AACR Conference on Proteases, Extracellular Matrix, and Cancer
AACR Conference on Proteases, Extracellular Matrix, and Cancer
Much of cancer research revolves around understanding the function of oncogenes and tumor suppressor gene products. These types of studies have revealed important information about the signaling pathways that control normal cell activities such as proliferation and apoptosis and about how genetic defects in the factors that control these processes can lead to tumor formation and progression. It has become clear, however, that defects in single genes, or even the sequential acquisition of mutations, are not the entire story in tumorigenesis, given that many cancer susceptibility genes show a high degree of tissue specificity in their association with neoplastic transformation. Mina Bissell of the Berkeley National Laboratory in California has been a pioneer in studying how the tissue context can support or prevent tumorigenesis of mutated cells.
A considerable body of evidence now indicates that cell-cell interactions, and cell interactions with the extracellular matrix (ECM), are essential organizing principles that help define the nature of the tissue context, and these interactions play important roles in regulating homeostasis and tissue specificity.
The ECM is a complex structural entity that surrounds and supports cells within mammalian tissues. It is composed of 3 major classes of biomolecules: structural proteins (such as collagen and elastin), specialized proteins (such as fibrillin, fibronectin, and laminin), and proteoglycans (protein cores that are attached to long chains of repeating disaccharide units termed glycosaminoglycans). The ECM has recently received considerable attention because of its importance in cell-cell signaling, wound repair, cell adhesion, and tissue function.
Polarization of Mammary Acini. Bissell has developed several systems for studying mechanisms of tissue organization and the way in which disruption of tissue integrity leads to malignancy. One highly complex tissue that she has studied is the mammary acinus. This 3-dimensional structure consists of luminal epithelial cells surrounded by a layer of myoepithelial cells. All these cells are surrounded by a specialized type of ECM called the basement membrane. Basement membranes are thin, extracellular, sheetlike structures that compartmentalize tissues. They are found beneath epithelia, endothelia, and surrounding individual cells, such as muscle fibers, neurons, and adipocytes. Laminin, type IV collagens, perlecan, and nidogens are some of the main components of mature basement membranes.
Tissue-specific signaling occurs bidirectionally between the cells of the mammary acinus and depends on the intact 3-dimensional structure of this tissue. The signals that determine the correct polarity of breast epithelial structures in vivo are not understood. Bissell has been studying the mammary acinus both in vivo in mice and in 3-dimensional cultures to learn how disruption of tissue organization can lead to tumor development.
When luminal epithelial cells are isolated from mouse acini and cultured on basement membrane proteins, a 3-dimensional structure forms in which luminal cells are on the inside, surrounded by basement membrane. Bissell found that culturing human primary luminal epithelial cells within collagen I gels led to formation of structures with no lumina, no basement membrane deposition, and reverse polarity. Addition of purified human myoepithelial cells isolated from normal glands corrected the inverse polarity and led to formation of double-layered acini with central lumina. Therefore, myoepithelial cells must receive some signal from collagen I that mediates proper tissue organization.
Laminin 1 Determines Polarity in Breast Cells. Bissell showed that myoepithelial cells produced a protein called laminin 1 that regulates polarity. In fact, laminin 1 was able to substitute for myoepithelial cells in reversing polarity. Furthermore, myoepithelial cells isolated from breast tumor samples did not express laminin 1 and either lacked the ability to interact with luminal epithelial cells or conveyed only correction of polarity in a fraction of acini. Breast carcinomas either were negative for laminin 1 (7/12 biopsies) or showed a focal, fragmented deposition of a less intensely stained basement membrane (5/12 biopsies). Bissell concluded that (1) myoepithelial cells induce polarity because they are the only source of laminin 1 in the breast and (2) tumor-derived myoepithelial cells are deficient in their ability to induce polarity because they no longer produce functional laminin 1.
Stromelysin, Genomic Instability, and Tumorigenesis. Proteinases such as matrix metalloproteinases (MMPs) are also important factors in the regulation of the structure of the ECM and surrounding tissue. Defects in MMP activity have been shown to alter tissue structure and promote malignancy. MMPs are also involved in regulating structure in mammary tissue. These enzymes have been shown to be involved in mammary gland involution after lactation. Stromelysin 1 (also known as MMP-3), in conjunction with the growth factor KGF, induces branching in mammary glands isolated from mice, whereas MMP inhibitors known as tissue inhibitors of metalloproteinases (TIMPs) suppress the branching morphogenesis.
