Differential expression of decorin, EGFR and cyclin D1 during mammary gland carcinogenesis in TA2 mice with spontaneous breast cancer

The Tientsin Albino 2 (TA2) mouse is an inbred strain originating from the Kunming strain. It has a high incidence of spontaneous breast cancer without the need for external inducers or carcinogens. The morbidity in parous females is 84.1% within an average of 280 days after birthing a litter [1–3]. Until now, the mechanism of carcinogenesis has remained unclear. Gene expression arrays are commonly used in cancer research to identify differentially expressed candidate genes under two different conditions [4, 5]. The Affymetrix expression array is one of the most widely used commercially available oligonucleotide arrays and can determine the gene expression status of virtually the complete genome at the mRNA level.

Genomic imprinting is an epigenetic process that marks the parental origin of a subset of genes, resulting in the silencing of specific alleles [6]. To date, more than 70 imprinted genes have been described in the mouse http://www.mgu.har.mrc.ac.uk/imprinting/imprinting.html. Proteins encoded by these genes include growth factors and components of the extracellular matrix (ECM). Imprinted genes are involved in several cellular processes and perform a variety of functions, including cell cycle control, G-protein-coupled receptor signaling, and intracellular signaling, thereby influencing both pre- and postnatal growth and development through endocrine/paracrine pathways[6]. More recent data have shown that abnormal expression of several imprinted genes including decorin can cause tumorigenesis. Decorin is a maternally expressed imprinted gene that belongs to the small leucine-rich proteoglycan (SLRP) gene family and has been implicated in the control of cell proliferation [7, 8]. Reduced expression of decorin facilitates tumorigenesis and cell growth [9, 10]. Decorin is a functional component of the ECM, and is also considered to be a novel biological ligand for EGFR, which is frequently expressed at elevated levels in multiple cancers of epithelial origin. Interactions between these factors can inhibit cell growth during tissue remodeling and cancer development [11]. In addition to serving as a ligand for EGFR, decorin can bind to various forms of active TGF-β through its core protein and can neutralize the activity of TGF-β[12]. Abnormal expression of decorin has been found in many tumors, including lymphoma and human breast carcinoma [13, 14].

In this study, gene expression profiles of normal mammary glands and spontaneous breast cancer tissues from TA2 mice were detected by Affymetrix Mouse Genome 430 2.0 Arrays for the first time. The expression data were analyzed by the MAS5.0 [4], BGX[15], and Array2BIO[16] methods. Based on the candidate genes identified by expression profiling, we hypothesized that abnormal expression of decorin, EGFR, and cyclin D1 might induce carcinogenesis of mammary gland epithelial cells in TA2 mice.

Breast cancer is one of the most common malignant tumors in adult females and develops as a result of altered expression of multiple genes and abnormal cellular pathways. In recent years, accumulating data has shown that alterations of the stromal compartment can also influence tumor cell behavior through paracrine growth factor pathways[9]. Proteoglycans are the main constituents of the ECM, and their synthesis and degradation are regulated by many effectors that control the development and function of the mammary gland. We hypothesized that alterations in the composition and architecture of the ECM could promote carcinogenesis of breast epithelial cells. To test this hypothesis, we used tissue samples taken from TA2 mice. Gene expression arrays revealed that several imprinted genes, oncogenes and tumor suppressor genes were differentially expressed between normal mammary glands and spontaneous breast cancer tissues. Some of these genes encoded stromal constituents such as versican and decorin. Decorin is synthesized by the majority of mesenchymal cells [18]. However, it also interacts with a variety of other ECM components and can affect cell growth. It has been shown that decorin functionally inactivates the ErbB2 protein in breast carcinoma cells [18], leading to growth suppression and cytodifferentiation of mammary carcinoma cells. Reduced expression of decorin may facilitate cell growth, tumorigenesis and metastasis[9, 19]. In human breast cancer tissues, decorin levels were decreased 2-5-fold when compared to normal breast tissue[14]. Treatment with decorin protein reduced primary tumor growth by 70% and eliminated observable metastasis in an orthotopic mammary carcinoma animal model injected with a metastatic breast cancer cell line. Adenoviral overexpression of decorin caused primary tumor retardation of 70%, in addition to greatly reducing the observation of metastasis [20]. The expression arrays revealed that decorin was down-regulated in tumor tissues, so we speculate that loss of decorin expression may contribute to the high proliferation of mammary epithelial cells. As a component of the ECM, decorin can bind several growth factors and their receptors, such as EGFR. After binding EGFR, decorin can inhibit cell proliferation by up-regulating the expression of p21.

