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

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

Role of Periostin in Adhesion and Migration of Bone Remodeling Cells.

Periostin is an extracellular matrix protein highly expressed in collagen-rich tissues subjected to continuous mechanical stress. Functionally, periostin is involved in tissue remodeling and its altered function is associated to numerous pathological processes. In orthodontics, periostin plays key roles in the maintenance of dental tissues and it is mainly expressed in those areas where tension or pressing forces are taking place. In this regard, high expression of periostin is essential to promote migration and proliferation of periodontal ligament fibroblasts. However little is known about the participation of periostin in migration and adhesion processes of bone remodeling cells. In this work we employ the mouse pre-osteoblastic MC3T3-E1 and the macrophage-like RAW 264.7 cell lines to overexpress periostin and perform different cell-based assays to study changes in cell behavior. Our data indicate that periostin overexpression not only increases adhesion capacity of MC3T3-E1 cells to different matrix proteins but also hampers their migratory capacity. Changes on RNA expression profile of MC3T3-E1 cells upon periostin overexpression have been also analyzed, highlighting the alteration of genes implicated in processes such as cell migration, adhesion or bone metabolism but not in bone differentiation. Overall, our work provides new evidence on the impact of periostin in osteoblasts physiology

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

Abstract Background Wnt factors control cell differentiation through semi-independent molecular cascades known as the β-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.</p

Selection of stable MC3T3-E1 and RAW 264.7 transfectants.

<p>a) Western blot analysis of MC3T3-E1 and RAW 264.7 producing exogenous periostin. Control, cells transfected with an empty vector. Top, detection with an anti-periostin antibody. An anti-Actin antibody was used as a loading control. Bottom, representation of normalized expression using values from Image J densitometry of western-blots. C, control cells. P, periostin overexpressing cells. b) Recombinant periostin is secreted to the cell medium. Top, Cellular inmunolocalization of periostin in MC3T3-E1 transfectants; cells using a specific anti-myc primary antibody and an Alexa-488-conjugated secondary antibody (green). DAPI staining was used to detect nuclei (blue). Bottom, recombinant periostin is detected either with anti-periostin or in an anti-periostin inmunoprecipitate with an anti-myc antibody by western-blot in MC3T3-E1 cells conditioned medium. C and P indicate conditioned medium of control cells transfected with an empty vector and with a vector containing the full-length cDNA for periostin tagged with a c-myc epitope respectively.</p

Periostin overexpressiong compromises migration properties of MC3T3-E1 cells.

<p>(a) MC3T3-E1 periostin-overexpressing (P) and control cells (C) were allowed to migrate simultaneously over a 500 μm gap in standard culture dishes commonly employed for wound healing assays (untreated) or wells coated with type-I collagen or fibronectin. Pictures at starting (t = 0 h), 4 h, 8 h, 12 h, and 16h time points are included. Starting point is indicated with a straight blue line and final points with a dotted line: yellow for MC3T3-E1 control cells and red for clone P3 cells. (b) Graphical representation of migration rate measured at 8 h from three independent experiments. Student t test (<i>p < 0</i>.<i>05</i>, *; <i>p < 0</i>.<i>01</i>, **; <i>p < 0</i>.<i>005</i>, ***).</p

Microarray analysis in MC3T3-E1 periostin overexpressing cells and control MC3T3-E1 cells.

<p>Heat map represents the relative expression levels of selected genes (logFC2_P3vsC) as determined by hybridization with GeneChip Mouse Gene 2.0 Array. Left column indicates gene name and Right column indicates logFC_P3vsC value.</p

Western-blots detection of proteins whose genes are differentially expressed upon periostin overexpression.

<p>a) Proteins detected are indicated on the left and, on the right, molecular weight of detected bands. P2rx7: arrows indicate glycosilated form (top) and unglycosilated form (bottom). LIFR: the truncated form of 60 kDa is shown. b) Levels of p-Akt and p-Erk. C, control cells. P, periostin overexpressing cells.</p