MCF-7, T47D and MDA-MB-231 breast cancer cells and A549 lung cancer cell lines were routinely maintained in Dulbecco's Modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum and 50 μg/ml streptomycin and 100 U/ml penicillin (Life Technologies, Carlsbad, CA, USA). These cell lines were kindly provided by Dr Carlos López-Otín (Universidad de Oviedo). pcDNA3 plasmid containing full-length cDNA for FBLN5 (22) was kindly provided by Dr William P. Schiemann (Case Western Reserve University, Cleveland, OH, USA). This expression vector was transfected into cells using TransIT-X2 Dynamic Delivery System (Mirus Bio LLC, Madison, WI, USA) as recommended by the manufacturer. Cells stably expressing exogenous FBLN5 were selected in the presence of 500 μg/ml G418 (Sigma-Aldrich). In all experiments cells transfected with an empty vector were employed as a control.

Cell extracts were resolved by 10% polyacrylamide gel electrophoresis, transferred to a PVDF membrane and then probed with a rabbit anti-Fibulin-5 antibody (Origene Technologies Inc., Rockville, MD, USA). Rabbit anti-β-catenin antibodies were from Cell Signaling Technology (Danvers, MA, USA), rabbit anti-Ki-67 antibody was from Santa Cruz Biotechnology (Dallas, TX, USA) and mouse monoclonal anti-β-actin antibody (AC-40) was from Sigma-Aldrich Química S.A. (Madrid, Spain). Immunoreactive proteins were visualized using HPR-peroxidase labeled anti-rabbit antibody (Pierce, Dorchester, MA, USA). For immunocytochemical analysis, breast cancer cells expressing Fibulin-5 were fixed with 3.7 paraformaldehyde in phosphate-buffered saline buffer and cells were then blocked with 10% fetal bovine serum. To detect Ki-67, blocked slides were incubated overnight with the H-300 antibody from Santa Cruz Biotechnologies, followed by 1-h incubation with a secondary Alexa 546-conjugated antibody (Life Technologies, Carlsbad, CA, USA). In all samples, DAPI was added at 100 ng/ml to visualize DNA in the cell nucleus. Images were obtained using a fluorescence microscope (Axiovert).

Cell proliferation was assayed using the CellTiter 96 Non-radiactive Cell Proliferation assay kit from Promega Biotech Ibérica SL (Madrid, Spain). MCF-7, T47D and MBA-MB-231 cells (5×10³/well) were seeded in 96-well plates and six replicates per condition. Cell proliferation rates were measured on four days using an automated microtiter plate reader Power WaveWS (Bio-Tek Instruments, Inc., Winooski, VT, USA). Additionally, cell proliferation was also estimated as an average of Ki-67-positive nuclei in relation to the total number of nuclei per microscopic field (n=4) (23).

In vitro invasion potential was examined by using 24-well Matrigel-coated invasion chambers with an 8-μm pore size (BD Biosciences, San Jose, CA, USA). To this end, 5×10⁴ MCF-7 or T47D cells were allowed to migrate for 96 h using 10% fetal bovine serum as a chemoattractant. In the case of MDA-MB-231 cells, migration time was 24 h. At least three independent experiments were made for each condition. Non-invading cells on the upper surface were removed from the chambers using a cotton swab and cells that reached the lower surface were fixed with 100% methanol and stained with 0.5% crystal violet in 2% ethanol. Cells were counted in four randomly selected microscopic fields.

To evaluate mammosphere formation capacity of breast tumor cells overexpressing Fibulin-5, 4×10⁴ MCF-7 and T47D cells were plated in 6-well ultralow attachments plates (Corning Costar, Corning, NY, USA) and grown in MammoCult Basal Medium (Stemcell Technologies, Vancouver, Canada) supplemented with 10% MammoCult proliferation supplement, 4 μg/ml heparin and 0.5 μg/ml hydrocortisone. After 7 days, cells were collected and enzymatically dissociated as previously described (24). Then, individual dissociated cells were cultured in 96-well ultralow attachment (Corning Costar) at a density of 20 cells/well. Mammosphere formation was daily monitored to ensure that they derived from single cells. Number of mammospheres were determined after 7 days and mammospheres were again counted and graphically presented.

To evaluate the migratory capacity on ECM components, we employed the Radius™ 24-Well Cell Migration assay kit (Cell Biolabs Inc., San Diego, CA, USA), following the manufacturer' instructions. Briefly, 1.25×10⁴ MCF-7 and MDA-MB-231 cells overexpressing Fibulin-5 or transfected with an empty vector were seeded on wells coated with fibronectin and type-I collagen. Migration was monitored for 12 h using a time-lapse Zeiss Axio Observer Microscopy. Experiments were performed in triplicates and covered area was quantified at different times using ImageJ software.

