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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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Related in: MedlinePlus

Aberrant ROR2 hypermethylation in colon cancer. (A) The ROR2 promoter with its associated CpG island (top). CpG location, transcription start site (TSS) and ATG are indicated. Bisulphite sequencing primers, white arrows; Illumina Infinium probes (465 bp upstream and 322 bp downstream of the TSS), red and green lines, respectively. Lower panels show bisulphite genomic sequencing results of 12 individual clones in healthy colon epithelium, colonocytes and eight colon cancer cell lines (HCT116, SW480, LOVO, HT29, HCT15, DLD1, COLO205 and RKO), showing methylated (black squares) or unmethylated cytosines (white squares). (B) DNA methylation analysis of two CpG sites within the ROR2 promoter in healthy colon epithelium, colonocytes and eight colon cancer cell lines using Infinium methylation arrays. Relative methylation for each CpG site (-465 bp and + 322 bp) is represented on a scale from 0.0 to 1.0 (corresponding to a 0% and 100% likelihood of CpG hypermethylation). (C) Methylation-specific PCR (MSP) of ROR2 promoter in healthy colon tissue and colon cancer cell lines. A PCR band in lanes M or U indicates methylated or unmethylated genes, respectively. In vitro-methylated DNA (IVD) was used as a positive control for methylated DNA. (D) Methylation analysis of the ROR2 promoter using MSP of 36 primary colon tumours. Percentage of tumours with ROR2 promoter hypermethylation (unmethylated, white; methylated, red; top) and ethidium bromide staining of an MSP reaction of two representative tumours (unmethylated and methylated) resolved in an agarose gel (bottom).
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Figure 1: Aberrant ROR2 hypermethylation in colon cancer. (A) The ROR2 promoter with its associated CpG island (top). CpG location, transcription start site (TSS) and ATG are indicated. Bisulphite sequencing primers, white arrows; Illumina Infinium probes (465 bp upstream and 322 bp downstream of the TSS), red and green lines, respectively. Lower panels show bisulphite genomic sequencing results of 12 individual clones in healthy colon epithelium, colonocytes and eight colon cancer cell lines (HCT116, SW480, LOVO, HT29, HCT15, DLD1, COLO205 and RKO), showing methylated (black squares) or unmethylated cytosines (white squares). (B) DNA methylation analysis of two CpG sites within the ROR2 promoter in healthy colon epithelium, colonocytes and eight colon cancer cell lines using Infinium methylation arrays. Relative methylation for each CpG site (-465 bp and + 322 bp) is represented on a scale from 0.0 to 1.0 (corresponding to a 0% and 100% likelihood of CpG hypermethylation). (C) Methylation-specific PCR (MSP) of ROR2 promoter in healthy colon tissue and colon cancer cell lines. A PCR band in lanes M or U indicates methylated or unmethylated genes, respectively. In vitro-methylated DNA (IVD) was used as a positive control for methylated DNA. (D) Methylation analysis of the ROR2 promoter using MSP of 36 primary colon tumours. Percentage of tumours with ROR2 promoter hypermethylation (unmethylated, white; methylated, red; top) and ethidium bromide staining of an MSP reaction of two representative tumours (unmethylated and methylated) resolved in an agarose gel (bottom).

Mentions: Analysis of the region 1.0 kb upstream and 0.5 kb downstream of the ROR2 transcriptional start site identified a CpG island, suggesting a potential role for CpG methylation in the regulation of ROR2 expression. To study the possible aberrant epigenetic regulation of ROR2 in colon cancer, we used bisulphite sequencing of multiple clones to determine the methylation status of a ROR2 promoter DNA region of 315 bp that spans the ROR2 transcriptional start point in healthy colon tissue, in vitro-growing colonocytes and eight colon cancer cell lines (HCT116, SW480, LOVO, HT29, HCT15, DLD1, COLO205 and RKO) (Figure 1A). This showed that the ROR2 promoter was completely unmethylated in non-tumourigenic colon primary tissues and in vitro-growing colonocytes, whilst it was densely hypermethylated in most cancer cell lines analysed (HT29, HCT15, DLD1, COLO205 and RKO). These results were confirmed using the 27K Illumina Infinium methylation platform to study the DNA methylation status of two CpG positions (located 465 bp upstream and 322 bp downstream of the ROR2 transcription start site, respectively) within the ROR2 CpG island (Figure 1B) and using methylation-specific PCR (Figure 1C).


