benzyloxycarbonylleucyl-leucyl-leucine-aldehyde and Neoplasm-Metastasis

High insulin/IGF is a biologic link between diabetes and cancers, but the underlying molecular mechanism remains unclear. Here we report a previously unrecognized tumour-promoting mechanism for stress protein TRB3, which mediates a reciprocal antagonism between autophagic and proteasomal degradation systems and connects insulin/IGF to malignant promotion. We find that several human cancers express higher TRB3 and phosphorylated insulin receptor substrate 1, which correlates negatively with patient's prognosis. TRB3 depletion protects against tumour-promoting actions of insulin/IGF and attenuates tumour initiation, growth and metastasis in mice. TRB3 interacts with autophagic receptor p62 and hinders p62 binding to LC3 and ubiquitinated substrates, which causes p62 deposition and suppresses autophagic/proteasomal degradation. Several tumour-promoting factors accumulate in cancer cells to support tumour metabolism, proliferation, invasion and metastasis. Interrupting TRB3/p62 interaction produces potent antitumour efficacies against tumour growth and metastasis. Our study opens possibility of targeting this interaction as a potential novel strategy against cancers with diabetes.

Topics: Adaptor Proteins, Signal Transducing; Adenine; Animals; Autophagy; Cell Cycle Proteins; Cell Movement; Gene Expression Regulation, Neoplastic; Humans; Insulin; Insulin Receptor Substrate Proteins; Insulin-Like Growth Factor I; Leupeptins; Male; Mice; Mice, Inbred C57BL; Mice, Nude; Neoplasm Metastasis; Neoplasms, Experimental; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Repressor Proteins; Sequestosome-1 Protein; Tissue Array Analysis; Ubiquitin

The current treatment for advanced nonsmall cell lung cancer (NSCLC) remains unsatisfactory due to resistance to chemotherapy and ionizing radiation. The ubiquitin-proteasome system (UPS) regulates multiple cellular processes that are crucial for the proliferation and survival of all kinds of cells. Carbobenzoxyl-leucinyl-leucinyl-leucinal-H (MG132), a specific and selective reversible inhibitor of the 26S proteasome, represents a novel approach for cancer therapy. However, whether MG132 can potentiate the effect of radiation against the growth and metastasis of NSCLC is not clear. We found that MG132 inhibited the proliferation of human NSCLC cell lines (A549 and H1299) in a dose- and time-dependent manner by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Then MG132 at a nontoxic dose (100 nM) was selected for following studies. Pretreatment of A549 and H1299 cells with 100 nM MG132 before ionizing radiation (IR) potentiated the anticancer effect of IR. Moreover, pretreatment with 100 nM MG132 before IR-enhanced radiation induced cell cycle arrest by decreased CyclinD1 but increased Wee1 expression in A549 and H1299 cells. In addition, pretreatment of MG132 combined with IR significantly suppressed cell migration and invasion abilities in NSCLC cell lines, which was accompanied by decreased expression of matrix metalloproteinase (MMP)-2 and MMP-9 in NSCLC cell lines. Taken together, our results demonstrate that MG132 enhances the antigrowth and antimetastatic effects of irradiation in NSCLC cells by modulating expression of cell cycle and invasion- related genes.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cyclin D1; Humans; Leupeptins; Lung Neoplasms; Neoplasm Metastasis; NF-kappa B; Nuclear Proteins; Proteasome Inhibitors; Protein-Tyrosine Kinases; Radiation Tolerance

