Angiomotin (AMOT) is a membrane protein that is aberrantly expressed in a variety of solid tumors. Accumulating evidence support that AMOT is involved in the pathological processes of tumor proliferation, apoptosis, and invasion. However, the potential role of AMOT in the pathogenesis of diffuse large B-cell lymphoma (DLBCL) remains elusive. In the present study, we investigated the expression level and biological function of AMOT in DLBCL. AMOT expression was significantly reduced in DLBCL biopsy section, and low AMOT expression was associated with poor clinical prognosis. Overexpression of AMOT by lentivirus in human DLBCL cells induced cell viability inhibition concomitant with an increased percentage of cells in G1 phase and decreased percentage in S phase. Moreover, AMOT upregulation increased the sensitivity of DLBCL cells to doxorubicin. Furthermore, overexpression of AMOT led to reduced activation of key kinases for the DNA damage response (DDR). The above results indicated that AMOT acts as a tumor suppressor via inhibition of the DDR, thus reducing the viability while increasing the chemosensitivity in DLBCL. In summary, AMOT may be a novel potential target for DLBCL therapeutic intervention.
Diffuse large B-cell lymphoma (DLBCL) is the most common type of highly aggressive non-Hodgkin’s lymphoma. Though the long-term survival rate of DLBCL is ~60% after immunochemotherapy with R-CHOP , 30–40% of patients will present refractory or relapsed process after an initial response to therapy [2, 3]. Gene heterogeneity accounts for different clinical outcomes , therefore, the research of individualized treatment schemes based on new molecular targets and oncogenic pathways is the main research strategy at current.
Angiomotin (AMOT) was initially identified in 2001 as a medium that binding to angiostatin and regulating angiogenesis or endothelial cell migration [5,6,7]. Human AMOT gene is located in chromosome Xq23 with a 2025-bp open reading frame. AMOT-p130 and AMOT-p80 are two classic isoforms which are nearly identical at the C-terminal, while there is an extended glutamine-rich domain at the N-terminal of AMOT-p130 . These two splicing formations have different functions in regulating polarity rearrangements related to cell shaping and migration [8,9,10]. In murine, inactivation of AMOT led to failure in cell growth and migration in the anterior visceral endoderm, causing early embryonic lethal . Consistently, knockout of AMOT in zebrafish inhibited the proliferation rate of epithelial cells [12, 13] and led to defects in endothelial migration .
At present, the role of AMOT in several solid tumors is still controversial. AMOT expression was decreased in lung cancer, breast cancer, undifferentiated pleomorphic sarcoma, ovarian serous carcinoma, and clear cell renal cell carcinoma [15,16,17,18,19], and was related to poor prognosis of these diseases. Nevertheless, in epithelial ovarian cancer, prostate cancer, liver cancer, osteosarcoma, and sinonasal tumors, AMOT was overexpressed and promoted tumor cell proliferation or invasion [10, 20,21,22,23]. AMOT participated in the regulation of several signaling pathways, including Hippo, Wnt, ERK1/2, and VEGFR-2 pathways [12, 24,25,26,27,28,29]. However, the effect of AMOT in hematopoietic malignancies has not been reported.
Increased replication pressure and alteration of the DNA damage response (DDR) are crucial features of genetic instability and are closely related to tumorigenesis . DDR senses different types of damage and initiates DNA repair, involving transcriptional activation, cell cycle, senescence, and apoptosis . The ataxia-telangiectasia mutated (ATM) and ATM and RAD3-related (ATR) protein kinases are the main regulators of the DDR program. Despite their functional similarity, ATM is activated primarily during DNA double-strand breaks, phosphorylates checkpoint kinases 1 (Chk1) at S317 and S345, while ATR primarily regulates damaged replication forks, phosphorylates checkpoint kinases 2 (Chk2) at Thr68 [32, 33]. In some cases, AMOT was considered a substrate for ATM and ATR, but this finding has not been confirmed by further experiments .
In this study, we evaluated the expression level of AMOT in DLBCL. AMOT expression was upregulated to assess its effect on cell growth with or without doxorubicin treatment. The results showed that AMOT could act as a negative regulator of DDR signaling in DLBCL. As far as we know, this is the first report to confirm the regulatory effect of AMOT on the key enzymes of DDR in DLBCL.
Materials and methods
Patients and samples
Fifty-two specimens of newly diagnosed DLBCL and twenty-five specimens of reactive lymphoid hyperplasia (RLH) were collected at Jinan Central Hospital affiliated to Shandong University. According to the WHO classification, all patients were independently diagnosed by two experienced pathologists. Peripheral venous blood was collected from three healthy volunteers (N1, N2, and N3) and mononuclear cells were isolated by Ficoll centrifugation (TBD Science, Tianjin, China). The study was approved by the Medical Ethics Committee of Jinan Central Hospital affiliated to Shandong University. In accordance with the Declaration of Helsinki, the informed consent was obtained before all samples were collected.
