Background
Acute leukemia (AL), mainly consisting of acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), is currently the major cause of death in hematological malignancies, affecting patients of all ages. Despite ongoing improvements in the outcomes of patients with AL, only 30%-40% of adult ALL patients achieve long-term, disease-free survival due to drug resistance and disease relapse [
1,
2]. AML is a heterogeneous disease, and a substantial number of AML patients have quite a low cure rate even after hematopoietic stem cell transplantation [
3,
4]. Minimal residual disease (MRD), which is widely considered an independent prognostic factor and currently attracts much attention in treatment intervention, has become a vital challenge in the search for a cure for AL [
5-
7]. In addition, the leukemia niche is believed to play a critical role in the development of MRD.
The leukemia niche, composed of the osteoblastic and vascular bone marrow niche, provides a home for malignant cells and is responsible for disease relapse as well as treatment resistance. Previous studies have shown that stromal cells in the bone marrow microenvironment (BMM) play an important role in leukemia genesis and progress by secreting various chemicals and contacting signals [
8], for example the axis of VCAM-1/VLA-4 [
9], SDF-1/CXCR4 [
10], and Notch [
11], as shown
in vitro and
in vivo. Since mesenchymal stromal cells (MSCs), an important component of both the solid and hematologic tumor microenvironment [
12], give rise to different stromal cell lineages [
13]. In our study we used human bone marrow-derived MSCs (hBM-MSCs) to represent a relatively homogeneous BMM stromal cell population with hematopoiesis-supporting capabilities and immune-regulatory properties.
Galectin-3 (gal-3), a 30-kDa protein without enzymatic activity, is a member of the β-galactoside-specific lectin family. Gal-3 exhibits pleiotropic biological functions especially in tumors. It has roles in cell growth, apoptosis, adhesion, tumor angiogenesis, malignant cell metastasis, cancer-matrix interaction and also cancer drug resistance [
14,
15]. Recent evidence revealed that gal-3 was up-regulated in Ph
+ chronic myeloid leukemia (CML) and in pre-B ALL after conditioning with BM stromal cells [
16,
17]. Cheng and colleagues [
18] reported that in patients with AML, higher bone marrow
LGALS3(
gal-
3) gene expression was an independent unfavorable prognostic factor for overall survival.
However, the specific role of gal-3 in BMM-induced drug resistance of acute leukemia cells (ALCs) has not yet been investigated. The aim of our study was to identify the specific mechanism involved. We found that gal-3 was dramatically up-regulated in hBM-MSC-conditioned AL cell lines, accompanying activation of β-catenin signaling. Both gal-3 and β-catenin signaling were essential in promoting the survival of ALCs when treated with cytotoxic drugs. We also showed, for the first time, that gal-3 modulated β-catenin signaling by regulating GSK-3β phosphorylation and the PI3K/Akt pathway in hBM-MSC-conditioned ALCs.
Discussion
The present study demonstrates that gal-3 is specifically induced when acute leukemia cells (Reh, Sup-B15, Jurkat,Kasumi-1 and primary ALCs) are cultured with hBM-MSCs in vitro. Gal-3-shRNA largely eliminates hBM-MSC-induced gal-3 overexpression and reverses its protective effects against cytotoxic drugs in ALCs. Thus the induction of gal-3 is one of the pivotal underlying mechanisms of hBM-MSC-mediated protection of ALCs.
In view of the multiple biological functions of gal-3, we wondered how gal-3 performed its roles, especially in the leukemia BMM. Recent studies have suggested that gal-3 activates Wnt/β-catenin signaling in solid tumors, such as human colon and pancreatic cancer [
19,
20]. Wnt signaling plays an important role in maintaining normal hematopoiesis, and its degradation is causatively involved in the development of leukemia [
21-
23]. Yang
et al. [
24] indicated that Wnt signaling contributed to bone marrow stromal cell-mediated protection of ALL cells, which was in accordance with our results. However, the precise mechanisms involved remained unknown, especially in regard to the way in which gal-3 affected Wnt/β-catenin signaling between hBM-MSCs and acute leukemia. Our data were the first to reveal that gal-3 up-regulated β-catenin at the post-transcriptional level and activated its downstream signaling in the hBM-MSC-supported leukemia niche
in vitro.
