Single-cell transcriptomic heterogeneity between conduit and resistance mesenteric arteries in rats.

The endothelium contains morphologically similar cells throughout the vasculature, but individual cells along the length of a single vascular tree or in different regional circulations function dissimilarly. When observations made in large arteries are extrapolated to explain the function of endothelial cells (ECs) in the resistance vasculature, only a fraction of these observations are consistent between artery sizes. To what extent endothelial (EC) and vascular smooth muscle cells (VSMCs) from different arteriolar segments of the same tissue differ phenotypically at the single-cell level remains unknown. Therefore, single-cell RNA-seq (10x Genomics) was performed using a 10X Genomics Chromium system. Cells were enzymatically digested from large (>300 µm) and small (<150 µm) mesenteric arteries from nine adult male Sprague-Dawley rats, pooled to create six samples (3 rats/sample, 3 samples/group). After normalized integration, the dataset was scaled before unsupervised cell clustering and cluster visualization using UMAP plots. Differential gene expression analysis allowed us to infer the biological identity of different clusters. Our analysis revealed 630 and 641 differentially expressed genes (DEGs) between conduit and resistance arteries for ECs and VSMCs, respectively. Gene ontology analysis (GO-Biological Processes, GOBP) of scRNA-seq data discovered 562 and 270 pathways for ECs and VSMCs, respectively, that differed between large and small arteries. We identified eight and seven unique ECs and VSMCs subpopulations, respectively, with DEGs and pathways identified for each cluster. These results and this dataset allow the discovery and support of novel hypotheses needed to identify mechanisms that determine the phenotypic heterogeneity between conduit and resistance arteries.

The R-enantiomer of ketorolac reduces ovarian cancer tumor burden in vivo.

BACKGROUND: Rho-family GTPases, including Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42), are important modulators of cancer-relevant cell functions and are viewed as promising therapeutic targets. Based on high-throughput screening and cheminformatics we identified the R-enantiomer of an FDA-approved drug (ketorolac) as an inhibitor of Rac1 and Cdc42. The corresponding S-enantiomer is a non-steroidal anti-inflammatory drug (NSAID) with selective activity against cyclooxygenases. We reported previously that R-ketorolac, but not the S-enantiomer, inhibited Rac1 and Cdc42-dependent downstream signaling, growth factor stimulated actin cytoskeleton rearrangements, cell adhesion, migration and invasion in ovarian cancer cell lines and patient-derived tumor cells. METHODS: In this study we treated mice with R-ketorolac and measured engraftment of tumor cells to the omentum, tumor burden, and target GTPase activity. In order to gain insights into the actions of R-ketorolac, we also performed global RNA-sequencing (RNA-seq) analysis on tumor samples. RESULTS: Treatment of mice with R-ketorolac decreased omental engraftment of ovarian tumor cells at 18 h post tumor cell injection and tumor burden after 2 weeks of tumor growth. R-ketorolac treatment inhibited tumor Rac1 and Cdc42 activity with little impact on mRNA or protein expression of these GTPase targets. RNA-seq analysis revealed that R-ketorolac decreased expression of genes in the HIF-1 signaling pathway. R-ketorolac treatment also reduced expression of additional genes associated with poor prognosis in ovarian cancer. CONCLUSION: These findings suggest that R-ketorolac may represent a novel therapeutic approach for ovarian cancer based on its pharmacologic activity as a Rac1 and Cdc42 inhibitor. R-ketorolac modulates relevant pathways and genes associated with disease progression and worse outcome.

Evolution of a derived protein-protein interaction between HoxA11 and Foxo1a in mammals caused by changes in intramolecular regulation.

