Background
Gastric cancer (GC) is still an important public health problem, with the third leading cause of cancer-related mortality worldwide [
1]. The infection of Helicobacter pylori and Epstein-Barr (EB) virus is thought to be associated with carcinogenesis of gastric cancer [
2‐
6].
Although D2 resection is the standard treatment for gastric cancer, most of GC patients are diagnosed in a non-curable stage, especially in China. The understanding of the molecular mechanisms is helpful to develop effective treatment strategies. It has been known that several signaling pathways play critical roles in the development of gastric cancer. For example, there is an increasing interest in studying the role of the mammalian target of rapamycin (mTOR) in gastric cancer. We have also revealed that mTOR is frequently activated in GC and phosphorylated mTOR is associated with poor prognosis [
7].
mTOR can form one of two complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Activated mTORC1 phosphorylates two effector molecules, 4E-binding protein 1 (4EBP1) and S6 kinase 1 (S6K1), to stimulate protein synthesis supporting cell growth and cell metabolism and angiogenesis [
8‐
10].
As an oncoprotein, S-phase kinase associated protein 2 (Skp2) has also been reported activated in many types of cancers, included approximately 50% patients with GC [
11‐
13]. It is the key part of Skp-Cullin-F-box (SCF) complex and participates in cell proliferation, metabolism and tumorigenesis by promoting ubiquitination of p27 and p21 [
14‐
18]. Previously, we identified that macroh2A is a new Skp2 SCF substrate, whose ubiquitination by Skp2 inhibits the proliferation, colony formation and migration in breast cancer cells [
19]. However, the upstream regulation of Skp2 remains unclear [
20].
Recently, Jin et al. have found that Skp2 is a negative feedback regulator of amino-acid dependent mTORC1 signaling and activation of mTORC1 signaling can recruit Skp2 to RagA [
21]. This result indicates mTORC1 activity can influence Skp2 activity and it is possible that Skp2 phosphorylation mediated by mTORC1 [
22].
Here we evaluated the mechanism by which mTORC1 controls Skp2 stability at the phosphorylation level. Our findings may help to understand the role of mTOR-dependent networks and their relationship to compensatory pathways in GC tumorigenesis.
Methods
Cell lines, stable cell lines and plasmids
Human GC cell lines (BGC 823, MKN 45, SGC 7901 and MGC 803) and 293 T cell line were purchased from American Type Cell Culture (Manassas, VA). All cell lines were cultured in high-glucose RPIM-1640 or DMEM supplemented with 10% fetal bovine serum (Gibco) in a moist chamber with 5% CO2 at 37 °C.
Two cell lines (BGC 823 and MKN 45) were selected to generate stable cell lines in our research. The retroviral packaging system was purchased from Clontech. According to the manufacture’s introduction, the recombinant retroviruses expressing the vector pcDNA 3.1 and Skp2-wild-type (WT) or Skp2-S64A were generated. These retroviruses were infected to BGC 823 and MKN 45 and then were selected with 750ug/ml of G148 (Calbiochem). Mutation Skp2 was constructed by the Quik Change Site-Directed Mutagenesis Kit (Stratagene), and then verified by DNA sequencing. Follow the manufacture’s introduction, the plasmids containing pcDNA3.1-myc-mTOR, pcDNA3.1-FLAG-SKP2-wt and pcDNA3.1-FLAG-SKP2-S64A were constructed.
Transfections, cell synchronization and cycloheximide chase assay
Cell lines were transfected with variously plasmids with lipofectamine 2000 (Life technologies) according to the manufacture’s introductions. Generally, cells seeds at 2.0 × 105 cells per well in a 6-well culture dish or at 1.0 × 106 cells per 10-cm dish were transfected with various plasmids. For the synchronization experiment, 293 T cells were treated with 2 mM thymidine for 12 h, and washed and released into fresh medium tissue culture dishs. As indicated, transfection were performed during the last 4 h before the second thymidine treatment. After the double-thymidine block, the 293 T cells were re-plated and treated with cycloheximide (100ug/ml) for 0 h to 5 h. Then, the Skp2, mTOR and GAPDH were detected by immunoblotting.
Antibodies and reagents
Antibodies against Skp2, mTOR, phosphorylate-mTOR-ser2448, S6K, phosphorylate-S6K-ser389, 4EBP1, phosphorylate-4EBP1-ser65 were purchased from Santa Cruz Biotechnology Inc. Antibodies against FLAG, myc, His were purchased from Sigma-Aldrich. Phosphorylate-Skp2-ser64 was generated by Zoonbio Biotechnology Co.,Ltd. The mTOR inhibitor rapamycin were obtained from Sigma.
