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

Histone Deacetylase Inhibitors Activate Tristetraprolin Expression through Induction of Early Growth Response Protein 1 (EGR1) in Colorectal Cancer Cells

The RNA-binding protein tristetraprolin (TTP) promotes rapid decay of mRNAs bearing 3' UTR AU-rich elements (ARE). In many cancer types, loss of TTP expression is observed allowing for stabilization of ARE-mRNAs and their pathologic overexpression. Here we demonstrate that histone deacetylase (HDAC) inhibitors (Trichostatin A, SAHA and sodium butyrate) promote TTP expression in colorectal cancer cells (HCA-7, HCT-116, Moser and SW480 cells) and cervix carcinoma cells (HeLa). We found that HDAC inhibitors-induced TTP expression, promote the decay of COX-2 mRNA, and inhibit cancer cell proliferation. HDAC inhibitors were found to promote TTP transcription through activation of the transcription factor Early Growth Response protein 1 (EGR1). Altogether, our findings indicate that loss of TTP in tumors occurs through silencing of EGR1 and suggests a therapeutic approach to rescue TTP expression in colorectal cancer

The MCF10A stem cell model exhibits multi-lineage mammary differentiation.

<p>(A, upper panels) Confocal microscopy images of MCF10A collagen cultures stained with phalloidin (red) and DAPI (blue) 8 days after seeding. (A, lower panels) The images were segmented into ductal and lobular structures using CellProfiler and quantified (B). (C) 3D confocal reconstruction of a complex ductal-lobular tissue rudiment 12 days after seeding.</p

Schematic showing the steps in the calculation of a PEACS score.

<p>Step (1): calculate a median centroid for all perturbations (green star) in a given experiment. Step (2): calculate a centroid of a set of replicates for a single perturbation (blue star). Step (3): calculate the Euclidean distance (d) between the median centroid of all perturbations and the centroid of a set of replicates for a single perturbation. Step (4): calculate the standard error (S.E.) about the mean for a set of replicates for a single perturbation. For each set of perturbation replicates, the PEACS score is defined as the distance calculated in Step 3 divided by the S.E. calculated in Step 4. In the formula shown <i>k</i> is the number of SVD principal components, which is determined by a Scree plot; <i>repl</i> is the number of replicates for a given perturbation; </p><p></p><p></p><p></p><p><mi>u</mi></p><p><mi>i</mi></p><p><mi>j</mi></p><p></p><p></p><p></p> is the coefficient of the i^th SVD gene-expression vector for the j^th perturbation; and <p></p><p></p><p></p><p></p><p></p><p><mi>u</mi></p><mo>-</mo><p></p><p></p><p><mi>i</mi></p><p></p><p></p><p></p> is the average coefficient across all perturbation samples for the i^th SVD gene-expression vector.<p></p

RUNX1 is necessary for mammary stem cells to exit the bipotent state.

<p>(A) Experimental schematic and brightfield images of MCF10A cells stably transduced with a dox-inducible RUNX1 shRNA, grown in collagen culture for 7 days in the presence of dox. At day 7 half of the collagen gels were removed from dox, and the other half maintained in dox for an additional 4 days. Tissue rudiments maintained in dox remained as solid spheres, whereas spheres removed from dox rapidly sprouted ducts within 12–24 hrs. (B) Western blot confirming inducible RUNX1 inhibition by the dox inducible shRNA. MCF10A cells were cultured without dox for 7 days (lane 1), or with dox for 4 days followed either by culture without dox for an additional 3 days (lane 2) or culture with dox for an additional 3 days (lane 3). Also shown is quantification of the western blot normalized to GAPDH and the no dox control treatment, and quantification using qPCR. (C) Experimental schematic, brightfield images, and quantification of MCF10A dox-inducible shRUNX1 cells that were grown in the presence of dox for seven days, removed from collagen, dissociated into single cells, and reseeded into a new collagen pad in the presence or absence of dox. While control cells are unable to reseed structures, inducible shRUNX1 cells are able to reseed structures with high efficiency (reseeding capacity shown as structures formed per 7500 cells; * indicates p<0.05 relative to wild type by t-test. SEM is indicated n = 3). Dox-inducible shRUNX1 cells grown in the absence of dox are multipotent, with the capacity to form ducts, lobules, and complex ductal-lobular structures in 13 days.</p

Schematic of Perturbation-Expression Analysis of Cell States (PEACS).

<p>The PEACS pipeline consists of four steps: (1) perturb a heterogeneous population with a genetic or chemical perturbation, (2) profile gene expression, (3) factor the gene-expression matrix, and (4) analyze the factored gene-expression matrix to identify perturbations that altered cell-state proportions. Shown in the lower right is the SVD formula for matrix factorization.</p