The natural tumorcide Manumycin-A targets protein phosphatase 1α and reduces hydrogen peroxide to induce lymphoma apoptosis.

Numerous compounds for treating human disease have been discovered in nature. Manumycin-A (Man-A) is a natural, well-tolerated microbial metabolite and a potent experimental tumoricide. We recently showed that Man-A stimulated reactive oxygen species (ROS) which were upstream of serine/threonine (Ser/Thr) dephosphorylation and caspase-dependent cleavage of MEK and Akt in lymphoma apoptosis. Conversely, activation-specific, Ser/Thr phosphorylation of MEK and Akt proteins was stable in Man-A-resistant tumors suggesting that stimulation of Ser/Thr PPase activity might be required for Man-A tumoricidal activity. Pre-treatment with Calyculin-A, an equipotent inhibitor of PP1 and PP2A, blocked all downstream effects of Man-A whereas, the PP2A-selective inhibitor, Okadaic acid did not, suggesting that PP1 and not PP2A played a role in Man-A action. Phosphorylation of PP1α on Thr320 inhibits its activity. Hence, we posited that if PP1α was important for Man-A action, then Man-A treatment should promote dephosphorylation of PP1α on Thr320. Indeed, T320 was only dephosphorylated in the tumors that underwent apoptosis. Lastly, stable over-expression of a constitutively active PP1α mimetic (PP1αT320A mutant), elevated basal ROS levels and enhanced Man-A-stimulated apoptosis. Taken together, we conclude that PP1α is an important proximal effector of Man-A mediated lymphoma apoptosis and that the mechanisms of Man-A action warrant further investigation.

Reactive oxygen species-dependent destruction of MEK and Akt in Manumycin stimulated death of lymphoid tumor and myeloma cell lines.

Manumycin-A (Man-A) is a farnesyltransferase inhibitor (FTI), which was originally identified as an effective tumoricide against several cancers, especially ones harboring constitutively active Ras. However, it is becoming apparent that Man-A can stimulate tumor death independently of FTases. Antioxidant treatment blocked Man-A-stimulated DNA damage and reversed Man-A-inhibited tumor growth. However, the precise molecular details of how these reactive oxygen species (ROS) influence cell signaling modules are poorly understood. We examined how ROS may modulate death and survival pathways in a panel of tumor cells. Man-A treatment resulted in a massive induction of superoxide anion (.O(2) (-)) only in Man-A-sensitive tumors. Within 1 hr, Man-A caused the ROS-dependent activation of caspases 9 and 3. In this time-frame, the Ras-Raf target, MEK, and the survival protein Akt were dephosphorylated in ROS-dependent fashions and then cleaved in ROS and caspase-dependent manners. Pretreatment with ROS scavengers blocked the adverse effects of Man-A, including the processing of caspases and the cleavage of MEK and Akt. These events were noted before any losses in Ras activity or changes in its maturation could be detected. Finally, transfection with cDNAs encoding the antioxidant enzymes catalase, superoxide dismutase and thioredoxin reductase inhibited superoxide induction and apoptosis. Together, our data suggest that the elimination of tumors by Man-A can be independent of the inhibiting of Ras. However, one universal feature observed is the generation of death-triggering intracellular oxidants that appear to directly participate in the select targeting of growth and survival proteins that then either augment or ensure tumor cell death.

SHP-2 phosphatase is required for hematopoietic cell transformation by Bcr-Abl.

SHP-2 phosphatase forms a stable protein complex with and is heavily tyrosine-phosphorylated by the oncogenic tyrosine kinase Bcr-Abl. However, the role of SHP-2 in Bcr-Abl-mediated leukemogenesis is unclear. In the present report, we provide evidence that SHP-2 is required for hematopoietic cell transformation by Bcr-Abl. In vitro biological effects of Bcr-Abl transduction were diminished in SHP-2Delta/Delta hematopoietic cells, and the leukemic potential of Bcr-Abl-transduced SHP-2Delta/Delta cells in recipient animals was compromised. Further analyses showed that Bcr-Abl protein (p210) was degraded, and its oncogenic signaling was greatly decreased in SHP-2Delta/Delta cells. Treatment with proteasome inhibitors or reintroduction of SHP-2 restored p210 level in Bcr-Abl-transduced SHP-2Delta/Delta cells. Subsequent investigation revealed that SHP-2 interacted with heat shock protein 90, an important chaperone protein protecting p210 from proteasome-mediated degradation. The role of SHP-2 in the stability of p210 is independent of its catalytic activity. Blockade of SHP-2 expression in p210-expressing cells by antisense or small-interfering RNA approaches decreased p210 level, causing cell death. Inhibition of SHP-2 enzymatic activity by overexpression of catalytically inactive SHP-2 mutant did not destabilize p210 but enhanced serum starvation-induced apoptosis, suggesting that SHP-2 also plays an important role in downstream signaling of p210 kinase. These studies identified a novel function of SHP-2 and suggest that SHP-2 might be a useful target for controlling Bcr-Abl-positive leukemias.

