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Supplementary Figure S1 shows the strategy and result of co-essentiality module identificationfrom the DepMap CRISPR screen dataset and further characterization of the BIRC6 module. Supplementary Figure S2 shows the viability effect of BIRC6 depletion in BIRC6-dependent and -nondependent cancer cells lines as well asin nontransformed cells. Supplementary Figure S3 shows in vitro confirmations of the inducible BIRC6 depletion (by RNAi and CRISPR) systems as well as the effect of BIRC6 depletion on metastasis in xenograft models. Supplementary Figure S4 shows detailed results for the allele competition assay to evaluate the role of the BIR and UBC domains of BIRC6 and further evidence for the physical assembly of BIRC6 ubiquitin ligase complex. Supplementary Figure S5 shows evidence for the selective activation of the p-eIF2alpha/ATF4 arm of UPR (or ISR) upon BIRC6 depletion. Supplementary Figure S6 shows evidence supporting the importance of HRI-mediated ISR activation as a mechanism underlying the loss of viability caused by BIRC6 complex suppression. Supplementary Figure S7 shows evidence for the direct regulation of HRI ubiquitination and degradation by the BIRC6 complex Supplementary Figure S8 shows elevated expression of HRI mRNA in the tumor samples compared to the normal samples in large-scale datasets (TCGA, TARGET and GTEx). Supplementary Figure S9 shows the inefficiency of the gene expression and copy number of the functionally related genes in predicting BIRC6 dependency. Supplementary Table S1 lists the top 50 co-essentiality modules identified in the analysis explained in Supplementary Figure S1A. Supplementary Table S2 lists the key DNA and RNA sequences used in this study.

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

MOV file - 3.4MB, Kinetics of the assembly and disassembly of filopodia in cells cultured under monolayer conditions

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MOV file - 3.4MB, Kinetics of the assembly and disassembly of FLPs in cells cultured under MoT conditions

MOV file - 3.4MB, Direct conversion of FLP-associated integrin clumps into the ones constituting adhesion plaques

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In vivo and in vitro growth curves of D2A1-d and parental D2A1 cells

Summary of tumor incidences at limiting dilutions for D2A1-d + Zeb1 knockdown cells

Lung metastases can form after two weeks of continuous induction of the EMT program.

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.

Summary of tumor incidences at limiting dilutions for CD24negative and CD24positive cell populations

Supplementary Figure S1. Involvement of integrin:FAK-signaling in driving ERK activation and proliferation in vivo, related to Figure 1; Supplementary Figure S2. In vitro and in vivo behaviors of various mammary carcinoma cell types, related to Figure 1; Supplementary Figure S3. MoT culture as an in vitro model system to study signaling in DTCs, related to Figures 1 and 2; Supplementary Figure S4. ECM proteins in the lungs that surround recently-extravasated solitary DTCs, related to Figures 1 and 2; Supplementary Figure S5. KSR phosphorylation in nonaggressive/aggressive cancer cell types, related to Figure 2; Supplementary Figure S6. Restoration of ERK activation and proliferation by genetic depletion of Par-1, related to Figure 3; Supplementary Figure S7. Par-1:KSR:Ras/ERK signaling in various cell line models, related to Figure 3; Supplementary Figure S8. Par-1b phosphorylation and its effect on KSR:Ras/ERK signaling, related to Figure 3; Supplementary Figure S9. Function and expression of polarity-regulating proteins, related to Figure 4; Supplementary Figure S10. Distribution of polarity-regulating proteins in 3D conditions, related to Figure 4; Supplementary Figure S11. Restoration of ERK activation and proliferation by genetic depletion of Par-3, related to Figure 5; Supplementary Figure S12. Syndecans as crucial cell-surface sensors for the 3D ECM configuration, related to Figure 6; Supplementary Figure S13. Functional role of syndecans in regulating D2.1 cell behaviors in 3D conditions, related to Figure 6; Supplementary Fig. S14. Genetic inactivation of syndecans and its effect on aggressive cell behaviors, related to Figure 7; Supplementary Fig. S15. Concomitant operations of integrin:FAK-mediated pro-proliferative signaling and syndecan-mediated anti-proliferative signaling in the aggressive D2A1 cells, extended experimental results; Supplementary Fig. S16. Restoration of Par-3 and syndecan-1 expression in the B16F10 melanoma cells, extended experimental results; Supplementary Fig. S17. Evidence for genetic alterations to inactivate syndecan-mediated anti-proliferative signaling machinery in human cancers, extended analyses; Supplementary Fig. S18. Syndecan-mediated anti-proliferative signaling machinery that prevents the outgrowth of solitary DTCs, a summary illustration

Extended descriptions on experimental strategy

Detailed description of supplementary figures

LPS-induced inflammation involves the native but not the adaptive immune system.

