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ATG5 regulates mesenchymal stem cells differentiation and mediates chemosensitivity in acute myeloid leukemia
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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ATG5 regulates mesenchymal stem cells differentiation and mediates chemosensitivity in acute myeloid leukemia Ying Li, Yajing Jiang, Jingying Cheng, Jiao Ma, Qinghua Li, Tianxiang Pang* State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Disease, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2020 Accepted 12 February 2020 Available online xxx
Autophagy is related to the development of several tumors including acute myeloid leukemia (AML). Inhibition of autophagy in AML cells can make them more susceptible to chemotherapy. However, the influence of autophagy in mesenchymal stem cells (MSCs) remains inconclusive. In the present study, we demonstrated that the expression of ATG5 and autophagy were elevated in MSCs derived from AML patients (AML-MSCs) compared to healthy donors (HD-MSCs). After inhibiting autophagy by 3Methyladenine (3 MA) or silencing ATG5, the differentiation potential of AML-MSCs was decreased, the fraction of G0/G1 phase was increased while that of G2 phase was reduced, and the expression of CXCL12 was reduced in AML-MSCs. After co-culture of NB4 and THP1 with MSCs pretreated with 3 MA or ATG5 knockdown respectively, the sensitivity of AML cell lines to daunorubicin and doxorubicin was improved in a dose- and time-dependent manner compared to controls. The increased sensitivity of AML cells to genotoxic agents was related to ERK1/2 and AKT pathway. These results suggested ATG5 mediated potential differentiation capacities and cell cycle distribution of AML-MSCs, and targeting autophagy, especially ATG5 in AML-MSCs could improve the chemosensitivity of AML. © 2020 Elsevier Inc. All rights reserved.
Keywords: Acute myeloid leukemia Autophagy ATG5 Chemosensitivity Mesenchymal stem cells
1. Introduction AML is a common hematopoietic malignancy characterized by uncontrolled rapid expansion of immature myeloid cells in bone marrow. In recent decades, management for AML has improved vastly due to the growth of the number of drugs available for the treatment [1]. Despite this, primary induction failure and relapse remain great challenges in the treatment of AML for clinicians and researchers. MSCs are crucial components in bone marrow microenvironments. Evidence from several groups indicated that the disorder of MSCs could be an initiation of leukemia [2e4]. The dysfunctional MSCs suppress normal hematopoiesis, support the development of leukemia, contributes to chemoresistance and relapse in AML [5e9]. Crosstalk between MSCs and leukemic cells plays a crucial role in AML. However, interactions between AML cells and stromal
* Corresponding author. State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Disease, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing road 288, Tianjin, 300020, China. E-mail address: [email protected] (T. Pang).
cells have not been fully examined and are not well understood. Hence, investigating the alteration of MSCs could make it possible to help solve this problem and provide a novel target for the effort of improved management of AML. As a key molecule in autophagy, ATG5 is indispensable and silencing of ATG5 could cause the down-regulation of autophagy [10]. A few studies have reported that autophagy mediated by ATG5 is related to the development of numerous tumors, and ATG5 dependent autophagy could be targeted for the treatment of tumors [11e13]. Recent researches have reported that AML blast cells had a significantly higher autophagy activity than normal BM cells [14]. The elevated autophagy contributes to the chemoresistance of AML cells [15] and inhibiting autophagy in AML cells can make them more susceptible to chemotherapy [16e18]. However, the influence of autophagy in AML-MSCs is not known very well. And the impact of targeting ATG5 in AML-MSCs on leukemia cells is a research area that remains mostly unexplored. Thus, in the present study, we attempted to explore the impact of ATG5 on AML-MSCs. We hypothesize that silencing ATG5 could change the characteristics of AML-MSCs. And we hypothesize the supporting functions of AML-MSCs to leukemic cells were reduced by silencing ATG5. Further, the results may serve to find potential
https://doi.org/10.1016/j.bbrc.2020.02.091 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Li et al., ATG5 regulates mesenchymal stem cells differentiation and mediates chemosensitivity in acute myeloid leukemia, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.091
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2.4. Differentiation assays of MSCs Abbreviations 3 MA AML DNR DOX MSCs
MSCs were cultured in 24-well dishes then the medium was replaced with adipogenesis or osteogenesis differentiation induction medium when the cells reached 80% confluence. For adipogenesis differentiation, after 14 days of induced differentiation, the fatty drops were stained with Oil Red O. For osteogenesis differentiation, after 21 days of induced differentiation, the calcium nodules were stained with Alizarin Red.
3-Methyladenine acute myeloid leukemia daunorubicin doxorubicin mesenchymal stem cells
2.5. Western blotting targets for the treatment of AML. 2. Materials and methods 2.1. Cell culture MSCs were isolated from healthy donors or AML patients after informed consent and this study was approved by the Committee for Medical Care and Safety, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Science and Peking Union Medical College. The clinical characteristics of samples were listed in Table 1 and the detail information was shown in Supplementary Table 1. MSCs culture and phenotype identification followed methods previously published [19]. And MSCs were used in passages 2e4 for all experiments. NB4 and THP1 were cultured in RPMI-1640 (Gibco-BRL, Burlington, ON, Canada) medium supplemented with 10% FBS and respectively co-cultured with MSCs. 2.2. Lentiviral shRNA vectors and transfection The shATG5 sequences were integrated into GV248 plasmid. The GV248, pMD2.G and pSPAX2 plasmids were transfected into 293T cells with Lipofectamin™ 3000 (Invitrogen, CA, USA) according to the protocol. The culture medium was replaced by complete medium after 6 h. The supernatant was concentrated and collected after culturing 48 h. GV248-shATG5 (shMSCs) and the negative control (ncMSCs) lentiviral constructs were then transfected into MSCs separately in the presence of 8 mg/ml polybrene. The transfected cells were selected with puromycin. Knock-down efficiency of ATG5 was measured by real-time PCR and Western blot analysis. 2.3. Quantitative real-time PCR
2.6. Cytotoxicity assay Cells were seeded in 96 wells plates at a concentration of 1.5 105 cells/ml and were treated with daunorubicin (DNR) and doxorubicin (DOX), respectively. After 48 h of culture, 20 ml MTT (Sigma, America) was added in every well and then 100 ml solubilization buffer (10% SDS in 0.01 m HCl) was added after 4 h incubating. The absorbance at 570 nm was detected by microplate reader (BIO-TEK, America) after overnight incubating. 2.7. Flow cytometry To measure the percentage of apoptosis cells, we used the Annexin V- FITC apoptosis analysis kit (Sungene Biotech, Tianjin, China) and followed manufacturer protocols. Results were analyzed by FlowJo (vision 7.6.1). To determine cell cycle cell distribution, PI/RNase Staining Solution (Sungene Biotech, Tianjin, China) was used following manufacturer protocols. 2.8. Statistical analyses
Total RNA was extracted using Trizol Reagent (Invitrogen, Grand Island, NY) according to the manufacturer instructions, and cDNA was reversed transcribed used the Superscript II RT kit (Invitrogen, Grand Island, NY). Quantitative real-time PCR (qRT-RCR) was performed using the SYBR Green PCR kit (TaKaRa, Japan) on QuantStudio®5. We used GAPDH as a control for normalization. Primer sequences used for qRT-RCR and corresponding information were given in Supplementary Table 2.
