Background
Acute lymphoblastic leukemia (ALL) is an aggressive malignant disorder of lymphoid progenitor cells in both children and adults, and it is caused by genetic lesions in blood-progenitor cells [
1,
2]. Although improvements in contemporary therapy and development of new treatment strategies [
3‐
5] have led to dramatic increases in the cure rate in children, the relapse rate remains high and the prognosis of relapsed childhood ALL is poor [
6]. Hematopoietic stem cell transplantation (HSCT) seems to be curative for patients with ALL relapse and high-risk acute lymphoblastic leukemia [
7]. However, post-HSCT relapse is still an obstacle to therapeutic improvement [
8,
9]. Use of an antineoplastic immunosuppressive agent after transplantation should facilitate treatment. However, although immunosuppressive therapy can significantly increase the survival time of transplant patients, it also promotes tumor growth [
10].
Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), is a bacterial macrolide that was originally used as an antifungal agent [
11,
12]. The findings that rapamycin targets mTOR and is also antiproliferative led to its use as an anticancer agent [
10,
13]. Evidence indicates that the phosphatidylinositol 3-kinase (PI3K), Akt, mTOR signaling pathway (PI3K/Akt/mTOR) is dysregulated in hematologic malignancies and abnormally activated in childhood ALL. Most commonly, this abnormal activation is due to constitutive activation of Akt and provides a compelling rationale to target this pathway in ALL. Preclinical studies demonstrating significant activity against ALL has led to a number of clinical trials [
14]. Combination therapeutic strategies of using rapamycin with focal adhesion kinase (FAK) down-regulation may address the problem of resistance to mTOR-targeted monotherapy and improve the treatment effect.
FAK is a 125-kDa non-receptor tyrosine kinase that plays an important role in cell survival, proliferation, apoptosis, migration, and invasion [
15,
16]. FAK expression is higher in malignant cells than in the corresponding normal cells [
17], and a high expression of FAK is associated with enhanced blast migration and poor prognosis in acute myeloid leukemia (AML) [
18]. Furthermore, in our previous study, FAK down-regulation inhibited leukemogenesis in breakpoint cluster region/Abelson leukemia virus (BCR/ABL)-transformed ALL cells and increased apoptosis and drug efficacy in pro-B ALL cells. In vivo FAK down-regulation has also been shown to impair cell migration and inhibit leukemia progression [
19]. Interestingly, FAK is an upstream kinase of Akt [
20], indicating that FAK down-regulation might suppress rapamycin-induced Akt activation [
21,
22]. This possibility provided us with a rationale for combining rapamycin with FAK down-regulation therapy to treat ALL in patients who received HSCT.
In the present study, we found that FAK was activated in tumor cells of ALL patients. Either FAK down-regulation or rapamycin caused growth inhibition of a pro-B ALL cell line, and growth was more profoundly inhibited by a combination of FAK down-regulation and rapamycin. The combination enhanced the treatment effect of rapamycin, prolonged median survival time, and slowed the progression of leukemia in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice injected with REH (the human precursor B acute lymphoblastic leukemia cell line) cells. Collectively, our results suggest that the combination of rapamycin and FAK down-regulation may be a promising therapeutic strategy in ALL patients who received HSCT.
Discussion
In cancer therapy, combinatorial strategies are commonly used to treat malignancies that are resistant to standard treatment. Several combinations of rapamycin or rapalogs with other antitumor drugs have previously been investigated [
24‐
26]. Recently, it was found that the cytotoxic drug carboplatin could enhance the effect of the mTOR inhibitor everolimus, and this combination is already undergoing phase I evaluation [
27], which suggests that the clinical benefit from mTOR inhibitors could be maximized by combining them with other agents. In the present study, patients with ALL when compared to normal volunteers had varying degrees of FAK activation (Fig.
1a). In other studies, FAK was elevated in human AML, where increased FAK expression and activity were correlated with poor prognosis [
18]. In our previous study, FAK down-regulation enhanced the drug efficacy of imatinib in ALL cells [
19]. Herein, we assessed whether FAK could augment rapamycin efficacy in ALL cells, and the results were encouraging. In vitro, rapamycin combined with FAK down-regulation enhanced cell growth inhibition, G
0/G
1 cell cycle arrest, and cell apoptosis. In vivo, this combination slowed leukemia progression, indicating that FAK down-regulation can improve the efficacy of rapamycin in ALL treatment.
We supposed that rapamycin blocked the mTOR pathway, attenuating p70S6K activation and leading to Akt pathway activation. The antitumor activity of rapamycin is compromised by the hyperactivity of the feedback-loop-relevant PI3K/Akt signaling pathway [
24]. Thus, when FAK is down-regulated, rapamycin-induced Akt phosphorylation is inhibited, which might explain how FAK down-regulation enhances the efficacy of rapamycin. The results in Fig.
