Background
API-1 (4-amino-5,8-dihydro-5-oxo-8-β-D-ribofuranosyl-pyrido[2,3-
d]pyrimidine-6-carboxamide) is a recently identified novel Akt inhibitor. It inhibits Akt activity through binding to the pleckstrin homology domain of Akt and blocking its membrane translocation [
1]. API-1 possesses promising anticancer activity, evidenced by its ability to suppress cell growth, induce apoptosis and inhibit the growth of cancer xenografts, particularly those with activated Akt, in nude mice [
1]. We have recently shown that API-1 facilitates c-FLIP degradation, induces apoptosis and enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human non-small cell lung cancer (NSCLC) cells [
2]. c-FLIP degradation clearly contributes to the enhancement of TRAIL-induced apoptosis by API-1 [
2]. However, the mechanisms by which API-1 induces apoptosis in cancer cells and the additional mechanisms accounting for API-1-mediated augmentation of TRAIL-induced apoptosis are largely unknown.
In addition to the extrinsic death receptor-mediated apoptotic pathway, which is characterized by the oligomerization of cell surface death receptors and activation of caspase-8, the intrinsic apoptotic pathway that involves the disruption of mitochondrial membranes, release of cytochrome c and activation of caspase-9 is another critical apoptotic mechanism [
3]. It is known that the intrinsic apoptotic pathway is negatively regulated by anti-apoptotic Bcl-2 family members (e.g., Mcl-1, Bcl-2 and Bcl-X
L) and inhibitor of apoptosis proteins (IAPs; e.g., survivin). In general, downregulation of these anti-apoptotic proteins can trigger apoptosis or augment TRAIL-induced apoptosis [
4‐
6].
Among the anti-apoptotic Bcl-2 family members, Mcl-1 is known to be a short-lived protein that undergoes ubiquitination/proteasome-mediated degradation [
7]. One degradation mechanism involves glycogen synthase kinase 3 (GSK3), which phosphorylates Mcl-1 at S159, triggering Mcl-1 degradation [
8,
9]. It has been suggested that Mcl-1 phosphorylation at S159 facilitates the association of Mcl-1 with the E3 ligase β-transducin repeats-containing protein (β-TrCP), resulting in β-TrCP-mediated ubiquitination and degradation of Mcl-1. Recently two studies have suggested that phosphorylation at S159 enhances the association of Mcl-1 with the E3 ligase F-box/WD repeat-containing protein 7 (FBXW7), resulting in FBXW7-mediated ubiquitination and degradation of Mcl-1 [
10,
11].
In this study, we focused on revealing mechanisms by which API-1 induces apoptosis of cancer cells and uncovered GSK3-dependent Mcl-1 degradation as a critical mechanism accounting for induction of apoptosis by API-1. This mechanism also contributes to augmentation of TRAIL-induced apoptosis by API-1.
Methods
Reagents
API-1 (NSC177233) was obtained from the National Cancer Institute (Bethesda, MD). MK2206 was purchased from Active Biochem (Maplewood, NJ). They were dissolved in DMSO and stored at -80°C. Soluble recombinant human TRAIL was purchased from PeproTech, Inc. (Rocky Hill, NJ). The proteasome inhibitor MG132, the protein synthesis inhibitor cycloheximide (CHX) and the GSK3 inhibitor SB216763 were purchased from Sigma Chemical Co. (St. Louis, MO). The neddylation inhibitor MLN4924 was provided by Millennium Pharmaceuticals, Inc (Cambridge, MA). Expression plasmids in pCI vector carrying wild-type and mutant (S159A) human Mcl-1 were provided by Dr. X. Deng (Emory University, Atlanta, GA). Mouse monoclonal survivin and caspase-8 antibodies and rabbit polyclonal Bim, caspase-9 and PARP antibodies were purchased from Cell Signaling Technology (Danvers, MA). Mouse monoclonal caspase-3 antibody was purchased from Imgenex (San Diego, CA). Rabbit polyclonal Mcl-1, Bad, Bcl-XL and SKP1 and mouse monoclonal Bcl-2, Cul-1 and α-tubulin antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). GSK3α/β antibody was purchased from Upstate/Millipore (Billerica, MA). Mouse monoclonal Bax and rabbit polyclonal glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were purchased from Trevigen Inc. (Gaithersburg, MD). Both polyclonal and monoclonal actin antibodies were purchased from Sigma Chemical Co.
Cell lines and cell culture
The human NSCLC cell lines used in this study including those stably expressing ectopic Mcl-1 or survivin were described previously [
12‐
14]. A549 cells were recently authenticated by Genetica DNA Laboratories, Inc. (Cincinnati, OH) through analyzing short tandem repeat DNA profile; other cell lines have not been authenticated. HCT116/wild type (WT) and HCT116/FBXW7-KO cell lines were kindly provided by Dr. B. Vogelstein (Johns Hopkins University School of Medicine, Baltimore, MA). These cell lines were grown in monolayer culture in RPMI 1640 medium or McCoy’s medium supplemented with glutamine and 5% fetal bovine serum at 37°C in a humidified atmosphere consisting of 5% CO
2 and 95% air.
