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Mohamedi et al. J Cancer Metastasis Treat 2019;5:37 I http://dx.doi.org/10.20517/2394-4722.2018.81 Page 3 of 15

The roles of fibulins in tumor development have been widely discussed, and they show both pro- and anti-
[23]
tumor activities . In particular, fibulin-1 has been described to be overexpressed in various human cancers
and associated with processes such as invasion, migration, and in vivo tumor growth [24-26] . The role of
fibulin-1 in breast cancer depends on multiple factors. For example, four alternative forms of fibulin-1 have
[27]
been described (fibulin-1A, fibulin-1B, fibulin-1C and fibulin-1D), which differ at the carboxy terminal .
Fibulin-1D acts as a product of a tumor suppressor gene, whereas fibulin-1C (lacks only 21 amino acids of
the fibulin-like domain) behaves as an oncogenic protein [25,28] . Furthermore, the identification of fibulin-1
localization or fibulin-1-expressing cells are also important metrics since fibulin-1 is one of the genes
[29]
specifically expressed in normal fibroblasts but not in breast cancer-associated fibroblasts . The epigenetic
downregulation of fibulin-1 has also been described in various cancers, such as gastric cancer, colorectal
cancer, hepatocellular carcinoma, and bladder cancer, but this downregulation has not been described in
breast cancer [30-33] .

Herein, we describe the effects of the ADAMTS-1/fibulin-1 interaction on breast cancer cell properties
related to cancer progression and development. We found that cell proliferation, invasion and migration
are susceptible to being affected by this interaction as well as the capacity of tumorsphere formation, which
reflects the capability of cancer cells to form tumor focus. Finally, we detected very low levels of ADAMTS-1
expression and high levels of fibulin-1 expression in a human tissue array from breast cancer patients, which
can be considered a deviation towards a cancerous phenotype.

METHODS
Cells lines and cell culture conditions
Human MDA-MB-231 and murine MCF-7 breast cancer cell lines were kindly provided by Dr. Carlos
López-Otín (University of Oviedo, Spain). Cells were routinely maintained in Dulbecco’s Modified Eagle’s
medium (DMEM) containing 10% fetal bovine serum, 50 μg/mL streptomycin and 100 U/mL penicillin (Life
Technologies). For transfection experiments, vectors containing the full-length human cDNAs for fibulin-1
(kindly provided by Dr. Tatako Sasaki, Oita University, Japan) and Flag-tagged ADAMTS-1 (Origene) were
transfected into cells at 75% confluence using TransIT-X2 (Mirus) as recommended by the manufacturer.
Cells transfected with an empty vector were used as a control. Stably expressing transfectants were selected
with 500 μg/mL G418 (Sigma-Aldrich) and used for all the experiments.

Western blotting and immunoprecipitation
For western blot analysis, proteins were resolved by 10% polyacrylamide gel electrophoresis, transferred to
a nitrocellulose membrane and subsequently probed with the indicated antibodies. Primary antibodies for
the detection of ADAMTS-1 were purchased from Sigma-Aldrich (FLAG M2, for immunoprecipitation)
and Bioss (bs-1208R-A488, for immunohistochemistry and western blotting), and those used for the
detection of fibulin-1 were obtained from Novus (NBP1-84725, for immunohistochemistry) and Santa Cruz
Biotechnology (H-190, for western blotting).

The anti-actin antibody used as the load control was purchased from Sigma-Aldrich (AC-15). Immunoreactive
proteins were visualized using HRP-peroxidase-labeled anti-rabbit or anti-mouse secondary antibodies and
an electrochemiluminescence (ECL) detection system (Pierce). For immunoprecipitation, MCF-7 double-
transfected cells (ADAMTS-1 and fibulin-1) were resuspended in lysis buffer (100 mM Tris-HCl at pH 7.4,
150 mmol/L NaCl, 10 mmol/L EDTA, 1% desoxycholic acid, 1% Triton X-100, and 0.1% SDS containing one
complete protease inhibitor cocktail (one tablet/50 mL buffer; Roche Molecular Biochemicals) and incubated
for 2 h on ice with eventual stirring. Cell debris was removed by centrifugation in a bench-top centrifuge
for 15 min at 4 °C. Protein concentration was quantified using the BCA Protein Assay Kit from Pierce
Technology. For immunoprecipitation, 1 mg of protein extract was incubated with anti-FLAG M2 affinity gel