Bissell showed that recombinant stromelysin 1 induced branching morphogenesis in cultured mammary epithelial cells. Transgenic expression of stromelysin 1, however, induced tumor formation in mice. Analysis of these tumors using comparative genomic hybridization (a molecular cytogenetic method of screening a tumor for genetic changes) indicated that the genomes of these cells were highly unstable. Tumor formation was suppressed by coexpression of the TIMP-1 transgene. Thus, by altering the cellular microenvironment, stromelysin 1 can induce genomic instability and promote tumorigenesis. So can restoration of tissue structure overcome genomic instability and malignancy?
Integrins, Polarity, and Apoptosis. Bissell used the HMT-3522 breast cancer progression series to study the effects of tissue organization on tumor development. These cells are mammary cells that have been isolated at different stages of tumor progression -- nonmalignant, premalignant, and malignant stages. Bissell has associated overexpression of beta1 integrin, a receptor for ECM components, with the late stages of tumor progression. When a blocking antibody was used to disrupt beta1 integrin signaling in cultured malignant HMT-3522 cells, the cells reorganized into normal tissue-like acini. Surprisingly, blocking beta1 integrin signaling also down-regulated expression of the epidermal growth factor (EGF) receptor, indicating that there might be crosstalk between these 2 receptors.
Both these receptors are known to signal via the mitogen-activated protein kinase pathway, and Bissell showed that antibody-mediated inhibition of either of these receptors in the tumor cells, or inhibition of mitogen-activated protein kinase, induced a concomitant down-regulation of both receptors, followed by growth arrest and restoration of normal breast tissue morphogenesis. Furthermore, Bissell showed that when signaling through the EGF receptors ERB1 and ERB2 is activated in 2-dimensional cultures, cells become tumorigenic, but when they are activated in 3-dimensional cultures, nothing happens. Therefore, tissue structure is central to preventing tumor formation.
Finally, Bissell is also investigating the molecular mechanism whereby malignant and nonmalignant mammary epithelial cells become insensitive to apoptosis. Bissell treated mammary acini with pro-apoptotic agents such as TRAIL (tumor necrosis-related apoptosis-inducing ligand) and etoposide and found that only nonmalignant cells that were in the proper tissue conformation died; disorganized cells were resistant to death induction by these factors.
She showed that resistance to apoptosis requires ligation of beta4 integrin, which regulates tissue polarity, hemidesmosome formation, and nuclear factor-kappa B (NF-kappa B) activation. Expression of a mutant form of beta4 integrin that lacks the hemidesmosome targeting domain interferes with tissue polarity and NF-kappa B activation and permits apoptosis. These results indicate that integrin-induced polarity mediates resistance to apoptosis-inducing agents via effects on NF-kappa B.
Much of cancer research revolves around understanding the function of oncogenes and tumor suppressor gene products. These types of studies have revealed important information about the signaling pathways that control normal cell activities such as proliferation and apoptosis and about how genetic defects in the factors that control these processes can lead to tumor formation and progression. It has become clear, however, that defects in single genes, or even the sequential acquisition of mutations, are not the entire story in tumorigenesis, given that many cancer susceptibility genes show a high degree of tissue specificity in their association with neoplastic transformation. Mina Bissell of the Berkeley National Laboratory in California has been a pioneer in studying how the tissue context can support or prevent tumorigenesis of mutated cells.
A considerable body of evidence now indicates that cell-cell interactions, and cell interactions with the extracellular matrix (ECM), are essential organizing principles that help define the nature of the tissue context, and these interactions play important roles in regulating homeostasis and tissue specificity.
The ECM is a complex structural entity that surrounds and supports cells within mammalian tissues. It is composed of 3 major classes of biomolecules: structural proteins (such as collagen and elastin), specialized proteins (such as fibrillin, fibronectin, and laminin), and proteoglycans (protein cores that are attached to long chains of repeating disaccharide units termed glycosaminoglycans). The ECM has recently received considerable attention because of its importance in cell-cell signaling, wound repair, cell adhesion, and tissue function.
Polarization of Mammary Acini. Bissell has developed several systems for studying mechanisms of tissue organization and the way in which disruption of tissue integrity leads to malignancy. One highly complex tissue that she has studied is the mammary acinus. This 3-dimensional structure consists of luminal epithelial cells surrounded by a layer of myoepithelial cells. All these cells are surrounded by a specialized type of ECM called the basement membrane. Basement membranes are thin, extracellular, sheetlike structures that compartmentalize tissues. They are found beneath epithelia, endothelia, and surrounding individual cells, such as muscle fibers, neurons, and adipocytes. Laminin, type IV collagens, perlecan, and nidogens are some of the main components of mature basement membranes.
Tissue-specific signaling occurs bidirectionally between the cells of the mammary acinus and depends on the intact 3-dimensional structure of this tissue. The signals that determine the correct polarity of breast epithelial structures in vivo are not understood. Bissell has been studying the mammary acinus both in vivo in mice and in 3-dimensional cultures to learn how disruption of tissue organization can lead to tumor development.
