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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. PERK stress signaling is an important driver of cancer invasion and metastasis, but chemical inhibitors of PERK cause side effects. Here, the authors find that CREB3L1 is required for PERK's pro-metastatic function in breast cancer, and its inhibition suppresses cancer invasion and metastasis.

Inhibition of DOT1L, the H3K79 histone methyltransferase, increases cell reprogramming and substituted for KLF4 and c-Myc, showing that chromatin-modifying enzymes act not only as facilitators but also as barriers to reprogramming. Chromatin in iPS-cell formation A study of the role of chromatin-modifying enzymes in the reprogramming of human fibroblasts to induced pluripotent stem (iPS) cells suggests that such enzymes can act as facilitators — but also as barriers — to epigenetic remodelling. By knocking down 22 selected genes involved in DNA and histone methylation pathways, George Daley and colleagues identified both positive and negative regulators of iPS-cell generation. In particular, inhibition of DOT1L, the H3K79 histone methyltransferase, increased reprogramming and substituted for KLF4 and c-Myc, two of the factors needed in the reprogramming cocktail. The effect of DOT1L inhibition seems to be associated with increase in the reprogramming factors NANOG and LIN28. This work demonstrates that specific chromatin modifiers can be modulated to generate iPS cells more efficiently and with fewer exogenously introduced transcription factors. Generation of induced pluripotent stem cells (iPSCs) by somatic cell reprogramming involves global epigenetic remodelling 1 . Whereas several proteins are known to regulate chromatin marks associated with the distinct epigenetic states of cells before and after reprogramming 2 , 3 , the role of specific chromatin-modifying enzymes in reprogramming remains to be determined. To address how chromatin-modifying proteins influence reprogramming, we used short hairpin RNAs (shRNAs) to target genes in DNA and histone methylation pathways, and identified positive and negative modulators of iPSC generation. Whereas inhibition of the core components of the polycomb repressive complex 1 and 2, including the histone 3 lysine 27 methyltransferase EZH2, reduced reprogramming efficiency, suppression of SUV39H1, YY1 and DOT1L enhanced reprogramming. Specifically, inhibition of the H3K79 histone methyltransferase DOT1L by shRNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 and c-Myc (also known as MYC). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in two alternative factors, NANOG and LIN28, which play essential functional roles in the enhancement of reprogramming. Genome-wide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. These findings implicate specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrate how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.

The epithelial-to-mesenchymal transition (EMT) produces cancer cells that are invasive, migratory, and exhibit stem cell characteristics, hallmarks of cells that have the potential to generate metastases. Inducers of the EMT include several transcription factors (TFs), such as Goosecoid, Snail, and Twist, as well as the secreted TGF-β1. Each of these factors is capable, on its own, of inducing an EMT in the human mammary epithelial (HMLE) cell line. However, the interactions between these regulators are poorly understood. Overexpression of each of the above EMT inducers up-regulates a subset of other EMT-inducing TFs, with Twist, Zeb1, Zeb2, TGF-β1, and FOXC2 being commonly induced. Up-regulation of Slug and FOXC2 by either Snail or Twist does not depend on TGF-β1 signaling. Gene expression signatures (GESs) derived by overexpressing EMT-inducing TFs reveal that the Twist GES and Snail GES are the most similar, although the Goosecoid GES is the least similar to the others. An EMT core signature was derived from the changes in gene expression shared by up-regulation of Gsc, Snail, Twist, and TGF-β1 and by down-regulation of E-cadherin, loss of which can also trigger an EMT in certain cell types. The EMT core signature associates closely with the claudin-low and metaplastic breast cancer subtypes and correlates negatively with pathological complete response. Additionally, the expression level of FOXC1, another EMT inducer, correlates strongly with poor survival of breast cancer patients.

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.

