Mantle Cell Lymphoma: SAHA Blocks Akt/mTOR Pathway and Reduces Cyclin D1 Protein Levels by Affecting Translation.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase (HDAC) inhibitor. We have found that SAHA has profound antiproliferative activities against mantle cell lymphoma (MCL) cell lines (ED50 ≅ 1 μM SAHA for 3 days), and this was associated with a rapid decrease of Cyclin D1 protein as seen by western blot. For example, eight hours treatment with SAHA (5 μM), reduced the Cyclin D1 levels by 80 % in three of 3 MCL cell lines (Jeko1, SP49, SP53). In contrast, other HDAC inhibitors including valproic acid and trichostatin A, were unable to reduce protein levels of Cyclin D1 as rapidly or as completely as SAHA in these MCL cell lines. Also, a myelogenous leukemia cell line, K562 expressed low levels of Cyclin D1 and exposure of these cells to SAHA (8 hrs) had almost no effect on protein levels of Cyclin D1, although SAHA had profound antiproliferative effect on these cells. A hallmark of MCL cells is the t(11; 14)(q13; q32) chromosomal change which rearranges the enhancer region of the immunoglobulin heavy chain to the regulatory region of the cyclin D1 gene inducing overexpression of Cyclin D1 mRNA, leading to high expression of Cyclin D1 protein. Levels of Cyclin D1 mRNA (Northern blot) remained elevated in these cell lines during an 8 hr exposure to SAHA (5 μM). Also, stability of Cyclin D1 protein in these MCL lines as measured by pulse-chase assay and cyclohexamide treatment minimally changed during the 8 hours of exposure to SAHA in the MCL cell lines. Taken together, we speculated that translation of the Cyclin D1 may be slowed after treatment of the cells with SAHA. Since translation of Cyclin D1 protein is regulated by eIF4E, the binding activity of eIF4E to the cap site of mRNA was analyzed by immunoprecipitation using 7 methyl-GTP sepharose beads. Binding activity decreased rapidly after the treatment with SAHA. Since hypophosphorylated eIF4EBP binds eIF4E and blocks the function of eIF4E, activity of eIF4E is regulated by eIF4EBP and its phosphorylation status. Furthermore, Akt phosphorylates and activates mTOR which then phosphorylates eIF4EBP and prevents eIF4EBP from binding to eIF4E. We found that eIF4EBP, mTOR, and Akt proteins are markedly phosphorylated resulting in an activated Akt/mTOR pathway in the MCL cell lines. Levels of phospho-eIF4EBP, phosph-mTOR and phospho-Akt decreased rapidly after treatment with SAHA. Also, PI3K inhibitor, LY294002, transiently decreased the level of Cyclin D1 protein. In summary, our data suggest that 1) Akt/mTOR pathway is activated in MCL cells; 2) Translation of Cyclin D1 is dependent on this pathway in these cells; 3) Blockade of this pathway reduces the protein levels of Cyclin D1 rapidly by decreasing its translation; 4) SAHA blocks this pathway through a mechanism other than HDAC inhibition. This discovery may encourage a reappraisal of the mechanism of action of this interesting therapeutic compound and also focus attention to developing additional inhibitors of this pathway in cancers associated with dysregulated Cyclin D1.