SPAG6 silencing induces autophagic cell death in SKM‑1 cells via the AMPK/mTOR/ULK1 signaling pathway

Zhang,Meng; Luo,Jie; Luo,Xiaohua; Liu,Lin

doi:10.3892/ol.2020.11607

SPAG6 silencing induces autophagic cell death in SKM‑1 cells via the AMPK/mTOR/ULK1 signaling pathway

Authors:
- Meng Zhang
- Jie Luo
- Xiaohua Luo
- Lin Liu
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Affiliations: Department of Hematology, The First Affiliated Hospital of Chongqing Medical University, Yuzhong, Chongqing 400016, P.R. China
Published online on: May 13, 2020 https://doi.org/10.3892/ol.2020.11607
Pages: 551-560
Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

As a member of the cancer‑testis antigen family, sperm‑associated antigen 6 (SPAG6) has been reported to be associated with the pathogenesis of myelodysplastic syndromes (MDS). Previous studies have demonstrated that SPAG6 is upregulated in bone marrow from patients with MDS and MDS‑transformed acute myeloid leukemia and that knockdown of SPAG6 expression levels suppressed proliferation and promote apoptosis and differentiation in SKM‑1 cells. However, the association between SPAG6 and autophagy in SKM‑1 cells remains unclear. Hence, the aim of the present study was to investigate this association and its underlying mechanism. The present study used a short hairpin RNA (shRNA) lentivirus to silence SPAG6 expression levels in SKM‑1 cells and demonstrated that SPAG6 knockdown increased autophagy and apoptosis. Furthermore, pharmacologically inhibiting autophagy with chloroquine and 3‑methyladenine decreased SPAG6 knockdown‑mediated apoptosis, indicating that SPAG6 knockdown‑mediated autophagy promoted apoptosis in SKM‑1 cells. Additionally, compared with the expression levels in negative control‑shRNA lentivirus‑transfected SKM‑1 cells, the protein expression levels of phosphorylated AMP‑activated protein kinase (p‑AMPK) and phosphorylated unc‑51‑like autophagy activating kinase 1 (p‑ULK1) were upregulated, while phosphorylated mammalian target of rapamycin (p‑mTOR) protein expression was downregulated in SPAG6‑shRNA lentivirus‑transfected cells. Moreover, inhibiting AMPK expression levels with Compound C, a specific inhibitor of AMPK, attenuated SPAG6 knockdown‑induced autophagy and apoptosis, suggesting that AMPK‑mediated autophagy enhanced the pro‑apoptotic effect of SPAG6 knockdown in SKM‑1 cells. Taken together, the results of the present study demonstrated that SPAG6 silencing triggered autophagy via regulation of the AMPK/mTOR/ULK1 signaling pathway, which further contributed to the apoptosis of SKM‑1 cells induced by SPAG6 knockdown. Thus, the current results indicate that SPAG6 may be a potential therapeutic target against MDS, and that autophagy may represent a potential mechanism for the treatment of MDS.

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1	Corey SJ, Minden MD, Barber DL, Kantarjian H, Wang JC and Schimmer AD: Myelodysplastic syndromes: The complexity of stem-cell diseases. Nat Rev Cancer. 7:118–129. 2007. View Article : Google Scholar : PubMed/NCBI
2	Greenberg PL, Stone RM, Al-Kali A, Barta SK, Bejar R, Bennett JM, Carraway H, De Castro CM, Deeg HJ, DeZern AE, et al: Myelodysplastic syndromes, Version 2.2017, NCCN clinical practice guidelines in oncology. J Natl Compr Canc Netw. 15:60–87. 2017. View Article : Google Scholar : PubMed/NCBI
3	Saber W, Cutler CS, Nakamura R, Zhang MJ, Atallah E, Rizzo JD, Maziarz RT, Cortes J, Kalaycio ME and Horowitz MM: Impact of donor source on hematopoietic cell transplantation outcomes for patients with myelodysplastic syndromes (MDS). Blood. 122:1974–1982. 2013. View Article : Google Scholar : PubMed/NCBI
