High-order TRAIL oligomer formation in TRAIL-coated lipid nanoparticles enhances DR5 cross-linking and increases antitumour effect against colon cancer

doi:10.1016/j.canlet.2016.10.005

Cancer Letters

Volume 383, Issue 2, 28 December 2016, Pages 250-260

https://doi.org/10.1016/j.canlet.2016.10.005 Get rights and content

Highlights

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Liposomes with TRAIL show greater cytotoxicity than soluble TRAIL in colon cancer cells.
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Liposomes with TRAIL overcome resistance to soluble TRAIL in colon cancer cells.
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TRAIL anchored to liposome surface form high-order oligomers which in turn, induce a more potent clusterization of DR5.
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Liposomes with TRAIL show greater in vivo anti-tumour activity than soluble TRAIL.

Abstract

During the last years, a great effort has been invested into developing new TRAIL formulations with increased bioactivity, trying to overcome the resistance to conventional soluble TRAIL (sTRAIL) exhibited by many primary tumours. In our group, we have generated artificial lipid nanoparticles decorated with sTRAIL (LUV-TRAIL), emulating the physiological TRAIL-containing exosomes by which T-cells release TRAIL upon activation. We already demonstrated that LUV-TRAIL has greater cytotoxicity against both chemoresistant haematologic tumour cells and epithelial carcinoma cells compared to a form of sTRAIL similar to that used in clinical trials. In this study we have tested LUV-TRAIL in several human colon cancer cell lines with different sensitivity to sTRAIL. LUV-TRAIL significantly improved sTRAIL cytotoxicity in all colon cancer cell lines tested. Trying to ascertain the molecular mechanism by which LUV-TRAIL exhibited improved cytotoxicity, we demonstrated that TRAIL-coated lipid nanoparticles were able to activate DR5 more efficiently than sTRAIL, and this relied on LUV-TRAIL ability to promote DR5 clustering on the cell surface. Moreover, we show that TRAIL molecules are arranged in higher order oligomers only in LUV-TRAIL, which may explain their enhanced DR5 clustering ability. Finally, LUV-TRAIL showed significantly better antitumour activity than sTRAIL in an in vivo model using HCT-116 xenograft tumours in nude mice, validating its potential clinical application.

Introduction

Colorectal cancer (CRC) is one of the most frequent solid tumours in western countries. In fact, recent estimations indicate that CRC is the third most common cancer both in males and females [1]. The survival rate throughout all stages has improved mainly due to the implementation of screening programs, leading to the detection of CRC in earlier stages, together with the development of more effective treatment options. However, CRC still accounts for about 9% of the estimated cancer-related deaths [1].

Resistance of cancer cells to apoptosis is one of the so-called hallmarks of cancer [2]. Since impairments in the intrinsic apoptotic pathway are often involved in this resistance, targeting the extrinsic apoptotic pathway is especially attractive for cancer therapy [3], [4]. Among the death ligands capable of triggering apoptosis through the extrinsic pathway, CD95L showed a remarkable strong cytotoxic potential on cancer cells. However, disappointingly it also displayed a severe systemic toxicity, therefore making unrealistic its use as anti-cancer agent [5]. On the other hand, another death ligand, namely tumour necrosis factor (TNF)-related apoptosis–inducing ligand (TRAIL), was found to be able to induce apoptosis in tumour cells without affecting most normal cells [6], [7]. Hence, it was soon considered as potentially useful for anti-cancer therapy [8], [9]. However, despite the encouraging initial results, TRAIL-based therapies showed very limited therapeutic activity in phase II/III clinical trials carried out on a wide variety of human cancers [3], [10]. In order to overcome TRAIL resistance, better sensitization strategies [11], [12], [13], as well as novel TRAIL formulations with improved bioactivity can be of great usefulness for its future clinical use [14], [15], [16], [17].

TRAIL has four membrane-bound receptors in humans: TRAIL-R1/DR4, TRAIL-R2/DR5, TRAIL-R3/DcR1 and TRAIL-R4/DcR2 [18], [19], [20], [21], [22], [23], [24]. In addition, TRAIL has been described to be able to bind to the soluble receptor osteoprotegerin (OPG) [25]. Among these five receptors, only DR4 and DR5 are able to transduce the apoptotic signal upon TRAIL binding. Although they share high similarity, both receptors present certain functional differences. For example, DR5 has been described to present higher affinity for TRAIL [26]. However, apparently DR5 requires further clustering, so it can only be properly activated by membrane-bound TRAIL or by artificially cross-linked versions of the molecule [27], [28]. On the contrary, DR4 can readily be activated by soluble versions of TRAIL, although cross-linked versions of TRAIL proved again to be more active than soluble TRAIL [29]. Regarding the relative contribution of both receptors to TRAIL-induced apoptosis, DR4 has been described to be the pre-eminent pro-apoptotic receptor in haematological cells, while in epithelial cells expressing both receptors DR5 appears to be the main pro-apoptotic receptor [30], [31], [32], [33], [34]. However, the differential contribution of both receptors is still controversial.

