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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 growth of MDA-MB-231 xenografts.(A) MUC4 knockdown and control cells (0.1 × 106 cells/animal) were orthotopically implanted in mouse mammary fat pad of each mouse (right 3rd mammary gland). Tumor volumes were calculated every week. The MUC4 knockdown cells started to grow tumors during the 5th week, but control cells started to grow tumors during the 3rd week, p = 0.0001. (B) Eight weeks after implantation, mice were sacrificed and tumors were excised and weighed. Stable silencing of MUC4 was found to decrease tumor growth, p = 0.0001.
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pone-0054455-g005: MUC4 promotes growth of MDA-MB-231 xenografts.(A) MUC4 knockdown and control cells (0.1 × 106 cells/animal) were orthotopically implanted in mouse mammary fat pad of each mouse (right 3rd mammary gland). Tumor volumes were calculated every week. The MUC4 knockdown cells started to grow tumors during the 5th week, but control cells started to grow tumors during the 3rd week, p = 0.0001. (B) Eight weeks after implantation, mice were sacrificed and tumors were excised and weighed. Stable silencing of MUC4 was found to decrease tumor growth, p = 0.0001.

Mentions: As MUC4 knockdown was observed to augment proliferation, growth, migration, and invasion of MDA-MB-231 cells, we sought to investigate the effect of MUC4 knockdown on the tumorigenic and metastatic potential of MDA-MB-231 cells. Control and MUC4 knockdown cells were implanted orthotopically into mammary fat pads of two groups of female nude mice (n = 9). Control cells produced detectable tumors at week 3, while tumors resulting from MUC4 knockdown cells were detectable only after 5 weeks (Figure 5A). The tumor volume from MUC4 knockdown cells was significantly smaller (p = 0.0001) and the tumors excised at 8 weeks had markedly reduced weight, compared with tumors obtained from control cells (mean 0.171±0.05 g in MUC4 knockdown vs. 0.653±0.07 g in control cells) (Figure 5B). In addition, in vivo transgene expression in control cells was confirmed in excised tumors at the mRNA and protein levels (Figure S2A-B). Next, we determined the frequency of metastases in mice implanted with control or MUC4 knockdown cells. All mice, implanted with control cells, developed metastases to one or multiple sites. Metastasis was observed in 2 of nine mice of each organ such as lung, ovary, and peritoneum and; in 3 of nine of each site like mesenteric lymph nodes, and intestinal wall. In contrast no metastasis was observed in mice implanted with MUC4 knockdown cells. In an independent experiment, larger tumors were obtained by orthotopically implanting 3× more MUC4 knockdown cells (0.3 × 106). However, these tumors, while comparable in size to the previously obtained control tumors (0.75 g), were still incapable of distant metastasis, suggesting that the differences in the metastatic potential of control and MUC4 knockdown cells is independent of the size of the primary tumor (Table S1).


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 growth of MDA-MB-231 xenografts.(A) MUC4 knockdown and control cells (0.1 × 106 cells/animal) were orthotopically implanted in mouse mammary fat pad of each mouse (right 3rd mammary gland). Tumor volumes were calculated every week. The MUC4 knockdown cells started to grow tumors during the 5th week, but control cells started to grow tumors during the 3rd week, p = 0.0001. (B) Eight weeks after implantation, mice were sacrificed and tumors were excised and weighed. Stable silencing of MUC4 was found to decrease tumor growth, p = 0.0001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569463&req=5

pone-0054455-g005: MUC4 promotes growth of MDA-MB-231 xenografts.(A) MUC4 knockdown and control cells (0.1 × 106 cells/animal) were orthotopically implanted in mouse mammary fat pad of each mouse (right 3rd mammary gland). Tumor volumes were calculated every week. The MUC4 knockdown cells started to grow tumors during the 5th week, but control cells started to grow tumors during the 3rd week, p = 0.0001. (B) Eight weeks after implantation, mice were sacrificed and tumors were excised and weighed. Stable silencing of MUC4 was found to decrease tumor growth, p = 0.0001.
Mentions: As MUC4 knockdown was observed to augment proliferation, growth, migration, and invasion of MDA-MB-231 cells, we sought to investigate the effect of MUC4 knockdown on the tumorigenic and metastatic potential of MDA-MB-231 cells. Control and MUC4 knockdown cells were implanted orthotopically into mammary fat pads of two groups of female nude mice (n = 9). Control cells produced detectable tumors at week 3, while tumors resulting from MUC4 knockdown cells were detectable only after 5 weeks (Figure 5A). The tumor volume from MUC4 knockdown cells was significantly smaller (p = 0.0001) and the tumors excised at 8 weeks had markedly reduced weight, compared with tumors obtained from control cells (mean 0.171±0.05 g in MUC4 knockdown vs. 0.653±0.07 g in control cells) (Figure 5B). In addition, in vivo transgene expression in control cells was confirmed in excised tumors at the mRNA and protein levels (Figure S2A-B). Next, we determined the frequency of metastases in mice implanted with control or MUC4 knockdown cells. All mice, implanted with control cells, developed metastases to one or multiple sites. Metastasis was observed in 2 of nine mice of each organ such as lung, ovary, and peritoneum and; in 3 of nine of each site like mesenteric lymph nodes, and intestinal wall. In contrast no metastasis was observed in mice implanted with MUC4 knockdown cells. In an independent experiment, larger tumors were obtained by orthotopically implanting 3× more MUC4 knockdown cells (0.3 × 106). However, these tumors, while comparable in size to the previously obtained control tumors (0.75 g), were still incapable of distant metastasis, suggesting that the differences in the metastatic potential of control and MUC4 knockdown cells is independent of the size of the primary tumor (Table S1).

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