All results were analyzed by GraphPad Prism (vision 5.01). Every experiment had at least three replicates and the results were presented as mean ± standard error of mean. A student’s t-test was used to compare for significant differences between two groups. Pvalue < 0.05 was regarded as statistically significant. 3. Results 3.1. The expression of ATG5 and the basal level of autophagy were higher in AML-MSCs
Table 1 Characteristics of AML patients and healthy donors.
Number of samples Sex Female Male Age (years) Average Range
For western blotting analyses, total protein was extracted from MSCs using RIPA buffer (Beyotime, China) and separated by 10e12% SDS-PAGE. Protein was detected using antibodies against LC3b (Santa Cruz Biotechnology, America), ATG5 (Santa Cruz Biotechnology, America), p62 (Santa Cruz Biotechnology, America), AKT (Cell Signaling Technology, America), p-AKT (Cell Signaling Technology, America), ERK1/2 (Cell Signaling Technology, America), and p-ERK1/2(Cell Signaling Technology, America). We used GAPDH (Cell Signaling Technology, America) as a control for normalization. The relative intensity of protein bands was analyzed by Adobe Photoshop CS6.
AML
HD
28
20
11 17
7 13
40 23e53
34 18e53
First, we collected MSCs isolated from twenty healthy donors and twenty-eight AML patients. Expression of surface markers on MSCs indicated that CD34 and CD45 were negative and that CD73, CD90, and CD105 were positive (Supplementary Figure 1), which ultimately verified that the adherence cells were MSCs. Next, we compared the mRNA expression of ATG5 in HD-MSCs and AMLMSCs, and the results of qRT-PCR showed that AML-MSCs had a higher expression level than HD-MSCs (Fig. 1A, p ¼ 0.0086).
Please cite this article as: Y. Li et al., ATG5 regulates mesenchymal stem cells differentiation and mediates chemosensitivity in acute myeloid leukemia, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.091
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Fig. 1. Elevated expression of ATG5 influenced the characteristics of AML-MSCs. HD represents healthy donors, AML represents AML patients, Control represents MSCs pretreated with DMSO, 3 MA represents MSCs pretreated with 3 MA, nc represents MSCs transfected with negative control, sh represents MSCs transfected with shATG5. (A) mRNA of ATG5 was up regulated in AML-MSCs. (B) Cell cycle analysis of MSCs demonstrated that the proportion of cells in the G0/G1 phase was significantly increased and the proportion of cells in the G2 phase was significantly decreased in 3 MA-MSCs or shMSCs compared to controls. (C) mRNA of CXCL12 was significantly reduced by the addition of 50 mM 3 MA or silencing ATG5 in AML-MSCs. (D) The results of Western blot. Autophagy related genes expression (measured by protein level). Gray values of LC3b II and ATG5 from western blotting were up regulated and p62 was down regulated in AML-MSCs. (E) The potential differentiation capacities of AML-MSCs were decreased by the addition of 50 mM 3 MA. 3 MA-MSCs had less and little fatty drops. The mRNA expression of adipogenic markers PPARG and FABP4 were down regulated in 3 MA-MSCs. Alizarin red staining showed that 3 MA-MSCs had less calcium nodules compared to control. qRT-PCR analyses of osteogenic makers RUNX2 and ALP indicated that these were down regulated in 3 MA-MSCs. (F) The potential differentiation capacities of shMSCs were decreased. There were less and little fatty drops and less calcium nodules in shMSCs. mRNA expression of adipogenic markers and osteogenic makers were all down regulated in shMSCs. *p < 0.05 compared to control. **p < 0.01 compared to control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Please cite this article as: Y. Li et al., ATG5 regulates mesenchymal stem cells differentiation and mediates chemosensitivity in acute myeloid leukemia, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.091
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Besides, we also detected the protein expression of ATG5, LC3b II and p62 in HD-MSCs (n ¼ 3) and AML-MSCs (n ¼ 5) by Western blot (Fig. 1D). LC3b II (Fig. 1D, p ¼ 0.044) and ATG5 (Fig. 1D, p ¼ 0.017) expression were both upregulated and p62 expression (Fig. 1D, p ¼ 0.036) was down regulated in AML-MSCs. The results of Western blot suggested that autophagy was up-regulated in AMLMSCs. 3.2. Cell cycle distribution and potential differentiation capacities of AML-MSCs were altered by the inhibition of autophagy To explore the impact of autophagy on AML-MSCs, we inhibited autophagy by 50 mM 3 MA treatment for 24 h (Supplementary Figure 2A). Unexpectedly, 3 MA did not significantly influence the apoptosis of AML-MSCs, but it significantly influenced the cell cycle and the differentiation potential. AML-MSCs were arrested at the G0/G1 phase (Fig. 1B, p ¼ 0.020), and the G2 phase fraction was significantly reduced (Fig. 1B, p ¼ 0.020) after treating with 50 mM 3 MA. Additionally, compared to control, potential differentiation capacities of 3 MA treatment group (short for 3 MAMSCs) were reduced compared to controls. It showed that 3 MAMSCs had less and little fatty drops after 14 days of induction (Fig. 1E). mRNA of FABP4 (Fig. 1E, p ¼ 0.003) and PPARG (Fig. 1E, p ¼ 0.007) were significantly down regulated in 3 MA-MSCs. In terms of osteogenesis, 3 MA-MSCs had less calcium nodules after 21 days of induction (Fig. 1E). qRT-PCR showed that both ALP (Fig. 1E, p ¼ 0.025) and RUNX2 (Fig. 1E, p ¼ 0.038) were down regulated in 3 MA-MSCs. To further explore whether the function of autophagy in AMLMSCs was ATG5 dependent, we transfected sh-ATG5 plasmid in AML-MSCs. The results of qRT-PCR and Western blot showed that ATG5 was significantly down regulated (Supplementary Figure 2, p < 0.001), and autophagy was inhibited in shMSCs (Supplementary Figure 2). The proportion of cells in G0/G1 (Fig. 1B, p ¼ 0.016) phase was significantly increased and cells in the G2 (Fig. 1B, p ¼ 0.040) phase was significantly decreased in shMSCs. In addition, the adipogenesis and osteogenesis were both decreased in shMSCs (Fig. 1F). The mRNA of FABP4 (Fig. 1F, p ¼ 0.019), PPARG (Fig. 1F, p ¼ 0.031), RUNX2 (Fig. 1F, p ¼ 0.019) and ALP (Fig. 1F, p ¼ 0.034) were all down regulated in shMSCs. Taken together, inhibition of autophagy by 3 MA or silencing ATG5 in AML-MSCs could change the cell cycle distribution. And it could also reduce the potential differentiation capacities of AMLMSCs. 