Identification and Characterization of Signaling Pathways Underlying the Tumorigenic Activity of DeltaNp63alpha in Breast Cancer Stem Cells

Studies presented in this dissertation focus on the transcriptional targets and cellular functions of DeltaNp63&agr;, the predominant TP63 isoform expressed in epithelial stem cell compartments. TP63, a member of the p53 family of transcriptional factors, is required for the development and maintenance of stratified epithelia. Genetic analysis of TP63 indicates that the DeltaNp63 isoforms account for this activity by preserving long-term proliferative potential of adult stem cells. In squamous cell carcinomas, TP63 is commonly amplified, and DeltaNp63&agr; confers a potent survival advantage. Genome-wide occupancy studies demonstrate that DeltaNp63&agr; promotes bidirectional target gene regulation by binding 5,800 sites throughout the genome; however, the subset of targets mediating discreet activities of TP63 remains unclear. In the second chapter of this dissertation we characterize a novel genetic interaction between DeltaNp63&agr; and bone morphogenetic protein (BMP) signaling that governs stem cell dynamics in the normal mammary gland and breast tumor cell populations. Our initial studies clearly indicate hyper-activation of BMP signaling in diverse molecular subtypes of breast cancer, as well as in several mouse models of breast cancer. LDN193189, a small molecule inhibitor of BMP Type I Receptor kinases, suppresses BMP signaling in vitro and diminishes cancer stem cell activity. Limiting dilution analysis revealed that LDN193189 reduces tumor initiating ability and increases tumor latency in vivo. This work tested the in vivo efficacy of LDN193189 in a cancer setting for the first time.;The third chapter of this dissertation is centered on the identification of signaling crosstalk between p53 family members and Smad proteins. We demonstrate that P53 and P63 act as transcriptional partners for BMP and TGFbeta specific Smad effectors. P53 behaves as a repressive molecule to BMP inputs, whereas DeltaNp63&agr; synergistically enhances BMP induced transcription. Furthermore, we demonstrate that DeltaNp63&agr; and p53 can directly bind to a subset of canonical BMP targets genes. Using bioinformatic and biochemical approaches, we discovered that these proteins cooperate to regulate a global EMT transcriptional program in tumor cells. This work has large clinical implications as it predicts that tumor alterations in p53 family members alter cellular responsiveness to TGFbeta and BMP signals and may alter sensitivity to anti-TGFbeta or anti-BMP compounds.