Minimizar el exceso de aire en la combustión

Novel roles for FXR other than its role in metabolism were revealed recently in many non-enterohepatic tissues, including its role in cell growth regulation and carcinogenesis (Catalano et al., 2010). In FXR-null mice, hepatocarcinogenesis occurred spontaneously, including hepatocholangiocellular carcinoma and hepatocellular adenoma, consistent with a positive action of FXR in tumour growth (Kim et al., 2007b). Other studies produced data consistent with these findings: Zhang et al have shown that FXR down-regulation by miR-421 induced proliferation, migration, and invasion of hepatocellular carcinoma cells (HCC) (Zhang et al., 2012), while Modica et al demonstrated similar findings that FXR repressed intestinal carcinogenesis when it was activated by its ligands (Modica et al., 2008). In pancreatic cancer, FXR activation induced lymph node metastasis, leading to the proposal that FXR-antagonists may represent a novel class of therapeutics to retard pancreatic tumour development (Lee et al., 2011).

However, the exact mechanistic role that FXR plays in growth regulation, cancer, and apoptosis are still unclear and controversial. For example, studies have reported both positive and negative associations between expression of FXR and incidence of cancer, thus providing no definitive relationship (De Gottardi et al., 2006, Kim et al., 2007b, Journe et al., 2008, Modica et al., 2008, Maran et al., 2009, De Gottardi et al., 2004, Zhang et al., 2012, Lee et al., 2011). On this basis, the function of FXR in cancer, in general, and its role in pro-survival (e.g. migration, invasion and proliferation) or anti-survival (e.g apoptosis), specifically, remain an area that requires further study (Lee et al., 2011).

Recently, FXR expression was detected in breast cancer tissues and breast cancer cell lines in several studies (Silva et al., 2006, Swales et al., 2006, Journe et al., 2008). Further studies showed that FXR expression was related to proliferation of estrogen receptor-positive

luminal-like breast cancer in postmenopausal women, and FXR was also associated with tumour invasiveness and metastasis in this tissue (Journe et al., 2009).

As breast cancer occurrence has been linked to high-fat diets, it would be logical to presume that higher levels of circulating bile acids would also be present. This was indeed the case, with bile acids found in large amounts in the systemic circulation of postmenopausal women diagnosed with breast cancer (Costarelli and Sanders, 2002). Other studies showed that bile acids are implicated in other cancers such as esophageal and colon tumours (Zimber and Gespach, 2008, Debruyne et al., 2001). Swales et al demonstrated that activation of FXR by either CDCA or GW4064 induced apoptosis and inhibited the growth of breast cancer cell lines 7 and MDA-MB-468 (Swales et al., 2006), while FXR activation inhibited MCF-7 tamoxifen resistant growth (MCF-MCF-7TR) (Giordano et al., 2011). These data are consistent with the higher circulating levels of bile acids, and hence higher FXR activation, being a protective adaptation, acting to remove the tumour. However, not all studies are consistent with such a hypothesis: Silva et al showed that inhibition of FXR by the antagonist Z-guggulsterone induced apoptosis in MDA-MB-231 and prevented migration of these cells (Silva et al., 2006). Hence, the exact role of FXR in tumour progression is unclear, with evidence for both tumour-prevention and tumour-survival. It is thus important to undertake further studies to delineate the role of FXR activation in tumour aetiology.

Role of FXR in MMP regulation

Given the evidence that FXR activation may be important for the development of a pro-survival phenotype in tumours, it would be logical to hypothesis that FXR may interact with proteins involved in tumour growth, invasion and metastasis, such as the MMPs. However, few studies have examined the role of FXR in MMP regulation. Fiorucci et al. reported that in hepatic stellate cells (HSCs), the FXR-SHP regulatory cascade was involved in the regulation of tissue metalloproteinase inhibitor-1 (TIMP-1) and matrix metalloprotease (MMP) expression (Fiorucci et al., 2005). In addition, a study by Li, et al.,reported that the FXR agonist 6ECDCA decreased both MMP-2 and -9 activities and gene expression in the presence or absence of IL-1β in vascular smooth muscle cells (Yoyo T.Y. Li et al., 2006).

Both of these data are consistent with a protective effect of FXR activation, leading to a reduction in MMP activity and, hence, invasion potential. However, it has also been demonstrated that FXR activation induced cell motility in blood outgrowth endothelial cells (BOECs) (Das et al., 2006). FXR-mediated induction of SHP led to dissociation of the SP2/KLF6 complex, which is required to repress expression of MMP-9. This data would support a pro-survival role for FXR activation, through an increased expression of MMPs.

