Background
Chronic myeloid leukemia (CML), characterized by granulocytosis and splenomegaly, is a myeloproliferative disease and the disease course of CML is triphasic, starting with a chronic phase, progressing to an accelerated phase and ultimately ending in a terminal phase called blast crisis [
1]. BCR/ABL is derived from chromosomal translocation (relocation of the portion of c-ABL gene from chromosome 9 to the portion of BCR gene locus on chromosome 22 t (9;22)), yielding the Philadelphia (Ph) chromosome that is present in over 90% of CML [
2,
3]. The Philadelphia chromosome in CML gives rise to constitutively active protein tyrosine kinase product BCR-ABL, which is important because in patients with CML, there is clonal expansion of hematopoietic cells that express this fusion gene. Moreover, continued expression of BCR-ABL is required for sustained proliferation of leukemic cells in mouse models of CML [
4,
5]. Inhibition of BCR-ABL with kinase inhibitors such as imatinib mesylate in the treatment of Ph
+ CML is the current standard therapy, but it is highly effective in controlling but not curing the disease. This is largely due to the inability of these kinase inhibitors to kill leukemia stem cells (LSCs) responsible for disease relapse [
2]. This ‘native’ resistance of LSCs in CML to imatinib and other kinase inhibitors suggests that the kinase somehow turns on unique molecular pathways in LSCs through both kinase-dependent and, more importantly, kinase-independent mechanisms [
6].
To resolve the matter related to the drug resistance of LSCs in CML, it is essential to fully understand the molecular mechanisms in both kinase-dependent and kinase-independent pathways in CML. It is particularly crucial to identify the key genes that have significant roles in their survival and self-renewal. Emerging studies show that CD44 is an important biomarker of a cellular subpopulation (cancer stem cells, CSCs), which are capable of self-renewal and have the capacity for initiation, progression, invasion, metastasis, tumor recurrence, and resistance to chemo- and radiotherapy [
7]. CD44 denotes a large family of transmembrane glycoproteins that are expressed in a variety of cells and tissues and plays a critical role in a variety of cellular behaviors, including adhesion, migration, invasion, and survival [
8]. Daniela
et al. also found CD44 was indispensable for BCR-ABL-expressing leukemic stem cell to initiate CML and CD44 blockade decreased engraftment and impaired induction of CML-like myeloproliferative disease [
9].
The other key signal is Wnt/β-catenin, which are secreted signaling molecules that influence both development and cancer. Wnt/β-catenin regulates the differentiation of limbs, brain, kidney, and the reproductive tract in mice [
10,
11]. In addition to its importance in normal development, dysregulation of the Wnt/β-catenin pathway has potent oncogenic effects. Mutations in APC as well as β-catenin, a key mediator of Wnt/β-catenin signaling, are also found in a majority of sporadic colon cancers, hepatocellular carcinoma, as well as thyroid cancer, and ovarian cancer [
12]. The fact is that Wnt/β-catenin signaling is dysregulated in multiple solid cancers together with its observed influence on hematopoietic stem and progenitor cells [
13‐
16]. In blastcrisis CML patients, β-catenin is activated in myeloid progenitors and the activated β-catenin translocates to the nucleus [
4], where it interacts with lymphoid enhancer/T-cell transcription factors and regulates the expression of genes. Also, β-catenin has been shown to be involved in BCR-ABL leukemogenesis. BCR-ABL stabilizes β-catenin in myeloid cells through induction of tyrosine phosphorylation and activation of β-catenin in BCR-ABL-positive granulocyte-macrophage progenitors from blastic phase CML patients facilitates the acquisition by these cells of properties of LSCs [
1].
In this study, we used K562 chronic myeloid leukemia cells in vitro and in vivo to provide further evidence that CD44 and its target- β-catenin are essential for survival and self-renewal of CML cells.