Our group demonstrated that, in physiological conditions, TRAIL was indeed released by activated human T cells in its transmembrane form, inserted in the membrane of lipid microvesicles called exosomes [35], [36]. On this basis, we have generated artificial lipid nanoparticles containing membrane-bound TRAIL (LUV-TRAIL) resembling those natural exosomes. We already demonstrated that LUV-TRAIL was more effective inducing apoptosis than soluble TRAIL against leukemia cells that presented resistance to soluble TRAIL and chemotherapeutic drugs [37], [38] and also in epithelial-derived cancer cells [39], [40]. This increased cytotoxicity induced by LUV-TRAIL was due to a superior DR5 activation, which led to an enhanced DISC recruitment [40], [41].

In the present work, we have tested LUV-TRAIL in several human colon tumour cell lines with different sensitivity to soluble TRAIL, finding that LUV-TRAIL was more efficient than soluble TRAIL inducing apoptosis in all cancer cells tested. Finally, the in vivo antitumour potential of LUV-TRAIL against colon carcinoma cells was tested in a model of nude mice xenografted with the human colon carcinoma cell line HCT-116. This novel TRAIL formulation showed an enhanced antitumour ability, and could be potentially useful to improve therapy against colon cancer.

Section snippets

Generation of liposomes covered with soluble recombinant TRAIL

Large Unilamellar Vesicles (LUV) with soluble recombinant TRAIL (hereafter sTRAIL) anchored on their surface were generated as previously described [37], [42]. Briefly, after generating the lipid nanoparticles, a version of sTRAIL (rTRAIL-His₆, corresponding to amino acids 95–281, kindly provided by Dr. M. MacFarlane [22]) was attached to their surface by incubation at 37 °C for 30 min.

Cell culture

HCT-116, HT29 and CACO-2 cell lines were obtained from ATCC. Bax-deficient HCT-116 cells deficient (HCT-116-Bax

LUV-TRAIL show a significant higher cytotoxic ability compared with sTRAIL in human CRC cell lines

Firstly, we checked the surface expression of the TRAIL receptors DR4 and DR5 in all CRC cell lines studied (Fig. S2). finding that all cell lines expressed both pro-apoptotic TRAIL receptors. Noteworthy, all cell lines showed a higher expression of DR5 than DR4. Then, we characterized the overall sensitivity of the four CRC cell lines to both forms of TRAIL. For that, cell viability was analysed after carrying out dose-response assays using both sTRAIL and LUV-TRAIL (Fig. 1A). CRC cell lines

Discussion

Despite the initial optimism that TRAIL incited as a possible new and highly selective antitumour agent, the clinical effectiveness of TRAIL, both in monotherapy and in combined regimes, has been disappointing [3], [4], [10]. It is currently accepted that in order to overcome the poor responsiveness of some cancers to recombinant TRAIL formulations currently available [17] it appears as absolutely indispensable to improve its pro-apoptotic potential by the development of novel TRAIL

Acknowledgments

We gratefully acknowledge Dr. Gorka Basáñez for supporting in LUV-TRAIL generation. We are also indebted to Dr. Christoph Borner for providing HCT-Bax^−/− cells. Finally, we gratefully acknowledge Dr. Henning Walczak for support throughout the years.

This work was supported by Grants PI13/00416 (LML) from Instituto de Salud Carlos III, SAF2013-48626-C2-1-R (AA) from Ministerio de Economía y Competitividad. Additionally, this work has been also supported in part by Grant DPI2011-28262-C04-01 (LJF)

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¹: Current address: Cell Death, Cancer and Inflammation, University College of London, Grower Street, London, WC1E 6BT, United Kingdom.

²: Current address: Servicio de Inmunología, Hospital Clínico Universitario Lozano Blesa, Avda/San Juan Bosco 15, Zaragoza, 50009, Spain.

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Original ArticleHigh-order TRAIL oligomer formation in TRAIL-coated lipid nanoparticles enhances DR5 cross-linking and increases antitumour effect against colon cancer

Highlights

Abstract

Introduction

Section snippets

Generation of liposomes covered with soluble recombinant TRAIL

Cell culture

LUV-TRAIL show a significant higher cytotoxic ability compared with sTRAIL in human CRC cell lines

Discussion

Acknowledgments

Cell

Biochem. Pharmacol.

J. Biol. Chem.

Immunity

Cancer Lett.

Cancer Lett.

Trends Mol. Med.

Cancer Lett.

Immunity

Curr. Biol.

J. Biol. Chem.

FEBS Lett.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Curr. Opin. Pharmacol.

J. Biol. Chem.

Leuk. Res.

J. Immunol. Methods

Pharmacol. Ther.

Biotechnol. Annu. Rev.

Mol. Cell

Exp. Cell Res.

Cancer Cell

Exp. Cell Res.

Cancer statistics, 2014

CA Cancer J. Clin.

Getting TRAIL back on track for cancer therapy

Cell Death Differ.

Lethal effect of the anti-Fas antibody in mice

Nature

Directing cancer cells to self-destruct with pro-apoptotic receptor agonists

Nat. Rev. Drug Discov.

The TRAIL apoptotic pathway in cancer onset, progression and therapy

Nat. Rev. Cancer

Death receptors as targets in cancer

Br. J. Pharmacol.

State of art and recent developments of anti-cancer strategies based on TRAIL

Recent Pat. Anticancer Drug Discov.

On the TRAIL to successful cancer therapy? Predicting and counteracting resistance against TRAIL-based therapeutics

Oncogene

Onto better TRAILs for cancer treatment

Cell Death Differ.

Original Article
High-order TRAIL oligomer formation in TRAIL-coated lipid nanoparticles enhances DR5 cross-linking and increases antitumour effect against colon cancer