When luminal epithelial cells are isolated from mouse acini and cultured on basement membrane proteins, a 3-dimensional structure forms in which luminal cells are on the inside, surrounded by basement membrane. Bissell found that culturing human primary luminal epithelial cells within collagen I gels led to formation of structures with no lumina, no basement membrane deposition, and reverse polarity. Addition of purified human myoepithelial cells isolated from normal glands corrected the inverse polarity and led to formation of double-layered acini with central lumina. Therefore, myoepithelial cells must receive some signal from collagen I that mediates proper tissue organization.
Laminin 1 Determines Polarity in Breast Cells. Bissell showed that myoepithelial cells produced a protein called laminin 1 that regulates polarity. In fact, laminin 1 was able to substitute for myoepithelial cells in reversing polarity. Furthermore, myoepithelial cells isolated from breast tumor samples did not express laminin 1 and either lacked the ability to interact with luminal epithelial cells or conveyed only correction of polarity in a fraction of acini. Breast carcinomas either were negative for laminin 1 (7/12 biopsies) or showed a focal, fragmented deposition of a less intensely stained basement membrane (5/12 biopsies). Bissell concluded that (1) myoepithelial cells induce polarity because they are the only source of laminin 1 in the breast and (2) tumor-derived myoepithelial cells are deficient in their ability to induce polarity because they no longer produce functional laminin 1.
Stromelysin, Genomic Instability, and Tumorigenesis. Proteinases such as matrix metalloproteinases (MMPs) are also important factors in the regulation of the structure of the ECM and surrounding tissue. Defects in MMP activity have been shown to alter tissue structure and promote malignancy. MMPs are also involved in regulating structure in mammary tissue. These enzymes have been shown to be involved in mammary gland involution after lactation. Stromelysin 1 (also known as MMP-3), in conjunction with the growth factor KGF, induces branching in mammary glands isolated from mice, whereas MMP inhibitors known as tissue inhibitors of metalloproteinases (TIMPs) suppress the branching morphogenesis.
Bissell showed that recombinant stromelysin 1 induced branching morphogenesis in cultured mammary epithelial cells. Transgenic expression of stromelysin 1, however, induced tumor formation in mice. Analysis of these tumors using comparative genomic hybridization (a molecular cytogenetic method of screening a tumor for genetic changes) indicated that the genomes of these cells were highly unstable. Tumor formation was suppressed by coexpression of the TIMP-1 transgene. Thus, by altering the cellular microenvironment, stromelysin 1 can induce genomic instability and promote tumorigenesis. So can restoration of tissue structure overcome genomic instability and malignancy?
Integrins, Polarity, and Apoptosis. Bissell used the HMT-3522 breast cancer progression series to study the effects of tissue organization on tumor development. These cells are mammary cells that have been isolated at different stages of tumor progression -- nonmalignant, premalignant, and malignant stages. Bissell has associated overexpression of beta1 integrin, a receptor for ECM components, with the late stages of tumor progression. When a blocking antibody was used to disrupt beta1 integrin signaling in cultured malignant HMT-3522 cells, the cells reorganized into normal tissue-like acini. Surprisingly, blocking beta1 integrin signaling also down-regulated expression of the epidermal growth factor (EGF) receptor, indicating that there might be crosstalk between these 2 receptors.
Both these receptors are known to signal via the mitogen-activated protein kinase pathway, and Bissell showed that antibody-mediated inhibition of either of these receptors in the tumor cells, or inhibition of mitogen-activated protein kinase, induced a concomitant down-regulation of both receptors, followed by growth arrest and restoration of normal breast tissue morphogenesis. Furthermore, Bissell showed that when signaling through the EGF receptors ERB1 and ERB2 is activated in 2-dimensional cultures, cells become tumorigenic, but when they are activated in 3-dimensional cultures, nothing happens. Therefore, tissue structure is central to preventing tumor formation.
Finally, Bissell is also investigating the molecular mechanism whereby malignant and nonmalignant mammary epithelial cells become insensitive to apoptosis. Bissell treated mammary acini with pro-apoptotic agents such as TRAIL (tumor necrosis-related apoptosis-inducing ligand) and etoposide and found that only nonmalignant cells that were in the proper tissue conformation died; disorganized cells were resistant to death induction by these factors.
She showed that resistance to apoptosis requires ligation of beta4 integrin, which regulates tissue polarity, hemidesmosome formation, and nuclear factor-kappa B (NF-kappa B) activation. Expression of a mutant form of beta4 integrin that lacks the hemidesmosome targeting domain interferes with tissue polarity and NF-kappa B activation and permits apoptosis. These results indicate that integrin-induced polarity mediates resistance to apoptosis-inducing agents via effects on NF-kappa B.
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