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Detection of collagen triple helix repeat containing-1 and nuclear factor (erythroid-derived 2)-like 3 in colorectal cancer.

ABSTRACT: BACKGROUND:Collagen Triple Helix Repeat Containing-1 (CTHRC1) and Nuclear factor (erythroid-derived 2)-like 3 (NFE2L3) may be useful biomarker candidates for the diagnosis of colorectal cancer (CRC) since they have shown an increase messenger RNA transcripts (mRNA) expression level in adenomas and colorectal tumours when compared to normal tissues. METHODS:To evaluate CTHRC1 and NFE2L3 as cancer biomarkers, it was generated and characterised several novel specific polyclonal antibodies (PAb), monoclonal antibodies (MAbs) and soluble Fab fragments (sFabs) against recombinant CTHRC1 and NFE2L3 proteins, which were obtained from different sources, including a human antibody library and immunised animals. The antibodies and Fab fragments were tested for recognition of native CTHRC1 and NFE2L3 proteins by immunoblotting analysis and enzyme-linked immunosorbent assay (ELISA) in colorectal cell lines derived from tumour and cancer tissues. RESULTS:Both, antibodies and a Fab fragment showed high specificity since they recognised only their corresponding recombinant antigens, but not a panel of different unrelated- and related proteins.In Western blot analysis of CTHRC1, a monoclonal antibody designated CH21D7 was able to detect a band of the apparent molecular weight of a full-length CTHRC1 in the human colon adenocarcinoma cell line HT29. This result was confirmed by a double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with the monoclonal antibodies CH21D7 and CH24G2, detecting CTHRC1 in HT29 and in the colon adenocarcinoma cell line SW620.Similar experiments were performed with PAb, MAbs, and sFab against NFE2L3. The immunoblot analysis showed that the monoclonal antibody 41HF8 recognised NFE2L3 in HT29, and leukocytes. These results were verified by DAS-ELISA assay using the pairs PAb/sFab E5 and MAb 41HF8/sFab E5.Furthermore, an immunoassay for simultaneous detection of the two cancer biomarkers was developed using a Dissociation-Enhanced Lanthanide Fluorescent Immunoassay technology (DELFIA). CONCLUSIONS:In conclusion, the antibodies obtained in this study are specific for CTHRC1 and NFE2L3 since they do not cross-react with unrelated- and related proteins and are useful for specific measurement of native CTHRC1 and NFE2L3 proteins. The antibodies and immunoassays may be useful for the analysis of CTHRC1 and NFE2L3 in clinical samples and for screening of therapeutic compounds in CRC.

SUBMITTER: Palma M

PROVIDER: S-EPMC3293008 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Detection of collagen triple helix repeat containing-1 and nuclear factor (erythroid-derived 2)-like 3 in colorectal cancer.

Palma Marco M Lopez Lissett L García Margarita M de Roja Nuria N Ruiz Tamara T García Julita J Rosell Elisabet E Vela Carmen C Rueda Paloma P Rodriguez María-Jose MJ

BMC clinical pathology 20120209

<h4>Background</h4>Collagen Triple Helix Repeat Containing-1 (CTHRC1) and Nuclear factor (erythroid-derived 2)-like 3 (NFE2L3) may be useful biomarker candidates for the diagnosis of colorectal cancer (CRC) since they have shown an increase messenger RNA transcripts (mRNA) expression level in adenomas and colorectal tumours when compared to normal tissues.<h4>Methods</h4>To evaluate CTHRC1 and NFE2L3 as cancer biomarkers, it was generated and characterised several novel specific polyclonal antib ...[more]