Current models of developmental evolution suggest changes in gene regulation underlie the evolution of morphology. Despite the fact that protein complexes regulate gene expression, the evolution of regulatory protein complexes is rarely studied. Here, we investigate the evolution of a protein-protein interaction (PPI) between Homeobox A11 (HoxA11) and Forkhead box 01A (Foxo1a). Using extant and "resurrected" ancestral proteins, we show that the physical interaction between HoxA11 and Foxo1a originated in the mammalian stem lineage. Functional divergence tests and coimmunoprecipitation with heterologous protein pairs indicate that the evolution of interaction was attributable to changes in HoxA11, and deletion studies demonstrate that the interaction interface is located in the homeodomain region of HoxA11. However, there are no changes in amino acid sequence in the homeodomain region during this time period, indicating that the origin of the derived PPI was attributable to changes outside the binding interface. We infer that the amino acid substitutions in HoxA11 altered Foxo1a's access to the conserved binding interface at the HoxA11 homeodomain. We also found an expansion in the number of paired Hox/Fox binding sites in the genomes of mammalian lineage species suggesting the complex has a biological function. Our data indicate that the physical interaction between HoxA11 and Foxo1a evolved through noninterface changes that facilitate the PPI, which prevents inappropriate interactions, rather than through the evolution of a novel binding interface. We speculate that evolutionary changes of intramolecular regulation have limited pleiotropic effects compared with changes to interaction domains themselves.

The inflammatory response of human pancreatic cancer samples compared to normal controls.

Pancreatic ductal adenocarcinoma (PDAC) is a poor prognosis cancer with an aggressive growth profile that is often diagnosed at late stage and that has few curative or therapeutic options. PDAC growth has been linked to alterations in the pancreas microbiome, which could include the presence of the fungus Malassezia. We used RNA-sequencing to compare 14 matched tumor and normal (tumor adjacent) pancreatic cancer samples and found Malassezia RNA in both the PDAC and normal tissues. Although the presence of Malassezia was not correlated with tumor growth, a set of immune- and inflammatory-related genes were up-regulated in the PDAC compared to the normal samples, suggesting that they are involved in tumor progression. Gene set enrichment analysis suggests that activation of the complement cascade pathway and inflammation could be involved in pro PDAC growth.

Dominant Gene Expression Profiles Define Adenoid Cystic Carcinoma (ACC) from Different Tissues: Validation of a Gene Signature Classifier for Poor Survival in Salivary Gland ACC.

Adenoid cystic carcinoma (ACC) is an aggressive malignancy that most often arises in salivary or lacrimal glands but can also occur in other tissues. We used optimized RNA-sequencing to analyze the transcriptomes of 113 ACC tumor samples from salivary gland, lacrimal gland, breast or skin. ACC tumors from different organs displayed remarkedly similar transcription profiles, and most harbored translocations in the MYB or MYBL1 genes, which encode oncogenic transcription factors that may induce dramatic genetic and epigenetic changes leading to a dominant 'ACC phenotype'. Further analysis of the 56 salivary gland ACC tumors led to the identification of three distinct groups of patients, based on gene expression profiles, including one group with worse survival. We tested whether this new cohort could be used to validate a biomarker developed previously with a different set of 68 ACC tumor samples. Indeed, a 49-gene classifier developed with the earlier cohort correctly identified 98% of the poor survival patients from the new set, and a 14-gene classifier was almost as accurate. These validated biomarkers form a platform to identify and stratify high-risk ACC patients into clinical trials of targeted therapies for sustained clinical response.

Humanized Patient-derived Xenograft Models of Disseminated Ovarian Cancer Recapitulate Key Aspects of the Tumor Immune Environment within the Peritoneal Cavity.

