In order to accurately analyze gene function in transgenic mice, as well as to generate credible murine models of human diseases, the ability to regulate temporal- and spatial-specific expression of ...target genes is absolutely critical. Pioneering work in inducible transgenics, begun in the 1980s and continuing to the present, has led to the development of a variety of different inducible systems dedicated to this goal, the shared basis of which is the accurate conditional expression of a given transgene. Recent advances in inducible transgene expression in mice are discussed.
Expression of the cyclin-dependent kinase (Cdk) inhibitor (p27(Kip1)) is frequently reduced in human tumors, often correlating with poor prognosis. p27(Kip1) functions as a haploinsufficient tumor ...suppressor; however, the mechanism by which one allele of p27(Kip1) regulates oncogenic signaling in vivo is not well understood. We therefore investigated the mechanisms by which p27(Kip1) inhibits mammary tumor onset. Using the common background strain of FVB, p27(Kip1) heterozygosity (p27(+/-)) accelerated ErbB2-induced mammary tumorigenesis. We conducted microarray analyses of mammary tumors developing in mice with genetic haploinsufficiency for p27(Kip1) expressing a mammary-targeted ErbB2 oncogene. Global gene expression profiling and Western blot analysis of ErbB2/p27(+/-) tumors showed that the loss of p27(Kip1) induced genes promoting lymphangiogenesis, cellular proliferation, and collaborative oncogenic signaling (Wnt/beta-catenin/Tcf, Cdc25a, Smad7, and Skp2). Skp2 expression was induced by ErbB2 and repressed by p27(Kip1). Degradation of p27(Kip1) involves an SCF-type E3 ubiquitin ligase, including Skp2. The Skp2 component of the SCF(SKP2) complex that degrades p27(Kip1) was increased in ErbB2 tumors correlating with earlier tumor onset. In both murine and human ErbB2-overexpressing breast cancers, p27(Kip1) levels correlated inversely with Skp2. p27(Kip1) haploinsufficiency activated Wnt/beta-catenin/hedgehog signaling. Reintroduction of p27(Kip1) inhibited beta-catenin induction of Tcf-responsive genes (Siamosis, c-Myc, and Smad7). p27(Kip1) is haploinsufficient for ErbB2 mammary tumor suppression in vivo and functions to repress collaborative oncogenic signals including Skp2 and Wnt/beta-catenin signaling.
Expression of the cyclin-dependent kinase (Cdk) inhibitor (p27 super(Kip1)) is frequently reduced in human tumors, often correlating with poor prognosis. p27 super(Kip1) functions as a ...haploinsufficient tumor suppressor; however, the mechanism by which one allele of p27 super(Kip1) regulates oncogenic signaling in vivo is not well understood. We therefore investigated the mechanisms by which p27 super(Kip1) inhibits mammary tumor onset. Using the common background strain of FVB, p27 super(Kip1) heterozygosity (p27 super(+/-)) accelerated ErbB2-induced mammary tumorigenesis. We conducted microarray analyses of mammary tumors developing in mice with genetic haploinsufficiency for p27 super(Kip1) expressing a mammary-targeted ErbB2 oncogene. Global gene expression profiling and Western blot analysis of ErbB2/p27 super(+/-) tumors showed that the loss of p27 super(Kip1) induced genes promoting lymphangiogenesis, cellular proliferation, and collaborative oncogenic signaling (Wnt/ beta -catenin/Tcf, Cdc25a, Smad7, and Skp2). Skp2 expression was induced by ErbB2 and repressed by p27 super(Kip1). Degradation of p27 super(Kip1) involves an SCF-type E3 ubiquitin ligase, including Skp2. The Skp2 component of the SCF super(SKP2) complex that degrades p27 super(Kip1) was increased in ErbB2 tumors correlating with earlier tumor onset. In both murine and human ErbB2-overexpressing breast cancers, p27 super(Kip1) levels correlated inversely with Skp2. p27 super(Kip1) haploinsufficiency activated Wnt/ beta -catenin/hedgehog signaling. Reintroduction of p27 super(Kip1) inhibited beta -catenin induction of Tcf-responsive genes (Siamosis, c-Myc, and Smad7). p27 super(Kip1) is haploinsufficient for ErbB2 mammary tumor suppression in vivo and functions to repress collaborative oncogenic signals including Skp2 and Wnt/ beta -catenin signaling. (Cancer Res 2006; 66(17): 8529-41)
Constitutive beta -catenin/Tcf activity, the primary transforming events in colorectal carcinoma, occurs through induction of the Wnt pathway or APC gene mutations that cause familial adenomatous ...polyposis. Mice carrying Apc mutations in their germ line (Apc super(Min)) develop intestinal adenomas. Here, the crossing of Apc super(Min) with cyclin D1 super(-/-) mice reduced the intestinal tumor number in animals genetically heterozygous or nullizygous for cyclin D1. Decreased tumor number in the duodenum, intestines, and colons of Apc super(Min)/cyclin D1 super(+/-) mice correlated with reduced cellular proliferation and increased differentiation. Cyclin D1 deficiency reduced DNA synthesis and induced differentiation of colonic epithelial cells harboring mutant APC but not wild- type APC cells in vivo. In previous studies, the complete loss of cyclin D1 through homozygous genetic deletion conveyed breast tumor resistance. The protection of mice, genetically predisposed to intestinal tumorigenesis, through cyclin D1 heterozygosity suggests that modalities that reduce cyclin D1 abundance could provide chemoprotection.