RNAi treatment
All siRNAs were produced by GenePharma. According to the manufacturer’s protocol, 293 T cells were transfected with siRNA oligonucleotides using the Lipofectamine RNAiMAX transfection reagent (Invitrogen) for 48 h.
The siRNAs for mTOR are as follows:
mTOR no.1 sense:5’-GCAUCCAGCAGGAUAUCAATT-3’; antisense:5’-UUGAUAUCCUGCUGGAUGCTT-3’
mTOR no.2 sense:5’- GCUGUCAGCCUGUCAGAAUTT -3’; antisense:5’- AUUCUGACAGGCUGACAGCTT-3’
Immunoblotting and immunoprecipitation
Whole-cell extracts were lysed by lysis buffer (1 × Cell Lysis Buffer (Cell Signaling Technology) adding 1 mM phenylmethylsulphonyl fluoride (PMSF) immediately before use). Protein suspension liquid were boiled after addition 6 × SDS sample buffer for 5–10 min at 100 °C. In immunoprecipitation experiment, whole-cell were lysed by E1A lysis buffer (50 mM HEPES (pH 7.5), 250 mM NaCl, 5 mM EDTA, 0.1% NP-40, protease inhibitor cocktail (Roche)). Immunoprecipitation was carried out either by incubating appropriate antibody with cell lysate for 2–3 h, followed by incubating Protein-A/G beads overnight (Roche) or by incubating FLAG beads at 4 °C with lysate overnight. Beeds were washed with ice-cold PBS three times, resuspended in SDS loading buffer. The protein samples were resolved by 10% SDS-PAGE and transfered to membrane (Sigma-aldrich). Then, the blots were identified by various antibodies.
In vivo ubiquitination assay
In vivo ubiquitylation assays were performed as described19. In brief, 293 T cells were transfected with the indicated plasmids for 24 h, and were treated with 20 μM LY, or 100 nM Wortmannin together with 20 μM MG132 for 6 h prior to harvesting. The cell extracts were then incubated with nickel beads for 3 h, washed, and subjected to the Western blot analysis.
Cell growth and in vivo tumorigenesis assay
For cell growth assay, 5 × 103BGC823 stable cells with pcDNA, pcDNA-Skp2, or pcDNA-Skp2 S64A and pcDNA-Skp2 S64D were seeded in 12-wells in triplicate, harvested, and stained with trypan blue at different days. Numbers of viable cells were directly counted under the microscope. The individual clone was picked and verified by Western blot analysis.
For in vivo tumorigenesis assays, BGC823 stable cells (1 × 106) mixed with matrigel (1:1) were injected subcutaneously into the left flank of 6-week-old nude mice. Tumor size was measured weekly with a caliper, and the tumor volume was determined using the standard formula: L × W2 × 0.52, where W is the shortest diameter and L is the longest diameter.
Patients and specimens
GC samples and the adjacent nontumor samples were obtained from 169 GC patients who underwent radical resection for histologically confirmed gastric cancer from Sun Yat-sen University Cancer Center between January 2000 and December 2004. All patients had follow-up after surgery at 6 – 12 month intervals; the final date of follow-up was on Jan 1, 2015. The primary end point of our study was Overall survival (OS). Disease-free survival (DFS) was the time between the time of surgery and relapse or tumor-related death. Overall survival was defined as the period between the time of surgery and death.
Immunohistochemistry was done by means of a standard of protocol. Expression of p-mTOR and p-Skp2 were examined by at least two investigators who were blinded to clinical data. Immunostaining was classified based on staining intensity and percentage of p-mTOR-positive and p-Skp2-positive tumor cells. Staining intensity was determined as 0 (absent), 1 (weak), and 2 (strong). Expression levels were semi-quantified using an immunohistochemistry score (range, 0–200) calculated by the percentage of positive tumor cells. Patients with an immunochemistry score of >20 as p-m-TOR and p-Skp2 positive or high expression and those with a score of ≤20 were considered as p-mTOR and p-Skp2 negative or low expression.
Discussion
In this study, we showed that Skp2 could be regulated by ubiquitin-mediated degradation, which promoted by mTORC1, a central regulatory kinase in key cellular process. Specifically, we found that mTORC1 directly interacts with Skp2 in a phosphatase-independent manner. Conversely, both siRNA and mTOR inhibitor rapamycin disrupts the activity of Skp2. Taken together, these results indicate that the phosphorylation of Skp2 at Ser64 is mainly mediated by mTORC1.
