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Epigenetic repression of ROR2 has a Wnt-mediated, pro-tumourigenic role in colon cancer.

Lara E, Calvanese V, Huidobro C, Fernández AF, Moncada-Pazos A, Obaya AJ, Aguilera O, González-Sancho JM, Sánchez L, Astudillo A, Muñoz A, López-Otín C, Esteller M, Fraga MF - Mol. Cancer (2010)

Bottom Line: Wnt factors control cell differentiation through semi-independent molecular cascades known as the beta-catenin-dependent (canonical) and -independent (non-canonical) Wnt signalling pathways.Despite increasing evidence of the role of the non-canonical pathways in tumourigenesis, however, the underlying molecular mechanisms are poorly understood.Our data show the importance of epigenetic alterations of ROR2 in colon cancer, highlighting the close interconnection between canonical and non-canonical Wnt signalling pathways in this type of tumour.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology and Oncology, National Center for Biotechnology, CNB-CSIC, Cantoblanco, Madrid E-28049, Spain.

ABSTRACT

Background: Wnt factors control cell differentiation through semi-independent molecular cascades known as the beta-catenin-dependent (canonical) and -independent (non-canonical) Wnt signalling pathways. Genetic and epigenetic alteration of components of the canonical Wnt signalling pathway is one of the primary mechanisms underlying colon cancer. Despite increasing evidence of the role of the non-canonical pathways in tumourigenesis, however, the underlying molecular mechanisms are poorly understood.

Results: Here we report that the receptor tyrosine kinase-like orphan receptor 2 (ROR2), a transmembrane receptor for Wnt factors that activates non-canonical pathways, is frequently repressed by aberrant promoter hypermethylation in human colon cancer cell lines and primary tumours. By restoring ROR2 activity in colon cancer cells harbouring ROR2 promoter hypermethylation, we show that the role of ROR2 in colon cancer cells is mediated, at least in part, by canonical Wnt and that its epigenetic-dependent loss can be pro-tumourigenic.

Conclusions: Our data show the importance of epigenetic alterations of ROR2 in colon cancer, highlighting the close interconnection between canonical and non-canonical Wnt signalling pathways in this type of tumour.

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Promoter methylation-dependent ROR2 repression in colon cancer. (A) qRT-PCR analysis of ROR2 mRNA relative to GAPDH in three samples with unmethylated ROR2 (white) and four samples with ROR2 promoter hypermethylation (red; top panel). The bottom panel shows WB analysis of ROR2 and actin in normal colon epithelium. Two samples show unmethylated ROR2 (HCT116, SW480) and four samples show ROR2 promoter hypermethylation (HT29, HCT15, DLD1, RKO). (B) Relative ROR2 mRNA levels in two cancer cell lines with unmethylated ROR2 (white) and three samples with ROR2 promoter hypermethylation (red) after treatment with the demethylating drug 5-aza-2'-deoxycytidine. (C) qRT-PCR analysis of ROR2 mRNA in 20 primary colorectal tumours and the corresponding normal colon epithelium. Results are shown as the mean ± SD of three independent experiments. (D) Immunohistochemistry analysis of ROR2 in formalin-fixed, paraffin-embedded tissues. The images show protein expression in colorectal gland mucosa (arrow) in normal colon epithelium and lack of ROR2 expression in a poorly differentiated colon adenocarcinoma. (E) Relationship between ROR2 expression and promoter hypermethylation. qRT-PCR quantification of ROR2 relative to GAPDH expression in three ROR2-expressing tumours (U1-3) and three tumours with low ROR2 expression (M1-3; left panel). MSP analysis of ROR2 promoter methylation. A PCR band in lanes M or U indicates methylated or unmethylated, respectively. IVD is used as a positive control for methylated DNA and normal lymphocytes (NL) as negative control (upper right). WB analysis of ROR2 protein expression in the same tumour samples (lower right).
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Figure 2: Promoter methylation-dependent ROR2 repression in colon cancer. (A) qRT-PCR analysis of ROR2 mRNA relative to GAPDH in three samples with unmethylated ROR2 (white) and four samples with ROR2 promoter hypermethylation (red; top panel). The bottom panel shows WB analysis of ROR2 and actin in normal colon epithelium. Two samples show unmethylated ROR2 (HCT116, SW480) and four samples show ROR2 promoter hypermethylation (HT29, HCT15, DLD1, RKO). (B) Relative ROR2 mRNA levels in two cancer cell lines with unmethylated ROR2 (white) and three samples with ROR2 promoter hypermethylation (red) after treatment with the demethylating drug 5-aza-2'-deoxycytidine. (C) qRT-PCR analysis of ROR2 mRNA in 20 primary colorectal tumours and the corresponding normal colon epithelium. Results are shown as the mean ± SD of three independent experiments. (D) Immunohistochemistry analysis of ROR2 in formalin-fixed, paraffin-embedded tissues. The images show protein expression in colorectal gland mucosa (arrow) in normal colon epithelium and lack of ROR2 expression in a poorly differentiated colon adenocarcinoma. (E) Relationship between ROR2 expression and promoter hypermethylation. qRT-PCR quantification of ROR2 relative to GAPDH expression in three ROR2-expressing tumours (U1-3) and three tumours with low ROR2 expression (M1-3; left panel). MSP analysis of ROR2 promoter methylation. A PCR band in lanes M or U indicates methylated or unmethylated, respectively. IVD is used as a positive control for methylated DNA and normal lymphocytes (NL) as negative control (upper right). WB analysis of ROR2 protein expression in the same tumour samples (lower right).