The specific known target genes of Wnt signaling include c-Myc [
25], cyclin D1 [
26], Survivin [
27], gastrin [
28], MMP-7 [
29] and -2 [
20], and cyclooxygenase-2 [
30], most of which play an important role in cell survival, growth, self-renewal and motility. We therefore assessed the expression of these specific genes and detected increased transcription level of cyclin D1, survivin and c-Myc in ALCs conditioned by hBM-MSCs. However, once gal-3 was silenced, even though the cells were conditioned by hBM
-MSCs, transcription of these genes was not significantly up-regulated. The increase in expression of cyclin D1 shown in our results was far greater than c-Myc and survivin. Lin
et al. [
31] found that gal-3 could promote cyclin D1 expression by enhancing its promoter activity through SP1 and a cAMP-responsive element in human breast epithelial cells. It is still unclear whether this also applies to acute leukemia cells. Further study will be necessary to confirm whether there are other mechanisms involved in gal-3-mediated cyclin D1 expression other than Wnt signaling in the acute leukemia microenvironment.
Our results illustrated that gal-3 activated target genes of Wnt/β-catenin pathway (Cyclin D1, c-Myc and Survivin). However, gal-3 inhibition in ALC itself did not significantly affect the expression of these genes. This might be due to the fact that ALCs only expressed limited gal-3 spontaneously. As previous studies have reported, Wnt/β-catenin pathway was required for leukaemogenesis [
32] and a high frequency of leukemic cell lines were able to freely translocate cytosolic β-catenin to nucleus [
33]. So we suggest that gal-3 is not the predominant regulator of Wnt/β-catenin pathway in ALCs without stimulation of hBM-MSCs.
We also analyzed the expression of pho-Akt and pho-GSK-3β in ALCs conditioned by hBM-MSCs, and found that both pho-Akt and pho-GSK-3β increased before the accumulation of β-catenin, which was consistent with previous studies showing that PI3K-activated Akt can phosphorylate GSK-3β at Ser
9, thereby inactivating GSK-3β and triggering related signaling pathways [
34-
36]. In addition, Song [
19], Kobayashi [
20] and their colleagues reported that PI3K/Akt-inactivated GSK-3β might be the bridge between gal-3 and Wnt signaling in colon and pancreatic cancer. Taken together with our results, these findings suggest that Akt phosphorylation may be the first step that occurs after hBM-MSC-induced gal-3 up-regulation in ALCs, which subsequently promotes GSK-3β phosphorylation and supports β-catenin stabilization. Interestingly, the phosphorylation levels of β-catenin fluctuated behind GSK-3β after a time-lag, which indicates there may be a feedback loop between pho-GSK-3β and β-catenin stabilization.
Our results demonstrate that signals regulated by gal-3 are correlated with cell cycle progression and drug resistance, which may be major effects of leukemia microenvironments. This suggests the possibility that gal-3 could be a potential treatment target for acute leukemia, especially for minimal residual disease maintained by the leukemia niche. GCS-100, a citrus pectin-derived specific gal-3 antagonist, has proven to be effective in restoring or augmenting drug sensitivity in myeloma, large B-cell lymphoma and B-chronic lymphocytic leukemia [
37-
40]. Thus GCS-100, targeting the bone marrow microenvironment of acute leukemia, may be an innovative therapeutic molecule in the near future. Further
in vivo researches and clinical trials are still warranted.
Since gal-3 is expressed in many tissues and cells, we tested whether hBM-MSCs also expressed gal-3, and found that they did (data not shown). Liu
et al. [
41] showed that human umbilical cord MSCs expressed gal-3 both on the cell surface as well as in secreted form. Secreted gal-3 was critical for the immunomodulatory potency of hUC-MSCs. It was also mentioned that OP9 cells also secreted gal-3 and that some of the gal-3 detected on pre-B ALL cells was of stromal origin [
17]. The role of gal-3 derived from stromal cells and the potential mechanisms involved in its action in leukemia therefore deserve further study.
