Browsing by Author "Febbo, Phillip G"

Understanding the mechanisms whereby the sequence of the human genome is interpreted into diverse cellular phenotypes is a critical endeavor in modern biology. A major determinant of cellular phenotype is the spatial and temporal pattern gene expression, which is regulated in part by epigenomic properties such as histone post-translational modifications, DNA methylation, chromatin accessibility and the 3-dimensional architecture of the genome within the nucleus. These properties regulate the dynamic assembly of transcription factors and their co-regulatory proteins upon chromatin. To properly understand the interplay between the epigenomic framework of a cell and transcription factors, integrated analysis of transcription factor-DNA binding, chromatin status, and transcription is required. This work integrates information about chromatin accessibility, as measured by DNaseI hypersensitivity, transcription factor binding, as measured by chromatin immunoprecipitation, and transcription, as measured by microarray or transcriptome sequencing, to further understand the functional role of two important transcription factors, the androgen receptor (AR) and CTCF, in cancer cell line models. Data gathered from a prostate cancer cell line model demonstrate that the AR does not exclusively bind accessible chromatin upon ligand-activation, and induces significant changes in chromatin accessibility upon binding. Regions of quantitative change in chromatin accessibility contain motifs corresponding to potential collaborators for AR function, and are also significantly associated with AR-regulated transcriptional changes. Furthermore, base pair resolution of the DNaseI cleavage profile revealed three distinct patterns of AR-DNA interaction, suggesting multiple modes of AR interacting with the genome. A novel role for the nuclear receptor REV-ERBα in AR-mediated transcription was explored within the same model system. Though preliminary, results thus far indicate that REV-ERBα is required for AR-induced increases in target gene transcription in a manner that is likely dependent on HDAC3. Genetic knockdown of REV-ERBα resulted in notable changes in chromatin accessibility around AR-target genes both before and after AR activation. The function of CTCF was interrogated using stable knockdown in a breast cancer cell line model. CTCF knockdown led to widespread changes in chromatin accessibility that were dependent on DNA sequence. Further analysis suggested that AP-1 and FOXA1 are involved in CTCF function. Together, the work presented in this dissertation offers novel insight into the behavior of two critical transcription factors in cancer cell lines, and describe a framework of analysis that can be extended and applied to any transcription factor within any desired cellular context.

Castration resistant prostate cancer (CRPC) causes significant morbidity and mortality around the world and improving treatment options for patients with CRPC is a major concern for biomedical research. Because of the importance of activating mutations in the PI3K/AKT/mTOR pathway in prostate cancer, several mTOR inhibitors have been tested for efficacy in CRPC but despite promising preclinical findings, the results of clinical trials have been disappointing. The findings of several groups, including a clinical trial of RAD001 conducted at Duke, suggest that feedback upregulation of PI3K and autophagy may be potential mechanisms for resistance of CRPC to mTOR inhibitor therapy.

The main goal of this dissertation was to explore these mechanisms in vitro and to determine if combinations of PI3K inhibitors and different classes of mTOR inhibitors can overcome resistance to mTOR inhibitor monotherapy. In particular, we used immunoblotting, reverse phase protein microarrays, polysome profile analysis, cell cycle analysis, and several techniques for determining cell survival and proliferation to explore the differences in survival, proliferation, autophagy, and activity of the AKT, translation initiation, and autophagy cell signaling networks between prostate cancer cell lines treated with different combinations of mTOR and PI3K inhibitors. Our findings revealed that the combination of PI3K and mTOR inhibition leads to a synergistic inhibition of prostate cancer cell survival and cytostasis that is correlated decreased translation rates, hypophosphorylation of 4E-BP1, autophagy, and an uncoupling of normal signaling between AKT and mTOR. We were able produce an effect on cell survival similar to treatment with high doses of mTOR/PI3K inhibitor combinations by inhibiting cap-dependent translation using a non-phosphorylatable mutant of 4E-BP1. In contrast, knocking down two major autophagy genes had little to no effect on the survival of prostate cancer cells treated with PI3K/mTOR inhibitors but did protect from cell death caused by the UPR activator tunicamycin.

We conclude that treatment strategies that target PI3K, mTORC1 and mTORC2 simultaneously have the potential to be clinically useful in CRPC, probably due to the increased inhibition of eIF4E activity and cap-dependent translation when compared to monotherapy with allosteric mTORC1 inhibitors. Although autophagic cell death can be induced in prostate cancer cells, the autophagy observed after inhibition of PI3K and mTOR does not appear to contribute to cell death and is not a major resistance mechanism under these conditions. Nevertheless, we did observe different roles for autophagy in the survival of cells exposed to different types of stressors, and further elucidation of autophagy signaling networks may yet provide useful clinical targets.