Timothy K. Starr

Recurrent R-spondin fusions in colon cancer

Identifying and understanding changes in cancer genomes is essential for the development of targeted therapeutics. Here we analyse systematically more than 70 pairs of primary human colon tumours by applying next-generation sequencing to characterize their exomes, transcriptomes and copy-number alterations. We have identified 36,303 protein-altering somatic changes that include several new recurrent mutations in the Wnt pathway gene TCF7L2, chromatin-remodelling genes such as TET2 and TET3 and receptor tyrosine kinases including ERBB3. Our analysis for significantly mutated cancer genes identified 23 candidates, including the cell cycle checkpoint kinase ATM. Copy-number and RNA-seq data analysis identified amplifications and corresponding overexpression of IGF2 in a subset of colon tumours. Furthermore, using RNA-seq data we identified multiple fusion transcripts including recurrent gene fusions involving R-spondin family members RSPO2 and RSPO3 that together occur in 10% of colon tumours. The RSPO fusions were mutually exclusive with APC mutations, indicating that they probably have a role in the activation of Wnt signalling and tumorigenesis. Consistent with this we show that the RSPO fusion proteins were capable of potentiating Wnt signalling. The R-spondin gene fusions and several other gene mutations identified in this study provide new potential opportunities for therapeutic intervention in colon cancer.

A transposon-based genetic screen in mice identifies genes altered in colorectal cancer

Human colorectal cancers (CRCs) display a large number of genetic and epigenetic alterations, some of which are causally involved in tumorigenesis (drivers) and others that have little functional impact (passengers). To help distinguish between these two classes of alterations, we used a transposon-based genetic screen in mice to identify candidate genes for CRC. Mice harboring mutagenic Sleeping Beauty (SB) transposons were crossed with mice expressing SB transposase in gastrointestinal tract epithelium. Most of the offspring developed intestinal lesions, including intraepithelial neoplasia, adenomas, and adenocarcinomas. Analysis of over 16,000 transposon insertions identified 77 candidate CRC genes, 60 of which are mutated and/or dysregulated in human CRC and thus are most likely to drive tumorigenesis. These genes include APC, PTEN, and SMAD4. The screen also identified 17 candidate genes that had not previously been implicated in CRC, including POLI, PTPRK, and RSPO2.

A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma

We describe a system that permits conditional mobilization of a Sleeping Beauty (SB) transposase allele by Cre recombinase to induce cancer specifically in a tissue of interest. To demonstrate its potential for developing tissue-specific models of cancer in mice, we limit SB transposition to the liver by placing Cre expression under the control of an albumin enhancer/promoter sequence and screen for hepatocellular carcinoma (HCC)–associated genes. From 8,060 nonredundant insertions cloned from 68 tumor nodules and comparative analysis with data from human HCC samples, we identify 19 loci strongly implicated in causing HCC. These encode genes, such as EGFR and MET, previously associated with HCC and others, such as UBE2H, that are potential new targets for treating this neoplasm. Our system, which could be modified to drive transposon-based insertional mutagenesis wherever tissue-specific Cre expression is possible, promises to enhance understanding of cancer genomes and identify new targets for therapeutic development.

A modified sleeping beauty transposon system that can be used to model a wide variety of human cancers in mice.

Recent advances in cancer therapeutics stress the need for a better understanding of the molecular mechanisms driving tumor formation. This can be accomplished by obtaining a more complete description of the genes that contribute to cancer. We previously described an approach using the Sleeping Beauty (SB) transposon system to model hematopoietic malignancies in mice. Here, we describe modifications of the SB system that provide additional flexibility in generating mouse models of cancer. First, we describe a Cre-inducible SBase allele, RosaSBase(LsL), that allows the restriction of transposon mutagenesis to a specific tissue of interest. This allele was used to generate a model of germinal center B-cell lymphoma by activating SBase expression with an Aid-Cre allele. In a second approach, a novel transposon was generated, T2/Onc3, in which the CMV enhancer/chicken beta-actin promoter drives oncogene expression. When combined with ubiquitous SBase expression, the T2/Onc3 transposon produced nearly 200 independent tumors of more than 20 different types in a cohort of 62 mice. Analysis of transposon insertion sites identified novel candidate genes, including Zmiz1 and Rian, involved in squamous cell carcinoma and hepatocellular carcinoma, respectively. These novel alleles provide additional tools for the SB system and provide some insight into how this mutagenesis system can be manipulated to model cancer in mice.