Effects of a leukemia-associated gain-of-function mutation of SHP-2 phosphatase on interleukin-3 signaling.

Mutations in SHP-2 phosphatase that cause hyperactivation of its catalytic activity have been identified in human leukemias, particularly juvenile myelomonocytic leukemia, which is characterized by hypersensitivity of myeloid progenitor cells to granulocyte macrophage colony-stimulating factor and interleukin (IL)-3. However, the molecular mechanisms by which gain-of-function (GOF) mutations of SHP-2 induce hematopoietic malignancies are not fully understood. Our previous studies have shown that SHP-2 plays an essential role in IL-3 signal transduction in both catalytic-dependent and -independent manners and that overexpression (5-6-fold) of wild type (WT) SHP-2 attenuates IL-3-mediated hematopoietic cell function through accelerated dephosphorylation of STAT5. These results raised the possibility that SHP-2-associated leukemias are not solely attributed to the increased catalytic activity of GOF mutant SHP-2. GOF mutant SHP-2 must have gained additional capacities. To test this possibility, we investigated effects of a GOF mutation of SHP-2 (SHP-2 E76K) on hematopoietic cell function and IL-3 signal transduction by comparing with those of overexpressed WT SHP-2. Our results showed that SHP-2 E76K mutation caused myeloproliferative disease in mice, while overexpression of WT SHP-2 decreased hematopoietic potential of the transduced cells in recipient animals. The E76K mutation in the N-terminal Src homology 2 domain increased interactions of mutant SHP-2 with Grb2, Gab2, and p85, leading to hyperactivation of IL-3-induced Erk and phosphatidylinositol 3-kinase (PI3K) pathways. In addition, despite the substantial increase in the catalytic activity, dephosphorylation of STAT5 by SHP-2 E76K was dampened. Furthermore, catalytically inactive SHP-2 E76K with an additional C459S mutation retained the capability to increase the interaction with Gab2 and to enhance the activation of the PI3K pathway. Taken together, these studies suggest that in addition to the elevated catalytic activity, fundamental changes in physical and functional interactions between GOF mutant SHP-2 and signaling partners also play an important role in SHP-2-related leukemigenesis.

Critical roles of c-Kit tyrosine residues 567 and 719 in stem cell factor-induced chemotaxis: contribution of src family kinase and PI3-kinase on calcium mobilization and cell migration.

Stem cell factor (SCF) has crucial roles in proliferation, survival, and differentiation of hematopoietic stem cells and mast cells through binding to c-Kit receptor (KIT). Chemotaxis is another unique function of SCF. However, little is known about the intracellular signaling pathway of SCF/KIT-mediated cell migration. To investigate the signaling cascade, we made a series of 22 KIT mutants, in which tyrosine (Y) residue was substituted for phenylalanine (F) in the cytoplasmic domain, and introduced into BAF3 cells or 293T cells. On stimulation with SCF, BAF3 expressing KIT(WT)(WT) showed cell migration and Ca(2+) mobilization. Among 22 YF mutants, Y567F, Y569F, and Y719F showed significantly reduced cell migration and Ca(2+) mobilization compared to WT. In Y567F, Lyn activation on SCF stimulation decreased and C-terminal Src kinase (Csk) suppressed KIT-mediated Ca(2+) influx and cell migration, suggesting that Y567-mediated Src family kinase (SFK) activation leads to Ca(2+) influx and migration. Furthermore, we found that p38 mitogen-activated protein kinase (p38 MAPK) and Erk1/2 were also regulated by Y567/SFK and involved in cell migration, and that p38 MAPK induced Ca(2+) influx, thereby leading to Erk1/2 activation. In Y719F, the binding of phosphatidylinositol 3'-kinase (PI3K) to KIT was lost and KIT-mediated cell migration and Ca(2+) mobilization were suppressed by PI3K chemical inhibitors or dominant-negative PI3K, suggesting the involvement of Y719-mediated PI3K pathway in cell migration. Combination of Csk and the PI3K inhibitor synergistically reduced cell migration, suggesting the cooperation of SFK and PI3K. Taken together, these results indicate that 2 major KIT signaling pathways lead to cell migration, one is Y567-SFK-p38 MAPK-Ca(2+) influx-Erk and the other is Y719-PI3K-Ca(2+) influx.