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Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling.

Lee KM, Nam K, Oh S, Lim J, Kim YP, Lee JW, Yu JH, Ahn SH, Kim SB, Noh DY, Lee T, Shin I - Breast Cancer Res. (2014)

Bottom Line: We examined the effect of ECM1 on cell proliferation and cell signaling in vitro and in vivo.Through physical interaction with epidermal growth factor receptor (EGFR), ECM1 potentiated the phosphorylation of EGFR and extracellular signal-regulated kinase upon EGF treatment.Moreover, ECM1-induced galectin-3 cleavage through upregulation of matrix metalloproteinase 9 not only improved mucin 1 expression, but also increased EGFR and human epidermal growth factor receptor 3 protein stability as a secondary signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Hanyang University, 222 Wangshimni-ro, Seoul, 133-791, Republic of Korea. realwooma@gmail.com.

ABSTRACT

Introduction: Extracellular matrix protein 1 (ECM1) is a secreted glycoprotein with putative functions in cell proliferation, angiogenesis and differentiation. Expression of ECM1 in several types of carcinoma suggests that it may promote tumor development. In this study, we investigated the role of ECM1 in oncogenic cell signaling in breast cancer, and potential mechanisms for its effects.

Methods: In order to find out the functional role of ECM1, we used the recombinant human ECM1 and viral transduction systems which stably regulated the expression level of ECM1. We examined the effect of ECM1 on cell proliferation and cell signaling in vitro and in vivo. Moreover, tissues and sera of patients with breast cancer were used to confirm the effect of ECM1.

Results: ECM1 protein was increased in trastuzumab-resistant (TR) cells, in association with trastuzumab resistance and cell proliferation. Through physical interaction with epidermal growth factor receptor (EGFR), ECM1 potentiated the phosphorylation of EGFR and extracellular signal-regulated kinase upon EGF treatment. Moreover, ECM1-induced galectin-3 cleavage through upregulation of matrix metalloproteinase 9 not only improved mucin 1 expression, but also increased EGFR and human epidermal growth factor receptor 3 protein stability as a secondary signaling.

Conclusions: ECM1 has important roles in both cancer development and trastuzumab resistance in breast cancer through activation of EGFR signaling.

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Relationship of extracellular matrix protein 1 level to breast cancer cell proliferation. (A) Each cell line was treated with 200 ng/ml recombinant human extracellular matrix protein 1 (rhECM1) or 5 μg/ml anti-ECM1 antibodies, and cells were counted with a hemocytometer over the course of 3 days (*P < 0.05, **P < 0.005). Cont, Control; shC, Control short-hairpin RNA; shE/shECM1, Short-hairpin extracellular matrix protein 1 RNA; TR, Trastuzumab-resistant; Vec, Vector. (B) Cell cycles were analyzed by flow cytometry (*P < 0.05, **P < 0.005). (C) Each cell line was passaged by subcutaneous injection into the lower flank of each mouse. Tumor volume was measured every 3 days (n = 5 or 6 for each group). (D) Kaplan-Meier analyses for recurrence-free survival, overall survival and distant metastasis-free survival in the HER2-positive breast cancer patients. Data were obtained from the Kaplan-Meier Plotter Breast Cancer database [24]. Overall survival data from breast cancer patients were stratified by ECM1 levels in HER2-positive patient sera (n = 27).
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Fig2: Relationship of extracellular matrix protein 1 level to breast cancer cell proliferation. (A) Each cell line was treated with 200 ng/ml recombinant human extracellular matrix protein 1 (rhECM1) or 5 μg/ml anti-ECM1 antibodies, and cells were counted with a hemocytometer over the course of 3 days (*P < 0.05, **P < 0.005). Cont, Control; shC, Control short-hairpin RNA; shE/shECM1, Short-hairpin extracellular matrix protein 1 RNA; TR, Trastuzumab-resistant; Vec, Vector. (B) Cell cycles were analyzed by flow cytometry (*P < 0.05, **P < 0.005). (C) Each cell line was passaged by subcutaneous injection into the lower flank of each mouse. Tumor volume was measured every 3 days (n = 5 or 6 for each group). (D) Kaplan-Meier analyses for recurrence-free survival, overall survival and distant metastasis-free survival in the HER2-positive breast cancer patients. Data were obtained from the Kaplan-Meier Plotter Breast Cancer database [24]. Overall survival data from breast cancer patients were stratified by ECM1 levels in HER2-positive patient sera (n = 27).

