Background
Acute myeloid leukemia (AML), a rapidly progressing hematopoietic malignancy, is the most common form of acute leukemia in adults [
1]. Nowadays, extensive use of standard therapy induces complete remission (CR) in approximately 50 to 70% of AML patients, but about 76% of patients relapse or die eventually [
2‐
5], indicating that drug resistance has become a major obstacle to an optimistic prognosis. Therefore, it is of vital importance to explore the molecular rationale underlying drug resistance in AML.
Apoptosis induction underlies the therapeutic effects of most conventional antineoplastic agents. However, an expanding body of evidence has illustrated that dysregulated expression of anti-apoptotic molecules, such as BCL-2 and MCL-1, renders malignant cells resistant to the pro-apoptotic effects of cytotoxic agents [
6‐
8]. Inhibition of these molecules has increased chemosensitivity in AML, underlining the crucial role of apoptosis dysregulation in AML chemoresistance [
9‐
11]. Nevertheless, resistance to the currently available targeted inhibitors has also emerged afterwards, suggesting inter-complementary effects among anti-apoptotic molecules [
12‐
16]. Thus there arise demands for further exploration of new candidates in apoptosis regulation network in AML.
Tumor necrosis factor α-induced protein 8 (TNFAIP8), also called SCC-S2, GG2-1, MDC-3.13, NDED and OXI-α, containing a death effector domain, was first found in human head and neck squamous cell carcinoma [
17]. Its aberrant expression has been successively validated in cancers of breast, esophagus, lung, ovary, stomach and others [
17‐
25]. Subsequent research provided insight into the regulatory function of TNFAIP8 in cell apoptotic network. TNFAIP8 is capable of suppressing apoptosis by inhibiting caspase activation, thus promoting cisplatin resistance in cervical carcinoma [
24]. In addition, TNFAIP8 has been reported to promote p53 ubiquitination and decrease p53-dependent pro-apoptotic responses, promoting drug resistance to cisplatin and doxorubicin of NSCLC cells, respectively [
21,
23]. Moreover, TNFAIP8 has been found to be highly expressed in AML and acute lymphoblastic leukemia cell lines [
18]. However, detailed roles and mechanisms of TNFAIP8 in AML remain unclear.
In this study, we sought to investigate the role and molecular basis of TNFAIP8 in AML chemoresistance. We found that TNFAIP8 reduced cell apoptosis and increased chemoresistance in vitro. Mechanistically, we identified that ELF1 served as a positive regulator of TNFAIP8 transcription. And TNFAIP8 was found to enhance activity of ERK signaling pathway through interaction with Rac1, contributing to its anti-apoptotic effects on AML cells. Finally, by using a murine leukemia model, we found mice bearing murine AML cells with TNFAIP8 inhibition showed improved survival. Collectively, our data demonstrate that TNFAIP8 promotes chemoresistance and progression in AML and that targeting TNFAIP8 may be a promising strategy for AML treatment.
Methods
Patient samples
Bone marrow (BM) samples from 60 AML patients and 17 control donors were obtained at the Qilu Hospital of Shandong University, China. Samples were collected from AML patients at different stages of the disease, including patients with newly diagnosed AML (n = 28), patients with relapsed/refractory AML (n = 13) and patients with complete remission (n = 19). Control samples were obtained from donors without any malignant bone marrow disorder. Informed consent was obtained in accordance with the Declaration of Helsinki. All laboratory experiments with primary samples were approved by the Medical Ethics Committee of Qilu Hospital of Shandong University.
Cell lines and cell culture
Human leukemic cell lines THP1, U937, K562, K562/A02, K562/G01, HL60, HL60/ADR cells and human embryonic kidney 293 T cells were purchased from the Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China. Murine leukemic cell line C1498 was purchased from ATCC. THP1, U937, K562, K562/A02, K562/G01 cells were cultured in RPMI 1640 medium, HL60 and HL60/ADR cells were cultured in IMDM medium, 293 T cells were cultured in DMEM medium (with 10% heat-inactivated fetal calf serum, Gibco; penicillin and streptomycin, Invitrogen; 37 °C, 5% CO
2, in humidified incubator). Doxorubicin was added (final concentration of 0.5 μg/mL) to the complete culture medium of K562/A02 and HL60/ADR until 2 weeks before experiments. Multidrug resistant cell lines, HL60/ADR [
26] and K562/A02 [
27], and parental HL60 and K562 cell lines were used to investigate the effect of TNFAIP8 levels on chemoresistance in vitro. Cell line identity and purity were verified regularly by short tandem repeat profiling. The latest authentication of cell lines was conducted by Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (
July 16 to
August 23
, 2019).
