Amino acid starvation culture condition sensitizes EGFR-expressing cancer cell lines to gefitinib-mediated cytotoxicity by inducing atypical necroptosis

Saito,Yu; Moriya,Shota; Kazama,Hiromi; Hirasawa,Kazuhiro; Miyahara,Kana; Kokuba,Hiroko; Hino,Hirotsugu; Kikuchi,Hiroyuki; Takano,Naoharu; Hiramoto,Masaki; Tsukahara,Kiyoaki; Miyazawa,Keisuke

doi:10.3892/ijo.2018.4282

Amino acid starvation culture condition sensitizes EGFR-expressing cancer cell lines to gefitinib-mediated cytotoxicity by inducing atypical necroptosis

Authors:
- Yu Saito
- Shota Moriya
- Hiromi Kazama
- Kazuhiro Hirasawa
- Kana Miyahara
- Hiroko Kokuba
- Hirotsugu Hino
- Hiroyuki Kikuchi
- Naoharu Takano
- Masaki Hiramoto
- Kiyoaki Tsukahara
- Keisuke Miyazawa
View Affiliations

Affiliations: Department of Otolaryngology (Head and Neck Surgery), Tokyo Medical University, Tokyo 160-8402, Japan, Department of Biochemistry, Tokyo Medical University, Tokyo 160-8402, Japan, Department of Breast Oncology and Surgery, Tokyo Medical University, Tokyo 160-8402, Japan, Department of Joint Research for Basic Medical Science, Institute of Medical Science, Tokyo Medical University, Tokyo 160-8402, Japan, Department of Preventive Medicine and Public Health, Tokyo Medical University, Tokyo 160-8402, Japan
Published online on: February 23, 2018 https://doi.org/10.3892/ijo.2018.4282
Pages: 1165-1177
Copyright: © Saito et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The maintenance of the intracellular level of amino acids is crucial for cellular homeostasis. This is carried out via the regulation of both the influx from the extracellular environment and the recycling of intracellular resources. Since epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors, including gefitinib (GEF) have been reported to induce the apoptosis of several cancer cell lines, in the present study, we examined whether the cytotoxic effects of GEF are further enhanced under amino acid starvation (AAS) culture conditions. Under AAS culture conditions, the cell killing effect of GEF was synergistically pronounced in the EGFR-expressing cell lines, namely, CAL 27, Detroit 562, A549 and PANC-1 cells compared with those treated with either GEF or AAS alone. The addition of essential amino acids, but not non-essential amino acids to the cell culture medium resulted in the cancellation of this pronounced cytotoxicity. The knockdown of L-type amino acid transporter 1 (LAT-1) by siRNA also enhanced GEF-induced cytotoxicity. Therefore, the shortage of the intracellular amino acid pool appears to determine the sensitivity to GEF. Notably, this enhanced cytotoxicity is not mediated by the induction of apoptosis, but is accompanied by the pronounced induction of autophagy. The presence of necrostatin-1, an inhibitor of receptor-interacting serine/threonine-protein kinase 1 (RIPK‑1), but not that of Z-VAD-fmk, attenuated the cytotoxic effects of GEF under AAS culture conditions. Electron microscopy demonstrated that the CAL 27 cells treated with GEF under AAS culture conditions exhibited swelling of the cytosol and organelles with an increased number of autophagosomes and autolysosomes, but without chromatin condensation and nuclear fragmentation. Autophagic cell death was excluded as the inhibition of autophagy did not attenuate the cytotoxicity. These results strongly suggest the induction of necroptosis in response to GEF under AAS culture conditions. However, we could not detect any phosphorylation of RIPK-1 and mixed lineage kinase domain like pseudokinase (MLKL), as well as any necrosome formation. Therefore, the enhanced cytotoxic effect of GEF under AAS culture conditions is thought to be mediated by atypical necroptosis.

