Expression profiling of Wnt pathway genes in breast cancer
© BioMed Central 2005
Published: 17 June 2005
Wnt signaling, initially identified in early embryogenesis of Drosophila, is involved in a large set of cellular processes, including proliferation, differentiation, migration, and apoptosis. Canonical Wnt signaling is involved in cell fate choices, stem cell renewal and differentiation, whereas non-canonical signaling deals with morphological changes and tissue organization. Since the discovery of Wnt-1 as a virally-induced oncogene in mouse mammary tumors, Wnt signaling has become a center of interest in human breast carcinogenesis. Surprisingly, genetic aberrations of Wnt signaling have been revealed in different malignancies including colon cancer, liver cancer and hematological cancer, but not in breast cancer. Thus, epigenetic changes in Wnt signaling, rather than mutations, may be more relevant to breast cancers in humans. However, this aspect of Wnt signaling in breast cancer is not fully understood. We aimed to study the expression of key components of Wnt signaling in breast cancer. Here, we report the preliminary results obtained from our expression profiling studies for all known Wnt ligand, frizzled receptor, co-receptor and Lef/TCF transcription factor genes, using a set of cell lines and primary tumors.
We studied the expression profile of 34 Wnt pathway genes by the RT-PCR technique. RNAs were extracted from a telomerase-immortalized human mammalian epithelial cell line (HMEC), six breast cancer cell lines and 15 breast tumors. Tumor samples were selected following pathological analysis of fresh-frozen tissue slices. Corresponding cDNAs were synthesized and subjected to PCR amplification using specific pairs of primers. Equal loading of total cDNAs was checked by PCR analysis of the housekeeping gene GAPDH.
HMEC cells, used as a normal control, expressed many Wnt signaling genes, including 9/19 (48%) Wnt ligands, 7/9 (78%) frizzled receptors, LRP-5, LRP-6, as well as four LEF/TCF transcription factors. Expression patterns of frizzled receptors, LRP-5, LRP-6, and LEF/TCF transcription factors did not show major changes in breast cancer cell lines. The major change in Wnt signaling genes was observed at the level of ligand expression. The expression of Wnt-3a, Wnt-4, Wnt-6, Wnt-8b and Wnt-9a were upregulated in 50% or more breast cancer cell lines. Conversely, the expression of Wnt-5a, Wnt-9b and Wnt-16 was downregulated. Our ongoing studies with breast tumors indicate that Frizzled-1, Frizzled-2 and Frizzled-6 expression is also maintained in breast tumors. Moreover, upregulation of Wnt-4 and Wnt-9a, as well as downregulation of Wnt-5a expression, were observed in 79–100% of tumors.
These observations provide evidence for redundant expression of major genes involved in Wnt signaling in both normal and malignant breast cells. The expression of at least nine Wnt genes in HMEC strongly suggests that some Wnt ligands may provide autocrine or paracrine signaling to normal breast epithelial cells. Six Wnt genes were commonly expressed in both HMEC and breast cancer cell lines, suggesting that some Wnt ligands may not be significantly involved in malignant transformation of mammary epithelial cells. On the other hand, malignant cells have upregulated the expression of Wnt-3a, Wnt-4, Wnt-6, Wnt-8b and Wnt-9a genes that may play a positive role in malignancy. Wnt-3a and Wnt-4 are known to display transforming activity in mammary epithelial cells. The function of Wnt-8b in mammalian cells is not well known, but its Xenopus homolog displays strong axis-duplication activity, suggesting that it may also be a transforming Wnt. On the other hand, the expression of Wnt-5a, Wnt-9b and Wnt-16 was switched-off in malignant breast cells. Although the functions of Wnt-9b and Wnt-16 are not well known, Wnt-5a has been identified as a tumor suppressor in hematological malignancies, and acts as an antagonist of canonical Wnt signaling. Taken together, these results indicate that there is a switch in Wnt ligand expression pattern in breast cancer cells, and that this may provoke a functional switch in Wnt signaling from non-canonical to canonical pathways.