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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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MUC4 contributes to a altered phenotype.(A) The control or MUC4 knockdown cells were seeded in 2% Matrigel on top of a 100% Matrigel layer, and fed with media every 3 days. After 7 days, acini-like structures were photographed under a phase-contrast microscope. The acini-like structures (examples shown in the boxes) that were regular (smooth and spherical shape) or irregular (irregular outline, multi-lobular) were counted and plotted as a percentage of the total count (p = 0.0005 for regular and p = 0.002 for irregular). A minimum of 120 structures was counted for each of control cells or MUC4 knockdown cells. Reduced irregular outline, multi-lobular and increased smooth and spherical shape colonies were found in MUC4 knock down cells when compared with control cells. (B) Structures were stained with the anti-ZO-1 antibody. 4,6-diamidino-2-phenylindole (DAPI) was used for nuclei staining. Optical sections (0.7–0.9 µm) were captured using a laser scanning confocal microscope. The images presented here are the central planes of the acini. Bar = 20 µm.
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pone-0054455-g004: MUC4 contributes to a altered phenotype.(A) The control or MUC4 knockdown cells were seeded in 2% Matrigel on top of a 100% Matrigel layer, and fed with media every 3 days. After 7 days, acini-like structures were photographed under a phase-contrast microscope. The acini-like structures (examples shown in the boxes) that were regular (smooth and spherical shape) or irregular (irregular outline, multi-lobular) were counted and plotted as a percentage of the total count (p = 0.0005 for regular and p = 0.002 for irregular). A minimum of 120 structures was counted for each of control cells or MUC4 knockdown cells. Reduced irregular outline, multi-lobular and increased smooth and spherical shape colonies were found in MUC4 knock down cells when compared with control cells. (B) Structures were stained with the anti-ZO-1 antibody. 4,6-diamidino-2-phenylindole (DAPI) was used for nuclei staining. Optical sections (0.7–0.9 µm) were captured using a laser scanning confocal microscope. The images presented here are the central planes of the acini. Bar = 20 µm.

Mentions: Epithelial cells in the mammary gland maintain a polarized morphology, specialized cell-cell contacts, and attachment to the underlying basement membrane. The development and maintenance of this polarized structure are critical for the formation and function of mammary epithelial cells [35]. However, the pathogenesis of tumors originating from epithelial cells requires the disruption of this intact and well-organized structural design. We used a 3D Matrigel culture model [36] to determine the effect of MUC4 knockdown on the morphology of the resulting 3D structures. The control cells failed to polarize in Matrigel and formed large, disorganized colonies. MUC4 knockdown did not induce structural polarization, but resulted in the formation of more organized structures reminiscent of mammary gland acini (Figure 4A). Confocal imaging of 3D Matrigel structures for ZO-1, a tight junction protein, further confirmed that control cells predominantly form disorganized and larger 3D colonies in Matrigel (83%) compared with the MUC4 knockdown cells (23%; p = 0.002) (Figure 4B). These results indicate that MUC4 induces the transformation of MDA-MB-231 cells to a highly migratory phenotype, and that stable MUC4 knockdown partially reduces this phenomenon.


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 contributes to a altered phenotype.(A) The control or MUC4 knockdown cells were seeded in 2% Matrigel on top of a 100% Matrigel layer, and fed with media every 3 days. After 7 days, acini-like structures were photographed under a phase-contrast microscope. The acini-like structures (examples shown in the boxes) that were regular (smooth and spherical shape) or irregular (irregular outline, multi-lobular) were counted and plotted as a percentage of the total count (p = 0.0005 for regular and p = 0.002 for irregular). A minimum of 120 structures was counted for each of control cells or MUC4 knockdown cells. Reduced irregular outline, multi-lobular and increased smooth and spherical shape colonies were found in MUC4 knock down cells when compared with control cells. (B) Structures were stained with the anti-ZO-1 antibody. 4,6-diamidino-2-phenylindole (DAPI) was used for nuclei staining. Optical sections (0.7–0.9 µm) were captured using a laser scanning confocal microscope. The images presented here are the central planes of the acini. Bar = 20 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569463&req=5

pone-0054455-g004: MUC4 contributes to a altered phenotype.(A) The control or MUC4 knockdown cells were seeded in 2% Matrigel on top of a 100% Matrigel layer, and fed with media every 3 days. After 7 days, acini-like structures were photographed under a phase-contrast microscope. The acini-like structures (examples shown in the boxes) that were regular (smooth and spherical shape) or irregular (irregular outline, multi-lobular) were counted and plotted as a percentage of the total count (p = 0.0005 for regular and p = 0.002 for irregular). A minimum of 120 structures was counted for each of control cells or MUC4 knockdown cells. Reduced irregular outline, multi-lobular and increased smooth and spherical shape colonies were found in MUC4 knock down cells when compared with control cells. (B) Structures were stained with the anti-ZO-1 antibody. 4,6-diamidino-2-phenylindole (DAPI) was used for nuclei staining. Optical sections (0.7–0.9 µm) were captured using a laser scanning confocal microscope. The images presented here are the central planes of the acini. Bar = 20 µm.
Mentions: Epithelial cells in the mammary gland maintain a polarized morphology, specialized cell-cell contacts, and attachment to the underlying basement membrane. The development and maintenance of this polarized structure are critical for the formation and function of mammary epithelial cells [35]. However, the pathogenesis of tumors originating from epithelial cells requires the disruption of this intact and well-organized structural design. We used a 3D Matrigel culture model [36] to determine the effect of MUC4 knockdown on the morphology of the resulting 3D structures. The control cells failed to polarize in Matrigel and formed large, disorganized colonies. MUC4 knockdown did not induce structural polarization, but resulted in the formation of more organized structures reminiscent of mammary gland acini (Figure 4A). Confocal imaging of 3D Matrigel structures for ZO-1, a tight junction protein, further confirmed that control cells predominantly form disorganized and larger 3D colonies in Matrigel (83%) compared with the MUC4 knockdown cells (23%; p = 0.002) (Figure 4B). These results indicate that MUC4 induces the transformation of MDA-MB-231 cells to a highly migratory phenotype, and that stable MUC4 knockdown partially reduces this phenomenon.

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