Analysis of Metastasis in a Polyomavirus Middle T Mouse Model
Analysis of Metastasis in a Polyomavirus Middle T Mouse Model
Introduction: In order to study metastatic disease, we employed the use of two related polyomavirus middle T transgenic mouse tumor transplant models of mammary carcinoma (termed Met and Db) that display significant differences in metastatic potential.
Methods: Through suppression subtractive hybridization coupled to the microarray, we found osteopontin (OPN) to be a highly expressed gene in the tumors of the metastatic mouse model, and a lowly expressed gene in the tumors of the lowly metastatic mouse model. We further analyzed the role of OPN in this model by examining sense and antisense constructs using in vitro and in vivo methods.
Results: With in vivo metastasis assays, the antisense Met cells showed no metastatic tumor formation to the lungs of recipient mice, while wild-type Met cells, with higher levels of OPN, showed significant amounts of metastasis. The Db cells showed a significantly reduced metastasis rate in the in vivo metastasis assay as compared with the Met cells. Db cells with enforced overexpression of OPN showed elevated levels of OPN but did not demonstrate an increase in the rate of metastasis compared with the wild-type Db cells.
Conclusions: We conclude that OPN is an essential regulator of the metastatic phenotype seen in polyomavirus middle T-induced mammary tumors. Yet OPN expression alone is not sufficient to cause metastasis. These data suggest a link between metastasis and phosphatidylinositol-3-kinase-mediated transcriptional upregulation of OPN, but additional phosphatidylinositol-3-kinase-regulated genes may be essential in precipitating the metastasis phenotype in the polyomavirus middle T model.
Breast cancer is among the most common human cancers, affecting one in every eight women and accounting for an estimated 192,000 cases and more than 40,000 deaths in the United States during 2001. One of the significant predictors of breast cancer prognosis is regional and distant metastasis; yet the mechanism of metastasis and the ability to predict it are far from being fully understood.
From both a clinical and experimental perspective, a more detailed understanding of the mechanisms of metastasis is needed in order to identify better diagnostic markers and therapeutic approaches.
In order to study breast cancer, many investigators have used human derived cell lines that have yielded significant insight into the biology of breast cancer; yet these models remain an artificial in vitro system that may not reflect the in vivo biology. Over the past decade, the laboratory mouse has become the modern vehicle for human disease studies, and genetically engineered mice are particularly popular models for breast cancer (reviewed in ). The mouse offers an in vivo experimental system that can be manipulated and studied in great detail in order to understand the complex biology of cancer.
To study metastatic disease, we have employed the use of two related polyomavirus (PyV) middle T (mT) transgenic mouse mammary carcinoma transplant lines (termed Met and Db) that display significant differences in metastatic potential. The PyV-mT system is an ideal model to study mammary carcinoma because there is rapid mammary tumor formation with 100% penetrance, because the histopathology of the PyV-mT tumors mimics that of human breast carcinoma, and because, in many cases, the human and mouse derived tumors are indistinguishable. The PyV-mT transgene has also been used as an alternative, or a surrogate, for erbB2 in the mouse as the two molecules activate similar pathways. Desai and colleagues have recently shown that mammary tumors derived from PyV-mT mice and from erbB2 transgenic mice show striking similarities at the transciptome level.
Over the past few years c-erbB2 (HER2) has been shown to be a key molecule in human breast cancer, being overexpressed in 3040% of human breast cancer cases. PyV is capable of transforming cells by triggering signal transduction pathways that have been implicated as activated by erbB2, through interactions between its mT gene product and key cellular signaling proteins such as c-Src, Shc, and phosphatidylinositol 3-kinase (PI3-K), which have all been implicated as important in human breast cancer. Specifically, with respect to PI3-K, mT interacts with the 85 kDa regulatory subunit of PI3-K to activate PI3-K, which has been implicated as a key signal in carcinoma invasion.
The Met model, derived from transgenic mice constructed with the wild-type PyV-mT line, develops rapid mammary carcinoma in all animals with 100% pulmonary metastasis. In contrast, the Db model derived from animals with double site-directed mutations at amino acid residues 315 and 322 of the PyV-mT is decoupled from the PI3-K pathway. The Db model has 100% penetrance of mammary tumor but exhibits significantly fewer pulmonary metastases (9%). Similar metastatic rates were observed when Met and Db tumor lines were transplanted into syngeneic FVB mice. The site-directed mutations at residues 315 and 322 interfere with the recruitment of the p85 subunit of PI3-K, and thereby PI3-K is not recruited and activated. This subtle difference in the mT gene significantly affects the metastatic phenotype.
