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Supplementary Tables 3-6, Figure 5 from Transgenic Mice Expressing the p75 CCAAT-Displacement Protein/Cut Homeobox Isoform Develop a Myeloproliferative Disease–Like Myeloid Leukemia

The p75 CCAAT-displacement protein/Cut homeobox (CDP/Cux) isoform was previously reported to be overexpressed in human breast cancers. To investigate its oncogenic potential, we engineered two transgenic mouse lines expressing p75 CDP/Cux under the control of the mouse mammary tumor virus-long terminal repeat. The FVB strain of mouse is generally used in the generation of mouse models for breast cancer. The transgene was introduced into the hprt locus of 129/Ola embryonic stem cells and, following germ line passage, was backcrossed onto the FVB and C57BL/6 mouse strains. Here, we describe the phenotype of p75 CDP/Cux transgenic virgin female mice of the first backcross generations. We report that after a long latency period, ∼33% of mice from two independent transgenic lines and from backcrosses into either the FVB or the C57BL/6 strains succumbed to a similar disease characterized by splenomegaly, hepatomegaly, and frequent infiltration of leukocytes into nonhematopoietic organs like the kidneys and lungs. Although an excess of B or T cells was observed in three diseased mice, in 17 other cases, histologic and flow cytometry analyses revealed the expansion of a population of neutrophils in the blood, spleen, and bone marrow. The increase in neutrophils correlated with signs of anemia and thrombocytopenia, whereas there was no indication of a reactive process. Therefore, p75 CDP/Cux transgenic mice displayed heightened susceptibility to a disease defined as a myeloproliferative disease–like myeloid leukemia. These results indicate that the overexpression of p75 CDP/Cux could alter homeostasis in the hematopoietic compartment. (Cancer Res 2006; 66(19): 9492-501)

Supplementary Tables 1-2, Figure 1 from Mouse Mammary Tumor Virus p75 and p110 CUX1 Transgenic Mice Develop Mammary Tumors of Various Histologic Types

The p75 and p110 isoforms of the CUX1 homeodomain protein are overexpressed in breast tumors and cancer cell lines. To assess and compare the ability of these short CUX1 isoforms in driving mammary tumor development, we used site-specific transgenesis into the Hprt locus to generate transgenic mice expressing p75 or p110 CUX1 under the control of the mouse mammary tumor virus-long terminal repeat. We report that mammary tumors developed after a long latency period, and although various histopathologies were observed, the proportion of adenosquamous carcinomas was significantly higher in p75 CUX1 than in p110 CUX1 transgenic mice. Metastasis to the lung was observed in three p75 CUX1 transgenic mice. Comparisons between tumors and adjacent normal mammary glands revealed that transgenes were overexpressed in most but not all tumors, yet in all cases tested, CUX1 DNA binding was increased, suggesting that both higher expression and changes in post-translational modifications can contribute to stimulate transgene activity. Interestingly, higher expression of erbB2 mRNA was seen in most tumors, not only solid carcinomas but also adenosquamous carcinomas, whereas higher expression of various Wnt genes and activation of the β-catenin pathway was observed primarily in adenosquamous carcinomas. Activation of erbB2 expression appeared to represent a cooperating event that occurred independently of CUX1. In contrast, chromatin immunoprecipitation, short hairpin RNA–mediated knockdown, and reporter assays established that CUX1 is involved in the transcriptional regulation of several Wnt genes. Together, these results support the notion that oncogenic activity of CUX1 can facilitate the establishment of a Wnt/β-catenin autocrine loop. [Cancer Res 2009;69(18):7188–97]

The p75 and p110 isoforms of the CUX1 homeodomain protein are overexpressed in breast tumors and cancer cell lines. To assess and compare the ability of these short CUX1 isoforms in driving mammary tumor development, we used site-specific transgenesis into the Hprt locus to generate transgenic mice expressing p75 or p110 CUX1 under the control of the mouse mammary tumor virus-long terminal repeat. We report that mammary tumors developed after a long latency period, and although various histopathologies were observed, the proportion of adenosquamous carcinomas was significantly higher in p75 CUX1 than in p110 CUX1 transgenic mice. Metastasis to the lung was observed in three p75 CUX1 transgenic mice. Comparisons between tumors and adjacent normal mammary glands revealed that transgenes were overexpressed in most but not all tumors, yet in all cases tested, CUX1 DNA binding was increased, suggesting that both higher expression and changes in post-translational modifications can contribute to stimulate transgene activity. Interestingly, higher expression of erbB2 mRNA was seen in most tumors, not only solid carcinomas but also adenosquamous carcinomas, whereas higher expression of various Wnt genes and activation of the β-catenin pathway was observed primarily in adenosquamous carcinomas. Activation of erbB2 expression appeared to represent a cooperating event that occurred independently of CUX1. In contrast, chromatin immunoprecipitation, short hairpin RNA–mediated knockdown, and reporter assays established that CUX1 is involved in the transcriptional regulation of several Wnt genes. Together, these results support the notion that oncogenic activity of CUX1 can facilitate the establishment of a Wnt/β-catenin autocrine loop. [Cancer Res 2009;69(18):7188–97]