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Breast cancer antiestrogen resistance 3 (BCAR3) promotes cell motility by regulating actin cytoskeletal and adhesion remodeling in invasive breast cancer cells.

Wilson AL, Schrecengost RS, Guerrero MS, Thomas KS, Bouton AH - PLoS ONE (2013)

Bottom Line: Metastatic breast cancer is incurable.In order to improve patient survival, it is critical to develop a better understanding of the molecular mechanisms that regulate metastasis and the underlying process of cell motility.Taken together, these data establish that BCAR3 functions as a positive regulator of cytoskeletal remodeling and adhesion turnover in invasive breast cancer cells through its ability to influence the balance between Rac1 and RhoA signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology & Cancer Biology, University of Virginia, Charlottesville, Virginia, United States of America.

ABSTRACT
Metastatic breast cancer is incurable. In order to improve patient survival, it is critical to develop a better understanding of the molecular mechanisms that regulate metastasis and the underlying process of cell motility. Here, we focus on the role of the adaptor molecule Breast Cancer Antiestrogen Resistance 3 (BCAR3) in cellular processes that contribute to cell motility, including protrusion, adhesion remodeling, and contractility. Previous work from our group showed that elevated BCAR3 protein levels enhance cell migration, while depletion of BCAR3 reduces the migratory and invasive capacities of breast cancer cells. In the current study, we show that BCAR3 is necessary for membrane protrusiveness, Rac1 activity, and adhesion disassembly in invasive breast cancer cells. We further demonstrate that, in the absence of BCAR3, RhoA-dependent signaling pathways appear to predominate, as evidenced by an increase in RhoA activity, ROCK-mediated phosphorylation of myosin light chain II, and large ROCK/mDia1-dependent focal adhesions. Taken together, these data establish that BCAR3 functions as a positive regulator of cytoskeletal remodeling and adhesion turnover in invasive breast cancer cells through its ability to influence the balance between Rac1 and RhoA signaling. Considering that BCAR3 protein levels are elevated in advanced breast cancer cell lines and enhance breast cancer cell motility, we propose that BCAR3 functions in the transition to advanced disease by triggering intracellular signaling events that are essential to the metastatic process.

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BCAR3 promotes Rac1 activity.(A) BT549 cells transfected with siCtl (lane 1) or siB3-1 (lane 2) siRNA oligonucleotides were incubated for 72 hours, held in suspension for 90 minutes, then plated on 10 µg/ml fibronectin for 1 hour. GTP-bound Rac1 was isolated from whole cell lysates by incubation with PAK-1-binding domain agarose. Bound proteins (top panel) and total Rac1 (middle panel) were detected by immunoblotting with a Rac1 antibody, and BCAR3 knockdown was confirmed with a BCAR3-specific antibody (bottom panel). Quantification of the relative GTP-Rac1 level is shown. Data represent the mean ± SEM of 3 independent experiments (*, p<0.05). (B) BT549 cells were transfected with siCtl or siB3-1 oligonucleotides, incubated for 24 hours, followed by transfection with plasmids encoding Myc-RacL61 for an additional 48 hours. Cells were plated onto 10 µg/ml fibronectin-coated coverslips for 1–3 hours and processed for immunofluorescence as described in the methods. Actin is stained with Texas red-conjugated phalloidin (red) and Myc (RacL61) with fluorescein isothiocyanate (FITC) (green). Arrows indicate Myc-RacL61 expressing cells. Arrowheads indicate actin-rich stress fibers. The images shown are representative of 6 separate experiments. Scale bar = 15 µm. (C) The percentage of cells exhibiting actin-rich ruffles was determined for non-transfected and RacL61-expressing cells. Data represent the mean ± SEM of at least 36 cells per condition over to 2 separate experiments (*, p<0.05).
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pone-0065678-g002: BCAR3 promotes Rac1 activity.(A) BT549 cells transfected with siCtl (lane 1) or siB3-1 (lane 2) siRNA oligonucleotides were incubated for 72 hours, held in suspension for 90 minutes, then plated on 10 µg/ml fibronectin for 1 hour. GTP-bound Rac1 was isolated from whole cell lysates by incubation with PAK-1-binding domain agarose. Bound proteins (top panel) and total Rac1 (middle panel) were detected by immunoblotting with a Rac1 antibody, and BCAR3 knockdown was confirmed with a BCAR3-specific antibody (bottom panel). Quantification of the relative GTP-Rac1 level is shown. Data represent the mean ± SEM of 3 independent experiments (*, p<0.05). (B) BT549 cells were transfected with siCtl or siB3-1 oligonucleotides, incubated for 24 hours, followed by transfection with plasmids encoding Myc-RacL61 for an additional 48 hours. Cells were plated onto 10 µg/ml fibronectin-coated coverslips for 1–3 hours and processed for immunofluorescence as described in the methods. Actin is stained with Texas red-conjugated phalloidin (red) and Myc (RacL61) with fluorescein isothiocyanate (FITC) (green). Arrows indicate Myc-RacL61 expressing cells. Arrowheads indicate actin-rich stress fibers. The images shown are representative of 6 separate experiments. Scale bar = 15 µm. (C) The percentage of cells exhibiting actin-rich ruffles was determined for non-transfected and RacL61-expressing cells. Data represent the mean ± SEM of at least 36 cells per condition over to 2 separate experiments (*, p<0.05).

