Richard I. Feldman

AKT1/PKBα Kinase Is Frequently Elevated in Human Cancers and Its Constitutive Activation Is Required for Oncogenic Transformation in NIH3T3 Cells

Extensive studies have demonstrated that the Akt/AKT1 pathway is essential for cell survival and inhibition of apoptosis; however, alterations of Akt/AKT1 in human primary tumors have not been well documented. In this report, significantly increased AKT1 kinase activity was detected in primary carcinomas of prostate (16 of 30), breast (19 of 50), and ovary (11 of 28). The results were confirmed by Western blot and immunohistochemical staining analyses with phospho-Ser473 Akt antibody. The majority of AKT1-activated tumors are high grade and stage III/lV (13 of 16 prostate, 15 of 19 breast, and 8 of 11 ovarian carcinomas). Previous studies showed that wild-type AKT1 was unable to transform NIH3T3 cells. To demonstrate the biological significance of AKT1 activation in human cancer, constitutively activated AKT1 (Myr-Akt) was introduced into NIH3T3 cells. Overexpression of Myr-Akt in the stably transfected cells resulted in malignant phenotype, as determined by growth in soft agar and tumor formation in nude mice. These data indicate that AKT1 kinase, which is frequently activated in human cancer, is a determinant in oncogenesis and a potential target for cancer intervention.

Akt/protein kinase B signaling inhibitor-2, a selective small molecule inhibitor of Akt signaling with antitumor activity in cancer cells overexpressing Akt

Accumulated studies have shown that activation of the Akt pathway plays a pivotal role in malignant transformation and chemoresistance by inducing cell survival, growth, migration, and angiogenesis. Therefore, Akt is believed to be a critical target for cancer intervention. Here, we report the discovery of a small molecule Akt pathway inhibitor, Akt/protein kinase B signaling inhibitor-2 (API-2), by screening the National Cancer Institute Diversity Set. API-2 suppressed the kinase activity and phosphorylation level of Akt. The inhibition of Akt kinase resulted in suppression of cell growth and induction of apoptosis in human cancer cells that harbor constitutively activated Akt due to overexpression of Akt or other genetic alterations such as PTEN mutation. API-2 is highly selective for Akt and does not inhibit the activation of phosphatidylinositol 3′-kinase, phosphoinositide-dependent kinase-1, protein kinase C, serum- and glucocorticoid-inducible kinase, protein kinase A, signal transducer and activators of transcription 3, extracellular signal-regulated kinase-1/2, or c-Jun NH2-terminal kinase. Furthermore, API-2 potently inhibited tumor growth in nude mice of human cancer cells in which Akt is aberrantly expressed/activated but not of those cancer cells in which it is not. These findings provide strong evidence for pharmacologically targeting Akt for anticancer drug discovery.

Akt Phosphorylation and Stabilization of X-linked Inhibitor of Apoptosis Protein (XIAP)

Akt negatively regulates apoptotic pathways at a premitochondrial level through phosphorylation and modulation of proteins such as Bad, Forkhead proteins, and GSK-3β. Akt has also been shown to protect cell death at a post-mitochondrial level, although its downstream targets have not been well documented. Here, we demonstrate that Akt, including AKT1 and AKT2, interacts with and phosphorylates X-linked inhibitor of apoptosis protein (XIAP) at residue serine-87 in vitro and in vivo. Phosphorylation of XIAP by Akt protects XIAP from ubiquitination and degradation in response to cisplatin. Moreover, autoubiquitination of XIAP is also inhibited by Akt. Consistent with this, an XIAP mutant introduced into cells which mimics the Akt-phosphorylated form (i.e. XIAP-S87D) displays reduced ubiquitination and degradation as compared with wild type XIAP. The greater stability of XIAP-S87D in cells translated to increased cell survival after cisplatin treatment. Conversely, a mutant that could not be phosphorylated by Akt (XIAP-S87A) was more rapidly degraded and showed increased cisplatin-induced apoptosis. Furthermore, suppression of XIAP by either siRNA or adenovirus of antisense of XIAP induced programmed cell death and inhibited Akt-stimulated cell survival in ovarian cancer cells. These data identify XIAP as a new downstream target of Akt and a potentially important mediator of the effect of Akt on cell survival.

Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH. kinase/Akt pathway in human ovarian cancer

