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The role of MMP-1 in breast cancer growth and metastasis to the brain in a xenograft model.

Liu H, Kato Y, Erzinger SA, Kiriakova GM, Qian Y, Palmieri D, Steeg PS, Price JE - BMC Cancer (2012)

Bottom Line: Reduction of MMP-1 expression significantly attenuated brain metastasis and lung metastasis formation following injection of cells into the left ventricle of the heart and tail vein, respectively.There were significantly fewer proliferating cells in brain metastases of cells with reduced MMP-1 expression.Furthermore, reduced MMP-1 expression was associated with decreased TGFα release and phospho-EGFR expression in 231-BR and BR3 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Biology, The University of Texas, MD, Anderson Cancer Center, Houston, TX 77030, USA. hliu5@mdanderson.org

ABSTRACT

Background: Brain metastasis is an increasingly common complication for breast cancer patients; approximately 15- 30% of breast cancer patients develop brain metastasis. However, relatively little is known about how these metastases form, and what phenotypes are characteristic of cells with brain metastasizing potential. In this study, we show that the targeted knockdown of MMP-1 in breast cancer cells with enhanced brain metastatic ability not only reduced primary tumor growth, but also significantly inhibited brain metastasis.

Methods: Two variants of the MDA-MB-231 human breast cancer cell line selected for enhanced ability to form brain metastases in nude mice (231-BR and 231-BR3 cells) were found to express high levels of matrix metalloproteinase-1 (MMP-1). Short hairpin RNA-mediated stable knockdown of MMP-1 in 231-BR and 231-BR3 cells were established to analyze tumorigenic ability and metastatic ability.

Results: Short hairpin RNA-mediated stable knockdown of MMP-1 inhibited the invasive ability of MDA-MB 231 variant cells in vitro, and inhibited breast cancer growth when the cells were injected into the mammary fat pad of nude mice. Reduction of MMP-1 expression significantly attenuated brain metastasis and lung metastasis formation following injection of cells into the left ventricle of the heart and tail vein, respectively. There were significantly fewer proliferating cells in brain metastases of cells with reduced MMP-1 expression. Furthermore, reduced MMP-1 expression was associated with decreased TGFα release and phospho-EGFR expression in 231-BR and BR3 cells.

Conclusions: Our results show that elevated expression of MMP-1 can promote the local growth and the formation of brain metastases by breast cancer cells.

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MMP-1 shRNAs specifically inhibit MMP-1 expression in BR3 and BR cells. A, real-time PCR quantification of MMP-1 mRNA levels. Compared with MDA-MB-231 parental cells and variants LC3 (selected from experimental lung metastases), the brain metastasis-derived BR3 and BR cells showed high levels of MMP-1 expression. This data shown are representative of 3 independent experiments. B, MMP-1 shRNAs knocked down MMP-1 mRNA level in BR3 and BR cell lines, compared with shCtr and shNTC cells expressing control shRNA. This experiment was performed in triplicate. C, ELISA assays and D, western blotting showed that MMP-1 protein levels in conditioned media were significantly reduced in BR3 and BR expressing MMP-1 targeting shRNA. These experiments were performed in triplicate. E, MMP-1 shRNA did not affect the expression of MMP-2, TIMP-1, TIMP-2 and VEGF in sh1 cells. This data shown are representative of 3 independent experiments.
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Figure 1: MMP-1 shRNAs specifically inhibit MMP-1 expression in BR3 and BR cells. A, real-time PCR quantification of MMP-1 mRNA levels. Compared with MDA-MB-231 parental cells and variants LC3 (selected from experimental lung metastases), the brain metastasis-derived BR3 and BR cells showed high levels of MMP-1 expression. This data shown are representative of 3 independent experiments. B, MMP-1 shRNAs knocked down MMP-1 mRNA level in BR3 and BR cell lines, compared with shCtr and shNTC cells expressing control shRNA. This experiment was performed in triplicate. C, ELISA assays and D, western blotting showed that MMP-1 protein levels in conditioned media were significantly reduced in BR3 and BR expressing MMP-1 targeting shRNA. These experiments were performed in triplicate. E, MMP-1 shRNA did not affect the expression of MMP-2, TIMP-1, TIMP-2 and VEGF in sh1 cells. This data shown are representative of 3 independent experiments.

Mentions: Two variants of the MDA-MB-231 breast cancer cell line, 231-BR and 231-BR3, were established independently by two research groups, and have been shown to have enhanced brain-metastasizing potential [6,7]. Microarray analyses were performed by the Steeg laboratory to identify common differentially expressed genes; altered expression of 26 genes was seen in both brain metastasis-derived variants compared with the parental cell line. Of these, MMP-1 was the most highly expressed gene. The expression of MMP-1 gene in 231-BR cells increased 89-fold and in 231-BR3 cells increased 36-fold compared with parental MDA-MB-231 cells (data not shown). The increased expression was confirmed using real time PCR measurements (Figure 1A). Included in the comparison was a variant selected from experimental lung metastases (231-LC3 [6]), which did not express increased MMP-1; this suggested that the increase in expression is not a consequence of selection of cells from xenografted tumors in general, but may be linked to the formation of experimental brain metastases.


