Background
During epithelial-mesenchymal transition (EMT), epithelial cells lose their epithelial characteristics and acquire a mesenchymal phenotype. EMT is important during development, but also implicated in carcinoma cell progression and invasion and may contribute to the advancement of breast carcinoma to metastasis [
1,
2].
We have previously described a role for the transcription factor Nuclear factor I-C2 (NFI-C2) in breast tumor development where NFI-C2 prevents EMT, motility, invasiveness and tumor growth [
3]. NFI-C2 is lost during breast tumor progression and is virtually absent from lymph node metastases. Patients classified as stage II invasive breast cancer with NFI-C2 in their breast tumor cells have better prognosis compared to those without detectable NFI-C2 [
3]. In mammary epithelial cells, the amount of active NFI-C2 is regulated by prolactin, via Janus activated kinase 2 localized in the nucleus [
4]. NFI-C2 is known to activate p53 and to participate in the regulation of expression of milk genes during pregnancy [
5,
6]. We also identified a direct transcriptional repression by NFI-C2 of the transcription factor Forkhead box F1 (FoxF1). This finding provides a possible mechanism through which NFI-C2 inhibits EMT, since FoxF1 was shown to induce EMT and invasiveness, and forced expression of FoxF1 enhanced xenograft tumorigenesis in nude mice [
3]. This was the first demonstration of a role of FoxF1 in cancer. Previously known functions of FoxF1 includes activities in mesenchymal cells during development and importance for mesoderm differentiation, vasculogenesis and organogenesis [
7‐
10]. FoxF1 also promotes mesenchymal cell migration by transcriptionally regulating integrin β
3 [
11] and plays an important role in tumor stromal cells by stimulating cancer cell migration [
12].
Lysyl oxidase (LOX) is an extracellular matrix enzyme that catalyzes the cross-linking of collagens or elastin, thereby controlling the structure and tensile strength of the extracellular matrix. LOX is synthesized as a 48 kDa precursor, N-glycosylated and secreted as a 50 kDa pro-enzyme. In the extracellular compartment, pro-LOX is processed to the 32 kDa catalytically active LOX and an 18 kDa pro-peptide. LOX belongs to a gene family consisting of five members; LOX, LOX-like 1 (LOXL1), LOXL2, LOXL3 and LOXL4. All play important roles in regulating extracellular matrix remodeling and homeostasis. Recently, novel roles of LOX have been demonstrated including the ability to regulate gene transcription [
13], cell differentiation and tissue development [
14], and cell adhesion, motility and migration [
15‐
17]. This implicates a role for LOX during tumor progression. For example, LOX has been shown to have critical roles in EMT and invasiveness [
18]. LOX-mediated collagen crosslinking can also promote tumor progression and invasion by increasing extracellular matrix stiffness [
19]. Elevated LOX levels in breast cancer positively correlate with invasiveness and reduced metastasis-free and overall survival [
20,
21].
Here we show that LOX is downregulated by NFI-C2 and upregulated by FoxF1 and that FoxF1-mediated upregulation of LOX is responsible for the invasiveness caused by FoxF1 overexpression. Further, we show that FoxF1 suppresses Smad2/3 signaling through a FAK- and LOX-dependent mechanism.
Methods
Affymetrix microarray
Total RNA was prepared from three pools of NF1-C2S-, FoxF1-, or vector control expressing HC11 cells (GenElute Mammalian total RNA kit; Sigma-Aldrich, Stockholm, Sweden). RNA integrity was verified by electrophoresis on a bio-analyzer (model 2100; Agilent, Palo Alto, CA). Five micrograms of each RNA preparation was labeled and hybridized to a Mouse Gene ST 1.0 Array. Hybridization and scanning of the arrays were performed at SCIBLU Microarray Resource Centre (MARC; Lund, Sweden).
Antibodies
LOX (Novus Biologicals, NB100-2527), FoxF1 (Human Protein Atlas project, HPA003454 (mAb FoxF1 3454)), FAK-pY576 and FAK (Invitrogen), FAK-pY396 (Santa Cruz), α-tubulin (Sigma), Smad2-pSer465–467 (Calbiochem), Smad2/3 (Cell Signaling), p38-pT180-Y182 and p38 (Cell signaling), HDAC-1 (Santa Cruz), p130Cas-pY249 and p130Cas (Cell signaling).
