Shaokun Shu

Advances of AKT pathway in human oncogenesis and as a target for anti-cancer drug discovery.

AKT (also known as PKB) plays a central role in a variety of cellular processes including cell growth, motility and survival in both normal and tumor cells. The AKT pathway is also instrumental in epithelial mesenchymal transitions (EMT) and angiogenesis during tumorigenesis. AKT functions as a cardinal nodal point for transducing extracellular (growth factors including insulin, IGF-1 and EGF ) and intracellular (such as mutated/activated receptor tyrosine kinases, PTEN, Ras and Src) signals. It is positively regulated by phosphatidylinositol 3-kinase and inhibited by phosphatase PTEN. Deregulation of the PI3K/PTEN/AKT pathway is one of the most common altered pathways in human malignancy. In the past few years, significant advances have been made in the understanding of AKT signaling in human oncogenesis and the development of small molecule inhibitor of AKT pathway. Here, we will discuss the regulation and function of AKT as well as targeting AKT for anti-cancer drug discovery.

Phosphoinositide 3-Kinase/Akt Inhibits MST1-Mediated Pro-apoptotic Signaling through Phosphorylation of Threonine 120

The protein kinase mammalian sterile 20-like kinase 1 (MST1) is a mammalian homologue of the Drosophila hippo and plays a critical role in regulation of programmed cell death. MST1 exerts pro-apoptotic function through cleavage, autophosphorylation-Thr(183) and subsequent translocation to the nucleus where it phosphorylates a number of molecules, including LATS1/2, FOXO, JNK, and histone H2B. Here, we show that the cleavage of MST1 is inhibited by the phosphatidylinositol 3-kinase/Akt pathway. Akt interacts with MST1 and phosphorylates a highly conserved residue threonine 120 of MST1, which leads to inhibition of its kinase activity and nuclear translocation as well as the autophosphorylation of Thr(183). Phospho-MST1-Thr(120) failed to activate downstream targets FOXO3a and JNK. Further, inverse correlation between pMST1-Thr(120) and pMST1-Thr(183) was observed in human ovarian tumors. These findings indicate that the phosphorylation of MST1-Thr(120) by Akt could be a major mechanism of regulation of the Hippo/MST1 pathway by cell survival signaling.The protein kinase mammalian sterile 20-like kinase 1 (MST1) is a mammalian homologue of the Drosophila hippo and plays a critical role in regulation of programmed cell death. MST1 exerts pro-apoptotic function through cleavage, autophosphorylation-Thr183 and subsequent translocation to the nucleus where it phosphorylates a number of molecules, including LATS1/2, FOXO, JNK, and histone H2B. Here, we show that the cleavage of MST1 is inhibited by the phosphatidylinositol 3-kinase/Akt pathway. Akt interacts with MST1 and phosphorylates a highly conserved residue threonine 120 of MST1, which leads to inhibition of its kinase activity and nuclear translocation as well as the autophosphorylation of Thr183. Phospho-MST1-Thr120 failed to activate downstream targets FOXO3a and JNK. Further, inverse correlation between pMST1-Thr120 and pMST1-Thr183 was observed in human ovarian tumors. These findings indicate that the phosphorylation of MST1-Thr120 by Akt could be a major mechanism of regulation of the Hippo/MST1 pathway by cell survival signaling.

Deregulation of IKBKE Is Associated with Tumor Progression, Poor Prognosis, and Cisplatin Resistance in Ovarian Cancer

I-kappa-B kinase e (IKBKE; IKKepsilon) has been recently identified as a breast cancer oncogene, and its alteration appears to be an early event in breast cancer development. In this study, we demonstrated that IKKepsilon is frequently overexpressed and activated in human ovarian cancer cell lines and primary tumors. Of 96 ovarian cancer specimens examined, 63 exhibited elevated levels of IKKepsilon. Furthermore, alterations of IKKepsilon were associated with late-stage and high-grade tumors, suggesting a role of IKKepsilon in ovarian tumor progression rather than in tumor initiation. Overall survival in patients with elevated levels of IKKepsilon was significantly lower than patients whose tumors expressed normal levels of IKKepsilon. Moreover, both early and late-stage tumors that overexpressed IKKepsilon conferred a poor prognosis, as compared with those that did not possess elevated IKKepsilon levels. Notably, overexpression of IKKepsilon rendered cells resistant to cisplatin, whereas knockdown of IKKepsilon overcame cisplatin resistance in both A2780CP and C13 cells, which express high levels of endogenous IKKepsilon. Therefore, these data demonstrate for the first time that deregulation of IKKepsilon is a highly recurrent event in human ovarian cancer and could play a pivotal role in tumor progression and cisplatin resistance. IKKepsilon could also serve as a prognostic marker and potential therapeutic target for this malignancy.

