Profiling YB-1 responsive genes in basal-like breast cancer cells by ChIP-on-chip reveals direct binding to PIK3CAs

Purpose: Basal-like breast cancers (BLBC) are typically very aggressive and they are also prone to a high rate of recurrence. We recently identified the transcription factor Y-box binding protein-1 (YB-1) as an important component of BLBC by screening breast tumour tissue microarrays. YB-1 is well known for its ability to confer chemotherapy resistance and to promote the growth of a wide range of cancer cell types. The breast cancer cell line SUM149 was characterized by us as having all the hallmarks of BLBC including a lack of the estrogen, progesterone, and Her-2 receptors. We also find that they express high levels of the epidermal growth factor receptor, a YB-1 responsive gene. We performed a genome-wide promoter screen of the possible YB-1 responsive genes using the NimbleGen ChIP-on-chip (COC) platform to begin to understand how this oncogene regulates the growth of basal-like breast cancer cells. YB-1 was found to bind to ~6,700 human promoters. Several members of the phosphatidylinositol-3-kinase (PI3K) pathway were identified (p110a, p85, Akt-2, ILK). Of particular interest was the identification of PIK3CA, the gene that codes for the p110a catalytic subunit of PI3K. PI3K, a lipid kinase with important roles in neoplasia, phosphorylates membrane inositol lipids resulting in activation of downstream effectors that are involved in cellular responses such as cell proliferation, survival, metabolism, and cytoskeletal reorganization. Many studies have suggested importance of the role of PI3K signaling in various types of cancer. Approximately 20% of breast cancers harbour somatic activating mutations in PIK3CA, and about 8% have amplification of PIK3CA. The remainder of the cancers have elevated levels of PI3K via other mechanisms including increased transcription. Methods & Results: Here we propose that YB-1 overexpression in cancer leads to increased PIK3CA transcription via direct binding to the promoter. To extend our COC findings, we knocked down YB-1 with siRNA and observed a concomitant loss in p110a by immunoblotting. We have recently identified two alternate PIK3CA transcriptional start sites (exon 1a and 1b) and two alternate PIK3CA promoters (promoter 1a and 1b). We mapped potential YB-1 binding sites on PIK3CA promoters 1a and 1b, and identified five putative YB-1 binding sites within promoter 1a and one putative binding site within promoter 1b. We further performed traditional ChIP and EMSA to confirm the interaction of YB-1 with the putative binding sites on PIK3CA promoter, and determined that YB-1 binds to promoter 1a, but not to promoter 1b. Conclusion:The demonstration that YB-1 binds directly to the PIK3CA promoter and induces its activity identifies a novel mechanism whereby activation of YB-1 and subsequent up-regulation of PIK3CA may contribute to the pathophysiology, outcomes and therapeutic responsiveness of breast cancer.