Justin Y. Newberg

Transposon mutagenesis identifies genetic drivers of Braf V600E melanoma

Although nearly half of human melanomas harbor oncogenic BRAFV600E mutations, the genetic events that cooperate with these mutations to drive melanogenesis are still largely unknown. Here we show that Sleeping Beauty (SB) transposon-mediated mutagenesis drives melanoma progression in BrafV600E mutant mice and identify 1,232 recurrently mutated candidate cancer genes (CCGs) from 70 SB-driven melanomas. CCGs are enriched in Wnt, PI3K, MAPK and netrin signaling pathway components and are more highly connected to one another than predicted by chance, indicating that SB targets cooperative genetic networks in melanoma. Human orthologs of >500 CCGs are enriched for mutations in human melanoma or showed statistically significant clinical associations between RNA abundance and survival of patients with metastatic melanoma. We also functionally validate CEP350 as a new tumor-suppressor gene in human melanoma. SB mutagenesis has thus helped to catalog the cooperative molecular mechanisms driving BRAFV600E melanoma and discover new genes with potential clinical importance in human melanoma.

Coactivators enable glucocorticoid receptor recruitment to fine-tune estrogen receptor transcriptional responses

Nuclear receptors (NRs) are central regulators of pathophysiological processes; however, how their responses intertwine is still not fully understood. The aim of this study was to determine whether and how steroid NRs can influence each other’s activity under co-agonist treatment. We used a unique system consisting of a multicopy integration of an estrogen receptor responsive unit that allows direct visualization and quantification of estrogen receptor alpha (ERα) DNA binding, co-regulator recruitment and transcriptional readout. We find that ERα DNA loading is required for other type I nuclear receptors to be co-recruited after dual agonist treatment. We focused on ERα/glucocorticoid receptor interplay and demonstrated that it requires steroid receptor coactivators (SRC-2, SRC-3) and the mediator component MED14. We then validated this cooperative interplay on endogenous target genes in breast cancer cells. Taken together, this work highlights another layer of mechanistic complexity through which NRs cross-talk with each other on chromatin under multiple hormonal stimuli.

Transposon mutagenesis identifies genes and cellular processes driving epithelial-mesenchymal transition in hepatocellular carcinoma

Significance Epithelial-mesenchymal transition (EMT) contributes to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), but the genes driving EMT are poorly understood. Here, we describe a transposon mutagenesis screen that made it possible to identify 233 candidate cancer genes (CCGs) that are enriched for genes driving EMT in HCC. Twenty-three CCGs are predicted to function early in tumorigenesis, and alterations in these genes are associated with poor HCC patient survival. Validation studies showed that deregulation of the most highly mutated CCGs activates an EMT program that enhances HCC cell migration and also confers sorafenib resistance. Thus, transposon mutagenesis appears to provide an excellent resource for identifying genes regulating EMT in human HCC and for potentially identifying new drug targets for HCC. Epithelial-mesenchymal transition (EMT) is thought to contribute to metastasis and chemoresistance in patients with hepatocellular carcinoma (HCC), leading to their poor prognosis. The genes driving EMT in HCC are not yet fully understood, however. Here, we show that mobilization of Sleeping Beauty (SB) transposons in immortalized mouse hepatoblasts induces mesenchymal liver tumors on transplantation to nude mice. These tumors show significant down-regulation of epithelial markers, along with up-regulation of mesenchymal markers and EMT-related transcription factors (EMT-TFs). Sequencing of transposon insertion sites from tumors identified 233 candidate cancer genes (CCGs) that were enriched for genes and cellular processes driving EMT. Subsequent trunk driver analysis identified 23 CCGs that are predicted to function early in tumorigenesis and whose mutation or alteration in patients with HCC is correlated with poor patient survival. Validation of the top trunk drivers identified in the screen, including MET (MET proto-oncogene, receptor tyrosine kinase), GRB2-associated binding protein 1 (GAB1), HECT, UBA, and WWE domain containing 1 (HUWE1), lysine-specific demethylase 6A (KDM6A), and protein-tyrosine phosphatase, nonreceptor-type 12 (PTPN12), showed that deregulation of these genes activates an EMT program in human HCC cells that enhances tumor cell migration. Finally, deregulation of these genes in human HCC was found to confer sorafenib resistance through apoptotic tolerance and reduced proliferation, consistent with recent studies showing that EMT contributes to the chemoresistance of tumor cells. Our unique cell-based transposon mutagenesis screen appears to be an excellent resource for discovering genes involved in EMT in human HCC and potentially for identifying new drug targets.

Feed-forward inhibition of androgen receptor activity by glucocorticoid action in human adipocytes.

