Inhibition of cyclooxygenase-2 causes regression of gastric adenomas in trefoil factor 1 deficient mice.

Cyclooxygenase-2 (Cox-2) expression is a marker of reduced survival in gastric cancer patients, and inhibition of Cox-2 suppresses gastrointestinal carcinogenesis in experimental animal models. To investigate the role of Cox-2 in gastric carcinogenesis in vivo, we utilized trefoil factor 1 (Tff1) deficient mice, which model the neoplastic process of the stomach by developing gastric adenomas with full penetrance. These tumors express Cox-2 protein and mRNA, and we have now investigated the effects of genetic deletion of the mouse Cox-2 gene [also known as prostaglandin-endoperoxide synthase 2 (Ptgs2)] and a Cox-2 selective drug celecoxib. Our results show that genetic deletion of Cox-2 in the Tff1 deleted background resulted in reduced adenoma size and ulceration with a chronic inflammatory reaction at the site of the adenoma. To characterize the effect of Cox-2 inhibition in more detail, mice that had already developed an adenoma were fed with celecoxib for 8-14 weeks, which resulted in disruption of the adenoma that ranged from superficial erosion to deep ulcerated destruction accompanied with chronic inflammation. Importantly, mice fed with celecoxib for 16 weeks, followed by control food for 9 weeks, redeveloped a complete adenoma with no detectable inflammatory process. Finally, we determined the identity of the Cox-2 expressing cells and found them to be fibroblasts. Our results show that inhibition of Cox-2 is sufficient to reversibly disrupt gastric adenomas in mice.

PPARdelta is pro-tumorigenic in a mouse model of COX-2-induced mammary cancer.

Cyclooxygenase-2 (COX-2), overexpressed in inflammatory conditions and cancer, regulates angiogenesis and tumorigenesis via the production of biologically active prostanoids. Previously, we showed that COX-2 over-expression in the mammary gland of transgenic mice induces an angiogenic switch and transforms the mammary epithelium into invasive mammary carcinoma. Since COX-2-derived prostanoids can activate the nuclear receptor PPARdelta, we crossed Ppardelta(-/-) mice with COX-2 transgenic mice in the FVB/N background. PPARdelta was expressed constitutively in the mammary gland of virgin, pregnant and lactating mice. Mammary hyperplasia and tumorigenesis in the COX-2 transgenic mice was markedly reduced in the Ppardelta(-/-) mice compared to their wild type counterparts. Analysis of the mammary tissues indicated that immunoreactive Ki-67, cyclin D1 and phosphorylated histone 3 (Phospho H3) were reduced in Ppardelta(-/-) mice, suggesting that PPARdelta activation regulates cell proliferation in the mammary gland. We postulate that activation of the nuclear receptor PPARdelta by COX-2-derived prostanoids may be involved in the proliferation of mammary epithelial cells and therefore contribute to mammary cancer development.

Cytoplasmic HuR expression is a prognostic factor in invasive ductal breast carcinoma.

HuR is a ubiquitously expressed mRNA-binding protein. Intracellular localization of HuR is predominantly nuclear, but it shuttles between the nucleus and the cytoplasm. In the cytoplasm it can stabilize certain transcripts. Because nucleocytoplasmic translocation of HuR is necessary for its activity, it was hypothesized that cytoplasmic HuR expression in cancer cells could be a prognostic marker. To test the significance of HuR in carcinogenesis of the breast, we have investigated HuR expression in a mouse mammary gland tumor model and from 133 invasive ductal breast carcinoma specimens. HuR expression was elevated in the cyclooxygenase-2 transgene-induced mouse mammary tumors, and its expression was predominantly cytoplasmic in the tumor cells. In the human carcinoma samples, high cytoplasmic immunoreactivity for HuR was found in 29% (38 of 133) of the cases. Cytoplasmic HuR expression associated with high grade (P = 0.0050) and tumor size over 2 cm (P = 0.0082). Five-year distant disease-free survival rate was 42% [95% confidence interval (95% CI), 26-58] in cytoplasm-high category and 84% (95% CI, 76-91) in cytoplasm-negative or -low category (P < 0.0001), and high cytoplasmic expression of HuR was an independent prognostic factor in a Cox multivariate model (relative risk 2.07; 95% CI, 1.05-4.07). Moreover, high cytoplasmic HuR immunopositivity was significantly associated with poor outcome in the subgroup of node-negative breast cancer in a univariate analysis (P < 0.0007). Our results show that high cytoplasmic HuR expression is associated with a poor histologic differentiation, large tumor size, and poor survival in ductal breast carcinoma. Thus, HuR is the first mRNA stability protein of which expression associates with poor outcome in breast cancer.

VEGF-C and COX-2 expression in papillary thyroid cancer.

In papillary thyroid cancer (PTC), age appears to be the most important single prognostic factor. Another characteristic feature is the lack of association between survival and lymph node metastases. Earlier, we found that expression of cyclooxygenase-2 (COX-2) is higher in older PTC patients, in agreement with the finding that older patients have a worse prognosis. Recent findings suggest that COX-2 can up-regulate vascular endothelial growth factor-C (VEGF-C) expression. Here, we investigated whether expression of VEGF-C differs between young and older PTC patients and whether expression of VEGF-C and COX-2 are correlated. Our retrospective study comprised 106 PTC patients selected by age: those under 35 or over 55 at diagnosis. Paraffin-embedded tissue samples were analysed by immunohistochemistry for VEGF-C protein expression. Furthermore, we investigated by quantitative RT-PCR and enzyme immunoassay the relationship between VEGF-C and COX-2 expression in papillary thyroid cancer cells (NPA cells). VEGF-C expression was significantly increased with age. In the tumours from older lymph node-positive (N1) patients, VEGF-C expression was significantly higher than in the tumours from young N1 patients. Moreover, all patients who died of cancer or who developed distant metastases were old, and most tumours from these patients (4 of 5) expressed VEGF-C and had had nodal metastases at the time of primary operation. Immunohistochemically, expression of COX-2 and VEGF-C correlated strongly. In cell culture, this correlation was not so clear, because the COX-2 selective inhibitor, NS-398, did not reduce VEGF-C expression. However, as both COX-2 and VEGF-C were induced by the tumour promoter phorbol 12-myristate 13-acetate (PMA), the same factors may control them both.

