ABSTRACT/SUMMARY - Project 2 Fine-scale nucleosome repositioning of enhancers for hormone-independent genomic function The cognate receptors AR and ER can remain active for tumor progression after anti-hormone treatment for patients with prostate and breast cancers. Despite intensive efforts to elucidate the underlying mechanisms, little information is available concerning AR/ERα genomic function for promoting hormone resistance at the nucleosome level. In preliminary studies, we observed this genomic function is well orchestrated, relying on precise nucleosome organization within cis-bound enhancers for hormone-dependent transcription. Interestingly, we also found that this epigenetic mechanism can be hijacked by hormone-resistant cells to gain their growth and invasion advantages. Therefore, we hypothesize that altered nucleosome positions, or nucleosome repositioning, in and near AR/ERα-bound enhancers is being exploited for hormone-independent genomic function in advanced cancers. In Aim 1, we will conduct ChIP-ePENS and MNase-seq to comprehensively map nucleosome boundaries of AR/ERα-bound enhancers in a panel of hormone-sensitive and -resistant cancer cells. RNA-seq will be conducted to determine differential expression patterns of corresponding genes in these cell lines. The NucPat computational pipeline will be deployed to seamlessly process complex omics-seq data (Aim 2). We will use a Kernel Density Estimation algorithm to determine nucleosome positioning and spacing when AR or ERα establishes direct contact with its binding motif. Using a Hidden Markov model, we will identify active nucleosome states that maximize DNA-protein contact for AR/ERα genomic functions. In addition, pioneer factor FOXA1 and chromatin remodelers participate in this nucleosome repositioning even in the absence of agonists or in the presence of antagonists. To confirm this computational modeling in vivo, ChIP-ePENS and MNase-seq will be conducted in patient-derived xenograft (PDX) lines carrying hormone-sensitive and -resistant tumors (Aim 3). A nucleosome-phasing index (NPI) will be established to quantitatively assess the nucleosome states of AR/ERα redeployment in different PDX lines. This integrative omics analysis will be extended to a cohort of primary tumors, categorized into high- and low-risk groups. Again, we will calculate individual NPIs and correlate the data with clinicopathological features of patients. This translational study is intended to determine whether nucleosome phasing for AR/ERα redeployment is already present in high-risk primary tumors. Patients with this intrinsic phenotype are expected to have an adverse clinical outcome, irrespective of their anti-hormone treatments. Therefore, our proposed study will address a previously uncharacterized mechanism of hormone resistance and provide experimental evidence that nucleosome repositioning plays an integral role in redefining AR/ERα genomic function for advanced development of prostate and breast cancers.

NARRATIVE - Project 2 The proposed approach has the potential to improve upon gene expression profiling currently used to stratify breast or prostate cancer subtypes. In this regard, both ChIP-ePENS and MNase-seq detect relatively stable genomic events as transcription readouts rather than detecting transient gene expression profiles of RNA samples, which may have rapid fluctuations in clinical samples.