Summary Tissue homeostasis requires production of newly differentiated cells from resident adult stem cells. Central to this process is the expansion of undifferentiated intermediates known as transit-amplifying (TA) cells, but how stem cells are triggered to enter this proliferative TA state remains an important open question. Using the continuously growing mouse incisor as a model of stem cell-based tissue renewal, we found that the transcriptional cofactors YAP and TAZ are required both to maintain TA cell proliferation and to inhibit differentiation. Specifically, we identified a pathway involving activation of Integrin α3 in TA cells that signals through a LATS-independent FAK/CDC42/PP1A cascade to control YAP-S397 phosphorylation and nuclear localization. This leads to Rheb expression and potentiates mTOR signaling to drive proliferation of TA cells. These findings thus reveal a YAP/TAZ signaling mechanism that coordinates stem cell expansion and differentiation during organ renewal.
SUMMARY Gliomas comprise heterogeneous malignant glial and stromal cells. While blood vessel co-option is a potential mechanism to escape anti-angiogenic therapy, the relevance of glial phenotype in this process is unclear. We show that Olig2+ oligodendrocyte precursor-like glioma cells invade by single-cell vessel co-option and preserve the blood-brain barrier (BBB). Conversely, Olig2-negative glioma cells form dense perivascular collections and promote angiogenesis and BBB breakdown, leading to innate immune cell activation. Experimentally, Olig2 promotes Wnt7b expression, a finding that correlates in human glioma profiling. Targeted Wnt7a/7b deletion or pharmacologic Wnt inhibition blocks Olig2+ glioma single-cell vessel co-option and enhances responses to temozolomide. Finally, Olig2 and Wnt7 become upregulated after anti-VEGF treatment in preclinical models and patients. Thus, glial-encoded pathways regulate distinct glioma-vascular microenvironmental interactions.
The ETS (E26) protein Elk-1 serves as a paradigm for mitogen-responsive transcription factors. It is multiply phosphorylated by mitogen-activated protein kinases (MAPKs), which it recruits into pre-initiation complexes on target gene promoters. However, events preparatory to Elk-1 phosphorylation are less well understood. Here, we identify two novel, functional elements in Elk-1 that determine its stability and nuclear accumulation. One element corresponds to a dimerization interface in the ETS domain and the second is a cryptic degron adjacent to the serum response factor (SRF)-interaction domain that marks dimerization-defective Elk-1 for rapid degradation by the ubiquitin–proteasome system. Dimerization appears to be crucial for Elk-1 stability only in the cytoplasm, as latent Elk-1 accumulates in the nucleus and interacts dynamically with DNA as a monomer. These findings define a novel role for the ETS domain of Elk-1 and demonstrate that nuclear accumulation of Elk-1 involves conformational flexibility prior to its phosphorylation by MAPKs.
The use of microRNAs as biomarkers has been proposed for many diseases, including the diagnosis of melanoma. Although hundreds of microRNAs have been identified as differentially expressed in melanomas as compared to benign melanocytic lesions, a limited consensus has been achieved across studies, constraining the effective use of these potentially useful markers. In this study, we applied a machine learning-based pipeline to a dataset consisting of genetic features, clinical features, and next-generation microRNA sequencing from micro-dissected formalin-fixed paraffin embedded melanomas and their adjacent benign precursor nevi. We identified patient age and tumor cellularity as variables that frequently confound the measured expression of potentially diagnostic microRNAs. By employing the ratios of microRNAs that were either enriched or depleted in melanoma compared to the nevi as a normalization strategy, we developed a model that classified all the available published cohorts with an area under the receiver operating characteristic curve of 0.98. External validation on an independent cohort classified lesions with 81% sensitivity and 88% specificity and was uninfluenced by the tumor content of the sample or patient age.
LIMD1 is a tumour suppressor gene (TSG) down regulated in ∼80% of lung cancers with loss also demonstrated in breast and head and neck squamous cell carcinomas. LIMD1 is also a candidate TSG in childhood acute lymphoblastic leukaemia. Mechanistically, LIMD1 interacts with pRB, repressing E2F-driven transcription as well as being a critical component of microRNA-mediated gene silencing. In this study we show a CpG island within the LIMD1 promoter contains a conserved binding motif for the transcription factor PU.1. Mutation of the PU.1 consensus reduced promoter driven transcription by 90%. ChIP and EMSA analysis demonstrated that PU.1 specifically binds to the LIMD1 promoter. siRNA depletion of PU.1 significantly reduced endogenous LIMD1 expression, demonstrating that PU.1 is a major transcriptional activator of LIMD1.
