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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-associated gene expression, pathways, and interaction networks in MDA-MB-231 cells.Array data were validated using RT-PCR using specific primers: 20 ng mRNA from control and MUC4 knockdown cells were reverse transcribed and used for RT-PCR using MUC4 specific primers and the LightCycler SYBR Green 1 Master. The β-actin specific primers were used as control. CT values were calculated and plotted to verify the up-regulated and down-regulated genes.
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pone-0054455-g007: MUC4-associated gene expression, pathways, and interaction networks in MDA-MB-231 cells.Array data were validated using RT-PCR using specific primers: 20 ng mRNA from control and MUC4 knockdown cells were reverse transcribed and used for RT-PCR using MUC4 specific primers and the LightCycler SYBR Green 1 Master. The β-actin specific primers were used as control. CT values were calculated and plotted to verify the up-regulated and down-regulated genes.

Mentions: We investigated alterations at transcript level following MUC4 knockdown in MDA-MB-231 cells using human genome microarray analysis. A total of 175 genes exhibited a >2 fold differential expression in MUC4 knockdown cells compared with control cells. The top-scoring network of interactions among the differentially expressed genes in control versus MUC4 knockdown cells is shown along with the table that lists statistically significant enriched high-level functions (Figure S3). In agreement with the results presented in Figure 2C, the Erk1/2 and MAPK nodes were highly perturbed upon MUC4 silencing (Figure S3). Selected genes that exhibited the most differential expression in MUC4 knockdown cells are listed in Figure S4A-C. Analyses of the data revealed that several genes associated with cellular motility, proliferation, inflammatory response, and cellular signaling, were differentially regulated in MUC4 knockdown cells. Some important genes COL4A5, SMAD6, CXCL1, and DUSP2 were found to be up-regulated and several other genes A100A4, PDGFRB, CAV1, and CAV2 were found to be down-regulated and hence were validated (Figure 7). The results of the real time analyses were in complete agreement with the microarray data, indicating that these genes could be involved in mediating the modulation of signaling pathways by MUC4.


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-associated gene expression, pathways, and interaction networks in MDA-MB-231 cells.Array data were validated using RT-PCR using specific primers: 20 ng mRNA from control and MUC4 knockdown cells were reverse transcribed and used for RT-PCR using MUC4 specific primers and the LightCycler SYBR Green 1 Master. The β-actin specific primers were used as control. CT values were calculated and plotted to verify the up-regulated and down-regulated genes.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0054455-g007: MUC4-associated gene expression, pathways, and interaction networks in MDA-MB-231 cells.Array data were validated using RT-PCR using specific primers: 20 ng mRNA from control and MUC4 knockdown cells were reverse transcribed and used for RT-PCR using MUC4 specific primers and the LightCycler SYBR Green 1 Master. The β-actin specific primers were used as control. CT values were calculated and plotted to verify the up-regulated and down-regulated genes.
Mentions: We investigated alterations at transcript level following MUC4 knockdown in MDA-MB-231 cells using human genome microarray analysis. A total of 175 genes exhibited a >2 fold differential expression in MUC4 knockdown cells compared with control cells. The top-scoring network of interactions among the differentially expressed genes in control versus MUC4 knockdown cells is shown along with the table that lists statistically significant enriched high-level functions (Figure S3). In agreement with the results presented in Figure 2C, the Erk1/2 and MAPK nodes were highly perturbed upon MUC4 silencing (Figure S3). Selected genes that exhibited the most differential expression in MUC4 knockdown cells are listed in Figure S4A-C. Analyses of the data revealed that several genes associated with cellular motility, proliferation, inflammatory response, and cellular signaling, were differentially regulated in MUC4 knockdown cells. Some important genes COL4A5, SMAD6, CXCL1, and DUSP2 were found to be up-regulated and several other genes A100A4, PDGFRB, CAV1, and CAV2 were found to be down-regulated and hence were validated (Figure 7). The results of the real time analyses were in complete agreement with the microarray data, indicating that these genes could be involved in mediating the modulation of signaling pathways by MUC4.

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