Contributed equally
Although the introduction of tyrosine kinase inhibitors greatly improved the survival of patients with chronic myeloid leukemia (CML), drug resistance remains a problem. Thus, mechanism-based novel therapeutic targets warrant exploration. Recently, epidermal growth factor receptor kinase substrate 8 (EPS8), which has been identified as an oncogene and plays an important role in a broad spectrum of solid tumours, was reported to be related to poor prognosis or chemoresistance in acute leukemia patients. However, its role in CML remains unclear. In the present study, using q-RT-PCR, we demonstrated that CML patients expressed a higher level of EPS8 mRNA in bone marrow mononuclear cells than healthy controls. Then, to determine the effect of EPS8 on the biological functions of CML cells, EPS8 expression was knocked down in the human CML cell line K562. Reduced proliferation, increased apoptosis, impaired adhesion and migration were observed in K562 cells after EPS8 silencing. Notably, attenuation of EPS8 increased chemosensitivity both in imatinib-sensitive K562 cells and in the imatinib-resistant murine BCR-ABL+ 32D-p210BCR/ABL-T315I cells. Mechanistically, knockdown of EPS8 downregulated p-BCR/ABL and its downstream AKT/mTOR signalling pathway. Finally, knockdown of EPS8 attenuated K562 cell proliferation in BALB/c nude mice. These data indicated that EPS8 regulated the proliferation, apoptosis and chemosensitivity in BCR-ABL positive cells via the BCR-ABL/PI3K/AKT/mTOR pathway. Targeting EPS8 alone or combined with a tyrosine kinase inhibitor may be a promising alternative therapeutic strategy.
Chronic myeloid leukemia (CML) is a BCR-ABL oncogene-driven malignant disease, characterized by markedly elevated immature myeloid cells in bone marrow and peripheral blood. CML cells progress slowly in the chronic phase (CP) for about one to three years, and then proliferate more rapidly stepping into the accelerated phase (AP) with an increase in blast numbers. The accelerated phase lasts for only several months and eventually converts to acute leukemia in the blast crisis phase (BC) with more aggressive characteristics than
Epidermal growth factor receptor kinase substrate 8 (EPS8) is a cytoplasmic protein that acts as a substrate of receptor and non-receptor tyrosine kinases such as EGFR, FGFR, VEGFR and Src kinase. EPS8 functionally serves as an adaptor protein associating with diverse partner proteins to form complexes that regulate multiple signalling pathways. Physiologically, EPS8 forms a complex with Abi-1 and SOS-1 to regulate the Rac signalling pathway promoting cytoskeletal remodelling. EPS8 also plays a role in membrane flow, pseudopodium formation, morphogenesis of microvilli, stereocilia function and length, cellular adhesion and motility (
In the present study we performed q-RT-PCR to demonstrate that CML patients expressed higher EPS8 mRNA than healthy controls in bone marrow mononuclear cells. Then, we knocked down the expression of EPS8 in the CML cell line K562 and observed that attenuated EPS8 reduced proliferation, increased apoptosis, arrested the cell cycle at the G1 phase and reduced adhesion and migration. Notably, silencing EPS8 increased chemosensitivity both in the imatinib sensitive cell line K562 and the resistant cell line 32D-p210BCR/ABL-T315I. Mechanistically, knockdown of EPS8 downregulated p-BCR/ABL and its downstream AKT/mTOR signalling pathway. Notably, knockdown of EPS8 attenuated K562 cell proliferation in BALB/c nude mice. Collectively, these data revealed that EPS8 regulated the cell biology of CML. Targeting EPS8 alone or combined with TKI may be promising therapeutic strategies for refractory and relapsed CML patients.