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)

Aberrant ROR2 hypermethylation in colon cancer. (A) The ROR2 promoter with its associated CpG island (top). CpG location, transcription start site (TSS) and ATG are indicated. Bisulphite sequencing primers, white arrows; Illumina Infinium probes (465 bp upstream and 322 bp downstream of the TSS), red and green lines, respectively. Lower panels show bisulphite genomic sequencing results of 12 individual clones in healthy colon epithelium, colonocytes and eight colon cancer cell lines (HCT116, SW480, LOVO, HT29, HCT15, DLD1, COLO205 and RKO), showing methylated (black squares) or unmethylated cytosines (white squares). (B) DNA methylation analysis of two CpG sites within the ROR2 promoter in healthy colon epithelium, colonocytes and eight colon cancer cell lines using Infinium methylation arrays. Relative methylation for each CpG site (-465 bp and + 322 bp) is represented on a scale from 0.0 to 1.0 (corresponding to a 0% and 100% likelihood of CpG hypermethylation). (C) Methylation-specific PCR (MSP) of ROR2 promoter in healthy colon tissue and colon cancer cell lines. A PCR band in lanes M or U indicates methylated or unmethylated genes, respectively. In vitro-methylated DNA (IVD) was used as a positive control for methylated DNA. (D) Methylation analysis of the ROR2 promoter using MSP of 36 primary colon tumours. Percentage of tumours with ROR2 promoter hypermethylation (unmethylated, white; methylated, red; top) and ethidium bromide staining of an MSP reaction of two representative tumours (unmethylated and methylated) resolved in an agarose gel (bottom).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Aberrant ROR2 hypermethylation in colon cancer. (A) The ROR2 promoter with its associated CpG island (top). CpG location, transcription start site (TSS) and ATG are indicated. Bisulphite sequencing primers, white arrows; Illumina Infinium probes (465 bp upstream and 322 bp downstream of the TSS), red and green lines, respectively. Lower panels show bisulphite genomic sequencing results of 12 individual clones in healthy colon epithelium, colonocytes and eight colon cancer cell lines (HCT116, SW480, LOVO, HT29, HCT15, DLD1, COLO205 and RKO), showing methylated (black squares) or unmethylated cytosines (white squares). (B) DNA methylation analysis of two CpG sites within the ROR2 promoter in healthy colon epithelium, colonocytes and eight colon cancer cell lines using Infinium methylation arrays. Relative methylation for each CpG site (-465 bp and + 322 bp) is represented on a scale from 0.0 to 1.0 (corresponding to a 0% and 100% likelihood of CpG hypermethylation). (C) Methylation-specific PCR (MSP) of ROR2 promoter in healthy colon tissue and colon cancer cell lines. A PCR band in lanes M or U indicates methylated or unmethylated genes, respectively. In vitro-methylated DNA (IVD) was used as a positive control for methylated DNA. (D) Methylation analysis of the ROR2 promoter using MSP of 36 primary colon tumours. Percentage of tumours with ROR2 promoter hypermethylation (unmethylated, white; methylated, red; top) and ethidium bromide staining of an MSP reaction of two representative tumours (unmethylated and methylated) resolved in an agarose gel (bottom).
Mentions: Analysis of the region 1.0 kb upstream and 0.5 kb downstream of the ROR2 transcriptional start site identified a CpG island, suggesting a potential role for CpG methylation in the regulation of ROR2 expression. To study the possible aberrant epigenetic regulation of ROR2 in colon cancer, we used bisulphite sequencing of multiple clones to determine the methylation status of a ROR2 promoter DNA region of 315 bp that spans the ROR2 transcriptional start point in healthy colon tissue, in vitro-growing colonocytes and eight colon cancer cell lines (HCT116, SW480, LOVO, HT29, HCT15, DLD1, COLO205 and RKO) (Figure 1A). This showed that the ROR2 promoter was completely unmethylated in non-tumourigenic colon primary tissues and in vitro-growing colonocytes, whilst it was densely hypermethylated in most cancer cell lines analysed (HT29, HCT15, DLD1, COLO205 and RKO). These results were confirmed using the 27K Illumina Infinium methylation platform to study the DNA methylation status of two CpG positions (located 465 bp upstream and 322 bp downstream of the ROR2 transcription start site, respectively) within the ROR2 CpG island (Figure 1B) and using methylation-specific PCR (Figure 1C).

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