Background Three-dimensional (3D) cultures have proven invaluable for expanding human tissues for basic research and clinical applications. In both contexts, 3D cultures are most useful when they (1) support the outgrowth of tissues from primary human cells that have not been immortalized through extensive culture or viral infection and (2) include defined, physiologically relevant components. Here we describe a 3D culture system with both of these properties that stimulates the outgrowth of morphologically complex and hormone-responsive mammary tissues from primary human breast epithelial cells. Methods Primary human breast epithelial cells isolated from patient reduction mammoplasty tissues were seeded into 3D hydrogels. The hydrogel scaffolds were composed of extracellular proteins and carbohydrates present in human breast tissue and were cultured in serum-free medium containing only defined components. The physical properties of these hydrogels were determined using atomic force microscopy. Tissue growth was monitored over time using bright-field and fluorescence microscopy, and maturation was assessed using morphological metrics and by immunostaining for markers of stem cells and differentiated cell types. The hydrogel tissues were also studied by fabricating physical models from confocal images using a 3D printer. Results When seeded into these 3D hydrogels, primary human breast epithelial cells rapidly self-organized in the absence of stromal cells and within 2 weeks expanded to form mature mammary tissues. The mature tissues contained luminal, basal, and stem cells in the correct topological orientation and also exhibited the complex ductal and lobular morphologies observed in the human breast. The expanded tissues became hollow when treated with estrogen and progesterone, and with the further addition of prolactin produced lipid droplets, indicating that they were responding to hormones. Ductal branching was initiated by clusters of cells expressing putative mammary stem cell markers, which subsequently localized to the leading edges of the tissue outgrowths. Ductal elongation was preceded by leader cells that protruded from the tips of ducts and engaged with the extracellular matrix. Conclusions These 3D hydrogel scaffolds support the growth of complex mammary tissues from primary patient-derived cells. We anticipate that this culture system will empower future studies of human mammary gland development and biology.

Many tumors contain heterogeneous populations of cells, only some of which exhibit increased tumorigenicity and resistance to anticancer therapies. Evidence suggests that these aggressive cancer cells, often termed \"cancer stem cells\" or \"cancer stem-like cells\" (CSCs), rely upon developmental signaling pathways that are important for survival and expansion of normal stem cells. Here we report that, in analogy to embryonic mammary epithelial biology, estrogen signaling expands the pool of functional breast CSCs through a paracrine FGF/FGFR/Tbx3 signaling pathway. Estrogen or FGF9 pretreatment induced CSC properties of breast cancer cell lines and freshly isolated breast cancer cells, whereas contreatment of cells with tamoxifen or a small molecule inhibitor of FGFR signaling was sufficient to prevent the estrogen-induced expansion of CSCs. Furthermore, reduction of FGFR or Tbx3 gene expression was able to abrogate tumorsphere formation, whereas ectopic Tbx3 expression increased tumor seeding potential by 100-fold. These findings demonstrate that breast CSCs are stimulated by estrogen through a signaling pathway that similarly controls normal mammary epithelial stem cell biology.

Mammary morphogenesis is an orchestrated process involving differentiation, proliferation and organization of cells to form a bi-layered epithelial network of ducts and lobules embedded in stromal tissue. We have engineered a 3D biomimetic human breast that makes it possible to study how stem cell fate decisions translate to tissue-level structure and function. Using this advancement, we describe the mechanism by which breast epithelial cells build a complex three-dimensional, multi-lineage tissue by signaling through a collagen receptor. Discoidin domain receptor tyrosine kinase 1 induces stem cells to differentiate into basal cells, which in turn stimulate luminal progenitor cells via Notch signaling to differentiate and form lobules. These findings demonstrate how human breast tissue regeneration is triggered by transmission of signals from the extracellular matrix through an epithelial bilayer to coordinate structural changes that lead to formation of a complex ductal-lobular network. Mammary morphogenesis is a complex process. Here the authors describe how stem cells build a three-dimensional self-organizing multi-lineage tissue by showing that positional signals from the extracellular matrix through the collagen receptor DDR1 lead stem cells to differentiate into multi-lineage committed multi-layered progeny.

The NavDx® blood test analyzes tumor tissue modified viral (TTMV)-HPV DNA to provide a reliable means of detecting and monitoring HPV-driven cancers. The test has been clinically validated in a large number of independent studies and has been integrated into clinical practice by over 1000 healthcare providers at over 400 medical sites in the US. This Clinical Laboratory Improvement Amendments (CLIA), high complexity laboratory developed test, has also been accredited by the College of American Pathologists (CAP) and the New York State Department of Health. Here, we report a detailed analytical validation of the NavDx assay, including sample stability, specificity as measured by limits of blank (LOBs), and sensitivity illustrated via limits of detection and quantitation (LODs and LOQs). LOBs were 0–0.32 copies/μL, LODs were 0–1.10 copies/μL, and LOQs were <1.20–4.11 copies/μL, demonstrating the high sensitivity and specificity of data provided by NavDx. In-depth evaluations including accuracy and intra- and inter-assay precision studies were shown to be well within acceptable ranges. Regression analysis revealed a high degree of correlation between expected and effective concentrations, demonstrating excellent linearity (R2 = 1) across a broad range of analyte concentrations. These results demonstrate that NavDx accurately and reproducibly detects circulating TTMV-HPV DNA, which has been shown to aid in the diagnosis and surveillance of HPV-driven cancers.