Summary of incidence of metastasis formation at limiting dilutions in D2A1-d and parental D2A1 cells

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.

Targeting of mutated oncogenes has led to the identification of new targeted therapies. However, druggable oncogenes do not occur in most cancers. 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 793 cancer cell lines to identify selective co-essentiality modules and found that a ubiquitination ligase complex composed of UBA6, BIRC6, KCMF1 and UBR4, which encode an E1, E2 and two heterodimeric E3 subunits, respectively, is required for the survival of a subset of epithelial tumors, particularly subtypes of breast cancer. 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 and upregulation of the heme-regulated inhibitor (HRI), 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. Citation Format: Lisa D Cervia, Tsukasa Shibue, Benjamin Gaeta, Ashir A Borah, Lisa Leung, Naomi Li, Nancy Dumont, Alfredo Gonzalez, Nolan Bick, Mariya Kazachkova, Joshua M Dempster, John M Krill-Burger, Federica Piccioni, Namrata D Udeshi, Meagan E Olive, Steven A Carr, David E Root, James M McFarland, Francisca Vazquez, William C Hahn. A ubiquitination cascade regulating the integrated stress response and survival in carcinomas [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P3-09-01.

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

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.

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.

Targeting of mutated oncogenes has led to the identification of new targeted therapies. However, druggable oncogenes do not occur in most cancers. 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 793 cancer cell lines to identify selective co-essentiality modules and found that a ubiquitination ligase complex composed of UBA6, BIRC6, KCMF1 and UBR4, which encode an E1, E2 and two heterodimeric E3 subunits, respectively, is required for the survival of a subset of epithelial tumors. 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 and upregulation of the heme-regulated inhibitor (HRI), 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. Citation Format: Lisa D. Cervia, Tsukasa Shibue, Benjamin Gaeta, Ashir A. Borah, Lisa Leung, Naomi Li, Nancy Dumont, Alfredo Gonzalez, Nolan Bick, Mariya Kazachkova, Joshua M. Dempster, John M. Krill-Burger, Federica Piccioni, Namrata D. Udeshi, Meagan E. Olive, Steven A. Carr, David E. Root, James M. McFarland, Francisca Vazquez, William C. Hahn. A ubiquitination cascade regulating the integrated stress response and survival in carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 73.

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., The determination of whether cancer cell lines recapitulate the molecular features of corresponding patient tumours remains essential for the selection of appropriate cell line models for preclinical studies. The method developed here, Celligner, integrates cancer cell line and tumour RNA-seq datasets and reveals large differences in their concordance across cell lines and cancer types.

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., Large-scale screens of chemical and genetic vulnerabilities in cancer are typically limited to simple readouts of cell viability. Here, the authors develop a method for profiling post-perturbation transcriptional responses across large pools of cancer cell lines, enabling deep characterization of shared and context-specific responses.

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 (ROS), 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 trained 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 1,500+ compound perturbations across multiple doses, we validated predictions by orthogonal assay readouts, and by confirming mitotic arrest, ROS, and DNA damage phenotypes via PLK, proteasome, and aurora kinase/tubulin inhibition, respectively. We provide an intuitive web app to browse all predictions athttp://broad.io/cell-health-app. Our approach can be used to add cell health annotations to Cell Painting perturbation datasets.

Assays to study cancer cell responses to pharmacologic or genetic perturbations are typically restricted to using simple phenotypic readouts such as proliferation rate or the expression of a marker gene. Information-rich assays, such as gene-expression profiling, are generally not amenable to efficient profiling of a given perturbation across multiple cellular contexts. Here, we developed 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, and combine it with Cell Hashing to further multiplex additional experimental conditions, such as multiple 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 can be used to predict long-term cell viability from short-term transcriptional responses to treatment.