1b strongly support this hypothesis.
The present finding that the blockade of mTOR by rapamycin and down-regulation of FAK inhibit proliferation of REH cells and increase G
0/G
1 cell cycle arrest (Figs.
2b and
3a) are consistent with the findings of other groups. Recher et al. showed that rapamycin could inhibit the proliferation of acute myeloid leukemia cells and sensitize these cells to growth inhibition mediated by cytotoxic agents such as cytarabine [
28]. In another study, LY294002 was shown to enhance rapamycin-mediated inhibition of T-cell proliferation [
29]. On the other hand, Pan et al. found that Caco-2 cell proliferation was significantly decreased by inhibition of FAK gene expression [
30]. These results support our observations.
In our analyses, the induction of ALL cell apoptosis by rapamycin was mild. However, when rapamycin was combined with FAK down-regulation, the cell apoptosis effect was dramatically enhanced (Fig.
3b), indicating that the enhancement by FAK down-regulation of rapamycin’s antitumor efficacy is mainly due to apoptosis induction. Rapamycin treatment and/or FAK down-regulation can stimulate the expression of the pro-apoptosis genes, such as BIK, PUMA, BMF, BAX, and MCL-1S, and inhibit the expression of the anti-apoptosis genes such as BCL-2 and BCL-XL (Fig.
4). We suppose that using the new BCL-2 inhibitors in combination therapy will boost treatment efficacy [
31].
Using a murine model of leukemia induced by REH cells, we further investigated the effects of FAK down-regulation on rapamycin efficacy in vivo. All the mice died after injection of leukemic cells despite treatment with rapamycin for 7 days. But compared with rapamycin treatment alone, rapamycin combined with FAK down-regulation prolonged median survival by 14 days, reduced spleen size, and diminished peripheral leukocyte count in the model mice. These findings suggest that FAK down-regulation potentiates rapamycin-induced inhibition of leukemia progression in vivo.
Methods
Cell culture and clinical samples
The REH human acute lymphoblastic pro-B cell leukemia cell line (CRL8286, ATCC, USA) was cultured in RPMI1640 medium (Gibco, USA) supplemented with 10 % fetal bovine serum (Gibco, USA) in a CO
2 incubator. A total of 13 clinical samples, including samples from 8 patients with a primary diagnosis of acute lymphoblastic leukemia (ALL), 2 patients with relapsed ALL, and 3 normal samples from Sun Yat-sen Memorial Hospital, were included in this study. All cells were freshly isolated from the bone marrow of each individual. The clinical characteristics of these individuals are presented in Table
1. All patients provided informed consent, and the study was approved by the ethics committees of Sun Yat-sen Memorial Hospital.
Table 1
Patient clinical characteristics
#1 | 12 | Male | ALL newly diagnosed |
#2 | 3 | Female | ALL newly diagnosed |
#3 | 2 | Male | ALL newly diagnosed |
#4 | 7 | Female | ALL newly diagnosed |
#5 | 9 | Female | ALL newly diagnosed |
#6 | 7 | Female | ALL newly diagnosed |
#7 | 7 | Female | ALL newly diagnosed |
#8 | 8 | Male | ALL newly diagnosed |
#9 | 15 | Male | ALL relapse |
#10 | 13 | Female | ALL relapse |
NC1 | 14 | Female | Normal control |
NC2 | 15 | Male | Normal control |
NC3 | 7 | Male | Normal control |
Down-regulation of FAK with shRNA and establishment of stable transfected clones.
A short-hairpin RNA (shRNA)-expressing lentivirus-vector delivery system was applied as previously described [
34,
35]. The obtained lentiviruses, containing the GFP-FAK shRNA vector or a GFP-empty vector construct, were used for the transfection of REH cells. To establish stable transfected clones, the REH cells were sorted repeatedly based on a green fluorescent protein (GFP) expression using a flow cytometer (FACSAria, Becton Dickinson, CA) at 72 h after transfection, until the percentage of GFP-positive clones was greater than 99 %. The stably transfected clones were used for further experiments. Quantitative real-time PCR analysis revealed that the best silencing efficiency was achieved with the shRNA designated FAK X40-2 shRNA, and the FAK target sequence was 5′-GGAATGCTTCAAGTGTGCTT-3′.
Reagents
Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, was purchased from Sigma (USA). Rapamycin was dissolved in 100 % dimethyl sulfoxide (DMSO) (Sigma, USA) to a stock concentration of 25 mg/ml and stored at −20 °C.
