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Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma Yuta Tanaka a, Takato Takenouchi b, Mitsutoshi Tsukimoto a, * a
Department of Radiation Biosciences, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba, 278-0022, Japan Division of Animal Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 1-2 Ohwashi, Tsukuba, Ibaraki, 305-8634, Japan
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Article history: Received 14 January 2020 Accepted 30 January 2020 Available online xxx
Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a neuroprotective factor produced in response to endoplasmic reticulum (ER) stress induced by various stressors, but its involvement in the radioresistance of tumor cells is unknown. Here, we found that MANF is released after g-irradiation (2 Gy and 4 Gy) of B16 melanoma cells, and its release was suppressed by 4-phenylbutyric acid, an ER stress inhibitor. MANF was not released after low-dose (1 Gy) g-irradiation, but pretreatment of 1 Gy-irradiated cells with recombinant MANF enhanced the cellular DNA damage response and attenuated reproductive cell death. In MANF-knockdown cells, the DNA damage response and p53 activation by g-irradiation (2 Gy) were suppressed, and reproductive cell death was increased. MANF also activated the ERK signaling pathway. Our findings raise the possibility that MANF could be a new target for overcoming radioresistance. © 2020 Elsevier Inc. All rights reserved.
Keywords: MANF Melanoma Radioresistance DNA repair Radiation therapy
1. Introduction Melanoma is a type of skin cancer with high metastatic and proliferative potential, and has a poor prognosis unless it is detected early and surgically removed. Radiotherapy is often used as an adjuvant therapy, but advanced melanoma is often radioresistant [1e3]. The cancer-killing effect of radiation is due to DNA damage caused by reactive oxygen species (ROS) [4], but DNA damage repair can enable irradiated cells to survive, regrow, and acquire radioresistance. Therefore, the identification of factors associated with radioresistance is very important. One of the cellular responses to radiation is the unfolded protein response (UPR), which occurs because the endoplasmic reticulum (ER), an intracellular organelle that synthesizes and folds proteins, becomes dysfunctional under stress conditions [5,6]. This leads to inhibition of protein translation and activation of the ubiquitin/ proteasome protein degradation system [6]. If the stress is severe and prolonged, downstream signaling via the PERK (protein kinase R (PKR)-like ER kinase), ATF6 (activating transcription factor 6), and IRE1 (inositol-requiring enzyme 1a/b) pathways is excessively activated, inducing apoptosis [5,7]. The UPR is thought to interact
with the DNA damage response to promote the acquisition of radioresistance in cancer [8]. ER stress is also known to induce mesencephalic astrocytederived neurotrophic factor (MANF), a small, secreted protein with a selective neuroprotective action on dopaminergic neurons [9]. It also has a protective role against ER stress [10e14], and was shown to induce repair of damaged retinas in flies and mice via alternative activation of innate M2-type immune cells [15]. However, its receptors have not yet been identified [16]. Notably, MANF is widely expressed in various tissues and organs, and was initially known as ARMET (arginine-rich, mutated in early stage of tumors) [17]. The ARMET gene is mutated in a variety of human cancers [17]. However, the relationship between ARMET/MANF and melanoma has not been established. In this study, we investigated the role of MANF in the cellular responses of melanoma to radiation-induced DNA damage, including phosphorylation of ataxia telangiectasia mutation (ATM), formation of phosphorylated histone variant H2AX (gH2AX) foci, and accumulation of p53 binding protein 1 (53BP1), which occur within 1 h after irradiation [18e20]. Our results indicate that MANF is released by irradiated melanoma cells, and contributes to radioresistance by promoting the cellular DNA damage response.
* Corresponding author. E-mail address: [email protected] (M. Tsukimoto). https://doi.org/10.1016/j.bbrc.2020.01.167 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Tanaka et al., Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.167
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Fig. 1. Expression and release of MANF after g-irradiation of B16 cells. (A) MANF protein was detected in B16 cells by Western blotting at about 20 kDa (arrow). (BeD) To examine radiation-induced extracellular release of MANF protein, cells were irradiated with 1.0 (B), 2.0 (C) or 4.0 Gy (D) of g-rays and then incubated for 6 h. Western blotting was done at the indicated times (top), and the bands were quantitated with a densitometer (bottom). To evaluate significance, Mann-Whitney rank sum test was performed. Columns and bars show means ± S.E. (n ¼ 3). Typical data of 3 independent experiments are shown. Significant difference from non-irradiated cells in irradiated cells: * (P < 0.05). (E) Cells were pre-incubated for 1 h with 4-PBA (0.5 or 1 mM), irradiated with 2.0 Gy of g-rays, further incubated for 1 h, and subjected to Western blotting analysis (top). The bands were quantitated with a densitometer (bottom). Typical data of 3 independent experiments are shown. Columns and bars show means ± S.E. (n ¼ 3). Significant difference from non-irradiated cells: # (P < 0.05). Significant difference from irradiated cells in the absence of 4-PBA: * (P < 0.05).
Please cite this article as: Y. Tanaka et al., Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.167
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2. Materials and methods 2.1. Reagents Dulbecco’s modified Eagle’s medium (DMEM) was purchased from FUJIFILM Wako Pure Chemical Corporation. (Osaka, Japan). Gibco® fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific (U.S.A). The primary antibodies used were anti53BP1 rabbit polyclonal antibody (Novus, U.S.A.), anti-phosphohistone H2AX (Ser139) rabbit monoclonal antibody (Cell Signaling Technology, U.S.A.), anti-extracellular signal-regulated kinase (ERK) 1/2 mAb and anti-phospho-ERK 1/2 (Thr202/Tyr204) mAb (Cell Signaling Technology), rabbit anti-MANF antibody (ProSci Inc., U.S.A.), anti-p53 (D2H90) rabbit mAb (rodent specific) and anti-p-p53 (S15) rabbit Ab (Cell Signaling Technology). 4Phenylbutyric acid (4-PBA), an ER stress inhibitor, was obtained from Sigma-Aldrich.
