Inactivation of Apc in the mouse prostate causes prostate carcinoma.

Alterations of the Wnt/beta-catenin signaling pathway are positively associated with the development and progression of human cancer, including carcinoma of the prostate. To determine the role of activated Wnt/beta-catenin signaling in mouse prostate carcinogenesis, we created a mouse prostate tumor model using probasin-Cre-mediated deletion of Apc. Prostate tumors induced by the deletion of Apc have elevated levels of beta-catenin protein and are highly proliferative. Tumor formation is fully penetrant and follows a consistent pattern of progression. Hyperplasia is observed as early as 4.5 weeks of age, and adenocarcinoma is observed by 7 months. Continued tumor growth usually necessitated sacrifice between 12 and 15 months of age. Despite the high proliferation rate, we have not observed metastasis of these tumors to the lymph nodes or other organs. Surgical castration of 6-week-old mice inhibited tumor formation, and castration of mice with more advanced tumors resulted in the partial regression of specific prostate glands. However, significant areas of carcinoma remained 2 months postcastration, suggesting that tumors induced by Apc loss of function are capable of growth under conditions of androgen depletion. We conclude that the prostate-specific deletion of Apc and the increased expression of beta-catenin associated with prostate carcinoma suggests a role for beta-catenin in prostate cancer and offers an appropriate animal model to investigate the interaction of Wnt signaling with other genetic and epigenetic signals in prostate carcinogenesis.

Identification of a met-binding peptide from a phage display library.

PURPOSE: Aberrant c-Met expression has been implicated in most types of human cancer. We are developing Met-directed imaging and therapeutic agents. EXPERIMENTAL DESIGN: To seek peptides that bind specifically to receptor Met, the Met-expressing cell lines S114 and SK-LMS-1 were used for biopanning with a random peptide phage display library. Competition ELISA, fluorescence-activated cell sorting analysis, an internalization assay, and a cell proliferation assay were used to characterize a Met-binding peptide in vitro. To evaluate the utility of the peptide as a diagnostic agent in vivo, 125I-labeled peptide was injected i.v. into nude mice bearing s.c. xenografts of the Met-expressing and hepatocyte growth factor (HGF)/scatter factor-expressing SK-LMS-1/HGF, and total body scintigrams were obtained between 1 and 24 h postinjection. RESULTS: One Met-binding peptide (YLFSVHWPPLKA), designated Met-pep1, reacts with Met on the cell surface and competes with HGF/scatter factor binding to Met in a dose-dependent manner. Met-pep1 is internalized by Met-expressing cells after receptor binding. Met-pep1 inhibits human leiomyosarcoma SK-LMS-1 cell proliferation in vitro. In SK-LMS-1 mouse xenografts, tumor-associated activity was imaged as early as 1 h postinjection and remained visible in some animals as late as 24 h postinjection. CONCLUSIONS: Met-pep1 specifically interacts with Met: it is internalized by Met-expressing cells and inhibits tumor cell proliferation in vitro; it is a potential diagnostic agent for tumor imaging.

Decreased BMD and limb deformities in mice carrying mutations in both Lrp5 and Lrp6.

UNLABELLED: Humans and mice lacking Lrp5 have low BMD. To evaluate whether Lrp5 and Lrp6 interact genetically to control bone or skeletal development, we created mice carrying mutations in both Lrp5 and the related gene Lrp6. We found that compound mutants had dose-dependent deficits in BMD and limb formation, suggesting functional redundancy between these two genes in bone and limb development. INTRODUCTION: Lrp5 and Lrp6 are closely related members of the low density lipoprotein receptor family and are co-receptors for Wnt ligands. While Lrp5 mutations are associated with low BMD in humans and mice, the role of Lrp6 in bone formation has not been analyzed. MATERIALS AND METHODS: To address whether Lrp5 and Lrp6 play complimentary roles in bone and skeletal development, we created mice with mutations in both genes. We inspected limbs of mice from the different genotypic classes of compound mutants to identify abnormalities. DXA and muCT were used to evaluate the effect of mutations in Lrp5 and Lrp6 on BMD and microarchitecture. RESULTS: Mice heterozygous for mutations in Lrp6 and either heterozygous or homozygous for a mutation in Lrp5 (Lrp6(+/-);Lrp5(+/-) or Lrp6(+/-);Lrp5(-/-)) display limb defects with incomplete penetrance and variable expression. DXA analysis showed that BMD decreased as mice progressively were more deficient in Lrp5 and Lrp6. Lrp6(+/-);Lrp5(-/-) mice were more severely affected than Lrp6(+/+);Lrp5(-/-) mice, whereas Lrp6(+/-);Lrp5(+/-) mice had statistically higher BMD than Lrp6(+/+);Lrp5(-/-) mice and lower BMD compared with wildtype mice and mice heterozygous for either mutation alone. CONCLUSIONS: Lrp6 and Lrp5 genetically interact in limb development in mice. Furthermore, heterozygosity for an inactivating mutation in Lrp6 further reduces BMD in both male and female mice lacking Lrp5.

A novel multipurpose monoclonal antibody for evaluating human c-Met expression in preclinical and clinical settings.

The inappropriate expression of the c-MET cell surface receptor in many human solid tumors necessitates the development of companion diagnostics to identify those patients who could benefit from c-MET targeted therapies. Tumor tissues are formalin fixed and paraffin embedded (FFPE) for histopathologic evaluation, making the development of an antibody against c-MET that accurately and reproducibly detects the protein in FFPE samples an urgent need. We have developed a monoclonal antibody (mAb), designated MET4, from a panel of MET-avid mAbs, based on its specific staining pattern in FFPE preparations. The accuracy of MET4 immunohistochemistry (MET4-IHC) was assessed by comparing MET4-IHC in FFPE cell pellets with immunoblotting analysis. The technical reproducibility of MET4-IHC possessed a percentage coefficient of variability of 6.25% in intra-assay and interassay testing. Comparison with other commercial c-MET antibody detection reagents demonstrated equal specificity and increased sensitivity for c-MET detection in prostate tissues. In cohorts of ovarian cancers and gliomas, MET4 reacted with ovarian cancers of all histologic subtypes (strong staining in 25%) and with 63% of gliomas. In addition, MET4 bound c-MET on the surfaces of cultured human cancer cells and tumor xenografts. In summary, the MET4 mAb accurately and reproducibly measures c-MET expression by IHC in FFPE tissues and can be used for molecular imaging in vivo. These properties encourage further development of MET4 as a multipurpose molecular diagnostics reagent to help to guide appropriate selection of patients being considered for treatment with c-MET-antagonistic drugs.