We previously demonstrated that AKT2, a member of protein kinase B family, is activated by a number of growth factors via Ras and PI 3-kinase signaling pathways. Here, we report the frequent activation of AKT2 in human primary ovarian cancer and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase (PI 3-kinase)/Akt pathway. In vitro AKT2 kinase assay analyses in 91 ovarian cancer specimens revealed elevated levels of AKT2 activity (>3-fold) in 33 cases (36.3%). The majority of tumors displaying activated AKT2 were high grade and stages III and IV. Immunostaining and Western blot analyses using a phospho-ser-473 Akt antibody that detects the activated form of AKT2 (AKT2 phosphorylated at serine-474) confirmed the frequent activation of AKT2 in ovarian cancer specimens. Phosphorylated AKT2 in tumor specimens localized to the cell membrane and cytoplasm but not the nucleus. To address the mechanism of AKT2 activation, we measured in vitro PI 3-kinase activity in 43 ovarian cancer specimens, including the 33 cases displaying elevated AKT2 activation. High levels of PI 3-kinase activity were observed in 20 cases, 15 of which also exhibited AKT2 activation. The remaining five cases displayed elevated AKT1 activation. Among the cases with elevated AKT2, but not PI 3-kinase activity (18 cases), three showed down-regulation of PTEN protein expression. Inhibition of PI 3-kinase/AKT2 by wortmannin or LY294002 induces apoptosis in ovarian cancer cells exhibiting activation of the PI 3-kinase/AKT2 pathway. These findings demonstrate for the first time that activation of AKT2 is a common occurrence in human ovarian cancer and that PI 3-kinase/Akt pathway may be an important target for ovarian cancer intervention.

Phosphatidylinositol 3-kinase/Akt pathway regulates tuberous sclerosis tumor suppressor complex by phosphorylation of tuberin.

Normal cellular functions of hamartin and tuberin, encoded by the TSC1 and TSC2tumor suppressor genes, are closely related to their direct interactions. However, the regulation of the hamartin-tuberin complex in the context of the physiologic role as tumor suppressor genes has not been documented. Here we show that insulin or insulin growth factor (IGF) 1 stimulates phosphorylation of tuberin, which is inhibited by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 but not by the mitogen-activated protein kinase inhibitor PD98059. Expression of constitutively active PI3K or active Akt, including Akt1 and Akt2, induces tuberin phosphorylation. We further demonstrate that Akt/PKB associates with hamartin-tuberin complexes, promoting phosphorylation of tuberin and increased degradation of hamartin-tuberin complexes. The ability to form complexes, however, is not blocked. Akt also inhibits tuberin-mediated degradation of p27kip1, thereby promoting CDK2 activity and cellular proliferation. Our results indicate that tuberin is a direct physiological substrate of Akt and that phosphorylation of tuberin by PI3K/Akt is a major mechanism controlling hamartin-tuberin function.

AKT2 Inhibition of Cisplatin-induced JNK/p38 and Bax Activation by Phosphorylation of ASK1 IMPLICATION OF AKT2 IN CHEMORESISTANCE

Cisplatin and its analogues have been widely used for treatment of human cancer. However, most patients eventually develop resistance to treatment through a mechanism that remains obscure. Previously, we found that AKT2 is frequently overexpressed and/or activated in human ovarian and breast cancers. Here we demonstrate that constitutively active AKT2 renders cisplatin-sensitive A2780S ovarian cancer cells resistant to cisplatin, whereas phosphatidylinositol 3-kinase inhibitor or dominant negative AKT2 sensitizes A2780S and cisplatin-resistant A2780CP cells to cisplatin-induced apoptosis through regulation of the ASK1/JNK/p38 pathway. AKT2 interacts with and phosphorylates ASK1 at Ser-83 resulting in inhibition of its kinase activity. Accordingly, activated AKT2 blocked signaling down-stream of ASK1, including activation of JNK and p38 and the conversion of Bax to its active conformation. Expression of nonphosphorylatable ASK1-S83A overrode the AKT2-inhibited JNK/p38 activity and Bax conformational changes, whereas phosphomimic ASK1-S83D inhibited the effects of cisplatin on JNK/p38 and Bax. Cisplatin-induced Bax conformation change was inhibited by inhibitors or dominant negative forms of JNK and p38. In conclusion, our data indicate that AKT2 inhibits cisplatin-induced JNK/p38 and Bax activation through phosphorylation of ASK1 and thus, plays an important role in chemoresistance. Further, regulation of the ASK1/JNK/p38/Bax pathway by AKT2 provides a new mechanism contributing to its antiapoptotic effects.

Activation of Phosphatidylinositol 3-Kinase/Akt Pathway by Androgen through Interaction of p85α, Androgen Receptor, and Src

Recent studies have demonstrated that the cell growth and antiapoptotic actions of androgen could be dissociated from the transcriptional activity of the receptor and were, instead, mediated by activation of a mitogen-activated protein kinase pathway. This finding suggests an important cellular function of androgen receptor (AR) outside the nucleus. In this report, we demonstrate that androgen activates phosphatidylinositol 3-kinase (PI3K) and Akt, including AKT1 and AKT2, in AR-positive cells. Androgen-induced cell growth and survival were inhibited by PI3K inhibitor and dominant-negative Akt. AR interacts with the p85α regulatory subunit of PI3K, and its binding affinity is increased after androgen stimulation. The sites of interaction on the two proteins were mapped to the C-terminal Src-homology 2 domain of p85α and N terminus of AR. Activation of PI3K/Akt by androgen was inhibited by dominant-negative Src. Neither N-terminaltruncated nor proline-rich region-deleted AR mutants, which are unable to bind to p85α and Src, respectively, was able to mediate androgen-induced PI3K/Akt activation. AR with deletion of C-terminal region including ligand binding domain, however, retains the ability to activate PI3K/Akt upon androgen stimulation, which supports the notion that nongenomic function of androgen is mediated by its interaction with membrane receptors (1, 3, 4). These findings indicate that a triple complex between AR, p85α, and Src is required for androgen-stimulated PI3K/Akt activation, and that the PI3K/Akt pathway, in addition to mitogen-activated protein kinase, mediates androgen-induced cell growth and cell survival.