SKI and SnoN proteins have been shown to inhibit TGF-β signaling, acting both as transcriptional co-repressors in the cell nucleus, and as sequestrators of SMAD proteins in the cytoplasm. TGF-β, on the other hand, induces rapid, proteasome-mediated, degradation of both proteins. How elevated SKI and SnoN protein levels co-exist with active autocrine TGF-β signaling in cancer cells is yet to be understood.. In this study, we found elevated SKI and SnoN protein levels in a panel of melanoma cell lines, as compared to normal melanocytes. There was no correlation between SKI protein content and the capacity of melanoma cells to invade Matrigel™, to form subcutaneous tumors, or to metastasize to bone after intracardiac inoculation into nude mice. Nor did we find a correlation between SKI expression and histopathological staging of human melanoma. TGF-β induced a rapid and dose-dependent degradation of SKI protein, associated with SMAD3/4 specific transcriptional response and induction of pro-metastatic target genes, partially prevented by pharmacologic blockade of proteasome activity. SKI knockdown in 1205Lu melanoma cells did not alter their invasive capacity or transcriptional responses to TGF-β, and did not allow p21 expression in response to TGF-β or reveal any growth inhibitory activity of TGF-β.. Despite high expression in melanoma cells, the role of SKI in melanoma remains elusive: SKI does not efficiently interfere with the pro-oncogenic activities of TGF-β, unless stabilized by proteasome blockade. Its highly labile nature makes it an unlikely target for therapeutic intervention.

Topics: Animals; Cell Line; Cell Line, Tumor; Cell Proliferation; Cyclin-Dependent Kinase Inhibitor p21; DNA-Binding Proteins; Gene Knockdown Techniques; Humans; Intracellular Signaling Peptides and Proteins; Leupeptins; Melanoma; Mice; Mice, Nude; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasm Transplantation; Proteasome Inhibitors; Proto-Oncogene Proteins; RNA Interference; Skin Neoplasms; Smad Proteins; Transcriptional Activation; Transforming Growth Factor beta; Up-Regulation

ACK1 (activated Cdc42-associated kinase 1) is a cytoplasmic tyrosine kinase implicated in trafficking through binding to epidermal growth factor (EGF) receptor and clathrin. Here, we have identified a new ACK1-binding partner, the E3 ubiquitin ligase Nedd4-2, which binds ACK1 via a conserved PPXY-containing region. We show that this motif also binds Nedd4-related proteins and several other WW domain-containing proteins, including the tumor suppressor oxidoreductase Wwox. In HeLa cells ACK1 colocalizes with Nedd4-2 in clathrin-rich vesicles, requiring this PPXY motif. Nedd4-2 strongly down-regulates ACK1 levels when coexpressed, and this process can be blocked by proteasome inhibitor MG132. ACK1 degradation via Nedd4 requires their mutual interaction and a functional E3 ligase; it is also driven by ACK1 activity. ACK1 is polyubiquitinated in vivo, and dominant inhibitory Nedd4 blocks endogenous ACK1 turnover in response to acute EGF treatment. Because EGF stimulation activates ACK1 ( Galisteo, M., Y., Y., Urena, J., and Schlessinger, J. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 9796-9801 ), our result suggest that EGF receptor-mediated ACK1 activation allows Nedd4-2 to drive kinase degradation. Thus the interplay between Nedd4-2-related E3 ligases that regulate ACK1 levels and Cbl that modifies EGF receptor impinges on cell receptor dynamics. These processes are particularly pertinent given the report of genomic amplification of the ACK1 locus in metastatic tumors.

Topics: Amino Acid Motifs; Animals; Chlorocebus aethiops; Clathrin; Clathrin-Coated Vesicles; COS Cells; Cysteine Proteinase Inhibitors; Down-Regulation; Endosomal Sorting Complexes Required for Transport; Enzyme Activation; Epidermal Growth Factor; ErbB Receptors; Gene Expression Regulation, Enzymologic; HeLa Cells; Humans; Leupeptins; Nedd4 Ubiquitin Protein Ligases; Neoplasm Metastasis; Neoplasms; Oxidoreductases; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; Protein Structure, Tertiary; Protein Transport; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-cbl; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Ubiquitination; WW Domain-Containing Oxidoreductase