Mentions: As BT-474 TR cells had a rapid proliferation ratio compared with parental BT-474 cells (Additional file 2: Figures S1C and S1D), we hypothesized that ECM1 can promote cell proliferation. To test the direct effect of ECM1 on cell proliferation, we treated BT-474 and MCF-7 cells with rhECM1. As determined by cell counting, this treatment increased the cell proliferation rate in these breast cancer cell lines (Figure 2A). To further test this effect, we overexpressed ECM1 in the BT-474 and MCF-7 cells and silenced ECM1 in BT-474 TR cells. Relative protein levels confirmed stable ECM1 expression (Additional file 3: Figure S2A), and this overexpression of ECM1 enhanced cell proliferation in the cell lines (Figure 2A, bottom). The knockdown of ECM1 by treatment with shRNA and anti-ECM1 antibodies in BT-474 TR cells reduced cell proliferation (Figure 2A, right). The anti-ECM1 antibodies also inhibited cell proliferation in other breast cancer cell lines (Additional file 3: Figure S2B). The MDA-MB-231 cell line, which overexpresses ECM1, showed especially high sensitivity to anti-ECM1 antibodies. To explore the mechanism for the cell-proliferative effect of ECM1, we analyzed the interrelationship of ECM1 expression with cell cycle progression. Cells overexpressing ECM1 showed an increased fraction of cells in S phase with a corresponding reduction of the fraction in G1 phase. Similarly, ECM1 knockdown led to the accumulation of cells in G1 phase with a decrease in the S-phase fraction (Figure 2B). Thus, ECM1 may positively regulate cell proliferation by affecting cell cycle progression. To observe the effect of ECM1 on cell proliferation in vivo, we tested the relationship of ECM1 expression to xenograft tumor formation in BALB/c nude mice. In BT-474 cells overexpressing ECM1, tumor formation efficiency was significantly increased, whereas tumorigenic potential of the BT-474 TR cells was significantly decreased after ECM1 silencing (Figure 2C). Moreover, the Kaplan-Meier plots [24] constructed using datasets from HER2-positive breast cancer patients demonstrated a significant association of ECM1 expression with poor outcome. Increasing ECM1 protein level predicted shorter distant metastasis-free survival in HER2-positive breast cancer patients (Figure 2D, left). Indeed, we found that high levels of ECM1 in serum were directly and inversely related to overall survival in HER2-positive breast cancer patients (Figure 2D, right). These data suggested that ECM1 overexpression in patients may contribute to bad outcomes in human breast cancer.Figure 2


Extracellular matrix protein 1 regulates cell proliferation and trastuzumab resistance through activation of epidermal growth factor signaling.

Lee KM, Nam K, Oh S, Lim J, Kim YP, Lee JW, Yu JH, Ahn SH, Kim SB, Noh DY, Lee T, Shin I - Breast Cancer Res. (2014)

Relationship of extracellular matrix protein 1 level to breast cancer cell proliferation. (A) Each cell line was treated with 200 ng/ml recombinant human extracellular matrix protein 1 (rhECM1) or 5 μg/ml anti-ECM1 antibodies, and cells were counted with a hemocytometer over the course of 3 days (*P < 0.05, **P < 0.005). Cont, Control; shC, Control short-hairpin RNA; shE/shECM1, Short-hairpin extracellular matrix protein 1 RNA; TR, Trastuzumab-resistant; Vec, Vector. (B) Cell cycles were analyzed by flow cytometry (*P < 0.05, **P < 0.005). (C) Each cell line was passaged by subcutaneous injection into the lower flank of each mouse. Tumor volume was measured every 3 days (n = 5 or 6 for each group). (D) Kaplan-Meier analyses for recurrence-free survival, overall survival and distant metastasis-free survival in the HER2-positive breast cancer patients. Data were obtained from the Kaplan-Meier Plotter Breast Cancer database [24]. Overall survival data from breast cancer patients were stratified by ECM1 levels in HER2-positive patient sera (n = 27).
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Related In: Results  -  Collection

