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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 promotes proliferation and growth of MDA-MB-231 cells.(A) Detection of MUC4 protein expression in control (MDA-MB-231-SCR) and MUC4 knockdown (MDA-MB-231-shMUC4) cells. Immunoblot showed reduced expression of MUC4 in MDA-MB-231-shMUC4 cells compared to control cells. Immunostained cells using human anti-MUC4 mouse monoclonal antibody (8G7) showed reduced expression of MUC4 in MDA-MB-231-shMUC4 cells compared to control cells. (B) In proliferation analyses, when the number of cells was plotted against the incubation period (hours), control cells showed a significantly higher proliferation rate than MUC4 knockdown cells, p = 0.03. Population doubling time of control cells was less than MUC4 knockdown cells when calculated from the number of cells growing in log phase (day 2 to 6) using the formula, Td = 0.693t/ln (Nt/N0). (C) Cells, following synchronization and serum re-stimulation and stained with Telford reagent (containing propidium iodide) and analyzed by FACS, showed that the number of MUC4 knockdown cells in the G1 phase was higher than control cells suggesting the inhibition of cell cycle progression. (D) The colony forming ability of control cells was higher than MUC4 knockdown cells under anchorage-dependent conditions. After staining, colonies of >50 µm in size were counted using Quantity One software, p = 0.003. Images of anchorage–dependent growth assays were shown at the bottom. (E) The colony forming ability of control cells was higher than MUC4 knockdown cells under anchorage-independent conditions. Colonies were counted and plotted. Columns: mean of triplicates; bars: SD, p = 0.0001. Phase-contrast images were recorded at 10× magnification. Microscopic images of colonies found in anchorage–independent growth assays. Higher magnification of a typical colony was shown in box on bottom right corner to emphasize a (i) big colony with migratory outer cells versus (ii) smaller and compact colonies. SCR are control and shMUC4 are MUC4 knockdown cells. All data presented are the average of 3 independent experiments.
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pone-0054455-g001: MUC4 promotes proliferation and growth of MDA-MB-231 cells.(A) Detection of MUC4 protein expression in control (MDA-MB-231-SCR) and MUC4 knockdown (MDA-MB-231-shMUC4) cells. Immunoblot showed reduced expression of MUC4 in MDA-MB-231-shMUC4 cells compared to control cells. Immunostained cells using human anti-MUC4 mouse monoclonal antibody (8G7) showed reduced expression of MUC4 in MDA-MB-231-shMUC4 cells compared to control cells. (B) In proliferation analyses, when the number of cells was plotted against the incubation period (hours), control cells showed a significantly higher proliferation rate than MUC4 knockdown cells, p = 0.03. Population doubling time of control cells was less than MUC4 knockdown cells when calculated from the number of cells growing in log phase (day 2 to 6) using the formula, Td = 0.693t/ln (Nt/N0). (C) Cells, following synchronization and serum re-stimulation and stained with Telford reagent (containing propidium iodide) and analyzed by FACS, showed that the number of MUC4 knockdown cells in the G1 phase was higher than control cells suggesting the inhibition of cell cycle progression. (D) The colony forming ability of control cells was higher than MUC4 knockdown cells under anchorage-dependent conditions. After staining, colonies of >50 µm in size were counted using Quantity One software, p = 0.003. Images of anchorage–dependent growth assays were shown at the bottom. (E) The colony forming ability of control cells was higher than MUC4 knockdown cells under anchorage-independent conditions. Colonies were counted and plotted. Columns: mean of triplicates; bars: SD, p = 0.0001. Phase-contrast images were recorded at 10× magnification. Microscopic images of colonies found in anchorage–independent growth assays. Higher magnification of a typical colony was shown in box on bottom right corner to emphasize a (i) big colony with migratory outer cells versus (ii) smaller and compact colonies. SCR are control and shMUC4 are MUC4 knockdown cells. All data presented are the average of 3 independent experiments.

Mentions: In a preliminary screening of breast cancer cells, we observed that the invasive MDA-MB-231 TNBC cell line expressed MUC4 when grown in α-MEM media (Figure 1A, left lane), while non-invasive TNBC cell lines BT-20 and MDA-MB-468 [31] were MUC4 negative (data not shown). Thus, we chose MDA-MB-231 cells for further studies and generated a line with stable knockdown of MUC4 using a retroviral construct [7] to elucidate the functional significance of MUC4 in TNBC pathophysiology. A stable line generated with non-targeted scrambled shRNA (SCR) was used as control for all experiments performed in this study. Knockdown of MUC4 was confirmed by real-time PCR (data not shown), immunoblotting, and immunofluorescence analyses (Figure 1A). Immunoblot analysis indicated a ∼95% down-regulation of MUC4 at the protein level in MUC4 knockdown cells (MDA-MB-231-shMUC4) as compared with control cells (MDA-MB-231-SCR) cells.