The aggressive clinical behavior of melanoma suggests that the developmental origins of melanocytes in the neural crest might be relevant to their metastatic propensity. Here we show that primary human melanocytes, transformed using a specific set of introduced genes, form melanomas that frequently metastasize to multiple secondary sites, whereas human fibroblasts and epithelial cells transformed using an identical set of genes generate primary tumors that rarely do so. Notably, these melanomas have a metastasis spectrum similar to that observed in humans with melanoma. These observations indicate that part of the metastatic proclivity of melanoma is attributable to lineage-specific factors expressed in melanocytes and not in other cell types analyzed. Analysis of microarray data from human nevi shows that the expression pattern of Slug, a master regulator of neural crest cell specification and migration, correlates with those of other genes that are important for neural crest cell migrations during development. Moreover, Slug is required for the metastasis of the transformed melanoma cells. These findings indicate that melanocyte-specific factors present before neoplastic transformation can have a pivotal role in governing melanoma progression.

The conserved Musashi (Msi) family of RNA binding proteins are expressed in stem/progenitor and cancer cells, but generally absent from differentiated cells, consistent with a role in cell state regulation. We found that Msi genes are rarely mutated but frequently overexpressed in human cancers and are associated with an epithelial-luminal cell state. Using ribosome profiling and RNA-seq analysis, we found that Msi proteins regulate translation of genes implicated in epithelial cell biology and epithelial-to-mesenchymal transition (EMT), and promote an epithelial splicing pattern. Overexpression of Msi proteins inhibited the translation of Jagged1, a factor required for EMT, and repressed EMT in cell culture and in mammary gland in vivo. Knockdown of Msis in epithelial cancer cells promoted loss of epithelial identity. Our results show that mammalian Msi proteins contribute to an epithelial gene expression program in neural and mammary cell types. All living things start life as a single cell, but many organisms develop into a collection of different, specialized cells. Most of the cells in an organism can only divide to make more of the same type of cell; however, stem cells are different because they can ‘differentiate’ and develop into several different cell types. A key step in the development of an embryo is called the epithelial-to-mesenchymal transition, in which an epithelial cell—a cell type that normally lines body surfaces and cavities—begins to crawl away from the tissue it is in and starts to differentiate. This transition also allows cancer cells to leave tumors and spread around the body, in a process known as metastasis. In mammals, two proteins called Musashi1 and Musashi2 are abundant in stem cells and brain cancers, but are rarely found in specialized tissues and cells. Katz, Li et al. now find that the Musashi proteins are also often overexpressed in human breast, lung, and prostate tumors. In addition, Musashi proteins are much less abundant in cells that have completed an epithelial-to-mesenchymal transition. When Katz, Li et al. artificially reduced the amounts of Musashi proteins in breast cancer cells, the cells migrated and dispersed, as if becoming mesenchymal cells. Furthermore, many of the genes normally used in epithelial cells were switched off. In comparison, artificially increasing the levels of Musashi proteins halted the movement of mesenchymal cells and led to increased levels of genes used in epithelial cells, as if they were reverting to epithelial cells. Therefore, it appears that the Musashi proteins prevent epithelial cells from developing mesenchymal properties. Katz, Li et al. investigated how Musashi proteins work at the molecular level by studying neural and mammary cells in mice. This revealed that Musashi proteins control the steps that lead to the epithelial-to-mesenchymal transition by binding to the tail end of the RNA molecules that include the instructions to make certain proteins. This affects how often these proteins can be made from the RNA molecules. Katz, Li et al. suggest that Musashi proteins may similarly control the behavior of progenitor and stem cells in many other tissues as well; however, further study is needed to confirm this.