The regulated expression of a diverse set of genes during thymocyte positive selection in vivo

A signal initiated by the newly formed Ag receptor is integrated with microenvironmental cues during T cell development to ensure positive selection of CD4+CD8+ progenitors into functionally mature CD4+ or CD8+ T lymphocytes. During this transition, a survival program is initiated, TCR gene recombination ceases, cells migrate into a new thymic microenvironment, the responsiveness of the Ag receptor is tuned, and the cells commit to a specific T lineage. To determine potential regulators of these processes, we used mRNA microarray analysis to compare gene expression changes in CD4+CD8+ thymocytes from TCR transgenic mice that have received a TCR selection signal with those that had not received a signal. We found 129 genes with expression that changed significantly during positive selection, the majority of which were not previously appreciated. A large number of these changes were confirmed by real-time PCR or flow cytometry. We have combined our findings with gene changes reported in the literature to provide a comprehensive report of the genes regulated during positive selection, and we attempted to assign these genes to positive selection process categories.

A Sleeping Beauty transposon-mediated screen identifies murine susceptibility genes for adenomatous polyposis coli (Apc)-dependent intestinal tumorigenesis

It is proposed that a progressive series of mutations and epigenetic events leads to human colorectal cancer (CRC) and metastasis. Furthermore, data from resequencing of the coding regions of human CRC suggests that a relatively large number of mutations occur in individual human CRC, most at low frequency. The functional role of these low-frequency mutations in CRC, and specifically how they may cooperate with high-frequency mutations, is not well understood. One of the most common rate-limiting mutations in human CRC occurs in the adenomatous polyposis coli (APC) gene. To identify mutations that cooperate with mutant APC, we performed a forward genetic screen in mice carrying a mutant allele of Apc (ApcMin) using Sleeping Beauty (SB) transposon-mediated mutagenesis. ApcMin SB-mutagenized mice developed three times as many polyps as mice with the ApcMin allele alone. Analysis of transposon common insertion sites (CIS) identified the Apc locus as a major target of SB-induced mutagenesis, suggesting that SB insertions provide an efficient route to biallelic Apc inactivation. We also identified an additional 32 CIS genes/loci that may represent modifiers of the ApcMin phenotype. Five CIS genes tested for their role in proliferation caused a significant change in cell viability when message levels were reduced in human CRC cells. These findings demonstrate the utility of using transposon mutagenesis to identify low-frequency and cooperating cancer genes; this approach will aid in the development of combinatorial therapies targeting this deadly disease.

Virulence genes are a signature of the microbiome in the colorectal tumor microenvironment

BackgroundThe human gut microbiome is associated with the development of colon cancer, and recent studies have found changes in the microbiome in cancer patients compared to healthy controls. Studying the microbial communities in the tumor microenvironment may shed light on the role of host–bacteria interactions in colorectal cancer. Here, we highlight the major shifts in the colorectal tumor microbiome relative to that of matched normal colon tissue from the same individual, allowing us to survey the microbial communities in the tumor microenvironment and providing intrinsic control for environmental and host genetic effects on the microbiome.MethodsWe sequenced the microbiome in 44 primary tumor and 44 patient-matched normal colon tissue samples to determine differentially abundant microbial taxa These data were also used to functionally characterize the microbiome of the cancer and normal sample pairs and identify functional pathways enriched in the tumor-associated microbiota.ResultsWe find that tumors harbor distinct microbial communities compared to nearby healthy tissue. Our results show increased microbial diversity in the tumor microenvironment, with changes in the abundances of commensal and pathogenic bacterial taxa, including Fusobacterium and Providencia. While Fusobacterium has previously been implicated in colorectal cancer, Providencia is a novel tumor-associated agent which has not been identified in previous studies. Additionally, we identified a clear, significant enrichment of predicted virulence-associated genes in the colorectal cancer microenvironment, likely dependent upon the genomes of Fusobacterium and Providencia.ConclusionsThis work identifies bacterial taxa significantly correlated with colorectal cancer, including a novel finding of an elevated abundance of Providencia in the tumor microenvironment. We also describe the predicted metabolic pathways and enzymes differentially present in the tumor-associated microbiome, and show an enrichment of virulence-associated bacterial genes in the tumor microenvironment. This predicted virulence enrichment supports the hypothesis that the microbiome plays an active role in colorectal cancer development and/or progression. Our results provide a starting point for future prognostic and therapeutic research with the potential to improve patient outcomes.

