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

Abstract

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 <1year. 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 2591and 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.

Novel systemic therapy against malignant pleural mesothelioma

“…Preclinical models demonstrated that arginine deprivation is synthetically lethal in MPM cells that do not express argininosuccinate synthetase 1 (ASS1) (161). Approximately 60% of MPM tumors have loss of ASS1.…”

Arginine Deprivation as a Targeted Therapy for Cancer

“…Nevertheless, Dillion et al have reported that all 51 hepatoma tumor samples are deficient in ASS by immunohistochemical staining [36] Antitumor Activity of ADI-PEG20 in other Cell Lines Certain mesothelioma cell lines (2591 and MSTO) do not express ASS and are sensitive to ADI-PEG20 while two other cell lines (226,2461) express high levels [17]. Immunohistochemical study showed 63% of mesothelioma express low levels of ASS [17]. Based on this study, clinical trials in mesothelioma have been initiated [Szlosarck, personal communication].…”

“…In fact, our preliminary data showed increased Beclin1 in these cell lines with Mel1220 having the highest Beclin1 levels. In mesothelioma, Szlosarek has shown that arginine depletion induces BAX conformation changes and mitochondrial inner membrane depolarization in ASS(−) cells which leads to apoptosis [17,39]. Thus, the mechanism(s) which lead to apoptosis is complex and may be different among all the cell lines which lack ASS expression.…”

“…Thus, depletion of this amino acid by asparaginase therapy is generally well tolerated since normal human cells do not require asparagine. Thus, eliminating a certain Several laboratories have reported that certain tumor cell lines such as melanoma, hepatocellular carcinoma, renal cell carcinoma and some mesotheliomas do not express ASS, and hence become auxotrophic for arginine [9,11,[15][16][17][18]. Consequently, arginine degrading enzyme has been shown by several laboratories to have antitumor effect in these tumors [9,13,[19][20][21][22][23][24][25].…”

“…Furthermore, this enzyme is active in physiologic pH and has a high affinity for arginine. In fact, ADI has been reported to have antitumor activity both in vitro and in vivo in melanoma and hepatocellular carcinoma cell lines and in other cell lines [15][16][17][18][31][32][33][34]. However, one of the drawbacks of ADI is that it is not produced by humans and, consequently, it is highly antigenic.…”