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Carcinogenesis. 2008 December; 29(12): 2243–2251.
Published online 2008 August 19. doi:  10.1093/carcin/bgn199
PMCID: PMC2621102
NIHMSID: NIHMS85518

Fibulin-5 initiates epithelial–mesenchymal transition (EMT) and enhances EMT induced by TGF-β in mammary epithelial cells via a MMP-dependent mechanism

Abstract

Epithelial–mesenchymal transition (EMT) is a normal physiological process that regulates tissue development, remodeling and repair; however, aberrant EMT also elicits disease development in humans, including lung fibrosis, rheumatoid arthritis and cancer cell metastasis. Transforming growth factor-β (TGF-β) is a master regulator of EMT in normal mammary epithelial cells (MECs), wherein this pleiotropic cytokine also functions as a potent suppressor of mammary tumorigenesis. In contrast, malignant MECs typically evolve resistance to TGF-β-mediated cytostasis and develop the ability to proliferate, invade and metastasize when stimulated by TGF-β. It therefore stands to reason that establishing how TGF-β promotes EMT may offer new insights into targeting the oncogenic activities of TGF-β in human breast cancers. By monitoring alterations in the actin cytoskeleton and various markers of EMT, we show here that the TGF-β gene target, fibulin-5 (FBLN5), initiates EMT and enhances that induced by TGF-β. Whereas normal MECs contain few FBLN5 transcripts, those induced to undergo EMT by TGF-β show significant upregulation of FBLN5 messenger RNA, suggesting that EMT and the dedifferentiation of MECs override the repression of FBLN5 expression in polarized MECs. We also show that FBLN5 stimulated matrix metalloproteinase expression and activity, leading to MEC invasion and EMT, to elevated Twist expression and to reduced E-cadherin expression. Finally, FBLN5 promoted anchorage-independent growth in normal and malignant MECs, as well as enhanced the growth of 4T1 tumors in mice. Taken together, these findings identify a novel EMT and tumor-promoting function for FBLN5 in developing and progressing breast cancers.

Introduction

Epithelial–mesenchymal transition (EMT) is a normal physiological process that occurs when polarized epithelial cells acquire a mesenchymal phenotype that resembles those of fully differentiated fibroblasts or myofibroblasts (13). Whereas physiological EMT is essential for embryonic development, for wound healing and for tissue homeostasis, remodeling and repair, pathological reactivation of EMT in adult tissues can engender disease development in humans, including chronic inflammation, lung fibrosis, rheumatoid arthritis and cancer cell metastasis (13). Transforming growth factor-β (TGF-β) was first described as inducer of EMT in normal mammary epithelial cells (MECs) (4) and now is recognized as a master regulator of EMT in a variety of cell types and tissues (5). Phenotypically, EMT stimulated by TGF-β proceeds through a highly coordinated spatiotemporal sequence of events that includes the (i) disassembly of cell–cell junctions; (ii) reorganization of the actin cytoskeletal; (iii) loss of epithelial polarity and (iv) remodeling of cell–matrix adhesions (5,6). Malignant cells also undergo EMT, but in doing so, they acquire additional characteristics that facilitate their ability to undergo intravasation and extravasation and to sustain metastatic growth in distant locales (1). Thus, oncogenic EMT usually manifests in a genetic and cellular context that is highly abnormal and distinct from that observed during physiologic EMT. Moreover, oncogenic EMT enables cancer cells to acquire invasive and metastatic phenotypes, and consequently, to promote the dissemination of cancer cells beyond their tissue of origin, which represents the most lethal and deadly aspect of cancer (6).

In addition to regulating the polarity and EMT status of MECs, TGF-β also functions as a multifunctional cytokine that regulates every stage of mammary gland development, during which it potently suppresses mammary tumorigenesis (68). In contrast, developing and progressing breast cancers frequently inactivate the tumor suppressing activities of TGF-β, an event that facilitates the growth and metastatic spread of malignant MECs stimulated by TGF-β. Although the molecular mechanisms that enable mammary tumorigenesis to convert the cellular response of MECs to TGF-β remain to be fully elucidated, it is tempting to speculate that this switch in TGF-β function may reflect its ability to initiate and stabilize EMT in malignant MECs.

