Tawnya L. Bowles

Arginine Deiminase as a Novel Therapy for Prostate Cancer Induces Autophagy and Caspase-Independent Apoptosis

Arginine deprivation as an anticancer therapy has historically been met with limited success. The development of pegylated arginine deiminase (ADI-PEG20) has renewed interest in arginine deprivation for the treatment of some cancers. The efficacy of ADI-PEG20 is directly correlated with argininosuccinate synthetase (ASS) deficiency. CWR22Rv1 prostate cancer cells do not express ASS, the rate-limiting enzyme in arginine synthesis, and are susceptible to ADI-PEG20 in vitro. Interestingly, apoptosis by 0.3 microg/mL ADI-PEG20 occurs 96 hours posttreatment and is caspase independent. The effect of ADI-PEG20 in vivo reveals reduced tumor activity by micropositron emission tomography as well as reduced tumor growth as a monotherapy and in combination with docetaxel against CWR22Rv1 mouse xenografts. In addition, we show autophagy is induced by single amino acid depletion by ADI-PEG20. Here, autophagy is an early event that is detected within 1 to 4 hours of 0.3 microg/mL ADI-PEG20 treatment and is an initial protective response to ADI-PEG20 in CWR22Rv1 cells. Significantly, the inhibition of autophagy by chloroquine and Beclin1 siRNA knockdown enhances and accelerates ADI-PEG20-induced cell death. PC3 cells, which express reduced ASS, also undergo autophagy and are responsive to autophagy inhibition and ADI-PEG20 treatment. In contrast, LNCaP cells highly express ASS and are therefore resistant to both ADI-PEG20 and autophagic inhibition. These data point to an interrelationship among ASS deficiency, autophagy, and cell death by ADI-PEG20. Finally, a tissue microarray of 88 prostate tumor samples lacked expression of ASS, indicating ADI-PEG20 is a potential novel therapy for the treatment of prostate cancer

Pancreatic cancer cell lines deficient in argininosuccinate synthetase are sensitive to arginine deprivation by arginine deiminase

Eukaryotic cells can synthesize the non‐essential amino acid arginine from aspartate and citrulline using the enzyme argininosuccinate synthetase (ASS). It has been observed that ASS is underexpressed in various types of cancers ASS, for which arginine become auxotrophic. Arginine deiminase (ADI) is a prokaryotic enzyme that metabolizes arginine to citrulline and has been found to inhibit melanoma and hepatoma cancer cells deficient of ASS. We tested the hypothesis that pancreatic cancers have low ASS expression and therefore arginine deprivation by ADI will inhibit cell growth. ASS expression was examined in 47 malignant and 20 non‐neoplastic pancreatic tissues as well as a panel of human pancreatic cancer cell lines. Arginine deprivation was achieved by treatment with a recombinant form of ADI formulated with polyethylene glycol (PEG‐ADI). Effects on caspase activation, cell growth and cell death were examined. Furthermore, the effect of PEG‐ADI on the in vivo growth of pancreatic xenografts was examined. Eighty‐seven percent of the tumors lacked ASS expression; 5 of 7 cell lines similarly lacked ASS expression. PEG‐ADI specifically inhibited growth of those cell lines lacking ASS. PEG‐ADI treatment induced caspase activation and induction of apoptosis. PEG‐ADI was well tolerated in mice despite complete elimination of plasma arginine; tumor growth was inhibited by ∼50%. Reduced expression of ASS occurs in pancreatic cancer and predicts sensitivity to arginine deprivation achieved by PEG‐ADI treatment. Therefore, these findings suggest that arginine deprivation by ADI could provide a beneficial strategy for the treatment of pancreatic cancer, a malignancy in which new therapy is desperately needed.

ERK/BCL-2 pathway in the resistance of pancreatic cancer to anoikis.