Bone marrow mononuclear cells were collected from patients with CML at the Department of Hematology of Zhujiang Hospital, Southern Medical University from 2013 to 2015. Some of the RNA samples were purchased from KingMed Diagnostics (Guangzhou, China). In total, 113 cases of CML (male n=60, female n=53) including 50 cases of chronic phase (CP), 21 cases of accelerated phase (AP) and 21 cases of blast crisis phase (BC) as well as 21 CML patients in complete remission (CR) and 21 normal control were enrolled. In these cases 82 CML patients had clinical data of their quantitative BCR-ABL-p210 level presented as the percentage of p210 to Abl as assessed by qRT-PCR and blast percentage in bone marrow. All the patients had signed informed consents. The study was approved by the Ethics Committee of Zhujiang Hospital, Southern Medical University.
The human K562, KBM5, MEG01 cell lines were purchased from Jennio Biotech Co. (Guangzhou, China) and maintained in the laboratory of the Department of Hematology, Zhujiang Hospital, Southern Medical University. The murine 32D-p210-T315I and 32D-p210-WT myeloid precursor cell lines were kindly provided as gifts by Professor Lin Qiu from the Chinese Academy of Medical Sciences. The cells were cultured in RPMI-1640 containing 10% fetal calf serum (FCS) at 37°C and 5% v/v CO2.
A shRNA targeting EPS8 (CCCTATTGAATAAGGAC) or a scrambled shRNA was inserted into the pLVX lentiviral vector carrying the puromycin resistance gene to construct the transfer vector pLVX-shRNA-EPS8-puro. Then, the pLVX-shRNA-EPS8-puro vector and the control pLVX-GFP-puro vector were transfected into 293T cells, along with the packaging vector to envelop the lentivirus. The K562 cells were transfected with the lentivirus carrying the pLVX-shRNA-EPS8-puro vector or control vector, followed by puromycin selection for ~3 weeks to establish the stably transfected K562-shRNA-EPS8 and K562-GFP cell lines. Similarly, two efficient shRNA sequences targeting EPS8 and a scrambled shRNA were synthesized to establish the stably transfected 32D-p210-T315I-shRNA1-EPS8, T315I-shRNA2-EPS8 and T315I-scramble cell lines, respectively.
Total RNA was prepared using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). RNase-free DNase I (Promega, Madison, WI, USA) was used to remove the genomic DNA. Reverse transcription was performed with the M-MLV Reverse transcriptase cDNA synthesis kit (Promega). Quantitative real-time PCR was performed with SYBR Green qPCR SuperMix (Invitrogen) on an ABI PRISM 7500 Sequence Detection System according to the standard SYBR-Green PCR protocol. 18S rRNA was used as the internal control. Triplicate reactions were performed as follows: 40 cycles of a two-step PCR (95°C for 15 sec, 60°C for 32 sec) after initial denaturation (95°C for 2 min). The data are presented as the fold change in expression, as determined with the 2−ΔΔCt method (
Cells (1×104 cells/well) were plated in 96-well plates in quadruplicate and cultured in 10% FCS-containing medium with or without the indicated concentrations of drugs. At 0, 20, 44 and 68 h, 10 µl of CCK-8 solution was added to the cells, which were then incubated for an additional 4 h. Then, the OD450 values were obtained using a microplate reader.
Cells (5×105) were collected, and 1.24 µl of Annexin V-APC was added to the cells. The cells were incubated at room temperature for 15 min, centrifuged at 1,000 × g for 5 min, and the supernatant was discarded. The cells were then resuspended in 0.5 ml of cold 1X binding buffer. Ten microliters of 7-ADD or propidium iodide (PI) was added on ice, and the cells were analyzed by flow cytometry. The percentage of apoptotic cells (Annexin V+) was quantified.
Cells (1×106) were collected and washed twice with cold PBS. Then, cold 70% ethanol was added to the cells, which were incubated overnight at 4°C. The following day, the cells were washed once with PBS, and 500 µl of PBS containing 50 µl/ml PI, 100 µg/ml RNase A, and 0.2% Triton X-100 were added, and the cells were incubated for 30 min at 4°C in the dark. DNA content was detected by flow cytometry.
