Subpopulation commensalism promotes Rac1-dependent invasion of single cells via laminin-332.

Phenotypic heterogeneity poses a significant hurdle for cancer treatment but is under-characterized in the context of tumor invasion. Amidst the range of phenotypic heterogeneity across solid tumor types, collectively invading cells and single cells have been extensively characterized as independent modes of invasion, but their intercellular interactions have rarely been explored. Here, we isolated collectively invading cells and single cells from the heterogeneous 4T1 cell line and observed extensive transcriptional and epigenetic diversity across these subpopulations. By integrating these datasets, we identified laminin-332 as a protein complex exclusively secreted by collectively invading cells. Live-cell imaging revealed that laminin-332 derived from collectively invading cells increased the velocity and directionality of single cells. Despite collectively invading and single cells having similar expression of the integrin α6ß4 dimer, single cells demonstrated higher Rac1 activation upon laminin-332 binding to integrin α6ß4. This mechanism suggests a novel commensal relationship between collectively invading and single cells, wherein collectively invading cells promote the invasive potential of single cells through a laminin-332/Rac1 axis.

CeDAR: incorporating cell type hierarchy improves cell type-specific differential analyses in bulk omics data.

Bulk high-throughput omics data contain signals from a mixture of cell types. Recent developments of deconvolution methods facilitate cell type-specific inferences from bulk data. Our real data exploration suggests that differential expression or methylation status is often correlated among cell types. Based on this observation, we develop a novel statistical method named CeDAR to incorporate the cell type hierarchy in cell type-specific differential analyses of bulk data. Extensive simulation and real data analyses demonstrate that this approach significantly improves the accuracy and power in detecting cell type-specific differential signals compared with existing methods, especially in low-abundance cell types.

MERTK activation drives osimertinib resistance in EGFR-mutant non-small cell lung cancer.

Acquired resistance is inevitable in non-small cell lung cancers (NSCLCs) treated with osimertinib (OSI), and the mechanisms are not well defined. The MERTK ligand GAS6 promoted downstream oncogenic signaling in EGFR-mutated (EGFRMT) NSCLC cells treated with OSI, suggesting a role for MERTK activation in OSI resistance. Indeed, treatment with MRX-2843, a first-in-class MERTK kinase inhibitor, resensitized GAS6-treated NSCLC cells to OSI. Both GAS6 and EGF stimulated downstream PI3K/AKT and MAPK/ERK signaling in parental cells, but only GAS6 activated these pathways in OSI-resistant (OSIR) derivative cell lines. Functionally, OSIR cells were more sensitive to MRX-2843 than parental cells, suggesting acquired dependence on MERTK signaling. Furthermore, MERTK and/or its ligands were dramatically upregulated in EGFRMT tumors after treatment with OSI in both xenograft models and patient samples, consistent with induction of autocrine/paracrine MERTK activation. Moreover, treatment with MRX-2843 in combination with OSI, but not OSI alone, provided durable suppression of tumor growth in vivo, even after treatment was stopped. These data identify MERTK as a driver of bypass signaling in treatment-naive and EGFRMT-OSIR NSCLC cells and predict that MRX-2843 and OSI combination therapy will provide clinical benefit in patients with EGFRMT NSCLC.

The novel BET degrader, QCA570, is highly active against the growth of human NSCLC cells and synergizes with osimertinib in suppressing osimertinib-resistant EGFR-mutant NSCLC cells.

Lung cancer remains the leading cause of cancer deaths worldwide despite advances in knowledge in cancer biology and options of various targeted therapies. Efforts in identifying innovative and effective therapies are still highly appreciated. Targeting bromodomain and extra terminal (BET) proteins that function as epigenetic readers and master transcription coactivators is now a potential cancer therapeutic strategy. The current study evaluates the therapeutic efficacies of the novel BET degrader, QCA570, in lung cancer and explores its underlying mechanisms. QCA570 at low nanomolar ranges effectively decreased the survival of a panel of human lung cancer cell lines with induction of apoptosis in vitro. As expected, it potently induced degradation of BET proteins including BRD4, BRD3 and BRD2. Moreover, it potently decreased Mcl-1 levels due to transcriptional suppression and protein degradation; this event is critical for mediating apoptosis induced by QCA570. Moreover, QCA570 synergized with osimertinib in suppressing the growth of osimertinib-resistant cells in vitro and in vivo, suggesting potential in overcoming acquired resistance to osimertinib. These preclinical findings support the potential of QCA570 in treatment of lung cancer either as a single agent or in combination with others.

Author Correction: Truncation of mutant huntingtin in knock-in mice demonstrates exon1 huntingtin is a key pathogenic form.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-19873-9.

Simultaneous Determination of Six Chromones in Saposhnikoviae Radix via Quantitative Analysis of Multicomponents by Single Marker.