Our laboratory was the first to identify the extracellular matrix protein fibulin-5 (FBLN5) as a novel target gene for TGF-β in fibroblasts (9) and endothelial cells (9,10) and to establish FBLN5 as a multifunctional signaling molecule that (i) regulates the proliferation, motility and invasion of normal and malignant cells both in vitro and in vivo (911); (ii) antagonizes endothelial cell activities coupled to angiogenesis both in vitro and in vivo (10,11) and (iii) inhibits the growth of fibrosarcomas in mice (11). We also observed tumorigenesis to significantly repress the synthesis of FBLN5 transcripts in a variety of human cancers, including those of the breast (9). Unfortunately, the identity of the FBLN5-expressing cell types targeted by mammary tumorigenesis remain unknown, as do the direct effects of FBLN5 on the behaviors of normal and malignant MECs. Interestingly, recent evidence indicates that Fibulin family members, including FBLN5, are expressed in a developmentally regulated manner to regions of EMT during arterial, endocardial cushion tissue, neural crest and mesenchymal tissue development (1214). Thus, FBLN5 may be an important and novel regulator of normal EMT during embryonic development, as well as an inducer of oncogenic EMT during the development and progression of human breast cancers. The aim of the present study was to establish the function of FBLN5 in regulating EMT in normal and malignant MECs and in regulating the growth of mammary tumors in mice.

Materials and methods

Immunohistochemistry

Normal and malignant human mammary tissue microarrays were purchased from US Biomax (catalog # BR241; Rockville, MD), Millipore (catalog # TMA1201 and TMA1010; Temecula, CA) and Biomeda (catalog #M90; Foster City, CA) and were deparaffinized in xylene and rehydrated through a graded series of alcohols prior to inactivating endogenous peroxidase activity by incubation in 3% hydrogen peroxide for 5 min at room temperature. Antigen retrieval was performed by pressure cooking the sections in 10 mM sodium citrate/0.5% Tween 20 (pH 6.0) for 10 min at 120°C, at which point non-specific binding sites were blocked by incubation of the sections in phosphate-buffered saline containing 0.1% Tween-20/1% fetal bovine serum for 1 h at room temperature. Afterward, anti-FBLN5 antibodies [Santa Cruz Biotechnology, Santa Cruz, CA; at a 1:100 dilution or rabbit polyclonal at 1:100 as described (15)] were applied to the tissue sections for 1 h at room temperature, followed by additional 1 h incubation with biotin-conjugated anti-rabbit secondary antibodies (1:200; Jackson Immuno Research, West Grove, PA). The resulting immunocomplexes were visualized using the VectaStain ABC Kit (Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine (Millipore) and subsequently were counterstained with hematoxylin prior to tissue section dehydration and mounting with Permount Mounting Media (Fisher Scientific, Pittsburgh, PA). Negative staining controls for these analyses comprised the use of adjacent tissue sections that were processed in parallel in the absence of primary antibody. The immunoreactivity against FBLN5 in normal (n = 36) and malignant (n = 79) mammary tissues was scored semiquantitatively in a blinded manner by five individuals who used the following scale: (i) 0–1 represented no-to-low staining; (ii) 1–2 represented moderate staining and (iii) 2–3 represented high staining. Data are presented as the % of normal or malignant mammary specimens exhibiting low (average staining intensity < 1), moderate (1 < average staining intensity < 2) or high (average staining intensity > 2) FBLN5 immunoreactivity.