3.3. Chemosensitivity of leukemic cells was increased after cocultured with 3 MA-MSCs or shMSCs respectively compared to controls CXCL12 secreted by MSCs plays a major role in crosstalk between MSCs and leukemic cells [20]. To explore whether the support effect of MSCs on leukemia was altered after the inhibition of autophagy, we detected the expression of CXCL12. Results suggested that CXCL12 was down regulated in 3 MA-MSCs (Fig. 1C, p ¼ 0.002) and shMSCs (Fig. 1C, p ¼ 0.002) compared to controls. To further explore the support effects of AML-MSCs to AML cell lines, a co-culture system was used. NB4 and THP1 were respectively cocultured with 3 MA-MSCs or control MSCs for 48 h, and then were treated with an incremental concentration of DNR or DOX. After 48 h of treatment, cells were assessed for viability by MTT. IC50 was measured by GraphPad using MTT data. After co-cultured with 3 MA-MSCs, NB4 (Fig. 2A) and THP1 (Fig. 2B) were both more sensitive to DNR and DOX in a dose-dependent manner compared to controls. And IC50 of DNR and DOX were listed in Table 2. Besides, we also measured cell viability at different time points
treated with 0.3 mM DNR or 0.6 mM DOX. The results suggested that the chemosensitivity of NB4 and THP1 co-cultured with 3 MAMSCs were both improved by a time-dependent manner (Fig. 2C and D). Besides, we had similar results from NB4 and THP1 co-cultured with shMSCs or ncMSCs respectively. NB4 (Fig. 2A) and THP1 (Fig. 2B) were more sensitive to DNR or DOX after co-culture with shMSCs respectively. And the detail information of IC50 was listed in Table 2. The chemosensitivity of NB4 (Fig. 2C) and THP1 (Fig. 2D) co-cultured with shMSCs were both improved by a time-dependent manner. 3.4. DNR and DOX induced more apoptosis in THP1 and NB4 cells co-cultured with 3 MA-MSCs or shMSCs respectively compared to controls AML cells lines were treated with DNR and DOX separately after co-cultured with MSCs. The results suggested that 0.3 mM DNR (Fig. 3A, 11.8 ± 1.068 vs. 41.77 ± 2.121, p < 0.001) and 0.6 mM DOX (Figs. 3A and 31.47 ± 1.791 vs. 50.63 ± 0.4096, p < 0.001) respectively induced more apoptosis in NB4 co-cultured with 3 MA-MSCs compared to control. While we had similar results from THP1, using 0.3 mM DNR (Figs. 3B and 8.717 ± 0.355 vs. 42.7 ± 0.953, p < 0.001) and using 0.6 mM DOX (Figs. 3B and 29.8 ± 1.071 vs. 49.61 ± 1.562, p < 0.001) induced more apoptosis in THP1 co-cultured with 3 MAMSCs compared to control respectively. Besides, results from NB4 and THP1 cells co-culture with ncMSCs or shMSCs respectively were consistent with the results mentioned above. 0.3 mM DNR (Figs. 3A and 14.5 ± 0.9738 vs. 35.77 ± 1.631, p < 0.001) and 0.6 mM DOX (Figs. 3A and 29.03 ± 1.241 vs. 49.90 ± 1.31, p < 0.001) induced more apoptosis in NB4 cocultured with shMSCs compared to control. In terms of THP1, using 0.3 mM DNR (Figs. 3B and 11.5 ± 1.091 vs. 32.34 ± 1.447, p < 0.001) and using 0.6 mM DOX (Figs. 3B and 23.96 ± 2.727 vs. 46.25 ± 5.254, p ¼ 0.019) induced more apoptosis in THP1 cocultured with shMSCs compared to control respectively. 3.5. Autophagy in mesenchymal stem cells mediated sensitivity of acute myeloid leukemia cells to chemotherapy via the ERK1/2 and PI3K/AKT pathways Since we observed the influence of autophagy and ATG5 in AMLMSCs, in the next experiments we sought to explore how autophagy and ATG5 influence the protective effects of AML-MSCs to AML cells. We screen out two pathways from several pathways that had been reported were related to the chemotherapy in AML. As Fig. 4 showed, the phosphorylation of ERK1/2 and phosphorylation of AKT were both down regulated in THP1 and NB4 after co-culture with 3 MA-MSCs or shMSCs respectively. This suggested that ERK1/ 2 and AKT pathways could be involved in the protective effects of AML-MSCs to AML cell lines. 4. Discussion To explore the alteration of MSCs in AML, we compared the mRNA profiles of MSCs (GSE122917) derived from healthy donors and de novo AML patients (short for AML-MSCs). Enrichment analyses of differently expressed genes had indicated that autophagy was up regulated in AML-MSCs. To verify the results of enrichment analyses, we detected mRNA and protein expression of several autophagy related genes in AML-MSCs. Among that the upregulation of ATG5 was the most obvious. Besides, the results of Western blot also suggested that autophagy was elevated in AMLMSCs. To further investigate the impacts of autophagy on AML-MSCs,
Please cite this article as: Y. Li et al., ATG5 regulates mesenchymal stem cells differentiation and mediates chemosensitivity in acute myeloid leukemia, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.091
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Fig. 2. The sensitivity of acute myeloid leukemia cells to chemotherapy was increased by the inhibition of autophagy. Control represents cells co-cultured with control MSCs; 3 MA represents cells co-cultured with 3 MA-MSCs; nc represents cells co-cultured with ncMSCs; sh represents cells co-cultured with shMSCs. (A) NB4 and (B) THP1 co-cultured with 3 MA-MSCs or shMSCs respectively were more sensitive to DNR or DOX compared to controls. It was dose-dependent. Cells were treated with 0.3 mM DNR or 0.6 mM DOX, and the cell viability was measured at 0, 24, 48, 72, 96 h. NB4 (C) and THP1 (D) were more sensitive to DNR or DOX after co-cultured with 3 MA-MSCs or shMSCs. It was time-dependent.