Mentions: To determine the role of the ROR2 promoter hypermethylation in gene expression, we used qRT-PCR and WB to compare ROR2 mRNA and protein levels in healthy colon epithelium and the HCT116 and SW480 cell lines, which do not show ROR2 promoter hypermethylation, with the HT29, HCT15, DLD1 and RKO lines, which present dense DNA methylation at the ROR2 promoter (Figure 2A). These experiments showed that ROR2 mRNA and protein were only detected in samples lacking ROR2 promoter hypermethylation. Although they showed dense ROR2 promoter hypermethylation, HT29 cells expressed residual levels of ROR2 mRNA, insufficient to detect ROR2 protein. To study the relationship between ROR2 promoter hypermethylation and ROR2 repression in more detail, we analysed ROR2 mRNA levels in colon cancer cell lines incubated with the demethylating drug 5-aza-2-deoxycytidine (Figure 2B). Treatment with this drug resulted in marked reactivation of ROR2 in cell lines displaying ROR2 promoter hypermethylation (HCT15, DLD1 and RKO), but not in those lacking hypermethylation (HCT116 and SW480) (Figure 2B). This implies that in vitro ROR2 promoter hypermethylation is directly associated with ROR2 repression.


Epigenetic repression of ROR2 has a Wnt-mediated, pro-tumourigenic role in colon cancer.

Lara E, Calvanese V, Huidobro C, Fernández AF, Moncada-Pazos A, Obaya AJ, Aguilera O, González-Sancho JM, Sánchez L, Astudillo A, Muñoz A, López-Otín C, Esteller M, Fraga MF - Mol. Cancer (2010)

