Vincent Keng, PhD

Research Techniques:

The development of human cancer is a complex process involving the acquisition of multiple genetic changes that confer a selective advantage on a tumor clone.  Many of the genes involved in these genetic changes have been identified based on mutation analyses in cancer model organisms, inheritance of a mutant form in cancer-prone families, or presence of recurrent translocation breakpoints.  However, a very large number of chromosome abnormalities have been detected for which no corresponding molecular explanation has been provided at the level of individual genes.  This includes a very large class of unbalanced chromosomal abnormalities in which there is a net loss or gain of genetic material.

Transposon-tagged mutagenesis has proven invaluable for functional genomic screens in organisms such as Drosophila melanogaster, but transposons such as Sleeping Beauty (SB) that are capable of transposing in mouse cells have only recently been identified. To explore the potential of this system for cancer gene identification we decided to test whether we could use it to model hepatocellular carcinoma (HCC) and other cancers. HCC is an aggressive cancer and patients frequently present with an advanced stage that has metastasized, leading to a poor prognosis and a 5-year survival rate below 5%. HCC is prevalent worldwide but differences in disease incidence reflect regional diversity where specific etiological factors and perhaps, ethnicity differ. Gender also influences risk with males showing a 4:1 increase in prevalence over females. HCC is commonly associated with chronic viral infection by hepatitis B or C viruses. The mechanisms by which these viruses contribute to tumor initiation and progression remain elusive. Other common factors associated with HCC are chronic alcohol consumption, aflatoxin-B1 contaminated food sources and all liver cirrhosis-inducing conditions. Important information regarding the underlying genetic mechanisms involved with this disease remains to be elucidated.

SB is a DNA-type transposable element that transposes in a “cut-and-paste” manner and integrates at “TA” dinucleotides. SB was originally identified as a long-dormant transposable element and its relevant transposase in the genomes of salmonid fish. Directed mutagenesis was used to correct mutations that silenced the activity of the transposase. SB is a two-component system, composed of the transposase transgene and a transposon vector, flanked by special inverted terminal repeats (IRs). When the transposon vector is present in a cell expressing the transposase transgene, the “cut-and-paste” transposition reaction occurs. The SB transposase enzyme recognizes specific binding sites within the IRs excises and then integrates the transposon elsewhere at a TA-dinucleotide.

Research Interests:

Somatic and germline insertional mutagenesis using transposable elements:

A) Hepatocellular carcinoma
Described here are two approaches that use tumor formation in vivo in mice to identify cancer genes that will complement genetic studies on human tumors. These approaches are both based on the Sleeping Beauty (SB) transposon. In one approach, SB transposase is used to deliver SB transposon vectors carrying specific genes and/or short-hairpin RNA constructs to hepatocytes in vivo in mice. In the other approach, SB transposon vectors are induced to jump in hepatocytes in vivo – the result of which is tumor development via insertional mutagenesis, which allows identification of the affected genes.

SB has been shown to be transpositionally active in zebrafish, vertebrate cell culture, the mouse germline (Keng et al., Nat. Methods 2005) and in mouse somatic tissues when delivered on plasmid DNA. Others and we have previously shown that somatic mutagenesis is possible and this is a useful system for identifying novel cancer-associated genes. In order to elucidate novel cancer-associated genes in solid tumors, we have generated a conditional SB expression system where the SB transposase (called SB11) is knocked into the Rosa26 locus but expression is initially blocked by a floxed-stop cassette. To obtain tissue-specific SB transposase expression, the floxed-stop cassette is removed by a tissue-specific Cre transgene. When these two components are combined with a transposon mutagenic vector (T2/Onc) capable of either disrupting tumor suppressor genes or misexpressing oncogenes, we have a powerful conditional SB somatic mutagenesis forward genetics screen for cancer-associated genes.

Hepatocelluar carcinoma (HCC) is the 3rd most common cancer worldwide and causes about 600,000 deaths per year. HCC is universally associated with liver damage and cirrhosis secondary to exposure to liver toxins such as alcohol and/or to hepatitis B or C viruses. HCC seems to develop along a path of cirrhosis to dysplastic lesions to invasive carcinoma. Early stages of the disease are not well known because most patients are detected with advanced disease. As such, no widely agreed upon system for HCC tumor staging exists. Treatment consists of surgical resection of the tumor or liver transplantation, but many patients present with advanced disease that cannot be treated surgically. Percutaneous ablation is effective in some patients. Systemic chemotherapy is ineffective for HCC as no drug or drug combination has been proven to enhance survival. To date immunotherapies, internal radiation, anti-androgens and tamoxifen have shown no consistent tumor response or survival benefit. In fact, no first line treatment is available for patients with advanced HCC, and as a result, HCC patients have a poor prognosis. Clearly, new treatment options must be vigorously sought for HCC. These are most likely to come from a better understanding of the alterations in genetic pathways operative in HCC.