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Table 2 IC50 of DNR and DOX in NB4 and THP1.
NB4 Control vs. 3 MA nc vs. sh THP1 Control vs. 3 MA nc vs. sh
DNR(mM)
DOX(mM)
0.4365 vs. 0.1799 0.4367 vs. 0.2651
0.5806 vs. 0.4869 0.5795 vs. 0.5135
0.4129 vs. 0.3230 0.4146 vs. 0.3325
0.7101 vs. 0.5985 0.7377 vs. 0.6150
Abbreviations: DNR, daunorubicin; DOX, doxorubicin; 3 MA, 3-Methyladenine.
we inhibited autophagy in AML-MSCs by 3 MA. The results showed that the cell cycle and differentiation potential were significantly altered after the inhibition of autophagy in AMLMSCs. In fact, under some circumstances, the influence of autophagy in cell cycle [21] and osteogenic differentiation [22e24] have been reported in other cells. To explore whether the functions of autophagy in AML-MSCs were ATG5 dependent, we knocked down the expression of ATG5 in AML-MSCs. As we expected, silencing ATG5 had similar results with 3 MA treatment.
Take this, we concluded that the influence of autophagy in AMLMSCs was ATG5 dependent. Further, we detected the expression of CXCL12, and the results suggested it was down regulated in 3 MA-MSCs and shMSCs compared to controls. Since the expression of CXCL12 in MSCs contributes to the survival of AML cells [20], we found support for our hypothesis that the protective effect of AML-MSCs to AML cells was reduced after the inhibition of autophagy. We used a co-culture system to evaluate the protective effect of AML-MSCs on AML cells. However, inhibition of autophagy in AML-MSCs only affected the support of AML-MSCs to AML cell lines in the presence of DNR or DOX. This might be influenced by the finding that apoptosis was not often observed in NB4 or THP1 cell lines in the absence of stimulation. Previously researchers have indicated that aberrant activation of ERK1/2 and PI3K/AKT pathways are involved in AML [25]. AML cells were more sensitive to chemotherapy after the inhibition of the two pathways [26,27]. In the present study, the results of western blotting revealed that the phosphorylation of ERK1/2 and AKT in AML cell lines co-cultured with 3 MA-MSCs or shMSCs. This suggested autophagy in MSCs might work in a supporting role for AML
Fig. 3. DNR and DOX induced more apoptosis in NB4 and THP1 co-cultured with 3 MA-MSCs or shMSCs respectively compared to controls. Control represents cells co-cultured with control MSCs; 3 MA represents cells co-cultured with 3 MA-MSCs; nc represents cells co-cultured with ncMSCs; sh represents cells co-cultured with shMSCs. 0.3 mM DNR and 0.6 mM DOX induced an increase in the frequency of observed apoptotic cells in (A) NB4 and (B) THP1 co-cultured with 3 MA-MSCs or shMSCs respectively when compared to results for co-cultures with controls. *p < 0.05 compared to control. ***p < 0.001 compared to control.
Please cite this article as: Y. Li et al., ATG5 regulates mesenchymal stem cells differentiation and mediates chemosensitivity in acute myeloid leukemia, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.091
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Fig. 4. P-ERK1/2 and p-AKT were down regulated in cells co-cultured with 3 MA-MSCs or shMSCs. Control represents cells co-cultured with control MSCs; 3 MA represents cells cocultured with 3 MA-MSCs; nc represents cells co-cultured with ncMSCs; sh represents cells co-cultured with shMSCs. Western blot results reported that p-ERK1/2 and p-AKT were down regulated in NB4 and THP1 co-cultured with 3 MA-MSCs or shMSCs respectively compared to controls. **p < 0.01 compared to control. ***p < 0.001 compared to control.
cell lines via activating the ERK1/2 and PI3K/AKT pathways in AML cell lines. In conclusion, we demonstrated that ATG5 and autophagy were up regulated in AML-MSCs. Inhibition of autophagy by 3 MA or
silencing ATG5 significantly altered the cell cycle distribution and decreased differentiation capacities of AML-MSCs. Moreover, the sensitivity of AML cells to genotoxic agents was improved after cocultured with 3 MA-MSCs or shMSCs compared to controls, and it
Please cite this article as: Y. Li et al., ATG5 regulates mesenchymal stem cells differentiation and mediates chemosensitivity in acute myeloid leukemia, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.091
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was related to ERK1/2 and AKT pathway. Declaration of competing interest The author reports no conflicts of interest in this work.
