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Oncogene (2002) 21, 1391 ± 1400 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Role of the AKT kinase in expansion of multiple myeloma clones: eects on cytokine-dependent proliferative and survival responses Jung-hsin Hsu1, Yijiang Shi1, Liping Hu1, Myrna Fisher1, Thomas F Franke2 and Alan Lichtenstein*,1 1Department of Medicine and Pathology, West LA VA-UCLA Medical Center and Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, California, CA 90073, USA; 2Department of Pharmacology, Columbia University, New York, NY 10032, USA IL-6 is an established growth factor for multiple bone marrow of myeloma patients and levels correlate myeloma tumor cells, stimulating proliferative and with disease activity (Bataille et al., 1989; Reibnegger et survival responses. Recent work indicates that IL-6 can al., 1991; (2) Monoclonal anti-IL-6 antibodies can activate the AKT kinase in myeloma cells. Thus, to test induce anti-tumor responses in patients (Klein et al., a potential role for AKT in IL-6-induced cellular 1991); Bataille et al., 1995), and; (3) IL-6 transgenic responses, we transfected myeloma cell lines with an mice are prone to developing MM (Suematsu et al., active `E40K' or dominant negative `PH' AKT construct 1992), while IL-6 knockout mice are resistant (Hilbert using an adenoviral vector. Transfection of the E40K et al., 1995). The source of IL-6 is from the marrow into myeloma cells resulted in enhanced tumor cell microenvironment which stimulates MM cells in a growth and expression of the PH dominant negative paracrine fashion (Klein et al., 1989). AKT resulted in both inhibition of the IL-6-dependent The crucial signal transduction pathways that induce proliferative response and a decrease in S phase IL-6 stimulatory eects on MM cells are under intensive distribution. While transfection of E40K protected study. Previous investigations have implicated the ERK myeloma cells from dexamethasone-induced apoptosis, (Ogata et al., 1997), Jun kinase (Chauhan et al., 1997; the dominant negative PH had no eect on the ability of Xu et al., 1998), and STAT (Catlett-Falcone et al., 1999) IL-6 to protect these cells from dexamethasone. These signaling cascades. However, recent studies suggest a results clearly demonstrate that AKT activation is signal pathway involving the AKT kinase may also be critical for the IL-6 proliferative response. In addition, important in expansion of myeloma clones. AKT is although the level of AKT activation can regulate encoded by c-akt, the cellular homologue of the viral sensitivity to dexamethasone-induced apoptosis, addi- oncogene, v-akt, originally identi®ed in an acute tional cytokine-induced AKT-independent pathways can transforming retrovirus causing T cell lymphomas in mediate IL-6 protection against dexamethasone. mice (Bellacosa et al., 1991). AKT is activated as a Oncogene (2002) 21, 1391 ± 1400. DOI: 10.1038/sj/ major target of phosphoinositol 3-kinase (PI 3-kinase)- onc/1205194 generated signals (Franke et al., 1995; Stokoe et al., 1997). Activation requires phosphorylation of AKT on Keywords: multiple myeloma; AKT; interleukin-6 threonine and serine residues (Klippel et al., 1997; Franke et al., 1995). A newly described membrane bound kinase, 3-phosphoinositide-dependent kinase Introduction (PDK1), phosphorylates AKT on threonine residues. The phospholipid second messengers of the PI 3-kinase In multiple myeloma (MM), enhanced proliferation reaction bind to the pleckstrin homology (PH) domain and increased resistance to apoptotic stimuli account of AKT (Franke et al., 1997) and alter its conformation for the expansion of the malignant clone. It is now so that threonine residues become accessible to PDK1 clear that interleukin-6 (IL-6) can function as a speci®c (Klippel et al., 1997). PDK1 is a constitutively active MM tumor growth factor due to its ability to stimulate kinase that is membrane-bound due to its own PH both proliferative and survival responses in these cells domain binding to low basal levels of phospholipids. In (Anderson et al., 1989; Lichtenstein et al., 1995). contrast, AKT is located in the cytosol of unstimulated Further support for IL-6 as a tumor growth factor in cells but translocates to the cell membrane following vivo includes: (1) IL-6 levels are elevated in plasma and stimulation (Andjelkovic et al., 1997). Thus, the up- stream PI 3-kinase-generated phospholipid messengers are crucial as they recruit AKT to the membrane (near PDK1) and also facilitate threonine phosphorylation by *Correspondence: A Lichtenstein, Hematology-Oncology, VA West PDK1. Following threonine phosphorylation, AKT LA Hospital, W111H, 11301 Wilshire Blvd, Los Angeles, CA 90073, then autophosphorylates itself at serine residues (Toker USA; E-mail: [email protected] Received 13 August 2001; revised 30 October 