Human DLBCL cell lines LY1 and LY8 were obtained from Dr. B. Hilda Ye (Albert Einstein College of Medicine, NY, USA), and human DLBCL cell line Val was provided by Pro. Wing C. Chan (City of Hope National Medical Center, CA, USA). Cells were maintained in IMDM medium (Gibco, Carlsbad, USA) containing 10% fetal bovine serum (HyClone, Logan, USA) and 1% penicillin/streptomycin mixture. All cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. All human cell lines were examined for mycoplasma infection periodically and authenticated using small tandem repeat profiling conducted by LGC standards and ATCC.
Formalin-fixed and paraffin-embedded tissue sections obtained from DLBCL and RLH patients were used to perform IHC. The RLH tissues were used as control, as previously described [34, 35]. After deparaffinized and hydrated, the antigen was retrieved in 10 mM sodium citrate buffer (pH = 6.0) under high-pressure for 10 min. Three percent solution of H2O2 was used at 37 °C for 30 min to block the samples from endogenous peroxidases. After that, sections were incubated with primary anti-AMOT (1:200, Abcam, 85143, Cambridge, UK) at 4 °C overnight, then rinsed by 1× PBS for 15 min, and treated with second antibody from (Golden Bridge, Beijing, China) at 37 °C for 30 min. After incubation with SABC from SP reagent kit, the sections were stained with DAB (Golden Bridge, Beijing, China), counterstained with hematoxylin and mounted with neutral gum. The criterion for a positive result is that the positive cells rate exceeds 10%. Two independent researchers scored the IHC staining at different time points.
Real-time quantitative polymerase chain reaction (qRT-PCR)
Total RNA was isolated from peripheral blood mononuclear cells (PBMCs) and cultured cells by RNAiso Plus (TaKaRa, Dalian, China) and assessed by UV spectrophotometry for the concentrations and purity. After reverse transcription of total RNA into cDNA using the reagents kit (TaKaRa, Dalian, China), the amplification reactions were carried out with specific primers by means of SYBR Green Premix Ex Taq II kit (Takara, Dalian, China) on a Light Cycler 480 real-time PCR system (Roche, Basel, CH). AMOT-specific primers were the following: forward, 5′-AGCTCCAGGCAGCATGTGAA-3′; reverse, 5′-CTGAAACGTTGGTGGGCTGA-3′. Primers of GAPDH and DDR members are listed in Table S1. GAPDH was used as an endogenous control. The relative quantification was calculated by the 2−∆∆Ct method.
Total protein from the PBMCs and cultured cells was extracted using RIPA Lysis buffer (Shenergy, Shanghai, China) containing protease inhibitor cocktail tablets (Shenergy, Shanghai, China) and phosphatase inhibitor cocktail (Bosterbio, Wuhan, China), according to the operating instructions. Protein concentrations were measured using a BCA assay (Shenergy, Shanghai, China), then equal amounts of total protein were separated by electrophoresis on a 7.5 or 10% polyacrylamide gel (Bio-Rad, USA). The protein extracts were transferred onto PVDF membranes (Millipore, Billerica, USA), and the membranes were blocked in tris-buffered saline (contained 0.05% Tween) with 5% defatted milk or 1% bovine serum album for 1 h. Then the membranes were incubated with primary antibodies at 4 °C overnight. The primary antibodies were used with the concentration of 1:1000 as follows: anti-AMOT (Abcam, 85143, Cambridge, UK), anti-p-ATM, anti-p-ATR, anti-p-Chk1, anti-p-Chk2, and anti-p-H2A.X (Cell Signaling Technology, 9947, MA, USA). Anti-GAPDH (1:1000, Golden Bridge, TA-08, Beijing, China) was used as the endogenous control. Goat anti-rabbit or goat anti-mouse secondary antibodies labeled with HRP (1:5000, Golden bridge, ZB-2301, ZB-2305, Beijing, China) were hybrid binding at room temperature for 1 h, and the final signals were detected with the AI600 Imaging System (General Electric, Boston, USA) using an ECL kit (Millipore, Billerica, MA, USA). Finally, Image J software was used to quantified the intensities of protein bands.
