Differential expression and alternatively spliced variants of NRP-1 and CC3/TIP30 in breast cancer-derived cell lines.
Joseph M. Oleniczak
Breast carcinoma is the most frequent malignancy occurring in women of Western developed countries (Bray et al. 2004). There are numerous genes that have been demonstrated to have a role in breast cancer. However, more genes need to be assessed in order to determine which genes have the greatest impact on this cancer.
The genes neuropilin-1 (NRP-1) and CC3/TIP30 were examined in this study to see if their expression levels varied between the breast cancer cell lines Sk-Br-3 and T-47D. NRP-1 is a 130 to 140 kDa single spanning transmembrane glycoprotein originally described as a receptor for both semaphorin and vascular endothelial growth factor (Fujiswasa et al. 1995). NRP-1 is up-regulated in a variety of aggressive human cancers including pancreatic (Parirkh et al. 2003) and colon cancer (Parirkh et al. 2004). Therefore, NRP-1 may also be up-regulated in aggressive invasive breast cancer cell lines. While there are some reports of NRP-1 expression in breast cancer, those results differ in the pattern of its expression and have only been tested in a few breast cancer cell lines (Baba et al. 2007).
The human CC3/TIP30 gene is a putative metastasis suppressor gene, based on the results of experimental studies using lung and colon cancer cell lines (Shtivelman 1997). There are at least two mechanisms by which CC3/TIP30 exerts this metastasis suppressing effect. One mechanism that as been shown is that CC3/TIP30 induces apoptosis in v-SCLC cells deprived of growth factors (Shtivelman 1997). Secondly, CC3/TIP30 has been shown to have an angiogenesis-inhibiting effect, since it reduces the expression of certain angiogenic modulators in various tumor cell lines (NicAmhlaoibh and Shtivelman 2001). Due to the ability of CC3/TIP30 to act as a metastasis suppressor it may have a role in the invasiveness of certain cancers, especially breast cancer. However, there are a only a few reports of CC3/TIP30 expression in breast cancer cells (Zhang et al. 2003a, Zhang et al. 2003b), and these studies assess a limited number of breast cancer cell lines.
In this study, I sought to investigate the expression pattern of NRP-1 and CC3/TIP30 in an invasive and noninvasive breast cancer cell line. The two cell lines used in this study were Sk-Br-3 and T-47D. Sk-Br-3 is an invasive breast cancer cell line (Trempe and Fogh 1973) and T-47D is a noninvasive breast cancer cell line. (Keydar et al. 1979). The goal of this study was to examine gene expression of NRP-1 and CC3/TIP30 in the Sk-Br-3 and T-47D cell lines to determine if expression patterns correlated with a particular cell line.
Figure 1-Expression of NRP-1 mRNA in the Sk-Br-3 and T-47D cells lines.
Figure 2-Expression of CC3/TIP30 mRNA in the Sk-Br-3 and T-47D cells lines.
Figure 3-PCR of second band from NRP-1 primer set 1. Dilutions of 1:10, 1:100, 1:1000
Figure 4-Alternative splicing sites for NRP-1 using primer set 1, indicating the removal of Exon 3.
1) NRP-1 mRNA is expressed more in the SK-BR-3 cell line compared to the T-47D cell line.
2) CC3/TIP30 mRNA is expressed more in the T-47D cell line compared to the SK-BR-3 cell line.
3) NRP-1 is alternatively spliced in the Sk-Br-3 and the T-47D cell lines such that it is missing Exon 3.
4) The expression level of the alternatively spliced form of NRP-1 missing Exon 3 is expressed equally in Sk-Br-3 and T-47D cell lines.
Thank you to Dr. Rubin, Bo Lui, and Leleesha Samaraweera for all of your help and insights.