2001; accepted 26 and Newton, 2000). Activated AKT may promote November 2001 oncogenesis by virtue of its ability to activate several AKT in myeloma J-h Hsu et al 1392 distinct downstream pathways which mediate prolifera- tive or survival responses. In fact, the ability of AKT to transform cells (Cheng et al., 1997), of AKT antisense to inhibit transformation (Cheng et al., 1996), the presence of ampli®cation/over-expression of AKT in some solid tumors (Cheng et al., 1992; Miwa et al., 1996) and the fact that the PTEN phosphatase, which inhibits AKT activation, is a known tumor suppressor gene (Wu et al., 1998), all implicate AKT in oncogenesis. Support for a role of AKT speci®cally in myeloma pathogenesis comes from recent studies which demon- strated that IL-6 can activate AKT in myeloma cells (Tu et al., 2000), that some myeloma cell lines harbor PTEN loss-of-function mutations with heightened AKT activation (Hyun et al., 2000), and that the inhibition of myeloma cell growth in vitro could be induced by selective inhibition of PI 3-kinase, the upstream activator of AKT (Tu et al., 2000). Furthermore, in a recent study, we demonstrated frequent AKT activation in situ in myeloma cells of patient bone marrow (Hsu et al., 2001). However, there Figure 1 IL-6 induces wortmannin-sensitive activation and has been no previous assessment of the direct eects of activity of AKT. (a) AKT in vitro kinase assay of AF-10 cells treated with IL-6 (0.5 ng/ml) for 15 (15') or 30 (30') min or for AKT activation or inhibition on myeloma cell growth. 15 min following pre-treatment with wortmannin (0.1 mM) We, thus, initiated the current study to test the eects (15'+W). Immunodetection of phosphorylated GSK-3 is shown. of dominant active or dominant negative AKT Immunoprecipitates were also immunoblotted for total AKT constructs on MM cell responses to IL-6. (lower panel). (b) Western blot for phosphorylated AKT (P-AKT) or total AKT (AKT) in AF-10 cells treated with IL-6 (0.5 ng/ml) or with IL-6 following pre-treatment with wortmannin (IL- 6+W). Duration of IL-6 treatment is shown in minutes. Control Results cells (c) incubated for 15 min in absence of IL-6 AKT activation in multiple myeloma cells transfected with inhibiting AKT activation and AKT phosphorylation active or dominant negative constructs as it had no eect on IL-6-induced phosphorylation of The AF-10 myeloma cell line was used to investigate the p42 and p44 ERK in the same cells (Tu et al., 2000). role of AKT activation in IL-6-induced proliferative The above ability of wortmannin to prevent IL-6- response. This cell line consistently demonstrates an induced AKT activation in AF-10 cells correlated with a approximate twofold increase in its proliferative rate signi®cant (P50.05) inhibitory eect of the drug on IL- when exposed to the MM growth factor IL-6 (Tu et al., 6-induced stimulation of proliferation. Whereas the 2000). In addition, we have previously detected AKT mean IL-6-dependent proliferation index (assayed at activation in AF-10 cells following stimulation with IL- 72 h) in control AF-10 cells was 2.2+/70.4 (mean+/7 6(Tuet al., 2000). A more systematic evaluation of of four experiments), wortmannin at 0.1 mM signi®- AKT activation in this cell line was undertaken to cantly (P50.05) inhibited IL-6-induced cell growth evaluate its kinetics and wortmannin-sensitivity. As (proliferation index of 1.3+/70.2). These data are shown in one of several in vitro kinase assays (Figure similar to the previously demonstrated inhibitory eect 1a), IL-6 activated AKT activity within 15 min of of wortmannin on IL-6-dependent AF-10 cell growth incubation and activity decreased by 30 min. Pre- (Tu et al., 2000) and support a role for PI 3-kinase in treatment with the PI 3-kinase inhibitor wortmannin the IL-6 proliferative response. To speci®cally investi- (at 0.1 mM) abrogated AKT activity. IL-6 and wort- gate the role of AKT in IL-6-stimulated proliferation, mannin had no eect on expression of total AKT we exploited the susceptibility of myeloma cells to (bottom panel). These data are consistent with AKT adenovirus transfection (Teoh et al., 1998; Prince et al., activation occurring downstream of IL-6-induced PI 3- 1998). We transfected cells with an active AKT kinase activation. Similar results were obtained by construct that exhibits enhanced activity or a dominant immunoblotting with phospho-speci®c anti-AKT anti- negative AKT that inhibits endogenous activity. As an body, which recognizes AKT when phosphorylated at active AKT, we utilized the AKT-E40K mutant that serine 473 (Figure 1b, one of ®ve separate experiments). possesses enhanced activity due to a point mutation In the immunoblot analysis, phosphorylated AKT was resulting in an increased anity of the PH domain for ®rst detected by 5 min, increased by 15 min, and was the second messenger phospholipids (Aoki et al., 1998).