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Transposon mutagenesis identifies candidate genes that cooperate with loss of transforming growth factor-beta signaling in mouse intestinal neoplasms.

ABSTRACT: Colorectal cancer (CRC) results from the accumulation of gene mutations and epigenetic alterations in colon epithelial cells, which promotes CRC formation through deregulating signaling pathways. One of the most commonly deregulated signaling pathways in CRC is the transforming growth factor ? (TGF-?) pathway. Importantly, the effects of TGF-? signaling inactivation in CRC are modified by concurrent mutations in the tumor cell, and these concurrent mutations determine the ultimate biological effects of impaired TGF-? signaling in the tumor. However, many of the mutations that cooperate with the deregulated TGF-? signaling pathway in CRC remain unknown. Therefore, we sought to identify candidate driver genes that promote the formation of CRC in the setting of TGF-? signaling inactivation. We performed a forward genetic screen in mice carrying conditionally inactivated alleles of the TGF-? receptor, type II (Tgfbr2) using Sleeping Beauty (SB) transposon mediated mutagenesis. We used TAPDANCE and Gene-centric statistical methods to identify common insertion sites (CIS) and, thus, candidate tumor suppressor genes and oncogenes within the tumor genome. CIS analysis of multiple neoplasms from these mice identified many candidate Tgfbr2 cooperating genes and the Wnt/?-catenin, Hippo and MAPK pathways as the most commonly affected pathways. Importantly, the majority of candidate genes were also found to be mutated in human CRC. The SB transposon system provides an unbiased method to identify Tgfbr2 cooperating genes in mouse CRC that are functionally relevant and that may provide further insight into the pathogenesis of human CRC.

SUBMITTER: Morris SM

PROVIDER: S-EPMC5316486 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Transposon mutagenesis identifies candidate genes that cooperate with loss of transforming growth factor-beta signaling in mouse intestinal neoplasms.

Morris Shelli M SM Davison Jerry J Carter Kelly T KT O'Leary Rachele M RM Trobridge Patty P Knoblaugh Sue E SE Myeroff Lois L LL Markowitz Sanford D SD Brett Benjamin T BT Scheetz Todd E TE Dupuy Adam J AJ Starr Timothy K TK Grady William M WM

International journal of cancer 20161107 4

Colorectal cancer (CRC) results from the accumulation of gene mutations and epigenetic alterations in colon epithelial cells, which promotes CRC formation through deregulating signaling pathways. One of the most commonly deregulated signaling pathways in CRC is the transforming growth factor β (TGF-β) pathway. Importantly, the effects of TGF-β signaling inactivation in CRC are modified by concurrent mutations in the tumor cell, and these concurrent mutations determine the ultimate biological eff ...[more]

PMID: 27790711

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Project description:Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10^6 reads corresponding to >10^4 insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors. Solid tumors in mice were generated by somatic transposon mutagenesis with a PiggyBac transposon system. Insertion sites of transposons in 11 tumors and 6 non-cancerous tail controls were determined by Illumina high-throughput sequencing. Insertions were determined both on 5' and 3' sides of the transposon (PB5 and PB3, respectively). Quantitative analysis of read numbers revealed enrichment of certain insertions in tumors, but not in controls, and these enriched insertions identify candidate cancer genes.

Dataset Information

Transposon mutagenesis identifies candidate genes that cooperate with loss of transforming growth factor-beta signaling in mouse intestinal neoplasms.

Publications

Transposon mutagenesis identifies candidate genes that cooperate with loss of transforming growth factor-beta signaling in mouse intestinal neoplasms.

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