Slug and Sox9 cooperatively determine the mammary stem cell state.

Regulatory networks orchestrated by key transcription factors (TFs) have been proposed to play a central role in the determination of stem cell states. However, the master transcriptional regulators of adult stem cells are poorly understood. We have identified two TFs, Slug and Sox9, that act cooperatively to determine the mammary stem cell (MaSC) state. Inhibition of either Slug or Sox9 blocks MaSC activity in primary mammary epithelial cells. Conversely, transient coexpression of exogenous Slug and Sox9 suffices to convert differentiated luminal cells into MaSCs with long-term mammary gland-reconstituting ability. Slug and Sox9 induce MaSCs by activating distinct autoregulatory gene expression programs. We also show that coexpression of Slug and Sox9 promotes the tumorigenic and metastasis-seeding abilities of human breast cancer cells and is associated with poor patient survival, providing direct evidence that human breast cancer stem cells are controlled by key regulators similar to those operating in normal murine MaSCs.

WRN helicase is a synthetic lethal target in microsatellite unstable cancers.

Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics1. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins2. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach3. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.

Distinct EMT programs control normal mammary stem cells and tumour-initiating cells.

Tumour-initiating cells (TICs) are responsible for metastatic dissemination and clinical relapse in a variety of cancers. Analogies between TICs and normal tissue stem cells have led to the proposal that activation of the normal stem-cell program within a tissue serves as the major mechanism for generating TICs. Supporting this notion, we and others previously established that the Slug epithelial-to-mesenchymal transition-inducing transcription factor (EMT-TF), a member of the Snail family, serves as a master regulator of the gland-reconstituting activity of normal mammary stem cells, and that forced expression of Slug in collaboration with Sox9 in breast cancer cells can efficiently induce entrance into the TIC state. However, these earlier studies focused on xenograft models with cultured cell lines and involved ectopic expression of EMT-TFs, often at non-physiological levels. Using genetically engineered knock-in reporter mouse lines, here we show that normal gland-reconstituting mammary stem cells residing in the basal layer of the mammary epithelium and breast TICs originating in the luminal layer exploit the paralogous EMT-TFs Slug and Snail, respectively, which induce distinct EMT programs. Broadly, our findings suggest that the seemingly similar stem-cell programs operating in TICs and normal stem cells of the corresponding normal tissue are likely to differ significantly in their details.

Metastatic colonization: settlement, adaptation and propagation of tumor cells in a foreign tissue environment.

Disseminated tumor cells must negotiate multiple situations that challenge their viability and/or proliferative capacity before they can successfully colonize distant organ sites. Thus, the shear stress caused by the blood flow may physically damage tumor cells during their translocation from primary tumors to distant organs via the circulation. In addition, the tissue microenvironment of distant organs is generally unfamiliar to tumor cells, limiting their proliferation within the parenchyma of these organs. Each of these situations involves various types of interactions between tumor cells and host components, which either support or inhibit the establishment and subsequent progression of metastases. The initial formation of micrometastases, as well as their subsequent growth--often termed colonization--therefore require complex adaptations by tumor cells to various host components, most of which are never encountered by these cells during their growth within primary tumor sites. These difficulties explain why the colonization of distant organs by disseminated tumor cells is an extraordinarily demanding task and thus inefficient, and suggests a number of potential targets that might be used in the future to interfere therapeutically with this process. Studying the details of tumor-host interactions at each of the steps leading up to successful metastatic colonization may therefore pave the way for designing therapeutic strategies to counteract the metastatic spread of malignant tumors.

A Ubiquitination Cascade Regulating the Integrated Stress Response and Survival in Carcinomas.