The importance of the immune microenvironment in ovarian cancer progression, metastasis, and response to therapies has become increasingly clear, especially with the new emphasis on immunotherapies. To leverage the power of patient-derived xenograft (PDX) models within a humanized immune microenvironment, three ovarian cancer PDXs were grown in humanized NBSGW (huNBSGW) mice engrafted with human CD34+ cord blood-derived hematopoietic stem cells. Analysis of cytokine levels in the ascites fluid and identification of infiltrating immune cells in the tumors demonstrated that these humanized PDX (huPDX) established an immune tumor microenvironment similar to what has been reported for patients with ovarian cancer. The lack of human myeloid cell differentiation has been a major setback for humanized mouse models, but our analysis shows that PDX engraftment increases the human myeloid population in the peripheral blood. Analysis of cytokines within the ascites fluid of huPDX revealed high levels of human M-CSF, a key myeloid differentiation factor as well as other elevated cytokines that have previously been identified in ovarian cancer patient ascites fluid including those involved in immune cell differentiation and recruitment. Human tumor-associated macrophages and tumor-infiltrating lymphocytes were detected within the tumors of humanized mice, demonstrating immune cell recruitment to tumors. Comparison of the three huPDX revealed certain differences in cytokine signatures and in the extent of immune cell recruitment. Our studies show that huNBSGW PDX models reconstitute important aspects of the ovarian cancer immune tumor microenvironment, which may recommend these models for preclinical therapeutic trials. Significance: huPDX models are ideal preclinical models for testing novel therapies. They reflect the genetic heterogeneity of the patient population, enhance human myeloid differentiation, and recruit immune cells to the tumor microenvironment.

Glioblastoma initiation, migration, and cell types are regulated by core bHLH transcription factors ASCL1 and OLIG2.

Glioblastomas (GBMs) are highly aggressive, infiltrative, and heterogeneous brain tumors driven by complex driver mutations and glioma stem cells (GSCs). The neurodevelopmental transcription factors ASCL1 and OLIG2 are co-expressed in GBMs, but their role in regulating the heterogeneity and hierarchy of GBM tumor cells is unclear. Here, we show that oncogenic driver mutations lead to dysregulation of ASCL1 and OLIG2, which function redundantly to initiate brain tumor formation in a mouse model of GBM. Subsequently, the dynamic levels and reciprocal binding of ASCL1 and OLIG2 to each other and to downstream target genes then determine the cell types and degree of migration of tumor cells. Single-cell RNA sequencing (scRNA-seq) reveals that a high level of ASCL1 is key in defining GSCs by upregulating a collection of ribosomal protein, mitochondrial, neural stem cell (NSC), and cancer metastasis genes - all essential for sustaining the high proliferation, migration, and therapeutic resistance of GSCs.

Heterogeneity of Circulating Tumor Cell Neoplastic Subpopulations Outlined by Single-Cell Transcriptomics.

Fatal metastasis occurs when circulating tumor cells (CTCs) disperse through the blood to initiate a new tumor at specific sites distant from the primary tumor. CTCs have been classically defined as nucleated cells positive for epithelial cell adhesion molecule and select cytokeratins (EpCAM/CK/DAPI), while negative for the common lymphocyte marker CD45. The enumeration of CTCs allows an estimation of the overall metastatic burden in breast cancer patients, but challenges regarding CTC heterogeneity and metastatic propensities persist, and their decryption could improve therapies. CTCs from metastatic breast cancer (mBC) patients were captured using the RareCyteTM Cytefinder II platform. The Lin- and Lin+ (CD45+) cell populations isolated from the blood of three of these mBC patients were analyzed by single-cell transcriptomic methods, which identified a variety of immune cell populations and a cluster of cells with a distinct gene expression signature, which includes both cells expressing EpCAM/CK ("classic" CTCs) and cells possessing an array of genes not previously associated with CTCs. This study put forward notions that the identification of these genes and their interactions will promote novel areas of analysis by dissecting properties underlying CTC survival, proliferation, and interaction with circulatory immune cells. It improves upon capabilities to measure and interfere with CTCs for impactful therapeutic interventions.

Oncogenic Orphan Nuclear Receptor NR4A3 Interacts and Cooperates with MYB in Acinic Cell Carcinoma.

