Stromal Signals Dominate Gene Expression Signature Scores That Aim to Describe Cancer Cell-intrinsic Stemness or Mesenchymality Characteristics.

Epithelial-to-mesenchymal transition (EMT) in cancer cells confers migratory abilities, a crucial aspect in the metastasis of tumors that frequently leads to death. In multiple studies, authors proposed gene expression signatures for EMT, stemness, or mesenchymality of tumors based on bulk tumor expression profiling. However, recent studies suggested that noncancerous cells from the microenvironment or macroenvironment heavily influence such signature profiles. Here, we strengthen these findings by investigating 11 published and frequently referenced gene expression signatures that were proposed to describe EMT-related (EMT, mesenchymal, or stemness) characteristics in various cancer types. By analyses of bulk, single-cell, and pseudobulk expression data, we show that the cell type composition of a tumor sample frequently dominates scores of these EMT-related signatures. A comprehensive, integrated analysis of bulk RNA sequencing (RNA-seq) and single-cell RNA-seq data shows that stromal cells, most often fibroblasts, are the main drivers of EMT-related signature scores. We call attention to the risk of false conclusions about tumor properties when interpreting EMT-related signatures, especially in a clinical setting: high patient scores of EMT-related signatures or calls of "stemness subtypes" often result from low cancer cell content in tumor biopsies rather than cancer cell-specific stemness or mesenchymal/EMT characteristics. SIGNIFICANCE: Cancer self-renewal and migratory abilities are often characterized via gene module expression profiles, also called EMT or stemness gene expression signatures. Using published clinical tumor samples, cancer cell lines, and single cancer cells, we highlight the dominating influence of noncancer cells in low cancer cell content biopsies on their scores. We caution on their application for low cancer cell content clinical cancer samples with the intent to assign such characteristics or subtypes.

Identification of a Notch transcriptomic signature for breast cancer.

BACKGROUND: Dysregulated Notch signalling contributes to breast cancer development and progression, but validated tools to measure the level of Notch signalling in breast cancer subtypes and in response to systemic therapy are largely lacking. A transcriptomic signature of Notch signalling would be warranted, for example to monitor the effects of future Notch-targeting therapies and to learn whether altered Notch signalling is an off-target effect of current breast cancer therapies. In this report, we have established such a classifier. METHODS: To generate the signature, we first identified Notch-regulated genes from six basal-like breast cancer cell lines subjected to elevated or reduced Notch signalling by culturing on immobilized Notch ligand Jagged1 or blockade of Notch by γ-secretase inhibitors, respectively. From this cadre of Notch-regulated genes, we developed candidate transcriptomic signatures that were trained on a breast cancer patient dataset (the TCGA-BRCA cohort) and a broader breast cancer cell line cohort and sought to validate in independent datasets. RESULTS: An optimal 20-gene transcriptomic signature was selected. We validated the signature on two independent patient datasets (METABRIC and Oslo2), and it showed an improved coherence score and tumour specificity compared with previously published signatures. Furthermore, the signature score was particularly high for basal-like breast cancer, indicating an enhanced level of Notch signalling in this subtype. The signature score was increased after neoadjuvant treatment in the PROMIX and BEAUTY patient cohorts, and a lower signature score generally correlated with better clinical outcome. CONCLUSIONS: The 20-gene transcriptional signature will be a valuable tool to evaluate the response of future Notch-targeting therapies for breast cancer, to learn about potential effects on Notch signalling from conventional breast cancer therapies and to better stratify patients for therapy considerations.

RosettaSX: Reliable gene expression signature scoring of cancer models and patients.

Gene expression signatures have proven their potential to characterize important cancer phenomena like oncogenic signaling pathway activities, cellular origins of tumors, or immune cell infiltration into tumor tissues. Large collections of expression signatures provide the basis for their application to data sets, but the applicability of each signature in a new experimental context must be reassessed. We apply a methodology that utilizes the previously developed concept of coherent expression of genes in signatures to identify translatable signatures before scoring their activity in single tumors. We present a web interface (www.rosettasx.com) that applies our methodology to expression data from the Cancer Cell Line Encyclopaedia and The Cancer Genome Atlas. Configurable heat maps visualize per-cancer signature scores for 293 hand-curated literature-derived gene sets representing a wide range of cancer-relevant transcriptional modules and phenomena. The platform allows users to complement heatmaps of signature scores with molecular information on SNVs, CNVs, gene expression, gene dependency, and protein abundance or to analyze own signatures. Clustered heatmaps and further plots to drill-down results support users in studying oncological processes in cancer subtypes, thereby providing a rich resource to explore how mechanisms of cancer interact with each other as demonstrated by exemplary analyses of 2 cancer types.

The Dimethylbismuth Cation: Entry Into Dative Bi-Bi Bonding and Unconventional Methyl Exchange.