Discussion
Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder that is characterized by a t (9; 22) translocation, which results in the expression of BCR-ABL fusion oncoproteins that are unique to the leukemic cells. This fusion oncoprotein is responsible for the increased activation of several downstream signaling pathways, which affect malignant cells’ behaviors and are necessary for oncogenesis and potential immunogenic [
17,
18]. The BCR-ABL tyrosine kinase inhibitor imatinib is effective as a single agent for the treatment of patients in all stages of CML, with the most encouraging results seen in patients in chronic phase (CP) disease. Hematologic and cytogenetic responses to imatinib for the treatment of CP CML have permitted imatinib to be registered as first-line treatment for newly diagnosed CML [
19,
20]. Despite the success of imatinib and other tyrosine kinase inhibitors (TKIs), CML remains largely incurable, and this is likely due to the treatment resistance of leukemic stem cells, which are responsible for rapid disease relapse after the discontinuation of therapy [
21,
22]. How to treat CML patients who are resistant to BCR-ABL tyrosine kinase inhibitors is an important and urgent issue for clinical hematology. So making further efforts to understand the molecular signals in CML is indispensable.
A number of studies have aimed at identifying the specific molecules expressed in CML stem cells that correlate with oncogenic behaviors. Among such candidates there is a major cell surface receptor CD44, which is a multifunctional transmembrane glycoprotein expressed in many cells and tissues [
23]. CD44 is often expressed as a variety of variant isoforms generated by an alternative splicing mechanism and the expression of certain cell surface receptor CD44 variant (CD44v) isoforms is known to be associated with many physiological and pathological processes [
24]. So we detected the expressions of CD44 in leukemic patients and the results showed that the expressions of CD44 in leukemic patients were higher than that in normal control. We also examined the CD44 level K562 chronic myeloid leukemia cells compared with the healthy control and the results were the same with that in patients’ samples. Liqing
et al. also found that CD44 was a key regulator of AML LSCs to maintain their stem cell properties and may provide a therapeutic strategy to eliminate quiescent AML LSCs [
25].
A growing body of literatures implicate that many signaling pathways including Wnt, Hedgehog, Notch and Bmi which regulate normal stem cell developmentare, are also classically associated with cancers. One particular interesting pathway that has also been shown to regulate both self-renewal of stem cell and oncogenesis in different organs is the Wnt/β-catenin signaling pathway [
26‐
29]. And we detected the expression of β-catenin in K562 after treatment with CD44shRNA. The results showed that inhibition of CD44 induced slightly decreased β-catenin level but dramatically increased the expression of p-β-catenin.
The relation between β-catenin and CD44 has been studied in many solid tumors such as breast cancer, prostate cancer and colon carcinoma. Sarkar
et al. found the prograstrin could up-regulate the expressions of β-catenin and CD44, and subsequently increase the proliferation
in vivo[
30]. From that study a relatively good correlation between CD44 and β-catenin expression pattern could be seen. The study of Han
et al. suggested that siRNA-mediated down-regulation of β-catenin elevated the E-cadherin expression but reduced the CD44 expressions, which inhibited the invasion and migration of colon cancer cells [
31]. Meanwhile, Wielenga
et al. found the expressions of CD44 family were overexpressed in the colorectal adenoma carcinoma, which may be regulated by β-catenin/Tcf-4 signaling pathway [
32]. But in leukemia genesis the relationship between CD44 and β-catenin was little studied. A report from Bjorklund
et al. suggested that CD44 played an important role in cell adhesion and resistance to lenalidomide in multiple myeloma, which could be mediated by β-catenin [
33]. In our study, we found in the CD44 silencing K562 cells the expressions of β-catenin were down-regulated. These different results may be form different cell functions in different cell lines. K562 cell lines are BCR-ABL positive cells, which constitutive express BCR-ABL fusion protein. And β-catenin and CD44 may play different functions in these cells. Krause
et al. found that CD44 was indispensable for induction of leukemia by BCR-ABL and was specifically required for leukemia stem cell that initiated CML [
9]. And a study of Hu
et al. suggested that β-catenin played an essential role on survival and drug-resistance of leukemia stem cell in mice with BCR-ABL-induced chronic myeloid leukemia [
6]. Meanwhile, accumulated evidence showed that there is a deregulation and cross talk among Wnt and other signaling pathway such as Notch in chronic myeloid leukemia [
34]. So, CD44 may have a cross talk with β-catenin through BCR-ABL and there may be a regulated loop between CD44 and β-catenin.
