Single-Cell Applications of Next-Generation Sequencing.

The single cell is considered the basic unit of biology, and the pursuit of understanding how heterogeneous populations of cells can functionally coexist in tissues, organisms, microbial ecosystems, and even cancer, makes them the subject of intense study. Next-generation sequencing (NGS) of RNA and DNA has opened a new frontier of (single)-cell biology. Hundreds to millions of cells now can be assayed in parallel, providing the molecular profile of each cell in its milieu inexpensively and in a manner that can be analyzed mathematically. The goal of this article is to provide a high-level overview of single-cell sequencing for the nonexpert and show how its applications are influencing both basic and applied clinical studies in embryology, developmental genetics, and cancer.

TEsmall Identifies Small RNAs Associated With Targeted Inhibitor Resistance in Melanoma.

MicroRNAs (miRNAs) are small 21-22 nt RNAs that act to regulate the expression of mRNA target genes through direct binding to mRNA targets. While miRNAs typically dominate small RNA (sRNA) transcriptomes, many other classes are present including tRNAs, snoRNAs, snRNAs, Y-RNAs, piRNAs, and siRNAs. Interactions between processing machinery and targeting networks of these various sRNA classes remains unclear, largely because these sRNAs are typically analyzed separately. Here, we present TEsmall, a tool that allows for the simultaneous processing and analysis of sRNAs from each annotated class in a single integrated workflow. The pipeline begins with raw fastq reads and proceeds all the way to producing count tables formatted for differential expression analysis. Several interactive charts are also produced to look at overall distributions in length and annotation classes. We next applied the TEsmall pipeline to sRNA libraries generated from melanoma cells responding to targeted inhibitors of the MAPK pathway. Targeted oncogene inhibitors have emerged as way to tailor cancer therapies to the particular mutations present in a given tumor. While these targeted strategies are typically effective for short intervals, the emergence of resistance is extremely common, limiting the effectiveness of single-agent therapeutics and driving the need for a better understanding of resistance mechanisms. Using TEsmall, we identified several microRNAs and other sRNA classes that are enriched in inhibitor resistant melanoma cells in multiple melanoma cell lines and may be able to serve as markers of resistant populations more generally.

Single-cell RNA-seq analysis identifies markers of resistance to targeted BRAF inhibitors in melanoma cell populations.

Single-cell RNA-seq's (scRNA-seq) unprecedented cellular resolution at a genome-wide scale enables us to address questions about cellular heterogeneity that are inaccessible using methods that average over bulk tissue extracts. However, scRNA-seq data sets also present additional challenges such as high transcript dropout rates, stochastic transcription events, and complex population substructures. Here, we present a single-cell RNA-seq analysis and klustering evaluation (SAKE), a robust method for scRNA-seq analysis that provides quantitative statistical metrics at each step of the analysis pipeline. Comparing SAKE to multiple single-cell analysis methods shows that most methods perform similarly across a wide range of cellular contexts, with SAKE outperforming these methods in the case of large complex populations. We next applied the SAKE algorithms to identify drug-resistant cellular populations as human melanoma cells respond to targeted BRAF inhibitors (BRAFi). Single-cell RNA-seq data from both the Fluidigm C1 and 10x Genomics platforms were analyzed with SAKE to dissect this problem at multiple scales. Data from both platforms indicate that BRAF inhibitor-resistant cells can emerge from rare populations already present before drug application, with SAKE identifying both novel and known markers of resistance. These experimentally validated markers of BRAFi resistance share overlap with previous analyses in different melanoma cell lines, demonstrating the generality of these findings and highlighting the utility of single-cell analysis to elucidate mechanisms of BRAFi resistance.

Suppression of protein tyrosine phosphatase N23 predisposes to breast tumorigenesis via activation of FYN kinase.

