Reduced argininosuccinate synthetase expression in refractory sarcomas: Impacts on therapeutic potential and drug resistance.
BACKGROUND
Treating drug-resistant sarcomas remains a major challenge. The present study aimed to identify a novel therapy for drug-resistant sarcomas based on metabolic errors involving argininosuccinate synthetase1 (ASS1).


RESULTS
ASS1 expression was reduced in Dox-resistant sarcoma cells. Immunohistochemistry and real-time PCR showed an inverse correlation between ASS1 and P-gp expressions. The inhibition of cellular proliferation with G1-arrest was shown to lead to autophagy with arginine deprivation. In addition, the combination of an autophagy inhibitor plus arginine deprivation was more effective than arginine deprivation alone. In cells with suppressed ASS1 expression, P-gp expression was upregulated as compared to that in negative controls.


CONCLUSIONS
These results indicate that the reduced ASS1 expression in Dox-resistant sarcomas may contribute to drug resistance in association with the expression of P-gp. ASS1 deficiency is a potential target for novel drug therapies. The combination of arginine-deprivation therapy and an autophagy inhibitor may have anti-tumor effects in refractory sarcomas.


METHODS
We assessed the expressions of ASS1 and P-glycoprotein (P-gp) in clinical specimens and cell lines of osteosarcoma (KHOS), doxorubicin (Dox)-resistant osteosarcoma (KHOSR2), epithelioid sarcomas (ES-X and VAESBJ) and alveolar soft part sarcoma (ASPS-KY). Each cell line was cultured in arginine-containing and arginine-free media. Cell growth was assessed using an XTT assay and flow cytometry. We analyzed the induction of autophagy in arginine-free medium. Moreover, we assessed the expression of P-gp after suppressing ASS1 in Dox-sensitive cells (MCF-7 and KHOS) and after transfecting ASS1 into Dox-resistant cells (ES-X, VAESBJ, ASPS-KY and KHOSR2).
Division of Musculoskeletal Oncology, National Cancer Center Hospital, Tokyo, Japan.|Department of Experimental Therapeutics, National Cancer Center Hospital, Tokyo, Japan.|Department of Orthopaedic Surgery, Juntendo University School of Medicine, Tokyo, Japan.|Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Tokyo, Japan.
Youngji
Y
Kim
Division of Musculoskeletal Oncology, National Cancer Center Hospital, Tokyo, Japan.
Eisuke
E
Kobayashi
Division of Musculoskeletal Oncology, National Cancer Center Hospital, Tokyo, Japan.|Department of Orthopaedic Surgery, Juntendo University School of Medicine, Tokyo, Japan.
Daisuke
D
Kubota
Department of Orthopaedic Surgery, Juntendo University School of Medicine, Tokyo, Japan.
Yoshiyuki
Y
Suehara
Department of Orthopaedic Surgery, Juntendo University School of Medicine, Tokyo, Japan.
Kenta
K
Mukaihara
Department of Orthopaedic Surgery, Juntendo University School of Medicine, Tokyo, Japan.
Keisuke
K
Akaike
Department of Hematopoietic Stem Cell Transplantation, National Cancer Center Hospital, Tokyo, Japan.|Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Tokyo, Japan.
Ayumu
A
Ito
Department of Orthopaedic Surgery, Juntendo University School of Medicine, Tokyo, Japan.
Kazuo
K
Kaneko
Division of Musculoskeletal Oncology, National Cancer Center Hospital, Tokyo, Japan.
Hirokazu
H
Chuman
Division of Musculoskeletal Oncology, National Cancer Center Hospital, Tokyo, Japan.
Akira
A
Kawai
Department of Experimental Therapeutics, National Cancer Center Hospital, Tokyo, Japan.|Exploratory Oncology Research and Clinical Trial Center, National Cancer Center, Tokyo, Japan.