2.2. Cell culture and irradiation Mouse melanoma B16 cells were grown in DMEM supplemented with 10% FBS, penicillin (100 units/mL) and streptomycin (100 mg/mL) in a humidified atmosphere of 5% CO2 in air at 37 C, as described previously [21]. B16 cells were irradiated with g-rays from a Gammacell 40 (137Cs source) (Nordin International, Inc.; 0.72 Gy/min) at room temperature for an indicated time. After irradiation, the cells were incubated in a humidified atmosphere of 5% CO2 in air at 37 C.
2.3. Production of recombinant MANF protein The secreted form of recombinant mouse MANF protein fused with an N-terminal His-tag (rMANF) was produced using the Brevibacillus In vivo Cloning (BIC) System (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. The DNA fragment corresponding to mouse MANF (22e179 aa) was amplified by PCR and inserted into the pBIC4 expression vector. A mouse liver PCR Ready cDNA kit (Maxim Biotech, Inc., Rockville, MD) was used for PCR amplification. The vector carrying the MANF gene was validated by DNA sequence analysis. rMANF expressed in the culture supernatant of Brevibacillus choshinensis transformants was purified on His GraviTrap columns (GE Healthcare). The eluate was extensively dialyzed against PBS with a Float-A-Lyzer Dialysis Device (3.5e5 kDa cutoff, REPLIGEN, Waltham, MA), and concentrated using an Amicon Ultra-15 filter (3 kDa cutoff, Merck, Darmstadt, Germany), and the solvent was changed to Dulbecco’s phosphatebuffered saline. The protein purity was analyzed by 15% SDSPAGE followed by Quick-CBB staining (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan). The protein concentration was measured using a BCA protein assay kit (Pierce, Rockford. IL).
Fig. 2. Effect of MANF treatment on radioresistance in B16 cells. (A) DNA repair response (focus formation). Cells were treated with MANF (1 mg/mL) for
3 h, irradiated with g-rays (1.0e4.0 Gy), incubated for 0.5 h, and then immunostained for 53BP1 and gH2AX. Co-localized foci of 53BP1 and gH2AX in nuclei were counted. The data represent means ± S.E. (n ¼ 63e82). (B) Cell survival (colony formation assay). Cells were irradiated with g-rays (1.0e4.0 Gy), incubated for 24 h, then seeded in 60 mm dishes (4.0 102 cells/dish), further incubated for a week, and stained with crystal violet. Stained colonies were counted (>50 cells/colony). The data represent means ± S.E. (n ¼ 6). Open columns show vehicle (DMSO) controls, and solid bars show MANF treatment. (C) Phosphorylation of ERK1/2 in whole-cell lysates. B16 cells were treated with MANF (1 mg/mL), incubated for 0.5e6 h, and immunostained for total ERK1/2 and phosphorylated ERK1/2 (top). The bands were quantitated with a densitometer, and the ratio of phosphorylated ERK1/2 was calculated (bottom). Columns and bars show mean ± S.E. (n ¼ 4). Statistically significant differences between DMSO treatment and MANF treatment: ### (P < 0.001) or ## (P < 0.01). Statistically significant difference from control cells: * (P < 0.05). n.s.: not significant.
Please cite this article as: Y. Tanaka et al., Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.167
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2.4. Immunofluorescence staining
2.5. Colony formation assay
DNA damage response was quantified by immunofluorescence staining of gH2AX and 53BP1, as described previously [21].
The survival rate was quantified by colony formation assay, as described previously [21].
Fig. 3. Effect of MANF knockdown on radioresistance in B16 cells. MANF knockdown was done by transfection of B16 cells with siRNA duplex oligonucleotides directed against mouse MANF. (A) Confirmation of MANF knockdown. At 48 h after transfection, MANF protein (20 kDa) was detected by Western blotting (top). The bands were quantitated with a densitometer (bottom). Columns and bars show mean ± S.E. (n ¼ 3). Significant difference from scramble siRNA-transfected cells: ** (P < 0.01). (B) Time course of extracellular MANF. At 48 h after transfection, cells were irradiated with g-rays of 2.0 Gy and incubated for 3 h. Medium was collected periodically for Western blotting analysis (top). The bands were quantitated with a densitometer (bottom). Columns and bars show mean ± S.E. (n ¼ 3). Significant difference from irradiated scramble siRNA-transfected cells: *** (P < 0.001) or * (P < 0.05). (C) Colony formation. At 48 h after transfection, cells were irradiated with g-rays (2.0 Gy; solid bars) or not irradiated (open bars), seeded in 60 mm dishes (4.0 102 cells/dish), incubated for 1 week, and stained with crystal violet. Stained colonies (>50 cells/colony) were counted. The data represent means ± S.E. (n ¼ 6). Typical dishes are shown (top), together with the calculated survival fraction (bottom). Significant difference from non-irradiated scramble siRNA-transfected cells: # (P < 0.05). Significant difference from irradiated scramble siRNA-transfected cells: ** (P < 0.01). (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. Tanaka et al., Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.167
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Fig. 4. Effect of MANF knockdown on radioresistance factor in B16 cells. MANF knockdown was done by transfection of B16 cells with siRNA duplex oligonucleotides directed against mouse MANF. (A, B) Effect on DNA repair response. (A) At 48 h after transfection, the cells were irradiated with 2.0 Gy of g-rays and incubated for 30 min, and focus formation was evaluated as a measure of DNA repair response. (B) At 48 h after transfection, the cells were irradiated with 4.0 Gy of g-rays and incubated for 24 h, and focus formation was evaluated as a measure of unrepaired DNA damage. Focus formation was evaluated by immunostaining, and co-localized foci of 53BP1 and gH2AX in nuclei were counted. The data represent means ± S.E. (A; n ¼ 35e52, B; n ¼ 49e80). Significant difference from non-irradiated scramble siRNA-transfected cells: ### (P < 0.001) or # (P < 0.05). Significant difference from irradiated scramble siRNA-transfected cells: *** (P < 0.001). (C, D) Effect on p53 phosphorylation. At 48 h after transfection, the cells were irradiated with 2.0 Gy of g-rays and incubated for 1 h (C) or 24 h (D). Phosphorylated p53 in whole-cell lysates was determined by immunoblotting (top). The data represent means ± S.E. (n ¼ 4). Columns and bars show the ratio of phosphorylated p53 to actin in scramble siRNA-transfected cells (open bars) and MANF-knockdown cells (solid bars). Values are mean ± S.E. (bottom). Significant difference from non-irradiated scramble siRNA-transfected cells: ### (P < 0.001). Significant difference from irradiated scramble siRNA-transfected cells: ** (P < 0.01) or * (P < 0.05).
