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MUC4 overexpression augments cell migration and metastasis through EGFR family proteins in triple negative breast cancer cells.

Mukhopadhyay P, Lakshmanan I, Ponnusamy MP, Chakraborty S, Jain M, Pai P, Smith LM, Lele SM, Batra SK - PLoS ONE (2013)

Bottom Line: Moreover, our studies also showed that knockdown of MUC4 in TNBC cells induced molecular changes suggestive of mesenchymal to epithelial transition.We further demonstrate that MUC4 is differentially over-expressed in invasive TNBC tissues compared to normal breast tissue.MUC4 mucin expression is associated with TNBC pathobiology, and its knockdown reduced aggressiveness in vitro, and tumorigenesis and metastasis in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

ABSTRACT

Introduction: Current studies indicate that triple negative breast cancer (TNBC), an aggressive breast cancer subtype, is associated with poor prognosis and an early pattern of metastasis. Emerging evidence suggests that MUC4 mucin is associated with metastasis of various cancers, including breast cancer. However, the functional role of MUC4 remains unclear in breast cancers, especially in TNBCs.

Method: In the present study, we investigated the functional and mechanistic roles of MUC4 in potentiating pathogenic signals including EGFR family proteins to promote TNBC aggressiveness using in vitro and in vivo studies. Further, we studied the expression of MUC4 in invasive TNBC tissue and normal breast tissue by immunostaining.

Results: MUC4 promotes proliferation, anchorage-dependent and-independent growth of TNBC cells, augments TNBC cell migratory and invasive potential in vitro, and enhances tumorigenicity and metastasis in vivo. In addition, our studies demonstrated that MUC4 up-regulates the EGFR family of proteins, and augments downstream Erk1/2, PKC-γ, and FAK mediated oncogenic signaling. Moreover, our studies also showed that knockdown of MUC4 in TNBC cells induced molecular changes suggestive of mesenchymal to epithelial transition. We also demonstrated in this study, for the first time, that knockdown of MUC4 was associated with reduced expression of EGFR and ErbB3 (EGFR family proteins) in TNBC cells, suggesting that MUC4 uses an alternative to ErbB2 mechanism to promote aggressiveness. We further demonstrate that MUC4 is differentially over-expressed in invasive TNBC tissues compared to normal breast tissue.

Conclusions: MUC4 mucin expression is associated with TNBC pathobiology, and its knockdown reduced aggressiveness in vitro, and tumorigenesis and metastasis in vivo. Overall, our findings suggest that MUC4 mucin promotes invasive activities of TNBC cells by altering the expression of EGFR, ErbB2, and ErbB3 molecules and their downstream signaling.

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Related in: MedlinePlus

MUC4 enhances migratory and invasive potential.(A) After 24 hours serum starvation, a wound was created with a plastic tip on plates containing control or MUC4 knockdown cells. Cells were incubated in complete media for 12 hours. Motility of cells was photographed under bright-field microscopy (left, 10× magnification). After 12 hours, the migration of control cells and MUC4 knockdown cells was measured (in µm2) using DatInf Measure setup wizard software (http://tucows.texasonline.net). Values were calculated and plotted (right). (B and C) Control and MUC4 knockdown cells were serum starved for 48 h and seeded on non-coated or Matrigel-coated membranes for motility (B) and invasion (C) assays, respectively, and incubated for 24 h. Medium containing 10% fetal bovine serum in the lower chamber was used as a chemo-attractant. Cells that did not migrate through the Matrigel and/or pores in the membrane were removed using a cotton swab, and cells on the other side of the membrane were stained and photographed under bright-field microscopy (10× magnification). The number of cells that migrated and invaded was higher in control than the MUC4 knockdown cells. Data presented are the average number of cells/field for 10 fields. Columns: average of three independent experiments; bars: SE, p = 0.002 and p = 0.001, respectively. Representative images of control and MUC4 knockdown cells were shon in both figures. (D) Phalloidin staining showed that visualized F-actin under a laser scanning microscope is reduced in MDA-MB-231-shMUC4 cells compared with the control cells. (E) Immunoblot analysis showed reduced phosphorylation of FAK in MUC4 knockdown cells compared with the control cells. (F) Immunoblot analysis showed reduced expression of mesenchymal markers such as vimentin and vitronectin; and increased expression of CK-18 in MUC4 knockdown cells compared to the control.
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pone-0054455-g003: MUC4 enhances migratory and invasive potential.(A) After 24 hours serum starvation, a wound was created with a plastic tip on plates containing control or MUC4 knockdown cells. Cells were incubated in complete media for 12 hours. Motility of cells was photographed under bright-field microscopy (left, 10× magnification). After 12 hours, the migration of control cells and MUC4 knockdown cells was measured (in µm2) using DatInf Measure setup wizard software (http://tucows.texasonline.net). Values were calculated and plotted (right). (B and C) Control and MUC4 knockdown cells were serum starved for 48 h and seeded on non-coated or Matrigel-coated membranes for motility (B) and invasion (C) assays, respectively, and incubated for 24 h. Medium containing 10% fetal bovine serum in the lower chamber was used as a chemo-attractant. Cells that did not migrate through the Matrigel and/or pores in the membrane were removed using a cotton swab, and cells on the other side of the membrane were stained and photographed under bright-field microscopy (10× magnification). The number of cells that migrated and invaded was higher in control than the MUC4 knockdown cells. Data presented are the average number of cells/field for 10 fields. Columns: average of three independent experiments; bars: SE, p = 0.002 and p = 0.001, respectively. Representative images of control and MUC4 knockdown cells were shon in both figures. (D) Phalloidin staining showed that visualized F-actin under a laser scanning microscope is reduced in MDA-MB-231-shMUC4 cells compared with the control cells. (E) Immunoblot analysis showed reduced phosphorylation of FAK in MUC4 knockdown cells compared with the control cells. (F) Immunoblot analysis showed reduced expression of mesenchymal markers such as vimentin and vitronectin; and increased expression of CK-18 in MUC4 knockdown cells compared to the control.