Systematic identification of signaling pathways required for the fitness of cancer cells will facilitate the development of new cancer therapies. We used gene essentiality measurements in 1,086 cancer cell lines to identify selective coessentiality modules and found that a ubiquitin ligase complex composed of UBA6, BIRC6, KCMF1, and UBR4 is required for the survival of a subset of epithelial tumors that exhibit a high degree of aneuploidy. Suppressing BIRC6 in cell lines that are dependent on this complex led to a substantial reduction in cell fitness in vitro and potent tumor regression in vivo. Mechanistically, BIRC6 suppression resulted in selective activation of the integrated stress response (ISR) by stabilization of the heme-regulated inhibitor, a direct ubiquitination target of the UBA6/BIRC6/KCMF1/UBR4 complex. These observations uncover a novel ubiquitination cascade that regulates ISR and highlight the potential of ISR activation as a new therapeutic strategy. SIGNIFICANCE: We describe the identification of a heretofore unrecognized ubiquitin ligase complex that prevents the aberrant activation of the ISR in a subset of cancer cells. This provides a novel insight on the regulation of ISR and exposes a therapeutic opportunity to selectively eliminate these cancer cells. See related commentary Leli and Koumenis, p. 535. This article is highlighted in the In This Issue feature, p. 517.

Integrin beta1-focal adhesion kinase signaling directs the proliferation of metastatic cancer cells disseminated in the lungs.

The development of metastases is an extended and inefficient process involving multiple steps. The last of these involves the growth of micrometastases into macroscopic tumors. We show here that intravenously injected, nonmetastatic cancer cells cease proliferating after extravasating into the parenchyma of the lungs; this response is attributable to the cells inability to trigger adhesion-related signaling events when they are scattered sparsely within the extracellular matrix (ECM) of the parenchyma. We recapitulate this situation by culturing these nonmetastatic cells at low seeding density in ECM-derived gels in vitro, in which they undergo cell-cycle arrest resulting, in part, from insufficient activation of focal adhesion kinase (FAK). Metastatic cancer cells, in contrast, show sufficient FAK activation to enable their proliferation within ECM gels in vitro and continue cell-cycle progression within the lung parenchyma in vivo. Activation of FAK in these metastatic cells depends on expression of the beta(1) subunit of integrins, and proliferation of these cells after extravasation in the lungs is diminished by knocking down the expression of either FAK or integrin beta(1). These results demonstrate the critical role of integrin beta(1)-FAK signaling axis in controlling the initial proliferation of micrometastatic cancer cells disseminated in the lungs.

Global computational alignment of tumor and cell line transcriptional profiles.

Cell lines are key tools for preclinical cancer research, but it remains unclear how well they represent patient tumor samples. Direct comparisons of tumor and cell line transcriptional profiles are complicated by several factors, including the variable presence of normal cells in tumor samples. We thus develop an unsupervised alignment method (Celligner) and apply it to integrate several large-scale cell line and tumor RNA-Seq datasets. Although our method aligns the majority of cell lines with tumor samples of the same cancer type, it also reveals large differences in tumor similarity across cell lines. Using this approach, we identify several hundred cell lines from diverse lineages that present a more mesenchymal and undifferentiated transcriptional state and that exhibit distinct chemical and genetic dependencies. Celligner could be used to guide the selection of cell lines that more closely resemble patient tumors and improve the clinical translation of insights gained from cell lines.

Predicting cell health phenotypes using image-based morphology profiling.

Genetic and chemical perturbations impact diverse cellular phenotypes, including multiple indicators of cell health. These readouts reveal toxicity and antitumorigenic effects relevant to drug discovery and personalized medicine. We developed two customized microscopy assays, one using four targeted reagents and the other three targeted reagents, to collectively measure 70 specific cell health phenotypes including proliferation, apoptosis, reactive oxygen species, DNA damage, and cell cycle stage. We then tested an approach to predict multiple cell health phenotypes using Cell Painting, an inexpensive and scalable image-based morphology assay. In matched CRISPR perturbations of three cancer cell lines, we collected both Cell Painting and cell health data. We found that simple machine learning algorithms can predict many cell health readouts directly from Cell Painting images, at less than half the cost. We hypothesized that these models can be applied to accurately predict cell health assay outcomes for any future or existing Cell Painting dataset. For Cell Painting images from a set of 1500+ compound perturbations across multiple doses, we validated predictions by orthogonal assay readouts. We provide a web app to browse predictions: http://broad.io/cell-health-app. Our approach can be used to add cell health annotations to Cell Painting datasets.

Integration of interferon-alpha/beta signalling to p53 responses in tumour suppression and antiviral defence.