Western blotting and quantitative real-time PCR
The cells were lysed in radio immuno-precipitation assay (RIPA) buffer (Pierce, Rockford, IL, USA) with protease and phosphatase inhibitors (Roche, Beijing, China), and the supernatant was collected after centrifugation. Denatured proteins were fractionated via electrophoresis on a 10–12 % sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred to a methanol-activated polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was blocked for 2 h in Tris-buffered saline Tween-20 (TBST) containing 5 % bovine serum albumin and then incubated with a polyclonal mouse anti-FAK (Millipore, USA), rabbit anti-AKT (Cell Signaling Technology, Boston, MA, USA), rabbit anti-phospho-AKT (Ser473, Cell Signaling Technology, Boston, MA, USA), rabbit anti-GAPDH (Cell Signaling Technology, Boston, MA, USA), or rabbit anti-β-tubulin (Cell Signaling Technology, Boston, MA, USA) antibody overnight at 4 °C. One hour after incubation with the corresponding goat anti-mouse (Thermo) or goat anti-rabbit (Sigma) horseradish peroxidase-conjugated secondary antibody, the level of protein expression was detected using the enhanced chemiluminescence (ECL) method (Millipore, USA) according to the manufacturer’s instructions.
Total RNA was extracted using the TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocols. cDNA was prepared from 1 μg of total RNA using a reverse transcription-polymerase chain reaction (RT-PCR) kit (Takara, Japan) with oligodT according to the manufacturer’s instructions. cDNA samples were then analyzed via quantitative real-time PCR using SYBR Green (Takara, Japan) in an ABI Step One Real-Time PCR machine (Applied Biosystems, Foster City, CA), with 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The efficiency of cDNA synthesis was estimated using hGAPDH as a house-keeping gene. All data were analyzed via the comparative C
T method [
36], and all of the reactions were performed in triplicate.
Cell proliferation assays
REH-empty vector or REH-FAK shRNA cells (4 × 105/ml) were incubated with various concentrations of rapamycin for 48 or 72 h, respectively, in 96-well plates (Costor, USA). After culture, Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technologies, Shanghai, China) solution (10 μl) was added to each well, followed by incubation at 37 °C for an additional 2 h. The absorbance was measured at 450 and 630 nm using an absorbance reader. All experiments were performed in triplicate and were repeated at least three times.
Cell apoptosis and cell cycle analysis
Apoptosis was assessed through annexin V/propidium iodide (PI) staining. After transduced REH cells were treated with or without rapamycin (100 nM) for 30 h, they were stained with annexin V/PI (BD Biosciences, CA, USA) following the manufacturer’s instructions and then analyzed via flow cytometry. Cell cycle analysis was performed on transduced REH cells incubated with or without rapamycin (100 nM) for 48 h. The cells were subsequently collected and incubated with 0.5 ml of NP40/PI buffer and RNase (25 μg/ml) for 30 min at 37 °C. Analysis by flow cytometry was performed immediately thereafter.
In vivo experiments
Male NOD/SCID mice were purchased from the Huafukang Company (Beijing, China) and were maintained in the animal center of Sun Yat-sen University under specific pathogen-free conditions. The murine model of leukemia was established as previously described [
19]. Briefly, five million REH-FAK shRNA or REH-empty vector cells were injected into 6- to 8-week-old NOD/SCID mice via the tail vein. For the in vivo assessment of drug effects, 10 days after transplantation of the REH cells, the mice were treated daily with 1.5 mg/kg rapamycin (or DMSO for the control group) via intraperitoneal injection for 7 days. Survival was monitored daily. Full blood counts were performed once a week. Several mice were sacrificed 25 days after transplantation, and their spleens were removed. The animal study was approved by the ethics committees of the animal center of Sun Yat-sen University.
Statistical analysis
The results are expressed as the mean ± standard deviation (S.D.). The differences between groups were analyzed using Student’s t test when only two groups were compared or one-way analysis of variance (ANOVA) when more than two groups were compared. Log-rank p values were determined using the Kaplan–Meier method comparing survival curves. Values of p ≤ 0.05 were considered statistically significant.
Acknowledgements
This work was supported by the National Natural Science Foundation of China (81100370, 81570140).
The authors wish to thank Professor Chen Yueqin and Professor Su Peiqiang who provided us with a laboratory to carry out the experiments. We thank technician Wang Ying at the Second Affiliated Hospital of Sun Yat-sen University, who offered us the machine for full blood cell counting. We thank technician Wu Shouhai at the Department of Life Science of Sun Yat-sen University, who helped us operate the flow cytometer. We thank the volunteers of the Second Affiliated Hospital of Sun Yat-sen University, who were willing to donate their bone marrow for research purposes. We thank Dr. Zeng Chenwu for giving us the primer for the BCL-2 family. We thank Dr. Gao Wenjie for helping us modify the manuscript. We thank Dr. Liao Yadi for helping us with the statistical analysis.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
PS designed the study and carried out the cellular experiments and drafted the manuscript. LX participated in the design of the study and performed the in vivo experiments and helped to draft the manuscript. KL carried out the molecular experiments and helped with the in vitro and in vivo experiments. WW collected the clinical samples and helped with the statistical analysis. JF conceived of the study and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.