Cancer cells exhibit clonal diversity in phenotypic plasticity

Phenotypic heterogeneity in cancers is associated with invasive progression and drug resistance. This heterogeneity arises in part from the ability of cancer cells to switch between phenotypic states, but the dynamics of this cellular plasticity remain poorly understood. Here we apply DNA barcodes to quantify and track phenotypic plasticity across hundreds of clones in a population of cancer cells exhibiting epithelial or mesenchymal differentiation phenotypes. We find that the epithelial-to-mesenchymal cell ratio is highly variable across the different clones in cancer cell populations, but remains stable for many generations within the progeny of any single clone - with a heritability of 0.89. To estimate the effects of combination therapies on phenotypically heterogeneous tumours, we generated quantitative simulations incorporating empirical data from our barcoding experiments. These analyses indicated that combination therapies which alternate between epithelial- and mesenchymal-specific treatments eventually select for clones with increased phenotypic plasticity. However, this selection could be minimized by increasing the frequency of alternation between treatments, identifying designs that may minimize selection for increased phenotypic plasticity. These findings establish new insights into phenotypic plasticity in cancer, and suggest design principles for optimizing the effectiveness of combination therapies for phenotypically heterogeneous tumours.National Science Foundation (U.S.) (Grant 1122374)National Institutes of Health (U.S.) (Grant 2T32GM007287-36

The endoplasmic reticulum may be an Achilles' heel of cancer cells that have undergone an epithelial-to-mesenchymal transition

In a recent report published in Cancer Discovery we identified a novel vulnerability of cancer cells that have undergone an epithelial–mesenchymal transition (EMT) and established that the PERK branch of the unfolded protein response is constitutively activated upon EMT. In this commentary, we summarize and provide context for our findings. Keywords: EMT; ER stress; UPRNational Science Foundation (U.S.) (Grant 1122374

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

Cancer-specific PERK signaling drives invasion and metastasis through CREB3L1

PERK signaling is required for cancer invasion and there is interest in targeting this pathway for therapy. Unfortunately, chemical inhibitors of PERK's kinase activity cause on-target side effects that have precluded their further development. One strategy for resolving this difficulty would be to target downstream components of the pathway that specifically mediate PERK's pro-invasive and metastatic functions. Here we identify the transcription factor CREB3L1 as an essential mediator of PERK's pro-metastatic functions in breast cancer. CREB3L1 acts downstream of PERK, specifically in the mesenchymal subtype of triple-negative tumors, and its inhibition by genetic or pharmacological methods suppresses cancer cell invasion and metastasis. In patients with this tumor subtype, CREB3L1 expression is predictive of distant metastasis. These findings establish CREB3L1 as a key downstream mediator of PERK-driven metastasis and a druggable target for breast cancer therapy.National Science Foundation (U.S.) (Grant 1122374

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

BCL11B Drives Human Mammary Stem Cell Self-Renewal In Vitro by Inhibiting Basal Differentiation

The epithelial compartment of the mammary gland contains basal and luminal cell lineages, as well as stem and progenitor cells that reside upstream in the differentiation hierarchy. Stem and progenitor cell differentiation is regulated to maintain adult tissue and mediate expansion during pregnancy and lactation. The genetic factors that regulate the transition of cells between differentiation states remain incompletely understood. Here, we present a genome-scale method to discover genes driving cell-state specification. Applying this method, we identify a transcription factor, BCL11B, which drives stem cell self-renewal in vitro, by inhibiting differentiation into the basal lineage. To validate BCL11B's functional role, we use two-dimensional colony-forming and three-dimensional tissue differentiation assays to assess the lineage differentiation potential and functional abilities of primary human mammary cells. These findings show that BCL11B regulates mammary cell differentiation and demonstrate the utility of our proposed genome-scale strategy for identifying lineage regulators in mammalian tissues. Miller et al. describe a strategy to identify candidate master regulators of cell lineage specification. This approach identified BCL11B as a key regulator of human mammary stem cell self-renewal in in vitro progenitor and differentiation assays. Using a combination of 2D and 3D primary cell culture techniques, they show that BCL11B drives stem cell self-renewal by inhibiting basal lineage commitment.National Science Foundation (U.S.) (Grant 1122374