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 promotes proliferation and growth of MDA-MB-231 cells.(A) Detection of MUC4 protein expression in control (MDA-MB-231-SCR) and MUC4 knockdown (MDA-MB-231-shMUC4) cells. Immunoblot showed reduced expression of MUC4 in MDA-MB-231-shMUC4 cells compared to control cells. Immunostained cells using human anti-MUC4 mouse monoclonal antibody (8G7) showed reduced expression of MUC4 in MDA-MB-231-shMUC4 cells compared to control cells. (B) In proliferation analyses, when the number of cells was plotted against the incubation period (hours), control cells showed a significantly higher proliferation rate than MUC4 knockdown cells, p = 0.03. Population doubling time of control cells was less than MUC4 knockdown cells when calculated from the number of cells growing in log phase (day 2 to 6) using the formula, Td = 0.693t/ln (Nt/N0). (C) Cells, following synchronization and serum re-stimulation and stained with Telford reagent (containing propidium iodide) and analyzed by FACS, showed that the number of MUC4 knockdown cells in the G1 phase was higher than control cells suggesting the inhibition of cell cycle progression. (D) The colony forming ability of control cells was higher than MUC4 knockdown cells under anchorage-dependent conditions. After staining, colonies of >50 µm in size were counted using Quantity One software, p = 0.003. Images of anchorage–dependent growth assays were shown at the bottom. (E) The colony forming ability of control cells was higher than MUC4 knockdown cells under anchorage-independent conditions. Colonies were counted and plotted. Columns: mean of triplicates; bars: SD, p = 0.0001. Phase-contrast images were recorded at 10× magnification. Microscopic images of colonies found in anchorage–independent growth assays. Higher magnification of a typical colony was shown in box on bottom right corner to emphasize a (i) big colony with migratory outer cells versus (ii) smaller and compact colonies. SCR are control and shMUC4 are MUC4 knockdown cells. All data presented are the average of 3 independent experiments.
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Related In: Results  -  Collection

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pone-0054455-g001: MUC4 promotes proliferation and growth of MDA-MB-231 cells.(A) Detection of MUC4 protein expression in control (MDA-MB-231-SCR) and MUC4 knockdown (MDA-MB-231-shMUC4) cells. Immunoblot showed reduced expression of MUC4 in MDA-MB-231-shMUC4 cells compared to control cells. Immunostained cells using human anti-MUC4 mouse monoclonal antibody (8G7) showed reduced expression of MUC4 in MDA-MB-231-shMUC4 cells compared to control cells. (B) In proliferation analyses, when the number of cells was plotted against the incubation period (hours), control cells showed a significantly higher proliferation rate than MUC4 knockdown cells, p = 0.03. Population doubling time of control cells was less than MUC4 knockdown cells when calculated from the number of cells growing in log phase (day 2 to 6) using the formula, Td = 0.693t/ln (Nt/N0). (C) Cells, following synchronization and serum re-stimulation and stained with Telford reagent (containing propidium iodide) and analyzed by FACS, showed that the number of MUC4 knockdown cells in the G1 phase was higher than control cells suggesting the inhibition of cell cycle progression. (D) The colony forming ability of control cells was higher than MUC4 knockdown cells under anchorage-dependent conditions. After staining, colonies of >50 µm in size were counted using Quantity One software, p = 0.003. Images of anchorage–dependent growth assays were shown at the bottom. (E) The colony forming ability of control cells was higher than MUC4 knockdown cells under anchorage-independent conditions. Colonies were counted and plotted. Columns: mean of triplicates; bars: SD, p = 0.0001. Phase-contrast images were recorded at 10× magnification. Microscopic images of colonies found in anchorage–independent growth assays. Higher magnification of a typical colony was shown in box on bottom right corner to emphasize a (i) big colony with migratory outer cells versus (ii) smaller and compact colonies. SCR are control and shMUC4 are MUC4 knockdown cells. All data presented are the average of 3 independent experiments.
Mentions: In a preliminary screening of breast cancer cells, we observed that the invasive MDA-MB-231 TNBC cell line expressed MUC4 when grown in α-MEM media (Figure 1A, left lane), while non-invasive TNBC cell lines BT-20 and MDA-MB-468 [31] were MUC4 negative (data not shown). Thus, we chose MDA-MB-231 cells for further studies and generated a line with stable knockdown of MUC4 using a retroviral construct [7] to elucidate the functional significance of MUC4 in TNBC pathophysiology. A stable line generated with non-targeted scrambled shRNA (SCR) was used as control for all experiments performed in this study. Knockdown of MUC4 was confirmed by real-time PCR (data not shown), immunoblotting, and immunofluorescence analyses (Figure 1A). Immunoblot analysis indicated a ∼95% down-regulation of MUC4 at the protein level in MUC4 knockdown cells (MDA-MB-231-shMUC4) as compared with control cells (MDA-MB-231-SCR) cells.

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