Swift elimination of undesirable cells is an important feature in tumour suppression and immunity. The tumour suppressor p53 and interferon-alpha and -beta (IFN-alpha/beta) are essential for the induction of apoptosis in cancerous cells and in antiviral immune responses, respectively, but little is known about their interrelationship. Here we show that transcription of the p53 gene is induced by IFN-alpha/beta, accompanied by an increase in p53 protein level. IFN-alpha/beta signalling itself does not activate p53; rather, it contributes to boosting p53 responses to stress signals. We show examples in which p53 gene induction by IFN-alpha/beta contributes to tumour suppression. Furthermore, we show that p53 is activated in virally infected cells to evoke an apoptotic response and that p53 is critical for antiviral defence of the host. Our study reveals a hitherto unrecognized link between p53 and IFN-alpha/beta in tumour suppression and antiviral immunity, which may have therapeutic implications.

Multiplexed single-cell transcriptional response profiling to define cancer vulnerabilities and therapeutic mechanism of action.

Assays to study cancer cell responses to pharmacologic or genetic perturbations are typically restricted to using simple phenotypic readouts such as proliferation rate. Information-rich assays, such as gene-expression profiling, have generally not permitted efficient profiling of a given perturbation across multiple cellular contexts. Here, we develop MIX-Seq, a method for multiplexed transcriptional profiling of post-perturbation responses across a mixture of samples with single-cell resolution, using SNP-based computational demultiplexing of single-cell RNA-sequencing data. We show that MIX-Seq can be used to profile responses to chemical or genetic perturbations across pools of 100 or more cancer cell lines. We combine it with Cell Hashing to further multiplex additional experimental conditions, such as post-treatment time points or drug doses. Analyzing the high-content readout of scRNA-seq reveals both shared and context-specific transcriptional response components that can identify drug mechanism of action and enable prediction of long-term cell viability from short-term transcriptional responses to treatment.

Therapeutic potential of proapoptotic molecule Noxa in the selective elimination of tumor cells.

The selective elimination of tumor cells by inducing apoptosis is one of the most important issues in cancer therapy. In this context, artificial expression of the p53 tumor-suppressor gene has been an attractive approach and numerous studies have shown its efficacy in combination with other therapies such as radiation or chemotherapy. One of the critical issues for current cancer gene therapy is how to induce apoptosis in cancer cells without affecting normal cells. In the present study, we examined the potential of Noxa, a BH3-only protein with proapoptotic activity that functions downstream of the p53-mediated apoptotic pathway, to selectively induce apoptosis in tumor cells. We found that upon infection of a recombinant adenovirus contrived to express the Noxa gene, apoptosis was induced in vitro in several human breast cancer cell lines, but not in normal mammary epithelial cell lines. Furthermore, intratumoral injection of the Noxa-expressing adenovirus resulted in marked shrinkage of the transplanted tumor derived from breast cancer cells without any notable adverse effect on the surrounding normal tissue. In contrast, the expression of Puma, another BH3-only protein that also functions downstream of the p53 pathway, induced apoptosis in both cancer and normal cells. Thus, our results suggest a mechanism wherein Noxa, but not Puma, selectively induces apoptosis in human tumor cells. These data provide a new prospect for cancer therapy by the Noxa-mediated selective elimination of malignant cells.

Syndecan-Mediated Ligation of ECM Proteins Triggers Proliferative Arrest of Disseminated Tumor Cells.

Systemic dissemination of tumor cells often begins long before the development of overt metastases, revealing the inefficient nature of the metastatic process. Thus, already at the time of initial clinical presentation, many patients with cancer harbor a myriad disseminated tumor cells (DTC) throughout the body, most of which are found as mitotically quiescent solitary cells. This indicates that the majority of DTCs fail, for still unknown reasons, to initiate rapid proliferation after entering foreign tissue, which likely contributes significantly to the inefficiency of metastasis formation. Here, we showed that extracellular matrix (ECM) components of the host parenchyma prevented proliferation of DTCs that had recently infiltrated foreign tissue by binding to syndecan receptors expressed on the surface of these cells. This led to the recruitment of the Par-3:Par-6:atypical PKC protein complex, a critical regulator of cell polarity, to the plasma membrane and release of Par-1 kinase into the cytosol. Cytosolic Par-1 bound, phosphorylated, and inactivated KSR scaffolding proteins ultimately inhibited Ras/ERK signaling and, in turn, cell proliferation. Inhibition of the syndecan-mediated signaling restored the proliferation of otherwise dormant DTCs, enabling these cells to efficiently colonize foreign tissues. Intriguingly, naturally aggressive cancer cells overcame the antiproliferative effect of syndecan-mediated signaling either by shutting down this signaling pathway or by activating a proproliferative signaling pathway that works independent of syndecan-mediated signaling. Collectively, these observations indicate that the proliferative arrest of DTCs is attributable, in part, to the syndecan-mediated ligation of ECM proteins. SIGNIFICANCE: This study identifies a novel signaling pathway that regulates the proliferative dormancy of individual disseminated tumor cells.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/23/5944/F1.large.jpg.