Acinic cell carcinoma (AcCC) is a morphologically distinctive salivary gland malignancy often associated with chromosome rearrangements leading to overexpression of the NR4A3 transcription factor. However, little is known about how NR4A3 contributes to AcCC biology. Detailed RNA-sequencing of 21 archived AcCC samples revealed fusion reads arising from recurrent t(4;9), t(9;12), t(8;9) or t(2;4) chromosomal translocations, which positioned highly active enhancers adjacent to the promoter of the NR4A3 gene or the closely related NR4A2 gene, resulting in their aberrant overexpression. Transcriptome analyses revealed several distinct subgroups of AcCC tumors, including a subgroup that overexpressed both NR4A3 and MSANTD3. A poor survival subset of the tumors with high-grade transformation expressed NR4A3 and POMC as well as MYB, an oncogene that is the major driver in a different type of salivary gland tumor, adenoid cystic carcinoma. The combination of NR4A3 and MYB showed cooperativity in regulating a distinct set of genes. In addition, the ligand binding domain of NR4A3 directly bound the Myb DNA binding domain. Transformation assays indicated that, while overexpressed NR4A3 was sufficient to generate transformed colonies, the combination of NR4A3 plus Myb was more potent, leading to anchorage-independent growth and increased cellular invasiveness. The results confirm that NR4A3 and NR4A2 are the main driver genes of AcCC and suggest that concurrent overexpression of NR4A3 and MYB defines a subset of AcCC patients with high-grade transformation that display exceptionally poor outcome.

Keep your fingers off my DNA: protein-protein interactions mediated by C2H2 zinc finger domains.

Cys2-His2 (C2H2) zinc finger domains (ZFs) were originally identified as DNA-binding domains, and uncharacterized domains are typically assumed to function in DNA binding. However, a growing body of evidence suggests an important and widespread role for these domains in protein binding. There are even examples of zinc fingers that support both DNA and protein interactions, which can be found in well-known DNA-binding proteins such as Sp1, Zif268, and Ying Yang 1 (YY1). C2H2 protein-protein interactions (PPIs) are proving to be more abundant than previously appreciated, more plastic than their DNA-binding counterparts, and more variable and complex in their interactions surfaces. Here we review the current knowledge of over 100 C2H2 zinc finger-mediated PPIs, focusing on what is known about the binding surface, contributions of individual fingers to the interaction, and function. An accurate understanding of zinc finger biology will likely require greater insights into the potential protein interaction capabilities of C2H2 ZFs.

The protein-binding potential of C2H2 zinc finger domains.

There are over 10,000 C2H2-type zinc finger (ZF) domains distributed among more than 1,000 ZF proteins in the human genome. These domains are frequently observed to be involved in sequence-specific DNA binding, and uncharacterized domains are typically assumed to facilitate DNA interactions. However, some ZFs also facilitate binding to proteins or RNA. Over 100 Cys2-His2 (C2H2) ZF-protein interactions have been described. We initially attempted a bioinformatics analysis to identify sequence features that would predict a DNA- or protein-binding function. These efforts were complicated by several issues, including uncertainties about the full functional capabilities of the ZFs. We therefore applied an unbiased approach to directly examine the potential for ZFs to facilitate DNA or protein interactions. The human OLF-1/EBF associated zinc finger (OAZ) protein was used as a model. The human O/E-1-associated zinc finger protein (hOAZ) contains 30 ZFs in 6 clusters, some of which have been previously indicated in DNA or protein interactions. DNA binding was assessed using a target site selection (CAST) assay, and protein binding was assessed using a yeast two-hybrid assay. We observed that clusters known to bind DNA could facilitate specific protein interactions, but clusters known to bind protein did not facilitate specific DNA interactions. Our primary conclusion is that DNA binding is a more restricted function of ZFs, and that their potential for mediating protein interactions is likely greater. These results suggest that the role of C2H2 ZF domains in protein interactions has probably been underestimated. The implication of these findings for the prediction of ZF function is discussed.

Transcriptomes define distinct subgroups of salivary gland adenoid cystic carcinoma with different driver mutations and outcomes.