To ensure the effects of CD44 on proliferation, we down-regulated the CD44 level by shRNA and found that the proliferation of K562 cells significantly decreased compared with that of the parental cells. The inhibition of proliferation was major from the CD44 down-regulation induced a G
0/G
1 arrest in cell cycle of K562 cells. These data indicate that the effects of CD44 on cell proliferation are partially contributable to the G
0/G
1 arrest of cell cycle in K562 cells. Cell cycle progression through G
1 to S and the G
2 to M transitions are major checkpoints in the control of cells’ proliferation. Cyclins, cyclin-dependent kianses(CDK), and cyclin-dependent kinase inhibitors (CDKIs) play important roles in the above processes[
35‐
37]. Among these regulators, Cyclin D1 is indispensable in regulating the G
1 checkpoint. The expression of Cyclin D1 was usually over-expressed in some kinds of tumor cells such as the invasive breast cancer and ductal carcinoma [
38]. On the other hand, these kinase activities of Cyclin/CDK are negatively mediated by CDKIs families such p21 [
39]. But the relationship between Cyclin D1 and p21 was not simply negative. Ashrafi
et al. found that in the breast cancer of wistar albino female rats, the expressions of Cyclin D1 and p21 were all highky up-regulated [
38]. In our study, we found the down-regulation of CD44 induced the decreased Cyclin D1 expression and increased p21 expression. Sengupta
et al. found that β-catenin, CyclinD1, HoxA10 and p21 play important role in the signaling network for the apparently diverse but mutually interconnected self-renewal-associated genetic programs of CML cells and this finding was consistent with our results [
34].
Methods
Cell culture and material
We collected born marrow samples of patients in Hospital of Blood Diseases. We chose 4 acute myeloid leukaemia (AML) patients, 4 chronic myeloid leukemia (CML) patients, 4 acute lymphoblastic leukemia (ALL) patients, 3 myeloproliferative neoplasm (MPN) patients, 3 polycythemia vera (PV) patients, 2 essential thrombocythemia (ET) patients and 2 healthy volunteers. All patients are newly diagnosed. In all CML patients, three patients were in chronic phase and one patient was in accelerated phase. And all AML patients were diagnosed as AML-M3. Inclusion criteria for our study were based on the European Leukemia Net (ELN) criteria. Clinical evaluation of patients was performed with physical examination and laboratory monitoring. All the patient samples were treated in accordance with the Helsinki Declaration. Before the start of treatment, each patient gave written informed consent.
K562 cells were grown in RPMI 1640 (Gibco-BRL Life Technologies, Inc. Burlington, ON, Canada) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humid atmosphere with 5% CO2.
RNA interference studies, real-time quantitative PCR and Western blotting
These analyses were performed as described previously [
40]. Trizol kit (Invitrogen, Grand Island, NY) was used to isolate total RNA and the concentration of total RNA was measured by spectrophotometer. Real-time quantitative PCR (see Table
1 for primers) was performed with ABI 7500 system Instrument with SYBR Green PCR kit (Takara, Japan).
GAPDH | 5′-GAAGGTGAAGGTCGGAGTC-3′ | 5′-GAAGATGGTGATGGGATTTC-3′ |
CD44 | 5′-ACCCCAACTCCATCTGTG C-3′ | 5′-TTCTGGACATAGCGGGTG-3′ |
p21 | 5′-CCCGTGAGCGATGGAACTTG-3′ | 5′-TGCCTCCTCCCAACTCATC-3′ |
Cyclin D1 | 5′-GCGGAGGAGAACAAACAGAT-3′ | 5′-TGAGGCGGTAGTAGGACAGG-3′ |
β-catenin | 5′-CATCATCGTGAGGGCTTACTG-3′ | 5′-TGAAGGCAGTCTGTCGTAATAG-3′ |
The total proteins of K562 cells were extracted and separated by SDS-PAGE. And the protein bands were detected using ECL Western blotting detection kit (GE healthcare, UK). For western blotting analysis, we purchased antibodies GAPDH from Santa Cruz Biotechnology (Santa Cruz, CA); Cyclin D1, p21, Bcl-xl from Cell Signaling Technology (Cell Signaling Technology, USA); CD44 antibody from R&D systems.