Semiquantitative real-time polymerase chain reaction assays

Total RNA from control and FBLN5-expressing NMuMG and 4T1 cells was purified using the RNeasy Plus Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s recommendations. In some experiments, green fluorescent protein (GFP)- or FBLN5-expressing NMuMG and 4T1 cells were stimulated with TGF-β1 (5 ng/ml) in the absence or presence (10 μM) of the type I MMP-2/3 (catalog # 444239; Calbiochem, San Diego, CA) or MMP-2/9 (catalog # 444241; Calbiochem) inhibitors. Afterward, complementary DNAs were synthesized by iScript reverse transcription (Bio-Rad, Hercules, CA), which then were diluted 10-fold in H2O and employed in semiquantitative real-time polymerase chain reaction (PCR) reactions (25 μl) that used the SYBR Green system (Bio-Rad) supplemented with 5 μl of diluted complementary DNA and 0.1 μM of oligonucleotide pairs listed below. PCR reactions were performed and analyzed on an Bio-Rad Mini-Opticon detection system, and differences in RNA concentrations were controlled by normalizing individual gene signals to their corresponding glyceraldehyde 3-phosphate dehydrogenase RNA signals. The oligonucleotide primer pairs used were as follows: (i) matrix metalloproteinase (MMP)-2, forward: 5′-TAACCTGGATGCTGTCGTGGA-3′ and reverse: 5′-GCCCAGCCAGTCTGATTTGAT-3′; (ii) MMP-3, forward: 5′-TGTTCCTGATGTTGGTGGCTT-3′ and reverse: 5′-TGTCTTGGCAAATCCGGTG-3′; (iii) tissue inhibitor of metalloproteinases-1, forward: 5′-AAGCCTCTGTGGATATGCCCA-3′ and reverse: 5′-AACCAAGAAGCTGCAGGCACT-3′; (iv) tissue inhibitor of metalloproteinases-2, forward: 5′-GTCCCATGATCCCTTGCTACA-3′ and reverse: 5′-TGATGCAGGCGAAGAACTTG-3′; (v) tissue inhibitor of metalloproteinases-3, forward: 5′-CCCTGGCTATCAGTCCAAACA-3′ and reverse: 5′-TGGCGTTGCTGATGCTCTT-3′; (vi) thrombospondin-1, forward: 5′-GAACTCATTGGAGGTGCACGA-3′ and reverse: 5′-TGGAACTTGTCATCCGGCA-3′; (vii) membrane-type (MT)1-MMP, forward: 5′-CCCAGATAAGCCCAAAAACCCCG-3′ and reverse: 5′-CCGCCAGAACCATCGCTCCTTG-3′; (viii) MT2-MMP, forward: 5′-CGGGTGACAAGGATGAGGGCAG-3′ and reverse: 5′-CAGAGCAACAGCAGCAAGGGGAC-3′; (ix) MT3-MMP, forward: 5′-TTTGATGGGGAGGGAGGATTTTTGG-3′ and reverse: 5′-GCGGTGGGGTCGTTGGAATGTTC-3′; (x) E-cadherin, forward: 5′-CCCTACATACACTCTGGTGGTTCA-3′ and reverse: 5′-GGCATCATCATCGGTCACTTTG-3′; (xi) FBLN5, forward: 5′-TCGCTATGGTTACTGCCAGCA-3′ and reverse: 5′-TTGGCAAGACCTTCCATCGTC-3′; (xii) Twist, forward: 5′-CGGGTCATGGCTAACCTG-3′ and reverse: 5′-CAGCTTGCCATCTTGGAGTC-3′ and (xiii) glyceraldehyde 3-phosphate dehydrogenase, forward: 5′-CAACTTTGGCATTGTGGAAGGGCTC-3′ and reverse: 5′-GCAGGGATGATGTTCTGGGCAGC-3′.

Retroviral plasmids, transgene expression and recombinant FBLN5 production

A bicistronic retroviral vector (pMSCV-IRES-GFP) encoding for murine FBLN5 was described previously (9) and used to infect normal murine NMuMG and malignant, metastatic murine 4T1 MECs as described previously (9). Cells expressing GFP were isolated and collected 48 h later on a MoFlo Cell Sorter (Beckman Coulter, Fullerton, CA) and subsequently were expanded to yield stable polyclonal populations of control (i.e. GFP) and FBLN5-expressing cells.

The synthesis of a bacterial expression vector encoding FBLN5 fused to the C-terminus of glutathione S-transferase, as well as the purification of recombinant FBLN5 from transformed Escherichia coli was described previously (11).

