Perturbation-Expression Analysis Identifies RUNX1 as a Regulator of Human Mammary Stem Cell Differentiation

The search for genes that regulate stem cell self-renewal and differentiation has been hindered by a paucity of markers that uniquely label stem cells and early progenitors. To circumvent this difficulty we have developed a method that identifies cell-state regulators without requiring any markers of differentiation, termed Perturbation-Expression Analysis of Cell States (PEACS). We have applied this marker-free approach to screen for transcription factors that regulate mammary stem cell differentiation in a 3D model of tissue morphogenesis and identified RUNX1 as a stem cell regulator. Inhibition of RUNX1 expanded bipotent stem cells and blocked their differentiation into ductal and lobular tissue rudiments. Reactivation of RUNX1 allowed exit from the bipotent state and subsequent differentiation and mammary morphogenesis. Collectively, our findings show that RUNX1 is required for mammary stem cells to exit a bipotent state, and provide a new method for discovering cell-state regulators when markers are not available.National Science Foundation (U.S.). Graduate Research Fellowship (1122374)Smith Family FoundationBreast Cancer Allianc

De-Differentiation Confers Multidrug Resistance Via Noncanonical PERK-Nrf2 Signaling

Malignant carcinomas that recur following therapy are typically de-differentiated and multidrug resistant (MDR). De-differentiated cancer cells acquire MDR by up-regulating reactive oxygen species (ROS)–scavenging enzymes and drug efflux pumps, but how these genes are up-regulated in response to de-differentiation is not known. Here, we examine this question by using global transcriptional profiling to identify ROS-induced genes that are already up-regulated in de-differentiated cells, even in the absence of oxidative damage. Using this approach, we found that the Nrf2 transcription factor, which is the master regulator of cellular responses to oxidative stress, is preactivated in de-differentiated cells. In de-differentiated cells, Nrf2 is not activated by oxidation but rather through a noncanonical mechanism involving its phosphorylation by the ER membrane kinase PERK. In contrast, differentiated cells require oxidative damage to activate Nrf2. Constitutive PERK-Nrf2 signaling protects de-differentiated cells from chemotherapy by reducing ROS levels and increasing drug efflux. These findings are validated in therapy-resistant basal breast cancer cell lines and animal models, where inhibition of the PERK-Nrf2 signaling axis reversed the MDR of de-differentiated cancer cells. Additionally, analysis of patient tumor datasets showed that a PERK pathway signature correlates strongly with chemotherapy resistance, tumor grade, and overall survival. Collectively, these results indicate that de-differentiated cells up-regulate MDR genes via PERK-Nrf2 signaling and suggest that targeting this pathway could sensitize drug-resistant cells to chemotherapy.Breast Cancer Research Program (U.S.) (Award No. W81XWH-12-BCRP-POSTDOC2)Breast Cancer Alliance (Young Investigator Grant)National Science Foundation (U.S.) (Graduate Research Fellowship Grant No. 1122374)Richard and Susan Smith Family Foundation (Excellence in Biomedical Research award

THE ROLE OF THE RNA-BINDING PROTEIN TRISTETRAPROLIN IN CERVICAL AND COLORECTAL CANCERS

Messenger RNA decay is a critical mechanism to control the expression of many inflammation- and cancer-associated genes. These transcripts are targeted for rapid degradation through AU-rich element (ARE) motifs present in the mRNA 3\u27 untranslated region (3\u27UTR). Tristetraprolin (TTP) is an RNA-binding protein that plays a significant role in regulating the expression of ARE-containing mRNAs. Through its ability to bind AREs and target the bound mRNA for rapid degradation, TTP can limit the expression of a number of critical genes frequently overexpressed in inflammation and cancer. For this reason, loss of TTP expression is a consistent feature associated with many human malignancies that serves as a critical mechanism for allowing overexpression of oncogenic transcripts. In chapter I, we demonstrate that TTP expression is lost in cervical cancer, and ectopic expression of TTP acts in an anti-proliferative capacity in cervical cancer cells through p53 stabilization and telomerase inhibition leading to cellular senescence. This growth-suppressive phenotype is the functional consequence of TTP\u27s ability to promote rapid ARE-mRNA decay of the cellular ubiquitin ligase E6-AP, which is a key player in human papillomavirus (HPV)-mediated cellular transformation and tumorigenesis. In Chapter II we describe that loss of TTP is observed during early stages of human colon carcinogenesis and TTP expression in colon cancer cell model dramatically inhibits cell growth and tumorigenecity. We show that the anti-tumor effects of TTP expression in colon cancer cells are, at least in part, a result of delayed cell cycle progression and an increase in cellular doubling time. Finally, we demonstrate in Chapter III that apparent lack of TTP in colon cancer is attributable to epigenetic control of gene expression via chromatin remodeling, and treatment with histone deacetylase (HDAC) inhibitors relieves this repression leading to activation of TTP expression. Taken together, our novel findings provide strong evidence for the protective role of TTP in cervical and intestinal epithelium that originates from its ability to control pathogenic expression of various ARE-mRNAs coding for growth and inflammation promoting factors. Conversely, loss of TTP expression as evidenced in tumors, promotes selective enrichment of oncogenic factors through aberrant mRNA stabilization and directly contributes to tumor development