phosphothreonine and Carcinoma

N,N'-dinitrosopiperazine (DNP) with organ specificity for nasopharyngeal epithelium, is involved in nasopharyngeal carcinoma (NPC) metastasis, though its mechanism is unclear. To reveal the pathogenesis of DNP-induced metastasis, immunoprecipitation was used to identify DNP-mediated phosphoproteins. DNP-mediated NPC cell line (6-10B) motility and invasion was confirmed. Twenty-six phosphoproteins were increased at least 1.5-fold following DNP exposure. Changes in the expression levels of selected phosphoproteins were verified by Western-blotting analysis. DNP treatment altered the phosphorylation of ezrin (threonine 567), vimentin (serine 55), stathmin (serine 25) and STAT3 (serine 727). Furthermore, it was shown that DNP-dependent metastasis is mediated in part through ezrin at threonine 567, as DNP-mediated metastasis was decreased when threonine 567 of ezrin was mutated. Strikingly, NPC metastatic tumors exhibited a higher expression of phosphorylated-ezrin at threonine 567 than the primary tumors. These findings provide novel insight into DNP-induced NPC metastasis and may contribute to a better understanding of the metastatic mechanisms of NPC tumors.

Topics: Blotting, Western; Carcinoma; Cell Death; Cell Line, Tumor; Cell Movement; Cytoskeletal Proteins; Electrophoresis, Gel, Two-Dimensional; Humans; Mass Spectrometry; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Neoplasm Invasiveness; Neoplasm Metastasis; Nitrosamines; Phosphoproteins; Phosphorylation; Phosphothreonine; Proteomics; Reproducibility of Results

Although cyclin D1 is overexpressed in a significant number of human cancers, overexpression alone is insufficient to promote tumorigenesis. In vitro studies have revealed that inhibition of cyclin D1 nuclear export unmasks its neoplastic potential. Cyclin D1 nuclear export depends upon phosphorylation of a C-terminal residue, threonine 286, (Thr-286) which in turn promotes association with the nuclear exportin, CRM1. Mutation of Thr-286 to a non-phosphorylatable residue results in a constitutively nuclear cyclin D1 protein with significantly increased oncogenic potential. To determine whether cyclin D1 is subject to mutations that inhibit its nuclear export in human cancer, we have sequenced exon 5 of cyclin D1 in primary esophageal carcinoma samples and in cell lines derived from esophageal cancer. Our work reveals that cyclin D1 is subject to mutations in primary human cancer. The mutations identified specifically disrupt phosphorylation of cyclin D1 at Thr-286, thereby enforcing nuclear accumulation of cyclin D1. Through characterization of these mutants, we also define an acidic residue within the C-terminus of cyclin D1 that is necessary for recognition and phosphorylation of cyclin D1 by glycogen synthase kinase-3 beta. Finally, through construction of compound mutants, we demonstrate that cell transformation by the cancer-derived cyclin D1 alleles correlates with their ability to associate with and activate CDK4. Our data reveal that cyclin D1 is subject to mutations in primary human cancer that specifically disrupt phosphorylation-dependent nuclear export of cyclin D1 and suggest that such mutations contribute to the genesis and progression of neoplastic growth.

Topics: Alleles; Amino Acid Substitution; Animals; Carcinoma; Cell Line; Cell Line, Tumor; Cell Nucleus; Cell Transformation, Neoplastic; Cyclin D; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclins; DNA Mutational Analysis; DNA, Neoplasm; Esophageal Neoplasms; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Mice; Mutation, Missense; Neoplasm Proteins; NIH 3T3 Cells; Phosphorylation; Phosphothreonine; Point Mutation; Protein Processing, Post-Translational; Protein Transport; Recombinant Fusion Proteins; Sequence Deletion; Spodoptera