Yamamoto,
et al. [
16] have reported gal-3 is predominantly expressed in CML cells, but not in acute leukemias. Our results showed that hBM-MSCs also induced the up-regulation of gal-3 in ALCs
in vitro. Furthermore, the expression level of gal-3 in refractory/relapsed AL patients is predominantly higher than that in primary ones. This may suggest gal-3 play a pivotal role in the maintance of MRDs and development of drug resistance. However, mechanisms about how the leukemic niche modulates expression of gal-3 remain little understood, since no mutation of the
LGALS3 gene has been detected. Since epigenetic alterations are important in development and maintenance of leukemia cells [
42], it is still unknown whether gal-3 is promoted by activation of its transcription factors, DNA methylation, histone modifications, or action of non-coding RNAs which target gal-3 [
43].
Methods
Cells and drugs
Human acute leukemia cell lines Reh (non-B non-T ALL) [
44], Sup-B15 (B-ALL) [
45], Jurkat (T-ALL) [
46], and Kasumi-1 (AML, FAB M2) [
47] were obtained from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Bone marrow was collected from healthy adult donors after they had provided informed consent, and the hBM-MSCs obtained were cultured in DMEM-low glucose (Gibco/Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco), identified as described in our previous report [
48]. Bone marrow mononuclear cells, of which more than 90% were malignant cells, from primary and refractory/relapsed acute leukemia (non-M3 AML and ALL) patients were purified by Ficoll-Paque isodensity gradient centrifugation (Tianjing, China). Idarubicin (IDA) and the Wnt signaling-specific inhibitor ICG-001 were obtained from Pfizer Inc., (New York, NY, USA) and from Selleck Chemicals, (Houston, TX, USA), respectively, and dissolved in phosphate-buffered saline (PBS) and dimethylsulfoxide (DMSO). Etoposide (VP-16) and the PI3K/Akt signaling inhibitor Ly294002 were purchased from Sigma-Aldrich (St Louis, MO, USA), and dissolved in DMSO. All of them were stored at −20°C.
Cell culture and co-culture
Reh and Sup-B15 cells were maintained in IMDM (Gibco) supplemented with 10% FBS (Gibco), while Jurkat and Kasumi-1 cells were cultured in RPMI-1640 medium (Gibco) containing 10% FBS (Gibco). Human BM-MSCs at passage 3 to 7, displaying a homogeneous mesenchymal immunophenotype and multipotent differentiation potential, were used for co-culture experiments. They were seeded into 12- or 6-well plates at a density of 4 × 104/mL. ALCs were added to the confluent hBM-MSC layer 1-2 days later at a ratio of 10:1. After ALCs adhered to the hBM-MSC layer, IDA or VP-16 was added. ICG-001 was added half an hour ahead of cytotoxic drugs. At indicated times, ALCs were collected, leaving the adherent stromal layer intact, and washed with PBS for subsequent analyses.
Short hairpin RNA (shRNA) preparation and transfection
The shRNA against gal-3 in a lentiviral vector with green fluorescent protein, as well as the corresponding control vector were designed and synthesized by GenePharma Inc. (Shanghai, China). High-titer lentivirus was produced in 293 T cells by transfection of the lentiviral expression vector and packaging vectors, psPAX2 and pMD2.G (obtained from
www.Addgene.org), using a calcium phosphate cell transfection kit according to the manufacturer’s instructions (Beyotime Institute of Biotechnology, Shanghai, China). The lentivirus was harvested 48 h later, filtered, enriched using 40% polyethylene glycol, and then used to infect acute leukemia cells. After transfection for 72 h, the efficiency was estimated by evaluation of EGFP expression by fluorescence microscopy and flow cytometry. The gal-3 specific shRNA sequence used in our study was 5′-GTACAATCATCGGGTTAAA-3′.
CCK-8 assay for cell viability
Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Laboratories (Kumamoto, Japan). Measurements were taken 48 h after drug exposure at the indicated concentrations. Absorbance was detected at 450/630 nm by a Benchmark microtiter plate reader (Bio-Rad Laboratories, Hercules, CA, USA). The relative cell viability was determined by (A
co − cultured
− A
medium
)/(A
cultured alone
− A
medium
) × 100 %.
