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In tumor cells, cyclin E deregulation results in the appearance of five low molecular weight (LMW) isoforms. When overexpressed in breast cancer cells, these forms of cyclin E induce genomic instability, resistance to inhibition by p21 and p27, and resistance to antiestrogen therapy. Additionally, the LMW forms of cyclin E strongly correlate with decreased survival in patients with breast cancer. However, the oncologic role of the LMW forms of cyclin E in breast cancer tumorigenesis is yet to be determined. To this end, we generated transgenic mice expressing full-length cyclin E alone (M46A), full-length and the EL4 isoforms (EL1/EL4), or the EL2/3 isoforms of cyclin E (T1) under the control of the mouse mammary tumor virus promoter. Compared with full-length cyclin E, LMW cyclin E overexpression induces delayed mammary growth during the pubertal phase and abnormal cell morphology during lactation. Both primary mammary tumor formation and metastasis were markedly enhanced in LMW cyclin E transgenic mice. LMW cyclin E overexpression in mammary epithelial cells of mice is sufficient by itself to induce mammary adenocarcinomas in 34 of 124 (27%) animals compared with 7 of 67 (10.4%) mice expressing only the full-length cyclin E (P < 0.05). In addition, metastasis was seen in 25% of LMW cyclin E tumor–bearing animals compared with only 8.3% of tumors in the full-length cyclin E background (P < 0.05). Moreover, LMW cyclin E overexpression selects for inactivation of p53 by loss of heterozygosity and spontaneous and frequent inactivation of ARF. Therefore, LMW cyclin E overexpression strongly selects for spontaneous inactivation of the ARF-p53 pathway in vivo, canceling its protective checkpoint function and accelerating progression to malignancy. [Cancer Res 2007;67(15):7212–22]

The inducible form of cyclooxygenase (COX), COX-2, is up-regulated in many epithelial cancers and its prostaglandin products increase proliferation, enhance angiogenesis, and inhibit apoptosis in several tissues. Pharmacologic inhibition and genetic deletion studies showed a marked reduction of tumor development in colon and skin. COX-2 has also been strongly implicated in urinary bladder cancer primarily by studies with nonselective COX- and COX-2-selective inhibitors. We now show that forced expression of COX-2, under the control of a keratin 5 promoter, is sufficient to cause transitional cell hyperplasia (TCH) in 17% and 75% of the heterozygous and homozygous transgenic lines, respectively, in an age-dependent manner. TCH was strongly associated with inflammation, primarily nodules of B lymphocytes; some T cells and macrophage infiltration were also observed. Additionally, transitional cell carcinoma was observed in ∼10% of the K5.COX-2 transgenic mice; no TCH or transitional cell carcinoma was observed in wild-type bladders. Immunohistochemistry for vascular proliferation and vascular endothelial growth factor showed significant increases above that in wild-type urinary bladders. Our results suggest that overexpression of COX-2 is sufficient to cause hyperplasia and carcinomas in the urinary bladder. Therefore, inhibition of COX-2 should continue to be pursued as a potential chemopreventive and therapeutic strategy.

p53 levels are tightly regulated in normal cells, and thus, the wild-type p53 protein is nearly undetectable until stimulated through a variety of stresses. In response to stress, p53 is released from its negative regulators, mainly murine double minute 2 (Mdm2), allowing p53 to be stabilized to activate cell-cycle arrest, senescence, and apoptosis programs. Many of the upstream signals that regulate wild-type p53 are known; however, limited information for the regulation of mutant p53 exists. Previously, we showed that wild-type and mutant p53R172H are regulated in a similar manner in the absence of Mdm2 or p16. In addition, this stabilization of mutant p53 is responsible for the gain-of-function metastatic phenotype observed in the mouse. In this report, we examined the role of oncogenes, DNA damage, and reactive oxygen species, signals that stabilize wild-type p53, on the stabilization of mutant p53 in vivo and the consequences of this expression on tumor formation and survival. These factors stabilized mutant p53 protein which oftentimes contributed to exacerbated tumor phenotypes. These findings, coupled with the fact that patients carry p53 mutations without stabilization of p53, suggest that personalized therapeutic schemes may be needed for individual patients depending on their p53 status. Cancer Res; 71(23); 7168–75. ©2011 AACR.

