Increased protein expression of the PTEN tumor suppressor in the presence of constitutively active Notch-1.

Mammalian Notch-1 is part of an evolutionarily conserved family of transmembrane receptors best known for involvement in cell fate decisions. Mutations that result in Notch-1 activation result in T-lineage oncogenesis. In other cell lineages, however, studies have indicated that cooperation with cellular signaling pathways, such as Ras, is necessary for Notch-mediated oncogenesis and in some settings, Notch-1 has been reported to function as a tumor suppressor. In order to test the hypothesis that the Notch-1 pathway exhibits cross-talk with Ras/Raf/MEK/ERK, the constitutively active cytoplasmic portion of Notch-1 was introduced into 293 HEK fibroblasts via retroviral transduction. ERK-1,-2 activation was markedly increased in cells expressing constitutively active Notch-1. These cells exhibited a more rounded morphology as compared to 293 cells transduced with an empty vector or parental 293 cells. These observations correlated with decreased total and phosphorylated focal adhesion kinase protein (FAK). Subsequent examination of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) revealed that total and phosphorylated PTEN protein was elevated in cells expressing constitutively active Notch-1. Loss of Akt phosphorylation was also observed in cells bearing activated Notch-1. Two potential binding sites for the Notch effector CBF-1 were identified in the human PTEN promoter sequence. A PTEN promoter luciferase reporter exhibited increased activity in the presence of Notch-1 signaling. These data indicate that Notch-1 can participate in cross-talk with other signaling pathways such as Ras/Raf/MEK/ERK through the regulation of the PTEN tumor suppressor.

Activated EGFR promotes the survival of B-lineage acute leukemia in the absence of stromal cells.

B-lineage acute leukemia (B-ALL) cells often require stromal cell support for optimal proliferation and apoptotic resistance. In addition, stromal cell contact can promote resistance to chemotherapeutic agents. However, the precise biochemical pathways within the leukemic cell that are activated by the bone marrow microenvironment which result promotion of cell proliferation and apoptotic protection are not fully characterized. We have recently reported that simultaneous inhibition of the MEK and PI3K pathways or the MEK and mTOR pathways promote rapid apoptosis of the stromal cell dependent B-lineage ALL cell line BLIN-2 in the presence of stromal cell support. These data indicated that stromal cell induced apoptotic protection is mediated by PI3K/mTOR and MEK in a mechanism(s) that suggests cross-talk or points of convergence. The EGF receptor (EGFR) has been reported to activate both MEK and PI3K. We report herein that use of the EGFR inhibitor, AG1478, inhibits BLIN-2 survival in the presence of stromal cells. FACS analysis revealed that EGFR is expressed on the surface of BLIN-2 cells. The addition of EGF to BLIN-2 cultures in the absence of stromal cells prolongs BLIN-2 survival. Similarly, introduction of a constitutively active form of EGFR, v-ErbB, into BLIN-2 prolongs the survival of BLIN-2 cells in the absence of stromal cell support. These data provide evidence that stimulation of the EGFR pathway is one mechanism by which the bone marrow microenvironment may contribute to the growth and survival of B-cell acute leukemia.