4	Sapiro R, Kostetskii I, Olds-Clarke P, Gerton GL, Radice GL and Strauss III JF: Male infertility, impaired sperm motility, and hydrocephalus in mice deficient in sperm-associated antigen 6. Mol Cell Biol. 22:6298–6305. 2002. View Article : Google Scholar : PubMed/NCBI
5	Siliņa K, Zayakin P, Kalniņa Z, Ivanova L, Meistere I, Endzeliņš E, Abols A, Stengrēvics A, Leja M, Ducena K, et al: Sperm-associated antigens as targets for cancer immunotherapy: Expression pattern and humoral immune response in cancer patients. J Immunother. 34:28–44. 2011. View Article : Google Scholar : PubMed/NCBI
6	Steinbach D, Schramm A, Eggert A, Onda M, Dawczynski K, Rump A, Pastan I, Wittig S, Pfaffendorf N, Voigt A, et al: Identification of a set of seven genes for the monitoring of minimal residual disease in pediatric acute myeloid leukemia. Clin Cancer Res. 12:2434–2441. 2006. View Article : Google Scholar : PubMed/NCBI
7	Jiang M, Chen Y, Deng L, Luo X, Wang L and Liu L: Upregulation of SPAG6 in myelodysplastic syndrome: Knockdown inhibits cell proliferation via AKT/FOXO signaling pathway. DNA Cell Biol. 38:476–484. 2019. View Article : Google Scholar : PubMed/NCBI
8	Nakagawa T, Matozaki S, Murayama T, Nishimura R, Tsutsumi M, Kawaguchi R, Yokoyama Y, Hikiji K, Isobe T and Chihara K: Establishment of a leukaemic cell line from a patient with acquisition of chromosomal abnormalities during disease progression in myelodysplastic syndrome. Br J Haematol. 85:469–476. 1993. View Article : Google Scholar : PubMed/NCBI
9	Yang B, Wang L, Luo X, Chen L, Yang Z and Liu L: SPAG6 silencing inhibits the growth of the malignant myeloid cell lines SKM-1 and K562 via activating p53 and caspase activation-dependent apoptosis. Int J Oncol. 46:649–656. 2015. View Article : Google Scholar : PubMed/NCBI
10	Li X, Yang B, Wang L, Chen L, Luo X and Liu L: SPAG6 regulates cell apoptosis through the TRAIL signal pathway in myelodysplastic syndromes. Oncol Rep. 37:2839–2846. 2017. View Article : Google Scholar : PubMed/NCBI
11	Yin J, Li X, Zhang Z, Luo X, Wang L and Liu L: SPAG6 silencing induces apoptosis in the myelodysplastic syndrome cell line SKM-1 via the PTEN/PI3K/AKT signaling pathway in vitro and in vivo. Int J Oncol. 53:297–306. 2018.PubMed/NCBI
12	White E and DiPaola RS: The double-edged sword of autophagy modulation in cancer. Clin Cancer Res. 15:5308–5316. 2009. View Article : Google Scholar : PubMed/NCBI
13	Mortensen M, Ferguson DJ, Edelmann M, Kessler B, Morten KJ, Komatsu M and Simon AK: Loss of autophagy in erythroid cells leads to defective removal of mitochondria and severe anemia in vivo. Proc Natl Acad Sci USA. 107:832–837. 2010. View Article : Google Scholar : PubMed/NCBI
14	Watson AS, Mortensen M and Simon AK: Autophagy in the pathogenesis of myelodysplastic syndrome and acute myeloid leukemia. Cell Cycle. 10:1719–1725. 2011. View Article : Google Scholar : PubMed/NCBI
15	Mortensen M, Watson AS and Simon AK: Lack of autophagy in the hematopoietic system leads to loss of hematopoietic stem cell function and dysregulated myeloid proliferation. Autophagy. 7:1069–1070. 2011. View Article : Google Scholar : PubMed/NCBI
16	Ali AM and Raza A: Two different ‘tales’ of ATG7: Clinical relevance to myelodysplastic syndromes. Mol Cell Oncol. 3:e12126862016. View Article : Google Scholar : PubMed/NCBI
17	Jiang H, Yang L, Guo L, Cui N, Zhang G, Liu C, Xing L, Shao Z and Wang H: Impaired mitophagy of nucleated erythroid cells leads to anemia in patients with myelodysplastic syndromes. Oxid Med Cell Longev. 2018:63280512018. View Article : Google Scholar : PubMed/NCBI