Systematic identification of signaling pathways required for the viability of cancer cells will facilitate the development of novel cancer therapies. We used gene essentiality measurements in 726 cancer cell lines to identify selective co-essentiality modules and found a functional ubiquitination cascade containing UBA6, BIRC6, KCMF1 and UBR4, which encode an E1, E2, and two heterodimeric E3 subunits, respectively, as a vulnerability in a subset of epithelial tumors. Suppressing BIRC6 in cancer cell lines that are dependent on this ubiquitination cascade led to a strong reduction in cell fitness in vitro, and to potent tumor regression and metastasis suppression in vivo. Mechanistically, BIRC6 suppression resulted in selective and robust activation of the integrated stress response (ISR) signaling via upregulation of the heme-regulated inhibitor (HRI). Using proteomic profiling, we found that HRI itself is a key degradation target of the UBA6/BIRC6/KCMF1/UBR4 cascade. These observations demonstrate a protein ubiquitination cascade regulating ISR and highlight the potential of this cascade as a novel therapeutic target for a subset of epithelial cancers. Citation Format: Lisa D. Cervia, Tsukasa Shibue, Benjamin Gaeta, Ashir Borah, Lisa Leung, Naomi Li, Nancy Dumont, Alfredo Gonzalez, Nolan Bick, Mariya Kazachkova, Joshua Dempster, John M. Krill-Burger, Namrata Udeshi, Meagan Olive, Steven A. Carr, David E. Root, Federica Piccioni, James M. McFarland, Francisca Vazquez, William C. Hahn. A ubiquitination cascade regulates the integrated stress response and epithelial cancer survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1950.

Summary paragraph Synthetic lethality, an interaction whereby the co-occurrence of two or more genetic events lead to cell death but one event alone does not, can be exploited to develop novel cancer therapeutics1. DNA repair processes represent attractive synthetic lethal targets since many cancers exhibit an impaired DNA repair pathway, which can lead these cells to become dependent on specific repair proteins2. The success of poly (ADP-ribose) polymerase 1 (PARP-1) inhibitors in homologous recombination-deficient cancers highlights the potential of this approach in clinical oncology3,4. Hypothesizing that other DNA repair defects would give rise to alternative synthetic lethal relationships, we asked if there are specific dependencies in cancers with microsatellite instability (MSI), which results from impaired DNA mismatch repair (MMR). Here we analyzed data from large-scale CRISPR/Cas9 knockout and RNA interference (RNAi) silencing screens and found that the RecQ DNA helicase WRN was selectively essential in MSI cell lines, yet dispensable in microsatellite stable (MSS) cell lines. WRN depletion induced double-strand DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models specifically required the helicase activity, but not the exonuclease activity of WRN. These findings expose WRN as a synthetic lethal vulnerability and promising drug target in MSI cancers.

Despite its increasing success and revolutionary impact on clinical oncology, Precision Cancer Medicine still has major roadblocks before it becomes applicable to a large proportion of patients. One such roadblock is the limited number of therapeutic targets available. Indeed, for the vast majority of cancer patients, we either do not know what their specific vulnerabilities are or do not have strategies to precisely target their vulnerabilities. In the Cancer Dependency Map Project (DepMap) at the Broad Institute, we aim to overcome these limitations through the use of genome-scale loss-of-function screens in a large panel of cancer cell lines combined with systematic molecular characterization of these cell lines. To date, we have conducted viability screens with genome-wide RNAi and CRISPR/Cas9 libraries on > 800 cell lines, all of which have also been comprehensively profiled with various omics approaches. In order to systematically identify and prioritize potential therapeutic targets, we created an analytical framework that uses a multifaceted approach to score gene dependencies based on the information extracted from screening outcomes, predictive models of sensitivity from all the genetic and molecular information, and the use of priors. To reproducibly validate the nominated targets, we also developed a toolbox of standardized assays that include confirmation of cell viability effects with orthogonal reagents/read-outs and efficient testing for in vivo efficacy across multiple cancer models. Using this approach, we have identified and validated several promising targets, including the WRN DNA helicase that is selectively essential in cancers with microsatellite instability (MSI). The data, framework, and toolbox developed here can inform the nomination and advancement of promising targets for drug development for Precision Cancer Medicine. Citation Format: Tsukasa Shibue, John M. Krill-Burger, Brenton R. Paolella, Benjamin Gaeta, Adhana Asfaw, Joshua M. Dempster, James M. McFarland, David E. Root, Jesse S. Boehm, Aviad Tsherniak, William C. Hahn, Francisca Vazquez. Systematic target prioritization and validation from genome-scale loss-of-function screens in large panels of human cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-100.

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.