2.6. Western blotting Protein was detected by immunoblotting as described previously [22]. Culture supernatant was concentrated on Amicon®Ultra Centrifugal Filters (10 K), then mixed with 4 Laemmli Sample Buffer (BioRAD) and 10 mM DTT, and incubated at 95 C for 10 min. Aliquots were subjected to 10% (for ERK1/2 and p53, p-p53 detection) or 12% (for MANF detection) SDS-PAGE. The primary
antibodies used were anti-ERK1/2 antibody and anti-phosphoERK1/2 (Thr202/Tyr204) antibody (1:1000) for detection of ERK1/ 2 activation, rabbit MANF antibody (1:1000) for detection of MANF, p53 (D2H90) rabbit antibody for detection of p53, and p-p53 (S15) rabbit antibody for detection of p53 activation. The secondary antibodies used were goat horseradish peroxidase-conjugated antirabbit IgG antibody (1:20,000) for detection of ERK1/2 activation, and goat horseradish peroxidase-conjugated anti-rabbit IgG
Please cite this article as: Y. Tanaka et al., Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.167
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antibody (1:5000) for detection of MANF and p53/p-p53 at room temperature for 1.5 h. As a loading control, the blots were incubated with peroxidase-conjugated anti-b-actin monoclonal antibody (FUJIFILM Wako Pure Chemical Corp.) (1:50,000) at room temperature for 60 min. 2.7. Small interfering RNA (siRNA) transfection SiRNA targeting MANF and negative control siRNA (TriFECTa Kit® DsiRNA Duplex) were purchased from Integrated DNA Technology. B16 cells (5.0 104 cells/well) were incubated in culture for 16 h, then transfected with siRNA duplex oligonucleotides (25 nM) for knockdown of mouse MANF by using Lipofectamine RNAiMAX (Invitrogen) and Opti-MEM Reduced Serum Medium (Invitrogen) according to the manufacturer’s instructions. Cells were used for Western blotting, immunofluorescence and colony formation assay at 48 h after transfection. 2.8. Statistics Results are expressed as the mean ± standard error (S.E.). The statistical significance of differences between the control and other groups was calculated by using Dunnett’s test or Mann-Whitney rank sum test. Calculations (Dunnett’s test) were done with the Instat version 3.0 statistical software package (Graph Pad Software). The criterion of significance was set at p < 0.05. 3. Results & discussion First, we confirmed that the neurotrophic factor MANF is expressed in B16 melanoma cells by means of western blotting (Fig. 1A). Although 1.0 Gy g-rays failed to induce release of MANF (Fig. 1B), increased release of MANF was observed in response to girradiation at a dose of 2.0 Gy (Fig. 1C) or 4.0 Gy (Fig. 1D) with statistical significance. However, in the presence of 0.5 or 1.0 mM 4PBA, which inhibits ER stress [23], the radiation-induced extracellular release of MANF was blocked with statistical significance (Fig. 1E). These results suggest that ER stress results in the release of MANF in melanoma cells. Furthermore, we also examined the change in amount of intracellular MANF after g-irradiation. Though 1.0 Gy g-rays failed to induce expression of MANF, expression of MANF was increased in response to g-irradiation at a dose of 2.0 Gy or 4.0 Gy with no statistical significance (Supplementary Fig. 1). It is considered that the intracellular MANF did not significantly change because the intracellular MANF was much higher than the extracellularly secreted MANF. When cells were treated with MANF for 3 h before irradiation, the DNA damage response, evaluated in terms of gH2AX-53BP1 focus formation in the nuclei (Fig. 2A), and reproductive cell death (Fig. 2B) in 2.0 or 4.0 Gy irradiated cells were not affected, presumably because release of MANF was induced at these radiation doses (Fig. 1C and D), and was protective. On the other hand, MANF treatment followed by 1.0 Gy of g-rays, which is insufficient to induce endogenous MANF release (see Fig. 1B), increased the DNA damage response and suppressed reproductive cell death (Fig. 2A and B). These results indicate that endogenous MANF contributes to radioresistance by promoting DNA damage repair in melanoma cells. In addition, MANF induced ERK1/2 activation in B16 melanoma cells (Fig. 2C). Thus, g-ray-induced release of MANF activates signaling pathways, such as ERK, that promote the malignant phenotype. Further studies will be needed to establish how MANF triggers downstream signal activation. Next, to confirm the involvement of MANF in radioresistance, B16 cells were transfected with siRNA targeting MANF. Expression of MANF protein was decreased in MANF knockdown cells to 50.3%
of that in scramble siRNA-transfected cells, as determined by Western blotting (Fig. 3A). The release of MANF at 1 h after girradiation (2.0 Gy) was significantly suppressed by MANF knockdown (Fig. 3B). Furthermore, reproductive cell death induced by girradiation (2.0 Gy) was significantly enhanced in MANF-KD cells (Fig. 3C). These results support the idea that g-ray-induced MANF release promotes the survival of melanoma cells. We next investigated the effect of MANF on various factors related to cancer cell survival. First, to clarify the involvement of MANF in the DNA damage response, we evaluated the effect of MANF knockdown on gH2AX-53BP1 focus formation at DNA damage sites in g-irradiated B16 melanoma cells. Focus formation (co-staining with gH2AX and 53BP1) was significantly reduced in MANF knockdown cells (Fig. 4A). In order to examine the longerterm effects, we also evaluated gH2AX-53BP1 focus formation at 24 h after g-irradiation (4.0 Gy) as a measure