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This work was supported by the Chinese Academy of Medical Sciences Initiative for Innovative Medicine (CAMS-I2M-2016-1017) and National Natural Science Foundation of China (81170510).
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Appendix A. Supplementary data
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Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.02.091.
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ATG5 regulates mesenchymal stem cells differentiation and mediates chemosensitivity in acute myeloid leukemia Ying Li, Yajing Jiang, Jingying Cheng, Jiao Ma, Qinghua Li, Tianxiang Pang* State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Disease, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2020 Accepted 12 February 2020 Available online xxx
Autophagy is related to the development of several tumors including acute myeloid leukemia (AML). Inhibition of autophagy in AML cells can make them more susceptible to chemotherapy. However, the influence of autophagy in mesenchymal stem cells (MSCs) remains inconclusive. In the present study, we demonstrated that the expression of ATG5 and autophagy were elevated in MSCs derived from AML patients (AML-MSCs) compared to healthy donors (HD-MSCs). After inhibiting autophagy by 3Methyladenine (3 MA) or silencing ATG5, the differentiation potential of AML-MSCs was decreased, the fraction of G0/G1 phase was increased while that of G2 phase was reduced, and the expression of CXCL12 was reduced in AML-MSCs. After co-culture of NB4 and THP1 with MSCs pretreated with 3 MA or ATG5 knockdown respectively, the sensitivity of AML cell lines to daunorubicin and doxorubicin was improved in a dose- and time-dependent manner compared to controls. The increased sensitivity of AML cells to genotoxic agents was related to ERK1/2 and AKT pathway. These results suggested ATG5 mediated potential differentiation capacities and cell cycle distribution of AML-MSCs, and targeting autophagy, especially ATG5 in AML-MSCs could improve the chemosensitivity of AML. © 2020 Elsevier Inc. All rights reserved.
Keywords: Acute myeloid leukemia Autophagy ATG5 Chemosensitivity Mesenchymal stem cells
1. Introduction AML is a common hematopoietic malignancy characterized by uncontrolled rapid expansion of immature myeloid cells in bone marrow. In recent decades, management for AML has improved vastly due to the growth of the number of drugs available for the treatment [1]. Despite this, primary induction failure and relapse remain great challenges in the treatment of AML for clinicians and researchers. MSCs are crucial components in bone marrow microenvironments. Evidence from several groups indicated that the disorder of MSCs could be an initiation of leukemia [2e4]. The dysfunctional MSCs suppress normal hematopoiesis, support the development of leukemia, contributes to chemoresistance and relapse in AML [5e9]. Crosstalk between MSCs and leukemic cells plays a crucial role in AML. However, interactions between AML cells and stromal
* Corresponding author. State Key Laboratory of Experimental Hematology, National Clinical Research Center for Blood Disease, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing road 288, Tianjin, 300020, China. E-mail address: [email protected] (T. Pang).
cells have not been fully examined and are not well understood. Hence, investigating the alteration of MSCs could make it possible to help solve this problem and provide a novel target for the effort of improved management of AML. As a key molecule in autophagy, ATG5 is indispensable and silencing of ATG5 could cause the down-regulation of autophagy [10]. A few studies have reported that autophagy mediated by ATG5 is related to the development of numerous tumors, and ATG5 dependent autophagy could be targeted for the treatment of tumors [11e13]. Recent researches have reported that AML blast cells had a significantly higher autophagy activity than normal BM cells [14]. The elevated autophagy contributes to the chemoresistance of AML cells [15] and inhibiting autophagy in AML cells can make them more susceptible to chemotherapy [16e18]. However, the influence of autophagy in AML-MSCs is not known very well. And the impact of targeting ATG5 in AML-MSCs on leukemia cells is a research area that remains mostly unexplored. Thus, in the present study, we attempted to explore the impact of ATG5 on AML-MSCs. We hypothesize that silencing ATG5 could change the characteristics of AML-MSCs. And we hypothesize the supporting functions of AML-MSCs to leukemic cells were reduced by silencing ATG5. Further, the results may serve to find potential
https://doi.org/10.1016/j.bbrc.2020.02.091 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Li et al., ATG5 regulates mesenchymal stem cells differentiation and mediates chemosensitivity in acute myeloid leukemia, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.091
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2.4. Differentiation assays of MSCs Abbreviations 3 MA AML DNR DOX MSCs
MSCs were cultured in 24-well dishes then the medium was replaced with adipogenesis or osteogenesis differentiation induction medium when the cells reached 80% confluence. For adipogenesis differentiation, after 14 days of induced differentiation, the fatty drops were stained with Oil Red O. For osteogenesis differentiation, after 21 days of induced differentiation, the calcium nodules were stained with Alizarin Red.