Cell proliferation assay
To assess the proliferation state of K562 cells after various treatments, MTT proliferation assay was performed according to the manufacturer’s instructions. Briefly, K562 cells were seed in 96-well plates at a density of 4 × 104 cells/ml for 1–3 days. A volume of 20 μl MTT labeling reagent (5 mg/ml) was added every day to each well and the plates were incubated at 37°C for 4 h. The resulting formazon crystals were solubilized by adding 100 ml of solubilization buffer (10% SDS in 0.01 m HCl) per well and the plates were incubated at 37°C overnight. The absorbance of the formazon measured at 575 nm was used to account for the proliferation state of cells. Trypan blue cell exclusion was also used to assess the cell viability and the cell number.
Flow cytometry
K562 cell surface staining, cell cycle, and apoptosis analysis was performed by flow cytometry with a BD LSRII flow cytometer.
For cell surface staining analysis, K562 Cells were pre-incubated with CD44 primary antibody for 1 h at 4°C. After three washes, the K562 cells were incubated with cy5-conjugated Goat Anti-Mouse IgG secondary antibody at room temperature. After analysis, the experiment was analyzed with software of Flowjo 7.6.
For cell cycle analysis, K562 cells in exponential growth phase were permeated with 75% ethanol for overnight at 4°C and stained with Propidium Iodide (PI) in the presence of 5 μg/ml RNase (Sigma) for 10 min. And cell cycle distribution (G0-G1, S and G2-M) was analyzed with DNA cell cycle analysis software (ModFit, Becton Dickinson).
Apoptosis was measured with a commercial kit (Tianjin Sungene Biotech, China) as recommended by the manufacturer. Approximately, 105 K562 cells were stained for 15 minutes with Annexin V- allophycocyanin (APC) and PI at room temperature in the dark. After analysis, the apoptotic K562 cells were analyzed with software of Flowjo 7.6.
K562 cells were harvested and washed three times with IMDM. The cell suspension (2 × 103 cells/ml) were cultured in semisolid methylcellulose medium (H4100, Stem Cell Technologies, Canada) supplemented with 10% FBS. Cells were incubated at 37°C with 5% CO2, and the total number of colonies was counted after 10 days by use of an inverted microscope.
Immunofluorescence assay
K562 cells were fixed with 4% paraformaldehyde for 30 min. K562 Cells were washed with ice-cold PBS, blocked with 0.5% BSA in PBS for 30 minutes and then pre-incubated with CD44 primary antibody overnight at 4°C. After three washes, the K562 cells were incubated with cy3-conjugated AffiniPure Goat Anti-Mouse IgG secondary antibody at room temperature and stained nuclei with 1 μg/μl DAPI. Then K562 cells were washed twice by PBS and the images were visualized with Bio-Rad 1024 confocal laser microscope.
Inoculation of nude mice
All animal experiments were performed in compliance with the guidelines of Laboratory Animal Care of National Institutes of Health for the care and use of laboratory animals. CD44 shRNA stable transfectants and its parental K562 cells were tested for their tumorigenic potential in vivo using nude mice. Five 4-week-old male BALB/C-nu/nu mice were included in each group. In subcutaneous models, 3 × 106 cells suspended in 0.1 ml PBS were injected into the right flank of each mouse at a single site. Tumor length and width were measured every week after injection. Volume was calculated as πLW2/6. All mice were kept in aseptic cages and killed 4 weeks after inoculation by cervical dislocation.
Statistical analysis
Each experiment was repeated at least three times. All data were summarized and represented as mean ± SD. The difference between means was statistically analyzed using the t-test. All statistical analyses were performed using GraphPad Prism software (San Diego, USA). p < 0.05 was considered as statistically significant.
Competing interests
We state here none of our authors has financial or other competing interest that might be construed as influencing the results or interpretation of our study.
Authors’ contributions
GQC, QHL and TXP provided the experimental design; GQC and LM provided the experiments; GQC, HJZ, JW, YJZ, HX, CJW and HRZ analyzed the interpretation of data; GQC wrote the article and all authors gave final approval of manuscript submitted.