Cell biological assays

The effect of FBLN5 on the behaviors of normal and malignant MECs and on their responses to TGF-β were determined as follows: (i) cell proliferation using 10 000 cells per well in a [3H]thymidine incorporation assay as described previously (911) and (ii) EMT induced by TGF-β1 (5 ng/ml) in the absence or presence (10 μM) of the type I MMP-2/3 or MMP-2/9 inhibitors as described previously (16). All images were captured on a Nikon Diaphot microscope.

The ability of FBLN5 and TGF-β to alter the anchorage-independent growth of normal and malignant MECs was performed as described previously (16). Briefly, duplicate cultures of control (i.e. GFP) or FBLN5-expressing NMuMG or 4T1 cells (10 000 cells per plate) were grown in 0.3% agar on a cushion of 0.6% agar in 60 mm plates. Normal and malignant MEC growth in the absence or presence of TGF-β1 (5 ng/ml) was allowed to proceed for 14 days, whereupon the number of colonies formed was quantified under a light microscope.

Alterations in MEC character also were monitored by immunoblotting quiescent and TGF-β-stimulated NMuMG cell lysates with antibodies against various EMT markers. In doing so, clarified whole-cell extracts were resolved on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels, transferred electrophoretically onto nitrocellulose membranes and blocked in 5% milk before incubation with the following primary antibodies (dilutions): (i) anti-E-cadherin (1:1000; BD Biosciences, San Jose, CA); (ii) anti-N-cadherin (1:250; Cell Signaling, Danvers, MA); (iii) anti-COX-2 (1:2000; Cayman Chemical Company, Ann Arbor, MI) and (iv) anti-β3 integrin (1:1000; Santa Cruz Biotechnology). The resulting immunocomplexes were visualized by enhanced chemiluminescence, and differences in protein loading were monitored by reprobing stripped membranes with anti-β-actin antibodies (1:1000; Rockland Immunology, Gilbertsville, PA).

Zymography

Altered MMP activity in NMuMG and 4T1 cells was determined by mixing control and FBLN5-expressing NMuMG and 4T1 cells (1.2 × 106 cells per well) in 0.5 ml collagen, which subsequently was solidified in 24-well plates. Twenty-four hours later, the medium was discarded and the collagen matrices were removed and pelleted by microcentrifugation before fractionating supernatants (20–80 μl/lane) through 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis supplemented with 0.1% gelatin (Sigma, St Louis, MO). Afterward, zymogram renaturing and developing were preformed using Novex Zymogram buffer system according to the instructions of the manufacturer (Invitrogen, Carlsbad, CA).

Tumor growth study

Control (GFP) and FBLN5-expressing 4T1 cells were resuspended in sterile phosphate-buffered saline and injected orthotopically at a density of either 50 000 or 12 500 cells per injection into the mammary fat pad of 6-week-old female Balb/C and Nude mice (four mice per condition; Jackson Laboratories, Bar Harbor, ME). Mice were monitored daily and primary tumors were measured with digital calipers (Fisher Scientific) between days 10 and 32. Tumor volumes were calculated using the following equation: tumor volume = 0.5 × x2 × y, where ‘x’ is the tumor width and ‘y’ is the tumor length. Thirty-two days post-inoculation, the mice were killed and their primary tumors were excised and weighed. Animal studies were performed in accordance with the animal protocol procedures approved by the Institutional Animal Care and Use Committee of University of Colorado.