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Fig2: Relationship of extracellular matrix protein 1 level to breast cancer cell proliferation. (A) Each cell line was treated with 200 ng/ml recombinant human extracellular matrix protein 1 (rhECM1) or 5 μg/ml anti-ECM1 antibodies, and cells were counted with a hemocytometer over the course of 3 days (*P < 0.05, **P < 0.005). Cont, Control; shC, Control short-hairpin RNA; shE/shECM1, Short-hairpin extracellular matrix protein 1 RNA; TR, Trastuzumab-resistant; Vec, Vector. (B) Cell cycles were analyzed by flow cytometry (*P < 0.05, **P < 0.005). (C) Each cell line was passaged by subcutaneous injection into the lower flank of each mouse. Tumor volume was measured every 3 days (n = 5 or 6 for each group). (D) Kaplan-Meier analyses for recurrence-free survival, overall survival and distant metastasis-free survival in the HER2-positive breast cancer patients. Data were obtained from the Kaplan-Meier Plotter Breast Cancer database [24]. Overall survival data from breast cancer patients were stratified by ECM1 levels in HER2-positive patient sera (n = 27).
Mentions: As BT-474 TR cells had a rapid proliferation ratio compared with parental BT-474 cells (Additional file 2: Figures S1C and S1D), we hypothesized that ECM1 can promote cell proliferation. To test the direct effect of ECM1 on cell proliferation, we treated BT-474 and MCF-7 cells with rhECM1. As determined by cell counting, this treatment increased the cell proliferation rate in these breast cancer cell lines (Figure 2A). To further test this effect, we overexpressed ECM1 in the BT-474 and MCF-7 cells and silenced ECM1 in BT-474 TR cells. Relative protein levels confirmed stable ECM1 expression (Additional file 3: Figure S2A), and this overexpression of ECM1 enhanced cell proliferation in the cell lines (Figure 2A, bottom). The knockdown of ECM1 by treatment with shRNA and anti-ECM1 antibodies in BT-474 TR cells reduced cell proliferation (Figure 2A, right). The anti-ECM1 antibodies also inhibited cell proliferation in other breast cancer cell lines (Additional file 3: Figure S2B). The MDA-MB-231 cell line, which overexpresses ECM1, showed especially high sensitivity to anti-ECM1 antibodies. To explore the mechanism for the cell-proliferative effect of ECM1, we analyzed the interrelationship of ECM1 expression with cell cycle progression. Cells overexpressing ECM1 showed an increased fraction of cells in S phase with a corresponding reduction of the fraction in G1 phase. Similarly, ECM1 knockdown led to the accumulation of cells in G1 phase with a decrease in the S-phase fraction (Figure 2B). Thus, ECM1 may positively regulate cell proliferation by affecting cell cycle progression. To observe the effect of ECM1 on cell proliferation in vivo, we tested the relationship of ECM1 expression to xenograft tumor formation in BALB/c nude mice. In BT-474 cells overexpressing ECM1, tumor formation efficiency was significantly increased, whereas tumorigenic potential of the BT-474 TR cells was significantly decreased after ECM1 silencing (Figure 2C). Moreover, the Kaplan-Meier plots [24] constructed using datasets from HER2-positive breast cancer patients demonstrated a significant association of ECM1 expression with poor outcome. Increasing ECM1 protein level predicted shorter distant metastasis-free survival in HER2-positive breast cancer patients (Figure 2D, left). Indeed, we found that high levels of ECM1 in serum were directly and inversely related to overall survival in HER2-positive breast cancer patients (Figure 2D, right). These data suggested that ECM1 overexpression in patients may contribute to bad outcomes in human breast cancer.Figure 2

Bottom Line: We examined the effect of ECM1 on cell proliferation and cell signaling in vitro and in vivo.Through physical interaction with epidermal growth factor receptor (EGFR), ECM1 potentiated the phosphorylation of EGFR and extracellular signal-regulated kinase upon EGF treatment.Moreover, ECM1-induced galectin-3 cleavage through upregulation of matrix metalloproteinase 9 not only improved mucin 1 expression, but also increased EGFR and human epidermal growth factor receptor 3 protein stability as a secondary signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Hanyang University, 222 Wangshimni-ro, Seoul, 133-791, Republic of Korea. realwooma@gmail.com.

ABSTRACT

Introduction: Extracellular matrix protein 1 (ECM1) is a secreted glycoprotein with putative functions in cell proliferation, angiogenesis and differentiation. Expression of ECM1 in several types of carcinoma suggests that it may promote tumor development. In this study, we investigated the role of ECM1 in oncogenic cell signaling in breast cancer, and potential mechanisms for its effects.

Methods: In order to find out the functional role of ECM1, we used the recombinant human ECM1 and viral transduction systems which stably regulated the expression level of ECM1. We examined the effect of ECM1 on cell proliferation and cell signaling in vitro and in vivo. Moreover, tissues and sera of patients with breast cancer were used to confirm the effect of ECM1.

Results: ECM1 protein was increased in trastuzumab-resistant (TR) cells, in association with trastuzumab resistance and cell proliferation. Through physical interaction with epidermal growth factor receptor (EGFR), ECM1 potentiated the phosphorylation of EGFR and extracellular signal-regulated kinase upon EGF treatment. Moreover, ECM1-induced galectin-3 cleavage through upregulation of matrix metalloproteinase 9 not only improved mucin 1 expression, but also increased EGFR and human epidermal growth factor receptor 3 protein stability as a secondary signaling.

Conclusions: ECM1 has important roles in both cancer development and trastuzumab resistance in breast cancer through activation of EGFR signaling.

Show MeSH
Related in: MedlinePlus