RESUMEN
Tumor metastasis is responsible for most cancer patients' deaths. Understanding the mechanism of metastasis is crucial for improving the cure rate for cancer. Here, we report that Gankyrin, a chaperone of ubiquitin-proteasome, has an essential role in breast cancer metastasis. We find that Gankyrin is highly overexpressed in human breast cancers and the expression correlates strongly with lymph node metastasis. Knocking down Gankyrin expression in highly metastatic human breast cancer cells significantly decreases cancer cell migration and invasion. Furthermore, we demonstrate that depletion of Gankyrin inhibits intrinsic Rac1 activity and induces large focal adhesions. Overexpression of Gankyrin accelerates focal adhesion turnover and increases cell migration. Notably, reduction of Gankyrin expression in mouse mammary tumor cell significantly decreases tumor metastasis to lung in animal models. Therefore, our findings suggest that Gankyrin is crucial for breast cancer metastasis and highlight the potential of Gankyrin as a therapeutic target for tumor metastasis.
Asunto(s)
Neoplasias de la Mama/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Animales , Neoplasias de la Mama/patología , Línea Celular , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Invasividad Neoplásica/patología , ARN Interferente Pequeño , Transducción de Señal/fisiología , TransfecciónRESUMEN
The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potent inducer of tumor cell apoptosis, but concerns of considerable liver toxicity limit its uses in human cancer therapy. Here, we show that i.v. injected Escherichia coli DH5alpha (E. coli DH5alpha) specifically replicates in solid tumors and metastases in live animals. E. coli DH5alpha does not enter tumor cells and suits for being the vector for soluble TRAIL (sTRAIL), which induces apoptosis by activating cell-surface death receptors. With the high 'tumor-targeting' nature, we demonstrate that intratumoral (i.t.) and intravenous injection of sTRAIL-expressing E. coli DH5alpha results in the tumor-targeted release of biologically active molecules, which leads to a dramatic reduction in the tumor growth rate and the prolonged survival of tumor-bearing mice. TRAIL delivery by E. coli DH5alpha did not cause any detectable toxicity to any organs, suggesting that E. coli DH5alpha-delivered sTRAIL protein therapy may provide a feasible and effective form of treatment for solid tumors.
Asunto(s)
Terapia Genética/métodos , Neoplasias/terapia , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Animales , Apoptosis/genética , Apoptosis/fisiología , Western Blotting , Línea Celular Tumoral , Escherichia coli/genética , Femenino , Citometría de Flujo , Vectores Genéticos/genética , Humanos , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/genética , Neoplasias/mortalidad , Reacción en Cadena de la Polimerasa , Tasa de Supervivencia , Ligando Inductor de Apoptosis Relacionado con TNF/genéticaRESUMEN
The solution to the protein folding problem lies in defining the relative energetic contributions of short-range and long-range interactions. In other words, the tendency of a stretch of amino acids to adopt a final secondary structural fold is context dependent. Our approach to this problem is to address whether an amino acid sequence, a "cassette," with a defined secondary structure in the three-dimensional structure of a native protein, can adopt a different conformation when placed into a different protein environment. Thus, we designed de novo a disulfide-bridged two-stranded alpha-helical parallel coiled coil, where each polypeptide chain consisted of 39 residues, as a "cassette holder." The 11-residue cassette would be inserted into the center of each polypeptide chain between the two nucleating alpha-helices to replace the control sequence. This Structural Cassette Mutagenesis model permits the analysis of short-range interactions within the inserted cassette as well as long-range interactions between the nucleating helices and the cassette region. The cassette holder, with a control sequence as the cassette, had a GdnHCl transition midpoint during denaturation of 5.6M. To demonstrate the feasibility of our model, an 11-residue beta-strand cassette from an immunoglobulin fold was inserted. The cassette was fully induced into the alpha-helical conformation with a [GdnHCl]1/2 value of 3.2M. To demonstrate the importance of short-range interactions (beta-sheet/alpha-helical propensities of amino acid side chains) in modulating structure and stability, a series of 1-5 threonine residues (highest beta-sheet propensity) were substituted into the solvent-exposed portions of the cassette in the alpha-helical conformation. Each successive substitution systematically decreased the stability of the coiled coil with peptide T4b (4 Thr residues) having a [GdnHCl]1/2 value of 2.2M. The single substitution of Ile in the hydrophobic core of the cassette with Ala or Thr had the most dramatic effect on protein stability (peptide 120T, [GdnHCl]1/2 value of 1.4M). Though these substitutions were able to modulate stability, they were not able to disrupt the alpha-helical conformation of the cassette, showing the importance of the nucleating alpha-helices on either side of the cassette in controlling conformation of the cassette. We have demonstrated the feasibility of our model protein to accept a beta-strand cassette. The effect of cassettes containing other beta-strands, beta-turns, loops, regions of undefined structure, and helical segments on conformation and stability of our model protein will also be determined.