Cancer Letters

Cancer Letters

Volume 370, Issue 2, 28 January 2016, Pages 250-259
Cancer Letters

Original Articles
Activation of the Farnesoid X-receptor in breast cancer cell lines results in cytotoxicity but not increased migration potential

https://doi.org/10.1016/j.canlet.2015.10.031Get rights and content

Highlights

  • We examine the impact of Farnesoid X-receptor activation in breast cancer.

  • FXR agonists are cytotoxic in multiple breast cancer cell lines.

  • Cytotoxicity is mediated through enhanced apoptosis and autophagy.

  • FXR agonists do not increase the migratory potential of breast cancer cells.

  • FXR agonists warrant further examination as novel cancer chemotherapeutics.

Abstract

Breast cancer is the commonest form of cancer in women, but successful treatment is confounded by the heterogeneous nature of breast tumours: Effective treatments exist for hormone-sensitive tumours, but triple-negative breast cancer results in poor survival. An area of increasing interest is metabolic reprogramming, whereby drug-induced alterations in the metabolic landscape of a tumour slow tumour growth and/or increase sensitivity to existing therapeutics. Nuclear receptors are transcription factors central to the expression of metabolic and transport proteins, and thus represent potential targets for metabolic reprogramming. We show that activation of the nuclear receptor FXR, either by its endogenous ligand CDCA or the synthetic GW4064, leads to cell death in four breast cancer cell lines with distinct phenotypes: MCF-10A (normal), MCF-7 (receptor positive), MDA-MB-231 and MDA-MB-468 (triple negative). Furthermore, we show that the mechanism of cell death is predominantly through the intrinsic apoptotic pathway. Finally, we demonstrate that FXR agonists do not stimulate migration in breast cancer cell lines, an important potential adverse effect. Together, our data support the continued examination of FXR agonists as a novel class of therapeutics for the treatment of breast cancer.

Introduction

Breast cancer represents one of the largest killers of women in the Western world, with a lifetime risk of one in eight [1]. In the past decades a number of successful therapeutics targeting breast cancer have been developed, used in both single and combination therapies. However, improvements in the five year survival rate have not been as high as hoped. This is to a large degree due to the heterogeneous nature of breast tumours, with multiple molecular landscapes being classified as a single disease. These diverse tumour phenotypes result in varied responses to therapeutic intervention, often leading to sub-optimal patient response [2]. To aid optimisation of treatment regimens, breast tumours are commonly classified into three clinically significant groups: hormone sensitive, Her2 positive and triple negative breast cancers (TNBC) [3]. Both hormone sensitive and Her2-positive tumours have a range of good therapeutic options, but TNBC tumours are characterised by a lack of molecular targets and tendency to develop drug resistance. Not surprisingly, TNBC represents the leading cause of death in breast cancer [4]. It is thus imperative to develop novel therapeutic options that would exploit tumour vulnerabilities, mitigate drug resistance and lead to improved patient response, and especially in the case of TNBC tumours.

Two areas of therapeutic intervention have received increasing attention in the past few years: Synthetic lethality and metabolic vulnerability. These related concepts exploit the greater understanding of network biology to predict synergistic combination therapies [5]. In synthetic lethality, the concept of ‘rescue pathways’ is exploited: In essence, targeting of a single species in a biological pathway is often negated by re-routing of the biological network to exploit a secondary pathway. In synthetic lethality, drug combinations are used to target both the primary and secondary pathways, producing a synergistic cytotoxicity [6]. In metabolic vulnerability, this theory of network targeting is further expanded, looking for novel agents that will work with existing therapeutics. Tumour cells have a high metabolic load, due to their requirement to make all the precursors required for constant cell proliferation [7]. As such, the development of drugs that target metabolism may reduce cell proliferation, and synergise with existing drugs to produce more effective combination therapies [8].