Using the SB transposon system described above, I have successfully modeled all stages of HCC using a liver cancer mouse model: from early onset of hyperplastic nodules to hepatic adenoma and finally HCC (Keng et al., Nat. Biotech. 2009). In some experimental animals with aggressive HCCs, lung metastases that had originated from the primary HCC were also detected. From the liver cancer screen described, I identified 19 candidate liver cancer genes that are in the process of being functionally validated. Importantly, the gene copy numbers of the human homologue of these candidate genes were affected in human HCC patients, indicating that the system is indeed modeling human HCC. Moreover, preliminary data obtained using the SB transposon system suggests that genes responsible for the metastatic process can also be identified.

In order to test candidate HCC-associated genes, I designed a novel reverse genetics model for testing these genes involving the use of fumaryl acetoacetate hydrolase (Fah) mutant mice and SB-based gene delivery. Using a novel vector that simultaneously expresses both Fah and any oncogene-of-interest, it can be stably incorporated into an expanding population of hepatocytes to test its oncogenicity. It is known that selective repopulation of the liver occurs when Fah is reintroduced back into these mutant mice, mimicking the selective advantage. Novel tumor suppressor genes can be introduced stably as short hairpin RNAs and tested in the same manner (Wangensteen et al., Hepatology 2008).

B) Malignant peripheral nerve sheath tumor
My other research interest also includes elucidating the genetic mechanisms of the neurofibromatosis type 1 (NF1) syndrome. NF1 is an autosomal dominant inherited disease which affects ~1 in 3500 individuals worldwide. Majority of patients develop plexiform and/or dermal neurofibromas. Of great concern is ~10% of these patients will further progress from benign neurofibromas to malignant peripheral nerve sheath tumors (MPNST), which has a poor prognosis. Schwann cells are believed to be the primary pathogenic cell source in neurofibromas because they show biallelic NF1 mutation. In addition to NF1 mutation, it is suspected that MPNST have at least several major unrecognized secondary genetic changes because they typically harbor complex karytotypes with chromosomal imbalances and abnormal breakpoints. In order to further understand the underlying genetic complexity of plexiform neurofibroma and MPNST, the SB transposon-based forward genetic screen can be used to elucidate mutations that can promote progressive neurofibroma and/or MPNST formation in the context of somatic inactivation of the Nf1 gene in Schwann cells and their precursors, using an conditional Nf1flox/flox allele. This also involves the use of a conditional SB expression system previously defined. Desert Hedgehog (Dhh) regulatory sequence driving Cre recombinase was used to elicit recombination of Nf1flox/flox allele and Rosa26-lsl-SB11 transgene, allowing for inactivation of the Nf1 gene and initiate expression of the SB transposase in Schwann cells, respectively. The hypothesis is that quadruple transgenic mice carrying Dhh-Cre:Nf1flox/flox:T2/Onc:Rosa26-lsl-SB11 will develop neurofibromas that will further progress to MPNST formation, due to somatic transposition of the mutagenic vector in Schwann cells or its precursor. To date, I have generated these transgenic mice and are currently being aged for analyses. Any tumors isolated will be screened for T2/Onc insertions using the transposon as a molecular tag. Genes identified in this screen will give a better understanding to the underlying genetic mechanisms of this disease.

Therefore, these newly identified cancer-associated genes will help to further understand the complex molecular mechanisms involved in this diseases and also be useful in future gene/drug therapeutic applications.

C) Other transposon systems
In addition to the SB transposon system, I am currently testing other DNA-type transposons for somatic and germline mutagenesis in mice. One such transposon is called Tol2. Preliminary results suggest that Tol2 is much more efficient than the current SB transposon system (Keng et al., Genetics 2009).