PMID: 22321245

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Project description:BackgroundCollagen triple helix repeat containing-1 (CTHRC1), which was firstly identified overexpressed in the adventitia and neointima of injured rat arteries, could inhibit collagen expression and increase cell migration. It was then found to be ubiquitously expressed in numerous cell types such as fibroblasts and smooth muscle cells, and aberrantly up-regulated in several malignant tumors. However, the functional role of CTHRC1 and its related mechanism in breast cancer still remains unclear.MethodsCTHRC1 expressions in breast cancer tissues and cells were assessed by qRT-PCR, western blot and immunohistochemistry. The relative expression level of miR-134, miR-155, miR-30c and miR-630 in breast cancer cells respectively was detected by qRT-PCR. Wild type (Wt) and Mutant type (Mut) CTHRC1 3'UTR sequences were cloned into a psi-CHECK2 reporter vector, and the relative luciferase activity was detected by dual-luciferase reporter assay in indicated cells. The effect of ectopic expression of miR-30c or gain and loss of CTHRC1 on cell viability, cell proliferation, cell cycle progression and apoptosis, cell invasion and migration was respectively detected by CCK-8 assay, colony formation assay, flow cytometry analysis, transwell invasion/migration assay. Protein levels of β-catenin, active β-catenin, normal and phosphorylated form of GSK-3β were detected by western blot in indicated cells. Immunofluorescence staining of β-catenin was performed to observe nuclear localization.ResultsWe found CTHRC1 was frequently up-regulated in human breast cancer cells and tissues. Then our cohort study and further meta-analysis validated high expression of CTHRC1 was associated with aggressive clinicopathological features and poor clinical outcome of breast cancer patients. In addition, CTHRC1 promoted cell proliferation, invasion and migration and suppressed cell apoptosis in breast cancer, which might be by activating GSK-3β/β-catenin signaling and inhibiting Bax/Caspase-9/Caspase-3 signaling respectively; and these biological functions of CTHRC1 could be directly negatively regulated by miR-30c.ConclusionTaken together, we identified the role of miR-30c/CTHRC1 axis in breast cancer progression and demonstrated CTHRC1 may serve as a prognostic biomarker and therapeutic target for breast cancer.

Project description:BackgroundOesophageal cancer is one of the most common malignancies worldwide,and oesophageal squamous cell carcinoma (ESCC) is the predominant histological type both globally and in China. Collagen triple helix repeat containing 1 (CTHRC1) has been found to be upregulated in ESCC. However, its role in tumourigenesis and progression of ESCC remains unclear.MethodsUsing our previous ESCC mRNA profiling data, we screened upregulated genes to identify those required for proliferation. Immunohistochemistry was performed to determine the level of CTHRC1 protein expression in 204 ESCC patients. Correlations between CTHRC1 expression and clinicopathological characteristics were assessed. In addition, pyrosequencing and 5-aza-dC treatment were performed to evaluate methylation status of CTHRC1 promoter. In vitro and in vivo analyses were also conducted to determine the role of CTHRC1 in ESCC cell proliferation, migration and invasion, and RNA sequencing and molecular experiments were performed to study the underlying mechanisms.ResultsBased on mRNA profiling data, CTHRC1 was identified as one of the most significantly upregulated genes in ESCC tissues (n = 119, fold change = 20.5, P = 2.12E-66). RNA interference screening also showed that CTHRC1 was required for cell proliferation. Immunohistochemistry confirmed markedly high CTHRC1 protein expression in tumour tissues, and high CTHRC1 expression was positively correlated with advanced T stage (P = 0.043), lymph node metastasis (P = 0.023), TNM stage (P = 0.024) and poor overall survival (P = 0.020). Promoter hypomethylation at cg07757887 may contribute to increased CTHRC1 expression in ESCC cells and tumours. Forced overexpression of CTHRC1 significantly enhanced cell proliferation, migration and invasion, whereas depletion of CTHRC1 suppressed these cellular functions in three ESCC cell lines and xenografts. CTHRC1 was found to activate FRA-1 (Fos-related antigen 1, also known as FOSL1) through the MAPK/MEK/ERK cascade, which led to upregulation of cyclin D1 and thus promoted cell proliferation. FRA-1 also induced snail1-mediated MMP14 (matrix metallopeptidase 14, also known as MT1-MMP) expression to facilitate ESCC cell invasion, migration, and metastasis.ConclusionsOur data suggest that CTHRC1 may act as an oncogenic driver in progression and metastasis of ESCC, and may serve as a potential biomarker for prognosis and personalized therapy.

Dataset Information

Detection of collagen triple helix repeat containing-1 and nuclear factor (erythroid-derived 2)-like 3 in colorectal cancer.

Publications

Detection of collagen triple helix repeat containing-1 and nuclear factor (erythroid-derived 2)-like 3 in colorectal cancer.

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