IKKepsilon phosphorylation of estrogen receptor alpha Ser-167 and contribution to tamoxifen resistance in breast cancer.

IKKϵ has recently been identified as a breast cancer oncogene. Elevated levels of IKKϵ are associated with cell survival and growth. Here, we show that IKKϵ interacts with and phosphorylates estrogen receptor α (ERα) on serine 167 in vitro and in vivo. As a result, IKKϵ induces ERα transactivation activity and enhances ERα binding to DNA. Cyclin D1, a major target of ERα, is transcriptionally up-regulated by IKKϵ in a phospho-ERα-Ser-167-dependent manner. Further, overexpression of IKKϵ induces tamoxifen resistance, whereas knockdown of IKKϵ sensitizes cells to tamoxifen-induced cell death. These data suggest that ERα is a bona fide substrate of IKKϵ and IKKϵ plays an important role in tamoxifen resistance. Thus, IKKϵ represents a critical therapeutic target in breast cancer.

Regulation of Proapoptotic Mammalian ste20–Like Kinase MST2 by the IGF1-Akt Pathway

Background Hippo, a Drosophila serine/threonine kinase, promotes apoptosis and restricts cell growth and proliferation. Its mammalian homolog MST2 has been shown to play similar role and be regulated by Raf-1 via a kinase-independent mechanism and by RASSF family proteins through forming complex with MST2. However, regulation of MST2 by cell survival signal remains largely unknown. Methodology/Principal Findings Using immunoblotting, in vitro kinase and in vivo labeling assays, we show that IGF1 inhibits MST2 cleavage and activation induced by DNA damage through the phosphatidylinosotol 3-kinase (PI3K)/Akt pathway. Akt phosphorylates a highly conserved threonine-117 residue of MST2 in vitro and in vivo, which leads to inhibition of MST2 cleavage, nuclear translocation, autophosphorylation-Thr180 and kinase activity. As a result, MST2 proapoptotic and growth arrest function was significantly reduced. Further, inverse correlation between pMST2-T117/pAkt and pMST2-T180 was observed in human breast tumors. Conclusions/Significance Our findings demonstrate for the first time that extracellular cell survival signal IGF1 regulates MST2 and that Akt is a key upstream regulator of MST2.

IKKepsilon phosphorylation of ERalpha-Ser167 and contribution to tamoxifen resistance in breast cancer

IKKϵ has recently been identified as a breast cancer oncogene. Elevated levels of IKKϵ are associated with cell survival and growth. Here, we show that IKKϵ interacts with and phosphorylates estrogen receptor α (ERα) on serine 167 in vitro and in vivo. As a result, IKKϵ induces ERα transactivation activity and enhances ERα binding to DNA. Cyclin D1, a major target of ERα, is transcriptionally up-regulated by IKKϵ in a phospho-ERα-Ser-167-dependent manner. Further, overexpression of IKKϵ induces tamoxifen resistance, whereas knockdown of IKKϵ sensitizes cells to tamoxifen-induced cell death. These data suggest that ERα is a bona fide substrate of IKKϵ and IKKϵ plays an important role in tamoxifen resistance. Thus, IKKϵ represents a critical therapeutic target in breast cancer.

Phosphorylation and Activation of Androgen Receptor by Aurora-A

Aurora-A kinase is frequently overexpressed/activated in various types of human malignancy, including prostate cancer. In this study, we demonstrate elevated levels of Aurora-A in androgen-refractory LNCaP-RF but not androgen-sensitive LNCaP cells, which prompted us to examine whether Aurora-A regulates the androgen receptor (AR) and whether elevated Aurora-A is involved in androgen-independent cell growth. We show that ectopic expression of Aurora-A induces AR transactivation activity in the presence and absence of androgen. Aurora-A interacts with AR and phosphorylates AR at Thr282 and Ser293 in vitro and in vivo. Aurora-A induces AR transactivation activity in a phosphorylation-dependent manner. Ectopic expression of Aurora-A in LNCaP cells induces prostate-specific antigen expression and cell survival, whereas knockdown of Aurora-A sensitizes LNCaP-RF cells to apoptosis and cell growth arrest. These data indicate that AR is a substrate of Aurora-A and that elevated Aurora-A could contribute to androgen-independent cell growth by phosphorylation and activation of AR.