EMT, CSCs, and drug resistance: the mechanistic link and clinical implications.

The success of anticancer therapy is usually limited by the development of drug resistance. Such acquired resistance is driven, in part, by intratumoural heterogeneity - that is, the phenotypic diversity of cancer cells co-inhabiting a single tumour mass. The introduction of the cancer stem cell (CSC) concept, which posits the presence of minor subpopulations of CSCs that are uniquely capable of seeding new tumours, has provided a framework for understanding one dimension of intratumoural heterogeneity. This concept, taken together with the identification of the epithelial-to-mesenchymal transition (EMT) programme as a critical regulator of the CSC phenotype, offers an opportunity to investigate the nature of intratumoural heterogeneity and a possible mechanistic basis for anticancer drug resistance. In fact, accumulating evidence indicates that conventional therapies often fail to eradicate carcinoma cells that have entered the CSC state via activation of the EMT programme, thereby permitting CSC-mediated clinical relapse. In this Review, we summarize our current understanding of the link between the EMT programme and the CSC state, and also discuss how this knowledge can contribute to improvements in clinical practice.

Inflammation Triggers Zeb1-Dependent Escape from Tumor Latency.

The emergence of metastatic disease in cancer patients many years or decades after initial successful treatment of primary tumors is well documented but poorly understood at the molecular level. Recent studies have begun exploring the cell-intrinsic programs, causing disseminated tumor cells to enter latency and the cellular signals in the surrounding nonpermissive tissue microenvironment that maintain the latent state. However, relatively little is known about the mechanisms that enable disseminated tumor cells to escape cancer dormancy or tumor latency. We describe here an in vivo model of solitary metastatic latency in the lung parenchyma. The induction of a localized inflammation in the lungs, initiated by lipopolysaccharide treatment, triggers the awakening of these cells, which develop into macroscopic metastases. The escape from latency is dependent on the expression of Zeb1, a key regulator of the epithelial-to-mesenchymal transition (EMT). Furthermore, activation of the EMT program on its own, as orchestrated by Zeb1, is sufficient to incite metastatic outgrowth by causing carcinoma cells to enter stably into a metastasis-initiating cell state. Cancer Res; 76(23); 6778-84. ©2016 AACR.

Correlating intravital multi-photon microscopy to 3D electron microscopy of invading tumor cells using anatomical reference points.

Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior in vivo with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D) mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis.

The epithelial-mesenchymal transition factor SNAIL paradoxically enhances reprogramming.

Reprogramming of fibroblasts to induced pluripotent stem cells (iPSCs) entails a mesenchymal to epithelial transition (MET). While attempting to dissect the mechanism of MET during reprogramming, we observed that knockdown (KD) of the epithelial-to-mesenchymal transition (EMT) factor SNAI1 (SNAIL) paradoxically reduced, while overexpression enhanced, reprogramming efficiency in human cells and in mouse cells, depending on strain. We observed nuclear localization of SNAI1 at an early stage of fibroblast reprogramming and using mouse fibroblasts expressing a knockin SNAI1-YFP reporter found cells expressing SNAI1 reprogrammed at higher efficiency. We further demonstrated that SNAI1 binds the let-7 promoter, which may play a role in reduced expression of let-7 microRNAs, enforced expression of which, early in the reprogramming process, compromises efficiency. Our data reveal an unexpected role for the EMT factor SNAI1 in reprogramming somatic cells to pluripotency.

An integrin-linked machinery of cytoskeletal regulation that enables experimental tumor initiation and metastatic colonization.