The 96-well plates were coated with fibronectin (30 mg/l) and dried overnight. PBS (20 µl) containing 3% BSA was added to the 96-well plate at 37°C for 2 h, and then removed. Two hundred microliters of cells (0.5×106 cells/ml) suspended in RPMI-1640 and 10% FBS were added to the 96-well plates and incubated for 1.5 h; the plate was then washed gently. The cells that adhered to the bottom of the 96-well plate were observed under a microscope, and the number of adherent cells was assessed using the CCK-8 assay.
The cell migration assay was performed as previously described. Briefly, the cells were suspended in serum-free RPMI-1640 containing 0.1% BSA at a concentration of 1×106 cells/ml. A suspension of 0.1 ml of cells was added to the upper chamber, and the cells were allowed to migrate to the lower chamber, which contained RPMI-1640 and 10% FBS, for 6–8 h at 37°C in a 5% CO2 incubator. The cells that had migrated to the lower chamber were observed under a microscope. The number of cells in the lower chamber was assessed using the CCK-8 assay.
The cells (1×104 cells/100 µl/well) were plated onto 96-well culture plates in quadruplicate in the presence of different concentrations of either daunorubicin or imatinib for the indicated time-points. Cell viability was assayed by adding CCK-8 solution. The percentage of cell viability was evaluated by assessing the absorbance at 450 nm and normalized to the corresponding untreated control.
Total protein lysates were separated on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membranes. The blots were subjected to a standard immunodetection procedure using specific antibodies. The signals were enhanced with a chemiluminescence substrate and detected using an image analyser. The primary antibodies used were as follows: ERK (rabbit, polyclonal, 1:1,000; cat. no. ab196883), p-ERK (rabbit, polyclonal, 1:1,000; cat. no. ab214362) (both from Abcam, Cambridge, UK); STAT5 (rabbit, monoclonal, 1:1,000; cat. no. 94205), p-STAT5 (rabbit, polyclonal, 1:1,000; cat. no. 9351), Bcl-2 (rabbit, polyclonal, 1:1,000; cat. no. 2872) (all from CST, Danvers, MA, USA); Mcl-1 (rabbit, polyclonal, 1:1,000; cat. no. 16225-1-AP; Proteintech, Rosemont, IL, USA); PTEN (rabbit, monoclonal, 1:1,000; cat. no. ab32199), EPS8 (rabbit, polyclonal, 1:1,000; cat. no. ab96144) (both from Abcam); phospho-BCR/ABL (rabbit, polyclonal, 1:500; cat no. 3901), BCR/ABL (rabbit, polyclonal, 1:1,000; cat. no. 3902), β-actin (mouse, monoclonal, 1:1,000; cat. no. 3700), mTOR (rabbit, polyclonal, 1:1,000; cat. no. 2972), phospho-mTOR-s2448 (rabbit, polyclonal, 1:1,000; cat. no. 2971), Akt (rabbit, polyclonal, 1:1,000; cat. no. 9272), phospho-Akt-t308 (rabbit, polyclonal, 1:1,000; cat. no. 5110), phospho-Akt-s473 (rabbit, monoclonal, 1:1,000; cat. no. 4060), GSK3β (rabbit, monoclonal, 1:1,000, cat. no. 5676), phospho-GSK3β-s9 (rabbit, monoclonal, 1:1,000; cat. no. 9322) (all from CST); 4EBP1 (mouse, polyclonal, 1:1,000; cat. no. 60246-1-Ig; Proteintech); phospho-4EBP1-T37/46 (rabbit, polyclonal, 1:1,000; cat. no. 9459; CST); and GAPDH (mouse, monoclonal, 1:1,000; cat. no. KC-5G4; KangChen Bio-tech).
BALB/c nu/nu mice were purchased from Southern Medical University. Nude mice, 6–8 weeks, were implanted subcutaneously with 1×107 K562-scramble or K562-shRNA-EPS8 cells, irrespectively. Tumor volume was assessed every 3 days. Mice were sacrificed on day 24 after inoculation and the subcutaneous tumors were weighed. All the procedures were performed according to an approved Southern Medical University, Animal Care and Use Committee protocol.