EGFR on the cell surface is thought to play a pivotal role in cell proliferation, cell migration, and cell survival, but Marti et al.[21] also reported a nuclear distribution for EGFR, now called "nuclear EGFR," in primary adrenocortical carcinomas more than a decade ago. High levels of nuclear EGFR have subsequently been reported in many tumors, including those of the human breast, thyroid and cervix [22, 23]. Thus two different signaling pathways, cytoplasmic/traditional and nuclear, have been identified. The cytoplasmic EGFR pathway often leads to tumorigenesis, tumor proliferation, metastasis, chemoresistance and radioresistance through the activation of Ras, PI-3K and STATs. The nuclear EGFR signaling pathway can escape the traditional transduction cascades and has different functions that depend on down-stream signaling molecules. Nuclear EGFR interacts with the DNA-binding transcription factors E2F1 and STAT3, and can accelerate G1/S cell cycle progression by up-regulating the expression of cyclin D1 and B-Myb. Cyclin D1 is a well-known oncogene whose overexpression is found in many cancers and is related to tumor progression and metastasis. Consistent with this mechanism, nuclear accumulation of EGFR is also associated with increased cell proliferation [22]. Increased expression of nuclear EGFR has been linked to poor clinical outcomes in patients with breast carcinoma [24] and oropharyngeal squamous cell carcinoma [25]. In the present study, we found that EGFR was located on the cell surface of mammary gland epithelial cells in five-month-old TA2 mice, while no nuclear EGFR was detected. In contrast, nuclear EGFR was detected in epithelial cells from normal mammary glands removed from spontaneous breast cancer-bearing TA2 mice as well as in breast cancer cells from those animals. In order to confirm the function of nuclear EGFR, we detected the expression of cyclin D1. A positive correlation between nuclear EGFR and cyclin D1 expression was observed both in mammary gland samples and breast cancer samples of cancer-bearing TA2 mice. The same result has also been observed in a cohort of breast carcinoma patients[24]. Our results suggest that nuclear translocation of EGFR may occur with increasing age, and that nuclear EGFR can promote the expression of cyclin D1, leading to a high proliferation index in mammary epithelial cells. Proliferating cell nuclear antigen (PCNA), the maestro of the replication fork, is a cofactor of DNA polymerases [26, 27]. PCNA is now one of the most commonly used molecules to detect the proliferation index of tumor cells. Our results indicated that the mammary epithelial cells from cancer-bearing TA2 mice had a higher proliferation index (PCNA labeling index) than those of the five-month-old TA2 mice, and this was further confirmed by real-time PCR. In order to know whether nuclear EGFR could affect the expression of PCNA we also detected PCNA by immunohistochemical staining and real-time PCR. No correlation was found between PCNA and EGFR expression. Our results confirm that nuclear EGFR can indirectly up-regulate the expression of cyclin D1.

In the present study, expression profiles data showed that EGFR expression was down-regulated in cancer tissues compared with that of the matched mammary glands, in contrast to results previously reported for human breast cancer. In order to confirm our findings, we detected EGFR expression by real-time PCR and immunohistochemical staining. The results of real-time PCR and immunohistochemical staining were consistent with those of the gene arrays. As we know, EGFR is one of the prognostic factors and therapeutic targets for human breast cancers[22]. According to our results, EGFR may have different effect on the progression of breast cancer of TA2 mice and human beings. For TA2 mice, high level of EGFR played an important role in the carcinogenesis of its mammary gland epithelial cells, which needs further exploration.

In briefly, our data suggest that the expression of decorin, EGFR and cyclin D1 in mammary epithelial cells changes with increasing age. Anestric mammary epithelial cells from five-month-old mice expressed low levels of EGFR. The kinase activity of this EGFR may have been attenuated in part by decorin. With increased age, decreased expression of decorin, increased expression of EGFR, especially nuclear EGFR, and increased expression of cyclin D1 may be the crucial alterations causing increased proliferation of mammary epithelial cells and these may partly conduce to the carcinogenesis of mammary gland epithelial cell of TA2 mice, which needs further studies.