Mentions: Because Rac1 activity is required for membrane protrusions [19], we next investigated whether BCAR3 promotes membrane protrusiveness through its ability to modulate Rac1 activity [20]. To test this hypothesis, active GTP-bound Rac1 was measured in BT549 cells transfected with siCtl or siB3-1 oligonucleotides. While total Rac1 expression was equivalent in control and BCAR3-depleted cells, Rac1-GTP levels were significantly decreased in the absence of BCAR3 (Fig. 2A). To determine whether this decrease in Rac1 activity was responsible for the loss of protrusiveness seen in the absence of BCAR3, constitutively active Rac1 (Myc-RacL61) was transiently expressed in control and BCAR3-depleted cells and actin-rich membrane protrusions were visualized by immunofluorescence microscopy. As expected, BCAR3 depletion reduced the percentage of cells exhibiting protrusions in the absence of RacL61 (Fig. 2C). However, while expression of RacL61 in control cells did not have a significant effect on membrane protrusions (Fig. 2B, left panel, compare cell marked with arrow to adjacent cell), RacL61 expression in BCAR3-depleted cells significantly increased the percentage of cells containing membrane protrusions (Fig. 2B, right panel, compare cell marked with arrow to adjacent cells marked with arrowheads; Fig. 2C). Interestingly, BCAR3-depleted cells that did not express RacL61 (Fig. 2B, right panel, arrowheads) exhibited prominent actin-rich stress fibers that were not evident in control cells or BCAR3-depleted cells expressing constitutively active Rac1. Our group has reported this stabilization of stress fibers in the absence of BCAR3 previously [10]. Collectively, these data show that BCAR3 promotes membrane protrusions through a Rac1-dependent mechanism.


Breast cancer antiestrogen resistance 3 (BCAR3) promotes cell motility by regulating actin cytoskeletal and adhesion remodeling in invasive breast cancer cells.

Wilson AL, Schrecengost RS, Guerrero MS, Thomas KS, Bouton AH - PLoS ONE (2013)

BCAR3 promotes Rac1 activity.(A) BT549 cells transfected with siCtl (lane 1) or siB3-1 (lane 2) siRNA oligonucleotides were incubated for 72 hours, held in suspension for 90 minutes, then plated on 10 µg/ml fibronectin for 1 hour. GTP-bound Rac1 was isolated from whole cell lysates by incubation with PAK-1-binding domain agarose. Bound proteins (top panel) and total Rac1 (middle panel) were detected by immunoblotting with a Rac1 antibody, and BCAR3 knockdown was confirmed with a BCAR3-specific antibody (bottom panel). Quantification of the relative GTP-Rac1 level is shown. Data represent the mean ± SEM of 3 independent experiments (*, p<0.05). (B) BT549 cells were transfected with siCtl or siB3-1 oligonucleotides, incubated for 24 hours, followed by transfection with plasmids encoding Myc-RacL61 for an additional 48 hours. Cells were plated onto 10 µg/ml fibronectin-coated coverslips for 1–3 hours and processed for immunofluorescence as described in the methods. Actin is stained with Texas red-conjugated phalloidin (red) and Myc (RacL61) with fluorescein isothiocyanate (FITC) (green). Arrows indicate Myc-RacL61 expressing cells. Arrowheads indicate actin-rich stress fibers. The images shown are representative of 6 separate experiments. Scale bar = 15 µm. (C) The percentage of cells exhibiting actin-rich ruffles was determined for non-transfected and RacL61-expressing cells. Data represent the mean ± SEM of at least 36 cells per condition over to 2 separate experiments (*, p<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3675087&req=5