The relative rarity of salivary gland adenoid cystic carcinoma (ACC) and its slow growing yet aggressive nature has complicated the development of molecular markers for patient stratification. To analyze molecular differences linked to the protracted disease course of ACC and metastases that form 5 or more years after diagnosis, detailed RNA-sequencing (RNA-seq) analysis was performed on 68 ACC tumor samples, starting with archived, formalin-fixed paraffin-embedded (FFPE) samples up to 25 years old, so that clinical outcomes were available. A statistical peak-finding approach was used to classify the tumors that expressed MYB or MYBL1, which had overlapping gene expression signatures, from a group that expressed neither oncogene and displayed a unique phenotype. Expression of MYB or MYBL1 was closely correlated to the expression of the SOX4 and EN1 genes, suggesting that they are direct targets of Myb proteins in ACC tumors. Unsupervised hierarchical clustering identified a subgroup of approximately 20% of patients with exceptionally poor overall survival (median less than 30 months) and a unique gene expression signature resembling embryonic stem cells. The results provide a strategy for stratifying ACC patients and identifying the high-risk, poor-outcome group that are candidates for personalized therapies.

Recurrent Fusions in MYB and MYBL1 Define a Common, Transcription Factor-Driven Oncogenic Pathway in Salivary Gland Adenoid Cystic Carcinoma.

UNLABELLED: Adenoid cystic carcinoma (ACC), the second most common malignancy of salivary glands, is a rare tumor with a bleak prognosis for which therapeutic targets are unavailable. We used RNA sequencing (RNA-seq) to analyze low-quality RNA from archival, formaldehyde-fixed, paraffin-embedded samples. In addition to detecting the most common ACC translocation, t(6;9) fusing the MYB proto-oncogene to NFIB, we also detected previously unknown t(8;9) and t(8;14) translocations fusing the MYBL1 gene to the NFIB and RAD51B genes, respectively. RNA-seq provided information about gene fusions, alternative RNA splicing, and gene expression signatures. Interestingly, tumors with MYB and MYBL1 translocations displayed similar gene expression profiles, and the combined MYB and MYBL1 expression correlated with outcome, suggesting that the related MYB proteins are interchangeable oncogenic drivers in ACC. Our results provide important details about the biology of ACC and illustrate how archival tissue samples can be used for detailed molecular analyses of rare tumors. SIGNIFICANCE: Using RNA-seq to perform whole-transcriptome analysis of archival ACC tumor samples, we identified novel, recurrent gene fusions, detected alternative RNA splicing, and established gene expression signatures that provide detailed information about the biology of ACC tumors.

Phosphorylation of serine 526 is required for MEKK3 activity, and association with 14-3-3 blocks dephosphorylation.

MAPK/ERK kinase kinase 3 (MEKK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that functions upstream of the MAP kinases and IkappaB kinase. Phosphorylation is believed to be a critical component for MEKK3-dependent signal transduction, but little is known about the phosphorylation sites of this MAP3K. To address this question, point mutations were introduced in the activation loop (T-loop), substituting alanine for serine or threonine, and the mutants were transfected into HEK293 Epstein-Barr virus nuclear antigen cells. MEKK3-dependent activation of an NF-kappaB reporter gene as well as ERK, JNK, and p38 MAP kinases correlated with a requirement for serine at position 526. Constitutively active mutants of MEKK3, consisting of S526D and S526E, were capable of activating a NF-kappaB luciferase reporter gene as well as ERK and MEK, suggesting that a negative charge at Ser526 was necessary for MEKK3 activity and implicating Ser526 as a phosphorylation site. An antibody was developed that specifically recognized phospho-Ser526 of MEKK3 but did not recognize the S526A point mutant. The catalytically inactive (K391M) mutant of MEKK3 was not phosphorylated at Ser526, indicating that phosphorylation of Ser526 occurs via autophosphorylation. Endogenous MEKK3 was phosphorylated on Ser526 in response to osmotic stress. In addition, phosphorylation of Ser526 was required for MKK6 phosphorylation in vitro, whereas dephosphorylation of Ser526 was mediated by protein phosphatase 2A and sensitive to okadaic acid and sodium fluoride. Finally, the association between MEKK3 and 14-3-3 was dependent on Ser526 and prevented dephosphorylation of Ser526. In summary, Ser526 of MEKK3 is an autophosphorylation site within the T-loop that is regulated by PP2A and 14-3-3 proteins.