Results

Mammary tumorigenesis and EMT induces FBLN5 expression

We reported previously that tumorigenesis downregulates FBLN5 messenger RNA expression in a variety of human malignancies, including metastatic breast cancers (9). Although the identity of the FBLN5-expressing cells targeted by mammary tumorigenesis remains to be determined definitively, these findings pointed toward FBLN5 functioning as a tumor suppressor in mammary tissues. Along these lines, Oncomine analyses comparing the gene expression profiles of 40 breast carcinomas versus seven normal breast tissues also found FBLN5 messenger RNA expression to be reduced in mammary tumors as compared with their normal counterparts (Figure 1A). In stark contrast, we observed human breast cancers to contain abundant levels of FBLN5 protein, and in fact, to express more FBLN5 protein than did their normal counterparts (Figure 1B and C). This finding was unexpected and surprising because Fibulins, including FBLN5, typically are not expressed in epithelial cells; however, these molecules are readily expressed in a variety of mesenchymal and stromal components (1214). Thus, the dramatic increase in FBLN5 expression by breast cancer cells could suggest that a loss in the epithelial phenotype that occurs during MEC dedifferentiation may override the normal mechanisms operant in repressing FBLN5 production in normal, polarized MECs. A corollary states that measures capable of inducing EMT in MECs may enable the reactivation of FBLN5 expression. We tested this hypothesis by administering TGF-β to normal murine NMuMG MECs, which readily undergo EMT when treated with TGF-β (16). In accordance with our hypothesis, TGF-β stimulation of EMT in NMuMG cells significantly induced their expression of FBLN5 (Figure 1D), suggesting that FBLN5 may participate in EMT stimulated by TGF-β. Collectively, these findings indicate that the role played by FBLN5 during mammary tumorigenesis is more complicated than originally hypothesized (9), and suggest that FBLN5 expression may function as an inducer of EMT in normal and malignant MECs.

Fig. 1.
Mammary tumorigenesis and EMT induces FBLN5 expression. (A) The expression of FBLN5 messenger RNA (mRNA) in normal and malignant mammary tissues was accessed using the Oncomine Research Database (http://www.oncomine.org) and are plotted on a log scale. ...

FBLN5 initiates EMT and enhances EMT induced by TGF-β in normal and malignant MECs

We tested the aforementioned hypothesis by stably expressing FBLN5 in NMuMG cells and in malignant, metastatic 4T1 breast cells and subsequently monitored their ability to undergo EMT in the absence or presence of TGF-β. Figure 2A shows that stable expression of FBLN5 in NMuMG cells induced a partial EMT, as evidenced by the altered cortical actin architectures observed in resting cells. Indeed, actin cytoskeletal rearrangements (Figure 2B, red channel) were readily apparent in NMuMG that expressed FBLN5 (Figure 2B, green channel), which contrasted sharply with the normal cuboidal morphologies and cortical actin architectures exhibited by adjacent NMuMG cells that failed to express the FBLN5 transgene (Figure 2B). Moreover, FBLN5 expression greatly potentiated the ability of TGF-β to induce EMT in NMuMG cells (Figure 2A). Similarly, FBLN5 expression also enhanced the resting and TGF-β-stimulated EMT in 4T1 breast cancer cells (Figure 2C), suggesting that the oncogenic activities of FBLN5 are not restricted to NMuMG cells. Besides its effects on actin stress fiber formation, FBLN5 expression also enhanced the ability of TGF-β to suppress E-cadherin expression and to induce the expression of N-cadherin, β3 integrin and Cox-2 in NMuMG cells (Figure 2D). Moreover, we found that EMT stimulated by TGF-β (Figure 2E) or the process of mammary tumorigenesis (Figure 2F) both induced Twist expression, and more importantly, that FBLN5 significantly enhanced resting and TGF-β-stimulated Twist expression in normal and malignant MECs (Figure 2E and F). Collectively, these findings show that TGF-β induces FBLN5 expression in MECs in a manner reflecting its ability to stimulate EMT, and that FBLN5 expression promotes the acquisition of EMT in normal and malignant MECs.

Fig. 2.
FBLN5 initiates EMT and enhances EMT induced by TGF-β in normal and malignant MECs. Parental (i.e. GFP) and FBLN5-expressing NMuMG (A and B) and 4T1 (C) cells were incubated for 36 h in the absence or presence of TGF-β1 (5 ng/ml) as indicated. ...