Chemical inhibitors
ERK1/2 inhibitor SCH772984 (MedChemExpress) was dissolved in DMSO (2.5 μM in culture). Rac1 inhibitor EHOP-016 trihydrochloride (Selleck) was dissolved in DMSO (5 μM in culture).
Lentiviral transduction
Lentiviral constructs repressing TNFAIP8, expressing TNFAIP8 with Flag-tagged or expressing ELF1 were purchased from Genechem (Shanghai, China). Those repressing mouse TNFAIP8 were also purchased from Genechem (Shanghai, China) and were used to establish C1498 cell line constitutively repressing TNFAIP8 (Table
1). Cells were infected with lentivirus for 24 h and selected by puromycin.
Table 1
Target sequence for TNFAIP8 shRNAs
TNFAIP8 (homo) shRNA | TTGGATGAAGAGAACATAT |
Scrambled (homo) control | TTCTCCGAACGTGTCACGT |
Tnfaip8 (mus) shRNA1 | GCTGCCTTGTACAATCCCTTT |
Tnfaip8 (mus) shRNA2 | CCACAGGAACAATCAGTTCAA |
Scrambled (mus) control | TTCTCCGAACGTGTCACGT |
Quantitative reverse transcription PCR (RT-qPCR)
Total RNA was extracted using TRIzol (Invitrogen). Reverse transcription was performed with a M-MLV RTase cDNA Synthesis Kit (Takara, Japan). RT-qPCR was performed by an Applied Biosystem 7900HT System (ABI) with appropriate primers (Table
2), SYBR Green PCR Master Mix (Toyobo, Japan), and GAPDH or ACTB as internal controls. Each sample was amplified in a 10 μL reaction volume according to manufacturer’s instructions.
TNFAIP8 (homo)-Forward | GCCGTTCAGGCACAAAAGA |
TNFAIP8 (homo)-Reverse | GCACCTCACTACTTGTGTCGTCTATT |
ELF1 (homo)-Forward | AGAGTCTTCAGATCCATCGCTA |
ELF1 (homo)-Reverse | GGTTTTGCAGCTTTAGAATTCCC |
GAPDH (homo)-Forward | GGAGCGAGATCCCTCCAAAAT |
GAPDH (homo)-Reverse | GGCTGTTGTCATACTTCTCATGG |
Tnfaip8 (mus)-Forward | GGTATCCAAATCCATCGCCACCA |
Tnfaip8 (mus)-Reverse | CCAGCTCGTCTTGATTGAACTGA |
ACTB (mus)-Forward | TACTGAGCTGCGTTTTACACC |
ACTB (mus)-Reverse | TCCTGAGTCAAAAGCGCCAA |
Western blot
Cells were lysed in a protein solubilization buffer. Protein extracts were prepared with a Total Protein Extraction Kit according to the manufacturer’s instructions (BestBio, Shanghai, China). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Millipore). β-actin or GAPDH served as a loading control. Primary antibodies included anti-GAPDH, anti-β-actin (ZSGB-BIO, China), anti-TNFAIP8, anti-Rac1 (Proteintech), anti-Flag (Sigma), as well as anti-ERK1/2, anti-p-ERK1/2, anti-MEK, and anti-p-MEK (CST). Protein bands were visualized using a FluorChem E Chemiluminescent Western Blot Imaging System (Cell Biosciences).
Proliferation and IC50
To assess proliferation, cells were plated in 96-well plates and cultured in an incubator at 37 °C. At each time point for the next 3 days, 10 μL CCK-8 (BestBio, Shanghai, China) was added to each well, then cells were incubated for 4 h. Absorbance (450 nm) was measured by a Microplate Reader (Thermo Scientific).
To measure half maximal inhibitory concentration (IC50), cells were exposed to serial dilutions of doxorubicin (ADM), cytarabine (Ara-C) or idarubicin (IDA) (Sigma) for 48 h. Cell viability was determined by CCK-8 assays and IC50 values were calculated.
