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
]. 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 < 0.001, Fig. ). Fifty percent of Pten
TG female mice formed tumors by 3.5 months, 2.5 months earlier than Wnt-1 TG
mice (Fig. ). 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
+/- 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.
Figure 1 Pten heterozygosity accelerates tumor appearance in Wnt-1 TG females (A) and males (B). Percentages of mice that were tumor-free, as determined by weekly visual inspection and/or palpation, were plotted against the age when tumors were found. Genotypes (more ...)
By one year of age, approximately 15% of Wnt-1
TG males have been reported to develop mammary tumors [27
]. 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. ). 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. 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.
Figure 2 Example of LOH analysis. Blots of tumor and tail DNAs, digested with PstI, were probed with a 32P labeled Pten probe. Two bands could be observed, representing wild type and mutant alleles, as indicated at the left. Genotypes of mice are shown at the (more ...)
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. ), suggesting a more aggressive phenotype.
Figure 3 Representative H+E sections of mammary tumors from progeny females of the MMTV-Wnt-1 and Pten+/- cross. Sections were from tumors that were Wnt-1 TG+, Pten+/+ (A); Wnt-1 TG+, Pten+/- (B); and Wnt-1 TG+, Pten+/- with LOH (C and D). A 40x objective was (more ...)
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. ), 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. ).
Figure 4 Immunohistochemical staining for Pten. Sections of mammary glands (A, D) and tumors (B, C, E, F) were stained for Pten (A, B, D, E, F) or control rabbit Ig (C) and counterstained with hematoxylin. Genotypes of mice were Pten+/+ (A); Wnt-1 TG, Pten+/+ (more ...)
Inactivation of Pten
promotes membrane recruitment of AKT and its subsequent activation by phosphorylation at Ser473 and Thr308 [10
]. All three human isoforms of AKT have been found to be deregulated in human breast cancer cells [32
]. 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
+/- with LOH displayed staining of phosphorylated AKT (Fig. ). 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
+/+ mice, and tumors from Wnt-1
+/- mice that retained the wild type allele (Fig. ).
Figure 5 Immunohistochemical staining for phosphorylated Akt. Sections of tumors were stained for phospho-Akt (A, B, C) or control rabbit IgG (D) and counterstained with hematoxylin. Genotypes of mice were Wnt-1 TG+, Pten+/- (A, B); Wnt-1 TG+, Pten+/+ (C, D). (more ...)
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
+/+ 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. ). These squamous lesions were usually multi-focal and located both at the periphery of the adenocarcinomas and in other mammary glands free of adenocarcinomas.
mutations are usually found in advanced tumors, and the protein has been suggested to inhibit cell spreading, migration, and invasion [36
], it is possible that the tumors from Wnt-1
+/- mice may have a higher potential to metastasize than those from mice carrying a Wnt-1
transgene in a wild type Pten
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
+/- mice harbored metastatic foci at similar frequencies (5/10). (Statistical evaluation was not performed because the sample sizes were too small.)