FBLN5 promotes EMT in normal and malignant MECs in a MMP-dependent manner

A hallmark of EMT phenotypes is the acquisition of highly motile and invasive phenotypes (2,5), which often corresponds to elevated expression and activity of MMPs (17). Semiquantitative real-time PCR analyses showed a clear linkage between FBLN5 expression and that of MMPs (Figure 3). To more thoroughly explore this potential connection, we performed gelatin zymography on conditioned media collected from GFP- and FBLN5-expressing NMuMG cells before and after their stimulation with TGF-β. As shown in Figure 4A, FBLN5 expression significantly augmented resting and TGF-β-stimulated MMP-2 and -9 proteolytic activity. Moreover, administration of recombinant FBLN5 to NMuMG cells also enhanced their resting and TGF-β-stimulated MMP-9 protease activity (Figure 4B). Similar stimulatory effects of FBLN5 expression were observed in resting and TGF-β-stimulated 4T1 cells (Figure 4C). Functionally, FBLN5 enhanced the EMT phenotype in normal (Figure 5A) and malignant (Figure 5B) MECs, as well as significantly augmented their tonic and TGF-β-stimulated invasion through synthetic basement membranes (Figure 5C; data not shown). Importantly, pharmacological treatment of the cells with type I MMP-2/3 or MMP-2/9 inhibitors impaired the ability of FBLN5 and TGF-β to promote these events in normal (Figure 5A and C) and malignant (Figure 5B; data not shown) cells. Accordingly, semiquantitative real-time PCR analyses demonstrated that FBLN5 significantly enhanced the ability of TGF-β to repress the transcription of E-cadherin (Figure 5D), while simultaneously inducing that of Twist (Figure 5E). As above, both transcriptional responses mediated by FBLN5 were reversed by treating the cells with the type I MMP-2/3 or MMP-2/9 inhibitors (Figure 5D and E). Collectively, these findings identify an important mechanism that links FBLN5 to the increased expression and activation of MMPs 2 and 9 that function to enhance TGF-β stimulation of EMT and invasion in MECs.

Fig. 3.
FBLN5 alters the protease expression profiles in NMuMG cells. Parental (i.e. GFP) or FBLN5-expressing NMuMG cells were induced to undergo EMT by TGF-β1 as above. Afterward, total RNA was isolated and used to monitor changes in the expression of ...
Fig. 4.
FBLN5 enhances resting and TGF-β-stimulated MMP activation in normal and malignant MECs. Gelatin zymography was carried out on conditioned media harvested from parental (i.e. GFP) or FBLN5-expressing cells that were incubated in the absence or ...
Fig. 5.
FBLN5 promotes EMT in normal and malignant MECs in a MMP-dependent manner. Parental (i.e. GFP) or FBLN5-expressing NMuMG (A) or 4T1 (B) cells were induced to undergo EMT by addition of TGF-β1 (5 ng/ml) in the absence or presence of the type I ...

FBLN5 enhances the growth of mammary tumors in mice

Our findings thus far implicate FBLN5 as a promoter of mammary tumorigenesis, during which normal MECs lose their polarity and acquire the ability to grow in an anchorage-independent manner. Indeed, when compared with their control counterparts, we observed FBLN5 expression to significantly increase the ability of NMuMG and 4T1 cells to grow in soft agar, a response that was further enhanced by inclusion of TGF-β to these MEC cultures (Figure 6A and B). These findings suggest that FBLN5 may promote the growth of mammary tumors in mice. We tested this hypothesis by monitoring the growth of GFP- and FBLN5-expressing 4T1 cells following their orthotopic injection into the mammary fat pads of syngeneic Balb/C mice. In keeping with our hypothesis, we found the growth of 4T1 tumors to be increased significantly by the expression of FBLN5 (Figure 6C and D). Interestingly, although FBLN5 did elicit minor alterations in the resting rates of DNA synthesis in normal and malignant MECs, the expression of this extracellular matrix (ECM) protein failed to effect the overall proliferative response of these same cells when stimulated by TGF-β (Figure 6E and F). Thus, the ability of FBLN5 to promote the growth of malignant MECs in soft agar and mice does not appear to reflect intrinsic differences in the proliferation rates of these cells to FBLN5, but instead may be linked to its ability to enhance TGF-β stimulation of EMT, invasion and MMP expression in developing and progressing mammary tumors.

