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Search for Triple Negative Breast Cancer Signature Open Access

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Search for Triple Negative Breast Cancer SignatureAbstractBackground: Triple-negative breast cancer (TNBC), an aggressive form of breast cancer in women, commonly found in younger population, is characterized by the loss of Estrogen Receptor alpha (ERα), Progesterone Receptor (PR) and Human Epidermal growth factor Receptor 2 (HER-2). Hence, commonly used therapies targeting ER, PR and HER-2 receptors are ineffective against this subtype of breast cancer. The purpose of our study is to search for unique set of genes that might be responsible for the progression of TNBC and may or not be affected by the loss of ER and HER-2. Approach: Since absence of therapeutic response of TNBC is characterized by the loss of TN-receptors, we aimed to restore ER or HER-2 in TNBC and generate a set of isogenic non-TNBC clones. Receptors were stably transfected into three different TNBC cell lines and pooled clones were isolated using G418 selection. The basal level difference in the proliferation and invasiveness in clones were studied in comparison with clones that were transfected with empty vector. The responsiveness of transfected receptors to agonists and antagonists and their effect on cell proliferation and migration were also studied. Ultimately, selected clones generated in two cell lines were subjected to gene profiling studies to identify and validate the bona-fide TNBC gene expression signature as affected by ERα or HER-2 status. Results: Expression of the test receptors in the multiple pooled clones were confirmed using western blot. Baseline data from proliferation and invasiveness assays indicated that the clones with ERα or HER-2 behaved differently than parental cells with empty vector. The transfected receptors responded to antagonist and agonist treatment. The ERα stable clones showed decreased proliferation and increased migration in response to estrogen treatment. This effect was blocked when tamoxifen was added along with estrogen. HER-2 clones didn't show any significant response to heregulin or herceptin treatment in regards to proliferation. However, the cell surface expression of HER-2 was found to be effectively downregulated with herceptin treatment. Gene expression profiling using microarrays with a p≤ 0.05 and Fold Change ≥1.5 provided a comprehensive list of differentially expressed genes between control and ERα/HER-2 clones. Among these, there were 137 genes that were differentially expressed between pCDNA and ERα clones. 25 genes, that were downregulated in ERα clones, were selected for further validation. Between pCDNA and HER-2 clones, there were 99 genes that had different expression levels and 18 genes that were downregulated in HER-2 clones were selected for further validation. Conclusion: We have successfully generated stable clones expressing either ERα and HER-2 in TNBC cell lines. The stable clones in which the receptors were restored exhibited different biological behaviors than control cells and also responded to agonist and antagonist molecules. Comparative expression profiling between receptor positive and receptor negative clones helped us narrow down several potential candidates that could define TNBC signature. With our ongoing studies, we are validating the diagnostic and/or prognostic significance of these genes in human samples.

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