Context-dependent role of ATG4B as target for autophagy inhibition in prostate cancer therapy

https://doi.org/10.1016/j.bbrc.2013.10.117Get rights and content

Highlights

  • Autophagy is an emerging anticancer therapy target.

  • ATG4B inhibitors are being developed as a novel class of autophagy inhibitors.

  • ATG4B plays a cancer type-, treatment-, and context-dependent role.

  • Predictive markers will be instrumental to clinically develop ATG4B inhibitors.

Abstract

ATG4B belongs to the autophagin family of cysteine proteases required for autophagy, an emerging target of cancer therapy. Developing pharmacological ATG4B inhibitors is a very active area of research. However, detailed studies on the role of ATG4B during anticancer therapy are lacking. By analyzing PC-3 and C4-2 prostate cancer cells overexpressing dominant negative ATG4BC74A in vitro and in vivo, we show that the effects of ATG4BC74A are cell type, treatment, and context-dependent. ATG4BC74A expression can either amplify the effects of cytotoxic therapies or contribute to treatment resistance. Thus, the successful clinical application of ATG4B inhibitors will depend on finding predictive markers of response.

Introduction

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved process implicated in cellular homeostasis and response to stress [1], [2]. Briefly, cellular macromolecules or organelles are sequestered in autophagosomes, which in turn fuse with lysosomes to form autolysomes. The activation of lysosomal enzymes then mediates the degradation of the autolysosome content to procure cells with carbohydrates, nucleotides, amino acids, and fatty acids. By enabling the removal of damaged cellular components, and by providing building blocks for anabolic reactions or energy production, autophagy can contribute to cell survival. Conversely, unabated autophagy has been associated with cell death, although it is controversial if autophagy is a bona fide mechanism of cell death on its own [3].

While autophagy plays an important role in a broad range of disease conditions, its role in cancer is particularly complex [1], [4]. During early carcinogenesis the tumor suppressing activities of autophagy seem to prevail (e.g., mitigation of DNA damage). In contrast, autophagy may contribute to tumor cell survival in established tumors that are commonly characterized by severe hypoxia and reduced nutrient availability, and in tumors subjected to cytotoxic therapy. Therefore, autophagy inhibition is being studied in phase II clinical trials combining the autophagy inhibitor hydroxychloroquine with chemotherapy (www.clinicaltrials.gov) [5].

Hydroxychloroquine is a lysosomotropic 4-aminoquinoline that impairs the fusion of autophagosomes and lysosomes, and that inhibits the acidification of autolysosomes and thus lysosomal enzyme activation [5], [6], [7]. However, the clinical development of hydroxychloroquine as an anticancer agent is challenging due to poor pharmacokinetic properties for cancer indications, lack of specificity, side-effects that could be enhanced by concomitant chemotherapy, and concerns whether the tissue levels of hydroxychloroquine that can be safely achieved are high enough to significantly impair autophagy [6], [8], [9]. Hence, the development of more potent and specific autophagy inhibitors is a very active area of research [10], [11], [12], [13], [14], [15]. ATG4B (autophagy-related 4B, also named autophagin-1) is a particularly interesting drug target in this respect. ATG4B belongs to the autophagin family of cysteine proteases required for autophagy [16]. By controlling the lipidation status of the autophagosome membrane protein LC3 (microtubule-associated protein 1 light chain 3), ATGB4 controls autophagosome maturation. Briefly, pro-LC3 is cleaved by ATG4B to its LC3-I isoform (18 kDa), followed by ATG7 and ATG3 mediated conjugation of LC3-I with phopshatidyl-ethanolamine to yield LC3-II (16 kDa), which integrates into the crescent autophagosome double-membrane under the control of the ATG5–ATG12–ATG16L complex [17]. Autophagic activity can be semi-quantified by obtaining the LC3-II/LC3-I ratio under steady state conditions, and by analyzing the LC3-II/LC3-I ratio as well as the accumulation of LC3-II in the presence of chloroquine, which inhibits the degradation of LC3-II. The latter reveals information about the autophagic flux [18]. The protease activity of ATG4B is considered more readily druggable than the complex protein–protein interactions found in other components of the autophagy machinery such as the multi-protein autophagy initiation complex. Furthermore, one expects ATG4B inhibitors to be very well tolerated given that the phenotype of atg4b deficient mice is largely restricted to inner ear development abnormalities [19], [20].

Given the lack of detailed studies on the role of ATG4B during anticancer therapy [21], we describe herein the derivation of PC-3 and C4-2 prostate cancer cells expressing ATG4BC74A, a dominant negative mutant of ATG4B phenocopying the characteristics of atg4b deficient cells [19], [22].

Section snippets

Materials

Docetaxel (Sanofi) and doxorubicin (Novopharm) were obtained from the Odette Cancer Centre Pharmacy. Docetaxel was freshly reconstituted before each use as per manufacturer’s instructions. Doxorubicin saline stock solution was kept at 4 °C. Topotecan (gift of Dr. R.S. Kerbel) was reconstituted in DMSO and the stock solution was kept at −80 °C. All other reagents were obtained from Sigma–Aldrich unless otherwise indicated.

Cell lines and culturing

PC-3 (purchased from the American Type Culture Collection) and C4-2 (gift

Characterization of ATG4BC74A expressing PC-3 and C4-2 human prostate cancer cell lines

Following transduction with pmStrawberry-Atg4bC74A, we found a ratio of ATG4BC74A over endogenous ATG4B of around 2.5 (PC-3) and 3 (C4-2), which was not markedly affected by autophagy-promoting culture conditions such as hypoxia (1% O2), or by chloroquine treatment (Fig. 1A, C). The presence of ATG4BC74A considerably changed the expression pattern of LC3 isoforms (Fig. 1B/C, E/F). LC3-I was found to accumulate in ATG4BC74A expressing PC-3 and C4-2 compared to corresponding control cells. When

Discussion

Autophagy plays a dual role in therapeutic resistance, one of the major challenges of systemic anticancer therapy. By facilitating the removal of damaged cellular structures, and by supporting anabolic and repair processes in cells exposed to cytotoxic stress, autophagy can contribute to cell survival and thus to therapeutic resistance. On the other hand, autophagy may also contribute to cell death and hence to treatment response [4].

The dual role of autophagy is only one of the challenges in

Acknowledgments

We are grateful to Dr. M.M. Rahim Mobin for providing us with the retroviral transduction system. These studies were conducted with funds from Prostate Cancer Canada to U. Emmenegger.

References (34)

  • S. Shen et al.

    The end of autophagic cell death?

    Autophagy

    (2012)
  • E. White

    Deconvoluting the context-dependent role for autophagy in cancer

    Nat. Rev. Cancer

    (2012)
  • Z.J. Yang et al.

    The role of autophagy in cancer: therapeutic implications

    Mol. Cancer Ther.

    (2011)
  • Q. McAfee et al.

    Autophagy inhibitor Lys05 has single-agent antitumor activity and reproduces the phenotype of a genetic autophagy deficiency

    Proc. Natl. Acad. Sci. U.S.A.

    (2012)
  • J. Ducharme et al.

    Clinical pharmacokinetics and metabolism of chloroquine. Focus on recent advancements

    Clin. Pharmacokinet.

    (1996)
  • T. Kimura et al.

    Chloroquine in cancer therapy: a double-edged sword of autophagy

    Cancer Res.

    (2013)
  • S.M. Gorski et al.

    Targeting autophagy: the Achilles’ heel of cancer

    Autophagy

    (2012)
  • Cited by (0)

    View full text