SH-5, an AKT inhibitor potentiates apoptosis and inhibits invasion through the suppression of anti-apoptotic, proliferative and metastatic gene products regulated by IκBα kinase activation

Abstract

Because the phosphatidylinositol-3-kinase-AKT pathway is emerging as an important regulator of tumor cell survival, inhibitors of this pathway have enormous potential in cancer treatment. A specific inhibitor of AKT, [d-3-deoxy-2-O-methyl-myo-inositol-1-[(R)-2-methoxy-3-(octadecyloxy)propyl hydrogen phosphate]] (SH-5) has been recently synthesized, but little is known about its effects on cytokine signaling. We found that SH-5 potentiated the apoptosis induced by tumor necrosis factor (TNF), as indicated by intracellular esterase staining, annexin V staining, and caspase-3 activation. This effect of SH-5 correlated with downregulation of various gene products that mediate cell survival, proliferation, metastasis, and invasion, all known to be regulated by NF-κB. SH-5 also blocked NF-κB activation induced by TNF-α, lipopolysaccharide, phorbol ester, and cigarette smoke but not that activated by hydrogen peroxide and RANK ligand, indicating differential requirement of AKT. Inhibition of NF-κB correlated with abrogation of phosphorylation and degradation of IκBα through the inhibition of activation of IκBα kinase (IKK). This led to suppression of the phosphorylation and translocation of p65 and also of NF-κB reporter activity induced by TNFR1, TRADD, TRAF2, NIK, and IKKβ but not that induced by p65 transfection. Thus, our results clearly demonstrate that inhibition of AKT leads to potentiation of apoptosis through modulation of NF-κB signaling.

Introduction

AKT, a serine–threonine kinase also known as protein kinase B, is a central signaling molecule in the phosphatidylinositol-3-kinase (PI3K) pathway. [1], [2], [3]. This kinase is activated by mitogens and cytokines that function as survival factors. AKT mediates its effects by phosphorylating substrates that decrease the activity of pro-apoptotic proteins or increase the activity of anti-apoptotic proteins [4], [5], [6], [7]. Activation of PI3K/AKT signaling results in a disturbance of control of cell proliferation and apoptosis, resulting in competitive growth advantage for tumor cells. Blockade of the PI3K–AKT pathway has been found to sensitize various tumor cell types to apoptotic cell death induced by a variety of chemotherapeutic agents [8], [9]. Hence, this pathway is an attractive target for the development of novel anticancer strategies. However, the molecular mechanisms for such enhanced induction of tumor cell apoptosis by the combination of a PI3K–AKT inhibitor and anticancer agents have remained largely unknown.

In addition to directly phosphorylating and inactivating pro-apoptotic protein targets, AKT can stimulate signaling pathways that regulate the activity of transcription factor NF-κB (10–16). [10], [11], [12], [13], [14], [15]. NF-κB is a family of Rel domain-containing proteins present in the cytoplasm of all cells, where they are kept in an inactive state by a family of anchorin domain-containing proteins, which includes IκBα, IκBβ, IκBγ, IκBɛ, Bcl-3, p105, and p100. Under resting conditions, NF-κB consists of a heterotrimer of p50, p65, and IκBα in the cytoplasm; only when activated and translocated to the nucleus is the sequence of events leading to activation initiated. Most carcinogens, inflammatory agents, and tumor promoters, including cigarette smoke, phorbol ester, okadaic acid, H₂O₂, and tumor necrosis factor (TNF), have been shown to activate NF-κB. The activation of NF-κB involves the phosphorylation, ubiquitination, and degradation of IκBα and phosphorylation of p65, which in turn leads to the translocation of NF-κB to the nucleus where it binds to specific response elements in the DNA. The phosphorylation of IκBα is catalyzed by IκBα kinase (IKK), which is essential for NF-κB activation by most agents [16], [17], [18].

However, the mechanism(s) by which NF-κB-AKT interaction contributes to survival in tumor cells is unknown. In the current study, we used a recently discovered inhibitor of AKT, the phosphatidylinositol ether lipid analogue [d-3-deoxy-2-O-methyl-myo-inositol-1-[(R)-2-methoxy-3-(octadecyloxy)propyl hydrogen phosphate]] (SH-5) [19] to investigate the role of NF-κB as a putative mediator of the anti-apoptotic function of AKT in TNF-induced cell signaling. Our results demonstrate that AKT inhibitor potentiates the TNF-induced apoptosis through downregulation of NF-κB-regulated anti-apoptotic gene products and the NF-κB activation pathway.