trichostatin-a and Leukemia-Lymphoma--Adult-T-Cell

Several studies have revealed that endosomal sorting controls the steady-state levels of Notch at the cell surface in normal cells and prevents its inappropriate activation in the absence of ligands. However, whether this highly dynamic physiologic process can be exploited to counteract dysregulated Notch signaling in cancer cells remains unknown. T-ALL is a malignancy characterized by aberrant Notch signaling, sustained by activating mutations in Notch1 as well as overexpression of Notch3, a Notch paralog physiologically subjected to lysosome-dependent degradation in human cancer cells. Here we show that treatment with the pan-HDAC inhibitor Trichostatin A (TSA) strongly decreases Notch3 full-length protein levels in T-ALL cell lines and primary human T-ALL cells xenografted in mice without substantially reducing NOTCH3 mRNA levels. Moreover, TSA markedly reduced the levels of Notch target genes, including pTα, CR2, and DTX-1, and induced apoptosis of T-ALL cells. We further observed that Notch3 was post-translationally regulated following TSA treatment, with reduced Notch3 surface levels and increased accumulation of Notch3 protein in the lysosomal compartment. Surface Notch3 levels were rescued by inhibition of dynein with ciliobrevin D. Pharmacologic studies with HDAC1, 6, and 8-specific inhibitors disclosed that these effects were largely due to inhibition of HDAC6 in T-ALL cells. HDAC6 silencing by specific shRNA was followed by reduced Notch3 expression and increased apoptosis of T-ALL cells. Finally, HDAC6 silencing impaired leukemia outgrowth in mice, associated with reduction of Notch3 full-length protein in vivo. These results connect HDAC6 activity to regulation of total and surface Notch3 levels and suggest HDAC6 as a potential novel therapeutic target to lower Notch signaling in T-ALL and other Notch3-addicted tumors.

Topics: Animals; Apoptosis; Cell Line, Tumor; Cell Movement; Cell Proliferation; Gene Expression Regulation, Neoplastic; Histone Deacetylase 6; Humans; Hydroxamic Acids; Jurkat Cells; Leukemia-Lymphoma, Adult T-Cell; Lysosomes; Mice; Mice, Inbred NOD; Mice, SCID; Protein Transport; Receptor, Notch3; RNA, Messenger; RNA, Small Interfering; Signal Transduction; T-Lymphocytes

Adult T-cell leukemia/lymphoma (ATL) is a highly invasive and intractable T-cell malignancy caused by human T-cell leukemia virus-1 infection. We demonstrate herein that normal tissue-derived epithelial cells (NECs) exert protective effects on the survival of leukemic cells, which may partially account for high resistance to antileukemic therapies in patients with ATL. Viral gene-silenced, ATL-derived cell lines (ATL cells) dramatically escaped from histone deacetylase inhibitor-induced apoptosis by direct co-culture with NECs. Adhesions to NECs suppressed p21(Cip1) expression and increased a proportion of resting G0/G1 phase cells in trichostatin A (TSA)-treated ATL cells. ATL cells adhering to NECs down-regulated CD25 expression and enhanced vimentin expression, suggesting that most ATL cells acquired a quiescent state by cell-cell interactions with NECs. ATL cells adhering to NECs displayed highly elevated expression of the cancer stem cell marker CD44. Blockade of CD44 signaling diminished the NEC-conferred resistance of ATL cells to TSA-induced apoptosis. Co-culture with NECs also suppressed the expression of NKG2D ligands on TSA-treated ATL cells, resulting in decreased natural killer cell-mediated cytotoxicity. Combined evidence suggests that interactions with normal epithelial cells augment the resistance of ATL cells to TSA-induced apoptosis and facilitate immune evasion by ATL cells.

Topics: Adult; Apoptosis; Cell Adhesion; Cell Communication; Cell Cycle; Cell Line, Tumor; Cell Separation; Cell Survival; Coculture Techniques; Cytoprotection; Epithelial Cells; Histone Deacetylase Inhibitors; Humans; Hyaluronan Receptors; Hydroxamic Acids; Intracellular Space; Leukemia-Lymphoma, Adult T-Cell; Ligands; NK Cell Lectin-Like Receptor Subfamily K; Tumor Stem Cell Assay