Effective methods for bulk RNA-seq deconvolution using scnRNA-seq transcriptomes.

BACKGROUND: RNA profiling technologies at single-cell resolutions, including single-cell and single-nuclei RNA sequencing (scRNA-seq and snRNA-seq, scnRNA-seq for short), can help characterize the composition of tissues and reveal cells that influence key functions in both healthy and disease tissues. However, the use of these technologies is operationally challenging because of high costs and stringent sample-collection requirements. Computational deconvolution methods that infer the composition of bulk-profiled samples using scnRNA-seq-characterized cell types can broaden scnRNA-seq applications, but their effectiveness remains controversial. RESULTS: We produced the first systematic evaluation of deconvolution methods on datasets with either known or scnRNA-seq-estimated compositions. Our analyses revealed biases that are common to scnRNA-seq 10X Genomics assays and illustrated the importance of accurate and properly controlled data preprocessing and method selection and optimization. Moreover, our results suggested that concurrent RNA-seq and scnRNA-seq profiles can help improve the accuracy of both scnRNA-seq preprocessing and the deconvolution methods that employ them. Indeed, our proposed method, Single-cell RNA Quantity Informed Deconvolution (SQUID), which combines RNA-seq transformation and dampened weighted least-squares deconvolution approaches, consistently outperformed other methods in predicting the composition of cell mixtures and tissue samples. CONCLUSIONS: We showed that analysis of concurrent RNA-seq and scnRNA-seq profiles with SQUID can produce accurate cell-type abundance estimates and that this accuracy improvement was necessary for identifying outcomes-predictive cancer cell subclones in pediatric acute myeloid leukemia and neuroblastoma datasets. These results suggest that deconvolution accuracy improvements are vital to enabling its applications in the life sciences.

SIM2s directed Parkin-mediated mitophagy promotes mammary epithelial cell differentiation.

The functionally differentiated mammary gland adapts to extreme levels of stress from increased demand for energy by activating specific protective mechanisms to support neonatal health. Here, we identify the breast tumor suppressor gene, single-minded 2 s (SIM2s) as a novel regulator of mitophagy, a key component of this stress response. Using tissue-specific mouse models, we found that loss of Sim2 reduced lactation performance, whereas gain (overexpression) of Sim2s enhanced and extended lactation performance and survival of mammary epithelial cells (MECs). Using an in vitro model of MEC differentiation, we observed SIM2s is required for Parkin-mediated mitophagy, which we have previously shown as necessary for functional differentiation. Mechanistically, SIM2s localizes to mitochondria to directly mediate Parkin mitochondrial loading. Together, our data suggest that SIM2s regulates the rapid recycling of mitochondria via mitophagy, enhancing the function and survival of differentiated MECs.

Hepatoblastomas with carcinoma features represent a biological spectrum of aggressive neoplasms in children and young adults.

BACKGROUND & AIMS: Hepatoblastoma (HB) and hepatocellular carcinoma (HCC) are the predominant liver cancers in children, though their respective treatment options and associated outcomes differ dramatically. Risk stratification using a combination of clinical, histological, and molecular parameters can improve treatment selection, but it is particularly challenging for tumors with mixed histological features, including those in the recently created hepatocellular neoplasm not otherwise specified (HCN NOS) provisional category. We aimed to perform the first molecular characterization of clinically annotated cases of HCN NOS. METHODS: We tested whether these histological features are associated with genetic alterations, cancer gene dysregulation, and outcomes. Namely, we compared the molecular features of HCN NOS, including copy number alterations, mutations, and gene expression profiles, with those in other pediatric hepatocellular neoplasms, including HBs and HCCs, as well as HBs demonstrating focal atypia or pleomorphism (HB FPAs), and HBs diagnosed in older children (>8). RESULTS: Molecular profiles of HCN NOS and HB FPAs revealed common underlying biological features that were previously observed in HCCs. Consequently, we designated these tumor types collectively as HBs with HCC features (HBCs). These tumors were associated with high mutation rates (∼3 somatic mutations/Mb) and were enriched with mutations and alterations in key cancer genes and pathways. In addition, recurrent large-scale chromosomal gains, including gains of chromosomal arms 2q (80%), 6p (70%), and 20p (70%), were observed. Overall, HBCs were associated with poor clinical outcomes. CONCLUSIONS: Our study indicates that histological features seen in HBCs are associated with combined molecular features of HB and HCC, that HBCs are associated with poor outcomes irrespective of patient age, and that transplanted patients are more likely to have good outcomes than those treated with chemotherapy and surgery alone. These findings highlight the importance of molecular testing and early therapeutic intervention for aggressive childhood hepatocellular neoplasms. LAY SUMMARY: We molecularly characterized a class of histologically aggressive childhood liver cancers and showed that these tumors are clinically aggressive and that their observed histological features are associated with underlying recurrent molecular features. We proposed a diagnostic algorithm to identify these cancers using a combination of histological and molecular features, and our analysis suggested that these cancers may benefit from specialized treatment strategies that may differ from treatment guidelines for other childhood liver cancers.