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., National Institutes of Health (U.S.) (Grant P01-CA080111)

Summary 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., Highlights • Knockdown of SNAIL reduces and overexpression enhances reprogramming • SNAIL-YFP-positive fractions reprogram at higher efficiency • Let-7 decreases early in reprogramming, and expression of SNAIL reduces let-7 • SNAIL binds to the promoters of let-7 family members during reprogramming, Unternaehrer and colleagues show that SNAI1, a factor associated with epithelial-to-mesenchymal transition, promotes reprogramming of somatic cells to pluripotency, a counterintuitive discovery since reprogramming requires the opposite process, mesenchymal-to-epithelial transition. SNAI1 expression is temporally associated with downregulation of the microRNA let-7, and SNAI1 binds the let-7 promoter consistent with direct regulation.

Microsatellite instability (MSI), a class of genetic hypermutability that arises from impaired DNA mismatch repair (MMR), contributes to the development of many malignancies including colon, endometrial, gastric, and ovarian cancers. While immune checkpoint blockade (ICB) has been an effective therapy for many patients with MSI cancers, numerous patients with MSI malignancies do not respond to ICB or the use of these agents are limited by their toxicity. Hence, there is still a pressing need to develop further therapies against MSI cancers. One approach to develop novel therapeutics is to leverage synthetic lethality, a phenomenon whereby the simultaneous occurrence of two or more genetic events lead to cell death but one event alone does not. DNA repair processes represent attractive synthetic lethal targets since many cancers exhibit an impaired DNA repair pathway, which can lead these cancers to become dependent on specific repair proteins. The success of poly (ADP ribose) polymerase (PARP) inhibitors in homologous recombination-deficient cancers highlights the potential of this approach. Hypothesizing that other DNA repair defects would give rise to alternative synthetic lethal relationships, we asked if there are specific dependencies in MSI cancers. Here, we analyzed data from large-scale CRISPR/Cas9 and RNA interference (RNAi) functional genomic screens and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, but dispensable in microsatellite stable models. WRN silencing induced double-strand DNA breaks, activated a DNA-damage response, and promoted apoptosis and cell cycle arrest preferentially in MSI models. MSI cancer models specifically required WRN’s helicase activity, but not its exonuclease activity. These findings expose WRN as a synthetic lethal vulnerability and promising drug target for MSI cancers. Citation Format: Edmond M. Chan, Tsukasa Shibue, James McFarland, Benjamin Gaeta, Francisca Vazquez, Adam J. Bass. WRN helicase is a synthetic lethal target in microsatellite unstable cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-048.

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 β1-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 β1 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., National Institutes of Health (U.S.) (Grant P01-CA080111)

SummaryRegulatory 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.

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.

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.

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.

Numerous genotoxic stresses interfere with the integrity of the genome; hence, cells maintain various mechanisms to avoid genetic alterations that would otherwise cause cell transformation (Lengauer et al. 1998). Depending on the degree and/or the nature of DNA damage, cells undergo either cell cycle arrest or apoptosis in order to allow DNA repair or altruistic suicide of cells, respectively (Rich et al. 2000; Zhou and Elledge 2000). The tumor suppressor p53 is the most prominent factor for the maintenance of genome integrity; p53 is activated in response to DNA damage and plays a key role in determining the fate of cells (Vousden and Lu 2002), mainly through the transcriptional induction of target genes (Ko and Prives 1996). Among the p53-mediated cellular responses, the induction of apoptosis is central to tumor suppression (Schmitt et al. 2002). In this context, numerous target genes of p53 have been identified that may be involved in p53-dependent apoptosis (Vousden and Lu 2002). However, little is known about the details of this process, particularly with regard to the interrelationship among these target genes. Bax and Bak, which are “multidomain pro-apoptotic members” of the Bcl-2 family, have been shown to play critical roles in apoptotic response (Wei et al. 2001); they form oligomers in the mitochondrial outer membrane to activate the cascade of apoptotic events. Perhaps central to the p53-dependent apoptosis is the induction of genes encoding another class of this family termed BH3-only members, which contribute to this Bax/Bak function either directly by stimulating the oligomerization or indirectly by interacting with “pro-survival members” such as Bcl-2 and Bcl-XL, which otherwise suppress the Bax/Bak function (Bouillet and Strasser 2002). The p53 target genes of the BH3-only members include Noxa (Oda et al. 2000) and Puma (Nakano and Vousden 2001; Yu et al. 2001). However, up to what extent and how these genes contribute to the p53-dependent apoptosis are currently unknown. In the present study, we generated mice deficient in Noxa, and examined the cellular response in the context of DNA-damage-induced apoptosis. Our results demonstrate that Noxa is an integral mediator of the p53-dependent apoptosis, but its contribution depends on cell type and cell state.

