Dataset Information

A novel gammaretroviral shuttle vector insertional mutagenesis screen identifies SHARPIN as a breast cancer metastasis gene and prognostic biomarker.

ABSTRACT: Breast cancer (BC) is the second leading cause of malignancy among U.S. women. Metastasis results in a poor prognosis and increased mortality, but the molecular mechanisms by which metastatic tumors occur are not well understood. Identifying the genes that drive the metastatic process could provide targets for improved therapy and biomarkers to improve BC patient outcomes. Using a forward mutagenesis screen, BC cells mutagenized with a replication-incompetent gammaretroviral vector (?RV) were xenotransplanted into the mammary fat pad of immunodeficient mice. In this approach the vector provirus dysregulates nearby genes, providing a selective advantage to transduced cells to form metastases. Metastatic tumors were analyzed for proviral integration sites to identify nearby candidate metastasis genes. The ?RV has a transgene cassette that allows for rescue in bacteria and rapid identification of vector integration sites. Using this approach, we identified the previously described metastasis gene WWTR1 (TAZ), and three other novel candidate metastasis genes including SHARPIN. SHARPIN was independently validated in vivo as a BC metastasis gene. Analysis of patient data showed that SHARPIN expression predicts metastasis-free survival after adjuvant therapy. Our approach has broad potential to identify genes involved in oncogenic processes for BC and other cancers. We show here it can identify both known (WWTR1) and novel (SHARPIN) BC metastasis genes.

SUBMITTER: Bii VM

PROVIDER: S-EPMC4741842 | biostudies-other | 2015 Nov

REPOSITORIES: biostudies-other

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A novel gammaretroviral shuttle vector insertional mutagenesis screen identifies SHARPIN as a breast cancer metastasis gene and prognostic biomarker.

Bii Victor M VM Rae Dustin T DT Trobridge Grant D GD

Oncotarget 20151101 37

Breast cancer (BC) is the second leading cause of malignancy among U.S. women. Metastasis results in a poor prognosis and increased mortality, but the molecular mechanisms by which metastatic tumors occur are not well understood. Identifying the genes that drive the metastatic process could provide targets for improved therapy and biomarkers to improve BC patient outcomes. Using a forward mutagenesis screen, BC cells mutagenized with a replication-incompetent gammaretroviral vector (γRV) were xe ...[more]

PMID: 26506596

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Project description:In the last few decades, dengue virus, an arbovirus, has spread to over 120 countries. Although a vaccine has been approved in some countries, limitations on its effectiveness and a lack of effective antiviral treatments reinforce the need for additional research. The functions of several viral nonstructural proteins are essentially unknown. To better understand the functions of these proteins and thus dengue virus pathogenesis, we embarked on a genomewide transposon mutagenesis screen with next-generation sequencing to determine sites in the viral genome that tolerate 15-nucleotide insertions. Using this approach, we generated support for several published predicted transmembrane and enzymatic domains. Next, we created 7 mutants containing the 15-nucleotide insertion from the original selection and found 6 of them were capable of replication in both mammalian and mosquito tissue culture cells. Interestingly, one mutation had a significant impairment of viral assembly, and this mutation may lead to a better understanding of viral assembly and release. In addition, we created a fully infectious virus expressing a functionally tagged NS4B protein, which will provide a much-needed tool to elucidate the role of NS4B in viral pathogenesis.IMPORTANCE Dengue virus is a mosquito-borne virus distributed in tropical and subtropical regions globally that can result in hospitalization and even death in some cases. Although a vaccine exists, its limitations and a lack of approved antiviral treatments highlight our limited understanding of dengue virus pathogenesis and host immunity. The functions of many viral proteins are poorly understood. We used a previously published approach using transposon mutagenesis to develop tools to study these proteins' functions by adding insertions randomly throughout the viral genomes. These genomes were transferred into cells, and infectious progeny were recovered to determine sites that tolerated insertions, as only the genomes that tolerated insertions would be able to propagate. Using these results, we created viruses with epitope tags, one in the viral structural protein Capsid and one in the viral nonstructural protein NS4B. Further investigation of these mutants may elucidate the roles of Capsid and NS4B during dengue virus infections.

Dataset Information

A novel gammaretroviral shuttle vector insertional mutagenesis screen identifies SHARPIN as a breast cancer metastasis gene and prognostic biomarker.

Publications

A novel gammaretroviral shuttle vector insertional mutagenesis screen identifies SHARPIN as a breast cancer metastasis gene and prognostic biomarker.

Similar Datasets

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