TI - Results . AB - Wnt-1 TG mice have been used to assess the functions of a number of genetic lesions in breast carcinogenesis because tumors appear with predictable kinetics . The median age at which tumors are detected is 5-6 months in MMTV-Wnt-1 TG females [27 , 28 , 30 , 31] . After one mutated allele of Pten was bred into this transgenic line , tumors appeared significantly earlier than in sibling females harboring only a MMTV-Wnt-1 transgene ( P lt 0001 , Fig 1A ) . Fifty percent of Pten+/- , Wnt-1 TG female mice formed tumors by 3.5 months , 2.5 months earlier than Wnt-1 TG mice ( Fig 1A ) . While it took one year for the entire cohort ( 15 ) of Wnt-1 TG females with a wild type Pten background to develop tumors , all Wnt-1 TG , Pten+/- females showed tumors by 5 months of age . Five each of Pten+/- mice without a Wnt-1 transgene were monitored for 7 months and 10.5 months , but none developed any palpable mammary lesions . These observations differ from a recent report that one of the other two Pten+/- lines develops fibroadenomas and adenocarcinomas in the mammary tissue after 7 months of age [26] . The lack of a mammary phenotype in our studies could be due to differences in the targeting constructs , the genetic backgrounds , or even environmental agents . By one year of age , approximately 15% of Wnt-1 TG males have been reported to develop mammary tumors [27 , 28 , 31] . In our cross , one of the four tumors from Wnt-1 TG males , in an otherwise wild type background , was a mammary carcinoma ; the other three tumors were salivary in origin , with a similar histopathology to the mammary lesion . The appearance of tumors was greatly accelerated in the Pten heterozygous background ( Fig 1B ) . Fifty percent of males developed tumors by 5.5 months of age , and all developed tumors within 10 months . As in the case of Wnt-1 TG male mice without Pten mutations , the majority of tumors ( 14/22 ) arose in the salivary tissue ; only a small number of tumors ( 2/22 ) were mammary in origin , and , interestingly , some ( 6/22 ) were bilateral or unilateral muzzle tumors of epithelial origin , similar histopathologically to lesions found in mammary and salivary tissues . To determine if the wild type allele of Pten was inactivated in tumors arising in a Pten+/- background , 11 tumors from Wnt-1 TG , Pten+/- mice were examined for LOH by Southern hybridization . Seven tumors had lost the wild type Pten allele ( see Fig 2 for an example of the analysis ) . This result suggests that , in tumorigenesis , cells that lack a functional allele of Pten have a significant proliferative advantage over the cells that retain the normal copy of Pten . Histological examination showed that tumors from Wnt-1 TG mice , regardless of Pten status , were adenocarcinomas with cribriform , cystic and focal papillary growth patterns . However , 3 of the 6 tumors that were Wnt-1 TG , Pten +/- with LOH appeared to have a higher nuclear grade , more cellular stroma and a more infiltrative pattern compared to the tumors without a Pten defect ( Fig 3 ) , suggesting a more aggressive phenotype . In human cancer , a tumor suppressor gene can be inactivated by mutations , chromosomal loss , promoter silencing , and other means . To determine if the Pten protein was produced in Wnt-1 TG , Pten+/- tumors that did not undergo LOH , sections were analyzed using an immunohistochemical assay with a polyclonal rabbit antibody . Abundant amounts of Pten were detected in mammary ducts from wild type mice . Pten was detected , at reduced levels , in both hyperplastic ducts and mammary tumors that did not have LOH at the Pten allele ( Fig 4 ) , suggesting that Pten was , indeed , made from the second allele . Similar low levels of Pten were also detected in mammary ductal epithelial cells in Pten heterozygous mice that did not have a Wnt-1 transgene , suggesting that the Wnt-1 transgene does not influence Pten expression . As expected , Pten was not detected in tumors that had undergone LOH at the Pten locus (Fig 4E) . Inactivation of Pten promotes membrane recruitment of AKT and its subsequent activation by PHOSphorylation at Ser473 and Thr308 [10 , 11 , 13] . All three human isoforms of AKT have been found to be deregulated in human breast cancer cells [32,33,34] . To determine if activated AKT was increased in tumors that had a Pten defect , these sections were stained using an antibody that specifically detects AKT1 phosphorylaTED at Ser 473 ( and AKT2 and AKT3 PHOSphorylated at equivalent sites ) . Tumors that were Wnt-1 TG , Pten+/- with LOH displayed staining of PHOSphorylated AKT (Fig 5A) . Surprisingly , the staining was patchy , suggesting additional factors may be required for AKT activation . As expected , PHOSphorylated AKT was undetectable in hyperplastic ducts , in tumors from Wnt-1 TG , Pten+/+ mice , and tumors from Wnt-1 TG , Pten+/- mice that retained the wild type allele ( Fig 5B & C ) . Several apoptotic pathways may be inhibited upon activation of AKT , promoting maintenance and survival of tumor cells . Cellular proliferation may also be enhanced by activated AKT , which inhibits p27 and GSK-3 ( leading to stabilization of cyclin D1 ) [35] . However , tumors from Wnt-1 TG , Pten+/+ mice have low apoptotic rates ( 40 + - 16 per digital camera field using a 20x objective ) and high proliferation rates ( 53% + - 11 ) . Neither apoptotic nor proliferation rates were affected by the status of Pten when measured by the TUNEL ( terminal deoxynucleotidyltransferase-mediated UTP end labeling ) assay , which labels the ends of fragmented DNA present in apoptotic cells and immunohistochemical staining for Ki67 , a cell proliferation marker ( data not shown ) . Most Wnt-1 TG mice ( 38/49 ) developed only one palpable tumor regardless of Pten status . Fifty-three of 55 single sections from Pten+/+ glands that were free of a palpable mass did not show any lesions more advanced than ductal hyperplasia . However , at the time of sacrifice , the majority ( 50/56 ) of glands from Wnt-1 TG , Pten+/- mice had intra-ductal squamous lesions of various sizes ( Fig 3D ) . These squamous lesions were usually multi-focal and located both at the periphery of the adenocarcinomas and in other mammary glands free of adenocarcinomas . Since Pten mutations are usually found in advanced tumors , and the protein has been suggested to inhibit cell spreading , migration and invasion [36 , 37] , it is possible that the tumors from Wnt-1 TG , Pten+/- mice may have a higher potential to metastasize than those from mice carrying a Wnt-1 transgene in a wild type Pten background . Wnt-1 TG mice occasionally show lymphoid or pulmonary metastasis [27] , and serial sectioning of lung tissues from the animals reported here revealed microsocopic metastatic foci in two of five lungs from Wnt-1 TG mice bearing primary tumors 1.5-2.0 cm in size . However , Wnt-1 TG mice and the Wnt-1 TG , Pten+/- mice harbored metastatic foci at similar frequencies ( 5/10 ) . ( Statistical evaluation was not performed because the sample sizes were too small ) .