Tumor necrosis factor alpha plays a substantial role in a number of conditions such as inflammation, autoimmunity, insulin resistance and sleep. Three new single nucleotide polymorphisms, -1,031 T/C, -863 C/A and -857 T/C, were recently identified in the upstream 5'-flanking region of TNFA in the Japanese population. In the present study, we developed polymerase chain reaction (PCR)-preferential homoduplex formation assay for the single-step allele typing of TNFA, and determined the genotypes of 271 healthy unrelated Japanese individuals. Four haplotypes, -1,031/-863/-857 TCC, TCT, CAC and CCC, were found to constitute the majority, if not all, of the TNFA alleles of healthy Japanese population. These alleles were designated as TNFA-U01, -U02 -U03 and -U04, respectively, in the order of frequency. Based on HLA-A, -B and -DRB1 genotypes together with TNFA genotypes, multi-locus haplotypes were analyzed. Significant positive associations were observed between TNFA-U01 and A*3303, B*5201, B*4403, B*4601, B*0702, DRB1*1502, DRB1*0101, DRB1*1302, between TNFA-U02 and B*5401, B*3501, DRB1*0405, DRB1*0407, between TNFA-U03 and B*4006, B*4002, DRB1*0803, DRB1*0802, DRB1*0403, DRB1*0901, and between TNFA-U04 and B*4801. Four-locus haplotype estimation revealed that A*3303-B*4403-TNFA-U01-DRB1*1302, A*2402-B*5201-TNFA-U01-DRB1*1502 and A*2402-B*5401-TNFA-U02-DRB1*0405 constitute major extended haplotypes in Japanese. Interestingly, TNFA alleles previously shown to have a higher promoter activity (U02, U03) were found to form haplotypes with certain DRB1 alleles associated with T helper 1 (Th1)-dominant diseases such as rheumatoid arthritis, insulin dependent diabetes mellitus and Crohn's disease in Japanese. In contrast, TNFA allele with a low promoter activity (U01) is in linkage disequilibrium with the DRB1 alleles associated with T helper 2 (Th2)-dominant diseases such as atopic dermatitis and ulcerative colitis. These observations raise the possibility that TNFA upstream promoter region polymorphisms contribute to some of the HLA-disease associations.

The ability of carcinoma cells to acquire cell-biological phenotypes associated with advanced malignancies is increasingly associated with activation of the EMT (epithelial-mesenchymal transition) program. Among the acquired phenotypes are those of motility, invasiveness, the ability to disseminate, and the ability to form increased numbers of tumor-initiating cells/cancer stem cells (CSCs). We have investigated the regulation of the EMT program and found that a key intracellular determinant is the levels of the cAMP second messenger molecule. When elevated, it causes exit of more mesenchymal carcinoma cells into an epithelial state. The formation of the CSCs within tumors has long been posited to arise following the conversion of normal tissue SCs into CSCs. However, in our hands, in a transgenic model of breast carcinoma formation, the SCs reside in one cell layer of the mammary ducts while the CSCs arise in another, making the direct derivation of CSCs from normal SCs unlikely, at least in this experimental model of mammary carcinogenesis. Once the EMT program is activated, carcinoma cells are able to acquire attributes that not only enable their dissemination from the primary tumor but also affect their subsequent post-extravasation proliferation in the lung parenchyma. The basis of this proliferation derives from the EMT-induced ability of extravasated carcinoma cells to form mature focal adhesions and activate the mitogenic signaling pathway via focal adhesion kinase (FAK). Additional consequences of EMT action will be discussed. Citation Format: Diwakar Pattabiraman, Diwakar Pattabiraman, Tsukasa Shibue, Tsukasa Shibue, Xin Ye, Xin Ye, Robert A. Weinberg, Robert A. Weinberg, Robert A. Weinberg. Regulation of epithelial versus mesenchymal polarity and malignant progression. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr IA26.

Recently extravasated metastatic cancer cells use the Rif/mDia2 actin-nucleating/polymerizing machinery in order to extend integrin β1-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 β-parvin, two integrin:actin-bridging proteins that block cofilin-mediated actin-filament severing. Notably, the combined actions of Rif/mDia2 and ILK/β-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/β-parvin/cofilin pathway., National Institutes of Health (U.S.) (Grant P01 CA080111), National Institutes of Health (U.S.) (Grant U54-CA163109)

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.

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 β 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 β 1 . These results demonstrate the critical role of integrin β 1 -FAK signaling axis in controlling the initial proliferation of micrometastatic cancer cells disseminated in the lungs.