ABSTRACT
Elevated CUB-domain containing protein 1 (CDCP1) is predictive of colorectal cancer (CRC) recurrence and poor patient survival. While CDCP1 expression identifies stem cell populations that mediate lung metastasis, mechanisms underlying the role of this cell surface receptor in CRC have not been defined. We sought to identify CDCP1 regulated processes in CRC using stem cell populations, enriched from primary cells and cell lines, in extensive in vitro and in vivo assays. These experiments, demonstrating that CDCP1 is functionally important in CRC tumor initiation, growth and metastasis, identified CDCP1 as a positive regulator of Wnt signaling. Detailed cell fractionation, immunoprecipitation, microscopy, and immunohistochemical analyses demonstrated that CDCP1 promotes translocation of the key regulators of Wnt signaling, ß-catenin, and E-cadherin, to the nucleus. Of functional importance, disruption of CDCP1 reduces nuclear localized, chromatin-associated ß-catenin and nuclear localized E-cadherin, increases sequestration of these proteins in cell membranes, disrupts regulation of CRC promoting genes, and reduces CRC tumor burden. Thus, disruption of CDCP1 perturbs pro-cancerous Wnt signaling including nuclear localization of ß-catenin and E-cadherin.
Subject(s)
Antigens, Neoplasm/genetics , Cadherins/genetics , Cell Adhesion Molecules/genetics , Colorectal Neoplasms/genetics , beta Catenin/genetics , Active Transport, Cell Nucleus/genetics , Carcinogenesis/genetics , Cell Proliferation/genetics , Colorectal Neoplasms/pathology , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic/genetics , HCT116 Cells , Humans , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Wnt Signaling Pathway/geneticsSubject(s)
DNA/analysis , Luciferases/analysis , Luminescent Measurements , Plasmids/analysis , Transfection , BRCA1 Protein/genetics , DNA/genetics , Genes, Reporter , Humans , Luciferases/genetics , Luminescent Measurements/methods , MCF-7 Cells , Plasmids/genetics , Reproducibility of Results , Transfection/methodsABSTRACT
BACKGROUND: MicroRNAs are potent post-transcriptional regulators involved in all hallmarks of cancer. Mir-196a is transcribed from two loci and has been implicated in a wide range of developmental and pathogenic processes, with targets including Hox, Fox, Cdk inhibitors and annexins. Genetic variants and altered expression of MIR196A are associated with risk and progression of multiple cancers including breast cancer, however little is known about the regulation of the genes encoding this miRNA, nor the impact of variants therein. METHODS: Genomic data and chromatin interaction analysis were used to discover functional promoter and enhancer elements for MIR196A. Expression data were used to associate MIR196A with mechanisms of resistance, breast cancer subtypes and prognosis. RESULTS: Here we demonstrate that MIR196A displays complex and dynamic expression patterns, in part controlled by long-range transcriptional regulation between promoter and enhancer elements bound by ERα. Expression of this miRNA is significantly increased in drug-resistant models of hormone-receptor positive disease. The expression of MIR196A also proves to be a robust prognostic factor for patients with advanced and post-menopausal ER+ disease. CONCLUSION: This work sheds light on the normal and abnormal regulation of MIR196A and provides a novel stratification method for therapeutically resistant breast cancer.
Subject(s)
Breast Neoplasms/genetics , Estrogen Receptor alpha/genetics , MicroRNAs/genetics , Biomarkers, Tumor/biosynthesis , Biomarkers, Tumor/genetics , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Chromatin/genetics , Chromatin/metabolism , DNA Methylation , Disease Progression , Drug Resistance, Neoplasm , Estrogen Receptor alpha/metabolism , Female , Gene Expression Regulation, Neoplastic , Homeodomain Proteins/genetics , Humans , MCF-7 Cells , MicroRNAs/biosynthesis , Prognosis , Tamoxifen/pharmacologyABSTRACT
CUB domain containing protein 1 (CDCP1) is a transmembrane protein involved in progression of several cancers. When located on the plasma membrane, full-length 135kDa CDCP1 can undergo proteolysis mediated by serine proteases that cleave after two adjacent amino acids (arginine 368 and lysine 369). This releases from the cell surface two 65kDa fragments, collectively termed ShE-CDCP1, that differ by one carboxyl terminal residue. To evaluate the function of CDCP1 and its potential utility as a cancer biomarker, in this study we developed an enzyme-linked immunosorbent assay (ELISA) to reliably and easily measure the concentration of ShE-CDCP1 in biological samples. Using a reference standard we demonstrate that the developed ELISA has a working range of 0.68-26.5ng/ml, and the limit of detection is 0.25ng/ml. It displays high intra-assay (repeatability) and high inter-assay (reproducibility) precision with all coefficients of variation ≤7%. The ELISA also displays high accuracy detecting ShE-CDCP1 levels at ≥94.8% of actual concentration using quality control samples. We employed the ELISA to measure the concentration of ShE-CDCP1 in human serum samples with our results suggesting that levels are significantly higher in serum of colorectal cancer patients compared with serum from individuals with benign conditions (p<0.05). Our data also suggest that colorectal cancer patients with stage II-IV disease have at least 50% higher serum levels of ShE-CDCP1 compared with stage I cases (p<0.05). We conclude that the developed ELISA is a suitable method to quantify ShE-CDCP1 concentration in human serum.