LY1 and LY8 cells were respectively seeded into 96-well plates at a concentration of 1 × 105 cells per well on the day before transfection. According to the manufacturer’s protocol, appropriate doses of lentivirus (GeneChem, Shanghai, China) and transfection enhancer were co-cultivation with the cells. Both the Empty vector lentivirus (LV-Con) and AMOT overexpression lentivirus (LV-AMOT) expressed green fluorescent protein (GFP) and puromycin resistance genes. After 48 h, 5 μg/ml puromycin (GeneChem, Shanghai, China) was added to the culture medium to screen stably transfected viable cells. The infection efficiencies were evaluated by GFP and then validated by qRT-PCR and western blot assays.
Cell viability assay
LY1 and LY8 cells with designed treatment were seeded at a density of 8000 cells per well into 96-well plates. At the designated time, 10 μl/well of CCK-8 (Dojindo, Kumamoto, Japan) was incubated with the cells at 37 °C for 2 h, then followed by absorbance values detection at 450 nm by a microplate reader (Thermo Scientifc, Rockford, IL, USA).
Cell cycle analysis
Cells with designed treatment were harvested and fixed in ice-cold 70% ethanol for 30 min, then rinsed by 1×PBS, and resuspended in 50 μg/ml PI staining (BD Biosciences, Bedford, USA) according to the manufacturer’s instructions. After incubation in the dark room for 15 min, the cells were passed through a strainer to prepare single cell suspension. Then cell cycle distribution was detected by on Navios Flow Cytometer (Beckman Coulter, CA, USA) and analyzed by ModFit LT (Verity Software House, USA).
Cell apoptosis analysis
Cells with designed treatment were washed in ice-cold PBS and resuspended in 100 µl binding buffer, then incubated with 5 µl 7AAD and 5 µl Annexin V-PE (BD Biosciences, Bedford, USA) in a dark room for 15 min. Afterward, another 400 µl of binding buffer was added to each sample and mix well, then the cells were subjected to the Navios Flow Cytometer, and distinguished into four groups: Annexin V-PE-/7AAD- (viable cells), Annexin V-PE-/7AAD + (dead cells), Annexin V-PE+/7AAD-(early-stage apoptosis cells), and Annexin V-PE+/7AAD+ (late-stage apoptosis cells). The ratio of early-stage apoptotic cells was used to perform statistical analysis.
All experiments were independently repeated at least in triplicate. GraphPad Prism 7.0 (GraphPad Software, CA, USA) and SPSS17.0 (IBM, NY, USA) were used for statistical analysis, and data were presented as mean ± standard deviation (mean ± SD). The correlation between the expression of AMOT in tissue sections and the clinical parameters of DLBCL patients was analyzed by Chi-square test. Kaplan–Meier analysis was performed for subsistence analysis. Differences between groups were analyzed by ANOVA or unpaired t-tests. P < 0.05 was considered as statistically significant.
AMOT was downregulated in DLBCL and related to poor tumor prognosis
IHC staining was carried out to check the expression level of AMOT in DLBCL. The percentage of DLBCL samples positive for AMOT was 40.4% (21/52), lower than the percentage of 80% (20/25) of RLH tissues (Fig. 1A). The clinical data from DLBCL patients was collected and the relationship between decreased AMOT expression and clinical parameters of DLBCL were analyzed. As presented in Table 1, the expression of AMOT was negatively associated with serum lactate dehydrogenase (LDH, P = 0.023) and international prognostic index (IPI) score (P = 0.017). Furthermore, Kaplan-Meier analysis indicated that the median survival of patients was 5.04 years in AMOT-positive group, significantly longer than 1.94 years in AMOT-negative group (Fig. 1B). The results above suggest that AMOT may be a biomarker to assess the risk and clinical prognosis in DLBCL. Next, we verified the expression of AMOT in human DLBCL cell lines. As expected, DLBCL cells exhibited remarkably lower mRNA as well as protein levels of AMOT, compared to the control groups (Fig. 1C, D).
AMOT restrained DLBCL cell proliferation
To investigate the biological function of AMOT in vitro, LY1 and LY8 cells were stably transfected with LV-Con or LV-AMOT. The GFP expression ratio indicated that more than 80% of cells were successfully transfected with the lentivirus, as indicated by qRT-PCR and western blot assay (Fig. 2A, B). Compared with LV-Con groups, cell viability was significantly suppressed in LV-AMOT groups (Fig. 2C). We then used XAV939, a tankyrase inhibitor that stabilizes the concentration of AMOT protein, to treat the DLBCL cell lines. XAV939 inhibited the viability of DLBCL cells in a time-and concentration-dependent manner (Fig. 2D). Enhancement of the AMOT protein concentration were detected in LY1 and LY8 cells following incubation with XAV939 for 24 h (Fig. 2E).