of unrepaired DNA damage. As shown in Fig. 4B, DNA damage was still apparent at 24 h after g-irradiation in MANF-KD cells. Since p53 is a radioresistance factor [24,25], we also investigated activation of p53 in MANFknockdown cells. The activation of p53 after 1 h of g-irradiation was significantly suppressed, and persistence of the activated state was observed after 24 h in the knockdown cells (Fig. 4C and D). These results strongly support the view that MANF is involved in DNA damage response and cell survival after g-irradiation and contributes to radioresistance. It has been reported that ER stress response contributes to the resistance of glioblastoma to radiotherapy [26], and that MANF is upregulated by acute ER stress, including chemical substances, ischemic conditions, and epileptic seizures [11,27]. Furthermore, cerebral ischemia induces neuronal cell death by ER stress [28,29], and during this process, MANF expression is induced and has a protective effect against cell death [30]. Also, recombinant human MANF inhibits apoptosis of neurons induced by ER stress [30]. Our present findings that release of MANF is induced by g-irradiation, and contributes to radioresistance are consistent with these results. However, this is the first report to demonstrate a role of MANF in radioresistance of cancer and melanoma. As noted above, given the possibility that MANF contributes to the radioresistance of glioblastoma [11,26,27], we speculate that MANF might be a candidate therapeutic target for glioblastoma. In addition, further studies are needed for cancer types such as lung and breast cancers for which radiation therapy is generally used. On the other hand, MANF promotes a switch of macrophage phenotype from pro-inflammatory to anti-inflammatory [15]. Furthermore, there are reports that M1 macrophages (pro-inflammatory) are increased in MANF-knockout mice, while M2 macrophages (anti-inflammatory) are increased under conditions of liver fibrosis [31]. These reports suggest that MANF also plays a role in suppressing inflammatory responses and controlling immune functions. As regards the mechanism of MANF’s action, we found that it activates the ERK signaling pathway, which lies downstream of the B-Raf proto-oncogene serine/threonine-kinase (BRAF). Recently, it was shown that treatments targeting the (BRAF)V600 (Val600) mutation in melanoma patients by combining BRAF inhibitors with mitogen-activated protein kinase inhibitors improved survival [32]. In addition, MAPK activation is thought to be a major cause of melanoma radioresistance, and 50e60% of melanomas are reported to have BRAF-activating mutations [33]. We found here that MANF knockdown suppresses the activation of p53, which is also a radioresistance factor. Further studies will be needed to elucidate in detail the downstream signaling pathways from MANF. There appears to be a possibility that treatment with MANF inhibitors or anti-MANF antibody might be therapeutically useful in combination with existing melanoma treatments. It will be interesting in
Please cite this article as: Y. Tanaka et al., Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.167
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the future to study the role of MANF in other cancer types. Acknowledgement This work was supported in part by JSPS KAKENHI Grant number JP 16K08148 (Grant-in-Aid for Scientific Research (C)) (to TT and MT). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.167. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.167. References [1] A. Mahadevan, V.L. Patel, N. Dagoglu, Radiation therapy in the management of malignant melanoma, Oncology (Williston Park) 29 (2015) 743e751. [2] B.H. Burmeister, M.A. Henderson, J. Ainslie, et al., Adjuvant radiotherapy versus observation alone for patients at risk of lymph-node field relapse after therapeutic lymphadenectomy for melanoma: a randomised trial, Lancet Oncol. 13 (2012) 589e597. [3] K. Satyamoorthy, N.H. Chehab, M.J. Waterman, et al., Aberrant regulation and function of wild- type p53 in radioresistant melanoma cells, Cell Growth Differ. 11 (2000) 467e474. [4] W. Yuan, Y. Yuan, T. Zhang, et al., Role of Bmi-1 in regulation of ionizing irradiation-induced epithelial-mesenchymal transition and migration of breast cancer cells, PloS One 10 (2015), e0118799. [5] W. Kim, S. Lee, D. Seo, et al., Cellular stress responses in radiotherapy, Cells 8 (2019) 1105. [6] H. Yoshida, ER stress and diseases, FEBS J. 274 (2007) 630e658. [7] S. Kanemoto, K. Imaizumi, Endoplasmic reticulum stress and diseases, J. Jpn. Biochem. Soc. 90 (2018) 51e59. [8] A. Nagelkerke, J. Bussink, A.J. van der Kogel, et al., The PERK/ATF4/LAMP3-arm of the unfolded protein response affects radioresistance by interfering with the DNA damage response, Radiother. Oncol. 108 (2013) 415e421. [9] P. Petrova, A. Raibekas, J. Pevsner, et al., MANF: a new mesencephalic, astrocyte-derived neurotrophic factor with selectivity for dopaminergic neurons, J. Mol. Neurosci. 20 (2003) 173e188. [10] N. Mizobuchi, J. Hoseki, H. Kubota, et al., ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element, Cell Struct. Funct. 32 (2007) 41e50. [11] A. Apostolou, Y. Shen, Y. Liang, et al., Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death, Exp. Cell Res. 314 (2008) 2454e2467. [12] A. Tadimalla, P.J. Belmont, D.J. Thuerauf, et al., Mesencephalic astrocytederived neurotrophic factor is an ischemia-inducible secreted endoplasmic reticulum stress response protein in the heart, Circ. Res. 103 (2008) 1249e1258.