3-Methyladenine acute myeloid leukemia daunorubicin doxorubicin mesenchymal stem cells
2.5. Western blotting targets for the treatment of AML. 2. Materials and methods 2.1. Cell culture MSCs were isolated from healthy donors or AML patients after informed consent and this study was approved by the Committee for Medical Care and Safety, Institute of Hematology and Blood Diseases Hospital, Chinese Academy of Medical Science and Peking Union Medical College. The clinical characteristics of samples were listed in Table 1 and the detail information was shown in Supplementary Table 1. MSCs culture and phenotype identification followed methods previously published [19]. And MSCs were used in passages 2e4 for all experiments. NB4 and THP1 were cultured in RPMI-1640 (Gibco-BRL, Burlington, ON, Canada) medium supplemented with 10% FBS and respectively co-cultured with MSCs. 2.2. Lentiviral shRNA vectors and transfection The shATG5 sequences were integrated into GV248 plasmid. The GV248, pMD2.G and pSPAX2 plasmids were transfected into 293T cells with Lipofectamin™ 3000 (Invitrogen, CA, USA) according to the protocol. The culture medium was replaced by complete medium after 6 h. The supernatant was concentrated and collected after culturing 48 h. GV248-shATG5 (shMSCs) and the negative control (ncMSCs) lentiviral constructs were then transfected into MSCs separately in the presence of 8 mg/ml polybrene. The transfected cells were selected with puromycin. Knock-down efficiency of ATG5 was measured by real-time PCR and Western blot analysis. 2.3. Quantitative real-time PCR
2.6. Cytotoxicity assay Cells were seeded in 96 wells plates at a concentration of 1.5 105 cells/ml and were treated with daunorubicin (DNR) and doxorubicin (DOX), respectively. After 48 h of culture, 20 ml MTT (Sigma, America) was added in every well and then 100 ml solubilization buffer (10% SDS in 0.01 m HCl) was added after 4 h incubating. The absorbance at 570 nm was detected by microplate reader (BIO-TEK, America) after overnight incubating. 2.7. Flow cytometry To measure the percentage of apoptosis cells, we used the Annexin V- FITC apoptosis analysis kit (Sungene Biotech, Tianjin, China) and followed manufacturer protocols. Results were analyzed by FlowJo (vision 7.6.1). To determine cell cycle cell distribution, PI/RNase Staining Solution (Sungene Biotech, Tianjin, China) was used following manufacturer protocols. 2.8. Statistical analyses
Total RNA was extracted using Trizol Reagent (Invitrogen, Grand Island, NY) according to the manufacturer instructions, and cDNA was reversed transcribed used the Superscript II RT kit (Invitrogen, Grand Island, NY). Quantitative real-time PCR (qRT-RCR) was performed using the SYBR Green PCR kit (TaKaRa, Japan) on QuantStudio®5. We used GAPDH as a control for normalization. Primer sequences used for qRT-RCR and corresponding information were given in Supplementary Table 2.
All results were analyzed by GraphPad Prism (vision 5.01). Every experiment had at least three replicates and the results were presented as mean ± standard error of mean. A student’s t-test was used to compare for significant differences between two groups. Pvalue < 0.05 was regarded as statistically significant. 3. Results 3.1. The expression of ATG5 and the basal level of autophagy were higher in AML-MSCs
Table 1 Characteristics of AML patients and healthy donors.
Number of samples Sex Female Male Age (years) Average Range
For western blotting analyses, total protein was extracted from MSCs using RIPA buffer (Beyotime, China) and separated by 10e12% SDS-PAGE. Protein was detected using antibodies against LC3b (Santa Cruz Biotechnology, America), ATG5 (Santa Cruz Biotechnology, America), p62 (Santa Cruz Biotechnology, America), AKT (Cell Signaling Technology, America), p-AKT (Cell Signaling Technology, America), ERK1/2 (Cell Signaling Technology, America), and p-ERK1/2(Cell Signaling Technology, America). We used GAPDH (Cell Signaling Technology, America) as a control for normalization. The relative intensity of protein bands was analyzed by Adobe Photoshop CS6.
AML
HD
28
20
11 17
7 13
40 23e53
34 18e53
First, we collected MSCs isolated from twenty healthy donors and twenty-eight AML patients. Expression of surface markers on MSCs indicated that CD34 and CD45 were negative and that CD73, CD90, and CD105 were positive (Supplementary Figure 1), which ultimately verified that the adherence cells were MSCs. Next, we compared the mRNA expression of ATG5 in HD-MSCs and AMLMSCs, and the results of qRT-PCR showed that AML-MSCs had a higher expression level than HD-MSCs (Fig. 1A, p ¼ 0.0086).
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Fig. 1. Elevated expression of ATG5 influenced the characteristics of AML-MSCs. HD represents healthy donors, AML represents AML patients, Control represents MSCs pretreated with DMSO, 3 MA represents MSCs pretreated with 3 MA, nc represents MSCs transfected with negative control, sh represents MSCs transfected with shATG5. (A) mRNA of ATG5 was up regulated in AML-MSCs. (B) Cell cycle analysis of MSCs demonstrated that the proportion of cells in the G0/G1 phase was significantly increased and the proportion of cells in the G2 phase was significantly decreased in 3 MA-MSCs or shMSCs compared to controls. (C) mRNA of CXCL12 was significantly reduced by the addition of 50 mM 3 MA or silencing ATG5 in AML-MSCs. (D) The results of Western blot. Autophagy related genes expression (measured by protein level). Gray values of LC3b II and ATG5 from western blotting were up regulated and p62 was down regulated in AML-MSCs. (E) The potential differentiation capacities of AML-MSCs were decreased by the addition of 50 mM 3 MA. 3 MA-MSCs had less and little fatty drops. The mRNA expression of adipogenic markers PPARG and FABP4 were down regulated in 3 MA-MSCs. Alizarin red staining showed that 3 MA-MSCs had less calcium nodules compared to control. qRT-PCR analyses of osteogenic makers RUNX2 and ALP indicated that these were down regulated in 3 MA-MSCs. (F) The potential differentiation capacities of shMSCs were decreased. There were less and little fatty drops and less calcium nodules in shMSCs. mRNA expression of adipogenic markers and osteogenic makers were all down regulated in shMSCs. *p < 0.05 compared to control. **p < 0.01 compared to control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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Besides, we also detected the protein expression of ATG5, LC3b II and p62 in HD-MSCs (n ¼ 3) and AML-MSCs (n ¼ 5) by Western blot (Fig. 1D). LC3b II (Fig. 1D, p ¼ 0.044) and ATG5 (Fig. 1D, p ¼ 0.017) expression were both upregulated and p62 expression (Fig. 1D, p ¼ 0.036) was down regulated in AML-MSCs. The results of Western blot suggested that autophagy was up-regulated in AMLMSCs. 