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Forward genetic screens in mice uncover mediators and suppressors of metastatic reactivation.

ABSTRACT: We have developed a screening platform for the isolation of genetic entities involved in metastatic reactivation. Retroviral libraries of cDNAs from fully metastatic breast-cancer cells or pooled microRNAs were transduced into breast-cancer cells that become dormant upon infiltrating the lung. Upon inoculation in the tail vein of mice, the cells that had acquired the ability to undergo reactivation generated metastatic lesions. Integrated retroviral vectors were recovered from these lesions, sequenced, and subjected to a second round of validation. By using this strategy, we isolated canonical genes and microRNAs that mediate metastatic reactivation in the lung. To identify genes that oppose reactivation, we screened an expression library encoding shRNAs, and we identified target genes that encode potential enforcers of dormancy. Our screening strategy enables the identification and rapid biological validation of single genetic entities that are necessary to maintain dormancy or to induce reactivation. This technology should facilitate the elucidation of the molecular underpinnings of these processes.

SUBMITTER: Gao H

PROVIDER: S-EPMC4246349 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Forward genetic screens in mice uncover mediators and suppressors of metastatic reactivation.

Gao Hua H Chakraborty Goutam G Lee-Lim Ai Ping AP Mavrakis Konstantinos J KJ Wendel Hans-Guido HG Giancotti Filippo G FG

Proceedings of the National Academy of Sciences of the United States of America 20141105 46

We have developed a screening platform for the isolation of genetic entities involved in metastatic reactivation. Retroviral libraries of cDNAs from fully metastatic breast-cancer cells or pooled microRNAs were transduced into breast-cancer cells that become dormant upon infiltrating the lung. Upon inoculation in the tail vein of mice, the cells that had acquired the ability to undergo reactivation generated metastatic lesions. Integrated retroviral vectors were recovered from these lesions, seq ...[more]

PMID: 25378704

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Project description:In Schizosaccharomyces pombe, systematic analyses of single transcription factor deletion or overexpression strains have made substantial advances in determining the biological roles and target genes of transcription factors, yet these characteristics are still relatively unknown for over a quarter of them. Moreover, the comprehensive list of proteins that regulate transcription factors remains incomplete. To further characterize Schizosaccharomyces pombe transcription factors, we performed synthetic sick/lethality and synthetic dosage lethality screens by synthetic genetic array. Examination of 2,672 transcription factor double deletion strains revealed a sick/lethality interaction frequency of 1.72%. Phenotypic analysis of these sick/lethality strains revealed potential cell cycle roles for several poorly characterized transcription factors, including SPBC56F2.05, SPCC320.03, and SPAC3C7.04. In addition, we examined synthetic dosage lethality interactions between 14 transcription factors and a miniarray of 279 deletion strains, observing a synthetic dosage lethality frequency of 4.99%, which consisted of known and novel transcription factor regulators. The miniarray contained deletions of genes that encode primarily posttranslational-modifying enzymes to identify putative upstream regulators of the transcription factor query strains. We discovered that ubiquitin ligase Ubr1 and its E2/E3-interacting protein, Mub1, degrade the glucose-responsive transcriptional repressor Scr1. Loss of ubr1+ or mub1+ increased Scr1 protein expression, which resulted in enhanced repression of flocculation through Scr1. The synthetic dosage lethality screen also captured interactions between Scr1 and 2 of its known repressors, Sds23 and Amk2, each affecting flocculation through Scr1 by influencing its nuclear localization. Our study demonstrates that sick/lethality and synthetic dosage lethality screens can be effective in uncovering novel functions and regulators of Schizosaccharomyces pombe transcription factors.

Dataset Information

Forward genetic screens in mice uncover mediators and suppressors of metastatic reactivation.

Publications

Forward genetic screens in mice uncover mediators and suppressors of metastatic reactivation.

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