Cell culture
The mouse mammary epithelial cell line HC11 was grown in RPMI 1640 supplemented with 10 % FCS, 1 % PEST, 5 μg/mL insulin and 10 μg/mL EGF. The human mammary epithelial cell line HB2 was grown in DMEM supplemented with 10 % FCS, 1 % PEST, 10 μg/mL insulin and 5 μg/mL hydrocortisone. Transfectants carrying the tetracycline repressor construct had 10 μg/mL blasticidin S and transfectants additionally carrying IRES-GFP constructs also had 0,5 μg/mL Geneticin added to the medium. Cells were grown at 37 °C and 5 % CO2.
Revers transcription PCR analysis
Total RNA was extracted from cells using Sigma-Aldrich GenElute Mammalian Total RNA Miniprep kit. Reverse transcription PCR was performed with Titan One Tube RT-PCR System kit from Roche Applied Science.
Reverse transcription-quantitative PCR analysis
cDNA was synthesized using a QuantiTect Reverse Transcription Kit (Qiagen) according to manufacturer’s instructions. Real-time PCR (RT-PCR) was performed using Qiagen kit for SYBR® Green-based real-time PCR and were run on a LightCycler 480 (Roche, Sweden). The following primers were used: Mm FoxF1; −5′ACATCAAGCAACAGCCTCTGTC3′- and −5′ATGTCTTGGTAGGTGACCTC3′-, Mm LOX; QT00098028 (Qiagen), Hs FoxF1; QT00029687 (Qiagen), Hs LOX: −5′CCACTATGACCTGCTTGATG3′- and −5′CATACGCATGATGTCCTGTG3′-. Melting curve analysis was performed to ensure that only one PCR product had been produced. A standard curve was generated for quantification and for estimating amplification efficiency using increasing concentrations of cDNA, and the amplification transcripts were quantified with the relative standard curve and normalized to the GAPDH reference gene.
RNA interference
Two 20-nucleotide small interfering RNA (siRNA) duplexes targeting LOX was used; GGCTGAAGGCCACAAAGCAA (Dharmacon), used in Fig.
3. Transfection was carried out using oligofectamine (Invitrogen) according to manufacturer’s instructions. And, CUGGCGCCAGACAAUCCAAUU (Dharmacon), used in Additional file
1: Figure S3. Transfection was carried out using HiPerfect (Qiagen) according to manufacturer’s instructions. A 21-nucleotide siRNA duplex was used for targeting p130Cas (QIAGEN). The sequence was CAGGAGGTGTCTCGTCCAATA. Transfection of siRNA duplex was carried out using oligofectamine (Invitrogen) according to manufacturer’s instructions.
Invasion assay
Invasion assays were performed using BD BioCoat Matrigel Invasion chambers with 8-mm pore size according to the manufacturer’s instructions (VWR International). After 48 h incubation, top cells were removed and bottom cells were counted.
Protein preparations
For whole-cell extract preparation, cells were treated with lysis buffer (150 mM NaCl, 50 mM Tris–HCl [pH 8], 1 % Triton X-100, 1 mM Na3VO4, 10 mM NaF and 1× Complete (Roche)) for 30 min at 4 °C. Preparations of nuclear extracts were made as described by Ausubel, F et al. 1987. Protein concentrations of the extracts were determined by using BioRad Protein Assay.
Western blot
The different extracts were electrophoresed through a NuPAGE 4 to 12 % Bis-Tris sodium dodecyl sulfate-polyacrylamide gel (Invitrogen) and subsequently electroblotted onto a Hybond-P filter (Amersham Bioscience).
Flow cytometry
Cells were detached with trypsin-EDTA. Single cell suspension were fixed in 4 % paraformaldehyde in PBS and permeabilazed with 0,5 % Triton X-100 in PBS on ice. mAb FoxF1 3454 and R-phytoerythrin-labeled goat anti rabbit secondary antibody were used. Dox-treated (i.e. GFP-expressing) cells incubated with secondary antibody only were used as controls for compensation of leakage of GFP fluorescence into the FL2 channel used to detect R-phytoerythrin fluorescence.