pone-0065678-g002: BCAR3 promotes Rac1 activity.(A) BT549 cells transfected with siCtl (lane 1) or siB3-1 (lane 2) siRNA oligonucleotides were incubated for 72 hours, held in suspension for 90 minutes, then plated on 10 µg/ml fibronectin for 1 hour. GTP-bound Rac1 was isolated from whole cell lysates by incubation with PAK-1-binding domain agarose. Bound proteins (top panel) and total Rac1 (middle panel) were detected by immunoblotting with a Rac1 antibody, and BCAR3 knockdown was confirmed with a BCAR3-specific antibody (bottom panel). Quantification of the relative GTP-Rac1 level is shown. Data represent the mean ± SEM of 3 independent experiments (*, p<0.05). (B) BT549 cells were transfected with siCtl or siB3-1 oligonucleotides, incubated for 24 hours, followed by transfection with plasmids encoding Myc-RacL61 for an additional 48 hours. Cells were plated onto 10 µg/ml fibronectin-coated coverslips for 1–3 hours and processed for immunofluorescence as described in the methods. Actin is stained with Texas red-conjugated phalloidin (red) and Myc (RacL61) with fluorescein isothiocyanate (FITC) (green). Arrows indicate Myc-RacL61 expressing cells. Arrowheads indicate actin-rich stress fibers. The images shown are representative of 6 separate experiments. Scale bar = 15 µm. (C) The percentage of cells exhibiting actin-rich ruffles was determined for non-transfected and RacL61-expressing cells. Data represent the mean ± SEM of at least 36 cells per condition over to 2 separate experiments (*, p<0.05).
Mentions: Because Rac1 activity is required for membrane protrusions [19], we next investigated whether BCAR3 promotes membrane protrusiveness through its ability to modulate Rac1 activity [20]. To test this hypothesis, active GTP-bound Rac1 was measured in BT549 cells transfected with siCtl or siB3-1 oligonucleotides. While total Rac1 expression was equivalent in control and BCAR3-depleted cells, Rac1-GTP levels were significantly decreased in the absence of BCAR3 (Fig. 2A). To determine whether this decrease in Rac1 activity was responsible for the loss of protrusiveness seen in the absence of BCAR3, constitutively active Rac1 (Myc-RacL61) was transiently expressed in control and BCAR3-depleted cells and actin-rich membrane protrusions were visualized by immunofluorescence microscopy. As expected, BCAR3 depletion reduced the percentage of cells exhibiting protrusions in the absence of RacL61 (Fig. 2C). However, while expression of RacL61 in control cells did not have a significant effect on membrane protrusions (Fig. 2B, left panel, compare cell marked with arrow to adjacent cell), RacL61 expression in BCAR3-depleted cells significantly increased the percentage of cells containing membrane protrusions (Fig. 2B, right panel, compare cell marked with arrow to adjacent cells marked with arrowheads; Fig. 2C). Interestingly, BCAR3-depleted cells that did not express RacL61 (Fig. 2B, right panel, arrowheads) exhibited prominent actin-rich stress fibers that were not evident in control cells or BCAR3-depleted cells expressing constitutively active Rac1. Our group has reported this stabilization of stress fibers in the absence of BCAR3 previously [10]. Collectively, these data show that BCAR3 promotes membrane protrusions through a Rac1-dependent mechanism.

Bottom Line: Metastatic breast cancer is incurable.In order to improve patient survival, it is critical to develop a better understanding of the molecular mechanisms that regulate metastasis and the underlying process of cell motility.Taken together, these data establish that BCAR3 functions as a positive regulator of cytoskeletal remodeling and adhesion turnover in invasive breast cancer cells through its ability to influence the balance between Rac1 and RhoA signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology & Cancer Biology, University of Virginia, Charlottesville, Virginia, United States of America.

ABSTRACT
Metastatic breast cancer is incurable. In order to improve patient survival, it is critical to develop a better understanding of the molecular mechanisms that regulate metastasis and the underlying process of cell motility. Here, we focus on the role of the adaptor molecule Breast Cancer Antiestrogen Resistance 3 (BCAR3) in cellular processes that contribute to cell motility, including protrusion, adhesion remodeling, and contractility. Previous work from our group showed that elevated BCAR3 protein levels enhance cell migration, while depletion of BCAR3 reduces the migratory and invasive capacities of breast cancer cells. In the current study, we show that BCAR3 is necessary for membrane protrusiveness, Rac1 activity, and adhesion disassembly in invasive breast cancer cells. We further demonstrate that, in the absence of BCAR3, RhoA-dependent signaling pathways appear to predominate, as evidenced by an increase in RhoA activity, ROCK-mediated phosphorylation of myosin light chain II, and large ROCK/mDia1-dependent focal adhesions. Taken together, these data establish that BCAR3 functions as a positive regulator of cytoskeletal remodeling and adhesion turnover in invasive breast cancer cells through its ability to influence the balance between Rac1 and RhoA signaling. Considering that BCAR3 protein levels are elevated in advanced breast cancer cell lines and enhance breast cancer cell motility, we propose that BCAR3 functions in the transition to advanced disease by triggering intracellular signaling events that are essential to the metastatic process.

Show MeSH
Related in: MedlinePlus