The isolation of simple, fundamentally important, and highly reactive organometallic compounds remains among the most challenging tasks in synthetic chemistry. The detailed characterization of such compounds is key to the discovery of novel bonding scenarios and reactivity. The dimethylbismuth cation, [BiMe2 (SbF6 )] (1), has been isolated and characterized. Its reaction with BiMe3 gives access to an unprecedented dative bond, a Bi→Bi donor-acceptor interaction. The exchange of methyl groups (arguably the simplest hydrocarbon moiety) between different metal atoms is among the most principal types of reactions in organometallic chemistry. The reaction of 1 with BiMe3 enables an SE 2(back)-type methyl exchange, which is, for the first time, investigated in detail for isolable, (pseudo-)homoleptic main-group compounds.

Aminotroponiminates: Impact of the NO2 Functional Group on Coordination, Isomerisation, and Backbone Substitution.

Aminotroponiminate (ATI) ligands are a versatile class of redox-active and potentially cooperative ligands with a rich coordination chemistry that have consequently found a wide range of applications in synthesis and catalysis. While backbone substitution of these ligands has been investigated in some detail, the impact of electron-withdrawing groups on the coordination chemistry and reactivity of ATIs has been little investigated. We report here Li, Na, and K salts of an ATI ligand with a nitro-substituent in the backbone. It is demonstrated that the NO2 group actively contributes to the coordination chemistry of these complexes, effectively competing with the N,N-binding pocket as a coordination site. This results in an unprecedented E/Z isomerisation of an ATI imino group and culminates in the isolation of the first "naked" (i. e., without directional bonding to a metal atom) ATI anion. Reactions of sodium ATIs with silver(I) and tritylium salts gave the first N,N-coordinated silver ATI complexes and unprecedented backbone substitution reactions. Analytical techniques applied in this work include multinuclear (VT-)NMR spectroscopy, single-crystal X-ray diffraction analysis, and DFT calculations.

ARP3 Controls the Podocyte Architecture at the Kidney Filtration Barrier.

Podocytes, highly specialized epithelial cells, build the outer part of the kidney filtration barrier and withstand high mechanical forces through a complex network of cellular protrusions. Here, we show that Arp2/3-dependent actin polymerization controls actomyosin contractility and focal adhesion maturation of podocyte protrusions and thereby regulates formation, maintenance, and capacity to adapt to mechanical requirements of the filtration barrier. We find that N-WASP-Arp2/3 define the development of complex arborized podocyte protrusions in vitro and in vivo. Loss of dendritic actin networks results in a pronounced activation of the actomyosin cytoskeleton and the generation of over-maturated but less efficient adhesion, leading to detachment of podocytes. Our data provide a model to explain podocyte protrusion morphology and their mechanical stability based on a tripartite relationship between actin polymerization, contractility, and adhesion.

Single cell polarity in liquid phase facilitates tumour metastasis.

Dynamic polarisation of tumour cells is essential for metastasis. While the role of polarisation during dedifferentiation and migration is well established, polarisation of metastasising tumour cells during phases of detachment has not been investigated. Here we identify and characterise a type of polarisation maintained by single cells in liquid phase termed single-cell (sc) polarity and investigate its role during metastasis. We demonstrate that sc polarity is an inherent feature of cells from different tumour entities that is observed in circulating tumour cells in patients. Functionally, we propose that the sc pole is directly involved in early attachment, thereby affecting adhesion, transmigration and metastasis. In vivo, the metastatic capacity of cell lines correlates with the extent of sc polarisation. By manipulating sc polarity regulators and by generic depolarisation, we show that sc polarity prior to migration affects transmigration and metastasis in vitro and in vivo.

Vps34 deficiency reveals the importance of endocytosis for podocyte homeostasis.

The molecular mechanisms that maintain podocytes and consequently, the integrity of the glomerular filtration barrier are incompletely understood. Here, we show that the class III phosphoinositide 3-kinase vacuolar protein sorting 34 (Vps34) plays a central role in modulating endocytic pathways, maintaining podocyte homeostasis. In mice, podocyte-specific conditional knockout of Vps34 led to early proteinuria, glomerular scarring, and death within 3-9 weeks of age. Vps34-deficient podocytes exhibited substantial vacuolization and foot process effacement. Although the formation of autophagosomes and autophagic flux were impaired, comparisons between podocyte-specific Vps34-deficient mice, autophagy-deficient mice, and doubly deficient mice suggested that defective autophagy was not primarily responsible for the severe phenotype caused by the loss of Vps34. In fact, Rab5-positive endosomal compartments, endocytosis, and fluid-phase uptake were severely disrupted in Vps34-deficient podocytes. Vps34 deficiency in nephrocytes, the podocyte-like cells of Drosophila melanogaster, resulted in a block between Rab5- and Rab7-positive endosomal compartments. In summary, these data identify Vps34 as a major regulator of endolysosomal pathways in podocytes and underline the fundamental roles of endocytosis and fluid-phase uptake for the maintenance of the glomerular filtration barrier.