View Figures

View References

1	Kuma A and Mizushima N: Physiological role of autophagy as an intracellular recycling system: With an emphasis on nutrient metabolism. Semin Cell Dev Biol. 21:683–690. 2010. View Article : Google Scholar : PubMed/NCBI
2	Segawa H, Fukasawa Y, Miyamoto K, Takeda E, Endou H and Kanai Y: Identification and functional characterization of a Na⁺-independent neutral amino acid transporter with broad substrate selectivity. J Biol Chem. 274:19745–19751. 1999. View Article : Google Scholar : PubMed/NCBI
3	Yanagida O, Kanai Y, Chairoungdua A, Kim DK, Segawa H, Nii T, Cha SH, Matsuo H, Fukushima J, Fukasawa Y, et al: Human L-type amino acid transporter 1 (LAT1): Characterization of function and expression in tumor cell lines. Biochim Biophys Acta. 1514:291–302. 2001. View Article : Google Scholar : PubMed/NCBI
4	Oda K, Hosoda N, Endo H, Saito K, Tsujihara K, Yamamura M, Sakata T, Anzai N, Wempe MF, Kanai Y, et al: L-type amino acid transporter 1 inhibitors inhibit tumor cell growth. Cancer Sci. 101:173–179. 2010. View Article : Google Scholar
5	Sakata T, Ferdous G, Tsuruta T, Satoh T, Baba S, Muto T, Ueno A, Kanai Y, Endou H and Okayasu I: L-type amino-acid transporter 1 as a novel biomarker for high-grade malignancy in prostate cancer. Pathol Int. 59:7–18. 2009. View Article : Google Scholar : PubMed/NCBI
6	Mizushima N, Levine B, Cuervo AM and Klionsky DJ: Autophagy fights disease through cellular self-digestion. Nature. 451:1069–1075. 2008. View Article : Google Scholar : PubMed/NCBI
7	Galluzzi L, Pietrocola F, Levine B and Kroemer G: Metabolic control of autophagy. Cell. 159:1263–1276. 2014. View Article : Google Scholar : PubMed/NCBI
8	White E: Deconvoluting the context-dependent role for autophagy in cancer. Nat Rev Cancer. 12:401–410. 2012. View Article : Google Scholar : PubMed/NCBI
9	Han W, Pan H, Chen Y, Sun J, Wang Y, Li J, Ge W, Feng L, Lin X, Wang X, et al: EGFR tyrosine kinase inhibitors activate autophagy as a cytoprotective response in human lung cancer cells. PLoS One. 6:e186912011. View Article : Google Scholar : PubMed/NCBI
10	Fung C, Chen X, Grandis JR and Duvvuri U: EGFR tyrosine kinase inhibition induces autophagy in cancer cells. Cancer Biol Ther. 13:1417–1424. 2012. View Article : Google Scholar : PubMed/NCBI
11	Sugita S, Ito K, Yamashiro Y, Moriya S, Che XF, Yokoyama T, Hiramoto M and Miyazawa K: EGFR-independent autophagy induction with gefitinib and enhancement of its cytotoxic effect by targeting autophagy with clarithromycin in non-small cell lung cancer cells. Biochem Biophys Res Commun. 461:28–34. 2015. View Article : Google Scholar : PubMed/NCBI
12	Mukai S, Moriya S, Hiramoto M, Kazama H, Kokuba H, Che XF, Yokoyama T, Sakamoto S, Sugawara A, Sunazuka T, et al: Macrolides sensitize EGFR-TKI-induced non-apoptotic cell death via blocking autophagy flux in pancreatic cancer cell lines. Int J Oncol. 48:45–54. 2016. View Article : Google Scholar : PubMed/NCBI
13	Wei Y, Zou Z, Becker N, Anderson M, Sumpter R, Xiao G, Kinch L, Koduru P, Christudass CS, Veltri RW, et al: EGFR-mediated Beclin 1 phosphorylation in autophagy suppression, tumor progression, and tumor chemoresistance. Cell. 154:1269–1284. 2013. View Article : Google Scholar : PubMed/NCBI
14	Sordella R, Bell DW, Haber DA and Settleman J: Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways. Science. 305:1163–1167. 2004. View Article : Google Scholar : PubMed/NCBI
15	Ciardiello F and Tortora G: EGFR antagonists in cancer treatment. N Engl J Med. 358:1160–1174. 2008. View Article : Google Scholar : PubMed/NCBI
16	Tan X, Thapa N, Sun Y and Anderson RA: A kinase-independent role for EGF receptor in autophagy initiation. Cell. 160:145–160. 2015. View Article : Google Scholar : PubMed/NCBI
17	Moriya S, Che XF, Komatsu S, Abe A, Kawaguchi T, Gotoh A, Inazu M, Tomoda A and Miyazawa K: Macrolide antibiotics block autophagy flux and sensitize to bortezomib via endoplasmic reticulum stress-mediated CHOP induction in myeloma cells. Int J Oncol. 42:1541–1550. 2013. View Article : Google Scholar : PubMed/NCBI