Because disruption of the PI3-K pathway in this model suppresses metastasis, and because of the purported role of PI3-K in carcinoma cell invasion, we wanted to identify the key regulators that are differentially expressed between the Met tumors and the Db tumors, as well as to validate their role in metastasis. We utilized suppression subtractive hybridization (SSH) coupled to the microarray in order to initially identify targets. SSH is a technique that generates cDNA libraries of transcripts that are differentially expressed between two populations of cells, normalizing for variable mRNA abundance by enrichment of rare transcripts.
Using SSH in conjunction with microarrays, we identified osteopontin (OPN) as a very highly expressed gene that was expressed more than threefold higher in the Met tumor lines compared with the Db tumor lines. Immunohistochemical analysis of the tumors confirmed the presence of OPN with a marked cytoplasmic stain in the Met tumor cells and an absence of staining in the Db tumor cells. Cell lines were developed from these tumors, and they showed differential expression of OPN in similar proportions. Furthermore, OPN expression directly correlated with the ability of the tumor-derived cell lines to migrate in response to serum. Correspondingly, antisense-mediated attenuation of OPN expression reduced the migratory capacity of Met cells (in a cell line termed Met-As), while enforced OPN expression in Db cells stimulated migration (in a cell line termed Db-S). In in vivo metastasis experiments, the wild-type Met cells showed a high rate of metastasis to the lungs of recipient mice, whereas the Met-As cells did not metastasize to the lungs of recipient mice. In contrast, the Db cells manifested a metastasis rate that was significantly lower than that of wild-type Met cells. However, the Db-S cells, with elevated OPN levels, showed no increase in the metastasis rate when compared with the wild-type Db cells.
Scanning electron microscopy (SEM) of the OPN antisense transfected Met line shows morphologic change consistent with a less metastatic phenotype, while the OPN-transfected Db cells show morphologic change consistent with a more metastatic phenotype. Given this evidence, we conclude that PyV-mT induces a metastatic phenotype, at least in part, through the PI3-K-mediated transcriptional upregulation of the matrix protein OPN, although enforced expression of OPN in the lowly metastatic Db cells does not increase their metastasis rate. We therefore conclude that OPN is essential for metastasis in the PyV-mT system, but that elevated OPN expression alone is not sufficient to induce metastasis.
Introduction: In order to study metastatic disease, we employed the use of two related polyomavirus middle T transgenic mouse tumor transplant models of mammary carcinoma (termed Met and Db) that display significant differences in metastatic potential.
Methods: Through suppression subtractive hybridization coupled to the microarray, we found osteopontin (OPN) to be a highly expressed gene in the tumors of the metastatic mouse model, and a lowly expressed gene in the tumors of the lowly metastatic mouse model. We further analyzed the role of OPN in this model by examining sense and antisense constructs using in vitro and in vivo methods.
Results: With in vivo metastasis assays, the antisense Met cells showed no metastatic tumor formation to the lungs of recipient mice, while wild-type Met cells, with higher levels of OPN, showed significant amounts of metastasis. The Db cells showed a significantly reduced metastasis rate in the in vivo metastasis assay as compared with the Met cells. Db cells with enforced overexpression of OPN showed elevated levels of OPN but did not demonstrate an increase in the rate of metastasis compared with the wild-type Db cells.
Conclusions: We conclude that OPN is an essential regulator of the metastatic phenotype seen in polyomavirus middle T-induced mammary tumors. Yet OPN expression alone is not sufficient to cause metastasis. These data suggest a link between metastasis and phosphatidylinositol-3-kinase-mediated transcriptional upregulation of OPN, but additional phosphatidylinositol-3-kinase-regulated genes may be essential in precipitating the metastasis phenotype in the polyomavirus middle T model.
Breast cancer is among the most common human cancers, affecting one in every eight women and accounting for an estimated 192,000 cases and more than 40,000 deaths in the United States during 2001. One of the significant predictors of breast cancer prognosis is regional and distant metastasis; yet the mechanism of metastasis and the ability to predict it are far from being fully understood.
From both a clinical and experimental perspective, a more detailed understanding of the mechanisms of metastasis is needed in order to identify better diagnostic markers and therapeutic approaches.
In order to study breast cancer, many investigators have used human derived cell lines that have yielded significant insight into the biology of breast cancer; yet these models remain an artificial in vitro system that may not reflect the in vivo biology. Over the past decade, the laboratory mouse has become the modern vehicle for human disease studies, and genetically engineered mice are particularly popular models for breast cancer (reviewed in ). The mouse offers an in vivo experimental system that can be manipulated and studied in great detail in order to understand the complex biology of cancer.
