Shi-Yong Sun

Activation of Akt and eIF4E Survival Pathways by Rapamycin-Mediated Mammalian Target of Rapamycin Inhibition

The mammalian target of rapamycin (mTOR) has emerged as an important cancer therapeutic target. Rapamycin and its derivatives that specifically inhibit mTOR are now being actively evaluated in clinical trials. Recently, the inhibition of mTOR has been shown to reverse Akt-dependent prostate intraepithelial neoplasia. However, many cancer cells are resistant to rapamycin and its derivatives. The mechanism of this resistance remains a subject of major therapeutic significance. Here we report that the inhibition of mTOR by rapamycin triggers the activation of two survival signaling pathways that may contribute to drug resistance. Treatment of human lung cancer cells with rapamycin suppressed the phosphorylation of p70S6 kinase and 4E-BP1, indicating an inhibition of mTOR signaling. Paradoxically, rapamycin also concurrently increased the phosphorylation of both Akt and eIF4E. The rapamycin-induced phosphorylation of Akt and eIF4E was suppressed by the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002, suggesting the requirement of PI3K in this process. The activated Akt and eIF4E seem to attenuate rapamycins growth-inhibitory effects, serving as a negative feedback mechanism. In support of this model, rapamycin combined with LY294002 exhibited enhanced inhibitory effects on the growth and colony formation of cancer cells. Thus, our study provides a mechanistic basis for enhancing mTOR-targeted cancer therapy by combining an mTOR inhibitor with a PI3K or Akt inhibitor.

Retinoids and their receptors in cancer development and chemoprevention

Retinoids play an important role in regulating the growth and differentiation of normal, premalignant and malignant cell types, especially epithelial cells, mainly through interaction with two types of nuclear receptors: retinoic acid receptors (RARalpha, beta and gamma) and retinoid X receptors (RXRalpha, beta and gamma). Vitamin A deficiency in experimental animals has been associated with a higher incidence of cancer and with increased susceptibility to chemical carcinogens. This is in agreement with the epidemiological studies indicating that individuals with a lower dietary vitamin A intake are at a higher risk to develop cancer. At the molecular level, aberrant expression and function of nuclear retinoid receptors have been found in various types of cancer including premalignant lesions. Thus, aberrations in retinoid signaling are early events in carcinogenesis. Retinoids at pharmacological doses exhibit a variety of effects associated with cancer prevention. They suppress transformation of cells in vitro, inhibit carcinogenesis in various organs in animal models, reduce premalignant human epithelial lesions and prevent second primary tumors following curative therapy for epithelial malignancies such as head and neck, lung, liver, and breast cancer.

c-Jun NH2-Terminal Kinase-Mediated Up-regulation of Death Receptor 5 Contributes to Induction of Apoptosis by the Novel Synthetic Triterpenoid Methyl-2-Cyano-3,12-Dioxooleana-1, 9-Dien-28-Oate in Human Lung Cancer Cells

Death receptor (DR) 4 or 5, on binding to its ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), triggers apoptosis via activating the caspase-8–mediated caspase cascade. Certain anticancer drugs up-regulate the expression of these receptors and thereby induce apoptosis or enhance TRAIL-induced apoptosis. In this study, we explored the ability of methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oate (CDDO-Me) to activate the extrinsic DR-mediated apoptotic pathway in human lung cancer cells. We found that CDDO-Me not only activated caspase-8 but also induced expression of DRs, particularly DR5, in a p53-independent mechanism. Correspondingly, CDDO-Me augmented TRAIL-induced apoptosis in these cells regardless of p53 status as evidenced by enhanced DNA fragmentation and activation of caspase cascades, suggesting that CDDO-Me–induced DRs are functionally active. Moreover, silencing of DR5 expression using small interfering RNA suppressed apoptosis induced by CDDO-Me alone or by combination of CDDO-Me and TRAIL, indicating that DR5 up-regulation is required for induction of apoptosis by CDDO-Me and for enhancement of TRAIL-induced apoptosis by CDDO-Me. CDDO-Me rapidly activated c-Jun NH2-terminal kinase (JNK) before DR up-regulation and caspase-8 activation. Moreover, application of the JNK-specific inhibitor SP600125 blocked CDDO-Me–induced increases in JNK activation, DR up-regulation, caspase-8 activation, and DNA fragmentation. These results show that activation of JNK pathway results in CDDO-Me–induced DR up-regulation, caspase-8 activation, and apoptosis. Collectively, we conclude that CDDO-Me induces apoptosis via the JNK-mediated DR up-regulation in human lung cancer cells.