Fig. 6.
FBLN5 enhances anchorage-independent growth of MECs in vitro and the growth of 4T1 tumors in mice. Parental (i.e. GFP) and FBLN5-expressing NMuMG (A) or 4T1 (B) cells were cultured in soft agar in the absence or presence of TGF-β1 (5 ng/ml) for ...

Discussion

TGF-β is a pluripotent cytokine that regulates tissue morphogenesis and differentiation by effecting cell proliferation and survival and by altering the production of ECM proteins within cell and tissue microenvironments (6,18). TGF-β also is a major inducer of physiological EMT during development and wound healing and of pathological EMT during fibrosis and tumorigenesis (5,6,19), during which oncogenic EMT is considered to be an important and essential evolutionary step in the development of metastatic disease (1,3). The ability of TGF-β to induce EMT was first observed to occur in normal MECs (4), but now is recognized to take place in a number of epithelial cell types and tissues, including those of the kidney, retina and lung (2022). Given the associations of EMT to cancer development and progression, together with the ability to TGF-β to promote both phenomena in malignant MECs, it stands to reason that increasing our knowledge of how TGF-β regulates physiological and pathological EMT will enhance the ability of science and medicine to chemotherapeutically target the oncogenic activities of TGF-β in developing and progressing breast cancers. To this end, we show for the first time that TGF-β significantly induces FBLN5 expression in MECs undergoing EMT (Figure 1C), and more importantly, that FBLN5 expression initiates EMT and enhances that stimulated by TGF-β via a MMP-dependent mechanism in normal and malignant MECs (Figures 2–5). Finally, our finding that human breast cancers significantly upregulate their expression of FBLN5 suggests that the inappropriate expression of this ECM protein actually may enhance the growth of developing mammary neoplasms. Accordingly, we observed the growth of 4T1 tumors in genetically normal mice to be enhanced significantly by FBLN5 (Figure 6), thereby associating a novel tumor-promoting function to this TGF-β gene target.

FBLN5 is a member of the Fibulin family of ECM proteins (1214) and functions in regulating epithelial, endothelial and fibroblast adhesion, proliferation and motility in a context-specific manner (912). FBLN5 also is a gene target for TGF-β in fibroblasts and endothelial cells (9,10), and its expression is (i) developmentally regulated and widespread in many adult tissues, including heart, spleen, kidney, lung, colon and ovary (9,12,23,24); (ii) upregulated in developing or injured blood vessels (23,24) and (iii) capable of enhancing wound closure in mice (25). In contrast, FBLN5 deficiency elicits lung and vasculature abnormalities in mice that arise from aberrant elastic fiber organization (26,27) that resembles cutis laxa syndrome, which in humans is linked to genetic defects in FBLN5 (28,29). Thus, FBLN5 mediates cell–cell and cell–matrix signaling coupled to the regulation of tissue development, remodeling and repair. Along these lines, TGF-β also is widely expressed during development to regulate EMT, particularly that occurring in the lung, kidney and mammary gland and that occurring during wound healing and tissue remodeling (5,6,19). Interestingly, circumstantial evidence also implicates a role for Fibulins, including FBLN5, in mediating EMT. Indeed, FBLNs 1 and 2 are expressed prominently in active EMT regions during embryonic development and organogenesis, particularly during formation of the neural tube and crest, skeletal muscle and the epicardium, endocardial cushion tissue and cardiac valves and septa (1214). Similarly, FBLN5 localizes to regions of EMT during arterial, endocardial cushion tissue, neural crest and mesenchymal tissue development (23,24). Thus, FBLN5 may be an important regulator of normal EMT during embryonic development, as well as abnormal EMT during cancer development, a supposition wholly supported by the current study.

