Investigation of the roles of novel apoptosis-controlling genes in breast cancer

Background

Normal mammary epithelial cells, like all other nucleated cells in the body, have the innate capacity to undergo programmed cell death by apoptosis. This process is controlled by external factors such as hormones and growth factors, as well as by cell–cell contacts and recognition of damaged DNA, and plays an essential role in maintaining stable cell numbers. In breast cancer cells, in common with most other cancer cells, the control of apoptosis is defective, so that the rate of cell death falls below that required to maintain a stable cell population size. The analysis of the molecular control of apoptosis is therefore very important in understanding breast cancer development and in producing novel therapies.

We have successfully used functional expression cloning [1] to identify novel genes playing controlling roles in the apoptosis process. We have used the effects of the genes on cell survival itself to isolate those genes that act at rate-limiting steps in the control of this process, and whose level of activity therefore determines whether the cell lives or dies. The genes we have identified include protein phosphatase 4 [2], Fau [3], vATPase E, PLAC8 and GAS5 [4]. Subsequently we have studied the effects of upregulation and downregulation of the activity of these genes on breast cancer cells. We have also analysed the levels of expression of these genes in normal breast epithelium and in breast cancer tissue in order to determine which of the genes are involved in the development of these cancers.

Methods

Gene expression in breast cancer cell lines was upregulated by transfection of full-length cDNAs in pcDNA3 or pCMVSPORT expression vectors. Downregulation was achieved by transfection of siRNAs (Ambion; Applied Biosystems, Warrington, UK). Real-time quantitative RT-PCR was used to monitor gene expression levels in the breast cancer cell lines, and also in breast cancer and matched normal breast tissue.

Results

Modulation of expression of several of the candidate genes, particularly Fau, altered the sensitivity of breast cancer cell lines to apoptosis. While expression of PLAC8 was not significantly altered in the breast tumour samples as a whole, several of the other genes, including Fau and vATPase E, did show significant changes in their levels of expression in breast tumour tissue, when compared with normal matched breast epithelial tissue from the same patients.

Conclusion

Several of the apoptosis-controlling genes identified by functional expression cloning affect the sensitivity of breast cancer cells to apoptosis, including that caused by DNA-damaging agents. Those genes that show differential expression may play particularly important roles in the development of breast cancer and in determining breast cancer resistance or sensitivity to cytotoxic therapy.