New Molecular Markers to Study Breast Cancer Progression
New Molecular Markers to Study Breast Cancer Progression
By Mary J.C. Hendrix, PhD, Elisabeth A. Seftor, and Dawn A. Kirschmann, PhD
The etiology of breast cancer tumorigenesis is currently unknown. However, it is thought to arise via a complex, multi-step process involving the accumulation of genetic alterations including amplification (e.g., c-myc and HER2/neu) and mutation or loss of tumor suppressor genes (e.g., p53 and maspin).1-3 Nevertheless, little is known about the function, molecular regulation, and interaction of genetic alterations in tumorigenesis, as well as the migration, invasion, and metastasis of breast carcinomas. In order to facilitate the discovery of genetic alterations, investigators have utilized subtractive hybridization and differential display methodologies to identify genes involved in breast cancer tumorigenesis.
Molecular Analysis of Breast Cancer
In several notable molecular studies, the experimental design consisted of identifying tumor suppressor genes and oncogenes by comparing gene expression in normal human mammary epithelial tissue or cell lines vs. breast carcinoma cell lines.4-13 These studies have identified stanniocalcin (regulator of calcium uptake),4 alpha 6 integrin,5 connexin 26 (gap junction protein),6,7 CaN 19 (S100 family of calcium-binding modulator proteins),6,8 glutathione-S-transferase p,6 maspin,9,10 a novel serine protease-like gene product,11 and a number of unknown genes,4,6,12 which were shown to be down-regulated in breast carcinoma cells by Northern blot analysis. In addition, several unknown genes have been identified as being up-regulated in breast carcinoma cells compared to normal mammary epithelial cells.4,12 Recently, a study has been reported in which differential display analysis was utilized to identify genes specifically involved in invasion and metastasis, rather than tumorigenesis. In this study, Liu and coworkers identified reticulocalbin (a protein containing 6 calcium-binding domains) and five other unknown genes that were up-regulated in MDA-MB-435 cells (invasive/metastatic phenotype) compared to MCF-7 cells (poorly invasive/non-metastatic phenotype).13
Identification of New Genes Associated with Tumor Cell Invasion and Metastasis
In a recently published study from our laboratory, we utilized differential display analysis as a tool to identify genes expressed by human breast cancer cells of varying invasive and metastatic potential.14 Specifically, our experimental strategy consisted of comparing two poorly invasive cell lines (MCF-7, MCF7neo) with two invasive/non-metastatic cell lines (MoVi-3, MoVi-10) and one highly invasive/metastatic cell line (MDA-MB-231) which constitutively expresses an interconverted phenotype. Using this approach, we were able to identify 44 PCR fragments that exhibited a differential gene expression pattern in the selected breast carcinoma cell lines. Thirteen of the 44 differentially expressed PCR fragments were further characterized with respect to gene expression in a larger panel of breast carcinoma cell lines with varying invasive and metastatic potential. Eight PCR fragments exhibited a cell line-specific expression, in which a hybridization signal was detected in only one of the breast carcinoma cell lines. Genes with cell line-specific expression patterns were not pursued further, since they were presumed not to be associated with the invasive and/or metastatic phenotype. However, five PCR fragments were identified which exhibited an expression pattern that was associated with the invasive and/or metastatic phenotype in breast carcinoma cell lines.
Among the identified genes associated with an invasive and/or metastatic phenotype were putative transcriptional regulators of gene expression: zinc finger proteins and HP1.15,16 Nil-2/ZEB/AREB6 zinc finger protein is unique in that seven zinc finger motifs exist, arranged in two widely separated clusters, with a homeodomain in the middle of these clusters. The Nil-2/ZEB/AREB6 zinc finger protein has been shown to regulate a diverse set of genes including interleukin 2,17 Na,K-ATPase a 1 subunit,18 IgH enhancer,19 delta-crystallin,20 and the 70-kDa heat shock gene.21 In addition, proteins containing homeodomains and zinc fingers have been shown to be involved in the transcriptional regulation of key developmental and differentiation processes22 and are thought to play a pivotal role in tumorigenesis.23,24 The gene(s) that the Nil-2/ZEB/ZREB6 zinc finger protein interacts within the breast carcinoma cells is currently unknown. Similarly, human heterochromatin-associated protein 1-Hs-alpha (HP1) has also been shown to possess DNA binding activity in vitro.25 Although the biological activity of HP1 in humans has not yet been characterized, HP1 is involved in heterochromatin binding and silencing of gene expression in Drosophila melanogaster.26,27 It is easy to presume that a loss of HP1 expression in cells would lead to the aberrant expression of gene(s) involved in cellular migration, invasion, and/or metastasis.
Another gene of interest is lysyl oxidase (LO), which is an extracellular copper enzyme that initiates crosslinking of collagens and elastin in the ECM—a crucial step in maintaining ECM stability.28 LO protein and mRNA expression have been shown to be decreased in human fibrosarcoma, melanoma, and choriocarcinoma cell lines,29,30 and in c-H-ras transformed mouse fibroblasts.31 Although these studies appear to contradict our observations, the authors of these reports did not specifically address LO expression in breast carcinoma cells. Peyrol and colleagues have observed that LO was expressed in invasive breast carcinoma tissues in the newly formed stroma surrounding carcinomatous ducts; however, little to no LO was expressed in carcinoma cells in their study.32 These results support the notion that the loss of LO expression may assist carcinoma cells in their invasion of basement membranes by decreasing the stability of the ECM through a lack of collagen/elastin crosslinking. However, in the Peyrol et al study, expression of LO in carcinoma cells in metastatic lesions was not specifically addressed. LO has also been observed intracellularly exhibiting a filamentous cytoskeletal protein-like structure in cultured smooth muscle cells and fibroblasts.33 Furthermore, LO has been shown to induce chemotactic and chemokinetic motility in human monocytes,34 thus suggesting that LO may play other critical roles in cell biology in addition to ECM maturation. Although LO mRNA was increased in breast carcinoma cell lines expressing the invasive and/or metastatic phenotype in our study, it is not currently known whether functionally active protein levels of LO are also increased, or whether LO contributes directly to the metastatic phenotype.
Conclusion
The roles played by LO, zinc finger protein, HP1, and other genes identified in this study in the complex cascade of tumor cell invasion and metastasis remain enigmatic. A putative interrelationship among these genes is shown in the Figure, which requires further study to elucidate their biological significance. Experimental expression of these genes in breast cancer cells with a poorly invasive/non-metastatic phenotype is required to test whether these genes can alter invasive and/or metastatic potential alone or in a specific combination. Identification of the regulation and biological significance of these genes is currently under investigation in our laboratory and may extend our understanding of the molecular/biochemical basis of tumor cell progression, and possibly contribute to the identification of new therapeutic targets for disease intervention. (Dr. Hendrix is Professor and Head of the Department of Anatomy and Cell Biology; Ms. Seftor is Senior Research Assistant, Department of Anatomy and Cell Biology; and Dr. Kirschmann is Assistant Research Scientist, Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA.)
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