Immunofluorescence
Cells were fixed in 4 % paraformaldehyde in PBS, permeabilized in 0,5 % Triton X-100 in PBS and blocked in 20 % FCS in PBS. After incubation with primary antibody diluted in 5 % FCS in PBS, the cells were incubated with TRITC-conjugated secondary antibody (Jackson ImmunoResearch) diluted in 5 % FCS in PBS. VectaShield/VectaShield-DAPI (3:2) was used for mounting, and the cells were viewed under a fluorescence equipped Zeiss Axioplan2 Imaging microscope.
Discussion
Invasion and metastasis are the most fatal aspects of cancer and can be facilitated by proteins that stimulate tumor cell attachment to the extracellular matrix and tumor cell motility.
Recently, it has become evident that LOX is involved in increased malignancy and invasiveness in a variety of human cancers. LOX is upregulated in invasive breast cancer cell lines and breast carcinomas [
32,
33] and has been shown to facilitate breast cancer cell migration by regulating cell-extracellular matrix adhesion formation [
16]. It has been demonstrated that LOX is a metastasis promoting gene as it is important for tumor progression to metastasis but not for tumor formation [
20]. In this study, we found LOX to be downregulated by NFI-C2 and upregulated by FoxF1. The increased invasion capacity of cells overexpressing FoxF1 could be reduced by inhibiting LOX activity or LOX expression. FoxF1 is highly expressed in invasive breast cancer cell lines compared to less invasive ones [
3]. However, the levels of FoxF1 in breast carcinomas have not been rigorously investigated due to the lack of specific antibodies. FoxF1 is repressed by NFI-C2, which is lost during mammary tumor progression and almost universally absent in lymph node metastases [
3]. Loss of NFI-C2 may be one event that facilitates metastatic dissemination and upregulation of factors like LOX. That FoxF1 upregulates LOX leading to increased invasive capacity implicates FoxF1 as a strong contributor to metastasis.
We show herein a signaling pathway where FoxF1-induced upregulation of LOX activates FAK, leading to suppression of Smad2 activity. Depletion of LOX diminished the activation of FAK and increased the phosphorylation of Smad2 and the levels of Smad2/3. However, inhibiting FAK only affected the activation of Smad2. This indicates that activation of LOX disrupts a possible scaffold function for Smad2/3 and that there are additional factors other than FAK that are involved in the regulation of Smad2/3 levels. Focal adhesions are multi-molecular complexes consisting of a range of scaffold and adaptor proteins. Elevated levels of the focal adhesion scaffold molecule p130Cas have been shown to reduce the activity of Smad2/3 [
30,
31]. p130Cas was upregulated by FoxF1 overexpression. However, suppression of Smad2/3 was not mediated by p130Cas in HC11 cells. Instead, our data suggest that FoxF1 upregulates p130Cas in a separate pathway, leading to activation of the p38 MAPK signaling pathway. It is commonly known that p130Cas is a downstream target of FAK [
34]. Inhibiting FAK by pharmacological treatment or LOX depletion increased the phosphorylation of p130Cas, suggesting the presence of an additional kinase that phosphorylates p130Cas when FAK is inhibited. This indicates cross-talk between the parallel pathways through which FoxF1 regulates Smad2/3 and p130Cas.
Upregulation of LOX, activation of FAK and subsequent suppression of Smad2 activation, as well as upregulation of p130Cas, were also observed in HB2 cells following short induction of FoxF1 expression. However, the p38 MAPK signaling pathway was not activated. One explanation for this could be the differences in epithelial characteristics between these cell types and a less advanced transformation to malignant phenotype of HB2 cells at that point. Long term expression of FoxF1 in HB2 cells was not possible owing to cell death after a few days of induction. It was therefore not possible to follow a potential FoxF1-induced EMT process in these cells. However, a dramatic change in morphology was observed, including formation of stress fibers and fibroblast-like shape, typical features during progression of EMT. When engineering inducible FoxF1-clones of HB2 cells we observed a less pronounced phenotype with a slower conversion to fibroblast-like morphology in low-expressing clones and a lower degree of cell death compared to high-expressing clones (data not shown). These observations are in accordance with earlier studies showing that FoxF1 effects are dose dependent [
35].