A breast cancer tissue array containing 78 samples of different types of human breast tumors and 3 normal tissues was obtained from the Institute of Oncology of Asturias Tumor Bank. Study subjects were newly diagnosed with breast cancer and histopathologically confirmed at the Hospital Universitario Central de Asturias (HUCA). Tumor-free samples were selected from healthy tissue areas and immediately frozen. Written informed consent was obtained from all patients prior to sample collection. The study was approved by the appropriate institutional review board according to national and EU guidelines. To perform the histological analysis, deparaffinized and rehydrated sections were processed for detection of Fibulin-5 and Ki-67 using the EnVision antibody complex kit (Dako Denmark A/S, Copenhagen, Denmark) following the manufacturer's recommendations. The rabbit anti-Fibulin-5 antibody (Abcam) was diluted 1:200 and the anti-Ki67 was a mouse monoclonal antibody (Dako, clone MIB-1) diluted 1:100. For control purposes, representative sections were processed as above but rabbit or goat non-immune serum, or blocking buffer, were used instead of the primary antibodies. For simultaneous detection of Fibulin-5 and Ki-67 double immunofluorescence coupled to laser confocal microscope was used. Non-specific binding was reduced by incubation for 30 min with a solution of 1% bovine serum albumin in TBS (Tris-buffered saline buffer). Sections were incubated overnight, at 4°C in a humid chamber with a 1:1 mixture of anti-fibulin-5 and anti-Ki-67 antibodies, diluted 1:200 and 1:100, respectively. After rinsing with TBS, sections were incubated for 1 h with Alexa Fluor 488-conjugated goat anti-rabbit IgG (AbD Serotec, Kidlington, UK), diluted 1:1,000 in TBS containing 5% mouse serum (AbD Serotec), then rinsed again, and incubated for another hour with CyTM3-conjugated donkey anti-mouse antibody (Jackson ImmunoResearch) diluted 1:50 in TBS. Double fluorescence was detected using a Leica DMR-XA automatic fluorescence microscope (Photonic Microscopy Service, University of Oviedo) coupled with a Leica Confocal software, version 2.5 (Leica Microsystems, Wetzlar, Germany) and the images captured were processed using ImageJ version 1.43g software, McMaster Biophotonics Facility (www.macbiophotonics.ca).

To evaluate the effect of Fibulin-5 on breast cancer prognosis, a Kaplan-Meier log-rank test survival plot was performed using the data available at www.kmplot.com (affy ID 203088_at) (25).

To perform statistical analysis, we employed GraphPad Prism 5.0 software and data are presented as means ± SE. Significant differences were determined with the Student-Welch t-test and P-values <0.05 were considered statistically significant (in the figures as: ^*P<0.05, ^**P<0.01, ^***P<0.005).

In the present study we wanted to examine if Fibulin-5 induces changes in the behavior of three commonly employed human breast cancer cells, the poorly invasive T47D and MCF-7 and the highly invasive MDA-MB-231 cell lines. Following transfection, production of recombinant Fibulin-5 was analyzed by western blot analysis using an anti-Fibulin-5 antibody (Fig. 1A). An immunoreactive band corresponding to Fibulin-5 was detected in cell extracts containing Fibulin-5, but not in control cells transfected with an empty vector. To assess whether Fibulin-5 could affect cell invasion, cells were allowed to invade using Matrigel-coated invasion chambers (Fig. 2B). After 96 h, invasion of T47D and MCF-7 was analyzed with the finding that the presence of Fibulin-5 reduced the invasive potential of both cell lines in comparison with control cells (45.5% reduction in the case of T47D cells; and 49.8% reduction in MCF-7 cells). MDA-MB-231 cells were allowed to invade for 24 h and cells producing recombinant Fibulin-5 showed 40.4% reduction in their invasive capacity when compared to control cells (Fig. 1B). These results suggest that Fibulin-5 strongly affects invasive capacity of breast cancer cells.

To assess the potential effect of Fibulin-5 on breast cancer cell proliferation we performed two different approaches. First, we measured cell proliferation on 4 consecutive days employing a MTT assay. As shown in Fig. 2A, presence of Fibulin-5 reduced the number of T47D, MCF-7 and MDA-MB-231 proliferating cells. It is noteworthy that the highly invasive MDA-MB-231 cells seemed to be more affected than the other two since difference in cell proliferation between MDA-MB-231 cells expressing recombinant Fibulin-5 and control cells was already evident on the second day of the assay. Second approach was to examine the presence of the proliferation marker Ki-67 in Fibulin-5-transfected breast cancer cells. Western blot analysis showed that expression level of Ki-67 was considerably reduced in breast cancer cell lines expressing Fibulin-5 when compared to control cells (Fig. 2B). Moreover, nuclear staining of the cells using an anti-Ki-67 antibody revealed that the number of nuclei showing Ki-67-positive staining was lower in cells expressing recombinant Fibulin-5 than in control cells. Thus, Ki-67-positive nuclei represented an average of 78, 64 and 72% in control MCF-7, T47D and MDA-MB-231 cells respectively, whereas 58, 50 and 44% of total nuclei showed positive staining in cells expressing exogenous Fibulin-5 (Fig. 2B). These data indicate that Fibulin-5 has an effect on breast cancer cell proliferation.