Cyclin D1 protein levels are elevated by mitogenic and oncogenic signaling pathways as cyclin D1 protein is rate-limiting for G1-phase cell cycle progression in many cell types as well as ...over-expressed in 20–30% of human breast cancers and elevated in human adenomatous polyps of the colon. Based on my hypothesis that the cyclin D1 protein is a critical downstream factor required in the initiation and progression of breast and colon cancers, I undertook investigations as to the oncogenic mechanism and requirement of cyclin D1 in the onset and progression of these diseases. Caveolin-1 is deleted in human cancers including breast tumors where it is also found mutated in approximately 16% of human breast cancers. Using the cyclin D1 promoter as a molecular probe to assay transcriptional events which regulate G1 progression, I found that cyclin D1 is regulated by caveolin-1 expression in a negative manner and that maximal inhibition of the cyclin D1 promoter was dependent on a T-cell factor/lymphoid enhancer factor-1 (TCF/LEF-1) response element in the cyclin promoter. Using transgenic and knockout mice to assess the specific role for p27Kip1 in neu-induced tumorigenesis, I identified p27Kip1 as being haplo-insufficient for neu-induced mammary tumor suppression in vivo and that inhibition of p27Kip1 function may be due in part to its down regulation by Skp2 and redistribution away from cyclin E and into cyclin D1 complexes. I have also examined the specific requirement of cyclin D1 in intestinal tumorigenesis. Mutations in the Apc tumor-suppressor gene are among the earliest events in colon cancer and subsequent activation of the Wnt pathway specifically targets and induces cyclin D1 expression. Using the ApcMin mouse model of human colorectal cancer, I found that reduction of cyclin D1 protein abundance in the presence of constitutive β-catenin/Tcf4-signaling increased cellular differentiation decreased DNA synthesis and importantly, reduced intestinal polyp formation. These studies identify cyclin D1 as a critical signaling target in both mammary and intestinal tumorigenesis. By understanding how cyclin D1 is regulated and the precise molecular functions associated with the protein, I aim to provide a rational basis for molecular approaches to inhibit cellular transformation.
Breast cancer arises from multiple genetic events that together contribute to the established, irreversible malignant phenotype. The development of inducible tissue-specific transgenics has allowed a ...careful dissection of the events required for induction and subsequent maintenance of tumorigenesis. Mammary gland targeted expression of oncogenic Ras or c-Myc is sufficient for the induction of mammary gland tumorigenesis in the rodent, and when overexpressed together the rate of tumor onset is substantially enhanced. In an exciting recent finding, D'Cruz et al discovered tetracycline-regulated c-Myc overexpression in the mammary gland induced invasive mammary tumors that regressed upon withdrawal of c-Myc expression. Almost one-half of the c-Myc-induced tumors harbored K-ras or N-ras gene point mutations, correlating with tumor persistence on withdrawal of c-Myc transgene expression. These findings suggest maintenance of tumorigenesis may involve a second mutation within the Ras pathway.
The Wnt/beta-catenin/Tcf and IkappaB/NF-kappaB cascades are independent pathways involved in cell cycle control, cellular differentiation, and inflammation. Constitutive Wnt/beta-catenin signaling ...occurs in certain cancers from mutation of components of the pathway and from activating growth factor receptors, including RON and MET. The resulting accumulation of cytoplasmic and nuclear beta-catenin interacts with the Tcf/LEF transcription factors to induce target genes. The IkappaB kinase complex (IKK) that phosphorylates IkappaB contains IKKalpha, IKKbeta, and IKKgamma. Here we show that the cyclin D1 gene functions as a point of convergence between the Wnt/beta-catenin and IkappaB pathways in mitogenic signaling. Mitogenic induction of G(1)-S phase progression and cyclin D1 expression was PI3K dependent, and cyclin D1(-/-) cells showed reduced PI3K-dependent S-phase entry. PI3K-dependent induction of cyclin D1 was blocked by inhibitors of PI3K/Akt/IkappaB/IKKalpha or beta-catenin signaling. A single Tcf site in the cyclin D1 promoter was required for induction by PI3K or IKKalpha. In IKKalpha(-/-) cells, mitogen-induced DNA synthesis, and expression of Tcf-responsive genes was reduced. Reintroduction of IKKalpha restored normal mitogen induction of cyclin D1 through a Tcf site. In IKKalpha(-/-) cells, beta-catenin phosphorylation was decreased and purified IKKalpha was sufficient for phosphorylation of beta-catenin through its N-terminus in vitro. Because IKKalpha but not IKKbeta induced cyclin D1 expression through Tcf activity, these studies indicate that the relative levels of IKKalpha and IKKbeta may alter their substrate and signaling specificities to regulate mitogen-induced DNA synthesis through distinct mechanisms.
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