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. 2013 Nov;142(2):341-53.
doi: 10.1007/s10549-013-2753-1. Epub 2013 Nov 7.

Upregulated WAVE3 expression is essential for TGF-β-mediated EMT and metastasis of triple-negative breast cancer cells

Molly A Taylor  1 , Gangarao Davuluri, Jenny G Parvani, Barbara J Schiemann, Michael K Wendt, Edward F Plow, William P Schiemann, Khalid Sossey-Alaoui
Affiliations

Upregulated WAVE3 expression is essential for TGF-β-mediated EMT and metastasis of triple-negative breast cancer cells

Molly A Taylor et al. Breast Cancer Res Treat. 2013 Nov.

Abstract

Breast cancer is the second leading cause of cancer death in women in the United States. Metastasis accounts for the death of ~90 % of these patients, yet the mechanisms underlying this event remain poorly defined. WAVE3 belongs to the WASP/WAVE family of actin-binding proteins that play essential roles in regulating cell morphology, actin polymerization, cytoskeleton remodeling, cell motility, and invasion. Accordingly, we demonstrated previously that WAVE3 promotes the acquisition of invasive and metastatic phenotypes by human breast cancers. Herein, we show that transforming growth factor-β (TGF-β) selectively and robustly induced the expression of WAVE3 in metastatic breast cancer cells, but not in their nonmetastatic counterparts. Moreover, the induction of WAVE3 expression in human and mouse triple-negative breast cancer cells (TNBCs) by TGF-β likely reflects its coupling to microRNA expression via a Smad2- and β3 integrin-dependent mechanism. We further demonstrate the requirement for WAVE3 expression in mediating the initiation of epithelial-mesenchymal transition (EMT) programs stimulated by TGF-β. Indeed, stable depletion of WAVE3 expression in human TNBC cells prevented TGF-β from inducing EMT programs and from stimulating the proliferation, migration, and the formation of lamellipodia in metastatic TNBC cells. Lastly, we observed WAVE3 deficiency to abrogate the outgrowth of TNBC cell organoids in 3-dimensional organotypic cultures as well as to decrease the growth and metastasis of 4T1 tumors produced in syngeneic Balb/C mice. Indeed, WAVE3 deficiency significantly reduced the presence of sarcomatoid morphologies indicative of EMT phenotypes in pulmonary TNBC tumors as compared to those detected in their parental counterparts. Collectively, these findings indicate the necessity for WAVE3 expression and activity during EMT programs stimulated by TGF-β; they also suggest that measures capable of inactivating WAVE3 may play a role in alleviating metastasis stimulated by TGF-β.

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Conflict of interest statement