The James Webb Space Telescope Mission

Twenty-six years ago a small committee report, building on earlier studies, expounded a compelling and poetic vision for the future of astronomy, calling for an infrared-optimized space telescope with an aperture of at least $4m$. With the support of their governments in the US, Europe, and Canada, 20,000 people realized that vision as the $6.5m$ James Webb Space Telescope. A generation of astronomers will celebrate their accomplishments for the life of the mission, potentially as long as 20 years, and beyond. This report and the scientific discoveries that follow are extended thank-you notes to the 20,000 team members. The telescope is working perfectly, with much better image quality than expected. In this and accompanying papers, we give a brief history, describe the observatory, outline its objectives and current observing program, and discuss the inventions and people who made it possible. We cite detailed reports on the design and the measured performance on orbit.Comment: Accepted by PASP for the special issue on The James Webb Space Telescope Overview, 29 pages, 4 figure

New methods to study human mammary development and breast cancer

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2017.Cataloged from PDF version of thesis.Includes bibliographical references.Breast cancer is fundamentally a disease of aberrant differentiation. Breast cancers arise from within the normal architecture of the mammary gland and resemble normal mammary epithelial cell types on a molecular and gene expression level. Many tumors become dependent on the signaling pathways that guide mammary differentiation and proliferation, and may be driven by transcription factors and signaling pathways that enforce cell state. It's no wonder then that many fundamental insights into breast cancer biology derive from study of the normal mammary gland. Mouse models of mammary gland development have helped identify many of the key genetic and hormonal drivers of mammary differentiation. However, these systems have some limitations. First, study of stem and progenitor cell differentiation decisions has been hampered by a lack of definitive markers of cell state. Second, validation of these pathways in human mammary tissue has been challenging due to a paucity of human model systems. This thesis describes work to overcome these limitations. Here I describe a computational method to identify regulators of cell state transitions without the need for definitive markers of cell state. Using this method, we identified RUNXI as a regulator of mammary stem cell differentiation, and demonstrated that RUNX1 is required for exit from the stem/progenitor state. RUNX1 inhibition expanded the pool of stem cells and blocked mammary morphogenesis. This thesis also describes the development of a 3D hydrogel culture system that supports the growth of primary human mammary epithelial tissues. The tissues exhibit all major cell types found in the mammary gland and are hormone responsive. We further adapted the culture system to study the early stages of breast cancer progression by injecting tumor cells into the tissues. Tumor cells interacted and intercalated with normal mammary epithelial cells before invading out of the tissues. We utilized this system to validate SMARCE1 as a regulator of human breast cancer progression. SMARCE1 expression is predictive of progression in early-stage epithelial tumors, and SMARCE1 is functionally required for basement membrane degradation. In our tissue model of tumor progression SMARCE1 was dispensable for cell growth and in situ spreading but was required for invasion.by Ethan S. Sokol.Ph. D

Supplementary Table 1: Phenotypic ratios and estimated population sizes of barcoded clones from Cancer cells exhibit clonal diversity in phenotypic plasticity

Supplementary table (.xlsx) with the phenotypic ratios and estimated population sizes of barcoded clone

Supplementary Figures and Supplementary Tables 2-3 from Cancer cells exhibit clonal diversity in phenotypic plasticity

PDF of supplementary Figures 1-4 (Tracking clones with DNA barcodes, Fluorescence activated cell sorting based on keratin expression, Growth rates of clones, and Flow cytometry of single cell clones) and supplementary tables 2-3 (the phenotypic ratios of single cell clones, and the table of primers and oligonucleotide sequences