Arginine deiminase PEG20 inhibits growth of small cell lung cancers lacking expression of argininosuccinate synthetase

Abstract

Background: Some cancers have been shown to lack expression of argininosuccinate synthetase (ASS), an enzyme required for the synthesis of arginine and a possible biomarker of sensitivity to arginine deprivation. Arginine deiminase (ADI) is a microbial enzyme capable of efficiently depleting peripheral blood arginine.

Methods: Argininosuccinate synthetase expression was assessed in human small cell lung cancer (SCLC) by immunohistochemistry (IHC), with expression also assessed in a panel of 10 human SCLC by qRT-PCR and western blot. Proliferation assays and analyses of apoptosis and autophagy assessed the effect of pegylated ADI (ADI-PEG20) in vitro. The in vivo efficacy of ADI-PEG20 was determined in mice bearing SCLC xenografts.

Results: Approximately 45% of SCLC tumours and 50% of cell lines assessed were negative for ASS. Argininosuccinate synthetase-deficient SCLC cells demonstrated sensitivity to ADI-PEG20, which was associated with the induction of autophagy and caspase-independent cell death. Arginine deiminase-PEG20 treatment of ASS-negative SCLC xenografts caused significant, dose-dependent inhibition of tumour growth of both small and established tumours.

Conclusion: These results suggest a role for ADI-PEG20 in the treatment of SCLC, and a clinical trial exploring this therapeutic approach in patients with ASS-negative SCLC by IHC has now been initiated.

Figure 1

Small cell lung cancer human tumours and cell lines frequently lack expression of ASS: ASS protein expression (brown color, arrows) in normal skin (A), colon carcinoma (B) and small cell lung cancer (C) at high power magnification, identified using anti-ASS antibody 195-21-1 (LICR). Expression of ASS protein was assessed by western blot in a panel of SCLC cell lines and compared with positive control SW1222 colon cancer cells (D). Argininosuccinate synthetase protein expression in cell lines was observed to generally correlate with mRNA expression as determined by qRT-PCR (E).

Figure 2

Arginine deiminase-PEG20 inhibits the in vitro proliferation of ASS-negative SCLC cells. Adherent (A) and non-adherent (B) cells were treated with ADI-PEG20 for 120 h before proliferation was assayed using the MTS assay for adherent cells or the BCA total protein assay for non-adherent cells.

Figure 3

Arginine deiminase induces apoptosis and autophagy in ASS-negative SK-LC-13 SCLC cells. Fluorescence-activated cell sorting analysis of sub-G₁ DNA content demonstrating apoptosis: cells were incubated in control media (A), 25 nM topotecan (B), 1.0 mIU ml⁻¹ ADI-PEG20 (C) and 10 mIU ml⁻¹ ADI-PEG20 (D) for 72 h before DNA staining with PI. An increase in LC3-II protein level following 24 h incubation with ADI-PEG20 or chloroquine (CQ)-positive control suggests autophagy (E). The apoptosis induced by ADI-PEG20 seems to be caspase independent, as only topotecan chemotherapy was observed to induce activation of caspase-3 in SK-LC-13 cells (F).

Figure 4

Silencing of ASS expression with siRNA. Relative expression of ASS mRNA expression determined by RT–PCR in SW1222 cells treated with ASS siRNA (A). Relative expression of ASS protein assessed by western blot in SW1222 cells treated with ASS siRNA (B). The MTS proliferation assay of ADI-PEG20-treated cells demonstrating susceptibility to anti-proliferative effects following loss of ASS expression (C).

Figure 5

Arginine deiminase-PEG20 inhibits the growth of moderately sized SK-LC-13 SCLC xenografts in BALB/c-nude mice. Growth curves of mice receiving PBS vehicle (•), a short 20 day course (dosing every 5 days) of ADI-PEG20 (▪), or continued dosing (every 5 days until group termination) of ADI-PEG20 (□) at doses of 1 IU per mouse (A), 2 IU per mouse (B) and 5 IU per mouse (C) as indicated. Tumour volumes at termination of the control group on day 33 are shown in (D). Serum levels of ADI-PEG20 (E), arginine (F) and citrulline (G) are shown for days 0, 12 and 40 of the study. Values are the same in short and extended dosing cohorts at day 0 and 12, as extended dosing was only initiated at day 20.

Figure 6

Arginine deiminase-PEG20 inhibits the growth of large SK-LC-13 SCLC xenografts in BALB/c-nude mice. Growth curves of mice receiving PBS vehicle (•), a short 20 day course of ADI-PEG20 (black arrows) (▪), or continued dosing of ADI-PEG20 (grey arrows) (▴) at doses of 1 IU per mouse (A), 2 IU per mouse (B) and 5 IU per mouse (C). Tumour volumes at termination of the control group on day 32 are shown in (D).