Mutations in the phosphatase and tensin homolog (PTEN) gene leading to PTEN protein deletion and subsequent activation of the PI3K/Akt signaling pathway are common in cancer. Here we show that PTEN inactivation in human T cell acute lymphoblastic leukemia (T-ALL) cells is not always synonymous with PTEN gene lesions and diminished protein expression. Samples taken from patients with T-ALL at the time of diagnosis very frequently showed constitutive hyperactivation of the PI3K/Akt pathway. In contrast to immortalized cell lines, most primary T-ALL cells did not harbor PTEN gene alterations, displayed normal PTEN mRNA levels, and expressed higher PTEN protein levels than normal T cell precursors. However, PTEN overexpression was associated with decreased PTEN lipid phosphatase activity, resulting from casein kinase 2 (CK2) overexpression and hyperactivation. In addition, T-ALL cells had constitutively high levels of ROS, which can also downmodulate PTEN activity. Accordingly, both CK2 inhibitors and ROS scavengers restored PTEN activity and impaired PI3K/Akt signaling in T-ALL cells. Strikingly, inhibition of PI3K and/or CK2 promoted T-ALL cell death without affecting normal T cell precursors. Overall, our data indicate that T-ALL cells inactivate PTEN mostly in a nondeletional, posttranslational manner. Pharmacological manipulation of these mechanisms may open new avenues for T-ALL treatment.

Interleukin (IL)-7 is essential for normal T cell development. Previously, we have shown that IL-7 increases viability and proliferation of T cell acute lymphoblastic leukemia (T-ALL) cells by up-regulating Bcl-2 and down-regulating the cyclin-dependent kinase inhibitor p27kip1. Here, we examined the signaling pathways via which IL-7 mediates these effects. We investigated mitogen-activated protein kinase (MEK)–extracellular signal-regulated kinase (Erk) and phosphatidylinositol-3-kinase (PI3K)–Akt (protein kinase B) pathways, which have active roles in T cell expansion and have been implicated in tumorigenesis. IL-7 induced activation of the MEK–Erk pathway in T-ALL cells; however, inhibition of the MEK–Erk pathway by the use of the cell-permeable inhibitor PD98059, did not affect IL-7–mediated viability or cell cycle progression of leukemic cells. IL-7 induced PI3K-dependent phosphorylation of Akt and its downstream targets GSK-3, FOXO1, and FOXO3a. PI3K activation was mandatory for IL-7–mediated Bcl-2 up-regulation, p27kip1 down-regulation, Rb hyperphosphorylation, and consequent viability and cell cycle progression of T-ALL cells. PI3K signaling was also required for cell size increase, up-regulation of CD71, expression of the glucose transporter Glut1, uptake of glucose, and maintenance of mitochondrial integrity. Our results implicate PI3K as a major effector of IL-7–induced viability, metabolic activation, growth and proliferation of T-ALL cells, and suggest that PI3K and its downstream effectors may represent molecular targets for therapeutic intervention in T-ALL.

Radiotherapy is a widely used treatment option in cancer. However, recent evidence suggests that doses of ionizing radiation (IR) delivered inside the tumor target volume, during fractionated radiotherapy, can promote tumor invasion and metastasis. Furthermore, the tissues that surround the tumor area are also exposed to low doses of IR that are lower than those delivered inside the tumor mass, because external radiotherapy is delivered to the tumor through multiple radiation beams, in order to prevent damage of organs at risk. The biological effects of these low doses of IR on the healthy tissue surrounding the tumor area, and in particular on the vasculature remain largely to be determined. We found that doses of IR lower or equal to 0.8 Gy enhance endothelial cell migration without impinging on cell proliferation or survival. Moreover, we show that low-dose IR induces a rapid phosphorylation of several endothelial cell proteins, including the Vascular Endothelial Growth Factor (VEGF) Receptor-2 and induces VEGF production in hypoxia mimicking conditions. By activating the VEGF Receptor-2, low-dose IR enhances endothelial cell migration and prevents endothelial cell death promoted by an anti-angiogenic drug, bevacizumab. In addition, we observed that low-dose IR accelerates embryonic angiogenic sprouting during zebrafish development and promotes adult angiogenesis during zebrafish fin regeneration and in the murine Matrigel assay. Using murine experimental models of leukemia and orthotopic breast cancer, we show that low-dose IR promotes tumor growth and metastasis and that these effects were prevented by the administration of a VEGF receptor-tyrosine kinase inhibitor immediately before IR exposure. These findings demonstrate a new mechanism to the understanding of the potential pro-metastatic effect of IR and may provide a new rationale basis to the improvement of current radiotherapy protocols.

In normal T-cell development interleukin-7 (IL-7) functions as an antiapoptotic factor by regulating bcl-2 expression in immature thymocytes and mature T cells. Similar to what occurs in normal immature thymocytes, prevention of spontaneous apoptosis by IL-7 in precursor T-cell acute lymphoblastic leukemia (T-ALL) cells correlates with up-regulation of bcl-2. IL-7 is also implicated in leukemogenesis because IL-7 transgenic mice develop lymphoid malignancies, suggesting that IL-7 may regulate the generation and expansion of malignant cells. This study shows that in the presence of IL-7, T-ALL cells not only up-regulated bcl-2 expression and escaped apoptosis but also progressed in the cell cycle, resulting in sequential induction of cyclin D2 and cyclin A. Down-regulation of p27^kip1 was mandatory for IL-7–mediated cell cycle progression and temporally coincided with activation of cyclin-dependent kinase (cdk)4 and cdk2 and hyperphosphorylation of Rb. Strikingly, forced expression of p27^kip1 in T-ALL cells not only prevented cell cycle progression but also reversed IL-7–mediated up-regulation of bcl-2 and promotion of viability. These results show for the first time that a causative link between IL-7–mediated proliferation and p27^kip1 down-regulation exists in malignant T cells. Moreover, these results suggest that p27^kip1 may function as a tumor suppressor gene not only because it is a negative regulator of cell cycle progression but also because it is associated with induction of apoptosis of primary malignant cells.