IKBKE Phosphorylation and Inhibition of FOXO3a: A Mechanism of IKBKE Oncogenic Function

Forkhead box O (FOXO) transcription factors are emerging as key regulators of cell survival and growth. The transcriptional activity and subcellular localization of FOXO are tightly regulated by post-translational modifications. Here we report that IKBKE regulates FOXO3a through phosphorylation of FOXO3a-Ser644. The phosphorylation of FOXO3a resulted in its degradation and nuclear-cytoplasmic translocation. Previous studies have shown that IKBKE directly activates Akt and that Akt inhibits FOXO3a by phosphorylation of Ser32, Ser253 and Ser315. However, the activity of Akt-nonphosphorytable FOXO3a-A3 (i.e., converting 3 serine residues to alanine) was inhibited by IKBKE. Furthermore, overexpression of IKBKE correlates with elevated levels of pFOXO3a-S644 in primary lung and breast tumors. IKBKE inhibits cellular function of FOXO3a and FOXO3a-A3 but, to a much less extent, of FOXO3a-S644A. These findings suggest that IKBKE regulates FOXO3a primarily through phosphorylation of SerS644 and that IKBKE exerts its cellular function, at least to some extent, through regulation of FOXO3a.

Abstract 1428: Upregulation of IKBKE promotes glioma cell migration and invasion by phosphorylation of Snail

IKBKE is a member of IκB kinase (IKK) family. Previous studies have shown that IKBKE is an oncogene in breast and ovarian cancer. In this study, we demonstrate that IKBKE is frequently upregulated in human glioma and regulates cell invasion, a process that causes incurable surgery for glioma. Increase expression of IKBKE was detected in 7 of 11 glioma cell lines and 41 of 57 primary tumors examined, which closely correlates with high grade tumors and patient poor overall survival. Further, ectopic expression of IKBKE induces and knockdown of IKBKE decreases cell migration and invasion. Notably, E-cadherin protein and mRNA levels were inversely regulated by IKBKE. Moreover, we demonstrated that IKBKE directly binds and phosphorylates Snail at Ser-165. The phosphorylation of Snail induces its stability and nuclear translocation and represses E-cadherin expression. Taken together, these findings indicate that IKBKE could play a pivotal role in gliomagenesis and invasion. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1428. doi:10.1158/1538-7445.AM2011-1428

Abstract 3836: Regulation of class III PI3K by class I PI3K/Akt inhibits autophagy through phosphorylation of UVRAG

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC It has been well documented that the class I PI3K (PI3KC1)/Akt pathway inhibits whereas class III PI3K (PI3KC3) induces autophagy. UVRAG forms a complex with Beclin1 and PI3KC3 leading to activation of autophagy process. In this study, we demonstrate that PI3KC1/Akt inhibits PI3KC3 kinase activity and autophagy induced by different stimuli, including mTOR inhibitor rapamycin, suggesting PI3KC1/Akt regulation of autophagy independent of mTOR. Further, Akt directly phosphorylates UVRAG on serine-509, which results in disruption of UVRAG-Beclin1-PI3KC3 complex, and inhibition of PI3KC3 and autophagy. Inhibition of PI3KC1 or depletion of Akt increases the ability of UVRAG to bind to Beclin 1. UVRAG-S509A, which is not phosphorylated by Akt, enhances whereas UVRAG-S509D, which mimics the phosphorylation by Akt, reduces the interaction between UVRAG and Beclin1. Notably, UVRAG-S509A abrogates the effect of Akt on PI3KC3 and autophagy. These findings establish the cross-talk between class I and class III PI3K and indicate that PI3KC1/Akt regulates autophagy through inhibition of PI3KC3 by phosphorylation of UVRAG. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3836.