Recently extravasated metastatic cancer cells use the Rif/mDia2 actin-nucleating/polymerizing machinery in order to extend integrin ß1-containing, filopodium-like protrusions (FLPs), which enable them to interact productively with the surrounding extracellular matrix; this process governs the initial proliferation of these cancer cells. Here, we identify the signaling pathway governing FLP lifetime, which involves integrin-linked kinase (ILK) and ß-parvin, two integrin:actin-bridging proteins that block cofilin-mediated actin-filament severing. Notably, the combined actions of Rif/mDia2 and ILK/ß-parvin/cofilin pathways on FLPs are required not only for metastatic outgrowth but also for primary tumor formation following experimental implantation. This provides one mechanistic explanation for how the epithelial-mesenchymal transition (EMT) program imparts tumor-initiating powers to carcinoma cells, since it enhances FLP formation through the activation of ILK/ß-parvin/cofilin pathway.

The outgrowth of micrometastases is enabled by the formation of filopodium-like protrusions.

UNLABELLED: Disseminated cancer cells that have extravasated into the tissue parenchyma must interact productively with its extracellular matrix components to survive, proliferate, and form macroscopic metastases. The biochemical and cell biologic mechanisms enabling this interaction remain poorly understood. We find that the formation of elongated integrin ß(1)-containing adhesion plaques by cancer cells that have extravasated into the lung parenchyma enables the proliferation of these cells via activation of focal adhesion kinase. These plaques originate in and appear only after the formation of filopodium-like protrusions (FLP) that harbor integrin ß(1) along their shafts. The cytoskeleton-regulating proteins Rif and mDia2 contribute critically to the formation of these protrusions and thereby enable the proliferation of extravasated cancer cells. Hence, the formation of FLPs represents a critical rate-limiting step for the subsequent development of macroscopic metastases. SIGNIFICANCE: Although the mechanisms of metastatic dissemination have begun to be uncovered, those involved in the establishment of extravasated cancer cells in foreign tissue microenvironments remained largely obscure. We have studied the behavior of recently extravasated cancer cells in the lungs and identified a series of cell biologic processes involving the formation of filopodium-like protrusions and the subsequent development of elongated, mature adhesion plaques, which contribute critically to the rapid proliferation of the micrometastatic cells and thus are prerequisites to the eventual lung colonization by these cells.

BH3-only proteins: integrated control point of apoptosis.

Bcl-2 family proteins play central roles in the regulation of most, if not all, apoptotic pathways, and hence this family plays a critical role in oncogenesis. The Bcl-2 homology 3 (BH3)-only members of this family are "proapoptotic," promoting apoptosis by sensing cellular stresses: that is, they are activated or induced in response to stress stimuli. These BH3-only proteins then interfere with the function of "prosurvival" Bcl-2 family members, thereby promoting the progression of apoptosis. It has long been recognized that the induction and activation of each of the BH3-only proteins are subject to the type of stress stimulus. Although it was originally assumed that all the BH3-only proteins exert similar effects on the downstream apoptotic machinery, recent studies have uncovered their distinct functional properties, indicating the operation of more intricate, versatile control mechanisms of apoptosis. In this review, we will summarize recent findings on the diversity in the activation and function of BH3-only proteins. In particular, we focus on the overlapping and individual roles of 2 BH3-only proteins, Puma and Noxa, in the context of the apoptotic response induced by the tumor suppressor p53.

Differential contribution of Puma and Noxa in dual regulation of p53-mediated apoptotic pathways.

The activation of tumor suppressor p53 induces apoptosis or cell cycle arrest depending on the state and type of cell, but it is not fully understood how these different responses are regulated. Here, we show that Puma and Noxa, the well-known p53-inducible proapoptotic members of the Bcl-2 family, differentially participate in dual pathways of the induction of apoptosis. In normal cells, Puma but not Noxa induces mitochondrial outer membrane permeabilization (MOMP), and this function is mediated in part by a pathway that involves calcium release from the endoplasmic reticulum (ER) and the subsequent caspase activation. However, upon E1A oncoprotein expression, cells also become susceptible to MOMP induction by Noxa, owing to their sensitization to the ER-independent pathway. These findings offer a new insight into differential cellular responses induced by p53, and may have therapeutic implications in cancer.