We compared transcriptomes of terminally differentiated mouse 3T3-L1 and human adipocytes to identify cell-specific differences. Gene expression and high content analysis (HCA) data identified the androgen receptor (AR) as both expressed and functional, exclusively during early human adipocyte differentiation. The AR agonist dihydrotestosterone (DHT) inhibited human adipocyte maturation by downregulation of adipocyte marker genes, but not in 3T3-L1. It is interesting that AR induction corresponded with dexamethasone activation of the glucocorticoid receptor (GR); however, when exposed to the differentiation cocktail required for adipocyte maturation, AR adopted an antagonist conformation and was transcriptionally repressed. To further explore effectors within the cocktail, we applied an image-based support vector machine (SVM) classification scheme to show that adipocyte differentiation components inhibit AR action. The results demonstrate human adipocyte differentiation, via GR activation, upregulates AR but also inhibits AR transcriptional activity.

Transposon mutagenesis identifies genes that cooperate with mutant Pten in breast cancer progression

Significance Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Despite extensive cancer genome-sequencing efforts, there is still an incomplete understanding of the genetic networks driving TNBC. Here, we used Sleeping Beauty transposon mutagenesis to identify genes that cooperate with mutant Pten in the induction of TNBC. We identified 12 candidate TNBC trunk drivers and a larger number of progression genes. Subsequent functional validation studies identified eight human TNBC tumor suppressor genes, including the GATA-like transcriptional repressor TRPS1, which was shown to inhibit lung metastasis by deregulating the expression of multiple serpin and epithelial-to-mesenchymal transition (EMT) pathway genes. Our study provides a better understanding of the genetic forces driving TNBC and discovered genes with clinical importance in TNBC. Triple-negative breast cancer (TNBC) has the worst prognosis of any breast cancer subtype. To better understand the genetic forces driving TNBC, we performed a transposon mutagenesis screen in a phosphatase and tensin homolog (Pten) mutant mice and identified 12 candidate trunk drivers and a much larger number of progression genes. Validation studies identified eight TNBC tumor suppressor genes, including the GATA-like transcriptional repressor TRPS1. Down-regulation of TRPS1 in TNBC cells promoted epithelial-to-mesenchymal transition (EMT) by deregulating multiple EMT pathway genes, in addition to increasing the expression of SERPINE1 and SERPINB2 and the subsequent migration, invasion, and metastasis of tumor cells. Transposon mutagenesis has thus provided a better understanding of the genetic forces driving TNBC and discovered genes with potential clinical importance in TNBC.

Analyzing tumor heterogeneity and driver genes in single myeloid leukemia cells with SBCapSeq

A central challenge in oncology is how to kill tumors containing heterogeneous cell populations defined by different combinations of mutated genes. Identifying these mutated genes and understanding how they cooperate requires single-cell analysis, but current single-cell analytic methods, such as PCR-based strategies or whole-exome sequencing, are biased, lack sequencing depth or are cost prohibitive. Transposon-based mutagenesis allows the identification of early cancer drivers, but current sequencing methods have limitations that prevent single-cell analysis. We report a liquid-phase, capture-based sequencing and bioinformatics pipeline, Sleeping Beauty (SB) capture hybridization sequencing (SBCapSeq), that facilitates sequencing of transposon insertion sites from single tumor cells in a SB mouse model of myeloid leukemia (ML). SBCapSeq analysis of just 26 cells from one tumor revealed the tumors major clonal subpopulations, enabled detection of clonal insertion events not detected by other sequencing methods and led to the identification of dominant subclones, each containing a unique pair of interacting gene drivers along with three to six cooperating cancer genes with SB-driven expression changes.

Automated Microscopy and Image Analysis for Androgen Receptor Function

Systems-level approaches have emerged that rely on analytical, microscopy-based technology for the discovery of novel drug targets and the mechanisms driving AR signaling, transcriptional activity, and ligand independence. Single cell behavior can be quantified by high-throughput microscopy methods through analysis of endogenous protein levels and localization or creation of biosensor cell lines that can simultaneously detect both acute and latent responses to known and unknown androgenic stimuli. The cell imaging and analytical protocols can be automated to discover agonist/antagonist response windows for nuclear translocation, reporter gene activity, nuclear export, and subnuclear transcription events, facilitating access to a multiplex model system that is inherently unavailable through classic biochemical approaches. In this chapter, we highlight the key steps needed for developing, conducting, and analyzing high-throughput screens to identify effectors of AR signaling.