18	Jacquel A, Luciano F, Robert G and Auberger P: Implication and regulation of AMPK during physiological and pathological myeloid differentiation. Int J Mol Sci. 19:E29912018. View Article : Google Scholar : PubMed/NCBI
19	Zhao M and Klionsky DJ: AMPK-dependent phosphorylation of ULK1 induces autophagy. Cell Metab. 13:119–120. 2011. View Article : Google Scholar : PubMed/NCBI
20	Nieminen AI, Eskelinen VM, Haikala HM, Tervonen TA, Yan Y, Partanen JI and Klefstrom J: Myc-induced AMPK-phospho p53 pathway activates Bak to sensitize mitochondrial apoptosis. Proc Natl Acad Sci USA. 110:E1839–E1848. 2013. View Article : Google Scholar : PubMed/NCBI
21	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
22	Klionsky DJ, Abdelmohsen K, Abe A, Abedin MJ, Abeliovich H, Acevedo Arozena A, Adachi H, Adams CM, Adams PD, Adeli K, et al: Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy. 12:1–222. 2016. View Article : Google Scholar : PubMed/NCBI
23	Yim NH, Hwang YH, Liang C and Ma JY: A platycoside-rich fraction from the root of Platycodon grandiflorum enhances cell death in A549 human lung carcinoma cells via mainly AMPK/mTOR/AKT signal-mediated autophagy induction. J Ethnopharmacol. 194:1060–1068. 2016. View Article : Google Scholar : PubMed/NCBI
24	Kim HI, Hong SH, Ku JM, Lim YS, Lee SJ, Song J, Kim TY, Cheon C and Ko SG: Scutellaria radix promotes apoptosis in non-small cell lung cancer cells via induction of AMPK-Dependent autophagy. Am J Chin Med. 47:691–705. 2019. View Article : Google Scholar : PubMed/NCBI
25	Wu Y, Wang X, Guo H, Zhang B, Zhang XB, Shi ZJ and Yu L: Synthesis and screening of 3-MA derivatives for autophagy inhibitors. Autophagy. 9:595–603. 2013. View Article : Google Scholar : PubMed/NCBI
26	Yin H, Yang X, Gu W, Liu Y, Li X, Huang X, Zhu X, Tao Y, Gou X and He W: HMGB1-mediated autophagy attenuates gemcitabine-induced apoptosis in bladder cancer cells involving JNK and ERK activation. Oncotarget. 8:71642–71656. 2017. View Article : Google Scholar : PubMed/NCBI
27	Kim J, Kundu M, Viollet B and Guan KL: AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat Cell Biol. 13:132–141. 2011. View Article : Google Scholar : PubMed/NCBI
28	Xing JJ, Hou JG, Ma ZN, Wang Z, Ren S, Wang YP, Liu WC, Chen C and Li W: Ginsenoside Rb3 provides protective effects against cisplatin-induced nephrotoxicity via regulation of AMPK-/mTOR-mediated autophagy and inhibition of apoptosis in vitro and in vivo. Cell Prolif. 52:e126272019. View Article : Google Scholar : PubMed/NCBI
29	Sperling AS, Gibson CJ and Ebert BL: The genetics of myelodysplastic syndrome: From clonal haematopoiesis to secondary leukaemia. Nat Rev Cancer. 17:5–19. 2017. View Article : Google Scholar : PubMed/NCBI
30	Schanz J, Tuchler H, Sole F, Mallo M, Luno E, Cervera J, Granada I, Hildebrandt B, Slovak ML, Ohyashiki K, et al: New comprehensive cytogenetic scoring system for primary myelodysplastic syndromes (MDS) and oligoblastic acute myeloid leukemia after MDS derived from an international database merge. J Clin Oncol. 30:820–829. 2012. View Article : Google Scholar : PubMed/NCBI
31	Li F, Zeng J, Gao Y, Guan Z, Ma Z, Shi Q, Du C, Jia J, Xu S, Wang X, et al: G9a inhibition induces autophagic cell death via AMPK/mTOR pathway in bladder transitional cell carcinoma. PLoS One. 10:e01383902015. View Article : Google Scholar : PubMed/NCBI
32	Mortensen M, Soilleux EJ, Djordjevic G, Tripp R, Lutteropp M, Sadighi-Akha E, Stranks AJ, Glanville J, Knight S, Jacobsen SE, et al: The autophagy protein Atg7 is essential for hematopoietic stem cell maintenance. J Exp Med. 208:455–467. 2011. View Article : Google Scholar : PubMed/NCBI