3.2. Cell cycle distribution and potential differentiation capacities of AML-MSCs were altered by the inhibition of autophagy To explore the impact of autophagy on AML-MSCs, we inhibited autophagy by 50 mM 3 MA treatment for 24 h (Supplementary Figure 2A). Unexpectedly, 3 MA did not significantly influence the apoptosis of AML-MSCs, but it significantly influenced the cell cycle and the differentiation potential. AML-MSCs were arrested at the G0/G1 phase (Fig. 1B, p ¼ 0.020), and the G2 phase fraction was significantly reduced (Fig. 1B, p ¼ 0.020) after treating with 50 mM 3 MA. Additionally, compared to control, potential differentiation capacities of 3 MA treatment group (short for 3 MAMSCs) were reduced compared to controls. It showed that 3 MAMSCs had less and little fatty drops after 14 days of induction (Fig. 1E). mRNA of FABP4 (Fig. 1E, p ¼ 0.003) and PPARG (Fig. 1E, p ¼ 0.007) were significantly down regulated in 3 MA-MSCs. In terms of osteogenesis, 3 MA-MSCs had less calcium nodules after 21 days of induction (Fig. 1E). qRT-PCR showed that both ALP (Fig. 1E, p ¼ 0.025) and RUNX2 (Fig. 1E, p ¼ 0.038) were down regulated in 3 MA-MSCs. To further explore whether the function of autophagy in AMLMSCs was ATG5 dependent, we transfected sh-ATG5 plasmid in AML-MSCs. The results of qRT-PCR and Western blot showed that ATG5 was significantly down regulated (Supplementary Figure 2, p < 0.001), and autophagy was inhibited in shMSCs (Supplementary Figure 2). The proportion of cells in G0/G1 (Fig. 1B, p ¼ 0.016) phase was significantly increased and cells in the G2 (Fig. 1B, p ¼ 0.040) phase was significantly decreased in shMSCs. In addition, the adipogenesis and osteogenesis were both decreased in shMSCs (Fig. 1F). The mRNA of FABP4 (Fig. 1F, p ¼ 0.019), PPARG (Fig. 1F, p ¼ 0.031), RUNX2 (Fig. 1F, p ¼ 0.019) and ALP (Fig. 1F, p ¼ 0.034) were all down regulated in shMSCs. Taken together, inhibition of autophagy by 3 MA or silencing ATG5 in AML-MSCs could change the cell cycle distribution. And it could also reduce the potential differentiation capacities of AMLMSCs. 3.3. Chemosensitivity of leukemic cells was increased after cocultured with 3 MA-MSCs or shMSCs respectively compared to controls CXCL12 secreted by MSCs plays a major role in crosstalk between MSCs and leukemic cells [20]. To explore whether the support effect of MSCs on leukemia was altered after the inhibition of autophagy, we detected the expression of CXCL12. Results suggested that CXCL12 was down regulated in 3 MA-MSCs (Fig. 1C, p ¼ 0.002) and shMSCs (Fig. 1C, p ¼ 0.002) compared to controls. To further explore the support effects of AML-MSCs to AML cell lines, a co-culture system was used. NB4 and THP1 were respectively cocultured with 3 MA-MSCs or control MSCs for 48 h, and then were treated with an incremental concentration of DNR or DOX. After 48 h of treatment, cells were assessed for viability by MTT. IC50 was measured by GraphPad using MTT data. After co-cultured with 3 MA-MSCs, NB4 (Fig. 2A) and THP1 (Fig. 2B) were both more sensitive to DNR and DOX in a dose-dependent manner compared to controls. And IC50 of DNR and DOX were listed in Table 2. Besides, we also measured cell viability at different time points
treated with 0.3 mM DNR or 0.6 mM DOX. The results suggested that the chemosensitivity of NB4 and THP1 co-cultured with 3 MAMSCs were both improved by a time-dependent manner (Fig. 2C and D). Besides, we had similar results from NB4 and THP1 co-cultured with shMSCs or ncMSCs respectively. NB4 (Fig. 2A) and THP1 (Fig. 2B) were more sensitive to DNR or DOX after co-culture with shMSCs respectively. And the detail information of IC50 was listed in Table 2. The chemosensitivity of NB4 (Fig. 2C) and THP1 (Fig. 2D) co-cultured with shMSCs were both improved by a time-dependent manner. 3.4. DNR and DOX induced more apoptosis in THP1 and NB4 cells co-cultured with 3 MA-MSCs or shMSCs respectively compared to controls AML cells lines were treated with DNR and DOX separately after co-cultured with MSCs. The results suggested that 0.3 mM DNR (Fig. 3A, 11.8 ± 1.068 vs. 41.77 ± 2.121, p < 0.001) and 0.6 mM DOX (Figs. 3A and 31.47 ± 1.791 vs. 50.63 ± 0.4096, p < 0.001) respectively induced more apoptosis in NB4 co-cultured with 3 MA-MSCs compared to control. While we had similar results from THP1, using 0.3 mM DNR (Figs. 3B and 8.717 ± 0.355 vs. 42.7 ± 0.953, p < 0.001) and using 0.6 mM DOX (Figs. 3B and 29.8 ± 1.071 vs. 49.61 ± 1.562, p < 0.001) induced more apoptosis in THP1 co-cultured with 3 MAMSCs compared to control respectively. Besides, results from NB4 and THP1 cells co-culture with ncMSCs or shMSCs respectively were consistent with the results mentioned above. 0.3 mM DNR (Figs. 3A and 14.5 ± 0.9738 vs. 35.77 ± 1.631, p < 0.001) and 0.6 mM DOX (Figs. 3A and 29.03 ± 1.241 vs. 49.90 ± 1.31, p < 0.001) induced more apoptosis in NB4 cocultured with shMSCs compared to control. In terms of THP1, using 0.3 mM DNR (Figs. 3B and 11.5 ± 1.091 vs. 32.34 ± 1.447, p < 0.001) and using 0.6 mM DOX (Figs. 3B and 23.96 ± 2.727 vs. 46.25 ± 5.254, p ¼ 0.019) induced more apoptosis in THP1 cocultured with shMSCs compared to control respectively. 3.5. Autophagy in mesenchymal stem cells mediated sensitivity of acute myeloid leukemia cells to chemotherapy via the ERK1/2 and PI3K/AKT pathways Since we observed the influence of autophagy and ATG5 in AMLMSCs, in the next experiments we sought to explore how autophagy and ATG5 influence the protective effects of AML-MSCs to AML cells. We screen out two pathways from several pathways that had been reported were related to the chemotherapy in AML. As Fig. 4 showed, the phosphorylation of ERK1/2 and phosphorylation of AKT were both down regulated in THP1 and NB4 after co-culture with 3 MA-MSCs or shMSCs respectively. This suggested that ERK1/ 2 and AKT pathways could be involved in the protective effects of AML-MSCs to AML cell lines. 4. Discussion To explore the alteration of MSCs in AML, we compared the mRNA profiles of MSCs (GSE122917) derived from healthy donors and de novo AML patients (short for AML-MSCs). Enrichment analyses of differently expressed genes had indicated that autophagy was up regulated in AML-MSCs. To verify the results of enrichment analyses, we detected mRNA and protein expression of several autophagy related genes in AML-MSCs. Among that the upregulation of ATG5 was the most obvious. Besides, the results of Western blot also suggested that autophagy was elevated in AMLMSCs. To further investigate the impacts of autophagy on AML-MSCs,
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Fig. 2. The sensitivity of acute myeloid leukemia cells to chemotherapy was increased by the inhibition of autophagy. Control represents cells co-cultured with control MSCs; 3 MA represents cells co-cultured with 3 MA-MSCs; nc represents cells co-cultured with ncMSCs; sh represents cells co-cultured with shMSCs. (A) NB4 and (B) THP1 co-cultured with 3 MA-MSCs or shMSCs respectively were more sensitive to DNR or DOX compared to controls. It was dose-dependent. Cells were treated with 0.3 mM DNR or 0.6 mM DOX, and the cell viability was measured at 0, 24, 48, 72, 96 h. NB4 (C) and THP1 (D) were more sensitive to DNR or DOX after co-cultured with 3 MA-MSCs or shMSCs. It was time-dependent.