Roles of AMOT on cell cycle, apoptosis, and DNA damage response proteins
We then sought to study whether AMOT can regulate the cell cycle and apoptosis of DLBCL cells using PI staining and annexin-V-based apoptotic assays. The results revealed that overexpressed AMOT increased the proportion of cells in the G1 phase in both LY1 (31.03 ± 1.0% in LV-Con vs. 36.31 ± 0.74% in LV-AMOT group, P = 0.002) and LY8 (29.69 ± 1.1% in LV-Con vs. 49.94 ± 0.64% in LV-AMOT group, P = 0.000) cells, with a concomitant decrease in the S phase (Fig. 3A). However, no significant change in the apoptosis rate after AMOT upregulation was observed in both LY1 and LY8 cells (Fig. 3B). Since the DDR was related to cell cycle arrest, the effect of AMOT on the activating phosphorylation of several key checkpoint proteins was examined by western blot assay. AMOT upregulation attenuated the activating phosphorylation of ATM (Ser1981), ATR (Ser428), Chk1 (Ser345), Chk2 (Thr68), and H2AX (Ser139) (Fig. 3C). However, the mRNA expression level of these genes did not change (Fig. 3D).
AMOT overexpression increased the response of DLBCL cells to doxorubicin
The regulation of AMOT to the DDR pathway led us to hypothesize that AMOT upregulation might sensitize DLBCL cells to DNA damaging reagents. To confirm our hypothesis, we incubated LV-AMOT and LV-Con cells with different concentrations of doxorubicin (80, 160, and 240 nM) for 48 h. Then the cell viability was evaluated by CCK-8 assay. The LV-AMOT group showed increased sensitivity to doxorubicin compared to the LV-Con group (Fig. 4A). Next, we incubated the cells with doxorubicin (160 nM) for 24, 48, and 72 h, and similarly, observed an increase in drug sensitivity in the LV-AMOT group (Fig. 4B). At last, we incubated the cells with doxorubicin (160 nM) for 24 h and performed cell cycle and apoptosis detection. Compared with the control, the LV-AMOT group showed an increased accumulation of cells in the G1 phase (Fig. 4C). As expected, under the treatment of doxorubicin, the apoptosis rate of cells in LV-AMOT group was statistically different from the control group (Fig. 4D).
AMOT upregulation attenuated DNA repair potential in response to doxorubicin in DLBCL cells
To investigate the potential mechanism by which AMOT enhances the sensitivity of DLBCL cells to chemotherapy, we incubated LV-AMOT and control cells with doxorubicin (160 nM) for 24 h, respectively, then checked the phosphorylation of ATM, ATR, Chk1, and H2AX proteins by western blot. The LV-AMOT groups were noted to have decreased levels of phosphorylated ATM, ATR, Chk1, Chk2, and H2AX (Fig. 5). We speculated that AMOT reduced the phosphorylation level of these key checkpoint proteins, thereby attenuating their response to doxorubicin-induced DNA damage.
Aberrant AMOT expression has been reported in solid tumors, but never in hematopoietic malignancy. For the first time, we elucidated in this study that AMOT may work as a potential tumor suppressor in the development of DLBCL. AMOT was suppressed during the pathogenesis of DLBCL, while overexpression of AMOT inhibited the progression of DLBCL in vitro. Furthermore, upregulation of AMOT led to increased sensitivity of DLBCL cells to DNA damaging reagents including doxorubicin. In terms of biological mechanism, AMOT could regulate the cell cycle by inhibiting DDR signalings in DLBCL cells. These encouraging findings may help to develop new treatment strategies and provide individualized governance in DLBCL.
In the present study, we identified for the first time that the expression of AMOT was remarkably repressed in DLBCL tissues and cell lines compared to reactive hyperplasia and PBMCs from healthy donors. Through the correlation analysis, we found that the reduction of AMOT was associated with high LDH and IPI scores, which were indicators of poor prognosis in patients with DLBCL. In the previous studies, AMOT had been reported as tumor suppressor in several solid malignancies . In advanced gastric cancer, lower AMOT-p130 was associated with higher TNM stage, venous invasion, shorter overall, and disease-free survival . The results demonstrate that AMOT may be a potential biomarker for prognostic evaluation of DLBCL in the context of individualized treatment. In the previous study, epigenetic mechanisms like promoter methylation or histone deacetylation may decrease the expression of AMOT in angiogenesis and sarcomagenesis [15, 38], so we speculate that an analogous mechanism may be involved in DLBCL. The specific mechanism requires further investigation.
