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DNA methylation and histone modifications regulate SOX11 expression in lymphoid and solid cancer cells.

Nordström L, Andersson E, Kuci V, Gustavsson E, Holm K, Ringnér M, Guldberg P, Ek S - BMC Cancer (2015)

Bottom Line: In contrast, SOX11 regulation in neoplastic tissues is more complex involving both DNA methylation and histone modifications.The possibility to re-express SOX11 in non-methylated tissue is of clinical relevance, and was successfully achieved in cell lines with low levels of SOX11 methylation.In breast cancer patients, methylation of the SOX11 promoter was shown to correlate with estrogen receptor status, suggesting that SOX11 may be functionally re-expressed during treatment with HDAC inhibitors in specific patient subgroups.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunotechnology, CREATE Health, Lund University, Lund, Sweden. lena.nordstrom@immun.lth.se.

ABSTRACT

Background: The neural transcription factor SOX11 is present at specific stages during embryo development with a very restricted expression in adult tissue, indicating precise regulation of transcription. SOX11 is strongly up-regulated in some malignancies and have a functional role in tumorgenesis. With the aim to explore differences in epigenetic regulation of SOX11 expression in normal versus neoplastic cells, we investigated methylation and histone modifications related to the SOX11 promoter and the possibility to induce re-expression using histone deacetylase (HDAC) or EZH2 inhibitors.

Methods: The epigenetic regulation of SOX11 was investigated in distinct non-malignant cell populations (n = 7) and neoplastic cell-lines (n = 42) of different cellular origins. DNA methylation was assessed using bisulfite sequencing, methylation-specific melting curve analysis, MethyLight and pyrosequencing. The presence of H3K27me3 was assessed using ChIP-qPCR. The HDAC inhibitors Vorinostat and trichostatin A were used to induce SOX11 in cell lines with no endogenous expression.

Results: The SOX11 promoter shows a low degree of methylation and strong enrichment of H3K27me3 in non-malignant differentiated cells, independent of cellular origin. Cancers of the B-cell lineage are strongly marked by de novo methylation at the SOX11 promoter in SOX11 non-expressing cells, while solid cancer entities display a more varying degree of SOX11 promoter methylation. The silencing mark H3K27me3 was generally present at the SOX11 promoter in non-expressing cells, and an increased enrichment was observed in cancer cells with a low degree of SOX11 methylation compared to cells with dense methylation. Finally, we demonstrate that the HDAC inhibitors (vorinostat and trichostatin A) induce SOX11 expression in cancer cells with low levels of SOX11 methylation.

Conclusions: We show that SOX11 is strongly marked by repressive histone marks in non-malignant cells. In contrast, SOX11 regulation in neoplastic tissues is more complex involving both DNA methylation and histone modifications. The possibility to re-express SOX11 in non-methylated tissue is of clinical relevance, and was successfully achieved in cell lines with low levels of SOX11 methylation. In breast cancer patients, methylation of the SOX11 promoter was shown to correlate with estrogen receptor status, suggesting that SOX11 may be functionally re-expressed during treatment with HDAC inhibitors in specific patient subgroups.

No MeSH data available.


Related in: MedlinePlus

Correlation between SOX11 promoter methylation and expression. The correlation between DNA methylation and gene expression was analyzed with RT-qPCR and western blot. In RT-qPCR, CT values >35 were considered below detection limit and corresponding SOX11 levels were set to zero. (A) MethyLight (x-axis) and melt curve analysis (see filled, grey or open diamonds) showed an inverse correlation between SOX11 mRNA and promoter methylation in lymphoma cell lines. (B) Likewise, inverse correlation between SOX11 mRNA and promoter methylation was seen for solid cancer cell lines. For clarity, SOX11 positive (western blot) cell line names are shaded. (C) Western blot analysis of SOX11 and GAPDH in Burkitt’s lymphoma, follicular lymphoma, diffuse large B-cell lymphoma and mantle cell lymphoma cell-lines. (D) Western blot analysis of SOX11 and GAPDH in breast cancer, ovarian cancer, lung cancer, brain cancer and neuroblastoma cell lines.
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Fig3: Correlation between SOX11 promoter methylation and expression. The correlation between DNA methylation and gene expression was analyzed with RT-qPCR and western blot. In RT-qPCR, CT values >35 were considered below detection limit and corresponding SOX11 levels were set to zero. (A) MethyLight (x-axis) and melt curve analysis (see filled, grey or open diamonds) showed an inverse correlation between SOX11 mRNA and promoter methylation in lymphoma cell lines. (B) Likewise, inverse correlation between SOX11 mRNA and promoter methylation was seen for solid cancer cell lines. For clarity, SOX11 positive (western blot) cell line names are shaded. (C) Western blot analysis of SOX11 and GAPDH in Burkitt’s lymphoma, follicular lymphoma, diffuse large B-cell lymphoma and mantle cell lymphoma cell-lines. (D) Western blot analysis of SOX11 and GAPDH in breast cancer, ovarian cancer, lung cancer, brain cancer and neuroblastoma cell lines.