A method, quantitative analysis of multicomponents by single marker (QAMS), was established and fully verified based on high-performance liquid chromatography (HPLC) for simultaneous determination of six chromone indicators of Saposhnikoviae Radix (SR). In the present study, cimifugin (C), 5-O-methylvisamminol (V), hamaudol (H), and their corresponding glycosides, prim-O-glucosylcimifugin (GC), 4'-O-ß-D-glucosyl-5-O-methylvisamminol (GV), and sec-O-glucosylhamaudol (GH), were selected as bioactive constituents and indicators for the quality evaluation of SR. GV was chosen as the unique reference standard, and relative correction factors (RCF) between GV and the other five chromones were calculated. The feasibility of QAMS for the analysis of chromones was investigated by comparing with the traditional external standard method (ESM). Furthermore, the method was proven to have accuracy (96.98%-102.50%), repeatability (RSD <3%), stability (RSD <3%), precision (RSD <3%), and desirable linearity (R 2 ≧0.9999). Subsequently, 55 batches of commercial SR from different regions were determined by QAMS, and their contents were analyzed by principal component analysis (PCA), correlation analysis, and hierarchical cluster analysis (HCA), respectively. Based on the results, a more refined quality standard of commercial SR was proposed: SR was qualified when the total contents of six chromones were greater than 3 mg·g-1. Furthermore, SR could initially be regarded as a superior medicine when it satisfied both conditions at the same time: the total content of GC, C, GV, V, GH, and H was greater than 8 mg·g-1, and the proportion of the total content of C, V, and H was greater than 10%. This study demonstrated that the quality of SR could be successfully evaluated by the developed QAMS method; meanwhile, valuable information was provided for improving the quality standard of SR.

Truncation of mutant huntingtin in knock-in mice demonstrates exon1 huntingtin is a key pathogenic form.

Polyglutamine expansion in proteins can cause selective neurodegeneration, although the mechanisms are not fully understood. In Huntington's disease (HD), proteolytic processing generates toxic N-terminal huntingtin (HTT) fragments that preferentially kill striatal neurons. Here, using CRISPR/Cas9 to truncate full-length mutant HTT in HD140Q knock-in (KI) mice, we show that exon 1 HTT is stably present in the brain, regardless of truncation sites in full-length HTT. This N-terminal HTT leads to similar HD-like phenotypes and age-dependent HTT accumulation in the striatum in different KI mice. We find that exon 1 HTT is constantly generated but its selective accumulation in the striatum is associated with the age-dependent expression of striatum-enriched HspBP1, a chaperone inhibitory protein. Our findings suggest that tissue-specific chaperone function contributes to the selective neuropathology in HD, and highlight the therapeutic potential in blocking generation of exon 1 HTT.

Lack of RAN-mediated toxicity in Huntington's disease knock-in mice.

Identification of repeat-associated non-AUG (RAN) translation in trinucleotide (CAG) repeat diseases has led to the emerging concept that CAG repeat diseases are caused by nonpolyglutamine products. Nonetheless, the in vivo contribution of RAN translation to the pathogenesis of CAG repeat diseases remains elusive. Via CRISPR/Cas9-mediated genome editing, we established knock-in mouse models that harbor expanded CAG repeats in the mouse huntingtin gene to express RAN-translated products with or without polyglutamine peptides. We found that RAN translation is not detected in the knock-in mouse models when expanded CAG repeats are expressed at the endogenous level. Consistently, the expanded CAG repeats that cannot be translated into polyglutamine repeats do not yield the neuropathological and behavioral phenotypes that were found in knock-in mice expressing expanded polyglutamine repeats. Our findings suggest that RAN-translated products do not play a major role in the pathogenesis of CAG repeat diseases and underscore the importance in targeting polyglutamine repeats for therapeutics.

The antitumor activity of naturally occurring chromones: A review.

Chromones are a group of compounds widely distributed in nature with wide range of biological activities, including antitumor, antimicrobial, antiviral, anti-inflammatory, antioxidant, and so on. This review mainly summarizes chromones with antitumor activity in nature and classifies them into three groups, including chromanones, simple chromones, and fused chromones. They exhibit antitumor activity through broad range of mechanisms including cytotoxicity, antimetastasis, antiangiogenesis, chemoprevention, immune regulation, etc.

Rapid characterisation and identification of compounds in Saposhnikoviae Radix by high-performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight mass spectrometry.

Saposhnikoviae Radix (SR), the dried root of Saposhnikovia divaricata (Turcz.) Schischk. (Umbelliferae), is commonly used as a traditional Chinese medicine. In this study, a rapid and accurate method was firstly, developed for the qualitative analysis of SR by high-performance liquid chromatography coupled with electrospray ionisation quadrupole time-of-flight mass spectrometry (HPLC-ESI-Q-TOF-MS/MS). A total of 45 compounds were identified or tentatively characterised, including 13 chromones, 28 coumarins and four others. Among them, 16 compounds were identified from SR for the first time. In addition, six chromones reference standards, including two isolated compounds of 3'-O-angeloylhamaudol and norcimifugin from the extraction of SR, were used to study the fragmentation pathways of chromones. The developed method was effective for characterising the compounds of SR, and the results of the study enriched the understanding of the chemical connotation.

mirTrans: a resource of transcriptional regulation on microRNAs for human cell lines.