The ETS domain transcription factor Elk-1 stimulates expression of immediate early genes (IEGs) in response to mitogens. These events require phosphorylation of Elk-1 by extracellular signal-regulated kinase (ERK) and phosphorylation-dependent interaction of Elk-1 with co-activators, including histone acetyltransferases and the Mediator complex. Elk-1 also recruits ERK to the promoters of its target genes, suggesting that ERK phosphorylates additional substrates in transcription complexes at mitogen-responsive promoters. Here we report that MED14, a core subunit of the Mediator, is a bona fide ERK substrate and identify serine 986 (S986) within a serine-proline rich region of MED14 as the major ERK phosphorylation site. Mitogens induced phosphorylation of MED14 on S986 at IEG promoters; RNAi knockdown of MED14 reduced CDK8 and RNA polymerase II (RNAPII) recruitment, RNAPII C-terminal domain phosphorylation and impaired activation of IEG transcription. A single alanine substitution at S986 reduced activation of an E26 (ETS)-responsive reporter by oncogenic Ras and mitogen-induced, Elk-1-dependent transcription, whereas activities of other transcriptional activators were unaffected. We also demonstrate that Elk-1 can associate with MED14 independently of MED23, which may facilitate phosphorylation of MED14 by ERK to impart a positive and selective impact on mitogen-responsive gene expression.
BACKGROUND Previous studies of solid tumors have been restricted in their ability to map how heterogeneous cell populations evolved within the tumor in three-dimensional (3D) space due to insufficient sampling, typically one sample per tumor, and a lack of knowledge of where within the tumor the sample was obtained. Knowledge of the extensivity of heterogeneity and how it is spatially distributed is crucial for assessing whether a therapeutic target is truly tumor-wide, and for exploring how mutations relate to heterogeneity in the local microenvironment. METHODS We developed a novel platform to integrate and visualize in 3D multi-omics data generated from each of 8–10 spatially mapped samples per tumor. Together, the 171 samples collected using this approach from 16 adult diffuse glioma at diagnosis and recurrence form a novel resource – the 3D Glioma Atlas. RESULTS By maximally sampling the tumor geography without excluding samples based on low cancer cell fraction (CCF), we identify a subpopulation of glioblastoma with pervasively lower CCF likely excluded by other atlases, such as the TCGA, that used stringent CCF cutoffs. Exome sequencing of 3D-mapped samples from lower-grade tumors revealed that clonal expansions are typically spatially segregated, implying minimal tumor-wide intermixing of genetically heterogenous cells. Heterogeneity is less spatially segregated for faster-growing high-grade tumors, suggesting that cell populations expand in these tumors differently. Recurrent low-grade tumors have greater intratumoral mutational heterogeneity than newly diagnosed tumors, though this did not translate into greater dissimilarity in gene expression profiles for recurrent tumors, suggesting minimal functional impact of this additional mutational diversity on gene expression. CONCLUSIONS The delineation of spatial patterns of heterogeneity that our work provides enables more informed interpretation of biopsies and greater insight into the factors shaping intratumoral variation of gene expression patterns. Ongoing work is exploring the spatial patterning of amplification events and the tumor microenvironment.
The use of microRNAs as biomarkers has been proposed for many diseases including the diagnosis of melanoma. Although hundreds of microRNAs have been identified as differentially expressed in melanomas as compared to benign melanocytic lesions, limited consensus has been achieved across studies, constraining the effective use of these potentially useful markers. In this study we quantified microRNAs by next-generation sequencing from melanomas and their adjacent benign precursor nevi.We applied a machine learning-based pipeline to identify a microRNA signature that separated melanomas from nevi and was unaffected by confounding variables, such as patient age and tumor cell content. By employing the ratios of microRNAs that were either enriched or depleted in melanoma compared to nevi as a normalization strategy, the classifier performed similarly across multiple published microRNA datasets, obtained by microarray, small RNA sequencing, or RT-qPCR. Validation on separate cohorts of melanomas and nevi correctly classified lesions with 83% sensitivity and 71-83% specificity, independent of variation in tumor cell content of the sample or patient age.
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