Mentions: In addition to enhanced proliferation, the aggressiveness of a malignant cell is determined by its migratory and invasive potential. MUC4 knockdown cells exhibited significant decrease in motility, trans-well migration, and invasion (p = 0.01, p = 0.002, and p = 0.001, respectively). The motility of cells, determined by their migration in the wound gap after 12 h, in the wound healing assay decreased by 18% following MUC4 knockdown (Figure 3A). Similarly, trans-well migration and Matrigel invasion (Figure 3B-C) of MUC4 knockdown cells was decreased by 58% and 65%, respectively. Since actin plays an important role in defining cell shape and orchestrating events related to cellular motility, we investigated the effect of MUC4 on actin cytoskeleton reorganization. Following cell staining with rhodamine-conjugated phalloidin, control cells exhibited more lamellipodial structures compared to MUC4 knockdown cells, which had reduced F-actin (Figure 3D) and decreased levels of phosphorylated (Y925) focal adhesion kinase (Figure 3E). These results strongly suggest that MUC4 facilitates the migratory and invasive potential of MDA-MB-231 cells by inducing the reorganization of actin filaments. Since alterations in cell motility and cytoskeleton reorganization are associated with epithelial-to-mesenchymal transition (EMT), we investigated whether MUC4 regulates EMT in MDA-MB-231 cells. MUC4 Knockdown resulted in increased expression of the epithelial marker CK-18, and decreased expression of mesenchymal markers vimentin and vitronectin (Figure 3F).


MUC4 overexpression augments cell migration and metastasis through EGFR family proteins in triple negative breast cancer cells.

Mukhopadhyay P, Lakshmanan I, Ponnusamy MP, Chakraborty S, Jain M, Pai P, Smith LM, Lele SM, Batra SK - PLoS ONE (2013)

MUC4 enhances migratory and invasive potential.(A) After 24 hours serum starvation, a wound was created with a plastic tip on plates containing control or MUC4 knockdown cells. Cells were incubated in complete media for 12 hours. Motility of cells was photographed under bright-field microscopy (left, 10× magnification). After 12 hours, the migration of control cells and MUC4 knockdown cells was measured (in µm2) using DatInf Measure setup wizard software (http://tucows.texasonline.net). Values were calculated and plotted (right). (B and C) Control and MUC4 knockdown cells were serum starved for 48 h and seeded on non-coated or Matrigel-coated membranes for motility (B) and invasion (C) assays, respectively, and incubated for 24 h. Medium containing 10% fetal bovine serum in the lower chamber was used as a chemo-attractant. Cells that did not migrate through the Matrigel and/or pores in the membrane were removed using a cotton swab, and cells on the other side of the membrane were stained and photographed under bright-field microscopy (10× magnification). The number of cells that migrated and invaded was higher in control than the MUC4 knockdown cells. Data presented are the average number of cells/field for 10 fields. Columns: average of three independent experiments; bars: SE, p = 0.002 and p = 0.001, respectively. Representative images of control and MUC4 knockdown cells were shon in both figures. (D) Phalloidin staining showed that visualized F-actin under a laser scanning microscope is reduced in MDA-MB-231-shMUC4 cells compared with the control cells. (E) Immunoblot analysis showed reduced phosphorylation of FAK in MUC4 knockdown cells compared with the control cells. (F) Immunoblot analysis showed reduced expression of mesenchymal markers such as vimentin and vitronectin; and increased expression of CK-18 in MUC4 knockdown cells compared to the control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569463&req=5