We next explored the potential functions of AMOT in the pathogenesis of DLBCL by gain-of-function assay. The results indicated that AMOT could inhibit cell viability, induce cell cycle arrest, and increase the sensitivity of cells to chemotherapeutics, instead of directly induce apoptosis in DLBCL. Tankyrases belonged to the poly (ADP-ribose) polymerase family which recognized its substrate through the tankyrase-binding domain  and induced the subcellular localization changes or degradation of its substrate . Tankyrase inhibitors could stabilize intracellular AMOT protein levels and consequently inhibited tumor cell proliferation by regulating downstream mechanisms [15, 41, 42]. In this study, XAV939 inhibited the proliferation of DLBCL cells, which may be related to the increase of AMOT protein concentration in the cytoplasm.
As is widely known, the ability of self-repair after DNA damage is closely related to the development of tumors. The carcinogenic mutations cause spontaneous DNA damage, which inhibits the progression of incipient cancer cells . The DNA damage checkpoints are important mediators of the tumorigenesis barrier, which is activated early in tumor development and later dysregulated. The cancer cells may rely on the DDR pathway to maintain genomic instability [30, 44]. ATM and ATR are essential components of DDR that are activated when cells are subjected to DNA replication stress or DNA double-strand breaks . Under these conditions, checkpoint kinase Chk1 and Chk2 are phosphorylated and inhibit the phosphorylation of cell division cycle (CDC) 25A protein, which subsequently regulates downstream kinases and blocks the cell cycle in S and G2 phases. This mechanism allows tumor cells to make adequate repairs and thereby promote their survival . The activated Chk1 transfers into the nucleus to prevent new source excitation . For hematopoietic tumors, Miroslav et al. had shown that Chk1 was a key protein in the development of B cell lymphoma . As a member of the histone H2A family, H2AX is a key promoter that amplifies signals in the early stages of DNA damage and continuously recruits repair factors to nuclear foci . The suppression of DNA damage checkpoints decreases DNA repair potential. This current study showed that AMOT could attenuate the phosphorylation activation of DNA damage checkpoint proteins, thereby inhibiting the ability of DLBCL cells to repair DNA damage. In this study, we observed that AMOT overexpression resulted in a shortened S phase of the cell cycle, but no significant change in the G2 phase, indicating that there may be other offsetting factors that require further study.
Many studies had demonstrated that ATM-Chk2 or ATR-Chk1 pathway inhibitors could enhance the sensitivity of various tumors, including lymphoma and leukemia, to radiation or chemotherapy drugs [43, 50,51,52]. Doxorubicin is an anti-mitotic cytotoxic drug that works primarily by interfering with DNA synthesis and disrupting DNA tertiary structure. In the current study, AMOT protein exhibits anti-cancer effect, and a decrease in the activity of key kinases in the DDR pathway has been observed. When doxorubicin was applied to AMOT-overexpressed DLBCL cells, an increased proportion of apoptosis was observed, suggesting an increased sensitivity to doxorubicin. Thus, AMOT may serve as a biomarker to predict doxorubicin treatment response.
In short, these findings suggest that AMOT downregulation is a frequent cancer-specific event. Upregulation of AMOT inhibited the proliferation rate and induced cell cycle arrest in DLBCL. More importantly, AMOT reinforced the chemosensitivity of DLBCL cells to DNA damaging agent. In terms of mechanism, AMOT inhibited the phosphorylation of DDR signaling proteins, thereby reducing the self-repair potential of DLBCL cells. This study clarified AMOT as a potential target for individualized treatment strategies.
Availability of data and materials
All data generated and analyzed during this study are included in this article and its supplementary information files.
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This study was funded by Translational Research Grant of National Clinical Research Center for Hematologic Diseases (NCRCH) (No. 2020ZKMB01); National Natural Science Foundation (No. 81800194, No. 82070203, No. 81770210, No. 81270598, and No. 81473486); Key Research and Development Program of Shandong Province (No. 2018CXGC1213); Technology Development Projects of Shandong Province (No. 2017GSF18189); Shandong Provincial Natural Science Foundation (No. ZR2018BH011); China Postdoctoral Science Foundation (No. 2020M672103); Development Project of Youth Innovation Teams in Colleges and Universities of Shandong Province (No. 2020KJL006); Technology Development Project of Jinan City (No. 201805065); Taishan Scholars Program of Shandong Province; Academic promotion programme of Shandong First Medical University; Shandong Provincial Engineering Research Center of Lymphoma.
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Sang, T., Yang, J., Liu, J. et al. AMOT suppresses tumor progression via regulating DNA damage response signaling in diffuse large B-cell lymphoma. Cancer Gene Ther (2021). https://doi.org/10.1038/s41417-020-00258-5