The data are reported as the mean ± standard deviations (SDs). One-way ANOVA followed by the Student-Newman-Keuls' pairwise multiple comparison test were used to compare the EPS8 mRNA expression among CML patients at different phases with healthy donors, as well as differences obtained in the proliferation, apoptosis, cell cycle, adhesion and migration assays. Spearman's rank correlation coefficient was used to analyze the correlation between EPS8 mRNA expression and clinical characteristics. A two-tailed independent Student's t-test was used to compare the means in the chemotherapy drug sensitivity assay. Differences were considered significant when P<0.05.
A published microarray data of 91 cases of CML patients reported by Radich
To evaluate the effect of EPS8 on the biological function of CML cells, we knocked down the expression of EPS8 in CML cell line K562. EPS8 expression was detected in three human CML cell lines. The K562 and KBM5 cells expressed a higher level of EPS8, while the MEG01 cells exhibited a lower expression (
To investigate the role of EPS8 in CML cells, we assessed the proliferation, apoptosis and cell cycle after EPS8 silencing. We first examined the proliferation of K562-shRNA-EPS8 cells and determined that knockdown of EPS8 reduced the proliferation of the K562 cells (
EPS8 plays an important role in cytoskeletal remodelling and cellular motility; therefore, we evaluated the effect of EPS8 on adhesion and migration of K562 cells. The K562 cells and their derived cells were plated on fibronectin-coated 96-well plates for 1.5 h and then washed. Less K562-shRNA-EPS8 cells remained on the fibronectin-coated plates compared with the control cells (
We then investigated the effect of EPS8 on the chemosensitivity of the K562 cells. Daunorubicin is a widespread used common chemotherapy drug to treat myeloid leukemia. In the present we determined that the viability of the K562-shRNA-EPS8 cells was significantly reduced in the presence of indicated concentrations of daunorubicin indicating that EPS8 knockdown increases their chemosensitivity (
To investigate whether knockdown of EPS8 can overcome imatinib resistance, we employed a murine imatinib-resistant cell line 32D-p210T325I-BCR/ABL (32D-p210-T315I), which was generated by transfecting myeloid precursor cells with the vector carrying prototype imatinib-resistant BCR/ABL point mutation T315I in CML patients. Compared to the imatinib sensitive cell line 32D-P210BCR/ABL (32D-p210-WT) which expressed native BCR/ABL protein p210, 32D-p210-T315I cells expressed a higher level of EPS8 protein (
In accordance with these results, we determined that knockdown of EPS8 notably increased apoptosis in imatinib-treated 32D-p210-T315I cells in a dose-dependent manner. Apoptosis was markedly increased in EPS8-shRNA cells compared with the control groups after imatinib treatment for 24 h (
To determine the molecular mechanism through which EPS8 regulates CML cells, key molecules in main signalling pathways involved in leukemogenesis were assessed. Notably, phosphorylated (p)-BCR-ABL was significantly decreased in EPS8-knockdown K562 cells compared with the control group, suggesting that EPS8 regulated BCR-ABL. Furthermore, our results illustrated that p-AKT and phosphorylation of its downstream proteins mTOR, eukaryotic translation initiation factor 4E binding protein 1 (EIF4EBP1) and glycogen synthase kinase 3β (GSK3β), as well as, p-STAT5 and p-ERK levels were reduced in K562-shRNA-EPS8 cells compared with the control cells. Since Mcl-1 and Bcl-2 function as anti-apoptotic markers in various cancers, we explored whether their expression was affected by EPS8. Indeed, we found that the expression of Mcl-1 and Bcl-2 was suppressed by EPS8 silencing. These data revealed that EPS8 inhibition-induced apoptosis was related with the reduction of anti-apoptotic factors and inhibition of proliferation and anti-apoptotic pathway activity (
