okadaic-acid and Melanoma

Elevated levels of the oncoprotein, osteopontin (OPN), are associated with poor outcome of several types of cancers including melanoma. We have previously reported an important involvement of DNAJB6, a member of heat-shock protein 40 (HSP40) family, in negatively impacting tumor growth. The current study was prompted by our observations reported here which revealed a reciprocal relationship between DNAJB6 and OPN in melanoma specimens. The 'J domain' is the most conserved domain of HSP40 family of proteins. Hence, we assessed the functional role of the J domain in activities of DNAJB6. We report that the J domain of DNAJB6 is involved in mediating OPN suppression. Deletion of the J domain renders DNAJB6 incapable of impeding malignancy and suppressing OPN. Our mechanistic investigations reveal that DNAJB6 binds HSPA8 (heat-shock cognate protein, HSC70) and causes dephosphorylation of glycogen synthase kinase 3β (GSK3β) at Ser 9 by recruiting protein phosphatase, PP2A. This dephosphorylation activates GSK3β, leading to degradation of β-catenin and subsequent loss of TCF/LEF (T cell factor1/lymphoid enhancer factor1) activity. Deletion of the J domain abrogates assembly of this multiprotein complex and renders GSK3β inactive, thus, stabilizing β-catenin, a transcription co-activator for OPN expression. Our in-vitro and in-vivo functional analyses show that silencing OPN expression in the background of deletion of the J domain renders the resultant tumor cells less malignant despite the presence of stabilized β-catenin. Thus, we have uncovered a new mechanism for regulation of GSK3β activity leading to inhibition of Wnt/β-catenin signaling.

Topics: Animals; beta Catenin; Cell Line, Tumor; Down-Regulation; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; HSC70 Heat-Shock Proteins; HSP40 Heat-Shock Proteins; Humans; Lymphoid Enhancer-Binding Factor 1; Melanoma; Mice; Mice, Nude; Molecular Chaperones; Neoplasm Transplantation; Nerve Tissue Proteins; Okadaic Acid; Oligonucleotide Array Sequence Analysis; Osteopontin; Phosphorylation; Protein Binding; Protein Interaction Domains and Motifs; Protein Phosphatase 2; Protein Processing, Post-Translational; Protein Structure, Tertiary; Skin Neoplasms; T Cell Transcription Factor 1; Transcription, Genetic; Transcriptome

Because melanoma tumors originate partly from excessive UV exposure but become relatively resistant to radiation, we have now compared the effects of okadaic acid, a phosphatase inhibitor, with that of the thymidine analog bromodeoxyuridine as sensitizers of DNA damage in B16 melanoma. We now show that 25 nM okadaic acid promotes DNA fragmentation in B16 melanoma, increasing cell detachment as well as pigmentation, a characteristic of melanocytic cell differentiation. At lower levels, okadaic acid synergizes with UV exposure to increase DNA fragmentation. Although bromodeoxyuridine also caused DNA damage, it did not increase pigmentation and it suppressed cell detachment. Okadaic acid was also more effective in promoting DNA laddering in growing versus quiescent melanocytes. Because DNA damaging effects of okadaic acid are mediated by different pathways from those used by nucleoside analogs, like bromodeoxyuridine, we propose their concurrent effect with radiation as sensitizers to DNA damage.

Topics: Animals; Antimetabolites, Antineoplastic; Arsenicals; Bromodeoxyuridine; Cell Adhesion; Cell Differentiation; Cycloheximide; DNA Damage; DNA Fragmentation; Enzyme Inhibitors; Melanoma; Mice; Okadaic Acid; Phosphoprotein Phosphatases; Pigmentation; Protein Synthesis Inhibitors; Radiation-Sensitizing Agents; Tumor Cells, Cultured; Ultraviolet Rays

Levamisole is an effective antihelminthic drug with immunomodulatory and anticancer activities in model systems. Combined with fluorouracil (5-FU) as adjuvant treatment following resection of Dukes' stage C colon carcinomas, levamisole significantly reduces mortality. However, neither 5-FU nor levamisole alone has a significant effect on survival in this patient group. Previously, we noted that in vitro levamisole potentiated the antiproliferative activity of 5-FU.. Because levamisole is known to inhibit alkaline phosphatases and has been reported to inhibit dephosphorylation of some membrane phosphoproteins, we studied the effects of levamisole analogues and of chemically unrelated inhibitors of phosphatases for their ability to potentiate 5-FU inhibition of tumor cell line proliferation in vitro.. Human cancer cell lines were exposed to drugs alone or in combination with 5-FU. Antiproliferative activity was measured by determining the extent of reduction of colony formation by the cell lines in test plates compared with control plates.. We found that potentiation of 5-FU cytotoxicity by levamisole and by p-hydroxytetramisole, a metabolite of levamisole, is mimicked by orthovanadate, an inhibitor of tyrosine phosphatases, but not by okadaic acid, an inhibitor of serine and threonine phosphatases, Furthermore, l-p-bromotetramisole, a synthetic analogue of levamisole that is 10-fold more potent in inhibition of alkaline phosphatase than levamisole, potentiates the antiproliferative activity of 5-FU to a greater extent than d-p-bromotetramisole, a stereoisomer of l-p-bromotetramisole with little antiphosphatase activity.. Inhibition of tyrosine phosphatases may be responsible for the potentiation by levamisole of the inhibitory activity of 5-FU in vitro.. Inhibition of dephosphorylation of regulatory phosphoproteins may be related to the therapeutic efficacy of the combination of levamisole and 5-FU in the adjuvant treatment of colon carcinoma and may underlie at least some of the multiple effects of levamisole on immune parameters.

Topics: Breast Neoplasms; Colonic Neoplasms; Drug Synergism; Ethers, Cyclic; Fluorouracil; Humans; Levamisole; Melanoma; Okadaic Acid; Phosphoric Monoester Hydrolases; Skin Neoplasms; Tumor Cells, Cultured; Tumor Stem Cell Assay; Vanadates