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Please cite this article as: Y. Tanaka et al., Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.167
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma Yuta Tanaka a, Takato Takenouchi b, Mitsutoshi Tsukimoto a, * a
Department of Radiation Biosciences, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda-shi, Chiba, 278-0022, Japan Division of Animal Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, 1-2 Ohwashi, Tsukuba, Ibaraki, 305-8634, Japan
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a b s t r a c t
Article history: Received 14 January 2020 Accepted 30 January 2020 Available online xxx
Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a neuroprotective factor produced in response to endoplasmic reticulum (ER) stress induced by various stressors, but its involvement in the radioresistance of tumor cells is unknown. Here, we found that MANF is released after g-irradiation (2 Gy and 4 Gy) of B16 melanoma cells, and its release was suppressed by 4-phenylbutyric acid, an ER stress inhibitor. MANF was not released after low-dose (1 Gy) g-irradiation, but pretreatment of 1 Gy-irradiated cells with recombinant MANF enhanced the cellular DNA damage response and attenuated reproductive cell death. In MANF-knockdown cells, the DNA damage response and p53 activation by g-irradiation (2 Gy) were suppressed, and reproductive cell death was increased. MANF also activated the ERK signaling pathway. Our findings raise the possibility that MANF could be a new target for overcoming radioresistance. © 2020 Elsevier Inc. All rights reserved.
Keywords: MANF Melanoma Radioresistance DNA repair Radiation therapy
1. Introduction Melanoma is a type of skin cancer with high metastatic and proliferative potential, and has a poor prognosis unless it is detected early and surgically removed. Radiotherapy is often used as an adjuvant therapy, but advanced melanoma is often radioresistant [1e3]. The cancer-killing effect of radiation is due to DNA damage caused by reactive oxygen species (ROS) [4], but DNA damage repair can enable irradiated cells to survive, regrow, and acquire radioresistance. Therefore, the identification of factors associated with radioresistance is very important. One of the cellular responses to radiation is the unfolded protein response (UPR), which occurs because the endoplasmic reticulum (ER), an intracellular organelle that synthesizes and folds proteins, becomes dysfunctional under stress conditions [5,6]. This leads to inhibition of protein translation and activation of the ubiquitin/ proteasome protein degradation system [6]. If the stress is severe and prolonged, downstream signaling via the PERK (protein kinase R (PKR)-like ER kinase), ATF6 (activating transcription factor 6), and IRE1 (inositol-requiring enzyme 1a/b) pathways is excessively activated, inducing apoptosis [5,7]. The UPR is thought to interact
with the DNA damage response to promote the acquisition of radioresistance in cancer [8]. ER stress is also known to induce mesencephalic astrocytederived neurotrophic factor (MANF), a small, secreted protein with a selective neuroprotective action on dopaminergic neurons [9]. It also has a protective role against ER stress [10e14], and was shown to induce repair of damaged retinas in flies and mice via alternative activation of innate M2-type immune cells [15]. However, its receptors have not yet been identified [16]. Notably, MANF is widely expressed in various tissues and organs, and was initially known as ARMET (arginine-rich, mutated in early stage of tumors) [17]. The ARMET gene is mutated in a variety of human cancers [17]. However, the relationship between ARMET/MANF and melanoma has not been established. In this study, we investigated the role of MANF in the cellular responses of melanoma to radiation-induced DNA damage, including phosphorylation of ataxia telangiectasia mutation (ATM), formation of phosphorylated histone variant H2AX (gH2AX) foci, and accumulation of p53 binding protein 1 (53BP1), which occur within 1 h after irradiation [18e20]. Our results indicate that MANF is released by irradiated melanoma cells, and contributes to radioresistance by promoting the cellular DNA damage response.
* Corresponding author. E-mail address: [email protected] (M. Tsukimoto). https://doi.org/10.1016/j.bbrc.2020.01.167 0006-291X/© 2020 Elsevier Inc. All rights reserved.
Please cite this article as: Y. Tanaka et al., Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.167
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Fig. 1. Expression and release of MANF after g-irradiation of B16 cells. (A) MANF protein was detected in B16 cells by Western blotting at about 20 kDa (arrow). (BeD) To examine radiation-induced extracellular release of MANF protein, cells were irradiated with 1.0 (B), 2.0 (C) or 4.0 Gy (D) of g-rays and then incubated for 6 h. Western blotting was done at the indicated times (top), and the bands were quantitated with a densitometer (bottom). To evaluate significance, Mann-Whitney rank sum test was performed. Columns and bars show means ± S.E. (n ¼ 3). Typical data of 3 independent experiments are shown. Significant difference from non-irradiated cells in irradiated cells: * (P < 0.05). (E) Cells were pre-incubated for 1 h with 4-PBA (0.5 or 1 mM), irradiated with 2.0 Gy of g-rays, further incubated for 1 h, and subjected to Western blotting analysis (top). The bands were quantitated with a densitometer (bottom). Typical data of 3 independent experiments are shown. Columns and bars show means ± S.E. (n ¼ 3). Significant difference from non-irradiated cells: # (P < 0.05). Significant difference from irradiated cells in the absence of 4-PBA: * (P < 0.05).
Please cite this article as: Y. Tanaka et al., Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.167
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2. Materials and methods 2.1. Reagents Dulbecco’s modified Eagle’s medium (DMEM) was purchased from FUJIFILM Wako Pure Chemical Corporation. (Osaka, Japan). Gibco® fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific (U.S.A). The primary antibodies used were anti53BP1 rabbit polyclonal antibody (Novus, U.S.A.), anti-phosphohistone H2AX (Ser139) rabbit monoclonal antibody (Cell Signaling Technology, U.S.A.), anti-extracellular signal-regulated kinase (ERK) 1/2 mAb and anti-phospho-ERK 1/2 (Thr202/Tyr204) mAb (Cell Signaling Technology), rabbit anti-MANF antibody (ProSci Inc., U.S.A.), anti-p53 (D2H90) rabbit mAb (rodent specific) and anti-p-p53 (S15) rabbit Ab (Cell Signaling Technology). 4Phenylbutyric acid (4-PBA), an ER stress inhibitor, was obtained from Sigma-Aldrich.