Evidence that the death receptor DR4 is a DNA damage-inducible, p53-regulated gene

DR4 (TRAIL‐R1), a member of the tumor necrosis factor receptor superfamily, is a cell surface receptor that triggers the apoptotic machinery upon binding to its ligand tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL). Although three other TRAIL receptors DR5, DcR1, and DcR2 are induced by DNA damage and are regulated by the wild‐type p53 tumor suppressor, it was not known whether these factors also affect DR4 expression. In this study, we found that DR4 expression is also enhanced by DNA damage whether induced by ionizing radiation or by chemotherapeutic agents. The induction was observed predominantly in cells containing wild‐type p53 and was similar to the regulation patterns of DR5 and Fas, two other members of the family which are known to be regulated by p53. Transfection of HPV 16 E6 gene into cells with wild‐type p53, which decreased the level of p53 protein, resulted in suppression of DR4 induction by DNA‐damaging agents. Conversely, introduction of exogenous wild‐type p53 through adenovirus infection has led to upregulation of endogenous DR4 in cells with mutant p53. Moreover, the transcription inhibitor actinomycin D abolished DNA‐damaging agent‐induced DR4 expression. Thus, DR4 appears to be a DNA damage‐inducible, p53‐regulated gene.

Perifosine inhibits mammalian target of rapamycin signaling through facilitating degradation of major components in the mTOR axis and induces autophagy.

Perifosine is an alkylphospholipid exhibiting antitumor activity as shown in both preclinical studies and clinical trials. This activity is partly associated with its ability to inhibit Akt activity. It has been shown that the mammalian target of rapamycin (mTOR) axis plays a critical role in regulation of cell proliferation and survival primarily through functioning both downstream and upstream of Akt. The current study reveals a novel mechanism by which perifosine inhibits Akt and the mTOR axis. In addition to inhibition of Akt, perifosine inhibited the assembly of both mTOR/raptor and mTOR/rictor complexes. Strikingly, perifosine reduced the levels of Akt and other major components including mTOR, raptor, rictor, 70-kDa ribosomal S6 kinase, and 4E-binding protein 1 in the mTOR axis by promoting their degradation through a GSK3/FBW7-dependent mechanism. These results thus suggest that perifosine inhibits the mTOR axis through a different mechanism from inhibition of mTOR signaling by classic mTOR inhibitors such as rapamycin. Moreover, perifosine substantially increased the levels of type II light chain 3, a hallmark of autophagy, in addition to increasing poly(ADP-ribose) polymerase cleavage, suggesting that perifosine induces both apoptosis and autophagy. The combination of perifosine with a lysosomal inhibitor enhanced apoptosis and inhibited the growth of xenografts in nude mice, suggesting that perifosine-induced autophagy protects cells from undergoing apoptosis. Collectively, we conclude that perifosine inhibits mTOR signaling and induces autophagy, highlighting a novel mechanism accounting for the anticancer activity of perifosine and a potential strategy to enhance the anticancer efficacy of perifosine by preventing autophagy.