Cell survival and apoptotic assays
Cells were seeded in 96-well cell culture plates and treated the next day with the given agents. Viable cell numbers were determined using sulforhodamine B (SRB) assay as described previously [
15]. Combination index (CI) for drug interaction (e.g., synergy) was calculated using the CompuSyn software (ComboSyn, Inc.; Paramus, NJ). Apoptosis was evaluated with an annexin V-PE apoptosis detection kit (BD Biosciences; San Jose, CA) according to the manufacturer’s instructions. We also detected caspases and PARP cleavage by Western blot analysis as described below as additional indicators of apoptosis.
Western blot analysis
Preparation of whole-cell protein lysates and Western blot analysis were described previously [
16,
17].
Small interfering RNA (siRNA) and transfection
GSK3α/β siRNA (#6301) was purchased from Cell Signaling Technology. FBXW7 siRNA that targets the sequence of 5′-AACACAAAGCTGGTGTGTGCA-3′ [
18] was synthesized from Qiagen (Valencia, CA) and used in our previous study [
13]. Cullin 1 (Cul1; sc-35126), SKP1 (sc-29482) and β-TrCP (sc-37178) siRNAs were purchased from Santa Cruz Biotechnology, Inc. siRNA transfection was performed with HiPerFect transfection reagent (Qiagen) or Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions.
Reverse transcription-PCR (RT-PCR)
To confirm knockdown efficiencies of β-TrCP and FBXW7 siRNA, Control, β-TrCP, FBXW7 and β-TrCP plus FBXW7 siRNAs were transfected into H1299 cells with Lipofectamine 2000. After 48 h, total RNA was then prepared from the cells by Trizol (Sigma). Reverse transcription was performed with iScript select cDNA synthesis kit (Bio-Rad; Hercules, CA), followed with PCR using primers as follows: β-TrCP, 5′-CCCCAACTGACATTACCC-3′ (forward) and 5′-TCGAATACAACGCACCAA-3′ (reverse) [
19]; FBXW7, 5′-AAAGAGTTGTTAGCGGTTCTCG-3′ (forward) and 5′-CCACATGGATACCATC AAACTG-3′ (reverse) [
20]; GAPDH, 5′-TGATGACATCAAGAAGGTGGTGAAG-3′ (forward) and 5′-TCCTTGGAGGCCATGTGGGCCAT-3′ (reverse) [
21]. Using the same assay, Mcl-l mRNA expression in cells exposed to API-1 was detected with the following primers: 5′-TAAGGACAAAACGGGACTGG-3′ (forward) and 5′-ACCAGCTCCTACTCCAGCAA-3′ (reverse) [
22].
Discussion
Mcl-1 is a well-known Bcl-2 family protein that negatively regulates apoptosis by binding and sequestering the pro-apoptotic proteins such as Bax, Bak, Noxa, and Bim [
7]. In this study, we found that API-1 rapidly and potently decreased Mcl-1 levels in NSCLC cell lines sensitive to API-1, but only minimally in API-1-insenstive cell lines. Moreover, enforced expression of ectopic Mcl-1 substantially protected NSCLC cells from undergoing apoptosis induced by API-1. These results clearly demonstrated that Mcl-1 reduction is an essential event for API-1 to induce apoptosis. In this study, API-1 also reduced the levels of survivin, another important anti-apoptotic protein that acts downstream of Mcl-1 as an endogenous inhibitor of caspases (e.g., caspase-9) [
26]. However, we failed to demonstrate its essential role in mediating induction of apoptosis by API-1 because API-1 decreased survivin levels in NSCLC cells regardless of their sensitivities to API-1 and was equally effective in killing both NSCLC cells carrying a vector control and those expressing ectopic survivin. Hence the finding of Mc1 reduction as a critical mechanism accounting for API-1-induced apoptosis is novel. The current study focuses on demonstrating the role of Mcl-1 suppression in API-1-induced apoptosis. However, this does not exclude other targets or mechanisms such as Bcl-1 reduction described in this study (Figure
1) and c-FLIP degradation reported previously [
2] that may account for API-1-induced apoptosis, particularly in a given cancer cell line. It is more likely that the effect of API-1 on induction of apoptosis is an outcome of the combination of multiple mechanisms including Mcl-1 reduction.