The role of MMP-1 in breast cancer growth and metastasis to the brain in a xenograft model.

Liu H, Kato Y, Erzinger SA, Kiriakova GM, Qian Y, Palmieri D, Steeg PS, Price JE - BMC Cancer (2012)

MMP-1 shRNAs specifically inhibit MMP-1 expression in BR3 and BR cells. A, real-time PCR quantification of MMP-1 mRNA levels. Compared with MDA-MB-231 parental cells and variants LC3 (selected from experimental lung metastases), the brain metastasis-derived BR3 and BR cells showed high levels of MMP-1 expression. This data shown are representative of 3 independent experiments. B, MMP-1 shRNAs knocked down MMP-1 mRNA level in BR3 and BR cell lines, compared with shCtr and shNTC cells expressing control shRNA. This experiment was performed in triplicate. C, ELISA assays and D, western blotting showed that MMP-1 protein levels in conditioned media were significantly reduced in BR3 and BR expressing MMP-1 targeting shRNA. These experiments were performed in triplicate. E, MMP-1 shRNA did not affect the expression of MMP-2, TIMP-1, TIMP-2 and VEGF in sh1 cells. This data shown are representative of 3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3526403&req=5

Figure 1: MMP-1 shRNAs specifically inhibit MMP-1 expression in BR3 and BR cells. A, real-time PCR quantification of MMP-1 mRNA levels. Compared with MDA-MB-231 parental cells and variants LC3 (selected from experimental lung metastases), the brain metastasis-derived BR3 and BR cells showed high levels of MMP-1 expression. This data shown are representative of 3 independent experiments. B, MMP-1 shRNAs knocked down MMP-1 mRNA level in BR3 and BR cell lines, compared with shCtr and shNTC cells expressing control shRNA. This experiment was performed in triplicate. C, ELISA assays and D, western blotting showed that MMP-1 protein levels in conditioned media were significantly reduced in BR3 and BR expressing MMP-1 targeting shRNA. These experiments were performed in triplicate. E, MMP-1 shRNA did not affect the expression of MMP-2, TIMP-1, TIMP-2 and VEGF in sh1 cells. This data shown are representative of 3 independent experiments.
Mentions: Two variants of the MDA-MB-231 breast cancer cell line, 231-BR and 231-BR3, were established independently by two research groups, and have been shown to have enhanced brain-metastasizing potential [6,7]. Microarray analyses were performed by the Steeg laboratory to identify common differentially expressed genes; altered expression of 26 genes was seen in both brain metastasis-derived variants compared with the parental cell line. Of these, MMP-1 was the most highly expressed gene. The expression of MMP-1 gene in 231-BR cells increased 89-fold and in 231-BR3 cells increased 36-fold compared with parental MDA-MB-231 cells (data not shown). The increased expression was confirmed using real time PCR measurements (Figure 1A). Included in the comparison was a variant selected from experimental lung metastases (231-LC3 [6]), which did not express increased MMP-1; this suggested that the increase in expression is not a consequence of selection of cells from xenografted tumors in general, but may be linked to the formation of experimental brain metastases.

Bottom Line: Reduction of MMP-1 expression significantly attenuated brain metastasis and lung metastasis formation following injection of cells into the left ventricle of the heart and tail vein, respectively.There were significantly fewer proliferating cells in brain metastases of cells with reduced MMP-1 expression.Furthermore, reduced MMP-1 expression was associated with decreased TGFα release and phospho-EGFR expression in 231-BR and BR3 cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Biology, The University of Texas, MD, Anderson Cancer Center, Houston, TX 77030, USA. hliu5@mdanderson.org

ABSTRACT

Background: Brain metastasis is an increasingly common complication for breast cancer patients; approximately 15- 30% of breast cancer patients develop brain metastasis. However, relatively little is known about how these metastases form, and what phenotypes are characteristic of cells with brain metastasizing potential. In this study, we show that the targeted knockdown of MMP-1 in breast cancer cells with enhanced brain metastatic ability not only reduced primary tumor growth, but also significantly inhibited brain metastasis.

Methods: Two variants of the MDA-MB-231 human breast cancer cell line selected for enhanced ability to form brain metastases in nude mice (231-BR and 231-BR3 cells) were found to express high levels of matrix metalloproteinase-1 (MMP-1). Short hairpin RNA-mediated stable knockdown of MMP-1 in 231-BR and 231-BR3 cells were established to analyze tumorigenic ability and metastatic ability.

Results: Short hairpin RNA-mediated stable knockdown of MMP-1 inhibited the invasive ability of MDA-MB 231 variant cells in vitro, and inhibited breast cancer growth when the cells were injected into the mammary fat pad of nude mice. Reduction of MMP-1 expression significantly attenuated brain metastasis and lung metastasis formation following injection of cells into the left ventricle of the heart and tail vein, respectively. There were significantly fewer proliferating cells in brain metastases of cells with reduced MMP-1 expression. Furthermore, reduced MMP-1 expression was associated with decreased TGFα release and phospho-EGFR expression in 231-BR and BR3 cells.

Conclusions: Our results show that elevated expression of MMP-1 can promote the local growth and the formation of brain metastases by breast cancer cells.

Show MeSH
Related in: MedlinePlus