18	Hirasawa K, Moriya S, Miyahara K, Kazama H, Hirota A, Takemura J, Abe A, Inazu M, Hiramoto M, Tsukahara K, et al: Macrolide antibiotics exhibit cytotoxic effect under amino acid-depleted culture condition by blocking autophagy flux in head and neck squamous cell carcinoma cell lines. PLoS One. 11:e01645292016. View Article : Google Scholar : PubMed/NCBI
19	Kim YC and Guan KL: mTOR: A pharmacologic target for autophagy regulation. J Clin Invest. 125:25–32. 2015. View Article : Google Scholar : PubMed/NCBI
20	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
21	Dalle Pezze P, Ruf S, Sonntag AG, Langelaar-Makkinje M, Hall P, Heberle AM, Razquin Navas P, van Eunen K, Tölle RC, Schwarz JJ, et al: A systems study reveals concurrent activation of AMPK and mTOR by amino acids. Nat Commun. 7:132542016. View Article : Google Scholar : PubMed/NCBI
22	Kondo Y, Kanzawa T, Sawaya R and Kondo S: The role of autophagy in cancer development and response to therapy. Nat Rev Cancer. 5:726–734. 2005. View Article : Google Scholar : PubMed/NCBI
23	Shimizu S, Kanaseki T, Mizushima N, Mizuta T, Arakawa-Kobayashi S, Thompson CB and Tsujimoto Y: Role of Bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes. Nat Cell Biol. 6:1221–1228. 2004. View Article : Google Scholar : PubMed/NCBI
24	Teng X, Degterev A, Jagtap P, Xing X, Choi S, Denu R, Yuan J and Cuny GD: Structure-activity relationship study of novel necroptosis inhibitors. Bioorg Med Chem Lett. 15:5039–5044. 2005. View Article : Google Scholar : PubMed/NCBI
25	Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, Cuny GD, Mitchison TJ, Moskowitz MA and Yuan J: Chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury. Nat Chem Biol. 1:112–119. 2005. View Article : Google Scholar
26	Kawahara A, Yamamoto C, Nakashima K, Azuma K, Hattori S, Kashihara M, Aizawa H, Basaki Y, Kuwano M, Kage M, et al: Molecular diagnosis of activating EGFR mutations in non-small cell lung cancer using mutation-specific antibodies for immunohistochemical analysis. Clin Cancer Res. 16:3163–3170. 2010. View Article : Google Scholar : PubMed/NCBI
27	Hosokawa N, Hara Y and Mizushima N: Generation of cell lines with tetracycline-regulated autophagy and a role for autophagy in controlling cell size. FEBS Lett. 581:2623–2629. 2007.PubMed/NCBI
28	Sun XM, MacFarlane M, Zhuang J, Wolf BB, Green DR and Cohen GM: Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J Biol Chem. 274:5053–5060. 1999. View Article : Google Scholar : PubMed/NCBI
29	Miyahara K, Kazama H, Kokuba H, Komatsu S, Hirota A, Takemura J, Hirasawa K, Moriya S, Abe A, Hiramoto M, et al: Targeting bortezomib-induced aggresome formation using vinorelbine enhances the cytotoxic effect along with ER stress loading in breast cancer cell lines. Int J Oncol. 49:1848–1858. 2016. View Article : Google Scholar : PubMed/NCBI
30	Hansen TE and Johansen T: Following autophagy step by step. BMC Biol. 9:392011. View Article : Google Scholar : PubMed/NCBI
31	Golden SH, Folsom AR, Coresh J, Sharrett AR, Szklo M and Brancati F: Risk factor groupings related to insulin resistance and their synergistic effects on subclinical atherosclerosis: The atherosclerosis risk in communities study. Diabetes. 51:3069–3076. 2002. View Article : Google Scholar : PubMed/NCBI
32	Lynch TJ, Bell DW, Sordella R, Gurubhagavatula S, Okimoto RA, Brannigan BW, Harris PL, Haserlat SM, Supko JG, Haluska FG, et al: Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 350:2129–2139. 2004. View Article : Google Scholar : PubMed/NCBI
33	Wang J, Chen X, Su L, Li P, Liu B and Zhu Z: LAT-1 functions as a promotor in gastric cancer associated with clinicopathologic features. Biomed Pharmacother. 67:693–699. 2013. View Article : Google Scholar : PubMed/NCBI
34	Gong YN, Guy C, Olauson H, Becker JU, Yang M, Fitzgerald P, Linkermann A and Green DR: ESCRT-III acts downstream of MLKL to regulate necroptotic cell death and its consequences. Cell. 169:286–300.e16. 2017. View Article : Google Scholar : PubMed/NCBI