Nuclear factor kappa B (NFkappaB) is a pro-malignant transcription factor with reciprocal effects on pro-metastatic and anti-metastatic gene expression. Interestingly, NFkappaB blockade results in the reciprocal induction of retinoic acid receptors (RARs). Given the established property of RARs as negative regulators of malignant progression, we postulated that reciprocal interactions between NFkappaB and RARs constitute a signaling module in metastatic gene expression and malignant progression. Using Line 1 tumor cells as a model for signal regulation of metastatic gene expression, we investigated the reciprocal interactions between NFkappaB and RARs in response to the pan-RAR agonist, all-trans retinoic acid (at-RA) and the pan-RAR antagonist, AGN193109.. At-RA [0.1-1 microM] dose-dependently activated RAR and coordinately trans-repressed NFkappaB, while AGN193109 [1-10 microM] dose-dependently antagonized the effects of at-RA. At-RA and AGN193109 reciprocally regulate pro-metastatic matrix metalloprotease 9 (MMP 9) and its endogenous inhibitor, the tissue inhibitor of metalloprotease 1 (TIMP 1), in a manner consistent with the putative roles of NFkappaB and RAR in malignant progression. Activation of RAR concurs with its ubiquitination and proteosomal degradation. Accordingly, the proteosome inhibitor, MG132 [5 microM], blocked RAR degradation, quelled RAR trans-activation and enhanced RAR trans-repression of NFkappaB.. We conclude that reciprocal interactions between NFkappaB and RARs constitute a signaling module in metastatic gene expression and malignant progression and propose that the dissociative effect of proteosome inhibitors could be harnessed towards enhancing the anticancer activity of retinoids.

Topics: Active Transport, Cell Nucleus; Cell Line, Tumor; Cell Nucleus; DNA-Binding Proteins; DNA, Neoplasm; Enzyme Activation; Gene Expression Regulation; Humans; Leupeptins; Ligands; Macromolecular Substances; Matrix Metalloproteinase 9; Neoplasm Metastasis; Neoplasm Proteins; NF-kappa B; Protein Transport; Receptors, Retinoic Acid; Repressor Proteins; Retinoids; Tissue Inhibitor of Metalloproteinase-1; Transcriptional Activation

CTLs specific for tumor antigens play a major role in immunity against cancer. Improved binding affinity of putative TAA peptides could enhance the in vivo immunogenicity of these self-altered self- tumor antigens. We examined here the efficacy of tumor vaccines composed of an altered peptide ligand of MUT-1, designated MUT-D, which exhibited significantly higher class-I allele K(b) binding affinity than its native counterpart MUT-1. The peptide was loaded on antigen presenting cells composed of the C57BL/6-syngeneic fibroblast cell line BLK.CL4. These cells were treated with proteasome inhibitor in order to shut off the degradation of proteins and the subsequent loading of endogenous peptides onto MHC class-I molecules, thus allowing for the pulsing of these cells with the modified peptide MUT-D. Proteasome-inhibited and modified peptide-loaded fibroblasts induced a peptide-specific CTL that significantly delayed primary tumor progression and protected the pre-immunized mice against the development of lung metastasis following the surgical removal of the primary tumor. Genetic modification of the fibroblasts to express the immunostimulatory cytokine IL-2 did not improve the APC function of the modified cells, nor did it result in augmentation of the potency of the vaccine. Our results suggest that the proteasome-inhibited fibroblasts pulsed with modified, high binder tumor-associated antigen peptide are good antigen-presenting cells and represent an effective form of tumor vaccine.

Topics: Animals; Antigens, Neoplasm; Cancer Vaccines; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Epitopes, T-Lymphocyte; Fibroblasts; Immunotherapy; Interleukin-2; Leupeptins; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Multienzyme Complexes; Neoplasm Metastasis; Neoplasm Transplantation; Oligopeptides; Proteasome Endopeptidase Complex; T-Lymphocytes, Cytotoxic