BACKGROUND Anoikis is a special type of programmed cell death after loss of cell-cell and cell-extracellular matrix interactions. Resistance to anoikis is likely involved in the process of metastasis, specifically during the tumor cell migration through lymph or vascular channels. We have previously shown that BCL-2 confers resistance to other forms of programmed cell death (i.e., apoptosis); furthermore, the extracellular signaling-regulated kinase (ERK) signaling pathway regulates BCL-2 expression. We therefore tested the hypothesis that pancreatic cancer cell lines are resistant to anoikis and this resistance is due to activation of ERK1/2 and subsequent overexpression of BCL-2. MATERIALS AND METHODS Pancreatic cancer cell lines (MIA-PaCa-2 and BxPC-3) were examined for cell death following loss of adherence to extracellular matrix. Subclones of the MIA-PaCa-2 cell line (either selected in vivo for increased metastatic potential [MIA-LM2] or overexpressing BCL-2 [MIA-BCL2]) were also examined for induction of anoikis following loss of extracellular matrix adherence. Finally, the effect of the ERK inhibitor (PD98059) on BCL-2 expression and induction of anoikis was examined. RESULTS Under conditions of loss of cell-extracellular matrix interaction, pancreatic cancer cells undergo varying amounts of anoikis. Basal levels of activated ERK and BCL-2 paralleled the sensitivity to induction of anoikis. The highly metastatic cell line, MIA-LM2, was more resistant to anoikis than the parental cell line. Inhibition of ERK down-regulated BCL-2 and was associated with restoration of sensitivity to anoikis. CONCLUSIONS Activation of a signaling pathway from ERK to overexpression of BCL-2 may confer resistance to anoikis, a critical step in the development of metastasis. Targeting the ERK/BCL-2 pathway may lead to sensitization of pancreatic cancer to anoikis, thereby decreasing the ability of these cells to metastasize.

Abstract 4847: Arginine deprivation by PEG-ADI induces autophagic cell death and enhances the tumor suppression effect of gemcitabine in pancreatic cancer

Metabolic stress has been recently recognized to induce autophagic cell death; we have recently shown that nearly 80% of pancreatic adenocarcinoma tumor specimens are auxotrophic for the semi-essential amino acid arginine due to transcriptional suppression of argininosuccinate synthase (ASS). We hypothesized that selective arginine deprivation would induce autophagy and furthermore, by targeting alternative death pathways would increase the cytotoxic response to the chemotherapy gemcitabine. Methods: The human pancreatic cell line MIA-PaCa-2 was shown to be deficient in ASS by western blot. Arginine deprivation was achieved by treatment of cells and xenografts with PEG-ADI, which shuttles arginine to citrulline. Cells were treated with either PEG-ADI, gemcitabine or both. Cell death was measured by MTT and FACS. Western blot was used to measure autophagy by LC3 processing and apoptosis by caspase-3 cleavage. RRM2 expression was measured by western blot in cells treated with PEG-ADI. ASS expression was measured by western blot in cells treated with gemcitabine. To test the in vivo effect arginine deprivation a mice xenograft model was created by subcutaneously injecting Mia-PaCa2 cells into the flanks of 28 mice (two tumors per mice). Tumors were allowed to mature at which time the mice were assigned to one of four treatment groups: PBS control (once weekly), ADI-PEG20 (5 IU once weekly), Gemcitabine (125mg/kg twice weekly), or Gemcitabine and ADI-PEG20. All treatments were administered via intraperitoneal injection and tumors were measured twice a week. Results: 1. MIA-PaCa-2 lacks ASS and is auxotrophic for arginine. 2. PEG-ADI induces autophagy and cell death 3. Gemcitabine has little in vitro cytotoxicity 4. The combination of gemcitabine and PEG-ADI is at least additive in vitro and in vivo. 5. PEG-ADI does not regulate RRM2 expression and gemcitabine does not regulate ASS expression. Conclusions: Therapy targeting autophagy may provide increased efficacy for traditional genotoxic chemotherapies in pancreatic cancer. PEG-ADI in combination with gemcitabine is a novel combination to be pursued in clinical trials Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4847.