Recent findings indicate that oncogenic TGF-β action, which enhances tumor cell invasion and metastasis, is initiated by imbalance between canonical and non-canonical TGF-β signaling systems. We present data indicating that the association of FoxF1 with invasion and metastasis can be a consequence of FoxF1 being involved in the regulation of TGF-β signaling and promotion of non-canonical TGF-β signaling: i) overexpression of FoxF1 affects the expression of many genes involved in TGF-β pathways; ii) FoxF1 upregulates TGF-β2; iii) TGF-β treatment increases FoxF1 expression; iv) FoxF1 expression suppresses Smad2 activity; v) constitutive overexpression of FoxF1 results in activation of the p38 MAPK signaling pathway. Future research will establish how FoxF1 is coupled to TGF-β action, and whether these factors cooperate to influence metastatic activity.
Imbalance between cell-matrix and cell-cell adhesions is implicated in tumor progression. ECM remodeling such as increased cross-linking of fibrillar ECM proteins including collagen and fibronectin leads to matrix stiffness, FAK activation and increased cell adhesion [
19]. Changes in ECM density can trigger EMT via formation of cell-matrix adhesions and disassembly of cell-cell adhesions, altering intracellular signaling in a way that enhances tumor cell migration and invasion [
36]. FoxF1 downregulates cell-cell adhesion components, e.g. E-cadherin and desmosomes (e.g. Dsc2, Pkp1, Dsg and Dsp), upregulates genes affecting cell-matrix adhesion, e.g. LOX and fibronectin (Table
1 and Additional file
2: Table S1), induces EMT [
3] and invasiveness (Fig.
3). Taken together, this suggests that FoxF1 contributes to metastasis. In apparent contradiction to these results, there are reports of FoxF1 acting as a tumor suppressor. Lo et al. demonstrated that overexpression of FoxF1 in breast cancer cells led to G
1 arrest with or without concomitant apoptosis, depending on cell type [
37]. FoxF1 exerts dose dependent effects that are also reliant on cell type, as discussed above. This can result in differences in the consequences of FoxF1 overexpression. Tamura et al. have shown that p53-induced FoxF1 decreases the invasive capability of cancer cells [
38]. FoxF1 may induce different effects depending on the tissue studied and on the cell- and signal-context with which FoxF1 is associated. The status of p53 could be a determinant of FoxF1 action. This study relies on HC11 cells which express a mutant p53, and HB2 cells which are immortalized with SV40 large T antigen leading to inactivation of Rb and p53. FoxF1 is normally expressed in mesenchymal cells [
39,
40], which is difficult to reconcile with a tumor suppressing function of FoxF1 in epithelial cells. Although there are no reports of FoxF1 being involved in developmental EMT, FoxF1 is expressed when EMT occurs during gastrulation and in the sclerotome when cells migrate to the notochord [
7]. Clarifying the role of FoxF1 in carcinogenesis would be of great importance in order to evaluate the potential of FoxF1 as a molecular target against breast cancer invasion and metastasis.
Acknowledgements
The authors thank Professor Fredrik Pontén at Uppsala University for kindly providing the FoxF1 antibody, Professor Peter Carlsson, Dr Dan Baeckström and Dr Jeanette Nilsson at University of Gothenburg, for revising the manuscript, and Elin Söderberg, Kajsa Askevik, Maria Johansson Rinta and Anna-Lena Leverin for technical support. This work was supported by the Swedish Cancer Society, Assar Gabrielsson Foundation, Lennanders Foundation and Carl Trygger Foundation.
Competing interests
The author declares that they have no competing interests.
Authors’ contributions
MKJ and GN conceived and designed the study. GN performed most of the experiments. GN wrote the manuscript and GN and MKJ prepared figures and tables. MKJ supervised the study and revised the manuscript. Both authors have read and approved the manuscript.
GN is currently positioned at the Department of Physiology, Institute of Neuroscience and Physiology, University of Gothenburg.