Previously, some kinases have been shown to phosphorylate Skp2 by controlling its stability. For example, phosphorylation of Skp2 by CDK2 at Ser64 and Ser72 can protect it from degradation [
24]. Akt1 and Pim-1 also appears capable of phosphorylating Skp2 at Ser72 [
25,
26]. However, Unlike Akt1, Pim-1 kinase could not regulate Skp2 subcellular localization by phosphorylation [
26]. It suggests that the regulating role of phosphorylation of Skp2 is decided by the different kinases [
27]. Moreover, the critical role of Skp2 phosphorylation in cancer cell has not yet been explored.
Indeed, mTORC1 has emerged as an important player in the regulation of Skp2. For example, Shapira et al. revealed that the mTOR inhibitor rapamycin significantly decreased Skp2 levels and enhanced the degradation of Skp2. p27 levels were also up-regulated in rapamycin-sensitive cells [
28] Therefore, clarifying the mechanism how mTORC1 regulates Skp2 has important implications for cancer therapy [
28,
29].
Currently, our results demonstrated that phosphorylation on Ser64 by mTORC1 is required for the stabilization of Skp2 levels in GC. In GC cells, we showed that the mTOR siRNA and Skp2 S64A mutation inhibited cell transformation and proliferation than WT in vitro and in vivo. Notably, in a panel of GC cell lines, there is a positive correlation between the elevated expression of mTOR and p-Skp2 (Ser64). These results implicate mTORC1 as an upstream regulatory factor for the Skp2 pathway at the posttranslational level and overexpression of mTORC1 may contribute to the activation of Skp2 oncogenic function in GC.
Recent studies have revealed that p-mTOR expression is upregulated in GCs [
30,
31]. However, the expression of p-Skp2 (Ser64) and their correlation in GCs remain to be investigated. In the current study, we firstly showed that p-Skp2 overexpression in advanced GCs and can predict poor survival outcome for patients. Importantly, 83 out of 169 cancer tissues examined contained high expression of p-mTOR, whereas 68 out of the 88 tissues containing active Skp2, supporting the conclusion that activation of mTOR signaling is involved in the overproduction of Skp2. On the other hand, p-Skp2 (S64) was undetected in 81 of the 86 specimens with low p-mTOR expression, this suggests that decreased p-mTOR can predict the inactivation of Skp2 by reducing phosphorylation of Skp2. Furthermore, our study demonstrated that the combined expression of p-mTOR and p-Skp2 is much better to predict the survival of GC patients than each of them. Collectively, the clinical data further strengthened the notion that phosphorylation of Skp2 by mTORC1 protect Skp2 from degradation and promotes Skp2 activation and is associated with the prognosis of GC patients.
Our results have important clinical implications in the treatment of GC for several reasons. First, it is helpful to identify which subgroup of GCs may respond the most to mTOR inhibitor. To date, some clinical trials involving rapalogues have been performed in GC patients, with negative results [
32]. For example, in 2013, a randomized clinical trial showed that mTOR inhibitor everolimus failed to improve the survival of GC patients [
33]. The main reason may be that the patients particularly dependent on mTOR signaling were not identified [
34]. Indeed, given that cells that express elevated Skp2 Ser64 have better sensitivity to rapamycin, Skp2 Ser64 expression would be a potential biomarkers in the treatment of GC patients using mTOR inhibitor. Secondly, our data also provide a new view on cancer therapy by preventing the phosphorylation of Skp2. Recently, Chan CH et al. have reported Skp2 inhibitor can exhibits potent antitumor effect in many cancers by targeting cancer stem cell [
35]. It will be interesting to explore the effect of Skp2 S64 phosphorylation on GC stem cell. Additionally, combination target-therapy have emerged and showed promising anticancer activities [
36]. Since constitutive activation of the mTORC1-Skp2 pathway frequently occurs in GCs and combined their expression has worse prognosis, it is very likely that the intervention by combining Skp2 inhibitors and mTOR inhibitors may be a much better approach for GC therapy.
Conclusions
In conclusion, the present study provides the first evidence of how Skp2 is regulated by mTORC1. mTORC1 may function to regulate Skp2 by serving as a priming kinase, which stablizes Skp2 and consequently leads to its oncogenic effects on gastric cancer tumorigenesis. Importantly, our study indicates that p-Skp2 (Ser64) expression is a hopeful biomarker for the subset of GC patients that benefit from mTOR inhibitor. In addition, our data also suggest that combining Skp2 inhibitors and mTOR inhibitors might represent a potentical therapeutic strategy in the treatment of GC. Further studies are required to test this possibility.
Acknowledgments
We thank Dr Shixun Lu and Longjun He for their technical support. We also thank Dr Xuan Li and Hailiang Zhang for performing the data analysis.