Aurora-A Kinase Regulates Telomerase Activity through c-Myc in Human Ovarian and Breast Epithelial Cells

Aurora-A kinase is frequently overexpressed/activated in human ovarian and breast cancers. A rat mammary tumor model study indicates that alterations of Aurora-A are early events during mammary tumor development (T. M. Goepfert et al., Cancer Res., 62: 4115–4122, 2002), suggesting that Aurora-A plays a pivotal role in transformation. However, the molecular mechanism by which Aurora-A induces ovarian and breast cell transformation remains elusive. Here we show that ectopic expression of Aurora-A induces telomerase activity in human ovarian and breast epithelial cell lines HIOSE118 and MCF-10A. The mRNA and promoter activities of human telomerase reverse transcriptase (hTERT) are stimulated by Aurora-A. Furthermore, we have demonstrated that the c-Myc binding sites of hTERT promoter are required for Aurora-A-induced hTERT promoter activity. Ectopic expression of Aurora-A up-regulates c-Myc. Knockdown of c-Myc by RNA interference attenuates Aurora-A-stimulated hTERT expression and telomerase activity. To our knowledge, these findings demonstrate, for the first time, that Aurora-A induces telomerase activity and hTERT by up-regulation of c-Myc and provides an additional mechanism for the role of Aurora-A in malignant transformation in addition to its cell cycle control.

In situ hybridization for gastrin-releasing peptide receptor (GRP receptor) expression in prostatic carcinoma

Bombesin‐like peptides (BLPs), which have been implicated in the regulation of growth of prostatic carcinoma cells, are a product of neuroendocrine cells frequently found in prostate tissue and are postulated to play a role in the initiation or progression of prostatic carcinoma. In this report, we examined the expression, in human prostate tissue, of mRNA encoding the 3 known receptors that respond to BLPs in humans, i.e., gastrin‐releasing peptide (GRP) receptor, neuromedin B (NMB) receptor and bombesin receptor subtype 3 (BRS‐3). Competitive rt‐PCR experiments demonstrated the widespread but variable expression of GRP receptor mRNA in fresh‐frozen specimens of prostatic carcinoma (12 cases) and benign prostatic hypertrophy (6 cases). NMB receptor mRNA expression was also widespread, but its level was less variable than GRP receptor message. In contrast, we could not detect BRS‐3 mRNA in most tissue samples by rt‐PCR. To address which cells in the prostate express the GRP receptor, we used in situ hybridization methods to stain selectively GRP receptor mRNA. GRP receptor mRNA was expressed predominantly in the luminal and basal epithelial cells in both histologically normal and cancerous glands within sections of normal (3 cases) and diseased (37 cases) tissue. GRP receptor mRNA staining in cancerous tissue ranged widely from very intense to not detectable (about 30% of the cases), while normal tissue consistently displayed a low level of message staining. Taken together, our results demonstrate expression of the GRP receptor in a high percentage of basal and/or luminal epithelial cells of normal and diseased prostate tissues. Int. J. Cancer (Pred. Oncol.) 79:82–90, 1998.

Positive Feedback Regulation between Akt2 and MyoD during Muscle Differentiation CLONING OF Akt2 PROMOTER

Akt2 is a member of the Akt/PKB family, which is involved in a variety of cellular events including cell survival, proliferation, and differentiation. During skeletal muscle differentiation, the Akt2 but not Akt1 expression was significantly increased. Microinjection of anti-Akt2 but not anti-Akt1 antibody efficiently abrogated myogenesis, indicating that Akt2 plays a specific role in muscle differentiation. It has been well documented that ectopic expression of MyoD is sufficient to induce muscle differentiation in myoblasts. However, the mechanism of induction of Akt2 during muscle differentiation and the significance of Akt2 protein in MyoD-induced myogenesis are largely unknown. In this study, we provide direct evidence that Akt2 is transcriptionally regulated by MyoD and activates MyoD-myocyte enhancer binding factor-2 (MEF2) transactivation activity. The Akt2 promoter was isolated and found to contain nine putative E-boxes (CANNTG), which are putative MyoD binding sites. Electrophoretic mobility shift analyses revealed that MyoD bound to eight of the sites. The expression of MyoD significantly enhanced Akt2 promoter activity and up-regulated Akt2 mRNA and protein levels. Moreover, Akt2 but not Akt1 was activated during differentiation. The expression of Akt2 activated MyoD-MEF2 transcriptional activity and induced myogenin expression. These data indicate that there is a positive feedback regulation loop between Akt2 and MyoD-MEF2 during muscle differentiation, which is essential for MyoD-induced myogenesis.