HepT1-derived murine models of high-risk hepatoblastoma display vascular invasion, metastasis, and circulating tumor cells.

Hepatoblastoma (HB) is the most common pediatric primary liver malignancy, and survival for high-risk disease approaches 50%. Mouse models of HB fail to recapitulate hallmarks of high-risk disease. The aim of this work was to generate murine models that show high-risk features including multifocal tumors, vascular invasion, metastasis, and circulating tumor cells (CTCs). HepT1 cells were injected into the livers or tail veins of mice, and tumor growth was monitored with magnetic resonance and bioluminescent imaging. Blood was analyzed with fluorescence-activated cell sorting to identify CTCs. Intra- and extra-hepatic tumor samples were harvested for immunohistochemistry and RNA and DNA sequencing. Cell lines were grown from tumor samples and profiled with RNA sequencing. With intrahepatic injection of HepT1 cells, 100% of animals grew liver tumors and showed vascular invasion, metastasis, and CTCs. Mutation profiling revealed genetic alterations in seven cancer-related genes, while transcriptomic analyses showed changes in gene expression with cells that invade vessels. Tail vein injection of HepT1 cells resulted in multifocal, metastatic disease. These unique models will facilitate further meaningful studies of high-risk HB. This article has an associated First Person interview with the first author of the paper.

Nuclear Receptor 4A2 (NR4A2/NURR1) Regulates Autophagy and Chemoresistance in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor prognosis and chemotherapy with gemcitabine has limited effects and is associated with development of drug resistance. Treatment of Panc1 and MiaPaca2 pancreatic cancer cells with gemcitabine induced expression of the orphan nuclear receptor 4A2 (NURR1) and analysis of the cancer genome atlas indicated the NURR1 is overexpressed in pancreatic tumors and is a negative prognostic factor for patient survival. Results of NURR1 knockdown or treatment with the NURR1 antagonist 1,1-bis(3΄-indolyl)-1-(p-chlorophenyl)methane (C-DIM 12) demonstrated that NURR1 was pro-oncogenic in pancreatic cancer cells and regulated cancer cell and tumor growth and survival. NURR1 is induced by gemcitabine and serves as a key drug-resistance factor and is also required for gemcitabine-induced cytoprotective autophagy. NURR1 regulated genes were determined by RNA sequencing of mRNAs expressed in MiaPaCa2 cells expressing NURR1 and in CRISPR/Cas9 gene edited cells for NURR1 knockdown and KEGG enrichment analysis of the differentially expressed genes showed that autophagy was the major pathway regulated by NURR1. Moreover, NURR1 regulated expression of two major autophagic genes ATG7 and ATG12 which are also overexpressed in pancreatic tumors and like NURR1 are negative prognostic factors for patient survival. Thus, gemcitabine-induced cytoprotective autophagy is due to the NURR1 - ATG7/ATG12 axis and this can be targeted and disrupted by NURR1 antagonist C-DIM12 demonstrating the potential clinical applications for combination therapies with gemcitabine and NURR1 antagonists.