Cell apoptosis and cell cycle analyses
Acute leukemia cells cultured alone or co-cultured with hBM-MSCs were exposed to IDA or VP-16 at the indicated concentrations for 48 h, then cells were harvested, washed and resuspended in PBS. Apoptotic cells among non-transfected ALCs were identified by staining with Annexin V-FITC/PI (BD Pharmingen, Franklin Lakes, NJ, USA), while those in transfected ALCs were identified by staining with Annexin V-PE/7AAD (BD Pharmingen) according to the manufacturer’s instructions. The stained cells were analyzed by fluorescence activated cell sorting (FACS) (Beckman Coulter, Brea, CA, USA).
ALCs for cell cycle analyses were collected after 48 h of co-culture with hBM-MSCs. All cells were stained using the cell cycle detection kit (KeyGen Biotech. Co. Ltd., Nanjing, China) and analyzed by FACS. Cells in S and G2M phases were considered proliferative.
Protein extraction and western blot analyses
Collected cells were lysed in lysis buffer containing 0.5 M Tris-HCl, pH 6.8, 2 mM EDTA, 10% glycerol, 2% SDS, 5% β-mercaptoethanol and protease inhibitors. Thirty to fifty micrograms of protein was separated by 10%-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electotransferred onto polyvinylidene fluoride membranes. The membranes were blocked in 5% bovine serum albumin (BSA) at room temperature for 2 h, incubated with primary antibodies overnight at 4°C, and then incubated with an IRDye secondary antibody (Li-Cor Biosciences, Lincoln, NE, USA) at room temperature for 1 h. The following antibodies were used: anti-gal-3 (Epitomics, Abcam, Cambridge, MA, USA), anti-β-catenin, anti-Akt, anti-pAkt (Cell Signaling Technology, Danvers, MA, USA), anti-pho-GSK-3β, anti-GSK-3β (Abcam). We also used anti-β-actin antibody (Sigma) as a control. Immunoreactive bands were visualized using an Odyssey infrared imaging system (Li-Cor). Signal intensity was quantified using Quantity One software (Bio-Rad) when necessary.
RNA isolation and PCR analyses
Total RNA from collected cells was extracted using Trizol reagent (Takara Bio Inc., Shiga, Japan). One thousand nanograms of total RNA was used in a 2-step quantitative reverse transcription-PCR (Takara). Real time PCR was performed with the Roche Applied Science LightCycler 480 II Real-Time PCR System using the SYBR Green gene expression assay (Takara), according to the manufacturer’s instructions. The following primer sets were used (Sangon, Shanghai, China): Gal-3, 5′-GCCTTCCACTTTAACCCACG-3′ (forward) and 5′-AACCGACTGTCTTTCTTCCCTTC-3′ (reverse); β-catenin, 5′-CTGAGGACAAGCCACAAGATTA-3′ (forward) and 5′-ATCCACCAGAGTGAAAAGAACG-3′ (reverse); Cyclin D1, 5′-TCTACACCGACAACTCCATCC-3′ (forward) and 5′-GCATTTTGGAGAGGAAGTGTTC-3′ (reverse); c-Myc, 5′-CCTCCACTCGGAAGGACTATC-3′ (forward) and 5′-TGTTCGCCTCTTGACATTCTC-3′ (reverse); Survivin, 5′-CACCGCATCTCTACATTCAAGA -3′ (forward) and 5′-CAAGTCTGGCTCGTTCTCAGT-3′ (reverse); and GAPDH, 5′-AGAAGGCTGGGGCTCATTTG-3′ (forward) and 5′-AGGGGCCATCCACAGTCTTC-3′ (reverse). Independent triplicate samples were used in our experiments.
Statistical analyses
Statistical analyses were performed using GraphPad Prism for Windows version 5.00 (GraphPad Software, San Diego, CA, USA) and SPSS 20.0. All data were presented as mean ± SD and statistical differences were evaluated using Student’s 2-tailed t-test (paired or unpaired, as appropriate) and Mann-Whitney U test (for data from AL patients). Differences were considered statistically significant at P < 0.05.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
KH, LL and HH were responsible for concept and design of the study. KH, YG and BW conducted the experiments. He Huang and XY contributed essential reagents and tools. LL and KH were responsible for data analysis. KH, YG and HH drafted the article. All authors made final approval of this article.