Expression analysis of novel biomarkers for breast cancer

Cancer Research UK (2007) [1] stated that the most common cancer for women in the United Kingdom is breast cancer. In 2004, about 20% of all breast cancer cases diagnosed would lead to death [2]. The accepted prognostic factors fail to establish accurately the outcome for breast cancer patients as a large proportion of those diagnosed with invasive breast carcinomas are given aggressive treatments even though many of them are unlikely to develop a life-threatening cancer even without therapies. Over the past decade, many genetic and molecular pathways have been associated with breast cancer. To progress towards personalized therapies, there is a need for novel bio-markers for diagnosis, for the detection of metastasis and as targets for new selective immunotherapies. The BUC genes (Breast UniGene Cluster) are novel breast-associated genes identified on the basis of their specific expression spectrum, which includes testis, normal breast and breast cancer tissue. During in silico analysis of the BUC gene sequence, we discovered that the BUC11 gene sequence shares significant similarity with the gene sequence of an unpublished gene that codes for a predicted protein (source data obtained from the NCBI website [3]).

siRNA was designed for specific BUC11 silencing. BUC11 siRNA efficacy was first tested using real-time RT-PCR following transfection and mRNA isolation. The transfection of breast cancer cell line MDA231 was carried out using INTERFERin siRNA Transfection reagent (Autogen Bioclear, Calne, UK). The experiment was performed in duplicate wells. Each experiment comprised cells with BUC11 gene-specific siRNA, cells with negative control siRNA, cells with INTERFERin alone and cells alone. On day 2, ³H-thymidine (Sigma-Aldrich, Gillingham, UK) was added to the cells. On day 3, cell suspensions were transferred to a filter plate, Microscint solution (Packard, Meriden, CT, USA) was added and the reading of the plate was performed. The procedure was repeated on day 7 and on day 10. To quantify gene expression at the mRNA level in breast tumours, conventional RT-PCR as well as real-time quantitative RT-PCR were carried out. Samples used in this study come from various invasive and noninvasive histological subtypes of breast cancer, different malignancies (for example, melanoma, testis cancer, mesothelioma) and normal tissues.

Regarding BUC11 gene knockdown, 72 hours following transfection, 89.7% of specific inhibition of BUC11 mRNA expression was observed (real-time RT-PCR results). Three days following transfection of MDA231 with BUC11 siRNA, cell proliferation was inhibited by 98%. This result is still observed 7 days following transfection. However, the inhibition of proliferation is no longer observed 10 days following transfection, which is not surprising due to the transient nature of transfection. The BUC11 gene was expressed in 90% of the breast cancer tissues tested. BUC11 mRNA was not (or at very low levels) expressed in the normal tissues tested (heart, liver, prostate, brain, uterus, spleen, skeletal muscle, lung, kidney, placenta, trachea, thyroid, spinal cord, salivary gland, thymus and peripheral blood mononuclear cell) except for normal testis and normal breast tissues. BUC11 mRNA was expressed in varying levels in the breast cancer samples tested. BUC11 mRNA was expressed at similar levels in the normal testis and testicular cancer tissues tested. BUC11 mRNA was only expressed in the breast cancer cell lines T47D and MDA231. Furthermore, BUC11 mRNA appears to be overexpressed in breast tumour compared with the normal counterpart in the early stages of the disease and down-regulated in more advanced aggressive breast cancers. Finally, BUC11 mRNA was not expressed in any of the other cancer samples tested (oesophageal, mesothelioma, melanoma, gastric and kidney).

BUC11 could potentially be a good candidate for the diagnosis and prognosis of breast cancer due to the correlation of BUC11 gene expression with the stage of breast cancer. siRNA silencing of BUC11 led to the inhibition of the proliferation of MDA231 breast cancer cells. This suggests that BUC11 might have a role in the proliferation of cancer cells in the breast. The tissue specificity of the BUC11 expression profile provides a rationale to consider BUC11 as a tissue-specific gene involved in the differentiation of breast and testis tissues. If the restricted expression spectrum is confirmed in a larger cohort of samples, BUC11 could be useful to detect micrometastasis in the lymph nodes or peripheral blood of breast cancer patients. Finally, BUC11 gene is not expressed in vital organs; thus it could potentially be a good target for vaccine strategies.