2.2. Cell culture and irradiation Mouse melanoma B16 cells were grown in DMEM supplemented with 10% FBS, penicillin (100 units/mL) and streptomycin (100 mg/mL) in a humidified atmosphere of 5% CO2 in air at 37 C, as described previously [21]. B16 cells were irradiated with g-rays from a Gammacell 40 (137Cs source) (Nordin International, Inc.; 0.72 Gy/min) at room temperature for an indicated time. After irradiation, the cells were incubated in a humidified atmosphere of 5% CO2 in air at 37 C.
2.3. Production of recombinant MANF protein The secreted form of recombinant mouse MANF protein fused with an N-terminal His-tag (rMANF) was produced using the Brevibacillus In vivo Cloning (BIC) System (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. The DNA fragment corresponding to mouse MANF (22e179 aa) was amplified by PCR and inserted into the pBIC4 expression vector. A mouse liver PCR Ready cDNA kit (Maxim Biotech, Inc., Rockville, MD) was used for PCR amplification. The vector carrying the MANF gene was validated by DNA sequence analysis. rMANF expressed in the culture supernatant of Brevibacillus choshinensis transformants was purified on His GraviTrap columns (GE Healthcare). The eluate was extensively dialyzed against PBS with a Float-A-Lyzer Dialysis Device (3.5e5 kDa cutoff, REPLIGEN, Waltham, MA), and concentrated using an Amicon Ultra-15 filter (3 kDa cutoff, Merck, Darmstadt, Germany), and the solvent was changed to Dulbecco’s phosphatebuffered saline. The protein purity was analyzed by 15% SDSPAGE followed by Quick-CBB staining (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan). The protein concentration was measured using a BCA protein assay kit (Pierce, Rockford. IL).
Fig. 2. Effect of MANF treatment on radioresistance in B16 cells. (A) DNA repair response (focus formation). Cells were treated with MANF (1 mg/mL) for
3 h, irradiated with g-rays (1.0e4.0 Gy), incubated for 0.5 h, and then immunostained for 53BP1 and gH2AX. Co-localized foci of 53BP1 and gH2AX in nuclei were counted. The data represent means ± S.E. (n ¼ 63e82). (B) Cell survival (colony formation assay). Cells were irradiated with g-rays (1.0e4.0 Gy), incubated for 24 h, then seeded in 60 mm dishes (4.0 102 cells/dish), further incubated for a week, and stained with crystal violet. Stained colonies were counted (>50 cells/colony). The data represent means ± S.E. (n ¼ 6). Open columns show vehicle (DMSO) controls, and solid bars show MANF treatment. (C) Phosphorylation of ERK1/2 in whole-cell lysates. B16 cells were treated with MANF (1 mg/mL), incubated for 0.5e6 h, and immunostained for total ERK1/2 and phosphorylated ERK1/2 (top). The bands were quantitated with a densitometer, and the ratio of phosphorylated ERK1/2 was calculated (bottom). Columns and bars show mean ± S.E. (n ¼ 4). Statistically significant differences between DMSO treatment and MANF treatment: ### (P < 0.001) or ## (P < 0.01). Statistically significant difference from control cells: * (P < 0.05). n.s.: not significant.
Please cite this article as: Y. Tanaka et al., Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.167
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2.4. Immunofluorescence staining
2.5. Colony formation assay
DNA damage response was quantified by immunofluorescence staining of gH2AX and 53BP1, as described previously [21].
The survival rate was quantified by colony formation assay, as described previously [21].
Fig. 3. Effect of MANF knockdown on radioresistance in B16 cells. MANF knockdown was done by transfection of B16 cells with siRNA duplex oligonucleotides directed against mouse MANF. (A) Confirmation of MANF knockdown. At 48 h after transfection, MANF protein (20 kDa) was detected by Western blotting (top). The bands were quantitated with a densitometer (bottom). Columns and bars show mean ± S.E. (n ¼ 3). Significant difference from scramble siRNA-transfected cells: ** (P < 0.01). (B) Time course of extracellular MANF. At 48 h after transfection, cells were irradiated with g-rays of 2.0 Gy and incubated for 3 h. Medium was collected periodically for Western blotting analysis (top). The bands were quantitated with a densitometer (bottom). Columns and bars show mean ± S.E. (n ¼ 3). Significant difference from irradiated scramble siRNA-transfected cells: *** (P < 0.001) or * (P < 0.05). (C) Colony formation. At 48 h after transfection, cells were irradiated with g-rays (2.0 Gy; solid bars) or not irradiated (open bars), seeded in 60 mm dishes (4.0 102 cells/dish), incubated for 1 week, and stained with crystal violet. Stained colonies (>50 cells/colony) were counted. The data represent means ± S.E. (n ¼ 6). Typical dishes are shown (top), together with the calculated survival fraction (bottom). Significant difference from non-irradiated scramble siRNA-transfected cells: # (P < 0.05). Significant difference from irradiated scramble siRNA-transfected cells: ** (P < 0.01). (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. Tanaka et al., Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.167
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Fig. 4. Effect of MANF knockdown on radioresistance factor in B16 cells. MANF knockdown was done by transfection of B16 cells with siRNA duplex oligonucleotides directed against mouse MANF. (A, B) Effect on DNA repair response. (A) At 48 h after transfection, the cells were irradiated with 2.0 Gy of g-rays and incubated for 30 min, and focus formation was evaluated as a measure of DNA repair response. (B) At 48 h after transfection, the cells were irradiated with 4.0 Gy of g-rays and incubated for 24 h, and focus formation was evaluated as a measure of unrepaired DNA damage. Focus formation was evaluated by immunostaining, and co-localized foci of 53BP1 and gH2AX in nuclei were counted. The data represent means ± S.E. (A; n ¼ 35e52, B; n ¼ 49e80). Significant difference from non-irradiated scramble siRNA-transfected cells: ### (P < 0.001) or # (P < 0.05). Significant difference from irradiated scramble siRNA-transfected cells: *** (P < 0.001). (C, D) Effect on p53 phosphorylation. At 48 h after transfection, the cells were irradiated with 2.0 Gy of g-rays and incubated for 1 h (C) or 24 h (D). Phosphorylated p53 in whole-cell lysates was determined by immunoblotting (top). The data represent means ± S.E. (n ¼ 4). Columns and bars show the ratio of phosphorylated p53 to actin in scramble siRNA-transfected cells (open bars) and MANF-knockdown cells (solid bars). Values are mean ± S.E. (bottom). Significant difference from non-irradiated scramble siRNA-transfected cells: ### (P < 0.001). Significant difference from irradiated scramble siRNA-transfected cells: ** (P < 0.01) or * (P < 0.05).
