Interaction between the ADAMTS-12 metalloprotease and fibulin-2 induces tumor-suppressive effects in breast cancer cells

Abstract

Balance between pro-tumor and anti-tumor effects may be affected by molecular interactions within tumor microenvironment. On this basis we searched for molecular partners of ADAMTS-12, a secreted metalloprotease that shows both oncogenic and tumor-suppressive effects. Using its spacer region as a bait in a yeast two-hybrid screen, we identified fibulin-2 as a potential ADAMTS-12-interacting protein. Fibulins are components of basement membranes and elastic matrix fibers in connective tissue. Besides this structural function, fibulins also play crucial roles in different biological events, including tumorigenesis. To examine the functional consequences of the ADAMTS-12/fibulin-2 interaction, we performed different in vitro assays using two breast cancer cell lines: the poorly invasive MCF-7 and the highly invasive MDA-MB-231. Overall our data indicate that this interaction promotes anti-tumor effects in breast cancer cells. To assess the in vivo relevance of this interaction, we induced tumors in nude mice using MCF-7 cells expressing both ADAMTS-12 and fibulin-2 that showed a remarkable growth deficiency. Additionally, we also found that ADAMTS-12 may elicit pro-tumor effects in the absence of fibulin-2. Immunohistochemical staining of breast cancer samples allowed the detection of both ADAMTS-12 and fibulin-2 in the connective tissue surrounding tumor area in less aggressive carcinomas. However, both proteins are hardly detected in more aggressive tumors. These data and survival analysis plots of breast cancer patients suggest that concomitant detection of ADAMTS-12 and fibulin-2 could be a good prognosis marker in breast cancer diagnosis.

Figure 1. ADAMTS-12 and fibulin-2 are two interacting proteins

(A) Schematic illustration of the interacting regions between ADAMTS-12 and fibulin-2 determined by yeast two-hybrid screen. TSP-1, thrombospondin-1-like domain; Cys-rich domain, cysteine-rich domain; PLAC, protease-lacunin; RGD, Arginine-Glycine-Aspartic Acid; EGF, ephitelial growth factor; CB-EGF, Calcium-binding EGF; BD, binding domain; AD, activating domain. (B) Western blot analysis of immunoprecipitated samples. Top, 293-EBNA control cells extracts (EBNAc) or 293-EBNA cell extracts producing ADAMTS-12-FLAG (EBNA-ADAMTS-12) were incubated with the indicated amounts of fibulin-2, immunoprecipitated with using anti-FLAG M2 affinity gel and further detection was performed with an anti-fibulin-2 antibody. Bottom, cell extracts were immunoprecipitated with an anti-fibulin-2 antibody and detection was performed with an anti-ADAMTS-12 antibody. F indicates that 1 μg of fibulin-2 was loaded in these lanes. Molecular weight marker is indicated on the right.

Figure 2. Selection of MCF-7 and MDA-MB-231 stable transfectants and invasion assay

(A) Western blot analysis of MCF-7 and MDA-MB-231 producing exogenous fibulin-2, ADAMTS-12 or both proteins simultaneously (ADAMTS-12/fibulin-2). These cells are referred as (fb), (ts) and (fb/ts) respectively in the body text. Control, cells transfected with an empty vector. Top, detection with an anti-fibulin-2 antibody. Bottom, detection with an anti-FLAG (ADAMTS-12) antibody. Samples were run in separated gels due to the similar molecular weights of fibulin-2 and ADAMTS-12. Actin was used as a loading control. Molecular weight markers are indicated on the right. (B) Immunoprecipitation of MCF-7 cell extracts producing fibulin-2 or both fibulin-2 and ADAMTS-12 using anti-FLAG M2 affinity gel. S, cell extracts. IP, immunoprepitated. Top, detection with an anti-fibulin-2 antibody. Bottom, detection with an anti-FLAG (ADAMTS-12) antibody. (C) Cell invasion assay using Matrigel-coated invasion chambers. Representative microscopic pictures of MCF-7 (top) and MDA-MB-231 (bottom) cells producing exogenous fibulin-2, ADAMTS-12 or both proteins simultaneously. Cells transfected with an empty vector were used as control. Cells that reached the lower surface were counted and graphically represented. Scale bar: 50 μm.

Figures 3. Interaction between fibulin-2 and ADAMTS-12 hinders migration of MCF-7 cells on ECM components

(A) MCF-7-(fb) (fibulin-2), MCF-7-(ts) (ADAMTS-12), and MCF-7-(fb/ts) (ADAMTS-12/fibulin-2) were allowed to migrate in uncoated wells or wells coated with type-I collagen, fibronectin or laminin I. MCF-7 cells transfected with an empty vector were used as control. Pictures of starting (t=0 h) and final (t=24 h) times are included. Starting point is indicated with a thin dotted line and final point with a thick dotted line. (B) Graphical representation of covered area after 24 h from three independent experiments.

Figure 4. Simultaneous production of fibulin-2 and ADAMTS-12 reduces self-renewal of mammosphere-forming units in MCF-7 cells

(A) Representative images of mammospheres derived from MCF-7-(fb) (fibulin-2), MCF-7-(ts) (ADAMTS-12), and MCF-7-(fb/ts) (ADAMTS-12/fibulin-2). Control, MCF-7 cells transfected with an empty vector. Sizes of some mammospheres are indicated. (B) Mammospheres were dissociated and passaged at a density of 20 cell/well in 96-well plates, and MFUs were counted and calculated as a percentage of mammospheres formed from the number of cell seeded.

Figure 5. Presence of fibulin-2 and ADAMTS-12 inhibits growth of subcutaneous tumors

(A) Nude mice were injected with MCF-7-(fb) (fibulin-2), MCF-7-(ts) (ADAMTS-12), MCF-7-(fb/ts) (ADAMTS-12/fibulin-2) or control MCF-7 cells, and tumors growth were weekly measured. (B) Representative subcutaneous tumors at the time of sacrifice (6 weeks).

Figure 6. FBLN2 and ADAMTS12 expression analysis in breast cancer samples

(A) representative tissue images for detection of ADAMTS-12 and fibulin-2 in healthy breast tissue and breast carcinoma samples. Arrows indicate detection of ADAMTS-12 and fibulin-2 in grade 1 breast carcinoma. Scale bar, 200 μm. (B) Kaplan-Meier survival plots showing a better outcome of breast patients with high expression level of both ADAMTS12 and FBLN2 (Hts+Hfb). Plots for high ADAMTS12 and low FBLN2 (Hts+Lfb); low ADAMTS12 and high FBLN2 (Lts+Hfb); and low ADAMTS12 and low FBLN2 (Lts+Lfb) are also included.