MCF-7 and T47D are breast tumor cell lines that show a strong ability to form mammospheres (26). To evaluate whether the presence of Fibulin-5 could alter this ability, we carried out mammosphere forming assays that showed that the presence of Fibulin-5 did not seem to affect the size or morphology of the mammospheres (Fig. 3). However, we found a significant decrease in the number of mammospheres derived from MCF-7 and T47D cells expressing recombinant Fibulin-5. Indeed, Fibulin-5 decreased ~50% the mammosphere forming units (MFU) in the case of MCF-7 cells and ~64% in the case of T47D cells in comparison with control cells and in two consecutive passages (Fig. 3). Overall, these data indicate that capacity for in vitro self-renewal of MCF-7 and T47D cells is constrained by the presence of Fibulin-5.

Next, we wanted to explore the possibility that Fibulin-5 affected breast cancer cell migration on type-I collagen and fibronectin, two extracellular matrix proteins that influence cell adhesion and migration properties of both the poorly invasive MCF-7 cells and the highly invasive MDA-MB-231 cells (27). After 12 h, MCF-7 expressing Fibulin-5 and control MCF-7 cells did not show differences in cell migration using wells coated with type-I collagen or fibronectin (Fig. 4). MDA-MB-231 cells also showed no differences between cells expressing Fibulin-5 and control cells in wells coated with fibronectin (Fig. 4). However, presence of Fibulin-5 affected the migratory capacity of MDA-MB-231 cells in wells coated with type-I collagen (Fig. 5). Thus after 12 h, 87.7% of area was covered by control MDA-MB-231 cells while only 53.9% was covered by MDA-MB-231 cells expressing Fibulin-5 (P<0.01). Differences in migration on type-I collagen were already evident at 4 h (48.2 vs. 5.7%; P<0.01). These data indicate that Fibulin-5 may act as a modulator of metastatic breast cancer cell migration through type-I collagen.

Then we asked whether the functional changes due to Fibulin-5 could be attributed to alterations in β-catenin phosphorylation. Western blot analysis revealed a decrease in the phosphorylation levels of the β-catenin residues Ser⁵⁵² and Ser⁶⁷⁵ in MCF-7 cells (Fig. 6). Attending to previous results on the effect of Fibulin-5 in Wnt/β-catenin signaling pathway in lung cancer (17), we evaluated whether the human lung carcinoma cell line A549 undergoes the same effect, with the finding that Fibulin-5 also affects phosphorylation of Ser⁵⁵² and Ser⁶⁷⁵ in this cell line (Fig. 6). Ser⁵⁵² and Ser⁶⁷⁵ residues are involved in the translocation of β-catenin from cell-cell contacts into the nucleus. In consequence, decrease in β-catenin phosphorylation could underlie the changes observed in the phenotype of breast cancer cells employed in the present study.

Taking into account our previous results on Fibulin-5 and Ki-67 in breast cancer cells, we next performed an immunohistochemical analysis to evaluate the simultaneous detection of Fibulin-5 and Ki-67 in breast cancer samples. To this end we employed a tissue array containing samples of the most common types of breast tumors as well as 3 normal breast tissues (Fig. 7). In normal samples, Fibulin-5 was present in both lobular and ductal epithelial cells, and the intensity of immunostaining varied among different segments of the organ. The density of Ki-67 positive nuclei profiles was very low or undetectable in wide segments of the tissue. In tumor samples, immunoreactivity of Fibulin-5 was very low or absent in those samples showing medium or high expression of Ki-67 independently of tumor type (Fig. 7A). In ductal carcinoma, Fibulin-5 showed high immunoreactivity in the most advance stages of the disease. In parallel, the density of Ki-67 immunoreactive nuclei profiles decreased. By contrast, in lobular carcinoma, the expression of Fibulin-5 and Ki-67 showed an opposite evolution. Double immunofluorescence for simultaneous detection of Fibulin-5 and Ki-67 was used to ascertain these results (Fig. 7B). In a representative section of poorly differentiated ductal carcinoma, abundant tumor cells displaying strong Fibulin-5 immunofluorescence were found which were devoid of Ki-67 immunofluorescence. Conversely, in sections of poorly differentiated lobular carcinoma Fibulin-5 was absent in tumor cells while they showed numerous Ki-67 positive nuclei profiles. This analysis also indicated that Fibulin-5 was not colocalized with Ki-67 in most samples examined. Moreover, Kaplan-Meier analysis using the data available at www.kmplot.com revealed that breast cancer patients with high levels of Fibulin-5 showed better prognosis than patients expressing low levels (Fig. 7C). This analysis suggests that FBLN5 gene expression is associated with better outcome in breast cancer patients.