Conflict of interest The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
WAVE3 expression is upregulated by TGF-β in a manner that correlates with metastatic potential of TNBCs. Murine 4T1 (a, b) and NME (c, d) breast cancer progression series or human MDA-MB-231 breast cancer cells (e, f) were stimulated with TGF-β1 (5 ng/ml) for 48 h to induce an EMT program. WAVE3 transcript levels were measured by semi-quantitative real-time PCR and normalized to GAPDH (a, c, e), while WAVE3 protein levels were determined by immunoblotting (b, d, f). TGF-β1 treatment upregulated WAVE3 mRNA and protein expression in metastatic 4T1 cells, but not in their nonmetastatic 67NR and 4T07 counterparts (a, b). Similarly, TGF-β also upregulated WAVE3 mRNA and protein in transformed NME and LM2 cells, but not in normal NMuMG cells as measured by semi-quantitative real-time PCR (b, c). Finally, TGF-β induced the synthesis of WAVE3 transcripts and protein in metastatic MDA-MB-231 cells (e, f). All data are representative of at least three independent experiments or are the mean (±SE; n = 3; *p <0.05; Student’s t test)
Fig. 2
Fig. 2
TGF-β induces WAVE3 expression in a Smad2- and β3 integrin-dependent manner in TNBCs. a Manipulating TGF-β signaling in 4T1 organotypic cultures negatively regulates miR-31 and miR-200 expression in metastatic TNBCs. b Knockdown of either Smad2 or β3 integrin, but not β1 integrin in 4T1 cells abrogated the induction of WAVE3 mRNA by TGF-β1 as measured by semi-quantitative real-time PCR, where mRNA signals were normalized to GAPDH (c). MDA-MB-231 (d) or 4T1 (e) cells rendered deficient in WAVE3 expression were stimulated with TGF-β1 (5 ng/ml) for 30 min, at which point the phosphorylation status of either Smad2 or Smad3 was monitored by immunoblot analysis as indicated. Differences in protein loading were determined by reprobing stripped membranes with antibodies against β-actin. All data are representative of three independent experiments or are the mean (±SE; n = 3; *p <0.05; Student’s t test)
Fig. 3
Fig. 3
WAVE3 is required for TGF-β-mediated EMT in TNBCs. a, b Control and WAVE3-deficient MDA-MB-231 cells were stimulated to undergo EMT by a 48-h treatment with TGF-β1 (5 ng/ml). Afterward, altered expression of E-cadherin, N-cadherin, fibronectin, or vimentin mRNA was determined by semi-quantitative real-time PCR. Individual transcript signals were normalized to GAPDH. c These same MDA-MB-231 derivatives were also prepared for immunoblot analyses to monitor the expression of fibronectin, vimentin, and β1 integrin as indicated. Differences in protein loading were controlled with anti-β-actin antibodies. d, e Control (i.e., shScram) and WAVE3-deficient MDA-MB-231 cells were seeded on gelatin-coated culture plates (1 μg/ml) and stimulated with TGF-β1 (5 ng/ml) for 48 h. Afterward, cells were fixed and stained for actin structures with Alexa Fluor 568-conjugated phalloidin (d). Arrowheads indicate the presence of lamellipodia membrane protrusions, while differences in lamellipodia formation are shown in panel e, which plots the % of cells with lamellipodia per field. All data are representative of three independent experiments (±SE; n = 3; *p <0.05; Student’s t test). f 4T1 cells were stimulated with TGF-β1 (5 ng/ml) to induce an EMT program. Boxed regions are shown as magnified insets on the right. All data are representative of three independent experiments or the mean (±SE; n = 3; *p <0.05; Student’s t test)
Fig. 4
Fig. 4
WAVE3 depletion abrogates TNBCs’ migration stimulated by TGF-β. a Basal levels of 4T1 cell invasion through a synthetic basic membrane were reduced by inactivation of WAVE3 function, while TGF-β1 (5 ng/ml) stimulation of invasion remained unaffected. Control and WAVE3-deficient MDA-MB-231 (b) or 4T1 (c) cells were induced to migrate into denuded wounds over a 24-h period in the absence or presence of TGF-β1 (5 ng/ml). All data are representative of three independent experiments or are the mean (±SE; n = 3; *p <0.05; Student’s t test)
Fig. 5
Fig. 5
WAVE3 depletion decreases TGF-β-mediated proliferation and 3D outgrowth of TNBCs. The proliferation of control (shScram) or WAVE3-deficient (shWAVE3) expressing MDA-MB-231 (a) or 4T1 (b) cells in response to TGF-β1 (5 ng/ml) as quantitated by a [3H]thymidine incorporation assay. The anti-proliferative activity of TGF-β1 (5 ng/ml) in 4T1 (c, d) and MDA-MB-231 (e) organoids was enhanced by WAVE3 inactivation. The outgrowth of 4T1 organoids in compliant 3D organotypic cultures was quantified by measuring bioluminescent signals 8 days after plating. All data are representative of three independent experiments or are the mean (±SE; n = 3; *p <0.05; Student’s t test)
Fig. 6
Fig. 6
WAVE3 deficiency decreases the growth and metastasis of TNBC tumors in mice. Primary tumor growth (a) and metastasis (b) of control and WAVE3-deficient 4T1 cells engineered to stably express firefly luciferase (Luc) were determined following their engraftment into the mammary fat pads of Balb/C mice (n = 5). c Depletion of WAVE3 inhibits the proliferative capacity of primary 4T1 tumors as measured by Ki67 staining, which was quantified using ImageJ. d These same 4T1 derivatives were also injected into the lateral tail vein for Balb/C mice, whose development of pulmonary tumors was measured by bioluminescence longitudinally every 3 days post-injection (top). Representative bioluminescent signals of pulmonary tumor formation 14 days post-injection are also shown (bottom). Following necropsy, the lungs were stained with H&E and the frequency of sarcomatoid phenotypes in these tissue samples was determined (e). (f) TGF-β upregulation of WAVE3 involves both the canonical (Smad2) and noncanonical (β3 integrin) signaling pathways, leading to the initiation of EMT programs that enhance TNBC cell proliferation, migration, and invasion, which culminates in driving the metastatic dissemination of TNBCs
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