33	Hu X, Yan R, Cheng X, Song L, Zhang W, Li K and Zhao S: The function of sperm-associated antigen 6 in neuronal proliferation and differentiation. J Mol Histol. 47:531–540. 2016. View Article : Google Scholar : PubMed/NCBI
34	Li X, Xu L, Sun G, Wu X, Bai X, Li J, Strauss JF, Zhang Z and Wang H: Spag6 mutant mice have defects in development and function of spiral ganglion neurons, apoptosis, and higher sensitivity to paclitaxel. Sci Rep. 7:86382017. View Article : Google Scholar : PubMed/NCBI
35	Maiuri MC, Criollo A and Kroemer G: Crosstalk between apoptosis and autophagy within the Beclin 1 interactome. EMBO J. 29:515–516. 2010. View Article : Google Scholar : PubMed/NCBI
36	Zhuang L, Ma Y, Wang Q, Zhang J, Zhu C, Zhang L and Xu X: Atg3 overexpression enhances bortezomib-induced cell death in SKM-1 cell. PLoS One. 11:e01587612016. View Article : Google Scholar : PubMed/NCBI
37	Romano A, Giallongo C, La Cava P, Parrinello NL, Chiechi A, Vetro C, Tibullo D, Di Raimondo F, Liotta LA, Espina V and Palumbo GA: Proteomic analysis reveals autophagy as pro-survival pathway elicited by long-term exposure with 5-azacitidine in high-risk myelodysplasia. Front Pharmacol. 8:2042017. View Article : Google Scholar : PubMed/NCBI
38	Gao L, Wang Z, Lu D, Huang J, Liu J and Hong L: Paeonol induces cytoprotective autophagy via blocking the Akt/mTOR pathway in ovarian cancer cells. Cell Death Dis. 10:6092019. View Article : Google Scholar : PubMed/NCBI
39	Maiuri MC, Zalckvar E, Kimchi A and Kroemer G: Self-eating and self-killing: Crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol. 8:741–752. 2007. View Article : Google Scholar : PubMed/NCBI
40	Martelli AM, Evangelisti C, Chappell W, Abrams SL, Basecke J, Stivala F, Donia M, Fagone P, Nicoletti F, Libra M, et al: Targeting the translational apparatus to improve leukemia therapy: Roles of the PI3K/PTEN/Akt/mTOR pathway. Leukemia. 25:1064–1079. 2011. View Article : Google Scholar : PubMed/NCBI
41	Zeng J, Liu W, Fan YZ, He DL and Li L: PrLZ increases prostate cancer docetaxel resistance by inhibiting LKB1/AMPK-mediated autophagy. Theranostics. 8:109–123. 2018. View Article : Google Scholar : PubMed/NCBI
42	Li TT, Zhu D, Mou T, Guo Z, Pu JL, Chen QS, Wei XF and Wu ZJ: IL-37 induces autophagy in hepatocellular carcinoma cells by inhibiting the PI3K/AKT/mTOR pathway. Mol Immunol. 87:132–140. 2017. View Article : Google Scholar : PubMed/NCBI
43	Chan EY, Kir S and Tooze SA: siRNA screening of the kinome identifies ULK1 as a multidomain modulator of autophagy. J Biol Chem. 282:25464–25474. 2007. View Article : Google Scholar : PubMed/NCBI
44	Zheng Q, Zhao Y, Guo J, Zhao S, Fei C, Xiao C, Wu D, Wu L, Li X and Chang C: Iron overload promotes mitochondrial fragmentation in mesenchymal stromal cells from myelodysplastic syndrome patients through activation of the AMPK/MFF/Drp1 pathway. Cell Death Dis. 9:5152018. View Article : Google Scholar : PubMed/NCBI
45	Basiorka AA, McGraw KL, Eksioglu EA, Chen X, Johnson J, Zhang L, Zhang Q, Irvine BA, Cluzeau T, Sallman DA, et al: The NLRP3 inflammasome functions as a driver of the myelodysplastic syndrome phenotype. Blood. 128:2960–2975. 2016. View Article : Google Scholar : PubMed/NCBI
46	Bullón P, Alcocer-Gómez E, Carrión AM, Marín-Aguilar F, Garrido-Maraver J, Román-Malo L, Ruiz-Cabello J, Culic O, Ryffel B, Apetoh L, et al: AMPK phosphorylation modulates pain by activation of NLRP3 inflammasome. Antioxid Redox Signal. 24:157–170. 2016. View Article : Google Scholar : PubMed/NCBI