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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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Related in: MedlinePlus

Extracellular matrix protein 1 activates extracellular signal-regulated kinase signaling by upregulating epidermal growth factor receptor and HER3. (A) At 24 hours after cell seeding, each cell line was treated with recombinant human extracellular matrix protein 1 (rhECM1; 200 ng/ml) or anti-ECM1 antibodies (5 μg/ml) and further incubated for 48 hours. Cells lysates were then analyzed by Western blotting. Cont, Control; ERK, Extracellular signal-regulated kinase; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1 short-hairpin RNA; TR, Trastuzumab-resistant; Vec, Vector; WT, Wild type. (B) Epidermal growth factor receptor (EGFR) and HER3 mRNA levels were determined by real-time PCR using primers specific for EGFR and HER3 (**P < 0.005, ***P < 0.0005). (C) Each cell line was treated with 100 μg/ml cycloheximide (CHX). Cell lysates were prepared at the indicated time points and analyzed on Western blots. Band intensities on the blots were quantified using 1DScan software (Scanalytics, Milwaukee, WI) and plotted versus time as the ratios of EGFR/actin and HER3/actin intensities (*P < 0.05).
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Fig4: Extracellular matrix protein 1 activates extracellular signal-regulated kinase signaling by upregulating epidermal growth factor receptor and HER3. (A) At 24 hours after cell seeding, each cell line was treated with recombinant human extracellular matrix protein 1 (rhECM1; 200 ng/ml) or anti-ECM1 antibodies (5 μg/ml) and further incubated for 48 hours. Cells lysates were then analyzed by Western blotting. Cont, Control; ERK, Extracellular signal-regulated kinase; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1 short-hairpin RNA; TR, Trastuzumab-resistant; Vec, Vector; WT, Wild type. (B) Epidermal growth factor receptor (EGFR) and HER3 mRNA levels were determined by real-time PCR using primers specific for EGFR and HER3 (**P < 0.005, ***P < 0.0005). (C) Each cell line was treated with 100 μg/ml cycloheximide (CHX). Cell lysates were prepared at the indicated time points and analyzed on Western blots. Band intensities on the blots were quantified using 1DScan software (Scanalytics, Milwaukee, WI) and plotted versus time as the ratios of EGFR/actin and HER3/actin intensities (*P < 0.05).

Mentions: Although we previously showed that ECM1 enhanced EGF-dependent activation of EGFR (Figure 3), we needed to check the effect of ECM1 on EGFR and HER3 expression because we found that EGFR and HER3 expression were increased in BT-474 TR cells compared with parental BT-474 cells (Additional file 3: Figure S2C). We first treated BT-474 and MCF-7 cells with rhECM1 for 48 hours (Figure 4A, right) and induced stable overexpression of ECM1 in the cells (Figure 4A, left). These treatments increased levels of EGFR, HER3 and phospho-ERK, proteins that were also upregulated in BT-474 TR cells. Conversely, knockdown of ECM1 in BT-474 TR cells reduced phospho-ERK levels. Consistent with these findings, treatment with anti-ECM1 antibodies (two different clones: N17 and P19) for 48 hours reduced expression of EGFR, HER3 and phospho-ERK in BT-474 TR cells (Figure 4A, middle).Figure 4


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)

Extracellular matrix protein 1 activates extracellular signal-regulated kinase signaling by upregulating epidermal growth factor receptor and HER3. (A) At 24 hours after cell seeding, each cell line was treated with recombinant human extracellular matrix protein 1 (rhECM1; 200 ng/ml) or anti-ECM1 antibodies (5 μg/ml) and further incubated for 48 hours. Cells lysates were then analyzed by Western blotting. Cont, Control; ERK, Extracellular signal-regulated kinase; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1 short-hairpin RNA; TR, Trastuzumab-resistant; Vec, Vector; WT, Wild type. (B) Epidermal growth factor receptor (EGFR) and HER3 mRNA levels were determined by real-time PCR using primers specific for EGFR and HER3 (**P < 0.005, ***P < 0.0005). (C) Each cell line was treated with 100 μg/ml cycloheximide (CHX). Cell lysates were prepared at the indicated time points and analyzed on Western blots. Band intensities on the blots were quantified using 1DScan software (Scanalytics, Milwaukee, WI) and plotted versus time as the ratios of EGFR/actin and HER3/actin intensities (*P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4308848&req=5

Fig4: Extracellular matrix protein 1 activates extracellular signal-regulated kinase signaling by upregulating epidermal growth factor receptor and HER3. (A) At 24 hours after cell seeding, each cell line was treated with recombinant human extracellular matrix protein 1 (rhECM1; 200 ng/ml) or anti-ECM1 antibodies (5 μg/ml) and further incubated for 48 hours. Cells lysates were then analyzed by Western blotting. Cont, Control; ERK, Extracellular signal-regulated kinase; shC, Control short-hairpin RNA; shE, Extracellular matrix protein 1 short-hairpin RNA; TR, Trastuzumab-resistant; Vec, Vector; WT, Wild type. (B) Epidermal growth factor receptor (EGFR) and HER3 mRNA levels were determined by real-time PCR using primers specific for EGFR and HER3 (**P < 0.005, ***P < 0.0005). (C) Each cell line was treated with 100 μg/ml cycloheximide (CHX). Cell lysates were prepared at the indicated time points and analyzed on Western blots. Band intensities on the blots were quantified using 1DScan software (Scanalytics, Milwaukee, WI) and plotted versus time as the ratios of EGFR/actin and HER3/actin intensities (*P < 0.05).
Mentions: Although we previously showed that ECM1 enhanced EGF-dependent activation of EGFR (Figure 3), we needed to check the effect of ECM1 on EGFR and HER3 expression because we found that EGFR and HER3 expression were increased in BT-474 TR cells compared with parental BT-474 cells (Additional file 3: Figure S2C). We first treated BT-474 and MCF-7 cells with rhECM1 for 48 hours (Figure 4A, right) and induced stable overexpression of ECM1 in the cells (Figure 4A, left). These treatments increased levels of EGFR, HER3 and phospho-ERK, proteins that were also upregulated in BT-474 TR cells. Conversely, knockdown of ECM1 in BT-474 TR cells reduced phospho-ERK levels. Consistent with these findings, treatment with anti-ECM1 antibodies (two different clones: N17 and P19) for 48 hours reduced expression of EGFR, HER3 and phospho-ERK in BT-474 TR cells (Figure 4A, middle).Figure 4

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