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Members of the PIAS (for protein inhibitor of activated STAT) family play critical roles in modulating the activity of a variety of transcriptional regulators. Zimp10, a novel PIAS-like protein, is a transcriptional coregulator and may be involved in the modification of chromatin through interactions with the SWI/SNF chromatin-remodeling complexes. Here, we investigate the biological role of Zimp10 in zimp10-deficient mice. Homozygosity for the Zimp10-targeted allele resulted in developmental arrest at approximately embryonic day 10.5. Analysis of knockout embryos revealed severe defects in the reorganization of the yolk sac vascular plexus. No significant abnormality in hematopoietic potential was observed in zimp10 null mice. Microarray and quantified reverse transcription-PCR analyses showed that the expression of the Fos family member Fra-1, which is involved in extraembryonic vascular development, was reduced in yolk sac tissues of zimp10 null embryos. Using fra-1 promoter/reporter constructs, we further demonstrate the regulatory role of Zimp10 on the transcription of Fra-1. This study provides evidence to demonstrate a crucial role for Zimp10 in vasculogenesis.

Amplification, overexpression, or rearrangement of the c-rel gene, encoding the c-Rel NF-kappaB subunit, has been reported in solid and hematopoietic malignancies. For example, many primary human breast cancer tissue samples express high levels of nuclear c-Rel. While the Rev-T oncogene v-rel causes tumors in birds, the ability of c-Rel to transform in vivo has not been demonstrated. To directly test the role of c-Rel in breast tumorigenesis, mice were generated in which overexpression of mouse c-rel cDNA was driven by the hormone-responsive mouse mammary tumor virus long terminal repeat (MMTV-LTR) promoter, and four founder lines identified. In the first cycle of pregnancy, the expression of transgenic c-rel mRNA was observed, and levels of c-Rel protein were increased in the mammary gland. Importantly, 31.6% of mice developed one or more mammary tumors at an average age of 19.9 months. Mammary tumors were of diverse histology and expressed increased levels of nuclear NF-kappaB. Analysis of the composition of NF-kappaB complexes in the tumors revealed aberrant nuclear expression of multiple subunits, including c-Rel, p50, p52, RelA, RelB, and the Bcl-3 protein, as observed previously in human primary breast cancers. Expression of the cancer-related NF-kappaB target genes cyclin D1, c-myc, and bcl-xl was significantly increased in grossly normal transgenic mammary glands starting the first cycle of pregnancy and increased further in mammary carcinomas compared to mammary glands from wild-type mice or virgin transgenic mice. In transient transfection analysis in untransformed breast epithelial cells, c-Rel-p52 or -p50 heterodimers either potently or modestly induced cyclin D1 promoter activity, respectively. Lastly, stable overexpression of c-Rel resulted in increased cyclin D1 and NF-kappaB p52 and p50 subunit protein levels. These results indicate for the first time that dysregulated expression of c-Rel, as observed in breast cancers, is capable of contributing to mammary tumorigenesis.

While the E2F transcription factors (E2Fs) have a clearly defined role in cell cycle control, recent work has uncovered new functions. Using genomic signature methods, we predicted a role for the activator E2F transcription factors in the mouse mammary tumor virus (MMTV)-polyomavirus middle T oncoprotein (PyMT) mouse model of metastatic breast cancer. To genetically test the hypothesis that the E2Fs function to regulate tumor development and metastasis, we interbred MMTV-PyMT mice with E2F1, E2F2, or E2F3 knockout mice. With the ablation of individual E2Fs, we noted alterations of tumor latency, histology, and vasculature. Interestingly, we noted striking reductions in metastatic capacity and in the number of circulating tumor cells in both the E2F1 and E2F2 knockout backgrounds. Investigating E2F target genes that mediate metastasis, we found that E2F loss led to decreased levels of vascular endothelial growth factor (Vegfa), Bmp4, Cyr61, Nupr1, Plod 2, P4ha1, Adamts1, Lgals3, and Angpt2. These gene expression changes indicate that the E2Fs control the expression of genes critical to angiogenesis, the remodeling of the extracellular matrix, tumor cell survival, and tumor cell interactions with vascular endothelial cells that facilitate metastasis to the lungs. Taken together, these results reveal that the E2F transcription factors play key roles in mediating tumor development and metastasis in addition to their well-characterized roles in cell cycle control.