Thymocyte Sensitivity and Supramolecular Activation Cluster Formation Are Developmentally Regulated: A Partial Role for Sialylation

TCR reactivity is tuned during thymic development. Immature thymocytes respond to low-affinity self-ligands resulting in positive selection. Following differentiation, T cells no longer respond to low-affinity ligands, but respond well to high-affinity (foreign) ligands. We show in this study that this response includes integrin activation, supramolecular activation cluster formation, Ca2+ flux, and CD69 expression. Because glycosylation patterns are known to change during T cell development, we tested whether alterations in sialylation influence CD8 T cell sensitivity to low affinity TCR ligands. Using neuraminidase treatment or genetic deficiency in the ST3Gal-I sialyltransferase, we show that desialylation of mature CD8 T cells enhances their sensitivity to low-affinity ligands, although these treatments do not completely recapitulate the dynamic range of immature T cells. These studies identify sialylation as one of the factors that regulate CD8 T cell tuning during development.

The role of KCNQ1 in mouse and human gastrointestinal cancers.

Kcnq1, which encodes for the pore-forming α-subunit of a voltage-gated potassium channel, was identified as a gastrointestinal (GI) tract cancer susceptibility gene in multiple Sleeping Beauty DNA transposon-based forward genetic screens in mice. To confirm that Kcnq1 has a functional role in GI tract cancer, we created ApcMin mice that carried a targeted deletion mutation in Kcnq1. Results demonstrated that Kcnq1 is a tumor suppressor gene as Kcnq1 mutant mice developed significantly more intestinal tumors, especially in the proximal small intestine and colon, and some of these tumors progressed to become aggressive adenocarcinomas. Gross tissue abnormalities were also observed in the rectum, pancreas and stomach. Colon organoid formation was significantly increased in organoids created from Kcnq1 mutant mice compared with wild-type littermate controls, suggesting a role for Kcnq1 in the regulation of the intestinal crypt stem cell compartment. To identify gene expression changes due to loss of Kcnq1, we carried out microarray studies in the colon and proximal small intestine. We identified altered genes involved in innate immune responses, goblet and Paneth cell function, ion channels, intestinal stem cells, epidermal growth factor receptor and other growth regulatory signaling pathways. We also found genes implicated in inflammation and in cellular detoxification. Pathway analysis using Ingenuity Pathway Analysis and Gene Set Enrichment Analysis confirmed the importance of these gene clusters and further identified significant overlap with genes regulated by MUC2 and CFTR, two important regulators of intestinal homeostasis. To investigate the role of KCNQ1 in human colorectal cancer (CRC), we measured protein levels of KCNQ1 by immunohistochemistry in tissue microarrays containing samples from CRC patients with liver metastases who had undergone hepatic resection. Results showed that low expression of KCNQ1 expression was significantly associated with poor overall survival.

TAPDANCE: An automated tool to identify and annotate transposon insertion CISs and associations between CISs from next generation sequence data

BackgroundNext generation sequencing approaches applied to the analyses of transposon insertion junction fragments generated in high throughput forward genetic screens has created the need for clear informatics and statistical approaches to deal with the massive amount of data currently being generated. Previous approaches utilized to 1) map junction fragments within the genome and 2) identify Common Insertion Sites (CISs) within the genome are not practical due to the volume of data generated by current sequencing technologies. Previous approaches applied to this problem also required significant manual annotation.ResultsWe describe Transposon Annotation Poisson Distribution Association Network Connectivity Environment (TAPDANCE) software, which automates the identification of CISs within transposon junction fragment insertion data. Starting with barcoded sequence data, the software identifies and trims sequences and maps putative genomic sequence to a reference genome using the bowtie short read mapper. Poisson distribution statistics are then applied to assess and rank genomic regions showing significant enrichment for transposon insertion. Novel methods of counting insertions are used to ensure that the results presented have the expected characteristics of informative CISs. A persistent mySQL database is generated and utilized to keep track of sequences, mappings and common insertion sites. Additionally, associations between phenotypes and CISs are also identified using Fisher’s exact test with multiple testing correction. In a case study using previously published data we show that the TAPDANCE software identifies CISs as previously described, prioritizes them based on p-value, allows holistic visualization of the data within genome browser software and identifies relationships present in the structure of the data.ConclusionsThe TAPDANCE process is fully automated, performs similarly to previous labor intensive approaches, provides consistent results at a wide range of sequence sampling depth, has the capability of handling extremely large datasets, enables meaningful comparison across datasets and enables large scale meta-analyses of junction fragment data. The TAPDANCE software will greatly enhance our ability to analyze these datasets in order to increase our understanding of the genetic basis of cancers.