Cyclooxygenase-2 expression and effect of celecoxib in gastric adenomas of trefoil factor 1-deficient mice.

Expression of cyclooxygenase-2 (Cox-2) is elevated in gastric adenocarcinomas and precursor lesions leading to this disease. Mice deficient for trefoil factor 1 (TFF1) develop a pyloric adenoma with full penetrance. Because inhibition of Cox-2 suppresses tumor growth in several animal models, we studied expression of Cox-2 and effect of a selective Cox-2 inhibitor celecoxib in gastrointestinal tissues of the TFF1-deficient mice. Cox-2 mRNA and protein were strongly expressed in the pyloric adenomas of the TFF1(-/-) mice as detected by in situ hybridization and immunohistochemistry. Nonneoplastic gastrointestinal tissues of wild-type or TFF1(-/-) mice expressed low or nondetectable levels of Cox-2. Celecoxib (1600 ppm p.o. for 3 months) caused ulceration and inflammation of the adenoma in all treated TFF1(-/-) mice (n = 7). This effect of the drug was adenoma specific, because no histological alterations were observed in the non-neoplastic gastric or intestinal tissues in the TFF1(-/-) or wild-type mice receiving the drug treatment. All untreated TFF1(-/-) mice had an adenoma (n = 7), but none demonstrated the combination of ulceration and inflammation. Our data show that Cox-2 is expressed in gastric adenomas of the TFF1(-/-) mice and suggest that inhibition of Cox-2 disturbs the integrity of the adenoma by promoting ulceration and inflammation. These findings support the effort to initiate clinical studies to investigate the effect of Cox-2 inhibitors as a chemotherapeutic modality for dysplasias of the stomach.

COX-2 inhibitors and genetic background reduce mammary tumorigenesis in cyclooxygenase-2 transgenic mice.

Cyclooxygenase-2 (COX-2) overexpression is a widely recognized feature of human breast cancer and inhibitors of the enzyme have antitumor effects in a subset of tumor settings. Previously, we demonstrated that direct overexpression of COX-2 under control of the mammary-specific MMTV promoter/enhancer, was itself oncogenic and lead to the induction of mammary tumors in multiparous, outbred CD1 mice. In the present study, we provide evidence that COX-2 dependent tumor progression can also be studied in FVB/N, an inbred strain widely used for analysis of breast cancer progression. In these mice, the human COX-2 transgene was strongly induced during pregnancy/lactation and mammary tumors developed after multiple pregnancies. However, crossing the COX-2 FVB/N mice with the C57BL6 strain resulted in loss of the mammary tumorigenic phenotype despite the fact that the human COX-2 gene was induced. Treatment of the COX-2 transgenic mice in the FVB/N strain with celecoxib (1600 ppm), a COX-2 selective inhibitor, resulted significant reduction in tumor size and multiplicity when compared to transgenic mice fed with control chow. SC-560 (20 ppm), a COX-1 selective inhibitor did not have significant effect on tumorigenesis. These studies suggest that FVB/N is a susceptible mouse strain well suited to the study of COX-2 mediated tumor progression and may provide a tool for the identification of interacting genes and therapeutic treatments for this clinically important target.

Empagliflozin reduces blood pressure in patients with type 2 diabetes and hypertension.

OBJECTIVE: To investigate the efficacy, safety, and tolerability of empagliflozin in patients with type 2 diabetes and hypertension. RESEARCH DESIGN AND METHODS: Patients (N = 825) with type 2 diabetes and hypertension (mean seated systolic blood pressure [SBP] 130-159 mmHg and diastolic blood pressure [DBP] 80-99 mmHg) were randomized (double blind) to 10 mg or 25 mg empagliflozin or placebo once daily for 12 weeks. RESULTS: At week 12, adjusted mean difference versus placebo in change from baseline in mean 24-h SBP (ambulatory blood pressure monitoring [ABPM]) was -3.44 mmHg (95% CI -4.78, -2.09) with 10 mg empagliflozin and -4.16 mmHg (-5.50, -2.83) with 25 mg empagliflozin (both P < 0.001). At week 12, adjusted mean difference versus placebo in change from baseline in mean 24-h DBP (ABPM) was -1.36 mmHg (95% CI -2.15, -0.56) with 10 mg empagliflozin and -1.72 mmHg (95% CI -2.51, -0.93) with 25 mg empagliflozin (both P < 0.001). Changes in office BP were consistent with ABPM. Adjusted mean difference versus placebo in change from baseline in HbA1c at week 12 was -0.62% (95% CI -0.72, -0.52) (-6.8 mmol/mol [95% CI -7.9, -5.7]) with 10 mg empagliflozin and -0.65% (95% CI -0.75, -0.55) (-7.1 mmol/mol [95% CI -8.2, -6.0]) with 25 mg empagliflozin (both P < 0.001). Empagliflozin was well tolerated. One patient on placebo and one patient on 10 mg empagliflozin reported events consistent with volume depletion. CONCLUSIONS: Empagliflozin was associated with significant and clinically meaningful reductions in BP and HbA1c versus placebo and was well tolerated in patients with type 2 diabetes and hypertension.