Activation of Akt by the phosphatidylinositol 3′-OH kinase (PI3K) results in the inhibition of proapoptotic signals and the promotion of survival signals (L. P. Kane et al., Curr. Biol. 9:601–604, 1999; G. J. Kops et al., Nature 398:630–634, 1999). Evidence supporting the importance of the PI3K/Akt signaling pathway in tumorigenesis stems from experiments with transgenic mice bearing polyomavirus middle T antigen under the control of the mouse mammary tumor virus long terminal repeat promoter. Mammary epithelium-specific expression of polyomavirus middle T antigen results in the rapid development of multifocal metastatic mammary tumors, whereas transgenic mice expressing a mutant middle T antigen decoupled from the phosphatidylinositol 3′-OH kinase (MTY315/322F) develop extensive mammary gland hyperplasias that are highly apoptotic. To directly assess the role of Akt in mammary epithelial development and tumorigenesis, we generated transgenic mice expressing constitutively active Akt (HAPKB308D473D or Akt-DD). Although expression of Akt-DD interferes with normal mammary gland involution, tumors were not observed in these strains. However, coexpression of Akt-DD with MTY315/322F resulted in a dramatic acceleration of mammary tumorigenesis correlated with reduced apoptotic cell death. Furthermore, coexpression of Akt-DD with MTY315/322F resulted in phosphorylation of the FKHR forkhead transcription factor and translational upregulation of cyclin D1 levels. Importantly, we did not observe an associated restoration of wild-type metastasis levels in the bitransgenic strain. Taken together these observations indicate that activation of Akt can contribute to tumor progression by providing an important cell survival signal but does not promote metastatic progression.

The effect of mammary gland-specific expression of the polyomavirus middle T antigen was examined by establishing lines of transgenic mice that carry the middle T oncogene under the transcriptional control of the mouse mammary tumor virus promoter/enhancer. By contrast to most transgenic strains carrying activated oncogenes, expression of polyomavirus middle T antigen resulted in the widespread transformation of the mammary epithelium and the rapid production of multifocal mammary adenocarcinomas. Interestingly, the majority of the tumor-bearing transgenic mice developed secondary metastatic tumors in the lung. Taken together, these results suggest that middle T antigen acts as a potent oncogene in the mammary epithelium and that cells that express it possess an enhanced metastatic potential.

The Ets2 transcription factor is regulated by mitogen-activated protein (MAP) kinase phosphorylation of a single threonine residue. We generated by gene targeting a single codon mutation in Ets2 substituting Ala for the critical Thr-72 phosphorylation site (Ets2A72), to investigate the importance of MAP kinase activation of Ets2 in embryo and tumor development. Ets2(A72/A72) mice are viable and develop normally. However, combining the Ets2A72 allele with a deletion mutant of Ets2 results in lethality at E11.5 and shows that Ets2A72 is a hypomorphic allele. Mammary tumors caused by transgenic polyomavirus middle T antigen, activated Neu(Erbb2), or the combination of Neu and transgenic VEGF (Neu; VEGF-25) were all restricted in Ets2(A72/A72) females. The Ets2(A72/A72) restriction on Neu; VEGF-25 tumor growth was associated with increased p21Cip1 expression. The size of tumors transplanted into fat pads of mice with Ets2 targeted alleles was correlated directly with Ets2 activity and fewer stromal cells expressing matrix metalloproteinase 9 (MMP-9). Decreased MMP-3 and MMP-9 mRNAs were confirmed in Ets2(A72/A72) macrophages. Activation of Ets2 at Thr-72 acts in the stroma, downstream of vascular endothelial growth factor production, in part through the regulation of macrophage proteases to support the progression of Neu- and polyomavirus middle-T-initiated mammary tumors.

Amplification of the Neu (ErbB-2 or HER-2) receptor tyrosine kinase occurs in 20 to 30% of human mammary carcinomas, correlating with a poor clinical prognosis. We have previously demonstrated that four (Y1144 Y1201, Y1227 and Y1253) of the five known Neu autophosphorylation sites can independently mediate transforming signals. The transforming potential of two of these mutants correlates with their capacity to recruit Grb2 directly to Y1144 (YB) or indirectly through Shc to Y1227 (YD). Here, we demonstrate that these transformation-competent neu mutants activate extracellular signal-regulated kinases and stimulate Ets-2-dependent transcription. Although the transforming potential of three of these mutants (YB, YD, and YE) was susceptible to inhibition by Rap1A, a genetic antagonist of Ras, the transforming potential of YC was resistant to inhibition by Rap1A. To further address the significance of these ErbB-2-coupled signaling molecules in induction of mammary cancers, transgenic mice expressing mutant Neu receptors lacking the known autophosphorylation sites (NYPD) or those coupled directly to either Grb2 (YB) or Shc (YD) adapter molecules were derived. In contrast to the NYPD strains, which developed focal mammary tumors after a long latency period with low penetrance, all female mice derived from YB and YD strains rapidly developed mammary tumors. Although female mice from several independent YB or YD lines developed mammary tumors, the YB strains developed lung metastases at substantially higher rates than the YD strains. These observations argue that Grb2 and Shc play important and distinct roles in ErbB-2/Neu-induced mammary tumorigenesis and metastasis.

Genomic DNAs from dimethylbenzanthracene-induced BALB/c mouse mammary tumors arising from the transplantable hyperplastic outgrowth (HPO) line designated DI/UCD transformed NIH 3T3 cells upon transfection. Transforming activity was attributed to the presence of activated Harvey ras-1 oncogenes containing an A----T transversion at the middle adenosine nucleotide in codon 61. DNAs from untreated DI/UCD HPO cells and radiation-induced and spontaneous mammary tumors from the DI/UCD HPO line failed to transform NIH 3T3 cells. The results indicated that the mutation activation of Harvey ras-1 oncogenes was specific to dimethylbenzanthracene treatment in the mouse mammary tumor system.