Apoptosis
Cells were treated with doxorubicin, cytarabine or idarubicin for 48 h, stained with Annexin V/PI (BestBio, Shanghai, China) or Annexin V/7-AAD (BestBio, Shanghai, China), and apoptosis was analyzed by flow cytometry (Beckman Coulter).
Luciferase assay
Twenty-four hours before transfection, cells were plated in 24-well dishes at 5 × 104 cells/well. Transfections included a constant amount of Renilla luciferase plasmid for internal control. Fourty-eight hours after transfection, cells were incubated with passive lysis buffer (15 min, 160 μL/well, Promega), then 25 μL of each lysate was subjected to a dual luciferase assay (Promega). Luciferase activity was measured using a luminometer (LB 96v, Berthold). Results of triplicate transfections were normalized to Renilla luciferase activity.
Chromatin immunoprecipitation (ChIP)
The SimpleChIP® Enzymatic Chromatin IP Kit (Cell Signaling Technology) was used to perform ChIP assays according to the manufacturer’s protocol. Chromatin fragments derived from K562, K562/A02, HL60 and HL60/ADR cells were immunoprecipitated with 5 μg ELF1 antibody (Proteintech). The 5′-upstream region of human TNFAIP8 gene (− 1154 to − 1142, TNFAIP8-promoter) was obtained by PCR of genomic DNA using the following primers: forward 5′- TTCTTCCAAACCCAGCTCAGAC - 3′; reverse 5′- AAACATACACAAGGTACGGAGG - 3′.
Co-immunoprecipitation (CoIP)
Total protein was incubated with anti-Flag or anti IgG antibodies (16 h, 4 °C, rotation), then protein A/G PLUS-agarose beads were added (20 μL, 1 h, 4 °C, rotation; Santa Cruz Biotechnology). Captured agarose beads-Ab-Ag complexes were washed (five times, PBS) and detected by western blot.
P21-activated kinase (PAK) pull-down
K562, HL60, K562/A02 and HL60/ADR cells were serum-starved (16 h), then treated with doxorubicin (4 h, 0.5 μg/mL for K562 and HL60, 5 μg/mL for K562/A02 and HL60/ADR). A Rac1 activation assay biochem kit (Cytoskeleton) was used to perform PAK pull-down assays according to the manufacturer’s protocol.
In vivo model of AML
Animal studies were conducted in compliance with institutional guidelines and were approved by the Medical Ethics Committee of Qilu Hospital of Shandong University. Female C57BL/6 mice (20; 8-week-old; Jinan pengyue laboratory animal breeding co., Ltd., Jinan, China) were intravenously injected with C1498 cells (2 × 105 cells/100 μL) transduced with a non-targeted short hairpin RNA (C1498: shNC, n = 10 mice) or TNFAIP8 shRNA (C1498: shTNFAIP8, n = 10 mice). Mice were monitored daily for evidence of leukemia.
On day 24, mice were euthanized and livers and spleens were measured. Leukemia infiltration into peripheral blood, bone marrow, spleen and liver was evaluated by flow cytometry detection of GFP-positive cells and hematoxylin and eosin staining. Liver, spleen and bone marrow tissues of mice were fixed (10% formaldehyde), paraffin-embedded, sectioned (4 μm), and stained with hematoxylin and eosin. Immunohistochemical detection of TNFAIP8 and p-ERK1/2 (CST) was performed on bone marrow samples. After heat-induced epitope retrieval, bone marrow samples were incubated with primary antibodies (4 °C, overnight), then with secondary antibody in a biotin-streptavidin HRP detection system, and finally 3,3′-diaminobenzidine was used for detection and visualization. Slides were observed by a microscope (Nikon, Ni-U). Survival was also followed and presented with a Kaplan-Meier survival plot.
Statistical analyses
Data are presented as mean ± standard deviation (SD). Differences between 2 groups were analyzed using an unpaired Student t-test. Differences between 3 or 4 groups were analyzed using one-way ANOVA or two-way ANOVA followed by LSD test with normally distributed data. The Mann-Whitney U test was used for cases with unequal variances. Survival was presented with a Kaplan-Meier survival plot. P < 0.05 was considered statistically significant.