pone-0054455-g003: MUC4 enhances migratory and invasive potential.(A) After 24 hours serum starvation, a wound was created with a plastic tip on plates containing control or MUC4 knockdown cells. Cells were incubated in complete media for 12 hours. Motility of cells was photographed under bright-field microscopy (left, 10× magnification). After 12 hours, the migration of control cells and MUC4 knockdown cells was measured (in µm2) using DatInf Measure setup wizard software (http://tucows.texasonline.net). Values were calculated and plotted (right). (B and C) Control and MUC4 knockdown cells were serum starved for 48 h and seeded on non-coated or Matrigel-coated membranes for motility (B) and invasion (C) assays, respectively, and incubated for 24 h. Medium containing 10% fetal bovine serum in the lower chamber was used as a chemo-attractant. Cells that did not migrate through the Matrigel and/or pores in the membrane were removed using a cotton swab, and cells on the other side of the membrane were stained and photographed under bright-field microscopy (10× magnification). The number of cells that migrated and invaded was higher in control than the MUC4 knockdown cells. Data presented are the average number of cells/field for 10 fields. Columns: average of three independent experiments; bars: SE, p = 0.002 and p = 0.001, respectively. Representative images of control and MUC4 knockdown cells were shon in both figures. (D) Phalloidin staining showed that visualized F-actin under a laser scanning microscope is reduced in MDA-MB-231-shMUC4 cells compared with the control cells. (E) Immunoblot analysis showed reduced phosphorylation of FAK in MUC4 knockdown cells compared with the control cells. (F) Immunoblot analysis showed reduced expression of mesenchymal markers such as vimentin and vitronectin; and increased expression of CK-18 in MUC4 knockdown cells compared to the control.
Mentions: In addition to enhanced proliferation, the aggressiveness of a malignant cell is determined by its migratory and invasive potential. MUC4 knockdown cells exhibited significant decrease in motility, trans-well migration, and invasion (p = 0.01, p = 0.002, and p = 0.001, respectively). The motility of cells, determined by their migration in the wound gap after 12 h, in the wound healing assay decreased by 18% following MUC4 knockdown (Figure 3A). Similarly, trans-well migration and Matrigel invasion (Figure 3B-C) of MUC4 knockdown cells was decreased by 58% and 65%, respectively. Since actin plays an important role in defining cell shape and orchestrating events related to cellular motility, we investigated the effect of MUC4 on actin cytoskeleton reorganization. Following cell staining with rhodamine-conjugated phalloidin, control cells exhibited more lamellipodial structures compared to MUC4 knockdown cells, which had reduced F-actin (Figure 3D) and decreased levels of phosphorylated (Y925) focal adhesion kinase (Figure 3E). These results strongly suggest that MUC4 facilitates the migratory and invasive potential of MDA-MB-231 cells by inducing the reorganization of actin filaments. Since alterations in cell motility and cytoskeleton reorganization are associated with epithelial-to-mesenchymal transition (EMT), we investigated whether MUC4 regulates EMT in MDA-MB-231 cells. MUC4 Knockdown resulted in increased expression of the epithelial marker CK-18, and decreased expression of mesenchymal markers vimentin and vitronectin (Figure 3F).

Bottom Line: Moreover, our studies also showed that knockdown of MUC4 in TNBC cells induced molecular changes suggestive of mesenchymal to epithelial transition.We further demonstrate that MUC4 is differentially over-expressed in invasive TNBC tissues compared to normal breast tissue.MUC4 mucin expression is associated with TNBC pathobiology, and its knockdown reduced aggressiveness in vitro, and tumorigenesis and metastasis in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska, USA.

ABSTRACT

Introduction: Current studies indicate that triple negative breast cancer (TNBC), an aggressive breast cancer subtype, is associated with poor prognosis and an early pattern of metastasis. Emerging evidence suggests that MUC4 mucin is associated with metastasis of various cancers, including breast cancer. However, the functional role of MUC4 remains unclear in breast cancers, especially in TNBCs.

Method: In the present study, we investigated the functional and mechanistic roles of MUC4 in potentiating pathogenic signals including EGFR family proteins to promote TNBC aggressiveness using in vitro and in vivo studies. Further, we studied the expression of MUC4 in invasive TNBC tissue and normal breast tissue by immunostaining.

Results: MUC4 promotes proliferation, anchorage-dependent and-independent growth of TNBC cells, augments TNBC cell migratory and invasive potential in vitro, and enhances tumorigenicity and metastasis in vivo. In addition, our studies demonstrated that MUC4 up-regulates the EGFR family of proteins, and augments downstream Erk1/2, PKC-γ, and FAK mediated oncogenic signaling. Moreover, our studies also showed that knockdown of MUC4 in TNBC cells induced molecular changes suggestive of mesenchymal to epithelial transition. We also demonstrated in this study, for the first time, that knockdown of MUC4 was associated with reduced expression of EGFR and ErbB3 (EGFR family proteins) in TNBC cells, suggesting that MUC4 uses an alternative to ErbB2 mechanism to promote aggressiveness. We further demonstrate that MUC4 is differentially over-expressed in invasive TNBC tissues compared to normal breast tissue.

Conclusions: MUC4 mucin expression is associated with TNBC pathobiology, and its knockdown reduced aggressiveness in vitro, and tumorigenesis and metastasis in vivo. Overall, our findings suggest that MUC4 mucin promotes invasive activities of TNBC cells by altering the expression of EGFR, ErbB2, and ErbB3 molecules and their downstream signaling.

Show MeSH
Related in: MedlinePlus