2.6. Western blotting Protein was detected by immunoblotting as described previously [22]. Culture supernatant was concentrated on Amicon®Ultra Centrifugal Filters (10 K), then mixed with 4 Laemmli Sample Buffer (BioRAD) and 10 mM DTT, and incubated at 95 C for 10 min. Aliquots were subjected to 10% (for ERK1/2 and p53, p-p53 detection) or 12% (for MANF detection) SDS-PAGE. The primary
antibodies used were anti-ERK1/2 antibody and anti-phosphoERK1/2 (Thr202/Tyr204) antibody (1:1000) for detection of ERK1/ 2 activation, rabbit MANF antibody (1:1000) for detection of MANF, p53 (D2H90) rabbit antibody for detection of p53, and p-p53 (S15) rabbit antibody for detection of p53 activation. The secondary antibodies used were goat horseradish peroxidase-conjugated antirabbit IgG antibody (1:20,000) for detection of ERK1/2 activation, and goat horseradish peroxidase-conjugated anti-rabbit IgG
Please cite this article as: Y. Tanaka et al., Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.167
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antibody (1:5000) for detection of MANF and p53/p-p53 at room temperature for 1.5 h. As a loading control, the blots were incubated with peroxidase-conjugated anti-b-actin monoclonal antibody (FUJIFILM Wako Pure Chemical Corp.) (1:50,000) at room temperature for 60 min. 2.7. Small interfering RNA (siRNA) transfection SiRNA targeting MANF and negative control siRNA (TriFECTa Kit® DsiRNA Duplex) were purchased from Integrated DNA Technology. B16 cells (5.0 104 cells/well) were incubated in culture for 16 h, then transfected with siRNA duplex oligonucleotides (25 nM) for knockdown of mouse MANF by using Lipofectamine RNAiMAX (Invitrogen) and Opti-MEM Reduced Serum Medium (Invitrogen) according to the manufacturer’s instructions. Cells were used for Western blotting, immunofluorescence and colony formation assay at 48 h after transfection. 2.8. Statistics Results are expressed as the mean ± standard error (S.E.). The statistical significance of differences between the control and other groups was calculated by using Dunnett’s test or Mann-Whitney rank sum test. Calculations (Dunnett’s test) were done with the Instat version 3.0 statistical software package (Graph Pad Software). The criterion of significance was set at p < 0.05. 3. Results & discussion First, we confirmed that the neurotrophic factor MANF is expressed in B16 melanoma cells by means of western blotting (Fig. 1A). Although 1.0 Gy g-rays failed to induce release of MANF (Fig. 1B), increased release of MANF was observed in response to girradiation at a dose of 2.0 Gy (Fig. 1C) or 4.0 Gy (Fig. 1D) with statistical significance. However, in the presence of 0.5 or 1.0 mM 4PBA, which inhibits ER stress [23], the radiation-induced extracellular release of MANF was blocked with statistical significance (Fig. 1E). These results suggest that ER stress results in the release of MANF in melanoma cells. Furthermore, we also examined the change in amount of intracellular MANF after g-irradiation. Though 1.0 Gy g-rays failed to induce expression of MANF, expression of MANF was increased in response to g-irradiation at a dose of 2.0 Gy or 4.0 Gy with no statistical significance (Supplementary Fig. 1). It is considered that the intracellular MANF did not significantly change because the intracellular MANF was much higher than the extracellularly secreted MANF. When cells were treated with MANF for 3 h before irradiation, the DNA damage response, evaluated in terms of gH2AX-53BP1 focus formation in the nuclei (Fig. 2A), and reproductive cell death (Fig. 2B) in 2.0 or 4.0 Gy irradiated cells were not affected, presumably because release of MANF was induced at these radiation doses (Fig. 1C and D), and was protective. On the other hand, MANF treatment followed by 1.0 Gy of g-rays, which is insufficient to induce endogenous MANF release (see Fig. 1B), increased the DNA damage response and suppressed reproductive cell death (Fig. 2A and B). These results indicate that endogenous MANF contributes to radioresistance by promoting DNA damage repair in melanoma cells. In addition, MANF induced ERK1/2 activation in B16 melanoma cells (Fig. 2C). Thus, g-ray-induced release of MANF activates signaling pathways, such as ERK, that promote the malignant phenotype. Further studies will be needed to establish how MANF triggers downstream signal activation. Next, to confirm the involvement of MANF in radioresistance, B16 cells were transfected with siRNA targeting MANF. Expression of MANF protein was decreased in MANF knockdown cells to 50.3%
of that in scramble siRNA-transfected cells, as determined by Western blotting (Fig. 3A). The release of MANF at 1 h after girradiation (2.0 Gy) was significantly suppressed by MANF knockdown (Fig. 3B). Furthermore, reproductive cell death induced by girradiation (2.0 Gy) was significantly enhanced in MANF-KD cells (Fig. 3C). These results support the idea that g-ray-induced MANF release promotes the survival of melanoma cells. We next investigated the effect of MANF on various factors related to cancer cell survival. First, to clarify the involvement of MANF in the DNA damage response, we evaluated the effect of MANF knockdown on gH2AX-53BP1 focus formation at DNA damage sites in g-irradiated B16 melanoma cells. Focus formation (co-staining with gH2AX and 53BP1) was significantly reduced in MANF knockdown cells (Fig. 4A). In order to examine the longerterm effects, we also evaluated gH2AX-53BP1 focus formation at 24 h after g-irradiation (4.0 Gy) as a measure of unrepaired DNA damage. As shown in Fig. 4B, DNA damage was still apparent at 24 h after g-irradiation in MANF-KD cells. Since p53 is a radioresistance factor [24,25], we also investigated activation of p53 in MANFknockdown cells. The activation of p53 after 1 h of g-irradiation was significantly suppressed, and persistence of the activated state was observed after 24 h in the knockdown cells (Fig. 4C and D). These results strongly support the view that MANF is involved in DNA damage response and cell survival after g-irradiation and contributes to radioresistance. It has been reported that ER stress response contributes to the resistance of glioblastoma to radiotherapy [26], and that MANF is upregulated by acute ER stress, including chemical substances, ischemic conditions, and epileptic seizures [11,27]. Furthermore, cerebral ischemia induces neuronal cell death by ER stress [28,29], and during this process, MANF expression is induced and has a protective effect against cell death [30]. Also, recombinant human MANF inhibits apoptosis of neurons induced by ER stress [30]. Our present findings that release of MANF is induced by g-irradiation, and contributes to radioresistance are consistent with these results. However, this is the first report to demonstrate a role of MANF in radioresistance of cancer and melanoma. As noted above, given the possibility that MANF contributes to the radioresistance of glioblastoma [11,26,27], we speculate that MANF might be a candidate therapeutic target for glioblastoma. In addition, further studies are needed for cancer types such as lung and breast cancers for which radiation therapy is generally used. On the other hand, MANF promotes a switch of macrophage phenotype from pro-inflammatory to anti-inflammatory [15]. Furthermore, there are reports that M1 macrophages (pro-inflammatory) are increased in MANF-knockout mice, while M2 macrophages (anti-inflammatory) are increased under conditions of liver fibrosis [31]. These reports suggest that MANF also plays a role in suppressing inflammatory responses and controlling immune functions. As regards the mechanism of MANF’s action, we found that it activates the ERK signaling pathway, which lies downstream of the B-Raf proto-oncogene serine/threonine-kinase (BRAF). Recently, it was shown that treatments targeting the (BRAF)V600 (Val600) mutation in melanoma patients by combining BRAF inhibitors with mitogen-activated protein kinase inhibitors improved survival [32]. In addition, MAPK activation is thought to be a major cause of melanoma radioresistance, and 50e60% of melanomas are reported to have BRAF-activating mutations [33]. We found here that MANF knockdown suppresses the activation of p53, which is also a radioresistance factor. Further studies will be needed to elucidate in detail the downstream signaling pathways from MANF. There appears to be a possibility that treatment with MANF inhibitors or anti-MANF antibody might be therapeutically useful in combination with existing melanoma treatments. It will be interesting in
Please cite this article as: Y. Tanaka et al., Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.167
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the future to study the role of MANF in other cancer types. Acknowledgement This work was supported in part by JSPS KAKENHI Grant number JP 16K08148 (Grant-in-Aid for Scientific Research (C)) (to TT and MT). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.167. Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2020.01.167. References [1] A. Mahadevan, V.L. Patel, N. Dagoglu, Radiation therapy in the management of malignant melanoma, Oncology (Williston Park) 29 (2015) 743e751. [2] B.H. Burmeister, M.A. Henderson, J. Ainslie, et al., Adjuvant radiotherapy versus observation alone for patients at risk of lymph-node field relapse after therapeutic lymphadenectomy for melanoma: a randomised trial, Lancet Oncol. 13 (2012) 589e597. [3] K. Satyamoorthy, N.H. Chehab, M.J. Waterman, et al., Aberrant regulation and function of wild- type p53 in radioresistant melanoma cells, Cell Growth Differ. 11 (2000) 467e474. [4] W. Yuan, Y. Yuan, T. Zhang, et al., Role of Bmi-1 in regulation of ionizing irradiation-induced epithelial-mesenchymal transition and migration of breast cancer cells, PloS One 10 (2015), e0118799. [5] W. Kim, S. Lee, D. Seo, et al., Cellular stress responses in radiotherapy, Cells 8 (2019) 1105. [6] H. Yoshida, ER stress and diseases, FEBS J. 274 (2007) 630e658. [7] S. Kanemoto, K. Imaizumi, Endoplasmic reticulum stress and diseases, J. Jpn. Biochem. Soc. 90 (2018) 51e59. [8] A. Nagelkerke, J. Bussink, A.J. van der Kogel, et al., The PERK/ATF4/LAMP3-arm of the unfolded protein response affects radioresistance by interfering with the DNA damage response, Radiother. Oncol. 108 (2013) 415e421. [9] P. Petrova, A. Raibekas, J. Pevsner, et al., MANF: a new mesencephalic, astrocyte-derived neurotrophic factor with selectivity for dopaminergic neurons, J. Mol. Neurosci. 20 (2003) 173e188. [10] N. Mizobuchi, J. Hoseki, H. Kubota, et al., ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element, Cell Struct. Funct. 32 (2007) 41e50. [11] A. Apostolou, Y. Shen, Y. Liang, et al., Armet, a UPR-upregulated protein, inhibits cell proliferation and ER stress-induced cell death, Exp. Cell Res. 314 (2008) 2454e2467. [12] A. Tadimalla, P.J. Belmont, D.J. Thuerauf, et al., Mesencephalic astrocytederived neurotrophic factor is an ischemia-inducible secreted endoplasmic reticulum stress response protein in the heart, Circ. Res. 103 (2008) 1249e1258.
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Please cite this article as: Y. Tanaka et al., Mesencephalic astrocyte-derived neurotrophic factor is a novel radioresistance factor in mouse B16 melanoma, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2020.01.167