Discussion
Evasion from apoptosis has been appreciated as one of the intrinsic mechanisms driving resistance to both traditional antineoplastic drugs and certain targeted therapies in malignant cells [
46]. Previous experimental and clinical studies have focused on BCL-2 family members in AML [
47]. However, the therapeutic values of narrowly targeting BCL-2 family in clinical settings have been compromised by occurrence of new resistance, partially due to functional compensation from non-targeted anti-apoptotic molecules [
12‐
16]. It is of vital importance to uncover novel candidates. In the current study, we characterized the role for TNFAIP8, a new anti-apoptotic molecule, in AML chemoresistance and investigated its underlying molecular basis. We showed that TNFAIP8 suppresses apoptosis and promotes chemoresistance in AML by interacting with Rac1 to activate the ERK pathway. We also demonstrated that TNFAIP8 inhibition is effective against AML both in vitro and in vivo.
We found TNFAIP8 expression is higher in resistant AML cell lines than sensitive AML cell lines. In addition, TNFAIP8 expression was increased in relapsed/refractory AML patients compared with newly-diagnosed AML patients, implicating a possible relationship between TNFAIP8 expression and chemotherapeutic response. Future investigation on the clinical prognostic values of TNFAIP8 is warranted. Interestingly, we then identified a previously undescribed role for ELF1 in transcriptional regulation of TNFAIP8. ELF1 has been implicated in transcription regulation of several tumor-promoting genes including
Tie2,
MEIS1,
CCL2, LUCAT1 [
48‐
51]. Besides, data from TCGA suggest an increased ELF1 expression and a positive correlation between ELF1 and TNFAIP8 in AML, further supporting the contribution of ELF1 to upregulated TNFAIP8 expression. Since the significance and role for ELF1 in AML remain elusive, future investigation is worthy of consideration, which may help in expanding our understanding of the dysregulated molecular networks in AML.
A previous study has demonstrated high TNFAIP8 expression in acute leukemia cell lines, whereas its exact roles in AML remain understudied [
18]. Here, we revealed the positive impact of TNFAIP8 on AML chemoresistance and found that TNFAIP8 suppression increased chemosensitivity through promoting chemotherapy-induced apoptosis in vitro. In addition, we found that mice bearing AML cells with TNFAIP8 suppression demonstrated lower leukemia infiltration and improved survival, providing in vivo experimental evidence for its therapeutic values. Together, our data suggest that TNFAIP8 might serve as a new candidate and justify investigating dual inhibition of TNFAIP8 and other anti-apoptotic molecules for mitigating AML chemoresistance in future studies.
Understanding the functional mechanism of TNFAIP8 in AML would greatly facilitate development of targeted therapy. Although the role of TNFAIP8 in negative regulation of apoptosis in malignancies have been well documented, the mechanisms by which TNFAIP8 functions vary among different cancer types and cell contexts [
17‐
25]. Our study interpreted a previously unknown link between TNFAIP8 and ERK. We found that, in AML, TNFAIP8 regulated apoptosis and proliferation accompanied by altered phosphorylation of ERK1/2 and inhibition of ERK activation partially abrogated the down-regulation on the apoptotic level by overexpressing TNFAIP8. ERK, acting as the main downstream effector in MAPK signaling pathway, plays an important role in cell proliferation and survival. Elevated ERK1/2 phosphorylation has been found in 83.3% of AML patients [
36], and inhibition of ERK1/2 induces cell cycle arrest and apoptosis in leukemic blasts [
34,
52]. Notably, ERK1/2 inhibition can increase cell sensitivity to chemotherapeutics in AML [
52,
53]. Given the significance of ERK signaling pathway in AML, our research provides novel insights that TNFAIP8 may promote AML chemoresistance by activating ERK signaling pathway. Rac1, a small GTPase, has been identified as a critical upstream mediator of the ERK pathway [
40‐
45]. We found that TNFAIP8 promoted ERK phosphorylation through modulating Rac1. Interestingly, Rac1 is overexpressed in primary AML cells, and has gained attention for its roles in AML initiation and chemoresistance [
54‐
56]. Our results shed light on the physical interaction between TNFAIP8 and Rac1. However, the mechanism underlying activation of TNFAIP8 on Rac1 remains unclear. Guanine nucleotide exchange factors (GEFs) have been shown to mediate the exchange of GDP for GTP by associating with membrane-bound Rac1, thereby activating Rac1 [
57]. Thus, there is good reason to hypothesize that TNFAIP8 serves as a platform for interaction between GEFs and Rac1. The possibility that TNFAIP8 binds other membrane proteins to facilitate the plasma membrane location of Rac1 also merits further exploration.
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