The molecular mechanisms underlying the biology and pathology of FBLN5 remain to be fully elucidated. However, FBLN5 does interact physically with a growing list of ECM and secreted proteins, many of which are implicated in promoting EMT and disease development in humans. Indeed, known FBLN5-binding proteins include (i) the integrins αvβ3, αvβ5 and α9β1 (10,23,26); (ii) apolipoprotein(a) (30); (iii) the lysyl oxidase (LOX) family members, LOXL1, LOXL2 and LOXL4 (31,32); (iv) tropoelastin (27); (v) the elastin-binding protein, Emilin-1 (33); (vi) latent TGF-β-binding protein 2 (34); (vii) extracellular superoxide dismutase (35) and (viii) fibrillin-1 (36). Interestingly, pharmacological treatments capable of inhibiting β1 integrin activity prevented TGF-β stimulation of EMT in normal MECs (37). Moreover, we recently established αvβ3 integrin as an essential mediator of oncogenic signaling by TGF-β in malignant MECs, including its ability to promote their EMT and invasion in vitro, as well as their growth and pulmonary metastasis in mice (3840). It should be noted that FBLN5 is unique amongst Fibulin family members by the presence of an integrin-binding RGD motif (12), and as such, future studies need to address the extent to which the binding of FBLN5 to integrins (i) contributes to its stimulation of EMT and mammary tumor growth and (ii) promotes the oncogenic activities of TGF-β. Due to the heterogenous nature of human breast cancers, it remains to be investigated as to whether aberrant and/or upregulated FBLN5 expression associates preferentially with distinct subtypes of human breast cancers, and if so, as to how this event impacts the manner in which these developing neoplasms sense and respond to TGF-β.

Future studies also need to address the potential role of LOXs in mediating the oncogenic activities of FBLN5 and TGF-β, both of which significantly induce the expression of LOX family members in normal and malignant MECs (M.A.Taylor and W.P.Schiemann, in preparation). LOX family members are a group of related copper-dependent amine oxidases that function in cross-linking collagens to elastin in the ECM, thereby increasing the tensile strength and structural integrity of tissues during embryonic development and organogenesis and during the maintenance of normal tissue homeostasis (41,42). Interestingly, FBLN5 interacts physically with the LOX family members LOXL1, LOXL2 and LOXL4 (31,32), while LOXL1-deficiency elicits elastogenic defects reminiscent of those observed in FBLN5-deficient mice (26,27,31). Thus, LOX family members may function coordinately with FBLN5 in mediating EMT and in promoting the development and progression of human breast cancers. Accordingly, aberrant LOX activity is associated with cancer progression, particularly the development of desmoplasia, whose ability to enhance tumor rigidity has been linked to the selection, expansion and dissemination of metastatic cells (43,44). With respect to breast cancer, elevated expression of LOX family members, particularly that of LOX, LOXL and LOXL2, correlates with increased malignancy and the acquisition of invasive/metastatic phenotypes and with the induction of EMT (41,42,4548). In particular, LOX expression recently was shown to be essential for hypoxia-induced metastasis of human MDA-MB-231 breast cancer cells in mice (45). Moreover, elevated LOX expression in human breast cancers was found most frequently in poorly differentiated, high-grade tumors and, consequently, was found to predict for increased disease recurrence and decreased patient survival (45). Clearly, the molecular connections potentially linking FBLN5 and LOX to the acquisition of oncogenic signaling by TGF-β needs to be examined in greater detail. Experiments designed to address these interesting issues are currently underway

Funding

National Institutes of Health (CA095519, CA114039 and CA129359); Komen Foundation (BCTR0706967 to W.P.S.); University of Colorado Cancer Center to Y.-H.L.

Acknowledgments

Members of the Schiemann Laboratory are thanked for critical reading of the manuscript. The technical expertise and support from members of the Flow Cytometry and Pathology Cores at the University of Colorado Cancer Center also is gratefully acknowledged.

Conflict of Interest Statement: None declared.

Glossary

Abbreviations

ECM
extracellular matrix
EMT
epithelial–mesenchymal transition
FBLN5
fibulin-5
GFP
green fluorescent protein
LOX
lysyl oxidase
MEC
mammary epithelial cell
MMP
matrix metalloproteinase
PCR
polymerase chain reaction
TGF-β
transforming growth factor-β

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