Cyclin E activates Cdk2, controls centrosome duplication, and regulates histone gene transcription. Cyclin E is deregulated in cancer and appears as low-molecular-weight (LMW) isoforms that correlate strongly with decreased survival in breast cancer patients. Transgenic mice overexpressing LMW-cyclin E have increased incidence of mammary tumors and distant metastasis when compared with mice that had full-length cyclin E. To specifically test the requirement for Cdk2 in LMW-cyclin E–mediated mammary tumorigenesis, we generated transgenic mice, which expressed LMW-cyclin E in a Cdk2-deficient background. We found that mammary gland development proceeds relatively normally in these animals, indicating that Cdk2 kinase activity is largely dispensable for this process. However, Cdk2-deficient mice were completely resistant to LMW-cyclin E–mediated mammary tumors. Cdk2 wild-type or heterozygous mice succumbed to mammary tumors with mean latencies of 16 and 19.5 months, respectively, but Cdk2 nullizygous littermates did not display tumors through 24 months. Similarly, continuous administration of two different Cdk inhibitors significantly delayed LMW-cyclin E–induced mammary tumor progression. Triple transgenic mice generated in a p53 heterozygous background also displayed no tumors. Finally, we found that Cdk2 silencing induced cell death in LMW-overexpressing breast cancer cell lines, but not in cell lines lacking LMW expression. Our findings establish a requirement for Cdk2 in LMW-cyclin E–mediated mammary tumorigenesis, arguing that human breast tumors overexpressing LMW-cyclin E are prime candidates for anti-Cdk2 therapy. Cancer Res; 71(9); 3377–86. ©2011 AACR.

High levels of the critical p53 inhibitor Mdm4 is common in tumors that retain a wild-type p53 allele, suggesting that Mdm4 overexpression is an important mechanism for p53 inactivation during tumorigenesis. To test this hypothesis in vivo, we generated transgenic mice with widespread expression of Mdm4. Two independent lines of transgenic mice, Mdm4Tg1 and Mdm4Tg15, developed spontaneous tumors, the most prevalent of which were sarcomas. To determine whether overexpression of Mdm4 also cooperated with p53 heterozygosity to induce tumorigenesis, we generated Mdm4Tg1 p53+/− mice. These mice had significantly accelerated tumorigenesis and a distinct tumor spectrum with more carcinomas and significantly fewer lymphomas than p53+/− or Mdm4Tg1 mice. Importantly, the remaining wild-type p53 allele was retained in most Mdm4Tg1 p53+/− tumors. Mdm4 is thus a bona fide oncogene in vivo and cooperates with p53 heterozygosity to drive tumorigenesis. These Mdm4 mice will be invaluable for in vivo drug studies of Mdm4 inhibitors. Cancer Res; 70(18); 7148–54. ©2010 AACR.

Cyclin E overexpression and generation of hyperactive LMW cyclin E is oncogenic and has been implicated in a variety of human cancers. We have developed a doxycycline-inducible LMW cyclin E transgenic mouse model to determine the dependence of established mammary adenocarcinomas on continued expression of LMW cyclin E. Mice harboring this TetO-LMW-E transgene, referred to as TLMW, were mated to MMTV-rtTA transgenic mice (MTB) to yield bitransgenic MTB/TLMW mice. Morphological examination of hematoxylin and eosin-stained sections showed that the mammary glands of induced MTB/TLMW mice following four days of doxycycline induction were hyperplastic compared with uninduced MTB/ TLMW animals. Consistent with this, immunohistochemistry using antibodies specific for BrdU revealed a 2-fold increase in BrdU incorporation in the mammary epithelium of induced bitransgenic females compared with uninduced mice (17.1+/−5.3% versus 30.9+/−4.9%, P=0.03). This data indicate that LMW cyclin E overexpression in the mammary epithelium increased proliferation. To determine whether sustained induction of transgene expression would result in mammary tumorigenesis in MTB/TLMW mice, a cohort of 27 bitransgenic females was treated with doxycycline beginning at 6 weeks of age. 41% of these mice developed tumors within 2 years of starting doxycycline with kinetics (T1/2= 20 months) resembling that described for MMTV-LMW cyclin E mice. Similarly, the predominant type of tumor observed for the inducible LMW forms was glandular adenocarcinomas with adenosquamous differentiation. Lung metastases are known to arise in a fraction of MMTV-LMW cyclin E transgenic mice harboring primary mammary tumors. Accordingly, we sacrificed a subset of mammary tumor bearing MTB/TLMW mice and examined organs at necropsy for evidence of metastatic spread. 4 of 7 mice examined had lung metastasizes (57%). Control mice without dox never developed mammary tumors. To shed further light on the molecular mechanisms of mammary tumorigenesis caused by LMW-E expression, we conducted DNA microarray analyses. Using the R package limma, 641 probes out of 30,854 probes were identified as differentially regulated by at least 2-fold by LMW-E in the tumors when compared to the contralateral mammary glands. Gene Ontology analysis identified genes on this list that were involved in a variety of processes, including cell cycle control of chromosome replication, and DNA methylation and transcriptional repression signaling. Based on histology and gene expression profile of the resultant mammary tumors, we propose that the MTB/TLMW is a murine model of breast cancer resembling the human invasive basal/triple negative breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3283. doi:1538-7445.AM2012-3283

