In vivo Loss of Expression of Argininosuccinate Synthetase in Malignant Pleural Mesothelioma Is a Biomarker for Susceptibility to Arginine Depletion

Clincial Cancer Research. 2006 Dec 1;12(23):7126-31. [Link]

Peter W. Szlosarek1,3, Astero Klabatsa2, Arben Pallaska6, Michael Sheaff2, Paul Smith4, Tim Crook4, Matthew J. Grimshaw5, Jeremy P. Steele3, Robin M. Rudd3, Frances R. Balkwill1 and Dean A. Fennell6

Authors’ Affiliations: 1 Cancer Research UK Translational Oncology Laboratory, Barts and The London and 2 Barts Mesothelioma Research, Institute of Pathology, Royal London Hospital, Queen Mary’s School of Medicine and Dentistry; 3 Lung and Mesothelioma Unit, Department of Medical Oncology, Gloucester House, St. Bartholomew’s Hospital, West Smithfield; 4 Cancer Genetics and Epigenetics, Breakthrough Breast Cancer Centre, Institute of Cancer Research; 5 Cancer Research UK Breast Cancer Biology Group, Guy’s Hospital, London, United Kingdom; and 6 Centre for Cancer Research and Cell Biology, Queen’s University Belfast and Northern Ireland Cancer Centre, Belfast, Northern Ireland


Purpose: 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.

Experimental Design: 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.