Given that EPS8 regulated the anti-apoptotic signalling and increased imatinib-induced apoptosis in imatinib-resistant 32D-p210-T315I cells, we next investigated the effect of EPS8 on BCR-ABL expression and its downstream PI3K/Akt signalling pathway in 32D-p210-T315I cells and their derived cells. In accordance with K562-shRNA-EPS8 cells, 32D-p210-T315I-shRNA-EPS8 cells exhibited decreased total BCR-ABL and p-BCR-ABL proteins. Furthermore, EPS8 silencing led to a decrease of p-Akt (T308, S476), p-4EBP1 (T37/46), p-mTOR (S2448) and p-GSK3-β (
Finally, we determined the efficacy of the inhibition of EPS8 on the proliferation of BCR-ABL positive cells
EPS8 is a cytoplasmic non-receptor kinase and is well recognized as an oncogene in a variety of solid tumours (
To determine the role in CML cells, we knocked down the expression of EPS8 in CML cell line K562 and determined that silencing of EPS8 led to reduced proliferation, increased apoptosis and cell cycle arrest in the G1 phase which was consistent with other studies on EPS8. Xu
One of the characteristics of leukemia cells is invasion to other organs. Cytoskeletal proteins have an important role in AML cell migration (
Gorsic
To explore the molecular mechanism involved in the EPS8-mediated effect on the response of CML cells to imatinib, we detected BCR-ABL and its downstream proteins in imatinib-sensitive cell line K562 and imatinib-resistant cell line 32D-p210-T315I and their derivatives. EPS8 is a critical signaling molecule and integrates multiple pathways. EPS8 has been reported to regulate PI3K/AKT/mTOR (
In conclusion, our data revealed that EPS8 regulated multiple biological functions such as proliferation, apoptosis, the cell cycle, drug sensitivity of CML cells possibly by mediating the regulation of the BCR-ABL/AKT/mTOR signalling pathway. Strategies that target EPS8 in CML patients may help to overcome resistance to tyrosine kinase inhibitors. The EPS8 inhibitor alone or combined with a tyrosine kinase inhibitor may be promising customized strategies to treat refractory and resistant CML patients.
The present study was supported by the Major Program for Health Medical Collaborative Innovation of Guangzhou (grant no. 201704020216), the Startup Project for Clinical Trials of Southern Medical University (grant no. LC2016ZD027), the Special Project for the Development of Science and Technology of Guangdong Province (grant no. 2016A020213005), the Competitive Allocation Project of the Comprehensive Strategic Cooperation of the Chinese Academy of Sciences of Guangdong Province (grant no. 2013B091500072), the National Science Foundation of China (no. 81372249 and 81300431) and the Youth Scientific Fund of Southern Medical University (no. PY2014N072).
EPS8 mRNA expression is elevated in CML patients. (A) Bone marrow mononuclear cells were collected from 113 cases of CML patients and 21 normal controls. qRT-PCR was performed to assess the expression of EPS8 mRNA. The ratio of EPS8 and 18srRNA was expressed using the 2−ΔΔCt method. The relative expression of EPS8 mRNA was compared among CP (n=50), AP (n=21), BC (n=21) of CML and healthy controls (n=21). (B and C) Correlation analysis was performed between EPS8 mRNA expression with (B) the BCR-ABL transcript or (C) the blast percentage in bone marrow. *P<0.05 vs. the healthy control; **P<0.01 vs. the healthy control. CML, chronic myeloid leukemia; CP, chronic phase; AP, accelerated phase; BC, blast crisis phase; ns, non-significant.
Effect of EPS8 on proliferation, apoptosis and the cell cycle of K562 cells. (A) The expression of EPS8 was revealed in human CML cell lines using western blotting. (B) The expression levels of the EPS8 protein in the K562-shRNA-EPS8 and the control cells were analysed by western blotting. (C) Cells were plated in 96-well plates and the OD values were assessed at indicated time-points using the CCK-8 assay. (D and E) The K562 cells and their derivatives were stained with Annexin V-APC and 7-AAD, and apoptosis was assessed by flow cytometry. (F) The K562 cells and their derivatives were stained with PI, and then the cell cycle was analysed by flow cytometry. Plots are representative of 3 independent experiments. *P<0.05 vs. the control. CML, chronic myeloid leukemia.
