P53 genotype as a determinant of ER expression and tamoxifen response in the MMTV-Wnt-1 model of mammary carcinogenesis.

Clinical studies show that estrogen receptor-α (ER) expressing tumors tend to have better prognosis, respond to antiestrogen therapy and have wild-type p53. Conversely, tumors with inactivating mutations in p53 tend to have worse outcomes and to be ER-negative and unresponsive to antihormone treatment. Previous studies from our laboratory have shown that p53 regulates ER expression transcriptionally, by binding the ER promoter and forming a complex with CARM1, CBP, c-Jun, RNA polymerase II and Sp1. In this study, the MMTV-Wnt-1 transgenic mouse model was used to demonstrate that p53 regulation of ER expression and function is not solely an in vitro phenomenon, but it is also operational in mammary tumorigenesis in vivo. The expression of ER and the ability to respond to tamoxifen were determined in mammary tumors arising in p53 wild type (WT) or p53 heterozygous (HT) animals carrying the Wnt-1 transgene. In p53 WT mice, development of ER-positive tumors was delayed by tamoxifen treatment, while tumors arising in p53 HT mice had significantly reduced levels of ER and were not affected by tamoxifen. P53 null tumors were also found in the p53 HT mice and these tumors were ER-negative. ER expression was upregulated in mouse mammary tumor cell lines following transfection with WT p53 or treatment with doxorubicin. These data demonstrate that p53 regulates ER expression in vivo, and affects response to tamoxifen. Results also provide an explanation for the concordant relationship between these prognostic proteins in human breast tumors.

Overexpression of cyclooxygenase-2 (COX-2) in the mouse urinary bladder induces the expression of immune- and cell proliferation-related genes.

The mechanisms whereby cyclooxygenase-2 (COX-2) overexpression may contribute to bladder carcinogenesis remain unknown. We recently developed a transgenic mouse model overexpressing COX-2 under the control of a bovine keratin 5 (BK5) promoter causing a high incidence of transitional cell hyperplasia (TCH) in the bladder with a proportion of lesions progressing to invasive carcinoma. Microarray gene analysis was employed to determine the effects of COX-2 overexpression on gene expression profiles in the urinary bladder. Statistical analysis revealed that 70 genes were upregulated and 60 were downregulated by twofold or more in bladders from transgenic compared to wild-type mice. Expression Analysis Systematic Explorer (EASE) analysis revealed that genes associated with Immune/Stress Response and Cell Cycle/Proliferation biological processes were overexpressed in the transgenic mice. Relevant downregulated genes included three transforming growth factor (TGF)-beta related genes, Tgfb2, Tgfb3, and Tgfbi. The growth factor epiregulin was the most highly induced gene among those validated by qRT-PCR in TCH of BK5.COX-2 mouse bladders in parallel with increased staining for Ki67. Prostaglandin E(2) (PGE(2)) directly induced the expression of epiregulin mRNA in bladders from wild-type FVB mice ex vivo. We further determined that recombinant epiregulin increased both cell proliferation and Erk phosphorylation in UMUC-3 bladder cancer cells. These results indicate that the response of the mouse urinary bladder to elevated COX-2 expression includes enhanced inflammatory response and induction of cell proliferation. The growth factor epiregulin may play a role in bladder carcinogenesis and may serve as a novel target for the prevention and treatment of bladder cancer.

Paracrine overexpression of insulin-like growth factor-1 enhances mammary tumorigenesis in vivo.

Insulin-like growth factor-1 (IGF-1) stimulates proliferation, regulates tissue development, protects against apoptosis, and promotes the malignant phenotype in the breast and other organs. Some epidemiological studies have linked high circulating levels of IGF-1 with an increased risk of breast cancer. To study the role of IGF-1 in mammary tumorigenesis in vivo, we used transgenic mice in which overexpression of IGF-1 is under the control of the bovine keratin 5 (BK5) promoter and is directed to either the myoepithelial or basal cells in a variety of organs, including the mammary gland. This model closely recapitulates the paracrine exposure of breast epithelium to stromal IGF-1 seen in women. Histologically, mammary glands from transgenic mice were hyperplastic and highly vascularized. Mammary glands from prepubertal transgenic mice had significantly increased ductal proliferation compared with wild-type tissues, although this difference was not maintained after puberty. Transgenic mice also had increased susceptibility to mammary carcinogenesis, and 74% of the BK5.IGF-1 mice treated with 7,12-dimethylbenz[a]anthracene (20 microg/day) developed mammary tumors compared with 29% of the wild-type mice. Interestingly, 31% of the vehicle-treated BK5.IGF-1 animals, but none of the wild-type animals, spontaneously developed mammary cancer. The mammary tumors were moderately differentiated adenocarcinomas that expressed functional, nuclear estrogen receptor at both the protein and mRNA levels. These data support the hypothesis that tissue overexpression of IGF-1 stimulates mammary tumorigenesis.

The resistance to the tumor suppressive effects of COX inhibitors and COX-2 gene disruption in TRAMP mice is associated with the loss of COX expression in prostate tissue.

Over-expression of cyclooxygenase-2 (COX-2) and prostaglandin E(2) has been demonstrated to play a significant role in the tumorigenesis of colon, lung, breast, bladder and skin. However, inconsistent and controversial reports on the expression and activity of COX-2 in prostate cancer raised the question of whether COX-2 plays a pivotal role in prostate carcinogenesis. To address this question, we examined the effects of COX-2 inhibition on prostate tumorigenesis in the transgenic adenocarcinoma mouse prostate (TRAMP) model. Three-week-old TRAMP mice were fed control, celecoxib- or indomethacin-supplemented diets for 27 weeks. A TRAMP/COX-2 knockout mouse model was also generated to determine the effects of the loss of the COX-2 gene on prostate tumorigenesis in TRAMP mice. These studies demonstrated that neither non-steroidal anti-inflammatory drugs (NSAIDs) nor genetic disruption of COX-2 was inhibitory in terms of tumor and metastases incidence, lobe weight or types of pathological lesions. A careful analysis of wild-type and TRAMP tissues was undertaken for the expression of cyclooxygenase-1 (COX-1) and COX-2 using immunoblotting, quantitative real time polymerase chain reaction (qRT-PCR) and immunohistochemistry approaches in TRAMP dorsal prostate tissue from 10- and 16-week-old, as well as tumor from 30-week-old mice. We found that the expression of COX-1 and COX-2 dramatically decreased during TRAMP carcinogenesis. Using the probasin promoter, a COX-2 over-expressing mouse model was also generated but failed to show any pathology in any of the prostate lobes. Collectively, our results suggest that COX-2 may not play a tumorigenic role during prostate carcinogenesis in the TRAMP model.