Once again, the literature is conflicted and further studies are required to fully understand the implications of FXR expression and activation in developing tumours.

Hypothesis and Objectives

The purpose of the present study was to test the hypothesis that FXR is a novel therapeutic target for breast cancer progression and metastasis.

GW4064 was identified to be a potential tool to characterize FXR function (Bass et al., 2009, Maloney et al., 2000), because it is selective agonist of FXR (Howarth et al., 2010). As bile acids activate many nuclear receptors for instance pregnane X receptor (PXR, NR1I2) and vitamin D receptor (VDR, NR1I1) (Xie et al., 2001, Makishima et al., 2002, Wang et al., 2002).

Objective 1: Measuring the effects of FXR ligands on cell viability.

 Treating all breast cancer and normal breast cell lines with FXR ligands natural

CDCA and synthetic GW4064 for 48 and 72 hours under 10% serum and serum free conditions and measuring the effects of FXR activation on cell viability by MTT assay.

Objective 2: Measuring the effects of FXR ligands on protein and mRNA levels of MMP-2, MMP-9, TIMP-1 and TIMP-2.

 Treating both cell lines MDA-MB-468 and MCF-7 with FXR ligands CDCA and GW4064 for 24 hours and preparing protein and RNA extracts and measure them by Western blotting, RTPCR, and also by ELISA in the conditioned media collected.

Objective 3: Measuring the effects of FXR ligands on MMP activity.

 By gelatin zymography and also by a fluorescence-based assay using MMP2/MMP-9 fluorescent substrate and conditioned media collected from cell viability experiments.

Objective 4: Measuring the effects of FXR ligands on cell migration

 Pre-treating breast cancer and normal breast cell lines with FXR agonist GW4064 for one hour prior to seeding in the transwells and incubating them for 72 hours and measuring by transwell migration assay, and also by using wound healing assay.

2 Materials and Methods

Materials

All materials were from Sigma-Aldrich (Poole, UK) and of molecular or cell-culture grade, as appropriate, unless otherwise stated.

Table 2-1 Materials and suppliers used

Material Supplier

Chemicals Used

GW4064 Tocris Biosciences, Abingdon, UK

Chenodeoxycholic Acid (CDCA) Sigma-Aldrich, Poole, UK Phorbol 12-myristate 13-acetate (PMA) Sigma-Aldrich, Poole, UK Epidermal growth factor (EGF) Sigma-Aldrich, Poole, UK Interleukin-1 beta (IL-1β) Sigma-Aldrich, Poole, UK Insulin-like growth factor 1 (IGF-1) Sigma-Aldrich, Poole, UK All-Trans Retinoic Acid (ATRA) Sigma-Aldrich, Poole, UK Gelatin Zymography & Western Blotting

Amicon Centrifugal Filter Units (10k filter)

Merck Millipore, Massachusetts, USA MMPs and TIMPs standards Enzo life sciences, Exeter, UK

Spectra Multicolour Broad Range Protein ladder

Fermentas, Loughborough, UK Protease Inhibitor Tablets Roche, Lewes, UK

Acrylamide stock solution 40% Microporous PVDF Western Blotting

Membrane (0.45μm)

Roche, Lewes, UK

Primary and secondary antibodies Santa Cruz Biotechnology, CA, USA β-Actin and TIMP-2 Primary antibodies Sigma-Aldrich, Poole, UK

Dried Skimmed Milk Marvel, Dublin, Ireland Pierce SuperSignal West pico

chemiluminescent Kit

Thermo scientific, IL, USA

Developer, Fixer, Hardener Champion Photochemistry, Essex, UK Restore Western Blot Stripping Buffer Thermo Scientific, IL, USA

X-Ray Film Thermo scientific, IL, USA

Real Time PCR

TRIzol® Reagent Invitrogen, Paisley, UK

Dnase, Dnase stop solution, buffers Promega, Southampton, UK Reverse Transcription kit, Master Mix

with low level Rox 2x

Primer Design, Southampton, UK Primers and probes sets Eurofins MWG Operon, Alabama, USA

Gelatin cleavage assay

MMP-2/MMP-9, MMP-1/MMP-9

Fluorogenic Substrates, Recombinant MMP-9 and MMP-2

Calbiochem by Merck Millipore, Nottingham, UK

Transwell Inserts 6.5 mm Corning Incorporated, USA Growth Factor Reduced Matrigel

Matrix

Table 2-2 Primers and TaqMan probes used for Real Time PCR Gene Primers and Probes Sequence 7500 Real Time PCR system software Applied Biosystem, California, USA

Tscratch Chair of computational Science, Zurich, Switzerland GraphPad PRISM v6.0 GraphPad Software Inc. California, USA

Methods