EPS8 increases the adhesion and migration of leukemia cells. (A and B) K562 cells and their derivatives (1×105 cells/well) were suspended in RPMI-1640 with 10% FBS and then added to a fibronectin-coated 96-well plate, incubated for 1.5 h and washed gently. The cells that adhered to the bottom of the plate were observed under a microscope. (A) The representative plots of the indicated strains under the microscope. (B) The number of adhered cells was assessed using the CCK-8 assay. (C and D) Transwell chambers were used to assess migration. Cells ~1×105 were placed on the upper layer of a cell permeable membrane in serum-free RPMI-1640 containing 0.1% bovine serum albumin and allowed to migrate to the lower chamber, which contained RPMI-1640 and 10% FBS, for 6–8 h at 37°C. The cells that had migrated to the lower chamber were observed under a microscope. (C) The representative plots of the indicated strains under the microscope. (D) The number of migrating cells was calculated using the CCK-8 assay. Plots are representative of 3 independent experiments. **P<0.01 vs. the control; ***P<0.001 vs. the control.
Knockdown of EPS8 increases sensitivity to chemotherapy. K562 cells and their derivatives (1×104 cells/100 µl/well) were added to 96-well culture plates and treated with the indicated concentrations of (A) daunorubicin and (B) imatinib for 48 h. Viability was assessed with the CCK-8 assay. (C) EPS8 expression in 32D-p210-WT and 32D-p210-T315I cells were assessed by western blotting. LV-EPS8-shRNA1 and LV-EPS8-shRNA2 vectors were transfected into 32D-p210-T315I cells, respectively. EPS8 expression was revealed in 32D-p210-T315I-shRNA1-EPS8, T315I-shRNA2-EPS8, and the control cell lines. (D and E) 32D-p210-T315I cells and their derived cells (1×104 cells/100 µl/well) were added to 96-well culture plates and treated with the indicated concentrations of imatinib for (D) 24 h and (E) 48 h, respectively. Viability was assessed with the CCK-8 assay. Plots are representative of 3 independent experiments. ***P<0.001 vs. the scramble control.
EPS8 knockdown increases imatinib induced apoptosis of 32D-p210-T315I cells. (A) The 32D-p210-T315I cells and their derived cells were stained with Annexin V-APC and 7-AAD, and apoptosis was assessed by flow cytometry. (B) Statistical analysis of the percentage of apoptotic cells in each group. Plots are representative of 3 independent experiments. ***P<0.001 vs. the scramble control.
Silencing of EPS8 downregulates the protein levels of BCR-ABL and its downstream Akt/mTOR signaling pathway. (A) K562, K562-scramble and K562-shRNA-EPS8 cells were lysed. Indicated proteins were blotted. GAPDH served as a loading control. (B) 32D-p210T315I-shRNA1-EPS8, T315I-shRNA2-EPS8, T315I-scramble and T315I cells were lysed. Indicated proteins were blotted. β-actin was used as a loading control. (C) A schematic diagram depicting the possible EPS8 regulation pathway in BCR-ABL positive cells. The solid arrow represents regulation, the dash arrow represents indirect regulation.
Knockdown of EPS8 attenuated the proliferation of K562 cells
Correlation analysis between EPS8 expression and the clinical features of CML patients.
Clinical features | Eps8 expression (mean rank) | P-value |
---|---|---|
Male (n=60) | 49.83 | 0.74 |
Female (n=53) | 48.99 | |
Correlation coefficient (Spearmans r) | ||
Age (years) | 0.019 | 0.85 |
p210 | 0.003 | 0.98 |
Blast percentage | 0.246 | 0.03 |
EPS8, epidermal growth factor receptor kinase substrate 8; CML, chronic myeloid leukemia.