35	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
36	Chen L, Meng Y, Guo X, Sheng X, Tai G, Zhang F, Cheng H and Zhou Y: Gefitinib enhances human colon cancer cells to TRAIL-induced apoptosis of via autophagy- and JNK-mediated death receptors upregulation. Apoptosis. 21:1291–1301. 2016. View Article : Google Scholar : PubMed/NCBI
37	Degterev A, Hitomi J, Germscheid M, Ch'en IL, Korkina O, Teng X, Abbott D, Cuny GD, Yuan C, Wagner G, et al: Identification of RIP1 kinase as a specific cellular target of necrostatins. Nat Chem Biol. 4:313–321. 2008. View Article : Google Scholar : PubMed/NCBI
38	Weinlich R, Oberst A, Beere HM and Green DR: Necroptosis in development, inflammation and disease. Nat Rev Mol Cell Biol. 18:127–136. 2017. View Article : Google Scholar
39	Li J, McQuade T, Siemer AB, Napetschnig J, Moriwaki K, Hsiao YS, Damko E, Moquin D, Walz T, McDermott A, et al: The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell. 150:339–350. 2012. View Article : Google Scholar : PubMed/NCBI
40	Zhang Y, Su SS, Zhao S, Yang Z, Zhong CQ, Chen X, Cai Q, Yang ZH, Huang D, Wu R, et al: RIP1 autophosphorylation is promoted by mitochondrial ROS and is essential for RIP3 recruitment into necrosome. Nat Commun. 8:143292017. View Article : Google Scholar : PubMed/NCBI
41	Ros U, Peña-Blanco A, Hänggi K, Kunzendorf U, Krautwald S, Wong WW and García-Sáez AJ: Necroptosis execution is mediated by plasma membrane nanopores independent of calcium. Cell Reports. 19:175–187. 2017. View Article : Google Scholar : PubMed/NCBI
42	Sun L, Wang H, Wang Z, He S, Chen S, Liao D, Wang L, Yan J, Liu W, Lei X, et al: Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase. Cell. 148:213–227. 2012. View Article : Google Scholar : PubMed/NCBI
43	Seto T, Kato T, Nishio M, Goto K, Atagi S, Hosomi Y, Yamamoto N, Hida T, Maemondo M, Nakagawa K, et al: Erlotinib alone or with bevacizumab as first-line therapy in patients with advanced non-squamous non-small-cell lung cancer harbouring EGFR mutations (JO25567): An open-label, randomised, multi-centre, phase 2 study. Lancet Oncol. 15:1236–1244. 2014. View Article : Google Scholar : PubMed/NCBI
44	Ichihara E, Hotta K, Nogami N, Kuyama S, Kishino D, Fujii M, Kozuki T, Tabata M, Harada D, Chikamori K, et al: Phase II trial of gefitinib in combination with bevacizumab as first-line therapy for advanced non-small cell lung cancer with activating EGFR gene mutations: The Okayama Lung Cancer Study Group Trial 1001. J Thorac Oncol. 10:486–491. 2015. View Article : Google Scholar : PubMed/NCBI
45	Huang D, Zheng X, Wang ZA, Chen X, He WT, Zhang Y, Xu JG, Zhao H, Shi W, Wang X, et al: The MLKL channel in necroptosis is an octamer formed by tetramers in a dyadic process. Mol Cell Biol. 37:e00497-e162017. View Article : Google Scholar
46	Goodall ML, Fitzwalter BE, Zahedi S, Wu M, Rodriguez D, Mulcahy-Levy JM, Green DR, Morgan M, Cramer SD and Thorburn A: The autophagy machinery controls cell death switching between apoptosis and necroptosis. Dev Cell. 37:337–349. 2016. View Article : Google Scholar : PubMed/NCBI
47	Bray K, Mathew R, Lau A, Kamphorst JJ, Fan J, Chen J, Chen HY, Ghavami A, Stein M, DiPaola RS, et al: Autophagy suppresses RIP kinase-dependent necrosis enabling survival to mTOR inhibition. PLoS One. 7:e418312012. View Article : Google Scholar : PubMed/NCBI
48	Gump JM, Staskiewicz L, Morgan MJ, Bamberg A, Riches DW and Thorburn A: Autophagy variation within a cell population determines cell fate through selective degradation of Fap-1. Nat Cell Biol. 16:47–54. 2014. View Article : Google Scholar
49	Thorburn J, Andrysik Z, Staskiewicz L, Gump J, Maycotte P, Oberst A, Green DR, Espinosa JM and Thorburn A: Autophagy controls the kinetics and extent of mitochondrial apoptosis by regulating PUMA levels. Cell Rep. 7:45–52. 2014. View Article : Google Scholar : PubMed/NCBI