Shigehisa
S
Kitano
D018435:ATP Binding Cassette Transporter, Subfamily B / Q000378:metabolism; D000293:Adolescent; D000328:Adult; D000368:Aged; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology / Q000627:therapeutic use; D001120:Arginine / Q000378:metabolism; D001124:Argininosuccinate Synthase / Q000235:genetics / Q000378:metabolism*; D001343:Autophagy / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002469:Cell Separation / Q000379:methods; D002648:Child; D015536:Down-Regulation; D004317:Doxorubicin / Q000494:pharmacology / Q000627:therapeutic use; D019008:Drug Resistance, Neoplasm; D005260:Female; D005434:Flow Cytometry; D059585:G1 Phase Cell Cycle Checkpoints; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D009364:Neoplasm Recurrence, Local / Q000201:enzymology* / Q000473:pathology; D060888:Real-Time Polymerase Chain Reaction; D012509:Sarcoma / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D015854:Up-Regulation
C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D000903:Antibiotics, Antineoplastic; D004317:Doxorubicin; D001120:Arginine; D001124:Argininosuccinate Synthase
P-glycoprotein; argininosuccinate synthetase; bone and soft-tissue sarcoma; drug-resistance; metabolic error
Clin Orthop Relat Res. 2000 Apr;(373):11-7
10810457
J Nutr. 2007 Jun;137(6 Suppl 2):1602S-1609S
17513435
Proc Natl Acad Sci U S A. 2014 Sep 30;111(39):14015-6
25228774
Int J Cancer. 2009 Sep 15;125(6):1454-63
19533750
Sci Signal. 2014 Apr 01;7(319):ra31
24692592
J Clin Oncol. 2003 Apr 15;21(8):1574-80
12697883
Nat Rev Cancer. 2010 Feb;10 (2):147-56
20075923
Eur J Pharmacol. 2009 Dec 25;625(1-3):220-33
19836374
Mol Cancer Ther. 2010 Mar;9(3):535-44
20159990
Eur J Cancer Clin Oncol. 1987 Mar;23(3):311-21
3595691
Cell Death Dis. 2012 Jul 05;3:e342
22764101
J Clin Oncol. 2015 Sep 20;33(27):3029-35
26304877
Invest New Drugs. 2013 Apr;31(2):425-34
22864522
Br J Cancer. 2012 Jan 17;106(2):324-32
22134507
Int J Cancer. 2007 Feb 15;120(4):897-905
17096330
Cancer. 1970 Nov;26(5):1029-41
5476785
BMC Cancer. 2014 Sep 20;14:686
25240403
Cancer. 2004 Feb 15;100(4):826-33
14770441
Int J Biochem Mol Biol. 2011;2(1):8-23
21494411
Clin Orthop Relat Res. 2005 Sep;438:144-8
16131883
Ann Surg Oncol. 2010 Dec;17(12):3229-33
20593242
Sarcoma. 2012;2012:907179
22666000
Blood. 2015 Jun 25;125(26):4060-8
25896651
Expert Rev Anticancer Ther. 2006 Jul;6(7):1075-85
16831079
Tumour Biol. 2014 Jan;35(1):161-9
23897555
Cancer. 2008 Aug 1;113(3):573-81
18521899
Nat Rev Cancer. 2005 Sep;5(9):726-34
16148885
Mod Pathol. 2001 Jul;14(7):655-63
11454997
J Cancer Res Clin Oncol. 2015 Aug;141(8):1363-9
25548129
Am J Surg Pathol. 1985 Apr;9(4):241-63
4014539
Cancer Cell. 2008 Jun;13(6):472-82
18538731
Eur J Cancer. 2003 Jul;39(11):1511-6
12855256
J Gastroenterol Hepatol. 2013 Feb;28(2):365-8
23339388
Cell Death Dis. 2013 Jan 17;4:e458
23328665
Clin Cancer Res. 2013 Jun 1;19(11):2861-72
23549872
J Cancer Res Clin Oncol. 1983;106 Suppl:55-67
6604058
Am J Clin Oncol. 1988 Feb;11(1):39-45
3341273
Lancet Oncol. 2014 Apr;15(4):415-23
24618336
Int J Cancer. 2005 May 10;114(6):854-62
15609299
Apoptosis. 2009 Nov;14 (11):1274-88
19757066
Nat Rev Cancer. 2002 Jan;2(1):48-58
11902585
Sci Rep. 2015 Apr 30;5:9783
25928182
J Clin Oncol. 2013 Mar 1;31(7):e111-3
23319692
Expert Opin Investig Drugs. 2006 Jul;15(7):815-22
16787144
Curr Mol Med. 2010 Jun;10(4):405-12
20459375
Dev Cell. 2004 Apr;6(4):463-77
15068787
Cancer. 1952 Jan;5(1):100-11
14886902