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Table 2 IC50 of DNR and DOX in NB4 and THP1.
NB4 Control vs. 3 MA nc vs. sh THP1 Control vs. 3 MA nc vs. sh
DNR(mM)
DOX(mM)
0.4365 vs. 0.1799 0.4367 vs. 0.2651
0.5806 vs. 0.4869 0.5795 vs. 0.5135
0.4129 vs. 0.3230 0.4146 vs. 0.3325
0.7101 vs. 0.5985 0.7377 vs. 0.6150
Abbreviations: DNR, daunorubicin; DOX, doxorubicin; 3 MA, 3-Methyladenine.
we inhibited autophagy in AML-MSCs by 3 MA. The results showed that the cell cycle and differentiation potential were significantly altered after the inhibition of autophagy in AMLMSCs. In fact, under some circumstances, the influence of autophagy in cell cycle [21] and osteogenic differentiation [22e24] have been reported in other cells. To explore whether the functions of autophagy in AML-MSCs were ATG5 dependent, we knocked down the expression of ATG5 in AML-MSCs. As we expected, silencing ATG5 had similar results with 3 MA treatment.
Take this, we concluded that the influence of autophagy in AMLMSCs was ATG5 dependent. Further, we detected the expression of CXCL12, and the results suggested it was down regulated in 3 MA-MSCs and shMSCs compared to controls. Since the expression of CXCL12 in MSCs contributes to the survival of AML cells [20], we found support for our hypothesis that the protective effect of AML-MSCs to AML cells was reduced after the inhibition of autophagy. We used a co-culture system to evaluate the protective effect of AML-MSCs on AML cells. However, inhibition of autophagy in AML-MSCs only affected the support of AML-MSCs to AML cell lines in the presence of DNR or DOX. This might be influenced by the finding that apoptosis was not often observed in NB4 or THP1 cell lines in the absence of stimulation. Previously researchers have indicated that aberrant activation of ERK1/2 and PI3K/AKT pathways are involved in AML [25]. AML cells were more sensitive to chemotherapy after the inhibition of the two pathways [26,27]. In the present study, the results of western blotting revealed that the phosphorylation of ERK1/2 and AKT in AML cell lines co-cultured with 3 MA-MSCs or shMSCs. This suggested autophagy in MSCs might work in a supporting role for AML
Fig. 3. DNR and DOX induced more apoptosis in NB4 and THP1 co-cultured with 3 MA-MSCs or shMSCs respectively compared to controls. Control represents cells co-cultured with control MSCs; 3 MA represents cells co-cultured with 3 MA-MSCs; nc represents cells co-cultured with ncMSCs; sh represents cells co-cultured with shMSCs. 0.3 mM DNR and 0.6 mM DOX induced an increase in the frequency of observed apoptotic cells in (A) NB4 and (B) THP1 co-cultured with 3 MA-MSCs or shMSCs respectively when compared to results for co-cultures with controls. *p < 0.05 compared to control. ***p < 0.001 compared to control.
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Fig. 4. P-ERK1/2 and p-AKT were down regulated in cells co-cultured with 3 MA-MSCs or shMSCs. Control represents cells co-cultured with control MSCs; 3 MA represents cells cocultured with 3 MA-MSCs; nc represents cells co-cultured with ncMSCs; sh represents cells co-cultured with shMSCs. Western blot results reported that p-ERK1/2 and p-AKT were down regulated in NB4 and THP1 co-cultured with 3 MA-MSCs or shMSCs respectively compared to controls. **p < 0.01 compared to control. ***p < 0.001 compared to control.
cell lines via activating the ERK1/2 and PI3K/AKT pathways in AML cell lines. In conclusion, we demonstrated that ATG5 and autophagy were up regulated in AML-MSCs. Inhibition of autophagy by 3 MA or
silencing ATG5 significantly altered the cell cycle distribution and decreased differentiation capacities of AML-MSCs. Moreover, the sensitivity of AML cells to genotoxic agents was improved after cocultured with 3 MA-MSCs or shMSCs compared to controls, and it
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was related to ERK1/2 and AKT pathway. Declaration of competing interest The author reports no conflicts of interest in this work.
[11]
[12]
Acknowledgements [13]
This work was supported by the Chinese Academy of Medical Sciences Initiative for Innovative Medicine (CAMS-I2M-2016-1017) and National Natural Science Foundation of China (81170510).
[14]
Appendix A. Supplementary data
[15]
Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.02.091.
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Please cite this article as: Y. Li et al., ATG5 regulates mesenchymal stem cells differentiation and mediates chemosensitivity in acute myeloid leukemia, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.02.091