Promoter methylation-dependent ROR2 repression in colon cancer. (A) qRT-PCR analysis of ROR2 mRNA relative to GAPDH in three samples with unmethylated ROR2 (white) and four samples with ROR2 promoter hypermethylation (red; top panel). The bottom panel shows WB analysis of ROR2 and actin in normal colon epithelium. Two samples show unmethylated ROR2 (HCT116, SW480) and four samples show ROR2 promoter hypermethylation (HT29, HCT15, DLD1, RKO). (B) Relative ROR2 mRNA levels in two cancer cell lines with unmethylated ROR2 (white) and three samples with ROR2 promoter hypermethylation (red) after treatment with the demethylating drug 5-aza-2'-deoxycytidine. (C) qRT-PCR analysis of ROR2 mRNA in 20 primary colorectal tumours and the corresponding normal colon epithelium. Results are shown as the mean ± SD of three independent experiments. (D) Immunohistochemistry analysis of ROR2 in formalin-fixed, paraffin-embedded tissues. The images show protein expression in colorectal gland mucosa (arrow) in normal colon epithelium and lack of ROR2 expression in a poorly differentiated colon adenocarcinoma. (E) Relationship between ROR2 expression and promoter hypermethylation. qRT-PCR quantification of ROR2 relative to GAPDH expression in three ROR2-expressing tumours (U1-3) and three tumours with low ROR2 expression (M1-3; left panel). MSP analysis of ROR2 promoter methylation. A PCR band in lanes M or U indicates methylated or unmethylated, respectively. IVD is used as a positive control for methylated DNA and normal lymphocytes (NL) as negative control (upper right). WB analysis of ROR2 protein expression in the same tumour samples (lower right).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Promoter methylation-dependent ROR2 repression in colon cancer. (A) qRT-PCR analysis of ROR2 mRNA relative to GAPDH in three samples with unmethylated ROR2 (white) and four samples with ROR2 promoter hypermethylation (red; top panel). The bottom panel shows WB analysis of ROR2 and actin in normal colon epithelium. Two samples show unmethylated ROR2 (HCT116, SW480) and four samples show ROR2 promoter hypermethylation (HT29, HCT15, DLD1, RKO). (B) Relative ROR2 mRNA levels in two cancer cell lines with unmethylated ROR2 (white) and three samples with ROR2 promoter hypermethylation (red) after treatment with the demethylating drug 5-aza-2'-deoxycytidine. (C) qRT-PCR analysis of ROR2 mRNA in 20 primary colorectal tumours and the corresponding normal colon epithelium. Results are shown as the mean ± SD of three independent experiments. (D) Immunohistochemistry analysis of ROR2 in formalin-fixed, paraffin-embedded tissues. The images show protein expression in colorectal gland mucosa (arrow) in normal colon epithelium and lack of ROR2 expression in a poorly differentiated colon adenocarcinoma. (E) Relationship between ROR2 expression and promoter hypermethylation. qRT-PCR quantification of ROR2 relative to GAPDH expression in three ROR2-expressing tumours (U1-3) and three tumours with low ROR2 expression (M1-3; left panel). MSP analysis of ROR2 promoter methylation. A PCR band in lanes M or U indicates methylated or unmethylated, respectively. IVD is used as a positive control for methylated DNA and normal lymphocytes (NL) as negative control (upper right). WB analysis of ROR2 protein expression in the same tumour samples (lower right).
Mentions: To determine the role of the ROR2 promoter hypermethylation in gene expression, we used qRT-PCR and WB to compare ROR2 mRNA and protein levels in healthy colon epithelium and the HCT116 and SW480 cell lines, which do not show ROR2 promoter hypermethylation, with the HT29, HCT15, DLD1 and RKO lines, which present dense DNA methylation at the ROR2 promoter (Figure 2A). These experiments showed that ROR2 mRNA and protein were only detected in samples lacking ROR2 promoter hypermethylation. Although they showed dense ROR2 promoter hypermethylation, HT29 cells expressed residual levels of ROR2 mRNA, insufficient to detect ROR2 protein. To study the relationship between ROR2 promoter hypermethylation and ROR2 repression in more detail, we analysed ROR2 mRNA levels in colon cancer cell lines incubated with the demethylating drug 5-aza-2-deoxycytidine (Figure 2B). Treatment with this drug resulted in marked reactivation of ROR2 in cell lines displaying ROR2 promoter hypermethylation (HCT15, DLD1 and RKO), but not in those lacking hypermethylation (HCT116 and SW480) (Figure 2B). This implies that in vitro ROR2 promoter hypermethylation is directly associated with ROR2 repression.

Bottom Line: Wnt factors control cell differentiation through semi-independent molecular cascades known as the beta-catenin-dependent (canonical) and -independent (non-canonical) Wnt signalling pathways.Despite increasing evidence of the role of the non-canonical pathways in tumourigenesis, however, the underlying molecular mechanisms are poorly understood.Our data show the importance of epigenetic alterations of ROR2 in colon cancer, highlighting the close interconnection between canonical and non-canonical Wnt signalling pathways in this type of tumour.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology and Oncology, National Center for Biotechnology, CNB-CSIC, Cantoblanco, Madrid E-28049, Spain.

ABSTRACT

Background: Wnt factors control cell differentiation through semi-independent molecular cascades known as the beta-catenin-dependent (canonical) and -independent (non-canonical) Wnt signalling pathways. Genetic and epigenetic alteration of components of the canonical Wnt signalling pathway is one of the primary mechanisms underlying colon cancer. Despite increasing evidence of the role of the non-canonical pathways in tumourigenesis, however, the underlying molecular mechanisms are poorly understood.

Results: Here we report that the receptor tyrosine kinase-like orphan receptor 2 (ROR2), a transmembrane receptor for Wnt factors that activates non-canonical pathways, is frequently repressed by aberrant promoter hypermethylation in human colon cancer cell lines and primary tumours. By restoring ROR2 activity in colon cancer cells harbouring ROR2 promoter hypermethylation, we show that the role of ROR2 in colon cancer cells is mediated, at least in part, by canonical Wnt and that its epigenetic-dependent loss can be pro-tumourigenic.

Conclusions: Our data show the importance of epigenetic alterations of ROR2 in colon cancer, highlighting the close interconnection between canonical and non-canonical Wnt signalling pathways in this type of tumour.

Show MeSH
Related in: MedlinePlus