Members of the nuclear receptor family of ligand-activated transcription factors generally regulate expression of genes that encode proteins involved in metabolic and transport processes [9]. As such, they may represent important targets to instigate metabolic reprogramming of tumour cells, leading to a reduction in supply of the components required for cell growth [10]. Nuclear receptor expression is widespread throughout the body [11], with a number being (over)expressed in breast tumours [12]. Indeed, two of the three major classifiers for breast cancer, the oestrogen receptor (ER; NR3A1) and the progesterone receptor (PR; NR3C3), are nuclear receptors, and disruption of their regulatory action is a successful treatment in receptor positive tumours [13], [14]. The Farnesoid X-receptor (FXR; NR1H4) is an adopted nuclear receptor with oxysterols such as primary and secondary bile acids as its endogenous ligands [15], [16]. Bile acids are the metabolic products of cholesterol, and can be toxic to the body at high concentrations. FXR acts to prevent accumulation of bile acids by inducing expression of a second nuclear receptor small heterodimer partner (SHP, NR0B2), which inhibits expression of CYP7A1, the rate-limiting enzyme in bile acid synthesis [17], [18]. The main site of bile acid production and secretion are the liver and intestine, respectively, and this is mirrored by high levels of FXR expression in these organs [19]. However, as approximately 70% of bile acids are re-absorbed by the body, they can reach micromolar concentrations in the plasma, which may underlie the expression of FXR in a number of other tissues [20]. FXR has been shown to be expressed in both normal breast tissue and breast tumours, and its activation in vitro stimulates apoptosis and inhibits aromatase [21]. These features suggest that FXR agonists may represent a novel class of breast cancer therapeutics due to their ability to alter the FXR-regulated metabolic network.

In the present work, we first fully delineate the molecular mechanisms by which FXR agonists activate apoptosis in breast cancer cell lines, demonstrating this to be through the intrinsic pathway. Next, we examine the ability of FXR agonists to stimulate migration in these cell lines, concluding that they do not possess this ability. Together, these data support the further examination of FXR agonists as cancer chemotherapeutics.

Section snippets

Materials

The breast tumour cell line MDA-MB-468 (ATCC-HTB-132), and the normal breast cell line MCF10A (ATCC-CRL-10317) were purchased from the ATCC (Teddington, UK), while the breast tumour cell lines MCF-7 (ECACC 86012803) and MDA-MB-231 (ECACC 92020424) were purchased from the ECACC (Porton Down, UK). CDCA and GW4064 were purchased from Sigma Aldrich (Poole, UK) and Tocris Biosciences (Abingdon, UK), respectively.

Cell culture

MCF7, MDA-MB-468 and MDA-MB-231 cells were grown in Dulbecco's modified eagle medium

Activation of FXR induces cell death in breast cancer cell lines

The nuclear receptor FXR is classically associated with bile acid homeostasis in the body and its target genes impact on the metabolic and transporter-mediated clearance of both bile acids and their precursors [15]. However, FXR activation has also been reported to elicit a number of other phenotypes, including cell death. To confirm this phenotype in breast cancer cell lines, we examined the effect of the endogenous ligand CDCA and the more potent and selective artificial ligand GW4064 [31].

Conflict of interest

KES is an employee of the Institute of Cancer Research. NA, RM, LM and NJP declare that they have no conflicts of interest.

Acknowledgements

NA was funded by the Kingdom of Saudi Arabia Ministry of Education (S7477).

References (49)

  • T.A. Kocarek et al.

    Use of dominant negative nuclear receptors to study xenobiotic-inducible gene expression in primary cultured hepatocytes

    J. Pharmacol. Toxicol. Methods

    (2002)
  • H. Zou et al.

    1 center dot cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9

    J. Biol. Chem

    (1999)
  • E.D. Strauch et al.

    Bile salts regulate intestinal epithelial cell migration by nuclear factor-kappa B-induced expression of transforming growth factor-beta

    J. Am. Coll. Surg

    (2003)
  • J. Silva et al.

    Lipids isolated from bone induce the migration of human breast cancer cells

    J. Lipid Res

    (2006)
  • J. Ferlay et al.

    Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012

    Int. J. Cancer

    (2015)
  • J. Crown et al.

    Emerging targeted therapies in triple-negative breast cancer

    Ann. Oncol

    (2012)
  • W.G. Kaelin

    The concept of synthetic lethality in the context of anticancer therapy

    Nat. Rev. Cancer

    (2005)
  • B. Al-Lazikani et al.

    Combinatorial drug therapy for cancer in the post-genomic era

    Nat. Biotechnol

    (2012)
  • N. Plant et al.

    Nuclear receptors: the controlling force in drug metabolism of the liver?

    Xenobiotica

    (2009)
  • H. Gronemeyer et al.

    Principles for modulation of the nuclear receptor superfamily

    Nat. Rev. Drug Discov

    (2004)
  • M. Uhlen et al.

    Tissue-based map of the human proteome

    Science

    (2015)
  • N.E. Hynes et al.

    ERBB receptors and cancer: the complexity of targeted inhibitors

    Nat. Rev. Cancer

    (2005)
  • X.J. Cui et al.

    Biology of progesterone receptor loss in breast cancer and its implications for endocrine therapy

    J. Clin. Oncol

    (2005)
  • M. Makishima et al.

    Identification of a nuclear receptor for bile acids

    Science

    (1999)
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    These authors contributed equally to the manuscript.

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