Selected Publications:

Keng, V.W., Ryan, B.J., Wangensteen, K.J., Baciunas, D., Schmedt, C., Ekker, S.C. & Largaespada, D.A. (2009) Tol2 as an alternative tool for high throughput insertional mutagenesis screens in mice. Genetics. 183, 1565-1573.

Kitada, K., Keng, V.W., Takeda, J. & Horie, K. (2009) Generating mutant rats using the Sleeping Beauty transposon system. Methods. 49, 236-242.

Keng, V.W., Villanueva, A., Chiang, D.Y., Dupuy, A.J., Ryan, B.J., Matise, I., Silverstein, K.A.T., Sarver, A., Starr, T.K., Akagi, K., Tessarollo, L., Collier, L.S., Powers, S., Lowe, S.W., Jenkins, N.A., Copeland, N.G., Llovet, J.M. & Largaespada, D.A. (2009) A conditional transposon-based insertional mutagenesis screen for hepatocellular carcinoma-associated genes in mice. Nat. Biotech. 27, 264-274.

Saito, E., Keng, V.W., Takeda, J. & Horie, K. (2008) Translation from nonautonomous type IAP retrotransposon is a critical determinant of transposition activity: Implication for retrotransposon-mediated genome evolution. Genome Res. 18, 859-868.
Wangensteen, K.J., Wilber, A., Keng, V.W., Chen, Y., Matise, I., Wangensteen, L., Steer, C.J., McIvor, R.S., Largaespada, D.A., Wang, X. & Ekker, S.C. (2008) A facile method for somatic, lifelong manipulation of multiple genes in the mouse liver. Hepatology 47, 1714-1724.

Takeda, J., Keng, V.W. & Horie, K. (2007) Germline mutagenesis mediated by Sleeping Beauty transposon system in mice. Genome Biol. 8 (Suppl 1): S14.

Horie, K., Saito, E., Keng, V.W., Ikeda, R., Ishihara, H. & Takeda, J. (2007) Retrotransposon influence the mouse transcriptome: Implication for the divergence of genetic traits. Genetics 176, 815-827.

Kitada, K., Ishishita, S., Tosaka, K., Takahashi, R., Ueda, M., Keng, V.W., Horie, K. & Takeda, J. (2007) Transposon-tagged mutagenesis in the rat. Nat. Methods 4, 131-133.

Ikeda, R., Kokubu, C., Yusa, K., Keng, V. W., Horie, K. & Takeda, J. (2007) Sleeping Beauty transposase has an affinity for heterochromatin conformation. Mol. Cell. Biol. 27, 1665-1676.

Yae, K., Keng, V. W., Koike, M., Yusa, K., Kouno, M., Uno, Y., Kondoh, G., Gotow, T., Uchiyama, Y., Horie, K. & Takeda, J. (2006) Sleeping Beauty transposon-based phenotypic analysis of mice: Lack of Arpc3 results in defective trophoblast outgrowth. Mol. Cell. Biol. 26, 6185-6196.

Keng, V. W., Yae, K., Hayakawa, T., Mizuno, S., Uno, Y., Yusa, K., Kokubu, C., Kinoshita, T., Akagi, K., Jenkins, N. A., Copeland, N. G., Horie, K. & Takeda, J. (2005) Region-specific saturation germline mutagenesis in mice using the Sleeping Beauty transposon system. Nat. Methods 2, 763-769.

Horie, K., Yusa, K., Yae, K., Odajima, J., Fisher, S.E., Keng, V. W., Hayakawa, T., Mizuno, S., Kondoh, G., Ijiri, T., Matsuda, Y., Plasterk, R.H. & Takeda, J. (2003) Characterization of Sleeping Beauty transposition and its application to genetic screening in mice.  Mol. Cell. Biol. 24, 9189-9207.

Keng, V. W., Yagi, H., Ikawa, M., Nagano, T., Myint, Z., Yamada, K., Tanaka, T., Sato, A., Muramatsu, I., Okabe, M., Sato, M. & Noguchi, T. (2000) Homeobox gene Hex is essential for onset of mouse embryonic liver development and differentiation of the monocyte lineage.  Biochem. Biophys. Res. Comm. 276, 1155-1161; erratum: 279, 739.

Keng, V. W., Fujimori, K. E., Myint, Z., Tamamaki, N., Nojyo, Y. & Noguchi, T. (1998) Expression of Hex mRNA in early murine postimplantation embryo development.  FEBS Lett. 426, 183-186.
 

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