The cell-specific information of transcriptional regulation on microRNAs (miRNAs) is crucial to the precise understanding of gene regulations in various physiological and pathological processes existed in different tissues and cell types. The database, mirTrans, provides comprehensive information about cell-specific transcription of miRNAs including the transcriptional start sites (TSSs) of miRNAs, transcription factor (TF) to miRNA regulations and miRNA promoter sequences. mirTrans also maps the experimental H3K4me3 and DHS (DNase-I hypersensitive site) marks within miRNA promoters and expressed sequence tags (ESTs) within transcribed regions. The current version of database covers 35 259 TSSs and over 2.3 million TF-miRNA regulations for 1513 miRNAs in a total of 54 human cell lines. These cell lines span most of the biological systems, including circulatory system, digestive system and nervous system. Information for both the intragenic miRNAs and intergenic miRNAs is offered. Particularly, the quality of miRNA TSSs and TF-miRNA regulations is evaluated by literature curation. 23 447 TSS records and 2148 TF-miRNA regulations are supported by special experiments as a result of literature curation. EST coverage is also used to evaluate the accuracy of miRNA TSSs. Interface of mirTrans is friendly designed and convenient to make downloads (http://mcube.nju.edu.cn/jwang/lab/soft/mirtrans/ or http://120.27.239.192/mirtrans/).

miR-124-3p functions as a tumor suppressor in breast cancer by targeting CBL.

BACKGROUND: The origin and development of breast cancer remain complex and obscure. Recently, microRNA (miRNA) has been identified as an important regulator of the initiation and progression of breast cancer, and some studies have shown the essential role of miR-124-3p as a tumor suppressor in breast tumorigenesis. However, the detailed role of miR-124-3p in breast cancer remains poorly understood. METHODS: Quantitative RT-PCR and western blotting assays were used to measure miR-124-3p and CBL expression levels in breast cancer tissues, respectively. Luciferase reporter assay was employed to validate the direct targeting of CBL by miR-124-3p. Cell proliferation and invasion assays were performed to analyze the biological functions of miR-124-3p and CBL in breast cancer cells. RESULTS: In the present study, we found that miR-124-3p was consistently downregulated in breast cancer tissues. Moreover, we showed that miR-124-3p significantly suppressed the proliferation and invasion of breast cancer cells. In addition, we investigated the molecular mechanism through which miR-124-3p contributes to breast cancer tumorigenesis and identified CBL (Cbl proto-oncogene, E3 ubiquitin protein ligase) as a direct target gene of miR-124-3p. Moreover, we found that ectopic expression of CBL can attenuate the inhibitory effect of miR-124-3p on cell proliferation and invasion in breast cancer cells. CONCLUSIONS: This study identified a new regulatory axis in which miR-124-3p and CBL regulate the proliferation and invasion of breast cancer cells.

Identifying cell-specific microRNA transcriptional start sites.

MOTIVATION: Identification of microRNA (miRNA) transcriptional start sites (TSSs) is crucial to understand the transcriptional regulation of miRNA. As miRNA expression is highly cell specific, an automatic and systematic method that could identify miRNA TSSs accurately and cell specifically is in urgent requirement. RESULTS: A workflow to identify the TSSs of miRNAs was built by integrating the data of H3K4me3 and DNase I hypersensitive sites as well as combining the conservation level and sequence feature. By applying the workflow to the data for 54 cell lines from the ENCODE project, we successfully identified TSSs for 663 intragenic miRNAs and 620 intergenic miRNAs, which cover 84.2% (1283/1523) of all miRNAs recorded in miRBase 18. For these cell lines, we found 4042 alternative TSSs for intragenic miRNAs and 3186 alternative TSSs for intergenic miRNAs. Our method achieved a better performance than the previous non-cell-specific methods on miRNA TSSs. The cell-specific method developed by Georgakilas et al. gives 158 TSSs of higher accuracy in two cell lines, benefitting from the employment of deep-sequencing technique. In contrast, our method provided a much higher number of miRNA TSSs (7228) for a broader range of cell lines without the limitation of costly deep-sequencing data, thus being more applicable for various experimental cases. Analysis showed that upstream promoters at - 2 kb to - 200 bp of TSS are more conserved for independently transcribed miRNAs, while for miRNAs transcribed with host genes, their core promoters (-200 bp to 200 bp of TSS) are significantly conserved. AVAILABILITY AND IMPLEMENTATION: Predicted miRNA TSSs and promoters can be downloaded from supplementary files. CONTACT: jwang@nju.edu.cn or jlee@nju.edu.cn or edgar.wingender@bioinf.med.uni-goettingen.de SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.