To study metastatic disease, we have employed the use of two related polyomavirus (PyV) middle T (mT) transgenic mouse mammary carcinoma transplant lines (termed Met and Db) that display significant differences in metastatic potential. The PyV-mT system is an ideal model to study mammary carcinoma because there is rapid mammary tumor formation with 100% penetrance, because the histopathology of the PyV-mT tumors mimics that of human breast carcinoma, and because, in many cases, the human and mouse derived tumors are indistinguishable. The PyV-mT transgene has also been used as an alternative, or a surrogate, for erbB2 in the mouse as the two molecules activate similar pathways. Desai and colleagues have recently shown that mammary tumors derived from PyV-mT mice and from erbB2 transgenic mice show striking similarities at the transciptome level.
Over the past few years c-erbB2 (HER2) has been shown to be a key molecule in human breast cancer, being overexpressed in 3040% of human breast cancer cases. PyV is capable of transforming cells by triggering signal transduction pathways that have been implicated as activated by erbB2, through interactions between its mT gene product and key cellular signaling proteins such as c-Src, Shc, and phosphatidylinositol 3-kinase (PI3-K), which have all been implicated as important in human breast cancer. Specifically, with respect to PI3-K, mT interacts with the 85 kDa regulatory subunit of PI3-K to activate PI3-K, which has been implicated as a key signal in carcinoma invasion.
The Met model, derived from transgenic mice constructed with the wild-type PyV-mT line, develops rapid mammary carcinoma in all animals with 100% pulmonary metastasis. In contrast, the Db model derived from animals with double site-directed mutations at amino acid residues 315 and 322 of the PyV-mT is decoupled from the PI3-K pathway. The Db model has 100% penetrance of mammary tumor but exhibits significantly fewer pulmonary metastases (9%). Similar metastatic rates were observed when Met and Db tumor lines were transplanted into syngeneic FVB mice. The site-directed mutations at residues 315 and 322 interfere with the recruitment of the p85 subunit of PI3-K, and thereby PI3-K is not recruited and activated. This subtle difference in the mT gene significantly affects the metastatic phenotype.
Because disruption of the PI3-K pathway in this model suppresses metastasis, and because of the purported role of PI3-K in carcinoma cell invasion, we wanted to identify the key regulators that are differentially expressed between the Met tumors and the Db tumors, as well as to validate their role in metastasis. We utilized suppression subtractive hybridization (SSH) coupled to the microarray in order to initially identify targets. SSH is a technique that generates cDNA libraries of transcripts that are differentially expressed between two populations of cells, normalizing for variable mRNA abundance by enrichment of rare transcripts.
Using SSH in conjunction with microarrays, we identified osteopontin (OPN) as a very highly expressed gene that was expressed more than threefold higher in the Met tumor lines compared with the Db tumor lines. Immunohistochemical analysis of the tumors confirmed the presence of OPN with a marked cytoplasmic stain in the Met tumor cells and an absence of staining in the Db tumor cells. Cell lines were developed from these tumors, and they showed differential expression of OPN in similar proportions. Furthermore, OPN expression directly correlated with the ability of the tumor-derived cell lines to migrate in response to serum. Correspondingly, antisense-mediated attenuation of OPN expression reduced the migratory capacity of Met cells (in a cell line termed Met-As), while enforced OPN expression in Db cells stimulated migration (in a cell line termed Db-S). In in vivo metastasis experiments, the wild-type Met cells showed a high rate of metastasis to the lungs of recipient mice, whereas the Met-As cells did not metastasize to the lungs of recipient mice. In contrast, the Db cells manifested a metastasis rate that was significantly lower than that of wild-type Met cells. However, the Db-S cells, with elevated OPN levels, showed no increase in the metastasis rate when compared with the wild-type Db cells.
Scanning electron microscopy (SEM) of the OPN antisense transfected Met line shows morphologic change consistent with a less metastatic phenotype, while the OPN-transfected Db cells show morphologic change consistent with a more metastatic phenotype. Given this evidence, we conclude that PyV-mT induces a metastatic phenotype, at least in part, through the PI3-K-mediated transcriptional upregulation of the matrix protein OPN, although enforced expression of OPN in the lowly metastatic Db cells does not increase their metastasis rate. We therefore conclude that OPN is essential for metastasis in the PyV-mT system, but that elevated OPN expression alone is not sufficient to induce metastasis.