Genomic and genetic variation in E2F transcription factor-1 in men with nonobstructive azoospermia

OBJECTIVE To identify gene dosage changes associated with nonobstructive azoospermia (NOA) using array comparative genomic hybridization (aCGH). DESIGN Prospective study. SETTING Medical school. PATIENT(S) One hundred ten men with NOA and 78 fertile controls. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) The study has four distinct analytic components: aCGH, a molecular karyotype that detects copy number variations (CNVs); Taqman CNV assays to validate CNVs; mutation identification by Sanger sequencing; and histological analyses of testicular tissues. RESULT(S) A microduplication at 20q11.22 encompassing E2F transcription factor-1 (E2F1) was identified in one of eight men with NOA analyzed using aCGH. CNVs were confirmed and in an additional 102 men with NOA screened using Taqman CNV assays, for a total of 110 NOA men analyzed for CNVs in E2F1. Eight of 110 (7.3%) NOA men had microduplications or microdeletions of E2F1 that were absent in fertile controls. CONCLUSION(S) E2F1 microduplications or microdeletions are present in men with NOA (7.3%). Duplications or deletions of E2F1 occur very rarely in the general population (0.011%), but E2F1 gene dosage changes, previously reported only in cancers, are present in a subset of NOA men. These results recapitulate the infertility phenotype seen in mice lacking or overexpressing E2f1.

Two-Step Forward Genetic Screen in Mice Identifies Ral GTPase-Activating Proteins as Suppressors of Hepatocellular Carcinoma

BACKGROUND & AIMS High-throughput sequencing technologies have identified thousands of infrequently mutated genes in hepatocellular carcinomas (HCCs). However, high intratumor and intertumor heterogeneity, combined with large numbers of passenger mutations, have made it difficult to identify driver mutations that contribute to the development of HCC. We combined transposon mutagenesis with a high-throughput screen of a small-hairpin RNA (shRNA) library to identify genes and pathways that contribute to HCC development. METHODS Sleeping beauty transposons were mobilized in livers of transgenic mice predisposed to develop hepatocellular adenoma and HCC owing to expression of the hepatitis B virus surface antigen. This whole-genome mutagenesis technique was used to generate an unbiased catalogue of candidate cancer genes (CCGs). Pooled shRNA libraries targeting 250 selected CCGs then were introduced into immortalized mouse liver cells and the cells were monitored for their tumor-forming ability after injection into nude mice. RESULTS Transposon-mediated mutagenesis identified 1917 high-confident CCGs and highlighted the importance of Ras signaling in the development of HCC. Subsequent pooled shRNA library screening of 250 selected CCGs validated 27 HCC tumor-suppressor genes. Individual shRNA knockdown of 4 of these genes (Acaa2, Hbs1l, Ralgapa2, and Ubr2) increased the proliferation of multiple human HCC cell lines in culture and accelerated the formation of xenograft tumors in nude mice. The ability of Ralgapa2 to promote HCC cell proliferation and tumor formation required its inhibition of Rala and Ralb. Dual inhibition of Ras signaling via Ral and Raf, using a combination of small-molecule inhibitor RBC8 and sorafenib, reduced the proliferation of HCC cells in culture and completely inhibited their growth as xenograft tumors in nude mice. CONCLUSIONS In a 2-step forward genetic screen in mice, we identified members of the Ral guanosine triphosphatase-activating protein pathway and other proteins as suppressors of HCC cell proliferation and tumor growth. These proteins might serve as therapeutic targets for liver cancer.

SBCDDB: Sleeping Beauty Cancer Driver Database for gene discovery in mouse models of human cancers

Abstract Large-scale oncogenomic studies have identified few frequently mutated cancer drivers and hundreds of infrequently mutated drivers. Defining the biological context for rare driving events is fundamentally important to increasing our understanding of the druggable pathways in cancer. Sleeping Beauty (SB) insertional mutagenesis is a powerful gene discovery tool used to model human cancers in mice. Our lab and others have published a number of studies that identify cancer drivers from these models using various statistical and computational approaches. Here, we have integrated SB data from primary tumor models into an analysis and reporting framework, the Sleeping Beauty Cancer Driver DataBase (SBCDDB, http://sbcddb.moffitt.org), which identifies drivers in individual tumors or tumor populations. Unique to this effort, the SBCDDB utilizes a single, scalable, statistical analysis method that enables data to be grouped by different biological properties. This allows for SB drivers to be evaluated (and re-evaluated) under different contexts. The SBCDDB provides visual representations highlighting the spatial attributes of transposon mutagenesis and couples this functionality with analysis of gene sets, enabling users to interrogate relationships between drivers. The SBCDDB is a powerful resource for comparative oncogenomic analyses with human cancer genomics datasets for driver prioritization.