phosphothreonine and Neoplasm-Metastasis

Abnormal expression of cyclin-dependent kinase 5 (CDK5) has been found in several human cancers, whereas the role of CDK5 in the malignant development of colorectal cancer (CRC) has not been well characterized. Here we investigated the role of CDK5 in CRC and found that its expression was much higher in CRC tissues than that in normal tissues with a higher expression level of CDK5 closely correlating to advanced American Joint Committee on Cancer (AJCC) stage, poor differentiation, increased tumor size and poor prognosis of CRC. Biological function experiments showed that CDK5 regulated CRC cell proliferation and metastasis ability. Whole-genome microarray analysis, co-immunoprecipitation, in vitro kinase assay, western blotting, luciferase reporter assays and electrophoretic mobility shift assay (EMSA) showed that CDK5 could directly phosphorylate ERK5 at threonine (Thr) 732 and finally modulate the oncogenic ERK5-AP-1 axis. Further researches showed that CDK5-ERK5-AP-1 axis could promote progression of CRC carcinogenesis and had a significant correlation in human CRC samples. In summary, this study revealed the functional and mechanistic links between CDK5 and the oncogenic ERK5-AP-1 signaling pathway in the pathogenesis of CRC. These findings suggest that CDK5 has an important role in CRC development and may serve as a potential therapeutic target for CRC.

Topics: Adaptor Proteins, Signal Transducing; Carcinogenesis; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; Cyclin-Dependent Kinase 5; Disease Progression; Down-Regulation; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Male; Middle Aged; Mitogen-Activated Protein Kinase 7; Neoplasm Invasiveness; Neoplasm Metastasis; Phosphorylation; Phosphothreonine; Signal Transduction; Survival Analysis; Transcription Factor AP-1; Up-Regulation

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

Tumor-derived microvesicles are rich in metastasis-related proteases and play a role in the interactions between tumor cells and tumor microenvironment in tumor metastasis. Because shed microvesicles may remain in the extracellular environment around tumor cells, the microvesicle membrane protein may be the potential target for cancer therapy. Here we report that chromosome segregation 1-like (CSE1L) protein is a microvesicle membrane protein and is a potential target for cancer therapy. v-H-Ras expression induced extracellular signal-regulated kinase (ERK)-dependent CSE1L phosphorylation and microvesicle biogenesis in various cancer cells. CSE1L overexpression also triggered microvesicle generation, and CSE1L knockdown diminished v-H-Ras-induced microvesicle generation, matrix metalloproteinase (MMP)-2 and MMP-9 secretion and metastasis of B16F10 melanoma cells. CSE1L was preferentially accumulated in microvesicles and was located in the microvesicle membrane. Furthermore, anti-CSE1L antibody-conjugated quantum dots could target tumors in animal models. Our findings highlight a novel role of Ras-ERK signaling in tumor progression and suggest that CSE1L may be involved in the "early" and "late" metastasis of tumor cells in tumorigenesis. Furthermore, the novel microvesicle membrane protein, CSE1L, may have clinical utility in cancer diagnosis and targeted cancer therapy.

Topics: Animals; Antibodies; Cell Line, Tumor; Cell-Derived Microparticles; Cellular Apoptosis Susceptibility Protein; Extracellular Signal-Regulated MAP Kinases; Humans; Male; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasms; Phosphorylation; Phosphothreonine; ras Proteins

MicroRNAs-221 and -222 are highly upregulated in several solid tumors, including melanomas. We demonstrate that the proto-oncogene ETS-1, involved in the pathogenesis of cancers of different origin, is a transcriptional regulator of miR-222 by direct binding to its promoter region. Differently from 293FT cells or early stage melanomas, where unphosphorylated ETS-1 represses miR-222 transcription, in metastatic melanoma the constitutively Thr-38 phosphorylated fraction of ETS-1 induces miR-222. Despite its stepwise decreased expression along with melanoma progression, the oncogenic activity of ETS-1 relies on its RAS/RAF/ERK-dependent phosphorylation status more than on its total amount. To close the loop, we demonstrate ETS-1 as a direct target of miR-222, but not miR-221, showing the novel option of their uncoupled functions. In addition, a spatial redistribution of ETS-1 protein from the nucleus to the cytoplasm is also evidenced in advanced melanoma cells. Finally, in vivo studies confirmed the contribution of miR-222 to the increased invasive potential obtained by ETS- silencing.

Topics: Animals; Cell Line, Tumor; Down-Regulation; Gene Expression Regulation, Neoplastic; Humans; Melanocytes; Melanoma; Mice; Mice, Nude; MicroRNAs; Neoplasm Metastasis; Phosphorylation; Phosphothreonine; Proto-Oncogene Mas; Proto-Oncogene Protein c-ets-1; Signal Transduction; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transcription, Genetic

Ezrin is highly expressed in metastatic tumors and is involved in filopodia formation as well as promotion of tumor metastasis. Thus, Ezrin may serve as a potential target for anti-metastatic therapy. This study demonstrates that berberine reduces filopodia formation of a nasopharyngeal carcinoma (NPC) cell line, 5-8F, at non-cytotoxic concentrations. Furthermore, invasion and motility of 5-8F cells are decreased in a dose- and time-dependent manner, resulting in 73.0% invasion and 67.0% motility inhibition at 20 mum. The inhibitory effects of berberine on 5-8F cell metastasis were further confirmed in a mouse model of metastasis. Berberine treatment in vivo resulted in a 51.1% inhibition of tumor metastasis to the lymph nodes and decreased Ezrin phosphorylation at threonine 567 in metastatic samples. Berberine suppressed the presence of phosphorylated Ezrin (phospho-Ezrin) in a dose- and time-dependent manner but had no effect on total Ezrin protein expression at non-cytotoxic concentrations. Furthermore, the inhibitory effects of berberine on phospho-Ezrin were dependent on the suppression of Rho kinase activity. Reduction of Ezrin phosphorylation at Thr(567) by berberine was associated with its inhibitory effect on filopodia formation in 5-8F cells. However, berberine did not effectively inhibit the motility and invasion of NPC cells containing Ezrin Thr(567) mutants. These results confirm that berberine inhibits Ezrin phosphorylation at Thr(567). Nonetheless, berberine reduces motility and invasion of cells and inhibits tumor metastasis. The reduction of Rho kinase-mediated Ezrin phosphorylation mediated by berberine may be a novel anti-metastatic pathway in NPC 5-8F cells.

Topics: Animals; Antineoplastic Agents; Berberine; Cell Line, Tumor; Cytoskeletal Proteins; Enzyme Activation; Female; Humans; Mice; Nasopharyngeal Neoplasms; Neoplasm Metastasis; Phosphorylation; Phosphothreonine; Pseudopodia; rho-Associated Kinases; Threonine