Disruption of the balanced modulation of reversible tyrosine phosphorylation has been implicated in the etiology of various human cancers, including breast cancer. Protein Tyrosine Phosphatase N23 (PTPN23) resides in chromosomal region 3p21.3, which is hemizygously or homozygously lost in some breast cancer patients. In a loss-of-function PTPome screen, our laboratory identified PTPN23 as a suppressor of cell motility and invasion in mammary epithelial and breast cancer cells. Now, our TCGA (The Cancer Genome Atlas) database analyses illustrate a correlation between low PTPN23 expression and poor survival in breast cancers of various subtypes. Therefore, we investigated the tumor-suppressive function of PTPN23 in an orthotopic transplantation mouse model. Suppression of PTPN23 in Comma 1Dß cells induced breast tumors within 56 wk. In PTPN23-depleted tumors, we detected hyperphosphorylation of the autophosphorylation site tyrosine in the SRC family kinase (SFK) FYN as well as Tyr142 in ß-catenin. We validated the underlying mechanism of PTPN23 function in breast tumorigenesis as that of a key phosphatase that normally suppresses the activity of FYN in two different models. We demonstrated that tumor outgrowth from PTPN23-deficient BT474 cells was suppressed in a xenograft model in vivo upon treatment with AZD0530, an SFK inhibitor. Furthermore, double knockout of FYN and PTPN23 via CRISPR/CAS9 also attenuated tumor outgrowth from PTPN23 knockout Cal51 cells. Overall, this mechanistic analysis of the tumor-suppressive function of PTPN23 in breast cancer supports the identification of FYN as a therapeutic target for breast tumors with heterozygous or homozygous loss of PTPN23.

NRF2 Promotes Tumor Maintenance by Modulating mRNA Translation in Pancreatic Cancer.

Pancreatic cancer is a deadly malignancy that lacks effective therapeutics. We previously reported that oncogenic Kras induced the redox master regulator Nfe2l2/Nrf2 to stimulate pancreatic and lung cancer initiation. Here, we show that NRF2 is necessary to maintain pancreatic cancer proliferation by regulating mRNA translation. Specifically, loss of NRF2 led to defects in autocrine epidermal growth factor receptor (EGFR) signaling and oxidation of specific translational regulatory proteins, resulting in impaired cap-dependent and cap-independent mRNA translation in pancreatic cancer cells. Combined targeting of the EGFR effector AKT and the glutathione antioxidant pathway mimicked Nrf2 ablation to potently inhibit pancreatic cancer ex vivo and in vivo, representing a promising synthetic lethal strategy for treating the disease.

SETD1A modulates cell cycle progression through a miRNA network that regulates p53 target genes.

Expression of the p53-inducible antiproliferative gene BTG2 is suppressed in many cancers in the absence of inactivating gene mutations, suggesting alternative mechanisms of silencing. Using a shRNA screen targeting 43 histone lysine methyltransferases (KMTs), we show that SETD1A suppresses BTG2 expression through its induction of several BTG2-targeting miRNAs. This indirect but highly specific mechanism, by which a chromatin regulator that mediates transcriptional activating marks can lead to the downregulation of a critical effector gene, is shared with multiple genes in the p53 pathway. Through such miRNA-dependent effects, SETD1A regulates cell cycle progression in vitro and modulates tumorigenesis in mouse xenograft models. Together, these observations help explain the remarkably specific genetic consequences associated with alterations in generic chromatin modulators in cancer.

TGF-ß/Smad signaling through DOCK4 facilitates lung adenocarcinoma metastasis.

The mechanisms by which TGF-ß promotes lung adenocarcinoma (ADC) metastasis are largely unknown. Here, we report that in lung ADC cells, TGF-ß potently induces expression of DOCK4, but not other DOCK family members, via the Smad pathway and that DOCK4 induction mediates TGF-ß's prometastatic effects by enhancing tumor cell extravasation. TGF-ß-induced DOCK4 stimulates lung ADC cell protrusion, motility, and invasion without affecting epithelial-to-mesenchymal transition. These processes, which are fundamental to tumor cell extravasation, are driven by DOCK4-mediated Rac1 activation, unveiling a novel link between TGF-ß and Rac1. Thus, our findings uncover the atypical Rac1 activator DOCK4 as a key component of the TGF-ß/Smad pathway that promotes lung ADC cell extravasation and metastasis.