Mentions: To explore the correlation between SOX11 promoter methylation and expression, each cell line was analyzed by RT-qPCR and western blot. An inverse correlation was observed between SOX11 promoter methylation and gene expression for both lymphoid and solid tumor cells (Spearman’s correlation ρ=-0.71; p<0.001 and ρ=-0.75; p<0.001, respectively) (Figure 3A and B). SOX11 protein was detected in 7/8 (88%) MCL cell-lines with an unmethylated promoter (Figure 3C), while only 6/14 (43%) solid cancer cell lines with an unmethylated promoter had detectable levels of the protein (Figure 3D). As expected, none of the cell lines with a methylated promoter expressed SOX11 mRNA or protein with the exception of BJAB. Using MS-MCA, we show that BJAB has a monoallelic methylation of the SOX11 promoter, explaining the observed co-existence of a methylated promoter and expression of mRNA/protein (Figure 2A and Figure 3A and C). Pyrosequencing was further used to investigate if specific CpG sites are important for SOX11 silencing in cell lines with low-to-intermediate methylation (as determined by MS-MCA and MethyLight). Data show that even at very low level of overall methylation, CpGs close to transcription start site are significantly methylated compared to expressing cell lines with a completely unmethylated promoter (Additional file 4). Finally, we demonstrate a correlation between SOX11 methylation, expression and subtypes in primary breast cancers. Breast cancer methylation data and RNA-seq data were downloaded from The Cancer Genome Atlas (TCGA) and show that SOX11 methylation is more abundant in estrogen recptor (ER) positive tumors (n=460) compared to ER negative tumors (n=139) (Figure 4A) with a strong anti-correlation between methylation and expression in each CpG site (Figure 4B and Table 2).Figure 3


DNA methylation and histone modifications regulate SOX11 expression in lymphoid and solid cancer cells.

Nordström L, Andersson E, Kuci V, Gustavsson E, Holm K, Ringnér M, Guldberg P, Ek S - BMC Cancer (2015)

Correlation between SOX11 promoter methylation and expression. The correlation between DNA methylation and gene expression was analyzed with RT-qPCR and western blot. In RT-qPCR, CT values >35 were considered below detection limit and corresponding SOX11 levels were set to zero. (A) MethyLight (x-axis) and melt curve analysis (see filled, grey or open diamonds) showed an inverse correlation between SOX11 mRNA and promoter methylation in lymphoma cell lines. (B) Likewise, inverse correlation between SOX11 mRNA and promoter methylation was seen for solid cancer cell lines. For clarity, SOX11 positive (western blot) cell line names are shaded. (C) Western blot analysis of SOX11 and GAPDH in Burkitt’s lymphoma, follicular lymphoma, diffuse large B-cell lymphoma and mantle cell lymphoma cell-lines. (D) Western blot analysis of SOX11 and GAPDH in breast cancer, ovarian cancer, lung cancer, brain cancer and neuroblastoma cell lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4403777&req=5