Tumor Growth Inhibition by Simultaneously Blocking Epidermal Growth Factor Receptor and Cyclooxygenase-2 in a Xenograft Model

Purpose: Our previous study revealed that simultaneously targeting epidermal growth factor receptor (EGFR) tyrosine kinase and cyclooxygenase-2 (COX-2) additively or synergistically inhibited growth of squamous cell carcinoma of the head and neck (SCCHN) in vitro. However, an in vivo efficacy of this combined treatment in SCCHN has not been studied. Experimental Design: Nude mice were pretreated with control (1% Tween 80), ZD1839 (50 mg/kg) alone, celecoxib (50 mg/kg) alone, or a combination of ZD1839 and celecoxib at the same dosages for 7 days before injection of a human SCCHN cell line Tu212. The animals were continuously treated with the agents 5 days a week for about 11 weeks. Results: Tumor growth in the combined treatment was significantly inhibited compared with the control (P < 0.001), ZD1839 (P = 0.005), or celecoxib (P < 0.001). At the same time, a dramatic delay of tumor progression was observed in the combined treatment compared with all other three groups. Molecular analysis showed that the combined treatment significantly decreased prostaglandin E metabolite production. The cooperative effect of these two agents in combination was also associated with down-regulation of phosphorylated EGFR, phosphorylated extracellular signal-regulated kinase, and phosphorylated signal transducers and activators of transcription 3 levels and reduction of vascular endothelial growth factor and Ki-67 expression. Specifically, gene silencing of both EGFR and COX-2 by small interfering RNA further confirmed the cooperative antitumor effect. Conclusion: The current results strongly suggest that a cooperative effect of the combined treatment on tumor progression is mediated through blocking both EGFR- and COX-2-related pathways. This combination regimen may provide a promising strategy for cancer therapy and chemoprevention in SCCHN.

Inhibition of Mammalian Target of Rapamycin Induces Phosphatidylinositol 3-Kinase-Dependent and Mnk-Mediated Eukaryotic Translation Initiation Factor 4E Phosphorylation

ABSTRACT The initiation factor eukaryotic translation initiation factor 4E (eIF4E) plays a critical role in initiating translation of mRNAs, including those encoding oncogenic proteins. Therefore, eIF4E is considered a survival protein involved in cell cycle progression, cell transformation, and apoptotic resistance. Phosphorylation of eIF4E (usually at Ser209) increases its binding affinity for the cap of mRNA and may also favor its entry into initiation complexes. Mammalian target of rapamycin (mTOR) inhibitors suppress cap-dependent translation through inhibition of the phosphorylation of eIF4E-binding protein 1. Paradoxically, we have shown that inhibition of mTOR signaling increases eIF4E phosphorylation in human cancer cells. In this study, we focused on revealing the mechanism by which mTOR inhibition increases eIF4E phosphorylation. Silencing of either mTOR or raptor could mimic mTOR inhibitors’ effects to increase eIF4E phosphorylation. Moreover, knockdown of mTOR, but not rictor or p70S6K, abrogated rapamycins ability to increase eIF4E phosphorylation. These results indicate that mTOR inhibitor-induced eIF4E phosphorylation is secondary to mTOR/raptor inhibition and independent of p70S6K. Importantly, mTOR inhibitors lost their ability to increase eIF4E phosphorylation only in cells where both Mnk1 and Mnk2 were knocked out, indicating that mTOR inhibitors increase eIF4E phosphorylation through a Mnk-dependent mechanism. Given that mTOR inhibitors failed to increase Mnk and eIF4E phosphorylation in phosphatidylinositol 3-kinase (PI3K)-deficient cells, we conclude that mTOR inhibition increases eIF4E phosphorylation through a PI3K-dependent and Mnk-mediated mechanism. In addition, we also suggest an effective therapeutic strategy for enhancing mTOR-targeted cancer therapy by cotargeting mTOR signaling and Mnk/eIF4E phosphorylation.

Mechanisms of apoptosis induced by the synthetic retinoid CD437 in human non-small cell lung carcinoma cells

