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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 up-regulates EGFR family receptors and induces downstream Erk1/2 and PKC-γ pathways.(A) Immunoblot analyses showed reduced expression of EGFR, ErbB2, and ErbB3 in MUC4 knockdown cells compared with control cells. (B) Reduced expression (using immunoblot) of Sprouty 2 was detected in MUC4 knockdown cell when compared with control cells. (C) Immunoblot showed that reduced phosphorylation of Erk1/2 and expression of PKC-γ in MUC4 knockdown cells compared with control cells. β-actin was used as a loading control. (D) Immunoblot analyses showed reduced expression of β-catenin and its target gene product cyclin D1in MUC4 knockdown compared with control cells.
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pone-0054455-g002: MUC4 up-regulates EGFR family receptors and induces downstream Erk1/2 and PKC-γ pathways.(A) Immunoblot analyses showed reduced expression of EGFR, ErbB2, and ErbB3 in MUC4 knockdown cells compared with control cells. (B) Reduced expression (using immunoblot) of Sprouty 2 was detected in MUC4 knockdown cell when compared with control cells. (C) Immunoblot showed that reduced phosphorylation of Erk1/2 and expression of PKC-γ in MUC4 knockdown cells compared with control cells. β-actin was used as a loading control. (D) Immunoblot analyses showed reduced expression of β-catenin and its target gene product cyclin D1in MUC4 knockdown compared with control cells.

Mentions: EGFR plays important roles in the proliferation of TNBC [33], while MUC4 has been demonstrated to stabilize another EGFR family member, ErbB2 [25]. Thus, we studied the effect of MUC4 knockdown on the status of EGFR family members and downstream signaling. Knockdown of MUC4 resulted in reduced expression of ErbB1 (EGFR) and ErbB3, whereas ErbB4 levels remained unchanged (Figure 2A). This result suggests that MUC4 may use an alternative mechanism to promote aggressiveness and metastasis of TNBC cells, because ErbB2 was present at low levels in TNBC cells. Sprouty 2 enhances EGFR stability by sequestering Cbl (Casitas B-lineage Lymphoma-an E3 ubiquitin ligase), and thus inhibiting ubiquitin-mediated degradation of EGFR [34]. We observed a decreased expression of Sprouty 2 in MUC4 knockdown cells compared with control cells (Figure 2B). Alteration of EGFR expression by MUC4 resulted in enhanced downstream signaling via Erk1/2 and PKC-γ pathways, as indicated by increased phosphorylation of Erk1/2 and increased expression of PKC-γ in control cells (Figure 2C). Furthermore, MUC4 knockdown resulted in decreased expression of cyclin D1 and its upstream regulator β-catenin, suggesting that MUC4 augments cell cycle progression possibly via cyclin D1(Figure 2D).


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 up-regulates EGFR family receptors and induces downstream Erk1/2 and PKC-γ pathways.(A) Immunoblot analyses showed reduced expression of EGFR, ErbB2, and ErbB3 in MUC4 knockdown cells compared with control cells. (B) Reduced expression (using immunoblot) of Sprouty 2 was detected in MUC4 knockdown cell when compared with control cells. (C) Immunoblot showed that reduced phosphorylation of Erk1/2 and expression of PKC-γ in MUC4 knockdown cells compared with control cells. β-actin was used as a loading control. (D) Immunoblot analyses showed reduced expression of β-catenin and its target gene product cyclin D1in MUC4 knockdown compared with control cells.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569463&req=5

pone-0054455-g002: MUC4 up-regulates EGFR family receptors and induces downstream Erk1/2 and PKC-γ pathways.(A) Immunoblot analyses showed reduced expression of EGFR, ErbB2, and ErbB3 in MUC4 knockdown cells compared with control cells. (B) Reduced expression (using immunoblot) of Sprouty 2 was detected in MUC4 knockdown cell when compared with control cells. (C) Immunoblot showed that reduced phosphorylation of Erk1/2 and expression of PKC-γ in MUC4 knockdown cells compared with control cells. β-actin was used as a loading control. (D) Immunoblot analyses showed reduced expression of β-catenin and its target gene product cyclin D1in MUC4 knockdown compared with control cells.
Mentions: EGFR plays important roles in the proliferation of TNBC [33], while MUC4 has been demonstrated to stabilize another EGFR family member, ErbB2 [25]. Thus, we studied the effect of MUC4 knockdown on the status of EGFR family members and downstream signaling. Knockdown of MUC4 resulted in reduced expression of ErbB1 (EGFR) and ErbB3, whereas ErbB4 levels remained unchanged (Figure 2A). This result suggests that MUC4 may use an alternative mechanism to promote aggressiveness and metastasis of TNBC cells, because ErbB2 was present at low levels in TNBC cells. Sprouty 2 enhances EGFR stability by sequestering Cbl (Casitas B-lineage Lymphoma-an E3 ubiquitin ligase), and thus inhibiting ubiquitin-mediated degradation of EGFR [34]. We observed a decreased expression of Sprouty 2 in MUC4 knockdown cells compared with control cells (Figure 2B). Alteration of EGFR expression by MUC4 resulted in enhanced downstream signaling via Erk1/2 and PKC-γ pathways, as indicated by increased phosphorylation of Erk1/2 and increased expression of PKC-γ in control cells (Figure 2C). Furthermore, MUC4 knockdown resulted in decreased expression of cyclin D1 and its upstream regulator β-catenin, suggesting that MUC4 augments cell cycle progression possibly via cyclin D1(Figure 2D).

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