Lung carcinogenesis occurs through a multi-step process, which involves the activation or over-expression of oncogenes and the inactivation or silencing of tumor-suppressor genes. Mouse models for human lung cancer have proven to be valuable tools for understanding the basic tumor biology as well as for the development and validation of new approaches to cancer prevention and therapy. Previously, we found that mice with a knockout (KO) of G-protein coupled receptor 5A (Gprc5a) develop lung tumors after a long latent period (12 to 24 months). To determine whether a tobacco carcinogen will enhance tumorigenesis in this model, we administered 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) i.p. to 2-months old Gprc5a-KO mice and sacrificed groups (n=5) of mice at 6, 9, 12, and 18 months later. Compared to control Gprc5a-KO mice, NNK-treated mice developed lung tumors at least 6 months earlier, exhibited 2- to 4-fold increased tumor incidence and multiplicity, and showed a dramatic increase in lesion size. A signature of differentially expressed genes derived by transcriptome analysis of epithelial cell lines from normal lungs of Gprc5a-KO mice and from NNK-induced adenocarcinoma was highly similar to differential expression patterns observed between normal and tumorigenic human lung cells. The signature also separated both mouse and human adenocarcinomas from normal lung tissues based on publicly available microarray datasets. A key feature of the signature, up-regulation of UBE2C, MCM2, and FEN-1, was validated in mouse normal lung and adenocarcinoma tissues and cells by immunohistochemistry and western blotting, respectively. Our findings support the following conclusions: 1) lung tumorigenesis in the Gprc5a-KO mouse model is augmented by NNK, 2) gene expression changes induced by tobacco carcinogen(s) are conserved between mouse and human lung epithelial cells, 3) the Gprc5a-KO mouse model recapitulates human lung adenocarcinoma. Thus, the Gprc5a-KO mouse model could be useful for cancer chemoprevention studies. Supported by the Samueal Waxman Cancer Research Foundation and the NCI Cancer Center Core Grant P30-CA16672. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2466.

A cell surface Mr approximately 90,000 glycoprotein (gp90) was found in higher amounts on brain-colonizing than on lung-colonizing murine B16 melanoma sublines. The possible role of gp90 in determining the brain-associated metastatic properties of B16 cells was examined by purifying the glycoprotein and studying the effects of anti-gp90 on the growth, adhesion, and organ colonization properties of B16 cells. The specificity of the anti-gp90 was demonstrated in immunoprecipitation studies where a cell surface- or metabolically labeled Mr approximately 90,000 glycoprotein of pI approximately 4 was exclusively found upon two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Immunoprecipitation analysis, enzyme-linked immunosorbent assays, and the lectin-binding properties of gp90 on lectin affinity columns indicated that it is a Mr approximately 180,000 disulfide-linked dimer, probably related to the transferrin receptor. B16 sublines selected for various organ colonization properties differentially expressed gp90, bound 125I-labeled transferrin, and responded differently to purified transferrin in proliferation assays in relation to their metastatic properties (B15b greater than O13 greater than F10 greater than F1). Anti-gp90 immunoglobulin G affected the growth of brain-colonizing B16-B15b more than B16-F1 cells, but had no effect on the adhesion of B16-B15b or -F1 cells to microvessel endothelial cells in vitro, and anti-gp90 immunoglobulin G F(ab')2 had little effect on the brain colonization properties of B16-B15b cells in syngeneic mice. In blocking assays, anti-gp90 inhibited the binding of 125I-labeled transferrin to B16-B15b cells in a dose-dependent manner. The results suggest that the differential growth-stimulating effects of transferrin on highly metastatic B16 melanoma cells may be due to their differential expression of a Mr approximately 90,000 glycoprotein that is related to the transferrin receptor. In organ sites, such as the brain, differential expression of a transferrin-like receptor may allow metastatic cells to respond to low concentrations of growth factors known to be present in certain organs.