Fig3: Correlation between SOX11 promoter methylation and expression. The correlation between DNA methylation and gene expression was analyzed with RT-qPCR and western blot. In RT-qPCR, CT values >35 were considered below detection limit and corresponding SOX11 levels were set to zero. (A) MethyLight (x-axis) and melt curve analysis (see filled, grey or open diamonds) showed an inverse correlation between SOX11 mRNA and promoter methylation in lymphoma cell lines. (B) Likewise, inverse correlation between SOX11 mRNA and promoter methylation was seen for solid cancer cell lines. For clarity, SOX11 positive (western blot) cell line names are shaded. (C) Western blot analysis of SOX11 and GAPDH in Burkitt’s lymphoma, follicular lymphoma, diffuse large B-cell lymphoma and mantle cell lymphoma cell-lines. (D) Western blot analysis of SOX11 and GAPDH in breast cancer, ovarian cancer, lung cancer, brain cancer and neuroblastoma cell lines.
Mentions: To explore the correlation between SOX11 promoter methylation and expression, each cell line was analyzed by RT-qPCR and western blot. An inverse correlation was observed between SOX11 promoter methylation and gene expression for both lymphoid and solid tumor cells (Spearman’s correlation ρ=-0.71; p<0.001 and ρ=-0.75; p<0.001, respectively) (Figure 3A and B). SOX11 protein was detected in 7/8 (88%) MCL cell-lines with an unmethylated promoter (Figure 3C), while only 6/14 (43%) solid cancer cell lines with an unmethylated promoter had detectable levels of the protein (Figure 3D). As expected, none of the cell lines with a methylated promoter expressed SOX11 mRNA or protein with the exception of BJAB. Using MS-MCA, we show that BJAB has a monoallelic methylation of the SOX11 promoter, explaining the observed co-existence of a methylated promoter and expression of mRNA/protein (Figure 2A and Figure 3A and C). Pyrosequencing was further used to investigate if specific CpG sites are important for SOX11 silencing in cell lines with low-to-intermediate methylation (as determined by MS-MCA and MethyLight). Data show that even at very low level of overall methylation, CpGs close to transcription start site are significantly methylated compared to expressing cell lines with a completely unmethylated promoter (Additional file 4). Finally, we demonstrate a correlation between SOX11 methylation, expression and subtypes in primary breast cancers. Breast cancer methylation data and RNA-seq data were downloaded from The Cancer Genome Atlas (TCGA) and show that SOX11 methylation is more abundant in estrogen recptor (ER) positive tumors (n=460) compared to ER negative tumors (n=139) (Figure 4A) with a strong anti-correlation between methylation and expression in each CpG site (Figure 4B and Table 2).Figure 3

Bottom Line: In contrast, SOX11 regulation in neoplastic tissues is more complex involving both DNA methylation and histone modifications.The possibility to re-express SOX11 in non-methylated tissue is of clinical relevance, and was successfully achieved in cell lines with low levels of SOX11 methylation.In breast cancer patients, methylation of the SOX11 promoter was shown to correlate with estrogen receptor status, suggesting that SOX11 may be functionally re-expressed during treatment with HDAC inhibitors in specific patient subgroups.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunotechnology, CREATE Health, Lund University, Lund, Sweden. lena.nordstrom@immun.lth.se.

ABSTRACT

Background: The neural transcription factor SOX11 is present at specific stages during embryo development with a very restricted expression in adult tissue, indicating precise regulation of transcription. SOX11 is strongly up-regulated in some malignancies and have a functional role in tumorgenesis. With the aim to explore differences in epigenetic regulation of SOX11 expression in normal versus neoplastic cells, we investigated methylation and histone modifications related to the SOX11 promoter and the possibility to induce re-expression using histone deacetylase (HDAC) or EZH2 inhibitors.

Methods: The epigenetic regulation of SOX11 was investigated in distinct non-malignant cell populations (n = 7) and neoplastic cell-lines (n = 42) of different cellular origins. DNA methylation was assessed using bisulfite sequencing, methylation-specific melting curve analysis, MethyLight and pyrosequencing. The presence of H3K27me3 was assessed using ChIP-qPCR. The HDAC inhibitors Vorinostat and trichostatin A were used to induce SOX11 in cell lines with no endogenous expression.

Results: The SOX11 promoter shows a low degree of methylation and strong enrichment of H3K27me3 in non-malignant differentiated cells, independent of cellular origin. Cancers of the B-cell lineage are strongly marked by de novo methylation at the SOX11 promoter in SOX11 non-expressing cells, while solid cancer entities display a more varying degree of SOX11 promoter methylation. The silencing mark H3K27me3 was generally present at the SOX11 promoter in non-expressing cells, and an increased enrichment was observed in cancer cells with a low degree of SOX11 methylation compared to cells with dense methylation. Finally, we demonstrate that the HDAC inhibitors (vorinostat and trichostatin A) induce SOX11 expression in cancer cells with low levels of SOX11 methylation.

Conclusions: We show that SOX11 is strongly marked by repressive histone marks in non-malignant cells. In contrast, SOX11 regulation in neoplastic tissues is more complex involving both DNA methylation and histone modifications. The possibility to re-express SOX11 in non-methylated tissue is of clinical relevance, and was successfully achieved in cell lines with low levels of SOX11 methylation. In breast cancer patients, methylation of the SOX11 promoter was shown to correlate with estrogen receptor status, suggesting that SOX11 may be functionally re-expressed during treatment with HDAC inhibitors in specific patient subgroups.

No MeSH data available.


Related in: MedlinePlus