In vivo loss of expression of argininosuccinate synthetase in malignant pleural mesothelioma is a biomarker for susceptibility to arginine depletion.
OBJECTIVE
Malignant pleural mesothelioma (MPM) is an increasing health burden on many societies worldwide and, being generally resistant to conventional treatment, has a poor prognosis with a median survival of <1 year. Novel therapies based on the biology of this tumor seek to activate a proapoptotic cellular pathway. In this study, we investigated the expression and biological significance of argininosuccinate synthetase (AS), a rate-limiting enzyme in arginine production.


METHODS
Initially, we documented down-regulation of AS mRNA in three of seven MPM cell lines and absence of AS protein in four of seven MPM cell lines. We confirmed that the 9q34 locus, the site of the AS gene, was intact using a 1-Mb comparative genomic hybridization array; however, there was aberrant promoter CpG methylation in cell lines lacking AS expression, consistent with epigenetic regulation of transcription. To investigate the use of AS negativity as a therapeutic target, arginine was removed from the culture medium of the MPM cell lines.


RESULTS
In keeping with the cell line data, 63% (52 of 82) of patients had tumors displaying reduced or absent AS protein, as assessed using a tissue microarray. Cell viability declined markedly in the AS-negative cell lines 2591 and MSTO but not in the AS-positive cell line, 28. This response was apparent by day 4 and maintained by day 9 in vitro. Arginine depletion induced BAX conformation change and mitochondrial inner membrane depolarization selectively in AS-negative MPM cells.


CONCLUSIONS
In summary, we have identified AS negativity as a frequent event in MPM in vivo, leading to susceptibility to cytotoxicity following restriction of arginine. A phase II clinical trial is planned to evaluate the role of arginine depletion in patients with AS-negative MPM.
Szlosarek
Peter W
PW
Cancer Research UK Translational Oncology Laboratory, Barts and The London, UK.
Klabatsa
Astero
A
Pallaska
Arben
A
Sheaff
Michael
M
Smith
Paul
P
Crook
Tim
T
Grimshaw
Matthew J
MJ
Steele
Jeremy P
JP
Rudd
Robin M
RM
Balkwill
Frances R
FR
Fennell
Dean A
DA
D001120:Arginine / Q000378:metabolism*; D001124:Argininosuccinate Synthase / Q000235:genetics*; D014408:Biomarkers, Tumor / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002899:Chromosomes, Human, Pair 9 / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics; D006801:Humans; D008654:Mesothelioma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D051336:Mitochondrial Membranes / Q000378:metabolism; D010997:Pleural Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics; D046888:Tissue Array Analysis / Q000379:methods; D051028:bcl-2-Associated X Protein / Q000378:metabolism
C494826:BAX protein, human; D014408:Biomarkers, Tumor; D012333:RNA, Messenger; D051028:bcl-2-Associated X Protein; D001120:Arginine; D001124:Argininosuccinate Synthase