The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) has been shown to induce apoptosis in various tumor cell lines including human non-small cell lung carcinoma (NSCLC) cells, which are resistant to the natural all-trans retinoic acid and to many synthetic receptor-selective retinoids. Although the mechanism of this effect was not elucidated, it was found to be independent of nuclear retinoid receptors. In the present study, we analysed the mechanisms by which CD437 induces apoptosis in two human NSCLC cell lines: H460 with wild-type p53 and H1792 with mutant p53. Both cell lines underwent apoptosis after exposure to CD437, although the cell line with wild-type p53 (H460) was more sensitive to the induction of apoptosis. CD437 increased the activity of caspase in both cell lines, however, the effect was much more pronounced in the H460 cells. The caspase inhibitors (Z-DEVD-FMK and Z-VAD-FMK) suppressed CD437-induced CPP32-like caspase activation and apoptosis in both cell lines. CD437 induced the expression of the p53 gene and its target genes, p21, Bax, and Killer/DR5, only in the H460 cells. These results suggest that CD437-induced apoptosis is more extensive in NSCLC cells that express wild-type p53, possibly due to the involvement of the p53 regulated genes Killer/DR5, and Bax although CD437 can also induce apoptosis by means of a p53-independent mechanism. Both pathways of CD437-induced apoptosis appear to involve activation of CPP32-like caspase.

Coupling of Endoplasmic Reticulum Stress to CDDO-Me–Induced Up-regulation of Death Receptor 5 via a CHOP–Dependent Mechanism Involving JNK Activation

The synthetic triterpenoid methyl-2-cyano-3,12-dioxoolean-1,9-dien-28-oate (CDDO-Me) is in phase I clinical trials as a novel cancer therapeutic agent. We previously showed that CDDO-Me induces c-Jun NH(2)-terminal kinase (JNK)-dependent death receptor 5 (DR5) expression and augments death receptor-induced apoptosis. The current study focused on addressing how CDDO-Me induces JNK-dependent DR5 expression. Analysis of DR5 promoter regions defines that the CCAAT/enhancer binding protein homologous protein (CHOP) binding site is responsible for CDDO-Me-induced transactivation of the DR5 gene. Consistently, CDDO-Me induced DR5 expression and parallel CHOP up-regulation. Blockade of CHOP up-regulation also abrogated CDDO-Me-induced DR5 expression. These results indicate that CDDO-Me induces CHOP-dependent DR5 up-regulation. Moreover, the JNK inhibitor SP600125 abrogated CHOP induction by CDDO-Me, suggesting a JNK-dependent CHOP up-regulation by CDDO-Me as well. Importantly, knockdown of CHOP attenuated CDDO-Me-induced apoptosis, showing that CHOP induction is involved in CDDO-Me-induced apoptosis. Additionally, CDDO-Me increased the levels of Bip, phosphorylated eukaryotic translation initiation factor 2alpha, inositol requiring kinase 1alpha, and activating transcription factor 4, all of which are featured changes during endoplasmic reticulum (ER) stress. Furthermore, salubrinal, an inhibitor of ER stress-induced apoptosis, inhibited JNK activation and up-regulation of CHOP and DR5 by CDDO-Me and protected cells from CDDO-Me-induced apoptosis. Thus, ER stress seems to be important for CDDO-Me-induced JNK activation, CHOP and DR5 up-regulation, and apoptosis. Collectively, we conclude that CDDO-Me triggers ER stress, leading to JNK-dependent, CHOP-mediated DR5 up-regulation and apoptosis.

Modulation of death receptors by cancer therapeutic agents.

Death receptors are important modulators of the extrinsic apoptotic pathway. Activating certain death receptors such as death receptors for tumor necrosis factor-related apoptosis-inducing factor (TRAIL) (i.e., DR4 and DR5) selectively kills cancer cells via induction of apoptosis while sparing normal cells. Thus, soluble recombinant TRAIL and agonistic antibodies to DR4 or DR5 have progressed to phase I and phase II clinical trials. Many cancer therapeutic drugs including chemotherapeutic agents have been shown to induce the expression or redistribution at the cell surface of death receptors including TRAIL death receptors. In addition, chemotherapeutic agents have also been shown to enhance induction of apoptosis by TRAIL or agonistic antibodies or overcome cell resistance to TRAIL or agonistic antibodies. Targeted induction of apoptosis by activation of the death receptor-mediated extrinsic apoptotic pathway should be an ideal therapeutic strategy to eliminate cancer cells. Therefore, death receptors, particularly TRAIL death receptors, have emerged as an important cancer therapeutic target. This article will focus on reviewing and discussing the modulation of death receptors by cancer therapeutic agents and its implications in cancer therapy.