 3. Statistical significance of differential expression between the proliferative and invasive phenotype cell cultures was determined by Student’s t-test. (C) Western blot for SOX9 in 10 melanoma cell cultures. GAPDH served as loading control. (D) Western blot for SOX9 of five proliferative melanoma cell cultures treated with 5-aza-2-deoxycytidine (Aza).
Mentions: From the methylation array, the area that had the most enrichment for methylation was about 2 kb upstream of the SOX9 transcriptional start site thus we validated the CpG island located there via sequencing of bisulfite-treated genomic DNA in the 10 melanoma cell cultures (Figure 2A). There are three predicted transcription factor binding sites in that upstream promoter region of SOX9 for MEF2, E2F, and HNF3B. We analyzed the DNA methylation status of a cluster of 17 CpGs across a 283-bp region and 15 CpGs across a 256-bp region of a CpG island, located approximately −2,500 bp to −2,000 bp upstream of the SOX9 transcriptional start site. The majority of CpGs in both regions of the SOX9 promoter were consistently methylated in the proliferative phenotype melanoma cell cultures and consistently unmethylated in the invasive phenotype melanoma cell cultures.Figure 2

Bottom Line: SOX9 overexpression results in decreased proliferation but increased invasion in vitro.Additional survival analysis on the targets of SOX9 reveals that most SOX9 downregulated genes have survival benefit for metastatic patients.A number of genes downregulated by SOX9 have a negative impact on patient survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Faculty of Medicine, University Hospital Zürich, and University of Zürich, Wagistrasse 14, CH-8952, Zürich, Switzerland. phil.cheng@usz.ch.

ABSTRACT

Background: Melanoma is the most fatal skin cancer displaying a high degree of molecular heterogeneity. Phenotype switching is a mechanism that contributes to melanoma heterogeneity by altering transcription profiles for the transition between states of proliferation/differentiation and invasion/stemness. As phenotype switching is reversible, epigenetic mechanisms, like DNA methylation, could contribute to the changes in gene expression.

Results: Integrative analysis of methylation and gene expression datasets of five proliferative and five invasion melanoma cell cultures reveal two distinct clusters. SOX9 is methylated and lowly expressed in the highly proliferative group. SOX9 overexpression results in decreased proliferation but increased invasion in vitro. In a B16 mouse model, sox9 overexpression increases the number of lung metastases. Transcriptional analysis of SOX9-overexpressing melanoma cells reveals enrichment in epithelial to mesenchymal transition (EMT) pathways. Survival analysis of The Cancer Genome Atlas melanoma dataset shows that metastatic patients with high expression levels of SOX9 have significantly worse survival rates. Additional survival analysis on the targets of SOX9 reveals that most SOX9 downregulated genes have survival benefit for metastatic patients.

Conclusions: Our genome-wide DNA methylation and gene expression study of 10 early passage melanoma cell cultures reveals two phenotypically distinct groups. One of the genes regulated by DNA methylation between the two groups is SOX9. SOX9 induces melanoma cell invasion and metastasis and decreases patient survival. A number of genes downregulated by SOX9 have a negative impact on patient survival. In conclusion, SOX9 is an important gene involved in melanoma invasion and negatively impacts melanoma patient survival.

Show MeSH
Related in: MedlinePlus