We noted that enforced expression of ectopic Mcl-1 blocked cleavage of not only caspase-9, but also caspase-8. It is very likely that caspase-8 activation caused by API-1 is secondary to activation of the intrinsic apoptotic pathway because caspase-8 can be activated by caspase-9 or caspase-3 [
27,
28]. However, we currently cannot rule out the possibility that caspase-8 activation is caused by Mcl-1 suppression if Mcl-1 has an uncovered role in direct suppression of the extrinsic apoptotic pathway.
We previously reported that API-1 effectively enhances TRAIL-induced apoptosis in human NSCLC cells involving induction of c-FLIP degradation [
2]. In this study, we found that enforced expression of ectopic Mcl-1 protected NSCLC cells from induction of apoptosis by the combination of API-1 and TRAIL. Hence, it is apparent that Mcl-1 reduction is also an important mechanism by which API-1 augments TRAIL-induced apoptosis.
Mcl-1 is known to undergo GSK3-dependent proteasomal degradation [
7,
23]. As an Akt inhibitor, it was plausible to speculate that API-1 reduces Mcl-1 levels through activating GSK3 followed by enhancing GSK3-dependent Mcl-1 degradation. In this study, API-1 did not alter Mcl-1 mRNA levels, suggesting that API-1-induced Mcl-1 reduction is likely to be a posttranscriptional event. Indeed, inhibition of proteasome with MG132 rescued Mcl-1 reduction induced by API-1. Moreover, API-1 decreased Mcl-1 stability. Hence, it is clear that API-1 induces proteasomal degradation of Mcl-1, leading to Mcl-1 reduction. We observed that decreased Mcl-1 levels were accompanied with an early and robust increase in Mcl-1 phosphorylation at S159/T163, primarily in in those NSCLC cell lines sensitive to API-1. Importantly, suppression of GSK3 inhibited Mcl-1 phosphorylation and reduction or degradation induced by API-1. In agreement, API-1 lost its activity on inducing degradation of mutant Mcl-1 (S159A), in which S159 could not be phosphorylated. Collectively, we conclude that API-1 decreases Mcl-1 levels through facilitating GSK3-dependent proteasomal degradation of Mcl-1.
In our study, we found that inhibition of GSK3 (e.g., with SB216763) antagonized the effects of API-1 on decreasing the survival of NSCLC cells and on inducing cleavage of caspases and PARP. Thus, it is clear that API-1 induces GSK3-dependent apoptosis as well. This reinforces the critical role of GSK3-dependent Mcl-1 reduction in mediating apoptosis induced by API-1. This finding also implies that, to prevent potential antagonism, API-1 should not be used in combination with any agents that may inhibit GSK3 activity in the treatment of cancer.
Although FBXW7 has been recently suggested to be a key E3 ligase that mediates GSK3-dependent Mcl-1 degradation [
10,
11], we found that knockdown or knockout of FBXW7 provided only limited protective effect against Mcl-1 reduction induced by API-1. However, this process does require activation of SCF E3 ligases since the inhibition of SCF complex formation by knockdown of Cul1, SKP1 or both drastically impaired the ability of API-1 to decrease Mcl-1 levels. Hence, FBXW7 may not be the major E3 ligase responsible for API-1-induced GSK3-dependent proteasomal degradation of Mcl-1, and additional SCF E3 ligase(s) may be involved in mediating GSK3-dependent degradation of Mcl-1 induced by API-1. Indeed, our further studies have shown that β-TrCP, another SCF E3 ligase involved in mediating GSK3-dependent degradation of Mcl-1 as suggested previously [
8], also contributes to API-1-induced Mcl-1 degradation since knockdown of β-TrCP provided a more drastic effect than FBXW7 knockdown on rescuing Mcl-1 reduction induced by API-1. Moreover, we found that co-knockdown of both β-TrCP and FBXW7 exhibited much more potent effects than knockdown of either single gene in preventing Mcl-1 reduction induced by API-1. Therefore, we believe that both β-TrCP and FBXW7 are involved in mediating GSK3-dependent Mcl-1 degradation induced by API-1. Our findings clearly suggest that two E3 ubiquitin ligases can cooperate to regulate the degradation of one protein.
Acknowledgements
We thank Dr. A. Hammond in our department for editing the manuscript and Dr. B. Vogelstein for providing FBXW7-deficient cells. We are also grateful to the high school students, Kevin Sun and Jerry Yue, from Parkview High School (Lilburn, GA) for assisting performance of some experiments. This study was supported by NIH R01 CA118450 (S-Y Sun) and R01 CA160522 (S-Y Sun). FR Khuri and S-Y Sun are Georgia Cancer Coalition Distinguished Cancer Scholars.
Competing interests
All authors declare that they have no competing interests.
Authors’ contributions
HR, JK, BG and PY designed and conducted experiments and data analysis. XD, MC and FRK participated in discussion of the data. XD provided some critical reagents. MC and SYS participated in experimental design, coordination, data analysis and draft of the manuscript. All authors read and approved the final manuscript.