Organoid models of human and mouse ductal pancreatic cancer.

Pancreatic cancer is one of the most lethal malignancies due to its late diagnosis and limited response to treatment. Tractable methods to identify and interrogate pathways involved in pancreatic tumorigenesis are urgently needed. We established organoid models from normal and neoplastic murine and human pancreas tissues. Pancreatic organoids can be rapidly generated from resected tumors and biopsies, survive cryopreservation, and exhibit ductal- and disease-stage-specific characteristics. Orthotopically transplanted neoplastic organoids recapitulate the full spectrum of tumor development by forming early-grade neoplasms that progress to locally invasive and metastatic carcinomas. Due to their ability to be genetically manipulated, organoids are a platform to probe genetic cooperation. Comprehensive transcriptional and proteomic analyses of murine pancreatic organoids revealed genes and pathways altered during disease progression. The confirmation of many of these protein changes in human tissues demonstrates that organoids are a facile model system to discover characteristics of this deadly malignancy.

G-protein-coupled receptor GPR161 is overexpressed in breast cancer and is a promoter of cell proliferation and invasion.

Triple-negative breast cancer (TNBC) accounts for 20% of breast cancer in women and lacks an effective targeted therapy. Therefore, finding common vulnerabilities in these tumors represents an opportunity for more effective treatment. Despite the growing appreciation of G-protein-coupled receptor (GPCR)-mediated signaling in cancer pathogenesis, very little is known about the role GPCRs play in TNBC. Using genomic information of human breast cancer, we have discovered that the orphan GPCR, G-protein-coupled receptor 161 (GPR161) is overexpressed specifically in TNBC and correlates with poor prognosis. Knockdown of GPR161 impairs proliferation of human basal breast cancer cell lines. Overexpression of GPR161 in human mammary epithelial cells increases cell proliferation, migration, intracellular accumulation of E-cadherin, and formation of multiacinar structures in 3D culture. GPR161 forms a signaling complex with the scaffold proteins ß-arrestin 2 and Ile Gln motif containing GTPase Activating Protein 1, a regulator of mammalian target of rapamycin complex 1 and E-cadherin. Consistently, GPR161 amplified breast tumors and cells overexpressing GPR161 activate mammalian target of rapamycin signaling and decrease Ile Gln motif containing GTPase Activating Protein 1 phosphorylation. Thus, we identify the orphan GPCR, GPR161, as an important regulator and a potential drug target for TNBC.

Chd5 requires PHD-mediated histone 3 binding for tumor suppression.

Chromodomain Helicase DNA binding protein 5 (CHD5) is a tumor suppressor mapping to 1p36, a genomic region that is frequently deleted in human cancer. Although CHD5 belongs to the CHD family of chromatin-remodeling proteins, whether its tumor-suppressive role involves an interaction with chromatin is unknown. Here we report that Chd5 binds the unmodified N terminus of H3 through its tandem plant homeodomains (PHDs). Genome-wide chromatin immunoprecipitation studies reveal preferential binding of Chd5 to loci lacking the active mark H3K4me3 and also identify Chd5 targets implicated in cancer. Chd5 mutations that abrogate H3 binding are unable to inhibit proliferation or transcriptionally modulate target genes, which leads to tumorigenesis in vivo. Unlike wild-type Chd5, Chd5-PHD mutants are unable to induce differentiation or efficiently suppress the growth of human neuroblastoma in vivo. Our work defines Chd5 as an N-terminally unmodified H3-binding protein and provides functional evidence that this interaction orchestrates chromatin-mediated transcriptional programs critical for tumor suppression.